Thesis
Toll-like receptor agonist decorated chitosan nanoparticles for
pulmonary DNA vaccination
HEUKING, Simon
Abstract
Ce travail de thèse vise à l´évaluation de nouvelles nanoparticules (NP) vectorisant un
antigène sous forme d'acide nucléique, fonctionnalisées par de puissants adjuvants pour une
vaccination par voie respiratoire. La vaccination par ADN est une nouvelle approche vaccinale
pour induire une réponse immunitaire spécifique contre un agent pathogène. Suite à son
administration, un vaccin à ADN permet la synthèse in vivo de l´antigène, puis sa présentation
sous forme de peptides antigéniques par les molécules du complexe majeur
d´histocompatibilité (CMH) de classe I. Par conséquent, les lymphocytes T CD8+ sont
stimulés, et déclenchent une réponse cytotoxique dirigée contre l´antigène. Malgré des
résultats pré-cliniques prometteurs de vaccination par ADN, les essais réalisés chez le
primate ou chez l'Homme n´ont pas permis l'induction d'une réponse immunitaire protectrice.
A cause de cela, plusieurs stratégies ont été proposées, comme le changement de la voie
d'administration, la complexation de l'ADN avec un polymère sous forme (nano)particulaire ou
l´ajout d´un adjuvant.
Reference
HEUKING, Simon. Toll-like receptor agonist decorated chitosan nanoparticles for
pulmonary DNA vaccination. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4242
URN : urn:nbn:ch:unige-119140
Available at:
http://archive-ouverte.unige.ch/unige:11914
Disclaimer: layout of this document may differ from the published version.
[ Downloaded 28/11/2016 at 12:10:45 ]
UNIVERSITE DE GENEVE
SECTION DES SCIENCES PHARMACEUTIQUES
LABORATOIRE DE PHARMACIE GALENIQUE
ET DE BIOPHARMACIE
FACULTE DES SCIENCES
PROFESSEUR GERRIT BORCHARD
Toll-like receptor agonist
decorated chitosan nanoparticles
for pulmonary DNA vaccination
THESE
présentée à la Faculté des Sciences de l´Université de Genève
pour obtenir le grade de Docteur en Sciences,
mention sciences pharmaceutiques
par
Simon HEUKING
de
Hünxe
(Allemagne)
Thèse N°4242
Genève
Atelier d´impression ReproMail
2010
2
To my families
in Germany,
Sweden
and Switzerland.
3
Stufen
Wie jede Blüte welkt
und jede Jugend dem Alter weicht,
blüht jede Lebensstufe,
blüht jede Weisheit auch und jede Tugend
zu ihrer Zeit und darf nicht ewig dauern.
Es muss das Herz bei jedem Lebensrufe
bereit zum Abschied sein und Neubeginne,
um sich in Tapferkeit und ohne Trauern
in and're, neue Bindungen zu geben.
Und jedem Anfang wohnt ein Zauber inne,
der uns beschützt und der uns hilft zu leben.
Wir sollen heiter Raum um Raum durchschreiten,
an keinem wie an einer Heimat hängen,
der Weltgeist will nicht fesseln uns und engen,
er will uns Stuf' um Stufe heben, weiten.
Kaum sind wir heimisch einem Lebenskreise
und traulich eingewohnt,
so droht Erschlaffen.
Nur wer bereit zu Aufbruch ist und Reise,
mag lähmender Gewöhnung sich entraffen.
Es wird vielleicht auch noch die Todesstunde
uns neuen Räumen jung entgegen senden:
des Lebens Ruf an uns wird niemals enden ...
Wohlan denn, Herz, nimm Abschied und gesunde !
Hermann Hesse
4
TABLE OF CONTENTS
Part I – Introduction
Chapter 1
Progress in chitosan-based vaccine delivery systems
7
Chapter 2
Aim of the thesis and study objectives
45
Part II – Preparation and in vitro characterization of TLRagonist functionalized DNA nanoparticles
Chapter 3
Toll-like receptor-2 agonist functionalized biopolymer for mucosal
vaccination
52
Chapter 4
Stimulation of macrophages using Toll-like receptor-2
(TLR-2) agonist decorated nanocarriers
79
Chapter 5
Functionalization with a TLR-7 agonist enhances the
immunogenicity of chitosan DNA nanoparticles in human THP-1
macrophages
109
Part III – In vitro and in vivo evaluation of TLR-agonist
functionalized nanoparticles for pulmonary DNA vaccination
Chapter 6
Fate of TLR-2 agonist functionalized pDNA nanocarriers upon
deposition at the bronchial epithelium in vitro
144
Chapter 7
Preliminary in vivo immunogenicity of TLR-2 agonist decorated
chitosan nanoparticles encapsulating a plasmid DNA vaccine against
Mycobacterium tuberculosis
168
Chapter 8
Discussion and future perspectives
196
Chapter 9
Summary
207
Chapter 10 Résumé
211
List of Abbreviations
215
Publications, oral presentations and awards
217
Curriculum vitae
219
Acknowledgments
220
5
Part I
Introduction
6
Chapter 1
Progress in chitosan-based
vaccine delivery systems
F. Esmaeili1,2,*, S. Heuking1,2,*, H.E. Junginger3 and G. Borchard1,2
Adapted from:
Journal of Drug Delivery and Science Technology (2010) 20, 53-61.
1School
of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
3Department
of Pharmaceutics, Faculty of Pharmaceutical Sciences, Naresuan
University, Phitsanulok, Thailand
*F.E. and S.H. have contributed equally to this review
7
Abstract
The biopolymer chitosan, derived from chitin by deacetylation, has been considered for
drug and vaccine carrier systems due to its biocompatibility, biodegradability, and
absence of toxicity. In addition, chitosan serves as the base for the synthesis of various
derivatives, having, e.g., an increased aqueous solubility at a wide pH range or offering
the opportunity for functionalization with targeting moieties. As the polymer was
described to display adjuvant properties, as well, chitosan and its derivatives are used in
vaccine delivery systems, alone or in combination with other polymers. This review is
highlighting the current status of the use of chitosan and its derivatives in vaccine
carrier design.
1. Chitosan
Chitosan is a cationic polymer derived from chitin and consists of randomly distributed
β-(1-4)-linked
D-glucosamine
(deacetylated
unit)
and
N-acetyl-D-glucosamine
(acetylated unit) monomers. Its properties are dependent on molecular weight and
degree of deacetylation [1,2]. Chitosan is a low cost, non-toxic, biodegradable and
biocompatible mucoadhesive polymer. Once the polymer is positively charged, it
transiently opens intercellular tight junctions, thereby enabling paracellular transport of
drugs [3]. Moreover, chitosan exhibits potential adjuvant properties to be exploited in
vaccine delivery [4]. With regard to the formulation of vaccines, the polymer may
encapsulate or adsorb antigens, giving rise to the formation of micro- or nanoparticles.
In addition, chitosan polymers are co-administered with the antigen in solution [5]. In
mucosal vaccination by the nasal or pulmonary route, chitosan, due to its mucoadhesive
properties, prolongs the residence time of the loaded antigen at mucosal sites, which is
suggested to increase antigenic uptake [6].
8
Because of its beneficial properties mentioned, chitosan attracted much attention as an
excipient for the preparation of vaccine delivery systems over the last decades, and has
been applied to the delivery of protein as well as plasmid DNA vaccines.
2. Chitosan for the formulation of protein and subunit vaccines
Peptide and protein subunit vaccines are single or multiple antigen(s) derived from a
pathogenic organism. Subunit vaccines often show a low immunogenicity, especially
when applied by mucosal routes. Adjuvant co-administration is therefore advised,
preferably within the same formulation or carrier system as the antigen itself. A range of
studies has been performed to identify and develop adjuvants especially suitable for the
mucosal route of immunization [7,8].
Many studies showed that chitosan, in soluble form as well as in particulate systems
does improve antigen uptake at mucosal epithelia, thereby allowing vaccine access to
subepithelial antigen-presenting cells (APC) and increasing local immune responses [911]. As an example, immunostimulatory effects of nasal co-administration of chitosan
and selected other adjuvants with recombinant Helicobacter pylori urease antigen were
examined by Moschos et al. [7]. In this study, the adjuvanticity of chitosan was evaluated
and compared to two well-defined adjuvants. The first of these adjuvants, cholera toxin
subunit B (CTB), is known as a potent mucosal adjuvant inducing strong humoral
responses [12]. The second, muramyl di-peptide (MDP), represents an adjuvant eliciting
cell-mediated immunity [13]. Results of these studies showed that immunomodulatory
effects of the H. pylori antigen were increased by chitosan as well as by MDP; in addition,
a combination of adjuvants administered with the vaccine also contributed to the
increased antigenicity of the recombinant antigen [7].
9
Related to this study, Xie et al. demonstrated the superiority of chitosan over cholera
toxin as an adjuvant for an H. pylori vaccine. Co-administration of chitosan and H. pylori
antigen did not only induce Th1 and Th2 immune responses, but also adjusted the
Th1/Th2 imbalance due to H. pylori infection [11]. In another study, Seferian and
Martinez studied the immunogenic activity of two new adjuvant formulations based on
chitosan to increase the immunogenic potential of bhCG (beta human chorionic
gonadotropin) [14]. bhCG is known as a target protein for immunocontraceptive
vaccines and tumor immunotherapy [15], while its immunogenicity, when administered
alone, is not pronounced. In vivo studies in BALB/c mice using bhCG in combination with
zinc-chitosan particles and/or an emulsion formulation containing chitosan resulted in
high and prolonged titers of antibodies as a result of B and T lymphocyte stimulation.
Many studies have been performed to develop an effective vaccine against
(myco)bacterial infections such as tuberculosis (TB), diphtheria, cholera and pertussis,
especially for mucosal vaccination.
Regarding TB vaccination, Zhu et al. have reported on the formulation of Ag85B–
MPT64190–198–Mtb8.4 (AMM) antigen in chitosan microspheres. AMM is a fusion
protein containing the structure of three antigenic epitopes of M. tuberculosis. In mice,
subcutaneously administered chitosan microspheres containing AMM produced
stronger humoral and cellular immune responses than a solution of AMM in phosphate
buffer saline (PBS) administered by the same route [9].
In view of nasal immunization, CRM197, a non-toxic mutant cross-reacting material of
diphtheria toxin (DT) was formulated with chitosan in solution or powder form and
CRM197´s in vivo immunogenic activity was studied [16]. Intranasal administration of
this formulation in mice as well as in guinea pigs yielded high titers of toxin-neutralizing
antibodies. Further studies revealed that intranasal administration of a powder
10
formulation of chitosan CRM197-based vaccine resulted in a boosted humoral and
cellular immune response in adult volunteers [16,17].
In a further investigation, van der Lubben et al. formulated chitosan microparticles
loaded with diphtheria toxoid (DT) and bovine serum albumin (BSA) by using a
precipitation/coacervation method [18]. Following oral administration of this
formulation
in
mice,
confocal
laser
scanning
microscopy
(CLSM)
and
immunohistochemistry revealed that these microparticles were taken up by intestinal
Peyer’s patches of the gut-associated lymphoid tissue (GALT) being an essential step in
oral vaccination [19].
Bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin have
been shown to induce very strong systemic and mucosal immune reactions against
antigens when co-administered nasally with chitosan [20,21]. In a study by Jabbal-Gill et
al. (1998), filamentous haemagglutinin (FHA) and recombinant pertussis toxin (rPT)
were formulated in a number of different chitosan-based nasal vaccines for pertussis as
single or as bivalent vaccines. Following intranasal administration of the chitosan
formulation in mice, immunological responses were monitored, and results revealed
that chitosan could enhance significantly serum IgG and secretory IgA levels after nasal
administration of FHA antigen or a mixture of FHA and rPT antigens, when compared to
the formulation lacking chitosan [20,21].
Moreover, chitosan microspheres containing Bordetella bronchiseptica dermonecrotoxin
(BBD) were formulated for nasal vaccination against atrophic rhinitis. In vitro
stimulation of murine macrophages (RAW264.7 cell line) by these particles resulted in
the gradual secretion of tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) from
macrophages, apparently induced by BBD released from the chitosan microspheres [22].
11
Interestingly, intranasal administration of the BBD-loaded chitosan microparticles in
mice was studied by Kang et al. [23]. BBD-specific IgA titers in the nasal cavity, as well as
those of systemic IgA and IgG were dependent on time and dose. Next to these studies,
intranasal administration of chitosan nanoparticles and emulsions containing cholera
toxin (CT) and bovine serum albumin (BSA) was addressed by Nagamoto et al. [24].
Their results revealed that these formulations could effectively target the NALT (nasalassociated lymphoid tissue) and induce a systemic immune effect.
In addition, BSA as a model antigen was also incorporated in chitosan microspheres,
prepared from chitosan base or chitosan chloride, using spray-drying [6]. This method
of preparation was shown to preserve the integrity of BSA, and administration of the
particles via the intranasal route in mice resulted in significantly more pronounced
immune responses than the administration of BSA in solution. Moreover, enhancement
of the IgG immune response was shown to be dependent on the type of chitosan used for
particle formation. Interestingly, microparticles prepared with chitosan base were found
to have a more pronounced effect on anti-BSA IgG secretions than those prepared from
chitosan chloride. This effect was suggested to be due to the higher permeation
enhancement potential of chitosan base than chitosan chloride across the nasal mucosa
[6,25], allowing chitosan vaccine carrier systems an improved access to subepithelial
antigen-presenting cells (APC).
One goal in the development of vaccine delivery systems has been to achieve prolonged
local delivery of immunomodulatory cytokines to enhance immune responses. As an
example, Zaharoff et al. studied the potential of chitosan to control the dissemination
and to improve the immunoadjuvant properties of GM-CSF [26].
Compared to lipid-based adjuvants and other vehicles, subcutaneous injection of
recombinant GM-CSF (rGM-CSF) formulated in chitosan solution resulted in longer local
12
retention up to nince days of rGM-CSF at the injection site. Prolongation of retention
time, in turn, resulted in a significant increase in antigen presenting cells (MHCII+ cells
and dendritic cells) and increase in overall vaccine´s immunogenicity [26].
McClure [27] described the feasibility of antigen delivery across the mucosal epithelium
for the induction of mucosal immune responses against Trichostrongylus colubriformis, a
non-blood-feeding intestinal nematode. Cellulose gel and chitosan gel or sponge
formulations were used as carrier systems, whereby delivery of Trichostrongylus native
and recombinant antigens across the rectal epithelium resulted in significant immune
responses even in the absence of mucosal adjuvants like cholera or E. coli toxins. In the
light of the results obtained, further investigations for the developpment of a mucosal
delivery system for vaccines against gastrointestinal helminths are warranted.
In general, most of studies performed used chitosan as an adjuvant or as a delivery
system for protein and subunit vaccines and demonstrated a potentiating effect of this
polymer on the immunogenicity of the vaccines applied.
3. Chitosan for the formulation of plasmid DNA vaccines
DNA vaccines consist of plasmid DNA of bacterial origin, bearing the genetic information
of one or more specific microbial antigenic epitopes [28]. In spite of extensive efforts to
translate results from animal studies into the clinic and into products for human use,
only a small number of DNA vaccines has recently been approved, all of them in the area
of veterinary medicine [29]. It is therefore the goal of currently ongoing studies to elicit
a sustained cellular and humoral immune response in a clinical setting. This challenge is
addressed by, on one hand, unraveling the biological concept of DNA vaccination in
humans, and on the other hand by designing improved formulations and vaccination
regimen.
13
Several studies on the improvement of the immunogenicity of DNA vaccines employed
cationic liposomes [30,31] and polymeric particulate delivery systems based on chitosan
[32] and poly (lactide-co-glycolide) (PLGA) [33-35]. These carrier systems were able to
enhance the uptake of DNA vaccines by antigen-presenting cells (APC) and to facilitate T
cell responses [36-38], which resulted in an improved immunogenicity [39,40],
especially after application by mucosal routes [41].
As chitosan is positively charged in weak acidic solutions, it readily forms associates
with highly negatively charged plasmid DNA by virtue of strong electrostatic interaction
[21,42]. In one of the first studies on orally applied gene delivery systems, Roy et al.
described the incorporation of a DNA vaccine against peanut allergy into chitosan
particles [42], which protected against allergy challenge. Illum et al. evaluated a flu
vaccine, formulated as chitosan-DNA nanoparticles. Intramuscular or intranasal
administration of these complexes in BALB/c mice resulted in antibody titers, which
were significantly elevated above those obtained by administration of DNA alone [32].
In a first study focusing on pulmonary delivery of a DNA vaccine against tuberculosis,
Bivas-Benita et al. [43] formulated chitosan nanoparticles containing a polyepitope DNA
vaccine
encoding
eight
different
HLA-A*0201-restricted
T-cell
epitopes
of
Mycobacterium tuberculosis. Induction of dendritic cell (DC) maturation by the DNAchitosan nanoparticles was shown in vitro. Secretion of IFN-γ from isolated splenocytes
post vaccination and incubated with M. tuberculosis homogenate was significantly
elevated in case of the pulmonary route of administration, as compared to intramuscular
injection. The same result was obtained when isolated splenocytes were challenged with
individual antigenic epitopes.
The same research group also used chitosan nanoparticles to deliver orally a DNA
vaccine encoding Toxoplasma gondii GRA-1 antigen, and chitosan microparticles for the
14
delivery of the GRA-1 protein, to the intestinal Peyer’s patches in mice. Their results
showed that the vaccination regime (prime/boost) and the type of vaccine (pDNA,
protein) are clearly the factors affecting the type of immune response [44]. Orally
administered GRA-1 loaded chitosan microparticles or GRA-1 protein expressiong
plasmid DNA loaded in chitosan nanoparticles could prime the immune response, when
anti-GRA antibodies in sera were analysed. In order to indicate the type of immune
response, IgG1 and IgG2a isotype ratios were determined. It was shown that priming
with plasmid DNA nanoparticles in mice induces IgG1 production, which is associated
with a Th2 response, while priming with GRA1 loaded microparticles reflects a mixed
Th1/Th2 immune response [44].
Topical application of DNA nanoparticulate vaccines was evaluated by Cui and Mumper
[45]. DNA chitosan nanoparticles, and DNA coated onto pre-formed cationic
chitosan/carboxymethylcellulose (CMC) nanoparticles were applied topically to mice.
Both vaccine systems were shown to induce increased titers of IgG up to 28 days,
showing that topical application of chitosan DNA vaccines allows significant stimulation
of the immune system [45].
Because of the promising results from studies using chitosan for the application of DNA
vaccines, in addition to its biocompatibility and its ability to readily form complexes or
particles with negatively charged DNA, future studies should be focusing on developing
safe, sustained release DNA vaccine carrier systems.
4. Vaccine formulation using chitosan in combination with other polymers
Cationic nanoparticles made from polyesters, such as poly(lactide) (PLA) and coated
with chitosan, have shown promising results for the delivery of DNA vaccines.
15
In one study [46], chitosan was compared to two other cationic agents,
poly(ethyleneimine)
(PEI)
and
poly(2-dimethyl-amino)ethyl
methacrylate
(pDMAEMA)), for the preparation of DNA-loaded PLA nanoparticles. All three
formulation groups could at least partially protect DNA from nuclease degradation,
although this effect was less pronounced for PLA–chitosan. PLA–pDMAEMA showed the
highest transfection efficiency in cell culture studies in vitro [46]. Kumar et al. [47] and
Davies et al. [48] also described on the use of similar chitosan-coated poly (lactic acid
glycolic acid) (PLGA) nanoparticles.
In an investigation by Fischer et al. [49], chitosan-coated PLGA microparticles were
prepared through a novel surfactant-free process, by which negatively charged PLGA
microparticles were coated with positively charged chitosan. In addition, targeting
moieties (antigenic and recombinant hepatits B surface, HBS) were grafted to chitosan´s
free amine groups onto the surface of these microparticules [49,50]. After nasal
administration, clearance rates of microparticles were shown to be lower than for
unmodified PLGA microspheres or lactose powder. The humoral immune response
following nasal administration of these modified systems was comparable to alum-HBS
antigen administered subcutaneously. In addition, modified microspheres not only
elicited significantly both systemic and mucosal humoral, but also a strong cellular
immune responses [50].
Another approach was based on the preparation of a complex between mannosebearing chitosan (m-chitosan) and hepatitis B virus, as reported by Zhou et al. [51].
Mannose receptors are expressed at a high density on the surface of antigen presenting
cells (APC), such as immature DC and mannose-bearing chitosan (MC) particles have
been shown to be able to target these cells. Interaction between targeting MC particles
and APC was followed by a sustained release of DNA, which induced an increased
16
cellular and humoral immune response compared to DNA alone. In addition, DNA
release was controlled by the biodegradation of chitosan and MC, with antigen release
from m-chitosan microspheres being quicker than from chitosan microspheres [51].
Alginate is another biopolymer, which has been used in combination with chitosan for
the preparation of vaccine delivery systems [52]. Proteins, cells, and DNA were
successfully incorporated into alginate matrices by a gelation process, while their
biological activity remained intact [53,54]. Ciofani et al. [55] reported on the
performance of polymeric alginate/chitosan nanoparticles for intracellular drug
delivery. A simple way to prepare chitosan-based vaccine delivery systems is to adsorb
the antigens onto the chitosan particle surface. Complexation of antigen-coated chitosan
particles with sodium alginate was used to increase the stability of these carrier systems
and also to prevent an immediate release of the adsorbed antigen [56]. Borges et al.
applied such a nanoparticulate system for the incorporation of BSA as a model antigen
and showed that alginate complexation with chitosan nanoparticles can increase the
stability of the particles, leading to a sustained release of the antigen and a better uptake
by Peyer's patches, rendering them an appropriate system for intestinal mucosal
vaccination [56].
5. Studies on interaction of chitosan particles with epithelial and immune cells
The exact mechanism of induction of the immune system by the majority of
immunomodulatory molecules remains yet to be elucidated. Chitosan, as an adjuvant,
was shown to induce polarized Th2 responses. Although the mechanism of this
induction is not well defined, results of the related studies showed that chitosan
polymer appears to specifically interact with APC and CD4+ T cells.
17
Such an interaction of chitosan particles with APC was demonstrated to enhance the
subsequent antigen presentation [56,57]. In the field of protein delivery, besides
interactions with the immune system and activation of macrophages and the
complement system, the absorption enhancing properties of chitosan represent an
added value [57]. In several studies, the superiority of chitosan powder enabling
absorption enhancing properties over its aqueous solutions was demonstrated [4,58]. As
an example, Hall et al. showed in a recent study that peptide antigen adsorbed to
chitosan could transiently activate T-cells to produce the immune cytokine IL-10 via
antigen-specific T cells [59].
IV
III
Polymeric
chitosan
II
nucleus
V
Expression of
antigenic
protein(s)
VI
Proteolysis
VII
MHC-I processing
I
Plasmid DNA
& presentation
VIII
T-cell
Figure 1: Schematic presentation of the uptake of plasmid DNA chitosan particles into
cells, successive expression of antigenic protein(s) in situ followed by MHC-I processing
and presentation to T-cells.
18
With regard to the application of DNA vaccines, if the delivery system can be targeted to
APC and induce endolysosomal DNA vaccine release, an effective T cell immunity may be
achieved at lower doses of plasmid DNA, which can be safer and more cost-effective
[43]. As mentioned before, mannose-bearing chitosan has the ability to target APC and
provide a sustained release of DNA vaccines [51]. Murthy et al. demonstrated that
chitosan particles are rapidly degraded within the lysosomal compartment after cellular
uptake, possibly resulting in dissociation of DNA from the polymer (Figure 1, I-III).
Antigens encoded by the DNA vaccines are then expressed and processed, finally
resulting in MHC-I restricted antigen presentation (Figure 1, IV-VIII) and a successive
immune response, which can be significantly higher than for DNA alone [60].
Moreover, some studies have shown that chitosan nanoparticles appeared to be
captured and transported by adsorptive transcytosis [61]. It was shown that the mucus
covering the nasal epithelium did not act as a diffusion barrier to the particles. On the
contrary, association of chitosan nanoparticles with mucus appeared to strongly
increase permeability of the particles through the mucus layer, compared to other
polymeric particles [61]. Behrens et al. also showed that internalization of chitosan
nanoparticles by epithelial and immune cells is a saturable, energy- and temperaturedependent process. Intra-duodenal administration of chitosan nanoparticles in rats
confirmed these in vitro results, and demonstrated that nanoparticles could be detected
in both epithelial cells and in immune cells comprising intestinal Peyer’s patches [61].
In conclusion, considering the ability of chitosan to modulate the immune system as an
adjuvant, as well as its role as a delivery system especially for DNA vaccines, its further
evaluation as vaccine carrier system can be supported [57].
19
6. Chitosan formulations for human use
Until now, solely a few studies have been performed in order to test chitosan-based
vaccine delivery in human subjects.
In 2003, Mills et al. evaluated a chitosan vaccine against diphtheria in healthy volunteers
[62]. Inactivated diphtheria toxoid (CRM197) in a chitosan delivery system was
administered intranasally in human healthy volunteers, which was well tolerated after
single immunization. The antitoxin neutralizing activity of this vaccine was comparable
to standard intramuscular diphtheria vaccine, while presence of chitosan could
potentiate the immunogenic responses significantly.
Huo et al. (2005) also tested another chitosan-based vaccine formulation in human
volunteers. A single intramuscular injection of a vaccine containing Neisseria
meningitidis serogroup C polysaccharide (MCP) conjugated with CRM197, in alum or
two nasal insufflations of the same vaccine powder mixed with chitosan, without alum,
was evaluated in healthy volunteers. Nasal vaccination was well tolerated, with fewer
symptoms. CRM197-specific IgG and diphtheria toxin-neutralizing levels were increased
after either nasal or intramuscular immunization, with balanced IgG1/IgG2 and higher
IgG4, while significant MCP-specific secretory IgA was detected in nasal washings only
after nasal immunization. These results showed that formulation of the MCP-CRM197
conjugate in chitosan could be an effective and inexpensive vaccine against N.
meningitides (serogroup C) and diphtheria [63]. Read et al. (2005) reported
immunization of healthy volunteers against chitosan-based influenza vaccine. Intranasal
administration of influenza virus subunit proteins in combination with chitosan
glutamate revealed that immunogenic responses for intranasal chitosan-based vaccine
was only slightly lower or similar to the results for standard intramuscular influenza
vaccine [64].
20
These kinds of studies support the development of mucosal vaccines against diphtheria
and other subunit chitosan-based vaccines especially when mucosal immunization is
desirable, such as human immunodeficiency virus infection [62].
7. Chitosan derivatives in vaccine carrier design
An important physico-chemical property of chitosan is the pKa value of 6.5 to 6.6 of the
primary amine group. This value does not vary significantly for different types of
chitosan of varying degrees of N-acetylation [65]. Chitosan´s amine groups (present in
the glucosamine unit) are therefore positively charged only in diluted acidic solutions
(pH < 5.5), rendering chitosan water-soluble. However, at physiological pH values (pH
7.2-7.4), amine groups are not protonated and chitosan precipitates from solution. In an
attempt to overcome this limitation, several derivatives were synthesized over the last
two decades and their potential use in drug carrier systems assessed. The molecular
structure of chitosan shows a free primary amine function, as well as a primary alcohol
function, both of which have been considered for chemical modification. Synthesis and
modification of chitosan polymers for vaccine delivery are well-described in literature
(see table 1 and figure 2), and will be presented here. For a comprehensive overview of
chitosan polymers in vaccine delivery we also recommend the excellent review by Arca
et al. [66], which was published during the edition of this review.
TMC polymer is a positively charged chitosan derivative, which is readily soluble in
water at pH values of 1-9 and at concentrations of up to 10% (w/v) [67]. In general, TMC
is prepared in a one- or two-step synthesis through nucleophilic substitution at the
primary amine group by methyl iodide (MeI) under basic conditions [68].
The degree of trimethylation (%DTM) is controlled by adjusting the reaction time and
number of reaction steps, and measured by 1H NMR spectroscopy [69]. This parameter
21
is of high relevance, as only TMC with a %DTM above 22% was shown to be capable of
opening tight junctions between epithelial cells in a transient manner, a prerequisite for
paracellular drug transport [64].
Table 1: Selected in vivo investigated chitosan derivatives for mucosal antigen delivery.
Derivative
MW[kDa]
TMC
DS
[%]
20
40
60
Delivery form
Delivery route
Ref.
110
81
79
Antigen/adjuvan
t
Ovalbumin
Ovalbumin
Ovalbumin
Solution
Solution
Solution
Nasal
Nasal
Nasal
73
TMC
n.s.
n.s.
CRM-MenC/LTK63
Solution
Nasal
74
TMC
15
37
63
94
Inactivated
influenza virus
Solution
Nasal
75
TMC
19
n.s.
CRM-MenC/LTK63
Microparticles,
solution
Nasal
76
TMC
25
177
H3N2 influenza
subunit
Nanoparticles
Nasal
77
TMC
57
n.s.
Tetanus toxoid
Nanoparticles
Nasal
78
TMC
50
n.s.
Diphtheria toxoid
Microparticles
Pulmonary
79
TMC
20
n.s.
Diphtheria toxoid
Microparticles,
solution
Nasal, oral
82
TMC
37
n.s.
H. pylori urease
Nanoparticles,
solution
Oral
83
TMC
15
n.s.
Ovalbumin
Nanoparticles
Oral
84
CTM
CDM
MCC
TMC
n.s.
n.s.
70
57
n.s.
n.s.
n.s.
pVAX(HBc)
Nanoparticles
Intramuscular
85
Tetanus toxoid
Nanoparticles,
solution
Nasal
80
MCC-TMC
70/5
7
5.9
n.s.
Tetanus toxoid
Nanoparticles
Nasal
86
n.s.
Multiple antigens
of B.
bronchiseptica
Microparticles
Nasal
87
MC
MW: molecular weight; DS: degree of substitution; Ref.: reference; TMC: N,N,N-trimethyl chitosan
polymer; CRM-MenC: C. menigococci conjugate vaccine; LTK63: E. coli enterotoxin LTK63; CTM: chitosanN-trimethylaminonethylacrylate chloride-methylacrylate; CDM: chitosan-N-dimethyl-aminoethylmethacrylate hydrochloride-methylmethacrylate; pVAX(HBc): plasmid DNA expressing the Hepatitis B
virus core antigen; MCC: Mono-N-carboxymethl chitosan; MC: Mannosylated chitosan; n.s.: not stated.
22
7.1. N,N,N-trimethyl chitosan (TMC)
On the other hand, Kean et al. [70] reported that an increase in %DTM of oligomeric and
polymeric TMC induces an increase in cytotoxicity in monkey kidney fibroblasts (COS-7)
and epithelial breast cancer (MCF-7) cells.
In addition, the degree of 3- and 6-hydroxy-methylation (%D3OM and %D6OM,
respectively), as well as dimethylation (%DDM) of TMC need to be determined following
synthesis, as it has been described that these three parameters are strongly correlated
to the cytotoxicity and physico-chemical properties of TMC. Jintapattanakit et al. [71]
reported that a ratio of %DDM/%DTM greater than unity causes a decrease in
mucoadhesion and cytotoxicity; as an example, for TMC of a DTM of 20% and a DDM of
20% (denoted TMC20-20), an IC50 value approximately 100-times lower than for
TMC20-60 was measured.
23
OH
OH
H3C
H3C
O
O
HO
OH
H3C
N+
H3C Cl-
OH
O
OH HO
H
n
CH3
OH
H
TMC
OH
NH
NH
H
n
S
O
H
MC
OH
H3C
O
O
HO
OH
O
O
HO
OH
NH
HO
H3C
n
O
O
O
MCC
H3C
Cl
OH
H3C
H3C
H3C
O
O
HO
p
NH
O
n
o
H3CO
+
N
-
CH3
R
R = H for CDM
R = CH3 for CTM
Figure 2: Molecular structure of a selection of chitosan derivatives (TMC = N,N,Ntrimethyl chitosan; MC = Mannosylated chitosan; MCC = Mono-N-carboxymethyl
chitosan; CTM = chitosan-N-trimethylaminonethylacrylate chloride-methylacrylate;
CDM
=
chitosan-N-dimethyl-aminoethylmethacrylate
hydrochloride-methylmethacrylate).
In addition, Verheul et al. [72] highlighted that 3- and 6-hydroxy-methylation of TMC
polymers cause lower cytotoxicity on Caco-2 cells when compared to the corresponding
O-methyl free TMC polymers.
With regard to the formulation of antigens, TMC is capable of forming complexes or
particles with negatively charged molecules by electrostatic interactions. This property
is employed for i) polyelectrolyte complexation (polyplex) or nanoparticle (in aqueous
sodium sulfate solution) formation with plasmid DNA (pDNA) encoding for the specific
24
antigen(s) in situ; ii) the association or coating of subunit antigen(s) with TMC and iii)
the encapsulation and/or absorption of subunit antigen(s) via ionic cross-linking with
tripolyphosphate (TPP).
Considering this versatility in vaccine formulation, TMC solutions as well as micro- or
nanosized particulate systems were extensively examined as carrier systems for
mucosally applied vaccines (see table 1), whereby most studies focused on the nasal,
pulmonary and oral routes of immunization [67].
Nasal delivery
Generally, DTM is used as a measure for the positive charge density of TMC, which
largely affects its permeation enhancing properties [69]. Boonyo et al. [73] studied the
immune stimulatory effect of three different TMC polymers (DTM 20, 40 and 60%) in
nasal application of ovalbumin in mice. In these studies, TMC with a DTM of 40% was
shown to be the more expedient delivery system/adjuvant owing to its higher induction
of mucosal and systemic antibodies at day 13 following immunization, when compared
to the other TMC polymers and polymeric chitosan. Although this superior effect of
TMC-40 became not significant at day 21, still ovalbumin in TMC-40 solution triggered
higher IgG and IgA responses than TMC with a DTM of 20 and 60%. In another study
involving an aqueous TMC solution, Baudner et al. [74] investigated the impact of TMC
solution (DTM not stated) on the immune response of a conjugate vaccine against group
C. meningococci (CRM-MenC) being co-administered with the mucosal adjuvant E. coli
enterotoxin LTK63. Interestingly, intranasal immunization enhanced antibody as well as
bactericidal responses in the presence of TMC, which were comparable to those induced
by subcutaneous vaccination using aluminium hydroxide as a reference Th2 adjuvant
and delivery system. Furthermore, Hagenaars et al. [75] coated whole inactivated
25
influenza virus (WIC) by simple mixing the antigen with two different TMC solutions
(%DTM 15 and 37, respectively), and evaluated their influence on the physico-chemical
and immunological properties of WIC. Around 10-15% of TMC polymer was associated
with the WIC antigen. As expected, only the TMC-37 coated vaccine enabled paracellular
transport across confluent Caco-2 cell monolayers. Following nasal immunization, the
TMC-37 coated vaccine elicited higher IgG levels than TMC-15 in mice. This effect was,
however, less pronounced than seen after intramuscular administration of the WIC
antigen alone. More noteworthy, both TMC coated nasal vaccines protected mice well
against influenza challenge, whereas nasally applied uncoated WIC did not.
However, in contrast to the studies mentioned above, TMC solutions might not be the
most suitable formulation type. Several studies indicated that particulate systems are
more advantageous with respect to immunogenicity and stability of the formulation,
among other factors [76,77]. Therefore, TMC-based micro- and nanoparticles were also
included in mucosal immunization studies. In a follow-up of the above-mentioned study
[78], CRM-MenC antigen was associated with LTK63 in TMC microparticles and their
stimulating effect was compared with the respective powder formulation. Intranasal and
subcutaneous application in mice did not result in significantly different serum and
antibody titres. However, only nasal vaccine delivery was able to generate mucosal
antibodies (immunoglobulin A, IgA). Moreover, the concept of using a particulate
delivery system was extended by Amidi et al. [79] by the formulation of a monovalent
influenza subunit H3N2 antigen in TMC nanoparticles. Particles had a size of about 650
nm and showed a positive surface charge. Nasal application of particles elicited more
secretions local IgA (in nasal washes) and serum IgG in mice when compared to
intranasal and intramuscular administration of the influenza antigen alone.
In contrast, Sayın et al. [80] did not detect high titres of local IgA in nasal and vaginal
26
secretions in mice after application of TMC nanoparticles loaded with tetanus toxoid
(TT). However, TT loaded TMC nanoparticles were capable to trigger significant levels of
serum IgG in a Th2 predominant way, which was assumed to be beneficial, as a systemic
immune response against TT was intended.
Pulmonary vaccine delivery
Besides nasal delivery, Amidi et al. [81] studied intratracheal immunization of guinea
pigs using TMC microparticles encapsulating a diphtheria toxoid (DT). Administration of
antigen-loaded TMC microparticles (MP) in guinea pigs resulted in a pronounced
secretion of systemic antibodies, with an IgG2/IgG1 ratio of approximately three.
Secrection of IgG and IgM were measured to be similar or even higher than those evoked
by subcutaneous administration of alum-associated DT. More interestingly, local S-IgA
antibodies were solely elicited after intratracheal application of DT-loaded TMC MP.
Oral vaccine delivery
The above mentioned diphtheria toxoid (DT) was also encapsulated in TMC
microparticles (MP) by van der Lubben et al. [82]. Particles had a size of around 2 µm
and were stable over a period of at least three months. In vivo studies in mice
demonstrated that orally administered MP can induce a strong systemic immune
response measured as significantly elevated IgG titers in the serum.
In another study [83], the antigenic protein urease of H. pylori was associated to TMC
nanoparticles. Mice immunized with these nanoparticles showed higher titres of serum
IgG, as well as intestinal IgA antibodies, when compared to the application of the antigen
in the absence of TMC. More recently, Slütter et al. [84] evaluated the use of ovalbuminloaded TMC nanoparticles in vivo in mice in a more systematic approach.
27
Similarly to van der Lubben et al. [82], oral application of TMC nanoparticles induced
considerably higher levels of serum IgG in comparison to intramuscular injection of the
model antigen. IgG response was slightly biased towards the Th1 type. In order to
elucidate the mechanism, the transport of ovalbumin-loaded TMC nanoparticles across
Caco-2/Raji-B cell co-culture was investigated. Caco-2 cell monolayers were previously
shown to differentiate to an M-cell phenotype on co-cultivation with human B-cells
(Raji). Stimulation of human dendritic cells (DC) by TMC nanoparticles was studied, as
well. It was demonstrated that TMC nanoparticles transported the antigenic ovalbumin
to a much higher extent across modified Caco-2 monolayers in comparison to the control
group. However, this increase in transport was independent of the presence of M-cells
capable of uptake and transcytosis of nanoparticles. On the other hand, only ovalbuminTMC nanoparticles facilitated maturation of human DCs into MHCII+/CD86+ DC, which is
considered a critical step towards antigen presentation and subsequent immune
activation of T lymphocytes.
7.2 Other chitosan derivatives
Beyond the broad application of TMC, several other water-soluble chitosan derivatives
have been used for the formulation of mucosal vaccines. In order to conserve the
positive charge of TMC, Jiang et al. [85] synthesized a series of water-soluble chitosan
derivatives by free radical polymerization.
After polymerization, chitosan-N-trimethylaminonethylacrylate chloride-methylacrylate
(CTM)
as
well
as
chitosan-N-dimethyl-aminoethylmethacrylate
hydrochloride-
methylmethacrylate (CDM) were obtained and used for complexation of the pDNA
vaccine pVAX(HBc) encoding a hepatitis B virus (HBV) core antigen (HBcAg).
28
Both chitosan derivatives formed nanoparticles of a size of 230-270 nm, a positive
surface charge and a complexation efficiency varying from 32 to 76%. After
intramuscular administration of CTM and CDM pDNA nanoparticles in mice, a much
stronger immune response to the pVAX(HBc) vaccine was elicited than for the vaccine
alone, shown by elevated IFN-γ production, high HBcAg-specific IgG titer and an HbsAgspecific cytotoxic T-lymphocyte response of murine splenocytes. Although no HBV
challenge study was performed, these studies indicated that both derivatives are
suitable pDNA vaccine delivery systems, with CTM appearing to be the better candidate.
A further water-soluble derivative of chitosan intended for vaccine delivery is mono-Ncarboxymethyl chitosan (MCC) [80]. Sayın and colleagues compared TMC to MCC in oral
delivery of a tetanus toxoid (TT) in particulate and dispersion form. MCC particles had a
small particle size of 40-90 nm and were negatively charged. In contrast to TT-bearing
chitosan and TMC nanoparticles, MCC particles did not elicit high levels of serum IgG in
mice after intranasal and subcutaneous administration. Interestingly, intranasal codelivery of MCC and TT in a dispersion triggered higher subclass IgG1 antibody
secretion than free TT. However, compared to chitosan and TMC, the IgG1 response to
MCC nanoparticles was significantly lower.
In a subsequent investigation, Sayın et al. [86] applied MCC and positively charged TMC
polymers to form nanosized particles incorporating TT. Intranasal administration in
mice of these TMC-MCC nanoparticles elicited systemic TT-specific IgG levels being
slightly superior to those of the control group (free TT applied subcutaneously). In
addition, MCC-TMC nanoparticles induced a Th2 predominant immune response (see
also section 5 of this review), when the ratio of IgG2a to IgG1 was calculated to be lower
than unity.
29
Local IgA antibody titers in vaginal and nasal secretions were not found to be significant
in comparison to systemic IgA titers. In order to evaluate further the benefit of this
vaccine carrier system, a challenge study in mice is strongly recommended.
Besides rendering polymeric chitosan water-soluble for its use at physiological pH,
another approach for vaccine delivery is the attachment of targeting moieties onto the
surface of chitosan particles. Jiang et al. [87] described the synthesis of mannosylated
polymeric chitosan (MC) for improved receptor-mediated uptake of particles,
presumably due to interaction of grafted mannose residues with the mannose receptor
predominantly expressed on macrophages and dendritic cells. Chitosan and MC
microparticles containing multiple antigens of B. bronchiseptica were applied nasally to
mice. Compared to chitosan microparticles, MC microparticles induced slighty higher
titers of local IgA (nasal and saliva washes) and serum IgA 10 weeks post immunization.
Moreover, immunization with both delivery systems yielded high survival rates of up to
70% seven days post challenge, although no significant difference between the MC and
chitosan groups was detected. In our lab, a new water-soluble chitosan derivative 6-Ocarboxymethyl-N,N,N-trimethyl chitosan polymer (CM-TMC) was synthesized, and used
for covalent attachment of a Toll-like receptor (TLR) agonist as adjuvant [88], as
described in chapter 2 of this thesis. This concept allows for the combination of an
innate immune system stimulator (adjuvant) and an antigen in the same delivery system
(i.e. chitosan particles), which is generally supposed to be needed for effective vaccines
[89]. As described in chapter 3 of this thesis, nanoparticles formed from this polymer
and green fluorescent protein (GFP) expressing DNA by self-assembly were shown to
elicit TLR-mediated chemokine IL-8 release from human macrophages [90].
30
8. Outlook
In the future, we expect the following progress in chitosan-based vaccine delivery
system taking place:
i)
More chitosan-based vaccine formulations are going into clinical trials. LigoCyte
Pharmaceutical, Inc. recently has introduced its product, Norwalk vaccine, into a
clinical phase I trial. The Norovirus vaccine is a needle-free, dry powder
formulation based on virus like particle (VLP) antigens, including the adjuvant
Monophosphoryl Lipid A, and chitosan to enhance nasal delivery [91].
ii)
More thorough investigations on chitosan’s adjuvant properties will help to
better define its potential in vaccine formulation.
iii)
New chitosan derivatives might emerge from the field of non-viral gene delivery
[92], e.g., PEGylated TMC for the field of DNA vaccine delivery [93].
iv)
Further improvements in vaccine design will be the attachment/inclusion of
specific adjuvants to antigen loaded chitosan formulations in order to trigger
well-defined parts of the immune system.
In a nutshell, as demonstrated in this review, chitosan and its derivatives are very
auspicious materials for the delivery of mucosal vaccines (e.g., for nasal, pulmonary and
oral administration), and merit further elaborate investigations as a vaccine delivery
platform.
31
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44
Chapter 2
Aim of the thesis
and study objectives
45
General
According to a recent report of the Initiative of Vaccine Research (WHO, 2005), three
major intracellular diseases (HIV/AIDS, malaria and tuberculosis) contribute to half of
the burden of all infectious diseases and cause death of over 15 million humans every
year worldwide.
With an increasing understanding of the human immune system and its molecular
answers to infections, it becomes more and more obvious that antibody-inducing
vaccines might be not the appropriate solution. For prevention of intracellular
infections, a vaccine initiating a potent cytotoxic T lymphocyte (CTL) response is
required. Plasmid deoxyribonucleic acid (DNA) vaccination might be an answer due to
its hallmark of inducing a strong CTL response in orchestration with CD4+ T helper cells
(cellular immunity), as well as its generation of antibodies (humoral immunity).
In comparison to current protein vaccines, plasmid DNA vaccination offers many
advantages, such as i) favoring a cellular immunity, ii) allowing the genetic construction
of multiple antigens of choice included in the same vector, iii) possessing an intrinsic
adjuvant, unmethylated 5´-deoxycytidine-phosphate-guanosine (CpG)-motifs, which are
stimulating the innate immune system via the Toll-like receptor-9 (TLR-9), iv)
prolonging the expression of the antigenic protein(s), which is continuously stimulating
the immune response, v) being easily produced, up-scaled and stored at higher
temperatures without causing loss of activity.
Despite promising results of plasmid DNA immunizations in preclinical trials, studies in
non-human primates and humans have failed so far in achieving protective immunity.
The main reason for this drawback was related to the low transfection efficacy of
plasmid DNA vaccines as a result of extra- (e.g., enzymatic degradation by DNases) and
intracellular (e.g., endosomal escape) transport barriers for plasmid DNA resulting in
46
low amounts of antigenic proteins expressed in situ. Consequently, amelioration
strategies were undertaken in relation to the targeted infectious disease, ranging from
plasmid DNA optimization over co-formulation with adjuvants to changing the route of
administration, to e.g., aerosol vaccination.
Aerosol delivery of vaccines is considered a promising route of immunization owing to
several clinical trials with measles vaccines, one being currently tested in clinical phase
II/III trial. Concerning an efficient delivery of DNA vaccine into the lung, particle-based
delivery systems are beneficial, because of their ability to protect the plasmid DNA from
enzymatic degradation and to facilitate DNA transfection in vivo. Next to the antigen
delivery system, a potent adjuvant capable of stimulating the innate immune system is
required for enhancing the overall poor immunogenicity of a DNA vaccine alone.
Moreover, adjuvant and antigen have to be co-delivered in the same particulate system
in order to elicit successfully an immune response against the targeted antigen.
Aim of thesis:
1. To prepare and to characterize TLR agonist functionalized polymers.
2. To use these new polymers for the formulation of DNA containing nanoparticles.
3. To study in vitro the benefit of TLR agonist functionalization of DNA
nanoparticles.
4. To evaluate the in vivo immunogenicity of TLR agonist functionalized DNA
nanoparticles.
47
Innovation of thesis
To our best knowledge, we are the first to provide a vaccine delivery system with a
covalent bond between the polymeric drug delivery system and Toll-like receptor
agonists. We submitted the detailed synthesis of CM-TMC-g-PEG-Pam3Cys co-polymer on
the 28th of October 2008 to the International Journal of Pharmaceutics.
In more detail, this thesis focused on the covalent coupling of different adjuvants (Tolllike receptor (TLR) agonists) onto a vaccine carrier system (polymeric chitosan)
followed by their detailed chemical analysis. TLR agonist functionalized chitosan
derivatives were then applied for the encapsulation of a plasmid DNA resulting in nanosized particles. TLR agonist functionalized DNA particles were investigated in vitro for
their transfection efficiency and immunogenicity in THP-1 macrophages. In terms of
pulmonary vaccination, we studied uptake and immune stimulatory capacity of adjuvant
functionalized DNA nanoparticles by using a triple cell culture model of the human
respiratory tract. Finally, the immunogenicity of TLR agonist functionalized
nanoparticles was evaluated in vivo for the delivery of a DNA vaccine encoding the
epitope Ag85A of Mycobacterium tuberculosis.
Organization of this thesis
This thesis consists of two experimental parts (part II and part III).
Part II describes the synthesis of novel TLR agonist chitosan derivatives being used for
the preparation of DNA nanoparticles and successive in vitro evaluation. Part III
comprises uptake studies in an in vitro model of the human respiratory tract followed by
an immunogenicity study in mice.
48
Part II
Within the first experimental part, chapter 3 describes the synthesis of a new watersoluble polymeric chitosan derivative, 6-O-carboxymethyl-N,N,N-trimethyl chitosan
(CM-TMC), which was employed as platform for the ligation of further TLR agonists. To
CM-TMC, we grafted a water-soluble TLR-2 agonist (NH2-PEG-Pam3Cys) through the use
of a poly(oxyethylene) (PEG) spacer (final product: co-polymer CM-TMC-g-PEGPam3Cys).
In chapter 4, we used the new co-polymer for preparation of DNA nanoparticles, which
were investigated for their physico-chemical properties, transfection efficiency and
immunogenicity in vitro. Chapter 5 presents the synthesis of a new TLR-7 agonist (9benzyl-8-hydroxyadenine, 8HA) functionalized co-polymer, CM-TMC-g-NH-PEG-8HA,
which was similarly used for DNA NP preparation followed by an in vitro analysis of
transfection efficiency and immunogenicity.
Part III
Chapter 6 reports on the uptake pattern of TLR-2 agonist nanoparticles in a triple cell
culture model involving two important species of antigen-presenting cells: human-blood
derived macrophages and dendritic cells. In chapter 7, we studied the in vivo adjuvant
potential of the CM-TMC-g-PEG-Pam3Cys co-polymer for the delivery of the plasmid DNA
vaccine pAg85A against Mycobacterium tuberculosis.
49
50
Part II
Preparation and
in vitro characterization
of TLR-agonist functionalized
DNA nanoparticles
51
Chapter 3
Toll-like receptor-2 agonist
functionalized biopolymer for
mucosal vaccination
S. Heuking1,2,#, A. Iannitelli1,2,3,#, A. di Stefano3 and G. Borchard1,2
Published in:
International Journal of Pharmaceutics (2009) 381, 97-105.
¹School of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
3Department
of Drug Science, University ”G. D'Annunzio”, Chieti, Italy
# Authors S.H. and A.I. contributed equally to this work
52
Abstract
The objective of this study was to provide a new water-soluble chitosan derivative being
functionalized with a Toll-like receptor-2 (TLR-2) agonist. At first, we synthesized the
water-soluble TLR-2 agonist ω-amido-[Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)propyl]-[R]–cysteinyl]-α-amino poly(ethylene glycol) (Pam3Cys-PEG-NH2), which was
characterized by 1H and 13C NMR as well as mass spectroscopy. Secondly, Pam3Cys-PEGNH2 was then successfully grafted to 6-O-carboxymethyl-N,N,N-trimethyl chitosan
polymers (CM-TMC) using EDC/NHS as condensing agents. The copolymer was analysed
by means of 1H and
13C
NMR and FTIR spectroscopy.
13C
NMR spectroscopy did not
deliver evidence that an amide bond was formed between CM-TMC and Pam3Cys-PEGNH2. However, 1H NMR and FTIR spectroscopy demonstrated clearly that successful
grafting took place. Based upon these results, this new TLR-2 functionalized biopolymer
merits further investigation as material for vaccine delivery systems.
1. Introduction
Chitosan is a linear, cationic polysaccharide consisting of randomly distributed β-(1-4)linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)
monomers. It is industrially produced by alkaline deacetylation of chitin, which is the
structural element in the exoskeleton of crustaceans. Due to many advantageous
properties, such as low toxicity, absorption enhancement of hydrophilic drugs and
mucoadhesive properties (van der Lubben et al. 2001a; Chopra et al. 2006), chitosan
attracted considerable attention as a novel excipient in mucosal drug and vaccine
delivery. Regarding the latter, chitosan polymers can easily encapsulate or adsorb
antigens via the formation of micro- or nanoparticles, which has been demonstrated,
e.g., for tetanus toxoid, diphtheria toxoid (van der Lubben et al. 2001b) and a plasmid
53
DNA encoding eight different antigenic epitopes of M. tuberculosis (Bivas-Benita et al.
2004) .
However, the majority of such vaccines lack most of the features of the original
pathogen, such as innate immune stimulation, and are therefore often poorly
immunogenic (O’Hagan et al. 2006). Hence, non-specific stimulators of the immune
system (adjuvants) are needed to render a vaccine more effective in view of eliciting
protective immunity (Pashine et al. 2005).
The present strategy for the development of new vaccines is to include highly purified
synthetic adjuvants, which are able to trigger well-defined elements of the immune
system.
Hereby, so-called Toll-like receptor (TLR) agonists have been lately considered as very
auspicious due to their ability to elicit a significant innate immune response which, in
turn, affects strongly the initiation of adaptive immunity (Iwasaki and Medzhitov 2004).
TLRs are a family of at least 10 receptors able to recognize and discriminate highly
conserved microbial structural motifs of bacteria, viruses, fungi and protozoae. Their
activation results in an immune response accompanied by increased levels of proinflammatory and immune-related cytokines. Maturation of dendritic cells and their
subsequent migration to regional lymph nodes followed by a facilitated presentation of
antigens to T-lymphocytes have been described (Iwasaki and Medzhitov 2004).
Among all TLRs, TLR-2 recognizes the broadest repertoire of pathogen-associated
molecular patterns (PAMPs) from a large variety of pathogens. TLR-2 is highly
expressed on the membrane ofby dendritic cells, which are considered as the most
potent cell type for antigen presentation (Wetzler 2003; Iwasaki and Medzhitov 2004;
Schmitt et al. 2008).
TLR-2 recognizes its ligands as heterodimer either in combination with TLR-1 or TLR-6.
54
A major difference between both heterodimer types is that TLR-1/TLR-2 enables
recognition of triacylated lipoproteins, whereas TLR-2/TLR-6 detects diacylated
lipoproteins and peptidoglycans (Wetzler 2003).
Notably, Nα-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys (4)
trihydrochloride (Pam3CSK4), a synthesized tripalmitoylated lipopeptide, is capable to
trigger TLR-2 as a TLR-1/TLR-2 agonist and possesses a strong adjuvant capacity
(Lombardi et al. 2008; Wedlock et al. 2008).
Moreover, Kleine et al. demonstrated that conjugates of the lipophilic Pam3Cys moiety
coupled to poly (ethylene glycol) (PEG) become water-soluble (up to 10mg/mL), while
retaining their immunological properties (Kleine et al. 1994).
In order to combine the adjuvant capacity of Pam3Cys moiety with the excellent mucosal
vaccine delivery features of chitosan polymers, we synthesized the pure diastereomeric
TLR-2
agonist
ω-amido-[Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]–
cysteinyl]-α-amino poly (ethylene glycol) (abbreviated Pam3Cys-PEG-NH2, molecular
weight ~3990 Da) and coupled it covalently to a new water-soluble chitosan polymer.
This concept would allow an ideal combination of adjuvant and (encapsulated or surface
adsorbed) antigens in the same particulate system (chitosan polymer), which is
supposed to be required for effective vaccines (O`Hagan et al. 2006; Schlosser et al.
2008).
As water-soluble chitosan derivative, we selected 6-O-carboxymethyl-N,N,N-trimethyl
chitosan polymer (CM-TMC, molecular weight ~200 kDa) and applied a relatively low
grafting percentage (Pam3Cys-PEG-NH2 to CM-TMC) of ≤ 5%, so that the main
characteristics of chitosan as a polymeric delivery system were preserved.
In order to synthesize CM-TMC we started firstly by trimethylation of chitosan polymer
giving N,N,N-trimethyl chitosan polymer (TMC). This chitosan derivative demonstrated
55
beneficial water-solubility at physiological pH values due to its permanent
quaternization (Sahni et al. 2008) and featured strong adjuvant properties when used as
vaccine delivery system, e.g., for a CRM-MenC conjugate vaccine (Baudner et al. 2004,
Baudner et al. 2005).
The successive synthesis of CM-TMC from TMC was described previously by Murata and
colleagues, however, neither the molecular weight of chitosan (e.g., oligomeric or
polymeric) used and conditions under which the chemical synthesis was performed
were mentioned (Murata et al. 1996; 1997).
Furthermore, Jansma et al. (2003) synthesized 6-O-carboxymethyl-N,N,N-trimethyl
chitosan oligomers (CM-TMO). However, oligomeric chitosan is readily water-soluble,
whereas polymeric chitosan solely dissolves in diluted acidic aqueous solutions (pH <
6.5) (Qin et al. 2006).
The aim of our study was to establish a reproducible method for the synthesis of CMTMC and to successively graft Pam3Cys-PEG-NH2 to polymeric CM-TMC.
2. Materials and methods
2.1 Materials
Chitosan (ChitoClear® cg110), of an intrinsic viscosity of 33 mPa·s, a molecular weight
(MW) of approximately 200 kDa, and a degree of deacetylation (DD) of 94% was
purchased from Primex, Iceland. Dialysis membrane Spectra/Por 4 (cut-off 12-14 kDa)
was obtained from Spectrum, USA. N,N’-Bis(fluorenylmethoxycarbonyl)-[R]-cystine-bistert-butyl ester was purchased from Bachem, Switzerland. α,ω-bis-amino poly(ethylene
glycol), PEG diamine (MW ~ 3000Da) was purchased from Iris Biotech GmbH, Germany.
All the other reagents and solvents were of analytical grade and supplied by SigmaAldrich, Switzerland.
56
2.2 Characterization of polymers
1H
NMR and
13C
NMR spectra were recorded on a Varian VXR 300 MHz or 500 MHz
spectrometer (Varian, Switzerland). Chitosan was dissolved in 1% v/v DCl/D2O,
chitosan derivatives in D2O and all other compounds in CDCl3. Chemical shifts are
reported in parts per million (δ) downfield from the internal standard tetramethylsilane
(Me4Si). MS spectra were recorded on an API 150 EX LC/MS System (Applied
Biosystems/MDS Sciex, Switzerland) equipped with a turbo ion spray ionization source.
The MALDI-Tof mass spectrometry was carried out on an Axima CFR+, Shimadzu mass
spectrometer,
using
2-(4-hydroxyphenylazo)-benzoic
acid
(HABA)
as
matrix.
Homogeneity was confirmed by thin layer chromatography (TLC) on silica gel Merck 60
F254 aluminium-backed plates. Solutions were routinely dried over anhydrous sodium
sulphate prior to evaporation. Chromatographic purifications were performed by
employing a Merck 60 70-230 and 230-400 mesh ASTM silica gel column. FTIR spectra
were recorded on a Perkin-Elmer 100 FTIR spectrometer (Perkin-Elmer, Switzerland) in
the range of 4000–400 cm-1 using KBr pellets (1% w/w of product in KBr).
2.3 Synthesis of TMC
Trimethylation of chitosan polymers was performed according to a previously published
method (Sieval et al. 1998) that was slightly modified. Briefly, chitosan polymer (2g)
was suspended in 1-methyl-2-pyrrolidinone (NMP, 80mL) with sodium hydroxide
(NaOH, 4.8g) at 60 °C under stirring in a round bottom flask. Sufficient NaOH (11 mL of
15% w/v; aqueous) was added in order to maintain an alkaline environment throughout
the reaction. Methylation was achieved through nucleophilic substitution by addition of
methyl iodide (MeI, 12 mL). 70 minutes later, the solution was taken and products were
precipitated by addition of sufficient volumes of a mixture of diethyl ether and ethanol
57
(ratio of 1:1 v/v). The wet product was again dissolved in 80 mL NMP at 60 °C and 10
mL of 15% w/v sodium hydroxide (NaOH, aqueous) was added to the solution. After
addition of 7 mL methyl iodide (MeI), the reaction was let under condensor for 35
minutes. Next, 0.6 g NaOH pellets and 5 mL MeI were added for further 35 minutes. TMC
was then precipitated by using 5-10 volumes diethyl ether:ethanol (1:1 v/v) and
centrifuged (1850 x g, 15 minutes). The finalized product was subsequently dissolved in
40 mL 10% NaCl solution to perform counter ion replacement and to prevent iodine
oxidation. After stirring overnight the TMC solution was dialysed over three days (twice
daily changing deionized water) and finally freeze-dried. The degree of trimethylation
(DTM), dimethylation (DDM), 3-hydroxy- (D3OM) and 6-hydroxy-methylation (D6OM)
were calculated as reported elsewhere (Polnok et al. 2004; Verheul et al. 2008). Yield
36%. 1H NMR (300 MHz, D2O): δ 2.0 (COCH3); δ 2.8 (N-(CH3)2 ); δ 3.3 (N+-(CH3)3); δ 3.4
(6O-CH3); δ 3.5 (3O-CH3); 4.8-6.0 (H-1, CH). DTM 61.4%; DDM 35.8%; DD 3.8%; D3OM
21.3%; D6OM 22.1%. 13C NMR (300 MHz, D2O): δ 42.1 (N-(CH3)2); δ 54.5 (N+-(CH3)3); δ
58.9 (C2); δ 61.1 (C6); δ 77.3 (C3); δ 96. 7 (C1). FTIR: 3429 (O-H stretching); 2925 (C-H
asymmetric stretching of methyl groups); 1642 (C=O stretching of amide groups); 1478
(C-H asymmetric bending of methyl groups); 1076 (C-N stretching of primary amine
groups).
2.4 Synthesis of CM-TMC
6-O-carboxymethylation of TMC was performed pursuant to a modification of a method
published by Jansma et al. (2003). TMC (0.5 g) was suspended in 50 mL of NMP and let
stirring overnight at room temperature. The next day, the pH was readjusted to a value
of 10 by using 15% aqueous NaOH solution and successively 2.2 g of chloroacetic acid
(20 mol equivalents (mol eq.) to TMC sugar monomers) were added. The pH was
58
maintained during the reaction at a value of 10 by sufficient addition of 15% aqueous
NaOH solution. After 3 hours, the product was precipitated by adding 5-10 volumes
ethanol and diethylether (1:1 v/v) and subsequently isolated by centrifugation (1850 x
g, 15 minutes). After re-dissolution in 50 mL of MilliQ water, CM-TMC were rendered
water-soluble by adjusting the pH to 5, the solution dialysed over the course of three
days (twice daily changing deionized water) and sterile filtrated before lyophilization.
The degree of carboxymethylation (DCM) of CM-TMC was estimated using the following
equation:
%DCM = ([(CH2)-CO)] / [H] x 1/2) x 100.
Hereby, [(CH2)-CO)] is the integral value of the methylene group of newly introduced
carboxymethyl function at 4.1 ppm and again [H] the integral value of the H-1 peaks
between 4.7 and 6.0 ppm.
Yield 73%. 1H NMR (300 MHz, D2O): δ 2.0 (COCH3); δ 2.8 (N-(CH3)2; δ 3.3 (N+-(CH3)3); δ
3.4 (6O-CH3); δ 3.5 (3O-CH3); δ 4.1 (CH2-CO); δ 4.8-6.0 (H-1, CH). DCM 17.2%; DTM
61.4%; DDM 35.8%; DD 3.8%; D3OM 21.3%; D6OM 22.1%. 13C NMR: (300 MHz, D2O) δ
42.1 (N-(CH3)2); δ 54.5 (N+-(CH3)3); δ 58.9 (C2); δ 61.1 (C6); δ 77.3 (C3); δ 96. 7 (C1); δ
175.2 (C=O of CH2-COOH). FTIR: 1725 (C=O stretching of COOH group), 1606 (C=O
asymmetric stretch vibration), 1384 (C=O symmetric stretch vibration).
2.5 Synthesis of ω-amido-[Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]–
cysteinyl]-α-amino poly(ethylene glycol) (abbreviated Pam3Cys-PEG-NH2, 7)
2.5.1 Nα-Fluorenylmethoxycarbonyl-S-[2,3-dihydroxy-(2R)-propyl]-[R]-cysteine tert-butyl
ester (2)
To a solution of 1 (2.58 g; 3.34 mmol) in CH2Cl2 (20.4 mL), zinc (1.48 g) and a freshly
59
prepared mixture of MeOH, 32% HCl (d = 1.16 g/mL) and concentrated H2SO4 (10.78
mL; 100:7:1) were added under vigorous stirring. After 15 min (S)-(-)-glycidol (1.16 mL;
32.42 mmol) was added. The mixture was stirred for 5 h at 40 °C. The solution was
evaporated to about half of its original volume and diluted with 5% KHSO4 (26 mL). This
mixture was kept at -4 °C for 16 h and then extracted with CH2Cl2. The organic phase
was dried over anhydrous Na2SO4, evaporated to dryness and the crude residue
chromatographed on silica gel with CHCl3, then CHCl3/MeOH (10:1) as eluant to yield 2
(2.74 g; 5.79 mmol) as a colourless oil. Yield 89%. Rf = 0.11, CHCl3; 0.73, CHCl3/MeOH
(10:1).
13C
NMR (CDCl3) δ 169 (Cys-CO); δ 155.7 (Fmoc-CO); δ 143.8 141.3 127.7 127
125.2 120 (Fmoc); δ 83.2 (tBu-Cq); δ 70.2 (S-glyceryl-CH); δ 67.3 (Fmoc-CH2-O); δ 63.4
(S-glyceryl-CH2-O); δ 54 (Cys-CH); δ 47.1 (Fmoc); δ 35 (Cys-CH2); δ 34.3 (S-glycerylCH2); δ 27.9 (COOtBu-CH3).
2.5.2
Nα-Fluorenylmethoxycarbonyl-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]-cysteine
tert-butyl ester (3)
2 (2.73 g; 5.78 mmol), palmitic acid (Pam-OH; 4.75 g; 18.54 mmol) and N,Ndiisopropylcarbodiimide (DIC; 3.46 mL; 22.35 mmol) were dissolved in dry
tetrahydrofuran (THF, 56.7 mL). N-Dimethylamino-pyridine (DMAP; 0.29 g; 2.37 mmol)
was added, and the mixture stirred for 2 h.
After the addition of glacial acetic acid (2.3 mL) the mixture was evaporated to dryness.
The residue was recrystallized from CH2Cl2/MeOH (1:3; 68 mL) at -20 °C. 3 (5.10 g; 5.37
mmol) was obtained as a colourless powder. Yield 93%. Rf = 0.72, CHCl3.
13C
NMR
(CDCl3) δ 173.5 173.4 (PamCO); δ 169 (Cys-CO); δ 155.7 (Fmoc-CO); δ 143.8 141.3 127.7
127 125.2 120 (Fmoc); δ 83.2 (tBu-Cq); δ 70.2 (S-glyceryl-CH); δ 67.3 (Fmoc-CH2-O); δ
63.4 (S-glyceryl-CH2-O); δ 54 (Cys-CH); δ 47.1 (Fmoc); δ 35 (Cys-CH2); δ 34.5 (Pam-CH2);
60
δ 34.3 (S-glyceryl-CH2); δ 32.2 29.9 29.7 29.5 (Pam-CH2); δ 27.9 (COOtBu-CH3); δ 25.8
25.1 22.9 (Pam-CH2); δ 14.3 (Pam-CH3). MS (API) m/z 950 M+.
2.5.3 S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]-cysteine tert-butyl ester (4)
To a solution of 3 (5.07 g; 5.34 mmol) in dry CH2Cl2 at 0 °C was added 1,8diazabicyclo[5.4.0]undec-7-ene (DBU). After 20 minutes the mixture was dried under
vacuum and chromatographed on silica gel, using as eluant CHCl3 then CHCl3/MeOH
(95:5) to give 4 (3.45 g; 4.73 mmol). Yield 89%. Rf = 0.13, CHCl3; 0.89, CHCl3/MeOH
(95:5). 13C NMR (CDCl3) δ 173.5 173.4 (PamCO); δ 169 (Cys-CO); δ 83.2 (tBu-Cq); δ 70.2
(S-glyceryl-CH); δ 63.4 (S-glyceryl-CH2-O); δ 54 (Cys-CH); δ 35 (Cys-CH2); δ 34.5 (PamCH2); δ 34.3 (S-glyceryl-CH2); δ 32.2 29.9 29.7 29.5 (Pam-CH2); δ 27.9 (COOtBu-CH3); δ
25.8 25.1 22.9 (Pam-CH2); δ 14.3 (Pam-CH3). MS (API) m/z 728 (M-H)+.
2.5.4 Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]-cysteine t-butyl ester (5)
Pam-OH (1.07 g; 4.16 mmol) was activated in dry CH2Cl2 (62.4 mL) with DIC (0.73 mL;
4.71 mmol) and hydroxybenzotriazole hydrate (HOBt; 0.64 g; 4.71 mmol) in dry DMF at
0 °C. After 30 min 4 (3.43 g; 4.71 mmol) was added. After being stirred for 15 h at room
temperature the solution was evaporated to dryness, the crude residue was dissolved in
CHCl3 and extracted with NaHCO3 5% and water. The organic phase was then
evaporated under vacuum and crystallized from CH2Cl2/MeOH (1:3; 73.8 mL) at -20 °C
affording 5 (2.73 g; 2.82 mmol) as a colourless powder. Yield 60%. Rf = 0.66, CHCl3. 13C
NMR (CDCl3) δ 173.6, 173.5, 173.4 (PamCO); δ 169 (Cys-CO); δ 83.2 (tBu-Cq); δ 70.2 (Sglyceryl-CH); δ 63.4 (S-glyceryl-CH2-O); δ 54 (Cys-CH); δ 35 (Cys-CH2); δ 34.5 (PamCH2); δ 34.3 (S-glyceryl-CH2); δ 32.2 29.9 29.7 29.5 (Pam-CH2); δ 27.9 (COOtBu-CH3); δ
61
25.8 25.1 22.9 (Pam-CH2); δ 14.3 (Pam-CH3). MS (API) m/z 967 (M-H)+.
2.5.5 Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]-cysteine (6)
To a solution of 5 (0.57 g; 0.59 mmol) in dry CH2Cl2 (0.35 mL) was added trifluoroacetic
acid (TFA; 0.59 mL; 7.64 mmol) and Et3SiH (0.23 mL; 1.47 mmol). After stirring for 6 h
at room temperature the solution was evaporated to dryness and the crude residue
repeatedly evaporated with diethyl ether to give 6 in quantitative yield. The product was
used without further purification. Rf = 0.38, CHCl3/MeOH (95:5). ).
13C
NMR (CDCl3) δ
174.6 (Cys-COOH); δ 173.6, 173.5, 173.4 (PamCO); δ 70.2 (S-glyceryl-CH); δ 63.4 (Sglyceryl-CH2-O); δ 54 (Cys-CH); δ 35 (Cys-CH2); δ 34.5 (Pam-CH2); δ 34.3 (S-glycerylCH2); δ 32.2 29.9 29.7 29.5 25.8 25.1 22.9 (Pam-CH2); δ 14.3 (Pam-CH3). MS (API) m/z
910 (M-H)+.
2.5.6 ω-amido-[Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl]-[R]–cysteinyl]-αamino poly(ethylene glycol) (7)
6 was activated in dry CH2Cl2 (4 mL) with DIC (0.06 mL; 0.4 mmol) and HOBt (0.05 g; 0.4
mmol) in dry DMF at 0 °C. After 30 min a solution of α, ω-bis-amino poly(ethylene
glycol) (1.2 g; 0.4 mmol) in dry CH2Cl2/DMF (1:1; 4 mL) at 0 °C was added. After being
stirred for 15 h at room temperature the reaction solution was evaporated to dryness,
the crude residue dissolved in CHCl3 and extracted with NaHCO3 5% and water.
The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum. The
crude
residue
was
chromatographed
on
silica
gel
(230-400
mesh)
with
CH2Cl2/MeOH/H2O (91:9:0.1) then CH2Cl2/MeOH/H2O (78:19:1) as eluant to yield 7
(0.41 g; 0.1 mmol) as a colourless powder. Yield 30%. Rf = 0.2; CH2Cl2/MeOH/H2O
(91:9:0.1). 1H NMR (300 MHz, D2O): δ 0.9 (CH3-Pam); δ 1.4 (CH2-Pam); δ 1.7 (C13-CH2) δ
62
2.2 (C14-CH2); δ ~3.6 (PEG-CH2); δ 4.5 (S-glyceryl-CH2-O); δ 5.0 (S-glyceryl-CH). 13C NMR
(CDCl3) δ 173.6, 173.5, 173.4 (PamCO); δ 170.4 (CysCO); δ 74.0 70.8 70.5 (PEG-CH2); δ
70.3 (S-glyceryl-CH); δ 63.8 (S-glyceryl-CH2-O); δ 52.1 (Cys-CH); δ 39.7 (PEG-CH2); δ
35.1 (Cys-CH2); δ 34.6 (Pam-CH2); δ 34.3 (S-glyceryl-CH2); δ 32.2 29.9 29.7 29.5 25.8
25.1 22.9 (Pam-CH2); δ 14.3 (Pam-CH3).
FTIR: 3429 (O-H stretching); 2918 (C-H asymmetric stretching of methyl groups); 1725
(C=O stretching of the COOH group); 1639 (C=O stretching of amide groups); 1470 (C-H
asymmetric bending of methyl groups); 1352 (C=O symmetric stretch vibration); 1102
(C-H stretching). MS (MALDI-Tof) m/z 3990 (M+).
2.6 Grafting of Pam3Cys-PEG-NH2 (7) to CM-TMC
7 (10.6 mg; 0.002 mmol, 0.2 mol equiv. to COOH) was completely dissolved in 1 mL of
MilliQ water by gently heating at 40 °C for 5 minutes. Separately, CM-TMC (degree of
trimethylation (DTM) 61.4%; degree of carboxymethylation (DCM) 17.2%; 20 mg; 0.089
mmol sugar units, 0.015 mmol COOH) was dissolved in MilliQ water (3 mL) at room
temperature and the pH adjusted to a value of 7±0.1. Next, EDC (5.7 mg, 0.03 mmol, 2
mol equiv. to COOH) and NHS (3.5 mg; 0.03 mmol, 2 mol equiv. to COOH) were added
and the pH re-adjusted to neutrality. The solution containing 7 was then added and
reaction was performed over 96 hours, whereby the pH was maintained at 7±0.1. In the
following, the solution was dialysed (membrane cut-off 12-14 kDa) over one week
(medium twice daily changed), sterile filtered and then freeze dried. The finalized
product (8) was then used for further investigations. The degree of grafting (DG) was
calculated by using the following equation: %DG = ([CH2-Pam] / [H] x 1/66) x 100,
whereby [(CH2-Pam)] is the integral value of the peak at 1.3 ppm, which is assigned to
methylene groups (3x11xCH2, 66H) of the palmitoyl moiety. [H] is the integral value of
63
H-1 peaks between 4.7 ppm and 6.0 ppm. A further approach of defining the %DG was
performed as follows:
%DG = ([PEG-CH2CH2] / [H] x 1/290.9) x 100.
Hereby, ([PEG-CH2CH2] is an average integral value of methylene groups of the PEG unit
(72.7xCH2CH2, 290.9H) at ~ 3.6 ppm. Both methods gave in accordance a grafting degree
of around 4.3%. Practical yield 87%. 1H NMR (300 MHz, D2O): δ 0.9 (CH3-Pam); δ 1.3
(CH2-Pam); δ 2.0 (COCH3); δ 2.8 (N-(CH3)2); δ 3.3 (N+-(CH3)3); δ 3.4 (6O-CH3); δ 3.5 (3OCH3); δ ~3.6 (CH2-CH2-CO); δ 4.1 (CH2-CO); δ 4.8-6.0 (H-1, CH); 13C NMR (300 MHz, D2O):
δ 22.3 36.1 (Pam-CH2); δ 42.1 (N-(CH3)2); δ 54.7 (N+-(CH3)3); δ 58.9 (C2); δ 61.1 (C6); ~
69.8 (PEG-CH2CH2); δ 77.3 (C3); δ 96. 7 (C1); δ 174.9 (C=O of COOH and CO-NH-PEG).
FTIR: 1725 (C=O stretching of the COOH group); 1600 (C=O asymmetric stretch
vibration); 1655 (C=O stretching of amide group); 1384 (C=O symmetric stretch
vibration).
3. Results and discussion
The aim of this study was at first to provide a water-soluble chitosan derivative with
auspicious properties for transmucosal vaccine delivery as well as the ability to
covalently bind targeting ligands. We therefore opted to synthesize TMC, which was in
turn modified to 6-O-carboxymethyl-N,N,N-trimethyl chitosan polymer (CM-TMC). To
our best knowledge, we report here for the first time a detailed synthesis of polymeric
CM-TMC.
This new chitosan derivative merges the promising vaccine delivery properties of TMC
together with the potential of covalent attachment of targeting ligands thanks to new
introduced carboxylic functions. In a second step, we synthesized a TLR-2 targeting
agonist (Pam3Cys-PEG-NH2) with the aid of modifications of by already published
64
methods (Metzger et al. 1991; Kleine et al. 1994). Finally, we grafted Pam3Cys-PEG-NH2
to CM-TMC by means of condensing agents (s. figure 1).
3.1 Synthesis of TMC
Regarding the characteristics of TMC, the degree of trimethylation (DTM) is assumed to
play an important role, whereby solely TMC having a DTM between 40% and 60% is
supposed to be beneficial as permeation enhancer of macromolecules (Sahni et al. 2008;
Hamman et al. 2003). In this study, we involved a TMC having a DTM of around 60%,
which was shown to facilitate transport of a peptide drug across the bronchial
epithelium in vivo (Florea et al. 2006).
65
HO
COOt-Bu
Fmoc
a
S
NH
Pam-O
b
HO
CH3
Pam-O
2
1
NH
Fmoc
COOt-Bu
RNH
2
Pam-O
d
c
COOt-Bu
3
R = -Fmoc
4
R = -H
Pam-O
Pam-O
Pam-O
f
Pam
NH
COOR
H
N
Pam NH
72
O
5 R = -t-Bu
e
NH2
O
7
6 R = -H
OR1
g
O
O
H 3C
CH3
O
R3
R2
n
8
O
R1 = -H, -CH3, -CH2COOH, -CH2
O
NH
O
NH
72
NH
Pam
+
R2 = -NHCOCH3, -N(CH3)2, -N (CH3)3
O-Pam
R3 = -OH, -OCH 3
O-Pam
Figure 1: Reaction scheme for the synthesis of CM20-TMC60-g-PEG-Pam3Cys.
Reagents and conditions: a) CH2Cl2, Zn/HCl/H2SO4, (S)-(+)-glycidol; b) Anhydrous THF,
Pam-OH, DIC, DMAP; c) Anhydrous CH2Cl2, DBU; d) Anhydrous CH2Cl2/DMF, Pam-OH,
DIC, HOBt; e) Anhydrous CH2Cl2, TFA, Et3SiH; f) Anhydrous CH2Cl2/DMF, PEG diamine,
DIC, HOBt; g) H2O, CM20-TMC60, EDC, NHS.
66
Moreover, the degrees of 3- (D3OM) and 6-hydroxy-methylation (D6OM), as well as
dimethylation (DDM) have to be taken into consideration. As reported elsewhere, they
influence cytotoxicity and physicochemical properties of TMC (Jintapattanakit et al.
2008; Verheul et al. 2008). A typical 1H NMR spectrum of TMC (61.4% trimethylated,
abbreviated TMC60) is shown in figure 2. Major peak assignments are as follows: δ = 3.3
ppm N+-(CH3)3, δ = 2.8 N-(CH3)2, δ = 3.4 6O-CH3, and δ = 3.5 3O-CH3 (Jintapattanakit et
al. 2008). In addition, the extent of di- and trimethylation of chitosan polymers was
analysed by 13C NMR spectroscopy, whereby the corresponding peaks appear at δ = 42.1
and 54.4 ppm, respectively (data not shown; Sieval et al. 1998).
Furthermore, FTIR spectroscopy of TMC60 showed the introduction of methyl groups,
which was observed at 2925 cm-1 (C-H asymmetric stretching of methyl groups) and
1478 cm-1 (C-H asymmetric bending of methyl groups).
3.2 Synthesis of CM-TMC
Regarding the carboxymethylation of chitosan, in most cases chloroacetic acid is used as
introducing reagent. Chen and Park investigated this reaction by using chloroacetic acid
in different mixtures of water and isopropanol under strong basic conditions (Chen and
Park 2003). Further on, Jansma et al. (1996) carboxymethylated trimethyl oligomers
(CM-TMO) using chloroacetic acid in NMP at pH 10, whereby the extent of
carboxymethylation was controlled by varying reaction time and equivalents of
chloroacetic acid used. We opted for the latter mild method and altered it for the
carboxymethylation of polymeric TMC60.
67
3-6
HO
HO
6
HO
HO
5
2
3
O
O
4
OH
O
HO
1
H2N
A
HN
O
2
1
H3C
7
7
CH3
11
HO
HO
O
6
5
2
N
H3C
8
CH3
8
8
N
H3C
O
10
O
HO
HN
CH3
O
O
O
O
1
+
3
H3C
8
O
O
4
HO
HO
CH3
CH3
9
3-6
H3C
7
10
11
1
9
7
B
HO
11
HO
O
6
2
5
+
3
N
H3C
8
CH3
8
CH3
8
1
O
O
O
4
HO
HO
8
CH3
O
12
10
O
HO
HN
O
H3C
12
O
O
O
O
N
H3C
9
H3C
CH3
1
7
CH3
3-6
10
11
9
7
C
Figure 2: 1H NMR spectra of a) chitosan (1% v/v DCl/D2O, 80 °C), b) TMC-60 and c)
CM20-TMC60 (both in D2O at 80 °C).
Figure 2c shows the
1H
NMR spectrum of a CM-TMC with a degree of
carboxymethylation (DCM) of 17.2% (abbreviated CM20-TMC60). The successful
introduction of carboxymethyl groups was observed at δ = 4.1 ppm (CH2-COO).
Moreover, it can be assumed that the carboxymethylation primarily occurred at the 6-O
position due to an exclusive peak appearance at δ = 4.1 ppm (Chen and Park 2003).
Besides, the new carboxylic functionality can also be noticed is similarly detected in the
13C
NMR spectrum (see figure 4a) at δ 175.2 ppm (C=O of CH2-COO). FTIR spectroscopy
(figure 3) similarly indicated an effective introduction of carboxylic moieties owing to
new bands at 1725 cm-1 (C=O stretching of COOH group), 1606 cm-1 (C=O asymmetric
68
stretch vibration) and 1384 cm-1 (C=O symmetric stretch vibration). The watersolubility of both chitosan derivatives, TMC60 and CM20-TMC60, was higher than 50
mg/mL.
A
B
%T
C
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
450.0
cm-1
Figure 3: FTIR spectra of a) chitosan, b) TMC60 and c) CM20-TMC60.
3.3 Synthesis Pam3Cys-PEG-NH2 (7)
The synthesis of the TLR-2 agonist 7 was performed by means of adapting already
published methods (Metzger et al. 1991; Kleine et al. 1994). Compound 7 was obtained
at high purity, as shown in 1H NMR (figure 5) and MALDI-Tof spectra (figure 6).
Moreover, the diastereomeric purity was confirmed by
13C
NMR spectroscopy (figure
4b). Finalized compound 7 was perfectly water-soluble after gently heating (40°C for 5
minutes) at concentrations up to 15 mg/mL.
69
3.4 Grafting of Pam3Cys-PEG-NH2 (7) to CM20-TMC60
In order to enable a covalent bond between amine moieties and carboxymethylated
chitosans, a number of different condensing reagents have been examined in the past.
Prabaharan
et
al.
(Prabaharan
et
al.
2007)
selected
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and in a later publication a
combination of EDC and N-hydroxysuccinimide (NHS) as condensing agents
(Prabaharan and Gong 2007), whereas Jansma et al. (2003) decided to use EDC together
with N-hydroxybenzotriazole (HOBt).
O
24
23
H3C
14
O
25
O
22
21
CH3
23
25
24
O
20
14
S
19
O
NH
13a
14
12
O
O
HO
6
1
+
3
N
8
O
8
N
6 2
C
7
9
9
10
1
CH3
H3C
H3C
H3C
CH3
O
O
13a+b
9
4 5
O
HN
CH3
8
CH3
H3C
O
O
O
HO
8
25
14
O
5
2
24
11
12
O
4
HO
HO
72
O
O
13b
16
CH3
17
NH
CH3
17
16
O
O
18
NH
16
15
7
O
H3C
2
27
2
2
1
11
O
8
7
O
14
3,4, 10
CH3
141
3
6
3
1
1
2
5
O
5
S
12
6
8
9
B
O
H3C
1
9
4
NH
10
NH
2
11
NH2
12
O
11
14
12
72
O
HO
12
13
HO
6
HO
HO
2
N
H3C
8
CH3
8
CH3
8
1
O
O
O
5
+
3
9
11
O
4
8
CH3
O
O
O
O
O
HN
O
H3C
10
H3C
13
O
HO
N
H3C
9
3
CH3
4 5
6 2
CH3
1
7
7
A
Figure 4: 13C NMR spectra of a) CM20-TMC60 (D2O) b) Pam3Cys-PEG-NH2 (CDCl3) and c)
copolymer CM20-TMC60-g-PEG-Pam3Cys (D2O).
70
In line with our study, we initially investigated the applicability of EDC/HOBt for
grafting compound 7 to CM20-TMC60 (data not shown). However, after purification (via
dialysis) we were not able to detect any grafting of Pam3Cys-PEG-NH2 to CM20-TMC60 by
NMR spectroscopy. Therefore we moved to EDC/NHS and tested their suitability as
condensing agents. In more detail, we added two mol equivalents (in correlation to
COOH groups) of EDC and NHS to CM20-TMC60 and let the reaction proceed in the
presence of 0.2 mol equivalents (in correlation to COOH groups) of Pam3Cys-PEG-NH2, to
achieve a theoretical grafting ratio of 5%. Following this method we were able to
determine a grafting success (%DG) of about 4.3%, as evidenced by
1H
NMR
spectroscopy (Fig. 5c).
8
HO
13
12
CH3
O
11
HO
O
6
5
2
N
8
10
N
H3C
O
H3C
8
O
HO
HN
CH3
8
CH3
H3C
O
O
O
O
1
+
3
O
O
4
HO
HO
O
9
CH3
12
CH3
H3C
3-6
7
1
O
7
CH3
141
3
11
O
6
3
1
2
A
9
2
5
O
H3C
11
10
7
O
14
S
B
8
O
H3C
2
9
4
1
NH
11
10
NH
2
14
O
O
O
24
23
H3C
14
O
25
NH2
12
11
12
72
1
8
22
O
21
23
O
20
CH3
25
24
14
S
19
O
13a
NH
O
HO
6
N
H3C
8
1
+
CH3
CH3
8
O
O
8
O
H3C
O
O
HN
H3C
10
N
H3C
9
25
14
O
O
O
HO
24
11
12
O
5
2
3
O
13b
4
HO
HO
72
O
CH3
17
NH
CH3
17
16
O
O
18
NH
16
15
14
12
O
CH3
9
3-6
CH3
1
12
11
10
C
2
9
7
1
7
Figure 5: 1H NMR spectra of a) CM20-TMC60 b) Pam3Cys-PEG-NH2 and c) CM20-TMC60-gPEG-Pam3Cys (all in D2O at 80 °C).
71
Interestingly, in this 1H NMR spectrum of CM20-TMC60-g-PEG-Pam3Cys, a decrease of the
peak integral at δ = 4.1 ppm (CH2-COO) was noticed. This change is likely the result of
the formation of an amide bond between CM20-TMC60 and Pam3Cys-PEG-NH2. Adjacent
Successive quantification of the DCM prior to and following the reaction depicts a
decrease of 4.9%, which is in good agreement with the supposed DG of 4.3%.
In addition, the
13C
NMR spectrum of CM20-TMC60-g-PEG-Pam3Cys (Fig. 4c) underlines
the functionalization of the polymeric backbone of CM20-TMC60 with the TLR-2 agonist.
Although not all atom signals of Pam3Cys-PEG-NH2 (Fig. 4b) were also recovered in the
spectrum of the copolymer, peaks at δ =22.3 and 36.1 ppm (Pam-CH2) as well as at ~
69.8 ppm (PEG-CH2CH2) are pointing at an effective grafting reaction (Mao et al. 2005).
A
B
A
A
Figure 6: MALDI-Tof spectra of a) α,ω-bis-amino poly(ethylene glycol) (MW ~ 3000 Da)
and b) Pam3Cys-PEG-NH2 (7, MW 3990 Da).
Besides, the peak at around 174.9 ppm was assigned to the new amide function together
with C=O peak for unreacted carboxylic moieties. This is consistent with
13C
NMR data
reported by Jeong et al. (2008). However, Jansma et al. showed contrarily that an amide
formation between oligomeric CM-TMO and tryptophan was observed at 167 ppm.
72
Considering this discrepancy together with the two facts that Pam3Cys-PEG-NH2 has
already three carbonylic functions by itself (Fig. 4b) and that it was solely applied at a
relatively low molar ratio to CM20-TMC60, a clear interpretation remains difficult.
Furthermore, we analysed the copolymer with FTIR spectroscopy (Fig. 7). The introduction of PEG polymer was confirmed on the basis of associated bands at 847 cm-1, 950
cm-1 and 2917 cm-1 (Jeong et al. 2008). In addition, the absorption band at 1606 cm-1
(C=O asymmetric stretch of carboxylate anion) shifted to 1641 cm-1 (C=O stretch of
amide), which reflects the formation of an amide bond (Prabaharan et al. 2007;
Prabaharan and Gong 2008).
A
A
B
%T
C
A
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
450.0
cm-1
Figure 7: FTIR spectra of a) CM20-TMC60, b) Pam3Cys-PEG-NH2 and c) CM20-TMC60-gPEG-Pam3Cys.
73
4. Conclusions
Assembling all results presented, it is concluded that the synthesis of the TLR-2 agonist
was accomplished at high purity. Secondly, Pam3Cys-PEG-NH2 in turn was successfully
grafted to CM20-TMC60, although
13C
NMR spectroscopy did not deliver clear results
regarding the formation of an amide bond. However, 1H NMR and FTIR spectroscopy are
of evidence of successful grafting taking place. In conclusion, this new copolymer merits
further investigations as a delivery system of vaccines, such as protein or recombinant
vaccines, due to its unique combination of immunomodulatory capacity and prominent
mucosal vaccine delivery characteristics.
Acknowledgments
Ph.D. Student Exchange Project, Regione Abruzzo-Italy.
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78
Chapter 4
Stimulation of macrophages using
Toll-like receptor-2 (TLR-2)
agonist decorated nanocarriers
S. Heuking1,2, S Adam-Malpel1,2, E. Sublet1,2, A. Iannitelli2,3, A. di Stefano3 and G.
Borchard1,2,*
Adapted from:
Journal of Drug Targeting (2009) 17, 662-670.
1School
of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
3Department
of Drug Science, University ”G. D'Annunzio”, Chieti, Italy
79
Abstract:
Chitosan from a vegetal source (Agaricus bisporus) and of GMP quality was used to
synthesize the derivative 6-0-carboxymethyl-N,N,N-trimethylchitosan (CM-TMC). Tolllike receptor-2 (TLR-2) agonist, Pam3Cys, was synthesized and coupled to CM-TMC
through a polyethylene glycol (PEG) spacer. Successively, Pam3Cys decorated
nanocarriers were prepared by complexation with plasmid DNA (pDNA) expressing
green fluorescence protein (GFP), and characterized with respect to their
physicochemical properties and protection of the included plasmid against DNase I
enzymatic degradation. GFP transfection studies demonstrated that TLR-2 agonist
decorated chitosan derivatives were able to transfect A549 cells. Moreover, in vitro
studies using phorbol 12-myristyl 13-acetate (PMA) stimulated macrophage-like THP-1
(mTHP-1) cells were focused on cytotoxicity of both polymers and particles, and their
potential to stimulate IL-8 release via the TLR-2 pathway. Our results showed that the
TLR-2 functionalized pDNA nanocarriers have the ability to complex and to protect
pDNA against enzymatic degradation. pDNA nanocarriers were of around 400 nm in
size, and displayed a positive zeta potential of 27.9 ± 1.6 mV. Chitosan, CM-TMC and
Pam3Cys-functionalized CM-TMC polymers displayed cytotoxicity on mTHP1 cells in a
concentration dependent manner, which decreased by 50-fold on complexation with
pDNA. In addition, decorated pDNA nanocarriers induced IL-8 secretions by mTHP-1
macrophages, which were increased by 10-fold compared to non-decorated carriers.
1. Introduction
Chitosan is typically produced in industry via alkaline N-deacetylation of chitin obtained
from wastes of crab shells. With regard to mucosal vaccination, chitosan has been shown
to be suitable as a carrier system due to its promising characteristics, such as adjuvant
80
activity (Lee et al., 2008), biodegradability, absorption enhancement of hydrophilic
drugs and mucoadhesivity (van der Lubben et al., 2001). Due to safety considerations
with respect to impurities of proteinaceous nature potentially present in chitosan of
animal origin, efforts have been undertaken to identify alternative sources. Hereby,
fungi (Agaricus bisporus) appeared to be of interest due to easy handling, harvesting and
controlled production, yielding a chitosan of high quality and purity (Di Mario et al.,
2008). As fungal chitosan shows no immunogenic contamination by animal proteins, it
may be regarded as a suitable material for the design of vaccination carrier systems.
Inhalation is considered an interesting route of vaccine application (Bivas-Benita et al.,
2005), as was shown, e.g., in several clinical measles vaccination trials (Dilraj et al.,
2000; Bennett et al., 2002). In addition, in previous animal studies it was shown that a
DNA vaccine, expressing several epitopes of M. tuberculosis, condensed by chitosan
(Bivas-Benita et al., 2004) or PLGA-PEI nanocarriers (Bivas-Benita et al., 2009) and
applied by the pulmonary route, was able to elicit an immune response superior to
intramuscular injection. In order to elicit a protective and long lasting immune response,
a safe and potent non-specific stimulator of the immune system (adjuvant) should be
considered (O`Hagan et al., 2006), especially for mucosal vaccination in the lung.
To address the necessity of including an adjuvant in the same formulation as the pDNA
vaccine, we recently described a method for the synthesis of a novel copolymer, CMTMC-g-PEG-Pam3Cys, based on chitosan from an animal source (Heuking et al., 2009).
This copolymer is composed of the water-soluble chitosan derivative 6-0carboxymethyl-N,N,N-trimethylchitosan
(CM-TMC),
to
which
the
water-soluble
(PEGylated) Toll-like receptor-2 (TLR-2) agonist ω-amido-[Nα-palmitoyl-oxy-S-[2,3bis(palmitoyl-oxy)-(2R)-propyl]-[R]–cysteinyl]-α-amino
poly
(ethylene
glycol)
(abbreviated Pam3Cys-PEG-NH2) was grafted.
81
We selected this particular PEGylated TLR-2 agonist due to the following reasons: i)
Kleine et al. (1994) demonstrated that conjugates of the lipophilic Pam3Cys moiety
coupled to poly (ethylene glycol) (PEG) retain their immunological properties; ii)
Pam3Cys-functionalized epitope peptides elicited strong local and systemic T-cell
responses after intranasal (Zeng et al., 2000) or after intravaginal (Zhang et al., 2009)
application; more interestingly, Zhang et al. (2009) were able to demonstrate that the
vaginal application of peptide epitope vaccines conjugated to Pam3Cys moieties elicits
protective immunity against Herpes simplex virus (HSV) in mice; iii) TLR-2 is abundantly
expressed in antigen-presenting cells (APC), such as dendritic cells and macrophages
(Muzio et al., 2000). In the framework of the pulmonary mucosal immune system,
macrophages in orchestration with dendritic and epithelial cells (Blank et al., 2007),
represent a first line of defence against invading pathogens due to their ability to
phagocytose, process and present antigens. Recognition of non-self is partially mediated
by pathogen recognition receptors (PRR), among which figures the family of Toll-like
receptors (TLR) (Iwasaki and Medzhitov 2004). One member of this family, TLR-2, is
expressed in the lung in macrophages, dendritic and epithelial cells (Nishimura and
Naito, 2005; Muzio et al., 2000). Differentiated THP-1 cells (mTHP-1 cells) showed much
promise as an in vitro model to elucidate the activation of the TLR-2 pathway in
macrophages. This human leukemia cell line is well-established and broadly used for the
investigation of the function of human macrophages (Schnoor et al., 2009). In view of the
TLR-2 pathway, Sadik et al. (2008) demonstrated that mTHP-1 cells are strongly
activated by the TLR-2 agonist Pam3Cys-Ser-(Lys)4. In more detail, they were able to
assess the extent of cellular activation by measuring the release of the chemokine IL-8,
which is an important regulator for successive leukocyte recruitment and trafficking to
the mucosal infection site.
82
In our study, we evaluated the potential of the new TLR-2 agonist functionalized
chitosan derivative for its application to mucosal vaccination. We prepared nanocarriers
by complex coacervation of CM-TMC-g-PEG-Pam3Cys with a model pDNA expressing the
reporter gene, green fluorescent protein (GFP). After physical characterization, and
assessment of cytotoxicty, we examined the ability of these nanocarriers to induce IL-8
secretion from differentiated mTHP-1 macrophages in vitro.
2. Materials and methods
2.1 Materials
N,N,N-trimethylated chitosan (trimethylation of 33.3%, abbreviated TMC35), prepared
from vegetal chitosan (degree of deacetylation of 87.7% and molecular weight of 108
kDa), was a kind gift by Kitozyme S.A. (Belgium). Dialysis membrane Spectra/Por 4 (cutoff
12-14,000Da)
was
obtained
from
Spectrum
(USA).
N,N’-
Bis(fluorenylmethoxycarbonyl)-[R]-cystine-bis-tert-butyl ester was purchased from
Bachem (Switzerland). α,ω-bis-amino poly(ethylene glycol), PEG diamine of a molecular
weight (MW) of approximately 3,000 Da was purchased from IRIS Biotech GmbH
(Germany). Pam3Cys-Ser-(Lys)4 (Pam3CSK4) was obtained from Invivogen (France) and
aliquoted in endotoxin-free water. RPMI 1640 cell culture medium, fetal calf serum
(FCS), mercaptoethanol and phorbol 12-myristate 13-acetate (PMA) were obtained from
Pan Biotech GmbH (Germany). Human IL-8 ELISA kit was from Assay Designs (France).
Agarose powder was obtained from Bio-Rad (France). pIRES-hrGFP II, a 5.5 kbp plasmid,
was received from Stratagene (France) and amplified by using an Endofree Plasmid
Maxi Kit (Qiagen, France). DNase I (amplification grade) was from Invitrogen (France).
All other reagents and solvents were of analytical grade and supplied by Sigma-Aldrich
(Switzerland).
83
2.2 Methods
2.2.1 Preparation of CM25-TMC35-g-PEG-Pam3Cys
6-0-carboxymethylation of TMC35 was performed according to a recently published
method (Heuking et al., 2009). Briefly, TMC35 was suspended in 1-methyl-2-pyrrolidone
(NMP) and the pH was adjusted to a value of 10.0 ± 0.1 by using 15% aqueous NaOH
solution. Next, chloroacetic acid (20 mol equivalents to TMC35 sugar monomers) was
added, and the pH was kept constant at a value of 10.0 ± 0.1. After 3h of reaction at room
temperature, the resulting 6-O-carboxymethyl-N,N,N-trimethylchitosan polymer (CM25TMC35) was precipitated with ethanol and diethylether (1:1 v/v) and centrifuged at
1850 x g for 15 minutes. CM25-TMC35 was then re-dissolved in MilliQ water and
rendered water-soluble by adjusting the pH to a value of 5.0 ± 0.1. Finally, the solution
was dialysed, sterile filtrated and lyophilized.
CM25-TMC35 was successively functionalized with the TLR-2 agonist NH2-PEG-Pam3Cys
(Heuking et al., 2009). Shortly, NH2-PEG-Pam3Cys was completely dissolved in MilliQ
water by gently heating at 40°C for 5 minutes. Separately, CM25-TMC35 was dissolved in
MilliQ water at room temperature and the pH adjusted to a value of 7.0 ± 0.1. Next, N-(3Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added and the pH re-adjusted to neutrality. The solution
containing NH2-PEG-Pam3Cys was then slowly added and the reaction was let stirring for
96 hours. Next, the solution was dialysed, sterile filtered and then freeze-dried.
2.2.2 Characterization of CM25-TMC35-g-PEG-Pam3Cys
The degree of 6-0-carboxymethylation (%DCM) and the degree of grafting (%DG) were
determined as described earlier (Heuking et al., 2009) by 1H NMR spectroscopy on a
Varian VXR 300 MHz spectrometer (Varian, Switzerland). Chitosan derivatives were
84
dissolved in D2O and all other compounds in CDCl3. FTIR spectra were recorded on a
Perkin-Elmer 100 FT-IR spectrometer (Perkin-Elmer, Switzerland) in the range of 4000–
400 cm-1 using KBr pellets (1% w/w of product in KBr). SEC-MALLS measurements
were performed using a TOSOH TSK Gel G3000PWXL-CP size exclusion column (TOSOH
Bioscience, Germany) with 0.2 M sodium acetate/0.3 M acetic acid (pH 4.4) as eluent
(0.3 mL/min). Waters Alliance HPLC system coupled to a differential refractive index
(RI) detector (Schambeck, Germany) and a light scattering detector (MiniDawn, Wyatt,
USA) was used for sample handling. Pullan standards ranging from 47,000 g/mol to
710,000 g/mol (PSS, Germany) were used for calibration. In addition, the solubility of
both chitosan derivatives (CM25-TMC35 and CM25-TMC35-g-PEG-Pam3Cys) was
determined in physiological phosphate buffered saline (PBS, pH 7.4) at a concentration
of 5.0 mg/mL. The transmittance (%T) of polymers was measured at λ = 600 nm by
using a Cintra 404 UV/VIS spectrometer (Switzerland). Polymers were considered
soluble for %T > 90% and very soluble for %T > 95 in comparison to the %T of PBS
alone (marked with + and ++, respectively).
2.2.3 Particle preparation
Polyplexes were formed according to the procedure described by Bivas-Benita et al.
(2004) with some modifications. More precisely, CM25-TMC35-g-PEG-Pam3Cys (at 3.10
mg/mL, average molecular weight per sugar unit of 290.8 Da, 3.6 μmol/mL -N+(CH3)3, N)
or CM25-TMC35 (at 2.21 mg/mL, average molecular weight per sugar unit of 207.1 Da,
3.6 μmol/mL -N+(CH3)3, N) were dissolved in MilliQ water. Separately, the plasmid
pIRES-hrGFP II (abbreviated pDNA; 1 μg of pDNA being equal to 3.1 nmol of phosphate
groups, P) was dissolved in 5 mM aqueous Na2SO4 at different concentrations in order to
yield N/P ratios of 3:1, 10:1 and 30:1. Both solutions were heated for 5 minutes at 55°C.
85
Next, the polymer solution was slowly added (approximately 1 drop per second) to the
pDNA solution and subsequently vortexed at high speed for 30 seconds. Attention was
paid to keep the final volume at 400 μL in order to obtain a narrow particle size
distribution. Particles were kept at room temperature for at least one hour prior to
further use.
2.2.4 Characterization of particles
2.2.4.1 Electrophoretic mobility analysis and DNase I protection assay
The ability of CM25-TMC35-g-PEG-Pam3Cys and CM25-TMC35 to bind and immobilize
pDNA was examined by agarose gel electrophoresis. Hereby, CM25-TMC35-g-PEGPam3Cys pDNA, CM25-TMC35 pDNA carriers (at N/P ratios of 3:1 to 30:1) were mixed
with bromophenol blue as loading dye. pDNA in the absence of polymers served as
control as well as a 1 kbp pDNA ladder (BioLabs, England). 20 μL of each sample was
applied in a 1.5 % agarose gel stained with 0.6 μg/mL of ethidium bromide.
Electrophoresis was then performed at 100 V for 20-30 minutes using TBE buffer. pDNA
migration was detected by means of an UV transilluminator (Bio-Rad, France).
In addition, the capability of polyplexes to protect incorporated pDNA from enzymatic
degradation via DNase I was assessed. pDNA in the absence of polymers, CM25-TMC35g-PEG-Pam3Cys pDNA and CM25-TMC35 pDNA carriers (both 20 μL, equivalent to 3 μg
pDNA, N/P ratio of 3:1) were incubated with 4 μL of DNase I solution (1U/μL in DNase I
buffer consisting of 200 mM TRIS-HCl (pH 8.4), 20 mM MgCl2 and 500 mM KCl) for 15
minutes at room temperature. The experiment was terminated by adding 4 μL of 25 mM
EDTA solution to the reaction mixture. The integrity of pDNA was then determined by
the electrophoretic mobility assay as described above.
86
2.2.4.2 Size and zeta potential of nanocarriers
Hydrodynamic diameters were measured by Photon Correlation Spectroscopy
(ZetaSizer 3000 HS, Malvern, Switzerland). For measurements, either 400 μL of
polyplexes were diluted with MilliQ water to 1.4 mL, or diluted at a ratio of 1:15 in plain
RPMI 1640 medium. Size distribution data were obtained by the number-averaged value
of three independent groups of ten measurements. In addition, zeta potential was
measured at least in triplicate via micro-electrophoresis by using an aqueous dip cell
(ZetaSizer 3000 HS, Malvern, Switzerland).
2.2.4.3 Loading efficiency
For the determination of encapsulated pGFP, 400 μL of nanoparticle suspension was
centrifuged at 16,000 x g for 30 min (Centrifuge 5417C/R, Eppendorf, Germany).
Unloaded pGFP in the supernatant was quantified with the PicoGreen assay (Invitrogen,
Switzerland) according to the manufacturer's procedures. The fluorescence was
measured with the FluoroMax spectrometer (Spex, Switzerland) at excitation and
emission wavelengths of 480 and 522 nm, respectively.
2.2.5 Cell lines and culture
All cell lines mentioned here are of human origin: A549, a cell line derived from a lung
carcinoma; Caco-2, a colorectal cancer cell line; Calu-3, a submucosal cell line derived
from a lung adenocarcinoma; HEK-293, an embryonic kidney cell line; SV-HUC-1, a
normal urothelial cell line; T24, a bladder cancer cell line and THP-1, a monocyte cell
line were purchased from ATCC (American Type Culture Collection, USA). 16HBE14o(HBE) cell line, passage number (PN) 80 was generously gifted by Dr. Carsten Ehrhardt,
Trinity College, University of Dublin, Ireland.
87
All cell lines were grown in specific medium supplemented with 10% (v/v) fetal bovine
serum and penicillin (100 U/mL) and streptomycin (100 µg/ml). Cells were cultured in
an incubator at 37ºC, 95% relative humidity, 5% CO2 and passaged at approximately
80% confluency. All cell culture reagents were provided by Invitrogen and SigmaAldrich (France).
THP-1 cells, a monocytic cell line, were cultured in RPMI 1640 medium, supplemented
with 10% heat-inactivated fetal calf serum (FCS) and 0.05 mM mercaptoethanol. Cells
were kept at a density below 106 cells/mL (PN<10) and were cultured as stated above.
THP-1 cells were seeded at a density of 2 x 105 cells per well in a 96-well plate unless
stated otherwise. For differentiation, PMA was added before seeding at a concentration
of 50 ng/mL and cells were incubated for 48h. After differentiation to mTHP-1 cells and
twice washing with phosphate buffered saline (PBS), cells were cultured for a recovery
period of 3h in the above mentioned medium and used for further experiments (Sadik et
al., 2008).
2.2.6 Isolation of TLR-2 mRNA, Reverse Transcriptase-Polymerase Chain Reaction (RTPCR) and Real Time Quantitative Polymerase Chain Reaction (qPCR)
All cell lines mentioned in 2.2.5 were detached, separately collected and centrifuged for
5 min at 1000 x g. The supernatant was removed and cells were snap frozen at –80°C.
Total RNA was isolated using the RNeasy Mini kit (Qiagen, France) according to
manufacturer’s instructions. Isolated RNA was quantified using a ND-1000
spectrophotometer (Nanodrop, France). RNA (1μg) was reverse transcribed using the
iScript cDNA Synthesis Kit (Bio-Rad, France) using conditions provided by the
manufacturer. 2 μL of RNase free water was used to prepare the negative control. The
reverse transcriptase was performed in a MyiQ apparatus (Bio-Rad, France) according
88
to the following protocol: 25°C for 5 minutes, 42°C for 30 minutes and 85°C for 5
minutes. In addition, qPCR analysis was performed in an MyIQ™ single-color Real-time
PCR detection system (Bio-Rad, France) thermal cycler to quantify TLR-2 and β-actin
mRNA levels. To validate each primer pair, a standard curve was plotted using known
concentrations of DNA expressing the target sequence. Oligonucleotides were used as
follows:
hβ-actin
forward
TCCCTGGAGAAGAGCTACGA,
hβ-actin
reverse
AGGAAGGAAGGCTGGAAGAG, hTLR-2 forward GGCCAGCAAATT ACCTGTGTG and hTLR-2
reverse AGGCGGACATCCTGAACCT. qPCR was performed in a total volume of 25 μl using
the iQ SYBR Green Supermix (Bio-Rad, France) and 300nM of each primer. Cycle
parameters were 95°C for 3 minutes, 40 times: 95°C for 10 seconds, 55°C to 60°C
(depending on the primer pair) for 30 seconds. All samples from the dilution series were
run in duplicates although a no-template control was prepared using Minversol water
(Aguettant, France). Data were analyzed using the Optical system software.
2.2.7 In vitro toxicity
Cytotoxicity in differentiated THP-1 cells (mTHP-1 cells) was evaluated using a
colorimetric assay based on a cell proliferation reagent WST-1 (Roche, Switzerland). The
rate of WST-1 cleavage by mitochondrial dehydrogenases correlates with the number of
viable cells in the culture. This allows a non-radioactive, spectrophotometric
quantification of cell viability. Following concentrations (100 µL per well) were applied
after 1:15 dilution with plain RPMI 1640 medium: polymers at 10 and 100 µg/mL; NH2PEG-Pam3Cys at 0.12 mg/mL; CM25-TMC35 at 0.14 mg/mL (pDNA nanocarrier, N/P 3:1),
CM25-TMC35-g-PEG-Pam3Cys at 0.2 mg/mL (pDNA nanocarrier, N/P 3:1) and pDNA at
40 μg/mL. 100 µL of each sample was incubated in a 96-well plate (at least in triplicates)
for 6h at 37ºC. The optical density was then read using a universal microplate
89
spectrophotometer reader, Power Wave XS (Biotek, France) at 690 and 450 nm. The
results were calculated by subtracting the absorbance at 690 nm from the one at 450
nm, thus data were expressed in δ OD values and corrected by the blank value.
2.2.8 GFP Transfection
A549 and mTHP-1 cells were seeded at 1 x 105 cells per well (24-well plate) and
cultured for 48h. After mTHP-1 cells were handled as above stated, the following DNA
formulations (2 µg /well) in the appropriate plain medium (for NP) or full medium (for
TransIT-LT1 formulation) were incubated with A549 and mTHP-1 cells: 1) CM25-TMC35
pDNA NP, 2) CM25-TMC35-g-PEG-Pam3Cys pDNA NP and 3) TransIT-LT1 pDNA
preparation (used pursuant to the manufacturer’s recommendations).
After five hours of exposure, formulations were removed (with the exception of TransITLT1 pDNA preparation) and full medium was added. Medium was then changed every
second day. Starting with day two until day six, cells were analysed by fluorescence
microscopy (Zeiss Axiovert 40 CFL, Germany) and pictures (Canon Powershot A640,
France) were taken at 5x magnification. Number of GFP-positive cells (transfection
efficiency) was assessed by using ImageJ 1.43 Software.
2.2.9 Interleukin-8 (IL-8) release by mTHP-1 cells
Human IL-8 TiterZyme ELISA kit was obtained from Assay Designs (USA). The following
concentrations (250 µL per well) were studied after 1:15 dilution with plain RPMI 1640
medium: Pam3Cys-Ser-(Lys)4 at 1.0 µg/mL, NH2-PEG-Pam3Cys at 2.6 µg/mL (equimolar to
1.0 µg/mL of Pam3Cys-Ser-(Lys)4), CM25-TMC35-g-PEG-Pam3Cys at 0.2 mg/mL (the
amount of NH2-PEG-Pam3Cys grafted is equimolar to 1.0 µg/mL of Pam3Cys-Ser-(Lys)4),
CM25-TMC35 at 0.14 mg/mL (equimolar to CM25-TMC35-g-PEG-Pam3Cys), both chitosan
90
derivatives were formulated with pDNA, either with CM25-TMC35 at 0.14 mg/mL or
CM25-TMC35-g-PEG-Pam3Cys at 0.2 mg/mL (both at N/P 3:1) and as control pDNA alone
(same amount as encapsulated in nanocarriers). A 96-well plate was incubated with
above mentioned samples (250 µL) in duplicates for 6h at 37ºC. Next, cell supernatants
of mTHP-1 cells were taken and used for quantification of human IL-8. The following
analysis of samples was performed in duplicates according to the manufacturer´s
instructions. Standard curve and sample concentrations were calculated with of the help
of Graph Pad Prism 5.02 software by using a four-parameter logistic curve fitting.
2.2.9 Data analysis and statistics
Obtained data were expressed as the mean ± standard error of the mean and compared
by a two-tailed Student's t-test using Graph Pad Prism 5.02 software (Graph Pad
Software, USA). Differences were considered significant at p<0.05.
3. Results and discussion
3.1 Synthesis and characterization of chitosan derivatives
In order to combine the adjuvant activity of Pam3Cys moiety with the beneficial
characteristics of chitosan polymers for mucosal pDNA vaccine delivery, we had
previously synthesized a new chitosan derivative (Heuking et al., 2009). Moreover, and
as described in this report, we applied the developed synthesis procedure to a chitosan
from a vegetal source. Firstly, vegetal chitosan was trimethylated yielding N,N,Ntrimethyl chitosan (TMC). TMC (synthesized from chitosan of animal origin)
demonstrated high water-solubility at physiological pH, as well as pronounced adjuvant
properties (Baudner et al., 2004). 1H NMR spectroscopy of vegetal TMC synthesized here
(Fig. 1B) revealed a degree of trimethylation (%DTM) of 33.3 % (abbreviated as TMC35),
91
whereas peaks can be assigned as published previously (Heuking et al., 2009).
A
B
C
Figure 1: 1H NMR spectra of A) Pam3Cys-PEG-NH2, B) TMC35 and C) CM20-TMC35-g-PEGPam3Cys (all in D2O at 80 °C).
The DTM is a crucial parameter, as only TMC polymers of a DTM higher than 22% are
able to open tight junctions of mucosal epithelia and are thus expedient for mucosal
delivery (Hamman et al., 2003). In addition, we quantified the degrees of 3- and 6hydroxy-methylation (%D3OM and %D6OM, respectively), as well as dimethylation
(%DDM) to be of 27.0, 30.0 and 54.4 %, respectively. These parameters were shown to
affect strongly cytotoxicity and physicochemical properties of TMC. More specific,
Jintapattanakit et al. (2008) reported that a ratio of %DDM / %DTM higher than 1
involves a decrease in mucoadhesivity and cytotoxicity. In addition, Verheul et al. (2008)
pointed out that O-methylated (%D3OM and %D6OM) TMC polymers caused lower
toxicity on Caco-2 cells when compared to O-methyl free TMC.
92
In a next step, we performed 6-0-carboxymethylation of TMC and determined the degree
of carboxymethylation (%DCM) to be of 23.2% (data not shown; polymer henceforth
abbreviated CM25-TMC35) by 1H NMR spectroscopy. In a next step, we conjugated the
TLR-2 agonist Pam3Cys-PEG-NH2 to polymeric CM25-TMC35 by using condensing agents.
Finally, we ascertained the degree of grafting (%DG) to be about 2.1% by 1H NMR
spectroscopy (Fig. 1C, Heuking et al., 2009). FTIR spectroscopy confirmed the successful
grafting (data not shown). Additionally, we analysed by SEC-MALLS for changes in the
molecular weight and found a shift from 98,330 Da for CM25-TMC35 to 250,900 Da for
CM25-TMC35-g-PEG-Pam3Cys, indicating that successful grafting occurred. Moreover,
both chitosan derivatives were highly soluble in PBS (pH 7.4) at 5.0 mg/mL
concentrations.
3.2 Nanocarrier preparation and characterization
Nanocarriers were formed at pH 7 and at low ionic strengths owing to complex
coacervation between negatively charged pDNA (phosphate groups, P) and positively
charged chitosan derivative (trimethylated amines, N). More precisely, nanocarriers of
CM25-TMC35 (used as control group) and CM25-TMC35-g-PEG-Pam3Cys were formed
with pDNA at different N/P ratios, ranging from 3:1-30:1.
Nanocarriers had a high encapsulation efficiency with 92.2 ± 1.6 % for CM25-TMC35
pDNA nanocarriers and 90.3 ± 2.3 % for CM25-TMC35-g-PEG-Pam3Cys pDNA
nanocarriers. Agarose gel electrophoresis (Fig. 2) demonstrated the capacity of CM25TMC35 and CM25-TMC35-g-PEG-Pam3Cys nanocarriers (N/P ratio of 3:1) to retain pDNA
(due to nanoparticle formation) and to protect pDNA against enzymatic degradation
with DNase I. Similar results were obtained with nanocarriers at N/P ratios of 10:1, as
well as 30:1 (data not shown).
93
Photon
Correlation
Spectroscopy
(PCS)
of
nanocarriers
pointed
at
narrow
hydrodynamic diameter distributions (table 1). Compared to CM25-TMC35 nanocarriers,
CM25-TMC35-g-PEG-Pam3Cys nanocarriers (at all N/P ratios studied) were slightly
bigger in their hydrodynamic diameter, which may be related to the protrusion of the
PEG polymers attached to the particle surface. In addition, conjugation of PEG polymers
reduced the observed zeta potential (table 1).
1
2
3
4
5
6
7
8
Figure 2: Agarose gel electrophoresis of pDNA ladder (lane 1), pDNA (lane 2), pDNA
incubated with DNase I (lane 3), CM25-TMC35 pDNA nanocarrier (lane 4), CM25-TMC35
pDNA nanocarrier incubated with DNase I (lane 5), CM25-TMC35-g-PEG-Pam3Cys pDNA
nanocarrier (lane 6), CM25-TMC35-g-PEG-Pam3Cys pDNA nanocarrier incubated with
DNase I (lane 7), pDNA ladder (lane 8). All nanocarriers were prepared at a N/P ratio of
3:1.
94
Interestingly, relating to the N/P ratio applied, the hydrodynamic diameter of
nanocarriers changed in an indirect proportional way, while the zeta potential changed
directly proportional. A comparable tendency was described by Mansouri et al. (2006)
for a chitosan-folate pDNA carrier system.
Table 1: Size distribution and zeta potential of nanocarriers (conjugate, CM25-TMC35-gPEG-Pam3Cys; 1after dilution 1:15 in plain RPMI 1640 medium).
Polymer
N/P
Size [nm]
ratio
Zeta potential
Poly-
[mV]
dispersity
CM25-TMC35
3:1
354.1 ± 87.3
32.3 ± 1.5
0.351
CM25-TMC-351
3:1
421.2 ± 125.3
17.3 ± 3.5
0.544
(96.3%)
2201.9 ± 877.2
(3.7%)
CM25-TMC35
10:1
291.1 ± 43.0
34.3 ± 7.3
0.531
CM25-TMC35
30:1
260.3 ± 75.2
36.8 ± 4.1
0.291
Conjugate
3:1
398.5 ± 112.1
27.9 ± 1.6
0.223
Conjugate1
3:1
452.1 ± 99.3 (97.8%)
15.2 ± 1.6
0.407
1901.4 ± 543.5
(2.2%)
Conjugate
10:1
327.2 ± 78.5
31.2 ± 1.4
0.458
Conjugate
30:1
281.2 ± 101.7
34.7 ± 5.3
0.433
However, after suspension of both, CM25-TMC35 as well as CM25-TMC35-g-PEG-Pam3Cys
nanocarrier systems 1:15 in RPMI 1640 medium (0.15 M ionic strength, pH 7.4), a bi-
95
modal hydrodynamic diameter distribution was detected. More than 90% of CM25TMC35 nanocarriers (N/P of 3:1) were of a size of 421.2 ± 125.3 nm, and showed a
tendency to aggregate (2201.9 ± 877.2 nm). Likewise, more than 90% of CM25-TMC35-gPEG-Pam3Cys nanocarriers (N/P of 3:1) increased in hydrodynamic diameter to 452.1 ±
99.3 nm with slightly smaller aggregates of 1901.4 ± 543.5 nm.
Gemershaus et al. (2008) observed similar characteristics of chitosan and chitosan
derivative pDNA polyplexes in DMEM culture medium. In addition, values for the zeta
potential of CM25-TMC35 and CM25-TMC35-g-PEG-Pam3Cys nanocarriers dropped
nearly by half, probably due the higher ionic strengh applied, from 32.3 ± 1.5 mV to 17.3
± 3.5 mV and 27.9 ± 1.6 mV to 15.2 ± 1.6 mV, respectively.
Moreover, chitosan pDNA vaccine delivery systems may transfect epithelial cells (Hu et
al., 2006; Huang et al. 2005). In the in vivo situation this translates into a transfection of
the pulmonary epithelium, production and secretion of the antigen, and its uptake by
antigen-presenting cells (cross-presentation). We therefore assessed the potential of
CM25-TMC35 and CM25-TMC35-g-PEG-Pam3Cys pDNA nanocarriers to transfect a human
alveolar epithelial cells, A549 (Forbes and Ehrhardt, 2005) and differentiated THP-1
macrophages. For comparison, we involved also the study of TransIT-LT1 (a
commercially available transfection reagent)-mediated pGFP transfection. All three
carrier systems had the ability to transfect A549 cells with the ranking of TransIT-LT1 >
CM25-TMC35 = CM25-TMC35-g-PEG-Pam3Cys (figure 3). In more detail, the use of
TransIT-LT1 induced at all time points (from day two until day six) a significant higher
protein expression than CM25-TMC35 and CM25-TMC35-g-NH-PEG-Pam3Cys mediated
transfection (p<0.05). No difference in transfection capacity was seen between CM25TMC35 and CM25-TMC35-g-NH-PEG-Pam3Cys pGFP delivery systems. It is noteworthy
that all chitosan-based delivery systems initiated a later onset of GFP expression in
96
comparison to TransIT-LT1 transfection. Considering the GFP transfection of mTHP-1
cells, the amount of GFP-positive cells between day two and day six was very low (below
2%, data not presented) for all formulations applied. Similar findings were already
published by Schnoor et al. (2009).
*
*
*
*
*
Figure 3: In vitro GFP transfection efficiency in A549 cells of CM25-TMC35 (red),
TransIT-LT1 transfection reagent (blue) and CM25-TMC35-g-PEG-Pam3Cys (green) pGFP
carrier systems. Statistical differences are denoted with * (p < 0.05) for TransIT-LT1
compared to LMC and CM25-TMC35-g-NH-PEG-8HA pGFP transfection, respectively.
3.3 mRNA expression of TLR-2 in different cell lines
In order to evaluate the ability of the copolymer pDNA nanocarrier to interact with the
TLR-2 receptor, we screened for the endogenous level of its expression by several
epithelial and non-epithelial cell lines using qPCR. In general, TLR-2 is expressed at the
cell surface in a large variety of cell types (Muzio et al., 2000). TLR-2 identifies its ligands
as heterodimer either combined with TLR-1 or TLR-6, whereby TLR1/TLR2
combination enables recognition of triacylated lipoproteins and TLR-2/TLR-6
recognizes diacylated lipoproteins and peptidoglycans (Wetzer, 2003).
97
In our hands, mTHP-1 cells exhibited the highest level of expression of TLR-2 (1200-fold,
normalized to β-actin expression, Fig. 4), when compared to Caco-2 or Calu-3 (250-fold)
or T24 (50-fold). 16HBE14o-, HEK-293 as well as A549 cell lines did not express TLR-2.
Similarly, Sadik et al. (2008) found also a strong expression of TLR-2 in mTHP-1 cells,
which allowed them to study the stimulation of TLR-2 agonist Pam3Cys-Ser-(Lys)4 by
measuring the release of the chemokine IL-8. Due to this study as well as the suitability
of mTHP-1 cells as an in vitro model of human macrophages (Schnoor et al., 2009), we
deployed this system for evaluating our TLR-2 agonist decorated pDNA nanocarrier.
Figure 4: mRNA expression, normalized to β-actin mRNA levels, of Toll-like receptor-2 in
different cell lines.
3.4 Cytotoxicity of chitosan derivatives and nanocarriers
As quaternization of chitosan polymers in general entails an increase in cellular toxicity
(Jintapattanakit et al., 2008), we evaluated the cytotoxicity of both new chitosan
98
derivatives (polymers and nanocarriers) as well as the TLR-2 agonist NH2-PEG-Pam3Cys
in differentiated THP-1 cells. Cytotoxicity was measured using the cell proliferation
assay, WST-1.
Both chitosan derivatives showed cytotoxicity dependent on the concentration applied
(IC50 > 10 µg/mL), however, when formulated with pDNA, the toxicity decreased by
more than 50-fold (Fig. 5).
120,0
100,0
cell survival [%]
80,0
60,0
40,0
20,0
A
iu
m
M
ed
pD
N
ju
g
at
e
C
N
C
Co
n
CM
TM
ys
N
C
m
3C
N
H2
-P
E
G
-P
a
ug
a
te
²
te
¹
C
on
j
ug
a
35
²
on
j
C
-T
M
C
M
25
C
C
M
25
-T
M
C
35
¹
0,0
Figure 5: Cytotoxicity of polymers and pDNA nanocarriers studied on mTHP-1 cells
using the following concentrations: 1at 100 μg/mL; 2at 10 μg/mL; NH2-PEG-Pam3Cys at
0.12 mg/mL; pDNA nanocarrier (NC) CM25-TMC35 at 0.14 mg/mL (N/P 3:1); NC
conjugate, CM25-TMC35-g-PEG-Pam3Cys at 0.2 mg/mL (N/P 3:1) and pDNA pDNA at 40
μg/mL.
In addition, the TLR-2 agonist NH2-PEG-Pam3Cys (applied at a molar equivalent to
99
grafted moieties) had only minor influence on the proliferation of mTHP-1 cells (cell
viability reduction of 25.4%).
3.5 IL-8 release upon stimulation of TLR-2 in mTHP-1 cells
The chemokine IL-8 (CXCL 8) is a proinflammatory cytokine, playing an important role
in the promotion of neutrophil recruitment (Baggiolini 2001). Interestingly,
differentiated THP-1 cells release high levels of IL-8 upon TLR-2 stimulation with 1
µg/mL of Pam3Cys-Ser-(Lys)4 (Sadik et al., 2008). We applied this particular in vitro
model in order to study our pDNA nanocarriers. Firstly, we investigated the potential of
NH2-PEG-Pam3Cys to trigger IL-8 production by applying an equimolar quantity to 1
µg/mL of Pam3Cys-Ser-(Lys)4. The results of this study were needed to ensure the
activity of the TLR-2 agonist grafted onto the surface of the nanocarriers by PEG spacer.
ns
70
**
IL-8 (ng/mL)
60
**
50
40
30
***
20
***
10
*
0
Medium
1.0 µg/mL of
Pam3CSK4
NH2-PEGPam3Cys
CM25-TMC35
Conjugate
Figure 6: IL-8 release studied by ELISA in mTHP-1 cell supernatants following polymer
exposure (conjugate, CM25-TMC35-g-PEG-Pam3Cys; Pam3CSK4, Pam3Cys-Ser-(Lys)4).
Significance was controlled by two-tailed Student's t-test via comparison with values
obtained from cell culture medium, unless indicated otherwise with horizontal bars.
100
Differences were considered significant for * p<0.05, ** p<0.01 and *** p<0.001. ns: not
significant.
As shown in Fig. 6, NH2-PEG-Pam3Cys elicited a strong IL-8 release (46.8 ng/mL), which
was not significantly different (p>0.05) from the value obtained for the reference TLR-2
agonist Pam3Cys-Ser-(Lys)4 (59.6 ng/mL).
Overall, we report here for the first time the immune stimulating effect of a PEGylated
Pam3Cys moiety, NH2-PEG-Pam3Cys, on mTHP-1 cells measured by IL-8 release.
Interestingly, Sadik et al. (2008) demonstrated that around 70% of IL-8 levels triggered
by Pam3Cys-Ser-(Lys)4 on mTHP-1 cells can be blocked via TLR-2 antibody co-incubation.
We assume therefore that the IL-8 inducing effect of NH2-PEG-Pam3Cys is also mainly
regulated by the same TLR-2 pathway.
In the past, the first study demonstrating immunogenic properties of PEGylated Pam3Cys
moieties were carried out by Kleine et al. (1994), whereby the adjuvant activity in vitro
in murine and human B lymphocytes as well as in vivo in mice was investigated. It was
shown that the water-soluble conjugate maintained its immunogenic properties after
PEGylation of the Pam3Cys moiety in vitro and in vivo when compared to Pam3Cys-Ser(Lys)4.
However, as TLR-2 expression was unknown at the time, those results were not brought
in correlation to the TLR-2 pathway. In line with our study, we found a significant
(p<0.001), almost 4-fold increase in IL-8 production caused by the 2.1 % grafting of NH2PEG-Pam3Cys to CM25-TMC35 (CM25-TMC35-g-PEG-Pam3Cys: 11.9 ng/mL; CM25-TMC35:
3.2 ng/mL). Interestingly, CM25-TMC35 polymer on itself appears to be able to trigger
release of IL-8, which is significantly different (p<0.05) from cell culture medium as the
respective control group (Fig. 6). This property of chitosan polymers was also reported
by Park et al. (2009). Herein, neutrophil-like HL60 cells were incubated with chitosan of
101
different degrees of acetylation and similarly to our study, the triggered secretion of IL-8
was analyzed.
As a result, they measured an IL-8 release up to approximately 1.5 ng/mL, which is in
good accordance with the level of IL-8 found (3.2 ng/mL) for CM25-TMC35 polymer
exposure in our study.
***
IL-8 (ng/mL)
20
***
10
***
**
0
Medium
pDNA
NP CM25-TMC35
NP Conjugate
Figure 7: IL-8 release studied by ELISA in mTHP-1 cell supernatants following pDNA
nanocarriers, as well as pDNA exposure (conjugate = CM25-TMC35-g-PEG-Pam3Cys).
Significance was checked by two-tailed Student's t-test via comparison with values
obtained from cell culture medium, unless indicated otherwise with horizontal bar.
Differences were considered significant for * p<0.05, ** p<0.01 and *** p<0.001. ns: not
significant.
Furthermore, the TLR-2 agonist decorated pDNA nanocarriers were about 10-fold more
potent than the CM25-TMC35 nanocarriers in view of eliciting IL-8 release from mTHP-1
cells (p<0.001, Fig. 7).
102
In our view, an explanation why this difference was not more pronounced lies in the
structure of the nanocarriers.
Due to the coacervation process between pDNA and the co-polymer, some of the TLR-2
targeting moieties may be buried inside the particles, and are thus not displayed on the
surface to interact with their target receptor.
4. Conclusions
The results obtained in this study point towards the possibility to synthesize TLR
agonist functionalized co-polymers, based on a chitosan obtained from vegetal sources.
Complexation with pDNA yielded decorated nanocarriers, which, by virtue of the TLR-2
agonist present at the particle surface, were able to induce IL-8 release in human
macrophages in vitro. In successive in vivo studies, the adjuvant effect of the described
nanocarriers will be evaluated. Moreover, we believe that TLR ligand functionalized
polymers, and nanocarriers systems, represent a technology platform to address the
modulation of TLR activity in a variety of diseases, including autoimmune and
inflammatory diseases, and cancer. Studies pertaining to such applications are currently
underway in our lab.
Acknowledgments
The authors would like to acknowledge Prof. Peter Speiser for the continuing inspiration
he provides for generations of scientists working in the field of drug nanocarrier
systems, and drug targeting.
103
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Chapter 5
Functionalization with a TLR-7
agonist enhances the
immunogenicity of chitosan DNA
nanoparticles in human THP-1
macrophages
S. Heuking1,2 and G. Borchard1,2
To be submitted
1School
of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
109
Abstract
In order to provide an adjuvant-equipped carrier system for plasmid DNA vaccines, we
grafted a TLR-7 agonistic moiety (9-benzyl-8-hydroxyadenine, 8HA) through a PEG
spacer onto a water-soluble chitosan derivative (CM-TMC). Successful grafting was
confirmed by spectroscopic (1H NMR, Mass, UV/VIS and FTIR) and chromatographic
(SEC-MALLS) methods. In the following, TLR-7 agonist functionalized nanoparticles
(NP) were prepared by aggregation with pDNA. NP were around 400 nm in size with a
positive surface charge and had the ability to transfect alveolar A549 cells. In addition,
TLR-7 agonist functionalization was shown to increase the IL-8 and IL-12 specific
immunogenicity in mTHP-1 macrophages significantly, when compared to nonfunctionalized NP.
1. Introduction
Recently, we reported on the synthesis of a new TLR-2 agonist functionalized chitosan
derivative (Heuking et al., 2009a), which was employed for the encapsulation of plasmid
DNA (pDNA) resulting in positively charged NP. Interestingly, TLR-2 agonist decoration
of pDNA nanoparticles (NP) enabled an approximately ten-fold higher induction (p<
0.001) of stimulatory cytokine IL-8 from differentiated human macrophages-like THP-1
cells (Heuking et al., 2009b). In an attempt to further study the potential of TLR ligands
as adjuvants for chitosan particle-based DNA vaccination, 8-hydroxyadenine derivatives
as TLR-7 agonists attracted our attention. Human TLR-7 located in the endosomal
compartment of innate immune cells is highly expressed in the lung, placenta and spleen
(Chuang and Ulevitch, 2000). Activation of TLR-7 involves the MyD88-dependent
signaling cascade and elicits the production of IFN-α, TNF-α and IL-12, favoring a cellmediated immunity (Hemmi et al., 2002). Natural ligands of TLR-7 consist of single-
110
stranded RNA oligonucleotides (ssRNA; Heil et al., 2004). In addition, small chemical
molecules, such as imidazoquinolines and 8-hydroxyadenine derivatives with significant
TLR-7 agonistic capacity were discovered recently (Hemmi et al., 2002; Lee et al., 2006).
In our study, a TLR-7 agonistic moiety, a 9-benzyl-8-hydroxyadenine derivative, was
selected and rendered water-soluble by PEG chain attachment at its C-2 position. In
turn, we grafted the TLR-7 targeting conjugate to a water-soluble chitosan derivative
(CM-TMC) using procedures established previously (Heuking et al., 2009a).
Subsequently, DNA incorporating nanoparticles were prepared, which were then
analysed for their DNA transfection efficiency and immunogenicity in THP-1
macrophages.
2. Materials and methods
2.1 Materials
N,N,N-trimethylated chitosan (trimethylation of 33.3%, abbreviated TMC35) was
prepared from vegetal chitosan (degree of deacetylation of 87.7% and molecular weight
of 108,000 Da), which was a kind gift by Kitozyme S.A. (Herstal, Belgium). Low
molecular weight chitosan (molecular weight below 10,000 Da and degree of
deacetylation of 85.3%) was obtained from Nicechem (Shanghai, China). Dialysis
membrane Spectra/Por 4 (cut-off 12-14,000Da) and Spectra/Por 7 (cut-off 1,000 Da)
were obtained from Spectrum (USA). α-amino-ω-t-butyloxycarbonylamino poly(ethylene
glycol) was purchased from IRIS Biotech GmbH (Germany). 2-chloro-9-benzyl-8hydroxyadenine was received from Accely (China). RPMI 1640 cell culture medium, fetal
calf serum (FCS), mercaptoethanol and phorbol 12-myristate 13-acetate (PMA) were
obtained from Pan Biotech GmbH (Germany). Human IL-12p40 ELISA kit was from BD
Biosciences (Switzerland). Human IL-8 ELISA kit was purchased from Assay Designs
111
(France). Agarose powder was obtained from Bio-Rad (France). pIRES-hrGFP II, a 5.5
kbp plasmid, was received from Stratagene (France) and amplified by using an Endofree
Plasmid Maxi Kit (Qiagen, France). DNase I (amplification grade) was from Invitrogen
(France). Heparin (175U/mg) was obtained from LKT labo (Switzerland). The
transfection reagent TransIT-LT1 was purchased from Mirus (France). All other
reagents and solvents were of analytical grade and supplied by Sigma-Aldrich
(Switzerland).
2.2 Characterization of polymers
1H
NMR spectra were recorded on a Varian VXR 300 MHz spectrometer (Varian,
Switzerland). Chitosan was dissolved in 1% v/v DCl/D2O, chitosan derivatives in D2O
and all other compounds in DMSO or CD3CN (concentrations of 20 mg/mL). Chemical
shifts are indicated in parts per million (δ) downfield from the internal standard
tetramethylsilane (Me4Si). Mass spectroscopy spectra were recorded on an API 150 EX
LC/MS System (Switzerland). The MALDI-TOF mass spectrometry was conducted on an
Axima CFR+, Shimadzu mass spectrometer (Switzerland). Homogeneity of synthesized
compounds was confirmed by thin layer chromatography (TLC) on silica gel Merck 60
F254 aluminium plates. FTIR spectra were recorded on a Perkin-Elmer 100 FT-IR
spectrometer (Perkin-Elmer, Switzerland) in the range of 4000–400 cm-1. SEC-MALLS
measurements were performed using a TOSOH TSK Gel G3000PWXL-CP size exclusion
column (TOSOH Bioscience, Germany) with 0.2 M sodium acetate/0.3 M acetic acid (pH
4.4) as eluent (0.3 mL/min). A Waters Alliance HPLC system coupled to a differential
refractive index (RI) detector (Schambeck, Germany) and a light scattering detector
(MiniDawn, Wyatt, USA) was used for sample handling. Pullan standards ranging from
47,000 g/mol to 710,000 g/mol (PSS, Germany) were used for calibration.
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2.3 Synthesis of ω-amido-[9-benzyl-8-hydroxyadenine-2yl]α-amino poly(ethylene glycol)
2.3.1 Synthesis of α-amido-[9-benzyl-8-hydroxyadenine-2yl]-ω-t-butyloxycarbonylamino
poly(ethylene glycol) 2
Synthesis of 2 was achieved according to Isobe et al. (2006) with some modifications.
First, 22.97 mg (83.3 mmol, five times excess of free amines) of 2-chloro-9-benzyl-8hydroxyadenine (abbreviated 8HA, 1) were dissolved in 20 mL n-butanol at 120°C
followed by the addition of 50 mg (16.7 mmol) of α-amino-ω-t-butyloxycarbonylamino
poly(ethylene glycol) (abbreviated Boc-NH-PEG-NH2). After 16h of stirring at 120°C, the
reaction mixture was cooled to room temperature and centrifuged at 1,850 x g for 15
minutes in order to remove unreacted (and at room temperature insoluble) 8HA. The
supernatant was taken and the procedure was repeatedtwice. The resulting supernatant
was then dialysed (cut-off 1,000 Da) over three days against deionized water (twice
daily changed), sterile filtered and lyophilized. Yellowish powder. Yield 64 %. 1H NMR
(300 MHz, CD3CN): δ 1.4 ((CH3)3-O); δ ~3.6 (PEG-CH2); δ 5.1 (NH2); δ 6.6 (PEG-NH); δ
7.3 (aromatic hydrogen); δ 8.1 (N-CH2). FTIR: 679, 770, 841, 908, 928, 1060, 1101, 1663,
1706, 2882, 3137, 3419. UV (λmax, 6M HCl): 259.9 nm.
2.3.2 Synthesis of ω-amido-[9-benzyl-8-hydroxyadenine-2yl]α-amino poly(ethylene glycol)
3
Compound 3 (abbreviated NH2-PEG-8HA) was obtained after deprotection of tert-butyl
cabarbamat (Boc) group by using the method of Shendage et al. (2004). Briefly, 100 mg
of 2 was dissolved in 20 mL of TFA:CH2Cl2 1:1 (v/v) and stirred at 1000 rpm for two
hours at room temperature. After evaporation of solvents, the residue was dissolved in
10 mL of deionized water and dialysed (membrane cut-off of 1,000 Da) over three days
(twice daily change of deionized water), sterile filtered and lyophilized. Yellowish
113
powder. Yield 94 %. 1H NMR (300 MHz, CD3CN): δ ~3.6 (PEG-CH2); δ 5.1 (NH2); δ 6.6
(PEG-NH); δ 7.3 (aromatic hydrogen); δ 8.1 (N-CH2). FTIR: 679, 770, 841, 908, 928,
1060, 1101, 1663, 1706, 2882, 3137, 3419. UV (λmax, 6M HCl): 258.8 nm. MS (API):
expected m/z 3072.6 M+, found m/z 3070.2 M+.
2.4 Synthesis of CM-TMC-g-NH-PEG-8HA (4)
TMC and CM-TMC were synthesized as described previously (Heuking et al., 2009a).
NH2-PEG-8HA was grafted to CM-TMC by using EDC/NHS condensing agents. Briefly, CMTMC (20 mg; 0.089 mmol sugar units, 0.015 mmol COOH) was dissolved in MilliQ water
(4 mL) at room temperature and the pH adjusted to a value of 7. Next, EDC (5.7 mg, 0.03
mmol, 2 mol equivalents to COOH) and NHS (3.5 mg; 0.03 mmol, 2 mol equivalents to
COOH) were added. NH2-PEG-8HA (8.2 mg, 0.003 mmol) was added and reaction was
performed during 120 hours, whereby the pH was maintained at neutrality. In the
following, the solution was dialysed (membrane cut-off 12-14,000 Da) over the course of
one week (medium changed twice daily), sterile filtered and then freeze dried. The
finalized product (4) was then submitted to further investigation. The degree of grafting
(DG) was assessed by using the following equation: %DG = ([PEG-CH2CH2] / (255.5 x
[H]) ) x 100. In orthogonal manner, UV/VIS spectroscopy (λmax = 260 nm) was utilized
for determination of %DG.
1H
NMR (300 MHz, D2O): δ 2.0 (COCH3); δ 2.8 (N-(CH3)2); δ 3.3 (N+-(CH3)3); δ 3.4 (6O-
CH3); δ 3.5 (3O-CH3); δ ~3.6 (CH2-CH2-CO); δ 4.1 (CH2-CO); δ 4.8-6.0 (H-1, CH). FTIR:
697, 770, 842, 908, 952, 1057, 1236, 1359, 1456, 1641, 1706, 2881, 3283. UV (λmax, 6M
HCl): 259.9 nm.
114
2.5 Solubility measurements of chitosan polymers
The solubility of both chitosan derivatives (CM-TMC and CM-TMC-g-NH-PEG-8HA) was
determined in physiological phosphate buffered saline (PBS, pH 7.4) at a concentration
of 5.0 mg/mL. The transmittance (%T) of polymers was measured at λ = 600 nm by
using a Cintra 404 UV/VIS spectrometer (Switzerland). Polymers were considered
soluble for %T > 90% and very soluble for %T > 95 in comparison to the %T of PBS
alone (marked with + and ++, respectively).
2.6 Nanoparticle preparation
Nanoparticles were formed according to the procedure described by Bivas-Benita et al.
(2004) and Köping-Hoggård et al. (2003) with some modifications. Briefly, CM-TMC-gNH-PEG-8HA (at 2.7 mg/mL, average molecular weight per sugar unit of 254.2 Da, 3.6
μmol/mL -N+(CH3)3, N) or CM-TMC (at 2.2 mg/mL, average molecular weight per sugar
unit of 207.1 Da, 3.6 μmol/mL -N+(CH3)3, N) were dissolved in Miniversol water
(France). Low molecular weight chitosan (LMC, average molecular weight per sugar
185.3 Da, 12 μmol/mL -N+(CH3)3, N) was dissolved at 2.0 mg/mL in 25 mM sodium
acetate buffer (pH5.2). Separately, the plasmid pIRES-hrGFP II (abbreviated pGFP; 1 μg
of pGFP being equal to 3.1 nmol of phosphate groups, P) was dissolved in 5 mM aqueous
Na2SO4 at a concentration of 390 μg/mL in order to yield N/P ratios of 3:1 (chitosan
polymers) or 10:1 (LMC). Both solutions were heated for 5 minutes at 55°C. Next, the
polymer solution was slowly added (approximately 1 drop per second) to the pGFP
solution and subsequently mixed at low speed for 30 seconds. Attention was paid to
keep the final volume below or at 400 μL in order to obtain a narrow particle size
distribution. Particles were kept at room temperature for at least one hour prior to
further use.
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2.7 Nanoparticle characterization
2.7.1 Loading efficiency
400 μL of nanoparticle suspension was centrifuged at 16,000 x g for 30 min (Centrifuge
5417C/R, Eppendorf, Germany). Unloaded pGFP plasmid in the supernatant was
quantified by PicoGreen assay (Invitrogen, Switzerland) according to the manufacturer's
procedures. The fluorescence was measured with a FluoroMax spectrometer (Spex,
Switzerland) at excitation and emission wavelengths of 480 and 522 nm, respectively.
2.7.2 Transmission electron microscopy (TEM)
The morphology of CM-TMC and CM-TMC-g-NH-PEG-8HA pGFP NP was analyzed by TEM
using an uranyl acetate staining. Samples were visualized by a FEI Tecnai G2 Sphera
electron microscope (Switzerland) at 200 kV.
2.7.3 Electrophoretic mobility analysis
First, the ability of CM-TMC and CM-TMC-g-NH-PEG-8HA to bind and immobilize pGFP
was examined by agarose gel electrophoresis. Hereby, CM-TMC-g-NH-PEG-8HA pGFP
andCM-TMC pGFP NP (N/P ratios of 3:1) were mixed with bromophenol blue as loading
dye. 20 μL of each sample was applied in a 1.5 % agarose gel stained with 0.6 μg/mL
ethidium bromide. Electrophoresis was then performed at 100 V for 35 minutes using
0.5x TBE buffer. pGFP migration was detected by an UV transilluminator (Bio-Rad,
France).
Second, the capability of NP to protect incorporated pGFP from enzymatic degradation
via DNase I was assessed. pGFP in the absence of polymers, CM-TMC and CM-TMC-g-NHPEG-8HA pGFP NP (both 20 μL, equivalent to 3 μg pGFP, N/P ratio of 3:1) were
incubated with 4 μL of DNase I solution (1U/μL in DNase I buffer consisting of 200 mM
116
TRIS-HCl (pH 8.4), 20 mM MgCl2 and 500 mM KCl) for 15 minutes at room temperature.
The experiment was terminated by adding 4 μL of 25 mM EDTA solution to the reaction
mixture. The integrity of pGFP was then determined by the electrophoretic mobility
assay as described above.
Third, the stability of DNA formulations was assessed by incubating CM-TMC and CMTMC-g-NH-PEG-8HA pGFP NP (20 μL, equivalent to 3 μg pGFP) with 5 μL/mL of heparin
for two hours at room temperature (Köping-Höggård et al., 2004), followed by an
electrophoretic mobility assay as described above. Moreover, we studied the in vitro
release of pGFP from NP by incubation in phosphate buffered saline (PBS, pH 7.4) at
37°C (Csaba et al., 2009). After one day and one week, CM-TMC pGFP NP, CM-TMC-g-NHPEG-8HA pGFP and LMC pGFP NP in PBS were centrifuged at 16,000 x g for 30 minutes
(Centrifuge 5417C/R, Eppendorf, Germany) and 15 μL of supernatant was submitted to
gel electrophoresis. For all experiments, pGFP in the absence of polymers and a 1 kbp
pDNA ladder (BioLabs, England) served as controls.
2.7.4 Size and zeta potential of nanoparticles
Hydrodynamic diameters were measured by Photon Correlation Spectroscopy
(ZetaSizer 3000 HS, Malvern, Switzerland). For each measurement, 400 μL of NP
suspension were diluted in PBS (pH 7.4) to a total of 1.4 mL. Size distribution data were
obtained by the number-averaged value of three independent groups of ten
measurements. In addition, zeta potential was measured at least in triplicate via microelectrophoresis by using an aqueous dip cell (ZetaSizer 3000 HS, Malvern, Switzerland).
117
2.8 Cell culture
THP-1 cells, a human monocytic cell line, and A549 cells, a cell line derived from a
human lung carcinoma, were purchased from ATCC (American Type Culture Collection,
USA). Cell lines were grown in specific medium supplemented with 10% (v/v) fetal
bovine serum and penicillin (100 U/mL) and streptomycin (100 µg/ml). Cells were
cultured in an incubator at 37°C, 95% relative humidity, 5% CO2 and passaged at
approximately 80% confluency. All cell culture reagents were provided by Invitrogen
and Sigma-Aldrich (France). For differentiation of THP-1 cells, seeding was performed at
a density of 2 x 105 cells per well (96-well plate, unless stated otherwise) in the presence
of PMA (50 ng/mL) followed by an incubation for 48h. After differentiation to mTHP-1
cells and twice washing with PBS, cells were cultured for a recovery period of 3h in the
above mentioned medium and used for further experiments (Sadik et al., 2008).
2.9 GFP Transfection
A549and mTHP-1 cells were seeded at 1 x 105 cells per well (24-well plate) and cultured
for 48h. After mTHP-1 cells were handled as stated above, the following pGFP
formulations (2 µg/well) in the appropriate plain medium (for NP) or full medium (for
TransIT-LT1 formulation) were incubated with A549 and mTHP-1 cells: 1) CM-TMC
pGFP NP, 2) CM-TMC-g-NH-PEG-8HA pGFP NP, 3) LMC pGFP NP and 4) TransIT-LT1
pGFP preparation (used pursuant to the manufacturer’s recommendations). After five
hours of exposure, formulations were removed (with the exception of TransIT-LT1 pGFP
preparation) and full medium was added. Medium was then changed every second day.
Starting with day two until day six, cells were analyzed by fluorescence microscopy
(Zeiss Axiovert 40 CFL, Germany) and pictures (Canon Powershot A640, France) were
taken at 5x magnification.
118
The percentage of GFP-positive cells (transfection efficiency) was assessed by using
ImageJ 1.43 software similar to Csaba et al. (2009).
2.10 hIL-12p40 and hIL-8 release by mTHP-1 cells
mTHP-1 cells were cultured in a 96-well plate as stated above. The following samples (in
duplicates, 250 µL per well) were studied after 1:15 dilution with plain RPMI 1640
medium: imiquimod (positive control) at 5 µg/mL (in 0.1% DMSO), 8HA at 5 µg/mL (in
0.1% DMSO), NH2-PEG-8HA at 55.5 µg/mL (equimolar amount of 8HA coupled to 5
µg/mL of 8HA), CM-TMC-g-NH-PEG-8HA at 0.21 mg/mL (the amount of NH2-PEG-8HA
grafted is equimolar to 5 µg/mL of 8HA), CM-TMC at 0.15 mg/mL (equimolar to CM-TMCg-NH-PEG-8HA). In addition, both chitosan derivatives were used for the formation of
DNA NP, either with CM-TMC at 0.15 mg/mL or CM-TMC-g-NH-PEG-8HA at 0.21 mg/mL
(both at N/P 3:1). 0.1% DMSO solution, pGFP alone (same amount as encapsulated in
NP) and 0.02% SDS (toxicity control) were used as further controls. After incubation
during 24h at 37°C, microscopic pictures were taken at 5x magnification (Zeiss Axiovert
40 CFL, Germany; Canon Powershot A640, France) in order to monitor toxicity.
Subsequently, cell supernatants of mTHP-1 cells were removed and stored at -80°C until
further analysis. For quantification of human IL-12p40 and IL-8, sandwich ELISA was
performed in triplicates (IL-12p40) or duplicates (IL-8) according to the manufacturer´s
instructions. Standard curve was calculated by a log log regression (R>0.95) and sample
concentrations were determined accordingly. Detection limits were 15.6 pg/mL for IL12p40 ELISA and 7.8 pg/mL for IL-8 ELISA.
119
2.12 Data analysis and statistics
Obtained data were expressed as the mean ± standard error of the mean and compared
by an one-way ANOVA using Origin 7.01 software. Differences were considered
significant at p<0.05.
3. Results and discussion
The aim of this study was to provide a water-soluble chitosan derivative being
functionalized with an immune stimulatory and TLR-7 agonistic (9-benzyl-8hydroxyadenine) moiety as potential adjuvant. Moreover, we applied this novel polymer
for the formation of NP delivering a model vaccine, in our case a plasmid DNA. The basis
of this concept is the general observation that according to O’Hagan et al. (2006) and
Schlosser et al. (2008), for an effective vaccination, the adjuvant (here: 9-benzyl-8hydroxyadenine moiety) and antigen (here: model plasmid DNA expressing GFP) needs
to be co-located within the same particulate system (here: chitosan-based
nanoparticles).
3.1 Synthesis of CM-TMC-g-PEG-8HA
Hirota et al. (2002) synthesized a series of 2-substituted 9-benzyl-8-hydroxyadenine
derivatives and analysed their capacity of inducing IFN-α in vitro and in vivo. Regarding
Structure-Activity-Relationships (SAR) of the compounds synthesized, they concluded
that: i) the 9-benzyl-position is essential for activity; ii) 9-benzyl-8-hydroxyadenine is the
simplest structure with IFN-inducing activity; iii) the introduction of alkyl-chain
substituents at the C-2 position strongly enhances the activity. In a follow-up study
(Isobe et al., 2006), the same group suggested that such 9-benzyl-8-hydroxyadenine
derivatives act through the TLR-7 signalling cascade, without presenting detailed
120
information in order to confirm this hypothesis. However, Lee et al. (2006) were able to
correlate a TLR-7 activating mechanism to a 9-benzyl-8-hydroxyadenine derivative,
namely 9-benzyl-8-hydroxy-2-(2-methoxy ethoxy)adenine (SM360320). In addition, a
further 9-benzyl-8-hydroxyadenine derivative and TLR-7 agonist, 2-(4-((6-amino-2(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)benzamido) acetic acid (CL264), was
identified. Considering above mentioned information, we selected the simplest and
potent TLR-7 agonistic moiety, 9-benzyl-8-hydroxyadenine, and coupled it through the
use of a PEG spacer to a NP forming vaccine carrier (polymeric chitosan). The PEG
spacer was thought to provide the TLR-7 agonist accessibility to its endosomal receptor
and maintain its agonistic properties. In more detail, we synthesized a 9-benzyl-8hydroxyadenine derivative PEGylated at its C-2 position, compound 3 (abbreviated NH2PEG-8HA), according to the scheme in figure 1.
Completeness of reaction was evidenced by 1H NMR (fig. 1) and in an orthogonal
manner by UV/VIS spectroscopy at λmax = 260 nm (data not presented). Both methods
indicated that complete amidation at the C-2 positon took place.
121
O
NH2
H3C
CH3
HO
O
H2N
m
NH
n
H3C
O
OCH3
O
CH3
Chitosan
OH
Boc-NH-PEG-NH2
NMP, NaI, NaOHaq,CH3I
1. 8HA, n-butanol
(60°C, 1h)
(120°C, 16h)
2. TFA / DCM 1:1
(RT, 2h)
+
N
NH2
HO
N
n
H 3C
N
N
O
N
NH
OCH3
O
HO
TMC
OH
NH
m 2
NMP, NaOHaq, ClCH2COOH
NH2-PEG-8HA
(RT, 3h)
+
N
CM-TMC, EDC, NHS
HO
n
H3 C
(RT, 120h)
O
R1
R3
O
n
H3 C
OCH3
O
CM-TMC
OH
OCH 3
O
R2
CM-TMC-g-NH-PEG-8HA
R1= -OH, -OCH3, -OCH2COOH, -OCH2CO-NH
NH
O
+
N
N
n
R2= -NHCOCH3, -NH(CH3)2, -N (CH3)3
OH
N
N
R3= -OH, -CH3
NH2
Figure 1: Reaction scheme for the synthesis of CM25-TMC35-g-NH-PEG-8HA.
In addition, NH2-PEG-8HA was obtained at high purity, as shown by
1H
NMR
spectroscopy (fig. 2C), TLC analysis and MALDI-TOF spectroscopy (data not shown).
NH2-PEG-8HA was water-soluble at concentrations even above 10 mg/mL, in contrast to
the unmodified 8HA.
In a next step, we synthesized a CM-TMC polymer with a degree of trimethylation
(%DTM) of 33.3% and carboxymethylation (%DCM) of 23.2%, as reported previously
(abbreviated CM25-TMC35; see figure 2C; Heuking et al., 2009b).
122
A
B
C
D
Figure 2: 1H NMR spectra of A) 9-benzyl-2-chloro-8-hydroxyadenine (8HA, DMSO-d6), B)
Boc-PEG-NH-8HA (CD3CN-d3) C) CM25-TMC35 and D) CM25-TMC35-g-NH-PEG-8HA
(both in D2O-d2 at 80 °C).
Finally, NH2-PEG-8HA was grafted to CM25-TMC35 by using EDC/NHS condensing agents
as mentioned above (Heuking et al., 2009b). For the quantification of the degree of
grafting (%DG), we firstly analysed using 1H NMR spectroscopy (Fig. 2D), but were only
able to observe the PEG backbone of the finalized product at around 3.6 ppm. This
phenomenon can be explained by the very low grafting ratio applied (< 3%), which
makes it difficult to detect any aromatic peaks between 7 and 8 ppm. As alternative
(orthogonal) method, we performed UV/VIS spectroscopy analysis (at λmax = 260 nm) of
CM25-TMC35-g-NH-PEG-8HA (data not shown). Chan et al. (2007) as well as Mansuri et
123
al. (2006) similarly analysed their chitosan-folate conjugates. Following this method, we
quantified a %DG of about 2.6%. Interestingly, after grafting the physical appearance
changed to a yellowish color, probably due to the presence of the π-electrons of the
aromatic ring system of 8HA in CM25-TMC35-g-NH-PEG-8HA (fig 3). In addition, SECMALLS measurements showed a unimodal distribution of molecular weight (MW) in the
eluogram (data not presented), together with an increase in molecular weight (MW;
table 1). Similar MW shifts were observed when PEG was grafted onto TMC polymers
(Mao et al., 2005).
Table 1: Aqueous solubility (SOL.), appearance of solution (AOS), molecular weight
(MW), particle size, ζ potential (ZP), polydispersity index (PDI) and loading efficiency
(LE) of chitosan-based DNA preparations used in this study.
Chitosan
SOLa
AOSb
derivative
CM25-TMC35
++
Trans-
MW
Size
ZP
(g/mol)
(nm)a
(mV)a
98,330
396.3 ±
17.2 ±
55.8
3.3
410.2 ±
19.5 ±
97.9
1.6
287.7 ±
16.5 ±
20.6
4.2
lucent
CM25-TMC35-g-
++
Yellowish
273,800
NH-PEG-8HA
LMC
++
Translucent
aanalyses
<10,000c
PDIa
LE
(%)a
0.451 ± 92.2 ±
0.055
1.6
0.576 ± 89.4 ±
0.039
3.8
0.266 ± 99.3 ±
0.040
1.7
were performed in triplicates; bsee figure 3; cnot analysed by SEC-MALLS.
Furthermore, we analyzed the co-polymer via FTIR spectroscopy (fig. 4D). Hereby, the
PEG-grafting was ascertained considering newly introduced bands at 842 cm-1, 952 cm-1
and 2881 cm-1 (Jeong et al., 2008). The absorption band at 1606 cm-1 (C=O asymmetric
124
stretch of COO- anion) was altered to 1641 cm-1 (C=O stretch of amide), which indicates
the formation of an amide bond (Prabaharan et al., 2007; Prabaharan and Gong, 2008).
Further subtle differences can be noted in the fingerprint area at 697 cm-1, 770 cm-1 and
908 cm-1, which are assigned to the 8HA moiety (fig. 4C).
Figure 3: Appearance of CM25-TMC35 (19.9 mg/mL, left) and CM25-TMC35-g-NH-PEG8HA (24.4 mg/mL, right) at equimolar concentrations.
A
B
C
D
E
Figure 4: FTIR spectra of A) NH2-PEG-8HA, B) Boc-NH-PEG-NH2, C) 8HA, D) CM25-TMC35g-NH-PEG-8HA and E) CM25-TMC35.
125
Taking all results together, the TLR-7 agonistic moiety 9-benzyl-8-hydroxyadenine was
successfully grafted through a PEG spacer to the CM25-TMC35 chitosan derivative,
yielding the water-soluble CM25-TMC35-g-NH-PEG-8HA co-polymer.
3.2 Characterization of CM25-TMC35-g-NH-PEG-8HA pGFP NP
The novel co-polymer was used to formulate NP by complex coacervation with a plasmid
DNA encoding for GFP (termed pGFP), employed as a model plasmid DNA (pDNA, 5,500
kbp) of medium size. It can be assumed that the application of prevalent, similar-sized
DNA vaccines does not yield NP of too dissimilar physico-chemical properties. In our
hands, pGFP NP formulations, either with CM25-TMC35 or CM25-TMC35-g-NH-PEG-8HA,
resulted in particles of a size of about 400 nm and positive surface charge of 15-20 mV
(table 1). The morphology of CM25-TMC35-g-NH-PEG-8HA pGFP NPs consisted mainly
of heterogenous rod-like structures (figure 5A) and was not different from CM25-TMC35
pGFP NP (figure 5B) in terms of size and shape.
1000 nm
A
1000 nm
B
Figure 5: TEM pictures of CM25-TMC35-g-NH-PEG-8HA pGFP NP (A) and CM25-TMC35
pGFP NP (B; both with a N/P ratio of 3:1). Horizontal bar indicates 1000 nm.
126
Rather polydisperse size distributions of polymeric chitosan pDNA NP were already
noticed by others (Lee et al., 2008; Erbacher et al., 1998), and was in line with
polydispersity indices (PDI) determined in our study (table 1). It was noticeable that
diameters of NP studied by TEM were smaller than those found by Photon Correlation
Spectroscopy (PCS), which might be explained by the fact that PCS is measuring the NP
size in the hydrated state in contrast to the dried state during TEM analysis (Lee et al.,
2008).
Furthermore, we studied the capacity of CM25-TMC35-g-NH-PEG-8HA NP to release
pDNA and applied a different pDNA NP system based on low molecular weight chitosan
(LMC; see properties in table 1) for comparison. LMC pDNA NP, as well as LMC pDNA
polyplexes were shown to release DNA easier and to transfect cells more efficiently,
which was probably due to less pronounced electrostatic interactions between
negatively charged pDNA and positively charged chitosan oligomer at acidic pH (KöpingHoggård et al., 2004; Strand et al., 2010).
Ladder
pGFP
CO NP
LMC NP
1 day
Ladder pGFP
CO NP LMC NP
1 week
CO NP LMC NP
*
Figure 6: pGFP release study of CM25-TMC35-g-NH-PEG-8HA (CO) as well as LMC pGFP
NP after one day and one week via electrophoretic mobility analysis. *Centrifuged NP of
release samples were re-suspended by gently vortexing and used as controls.
127
As shown in figure 6, both LMC- and CM25-TMC35-g-NH-PEG-8HA-based carrier systems
did not release significant amounts of pGFP in PBS medium at 37°C after one day and
one week.
However, when NP were challenged with anionic heparin (10-fold excess relative to
positive charges of amines), only LMC pGFP NP were able to partially release plasmid
DNA in supercoiled form, which indicates that the physical integrity of pGFP was not
altered during NP preparation. Similar observations were reported by Köping-Hoggård
et al. (2004) for LMC pDNA polyplexes. We can therefore assume that relatively stable
CM25-TMC35-g-NH-PEG-8HA pGFP NP were formed owing to permanent positive
trimethylamine charges in the co-polymer backbone. In addition, experiments with
agarose gel electrophoresis demonstrated that LMC and CM25-TMC35-g-NH-PEG-8HA
pGFP NP were able to protect pGFP against enzymatic degradation by DNase I (data not
shown).
Ladder
pGFP
pGFP
CO NP
CO NP
+
-
+
LMC NP LMC NP
OC
SC
Heparin
-
-
-
+
Figure 7: Anionic challenge of pGFP incorporating CO NP (CO, CM25-TMC35-g-NH-PEG8HA; N/P of 3:1) as well as LMC pGFP NP (N/P of 10:1) were exposed to heparin for 2h
at room temperature followed by electrophoretic mobility analysis.
128
Besides, pGFP NP of the non-functionalized polymer, CM25-TMC35, demonstrated
similar carrier properties in gel experiments as CM25-TMC35-g-NH-PEG-8HA (data not
presented).
3.3 pGFP transfection
After physico-chemical characterization of CM25-TMC35-g-NH-PEG-8HA pGFP NP, we
evaluated their transfection efficiency in A549 cells and differentiated THP-1
macrophage-like (mTHP-1) cells. For comparison purposes, we included also the study
of LMC- and TransIT-LT1 (a commercially available transfection reagent)-mediated
pGFP transfection. All three carrier systems had the ability to transfect A549 cells with
the ranking of TransIT-LT1 > LMC > CM25-TMC35-g-NH-PEG-8HA (figure 8 and figure 9).
In more detail, application of TransIT-LT1 resulted at all time points (from day two until
day six) in a significantly higher protein expression than LMC and CM25-TMC35-g-NHPEG-8HA (p<0.05). LMC was more efficient than CM25-TMC35-g-NH-PEG-8HA in
transfecting A549 cells, which might be explained by its easier dissociation in a strong
ionic environment (figure 7), as shown by the challenge incubation with heparin and
recent reports (Köping-Hoggård et al. 2003; Strand et al., 2010). However, the difference
between these pGFP delivery systems was not significant. CM25-TMC35 pGFP
transfection was not different in extent from CM25-TMC35-g-NH-PEG-8HA mediated
transfection (data not shown). It is worth mentioning that all chitosan-based delivery
systems initiated a later onset of GFP expression when compared to TransIT-LT1
transfection. A similar tendency was already noted by Csaba et al. (2009).
129
25,0
*
*
20,0
*
GFP+ cells [%]
*
*
15,0
10,0
5,0
0,0
d2
d3
d4
d5
d6
Figure 8: In vitro GFP transfection efficiency in A549 cells of LMC (red), TransIT-LT1
transfection reagent (blue) and CM25-TMC35-g-NH-PEG-8HA (green) pGFP carrier
systems. Statistical differences are denoted with * (p<0.05) for TransIT-LT1 compared
to LMC and CM25-TMC35-g-NH-PEG-8HA pGFP transfection, respectively.
Regarding the GFP transfection of mTHP-1 cells, the amount of GFP-positive cells was
(from day two until day six) very low (below 2%, data not presented) for all applied
formulations. Similar findings were already published by Schnoor et al. (2009),
concluding that mTHP-1 cells are difficult to transfect by common non-viral gene
delivery systems.
In addition, light microscopy pictures of A549 cells and mTHP-1 cells after incubation
were taken for all formulation groups and no toxic effects were observed in comparison
to 0.02% sodium lauryl sulfate treatment as control (pictures not shown). Thus, we can
assume that cytotoxicity of the CM25-TMC35-g-NH-PEG-8HA co-polymer does not play a
major role.
130
CM25-TMC35-g-NH-PEG-8HA
TransIT-LT1
LMC
pGFP alone
Figure 9: Fluorescence microscopy pictures (5x magnification) after six days of
transfection with CM25-TMC35-g-NH-PEG-8HA, LMC, TransIT-LT1 pGFP carrier systems
and pGFP alone as control.
3.4 IL-12p40 and IL-8 release from human THP-1 macrophages
In order to analyse the immunogenicity of the novel pDNA carrier system functionalized
with the 8HA moiety targeting TLR-7, we analysed at first the in vitro secretion of the T
helper cells type 1 (Th-1) cytokine IL-12p40, which is strongly related to TLR-7
agonistic activity (Hemmi et al., 2002).
In more detail, we selected the human macrophage-like cell line (mTHP-1) as cell culture
model, because we (Adam-Malpel et al., unpublished observation) and others (Gantier et
al., 2008; Yi et al., 2009) reported that THP-1 cells express the TLR-7.
131
In addition, mTHP-1 cells were shown to produce high amounts of the IL-12p40
cytokine (Feng et al., 2004). Furthermore, Salio et al. (2007) demonstrated that IFN-γ, a
further Th-1 associated cytokine, is secreted from THP-1 cells after exposure to TLR-7
agonists (R-837 and R-848). By application of this in vitro model, we found a relatively
weak (range of pg/mL concentrations), but still detectable immune response to our (co)polymers and pGFP containing NP (figure 10 and figure 11).
At first, we observed that PEG coupling to the C-2 position of 8HA significantly enhanced
the IL-12p40 stimulatory capacity (NH2-PEG-8HA: 37.7 ± 5.3 pg/mL; p<0.05). Successive
grafting
of
NH2-PEG-8HA
to
CM25-TMC35
increased
the
IL-12p40
related
immunogenicity (p<0.05) of the chitosan derivative to 17.7 ± 5.4 pg/mL (CM25-TMC35g-NH-PEG-8HA), although at this level no significance was found. Interestingly, the
positive control and well-known TLR-7 agonist imiquimod induced the highest IL-12p40
secretion (147.8 ± 3.5 pg/mL), which was around four-fold higher than those triggered
by NH2-PEG-8HA.
In line with our further study, functionalization of pGFP NP with the TLR-7 agonistic
moiety (9-benzyl-8-hydroxyadenine) caused a significant (p<0.05) increase in IL-12p40
secretion (CM25-TMC35 pGFP NP: 15.8 ± 1.0 pg/mL; CM25-TMC35-g-PEG-8HA: 34.0 ± 2.2
pg/mL). In addition, the plasmid pGFP alone caused also a relatively high IL-12p40
production (26.7 ± 0.3 pg/mL), which can be explained by its CpG motifs acting as TLR-9
agonist.
132
180,0
160,0
*
IL-12p40 [pg/ml]
140,0
120,0
100,0
*
80,0
*
60,0
*
40,0
NS
ND
20,0
0,0
Medium
control
8HA
NH2-PEG8HA
CM25TMC35
Conjugate
Imiquimod
Figure 10: IL-12p40 release from mTHP-1 cells owing to polymer exposure (8HA, 2chloro-9-benzyl-8-hydroxyadenine; conjugate, CM25-TMC35-g-NH-PEG-8HA). Detection
limit was 15,6 pg/mL and is marked with the horizontal bar (ND, not detectable).
Significance was controlled by one-way ANOVA by comparison to values obtained from
0.1% DMSO cell culture medium (unless indicated otherwise). Differences were
considered significant for * (p<0.05); NS, not significant.
Once pGFP was encapsulated in CM25-TMC35 NP, IL-12p40 levels dropped below the
limit of detection, but could be regained byNH2-PEG-8HA functionalization (figure 11).
Due to the fact that we observed only minor levels of IL-12p40, we extended our
immunogenicity study and analysed for IL-8 secretions. Primarily, the chemokine IL-8 is
involved in the attraction of leukocytes and their successive guidance to the mucosal site
of infection (chemotaxis).
133
50,0
NS
IL-12p40 [pg/ml]
40,0
30,0
*
*
*
NS
20,0
10,0
ND
0,0
Medium control
pGFP
CM25-TMC35
pGFP NP
Conjugate pGFP
NP
Figure 11: IL-12p40 release from mTHP-1 cells as a result of NP (N/P ratio 3:1)
exposure (conjugate, CM25-TMC35-g-NH-PEG-8HA). Detection limit was 15.6 pg/mL,
indicated by the horizontal bar (ND, not detectable). Significance was checked by oneway ANOVA by comparison to values obtained from 0.1% DMSO cell culture medium
(unless indicated otherwise). Differences were considered significant for * (p<0.05); ns:
not significant.
In terms of vaccine adjuvanticity, Sin et al. (2000) investigated the role of IL-8 in vivo by
co-injection of an IL-8 encoding pDNA with a pDNA vaccine encoding for Herpes simplex
virus type 2 (HSV-2). After lethal HSV-2 challenge, they observed an enhanced antigenspecific Th1 CD4+ T-cell response, which was correlated to a complete survival of mice
found up to 30 days post infection. In contrast, mice vaccinated with HSV-2 pDNA
vaccine alone died within ten days. In our laboratory, different epithelial cells lines
(A549, Caco-2 and 16HBE14o-) were found to secret IL-8 upon stimulation with the
TLR-7 agonist imiquimod (Adam-Malpel et al., unpublished observation).
134
In analogy, we presumed that also IL-8 secretions can be elicited from mTHP-1
macrophages owing to TLR-7 activation by 8HA derivatives.
Consequently, we analysed the supernatant of mTHP-1 cells after (co)-polymer and
pGFP NP exposure for IL-8 secretion, which was found to be more pronounced than the
concentrations found for IL-12p40 (range of ng/mL concentrations, figure 12 and 13).
25,0
*
IL-8 [ng/ml]
20,0
*
15,0
*
NS
10,0
*
*
*
5,0
*
0,0
Medium
8HA
NH2-PEG8HA
CM25TMC35
Conjugate
Imiquimod
Figure 12: IL-8 release from mTHP-1 cells owing to polymer exposure (8HA, 2-chloro-9benzyl-8-hydroxyadenine; conjugate, CM25-TMC35-g-NH-PEG-8HA). Detection limit was
7.8 pg/mL. Significance was checked by one-way ANOVA by comparison to values
obtained from 0.1% DMSO cell culture medium (unless indicated otherwise). Differences
were considered significant for * (p<0.05); ns: not significant.
In contrast to IL-12p40 results, PEG chain attachment at the C-2 position of 8HA did not
enhance the IL-8 stimulatory capacity of NH2-PEG-8HA, which was slightly weaker than
the one of 8HA (not significant).
135
Related to that, Weterings et al. (2009) noticed that modifications of the 2-chloro-9benzyl-8-hydroxyadenine molecule at its C-2 position with different 2-azidoalkoxy chains
entail a decrease of IL-12p40 secretions from murine dendritic cellsin dependence of
chain length. On the other hand, Hirota et al. (2002) reported for a similar molecule, 2hydro-9-benzyl-8-hydroxyadenine, relative low levels of IFN-α secretion in vitro and in
vivo, when compared to molecules with a C2-modifications (2-alkyloxy and 2alkylamino). Therefore, it may be concluded that modification at the C-2 position of 2chloro-9-benzyl-8-hydroxyadenine is important for immunological activity, but has to be
carefully performed in view of its effects on structure/activity relationship (SAR).
30,0
IL-8 [ng/ml]
25,0
*
*
20,0
15,0
10,0
*
*
pGFP
CM25TMC35
pGFP NP
5,0
0,0
Medium
Conjugate
pGFP NP
Figure 13: IL-8 release from mTHP-1 cells owing to NP (N/P ratio 3:1) exposure
(conjugate, CM25-TMC35-g-NH-PEG-8HA). Detection limit was 7.8 pg/mL. Significance
was checked by one-way ANOVA by comparison to values obtained from 0.1% DMSO
cell culture medium (unless indicated otherwise). Differences were considered
significant for * (p<0.05); ns: not significant.
136
Functionalization of CM25-TMC35with NH2-PEG-8HA significantly (p<0.05) enhanced IL8 secretions from mTHP-1 macrophages (CM25-TMC35: 2.86 ± 0.11 ng/mL versus CM25TMC35-g-NH-PEG-8HA: 10.06 ± 0.93 ng/mL). Imiquimod as a positive control induced
highest IL-8 secretions (16.4 ± 1.2 ng/mL), which were around three times higher than
those triggered by NH2-PEG-8HA.
Considering the IL-8 related immunogenicity of pGFP NP formulations, a comparable
tendancy to IL-12p40 secretions was noticed. Plasmid pGFP alone enabled a relatively
high IL-8 production (8.81 ± 0.95 pg/mL), but once the pGFP was encapsulated into
CM25-TMC35 pGFP NP a slight decrease was remarked (CM25-TMC35 pGFP NP: 6.77 ±
0.99 ng/mL, not significant). A similar trend regarding pGFP encapsulation and
reduction of IL-8 secretion was already noted previously (Heuking et al., 2009b).
Interestingly, functionalization of pGFP NP with the TLR-7 agonistic moiety (9-benzyl-8hydroxyadenine) gave rise to a significant (p<0.05) increase in IL-8 secretions (CM25TMC35-g-PEG-8HA: 17.96 ± 2.37 ng/mL), which can be related to a higher
immunogenicty.
In conclusion, results presented in this study clearly indicate the possibility of grafting a
TLR-7 agonistic moiety through a PEG spacer to a water-soluble chitosan derivative.
Subsequent aggregation with the plasmid pGFP resulted in nano-sized and positively
charged NP, which were able to transfect alveolar A549 cells. Moreover, TLR-7 agonist
functionalization was shown to increase significantly the IL-8 and IL-12 specific
immunogenicity in mTHP-1 macrophages, when compared to unfunctionalized NP.
Consequently, the novel CM25-TMC35-g-PEG-8HA carrier system merits further
investigation towards its application for mucosal DNA vaccination.
137
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Part III
In vitro and in vivo evaluation
of TLR-agonist functionalized
nanoparticles
for pulmonary DNA vaccination
143
Chapter 6
Fate of TLR-2 agonist functionalized
pDNA nanocarriers upon deposition
at the bronchial epithelium in vitro
S. Heuking1,2,3, B. Rothen-Rutishauser3, P. Gehr3 and G. Borchard1,2
To be submitted
1School
of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
3Institute
of Anatomy, University of Bern, Switzerland
144
Abstract
A TLR-2 agonist functionalized pDNA nanoparticle (NP) system for potential pulmonary
DNA vaccination was studied by using a three-dimensional (3D) cell model of the human
lung barrier. pDNA loaded NP were administered via microsprayer onto the 3D cell
culture model and uptake of NP by epithelial and immune cells (blood monocyte-derived
dendritic cells (MDDC) and macrophages (MDM)) was visualized by confocal laser
scanning microscopy (CLSM).
We were able to detect pDNA NP transfer to MDDC located at the basolateral side of the
epithelium. Although no significant difference in uptake pattern was observed for TLR-2
agonist modified and unmodified NP systems studied, ELISA of IL-8 and TNF-α
demonstrated clearly that TLR-2 agonist functionalization induces a higher immune
response with the ranking of CM25-TMC35-g-PEG-Pam3Cys pGFP NP > CM25-TMC35
pGFP NP > unloaded CM25-TMC35 NP. Consequently, the novel TLR-2 agonist
functionalized DNA carrier merits further investigation as novel material for DNA
vaccination.
1. Introduction
In general, plasmid DNA (pDNA) vaccines consist of a bacterial plasmid vector, which
contains the genetic information encoding for one or more antigenic protein(s). Plasmid
DNA vaccines are usually produced in bacteria (e.g., Escherichia coli), purified and
injected into the host (Huygen, 2005). When compared to gene therapy, vaccination
using pDNA is thought to be effective already at relatively low levels of gene expression
achieved. Today, the present strategy for the formulation of pDNA vaccines is to include
highly purified synthetic adjuvants, which are able to activate distinct parts of the
immune system, augmenting the vaccine´s immunogenicity. In terms of effective
145
vaccination, the adjuvant should be encapsulated together with the vaccine within the
same delivery vector (O´Hagan et al., 2006).
In order to address this necessity, we recently synthesized a novel copolymer, CM-TMCg-PEG-Pam3Cys, based on a chitosan polymer (Heuking et al., 2009a). To a new chitosan
derivative,
6-0-carboxymethyl-N,N,N-trimethylchitosan
(CM-TMC),
the
Toll-like
receptor-2 (TLR-2) agonist, Pam3Cys, an adjuvant activating the innate immune system,
was grafted through a polyethylene glycol (PEG) spacer. In a second step, Pam3Cys
functionalized nanoparticles (NP) were prepared by aggregation of CM-TMC-g-PEGPam3Cys with pDNA expressing the green fluorescence protein (GFP) (Heuking et al.,
2009b). Regarding the immunogenicity of the new carrier system, we observed that
TLR-2 agonist functionalized pDNA nanocarriers induced IL-8 secretion from
differentiated THP-1 human macrophages, which were increased by 10-fold compared
to non-functionalized carriers (Heuking et al., 2009b).
Aerolization deposition of vaccines is regarded as a promising route of immunization
(Bivas-Benita et al., 2005) owing to several clinical trials with measles vaccines (Dilraj et
al., 2000; Bennett et al., 2002), one of which in clinical phase II/III (Simon et al., 2010).
It is generally assumed that mimicking the natural way of infection by applying vaccines
to the respiratory tract represents an auspicious strategy for the prevention of lung
infections (e.g., influenza, measles and tuberculosis). Further advantages are:
i)
delivery of vaccines into the respiratory tract elicits the secretion of local
antibodies (IgA), which in turn are capable of crossing epithelia and
preventing further entrance of pathogens (Brandtzaeg, 2007);
ii)
the particular non-invasive nature of pulmonary antigen delivery circumvents
the common use of needles, which are the major cause for unsafe injections
especially in developing countries (Miller et al., 1998);
146
iii)
use of pulmonary dry powder vaccines may circumvent the common
imperative of an intact cold chain for vaccine storage;
iv)
no specially trained medical personnel is required for the administration of
vaccines by inhalers.
Next to these benefits, pulmonary delivery of pDNA vaccines in nanoparticulate form
gave rise to immune responses in vivo superior to intramuscular injection (Bivas-Benita
et al., 2004; Bivas-Benita et al., 2009).
Considering the above, we intended to evaluate our TLR-2 agonist functionalized carrier
system further for pulmonary pDNA vaccination. Hereby, next to animal and ex vivo
models, a three-dimensional (3D) cell model of the human lung barrier tract was shown
to be a valuable tool for the study of the human airway system (Rothen-Rutishauser et
al., 2005; Rothen-Rutishauser et al., 2008). This cell culture model allows in particular
the study of particle uptake by immune cells, i.e. human blood monocyte-derived
dendritic cells (MDDC) and macrophages. We administered pDNA loaded nanoparticles
via microsprayer onto this 3D cell culture model and examined the uptake of NP by
epithelial and immune cells. In addition, we monitored changes in immune responses
after NP exposure by measuring secretion of IL-8 and TNF-α.
2. Materials and methods
2.1 Triple cell culture
The triple cell culture system was set up as previously published (Rothen-Rutishauser et
al., 2005; Blank et al., 2007). Briefly, bronchial 16HBe14o- (HBE) cells (passage numbers
2.45–2.80) were maintained in MEM 1x, with Earle's Salts, 25 mM HEPES, without LGlutamine (Gibco BRL Life Technologies Invitrogen AG, Basel, Switzerland)
supplemented with 1% L-Glutamine (LabForce AG, Nunningen, Switzerland), 1%
147
penicillin/streptomycin (Gibco BRL, Switzerland), and 10% fetal calf serum (PAA
Laboratories, Lucerna-Chem AG, Lucerne, Switzerland) on transparent BD Falcon cell
culture inserts (pores with 3.0 µm diameter, PET membranes for 6-well plates; BD
Biosciences) treated with fibronectin coating solution containing bovine serum albumin,
0.1 mg/ml (Sigma, Fluka Chemie GmbH, Buchs, Switzerland) with 1% bovine collagen,
Type I (BD Biosciences, Basel, Switzerland) and 1% human fibronectin (BD Biosciences)
in LHC Basal Medium (Lucerna Chemie AG, Switzerland).
MDM and MDDC were derived from human blood monocytes as already described
(Rothen-Rutishauser et al., 2005). Briefly, peripheral blood monocytes were isolated
from buffy coats (Blood Donation Service, Bern, Switzerland) and cultured in the same
medium as used for the epithelial cells except for the supplementation of 5% human
serum (Blood Donation Service) instead of 10% fetal calf serum. Regarding the
generation of MDDC, monocytes were cultured for seven days in medium supplemented
with 34 ng/ml of IL-4 (Sigma, Fluka Chemie GmbH, Switzerland) and with 50 ng/ml of
GM-CSF (R&D Systems, Oxon, UK), whereas MDM were obtained without any additional
supplements. Epithelial cells were then cultured for seven days before MDM were added
at the apical and MDDC at the basolateral side of the epithelial monolayer.
Next, the triple cell co-cultures were kept overnight in medium supplemented with 1%
L-Glutamine, 1% penicillin/streptomycin, and 5% heat-inactivated (pooled) human
serum (= full medium) at 37°C in a 5% CO2 humidified atmosphere. The following day
the medium was removed completely from the apical chamber while 1.2 ml of medium
were kept in the basolateral well to feed the cultures from the basal side of the insert.
The cells were exposed to air for 24 h at 37°C in a 5% CO2 humidified atmosphere.
148
2.2 NP preparation and characterization
Synthesis
of
6-0-carboxymethyl-N,N,N-trimethylchitosan
with
a
degree
of
carboxymethylation of 23.2% and degree of trimethylation of 33% (abbreviated CM25TMC35) and subsequent synthesis of CM25-TMC35-g-PEG-Pam3Cys was performed as
described earlier (Heuking et al., 2009b). In addition to our previous publication, we
analyzed the molecular weight of the chitosan derivative by SEC-MALLS. Hereby,
measurements were performed using a TOSOH TSK Gel G3000PWXL-CP size exclusion
column (TOSOH Bioscience, Germany) with 0.2 M sodium acetate/0.3 M acetic acid (pH
4.4) as eluent (0.3 mL/min). A Waters Alliance HPLC system coupled to a differential
refractive index (RI) detector (Schambeck, Germany) and a light scattering detector
(MiniDawn, Wyatt, USA) was used for sample handling. Pullan standards ranging from
47,000 g/mol to 710,000 g/mol (PSS, Germany) were used for calibration. Next, in order
to fluorescently label NP, we selected an Alexa Fluor 488 sulfodichlorophenol dye
(A30052, Invitrogen, France). The Alexa Fluor 488 dye was coupled to free amine
moieties of CM25-TMC35 and CM25-TMC35-g-PEG-Pam3Cys (at 1% molar ratio of sugar
units), respectively, according to manufacturer´s recommendation. Emission spectra
(excitation at λ = 495 nm) of labeled chitosan derivative were taken using a FluoroMax
spectrometer (Spex, Switzerland) and no quenching was seen. Successively,
nanoparticles were formed according to our published method (Heuking et al., 2009b).
Briefly, CM25-TMC35-g-PEG-Pam3Cys (at 3.10 mg/mL, average molecular weight per
sugar unit of 290.8 Da, 3.6 μmol/mL -N+(CH3)3, N) or CM25-TMC35 (at 2.21 mg/mL,
average molecular weight per sugar unit of 207.1 Da, 3.6 μmol/mL -N+(CH3)3, N) were
dissolved in Miniversol water. Separately, the plasmid pIRES-hrGFP II (abbreviated
pGFP; 1 μg of pGFP being equal to 3.1 nmol of phosphate groups, P) was dissolved in 5
mM aqueous sodium sulfate solution at a concentration of 390 μg/mL in order to yield
149
N/P ratios of 3:1. Both solutions were heated for 5 minutes at 55°C. In the following, the
polymer containing solution was slowly added (approximately 1 drop per second) to the
pGFP solution and vortexed at low speed for 30 seconds. Attention was paid to keep the
final volume below or at 400 μL in order to obtain a narrow particle size distribution.
Moreover, "empty" CM25-TMC35 nanoparticles (control group) were prepared with the
help of the cross-linking agent pentasodium tripolyphosphate (TPP) at a molar ratio of
3:1 (positive amines N+(CH3)3 to TPP molecules) at room temperature via dropwise
addition of TPP solution (0.58 mg/mL) to slowly vortexed polymer solution. The final
volume was similarly kept at ≤ 400 μL. All particle formulations were kept at room
temperature for at least one hour prior to further use. After preparation, hydrodynamic
diameters of labelled NP were measured by Photon Correlation Spectroscopy (ZetaSizer
3000 HS, Malvern, Switzerland). For each set of measurements, 400 μL of nanoparticle
suspensions were diluted in PBS (pH 7.4) to a total of 1.4 mL. Size distribution data were
obtained by the number-averaged value of three independent groups of ten
measurements. In addition, the zeta potential was measured at least in triplicate via
micro-electrophoresis by using an aqueous dip cell (ZetaSizer 3000 HS, Malvern,
Switzerland). The loading efficiency of NP was determined by centrifugation of 400 μL of
nanoparticle suspension at 16,000 x g for 30 min (Centrifuge 5417C/R, Eppendorf,
Germany) and quantification of the unloaded pGFP in the supernatant by PicoGreen
assay (Quant-iT PicoGreen, Invitrogen, France) according to the manufacturer's
specifications. Fluorescence was measured with a FluoroMax spectrometer (Spex,
Switzerland) at excitation and emission wavelengths of 480 and 522 nm, respectively.
2.5 Exposure
For exposure, NP suspensions were diluted 1:4 in plain cell culture medium in order to
150
yield 4 μg of pGFP in 150 μL medium. After exposure of cell cultures to air for 24 h, for
each exposure, the respective insert was taken out and placed shortly into another 6well plate (filled with 1.2 mL of full medium). The diluted NP suspensions (150 μL per 6well plate) were then sprayed on the apical surface of the co-culture by using a
MicroSprayer (model IA-1C, 10" long 0.64-mm tube, PennCentury, USA). After exposure,
the insert was placed back into its original position within the 6-well plate, cultured for
another 24h and fixated for microscopic analysis.
2.6 Cell labeling and CLSM
Co-cultures were fixed and stained as previously reported (Rothen-Rutishauser et al.,
2005). Antibodies were diluted in PBS as follows: mouse anti human CD14 1:20 (Clone
UCHM-1, C 7673, Sigma, Switzerland), mouse anti-human CD86 1:20 (Clone HB15e,
36931A, PharMingen, BD Biosciences, Switzerland), goat anti-mouse cyanine 5 1:50
(AP124S, Chemicon, Switzerland), DAPI at 1 μg/mL (Molecular Probes, Switzerland) and
rhodamine phalloidin 1:50 (R-415; Molecular Probes, Switzerland).
A Zeiss LSM 510 Meta with an inverted Zeiss microscope (Axiovert 200M, lasers: HeNe
633 nm, HeNe 543 nm, and Ar 488 nm; Carl Zeiss AG, Switzerland) was used. Image
processing and visualization was performed using IMARIS (Bitplane AG, Switzerland), a
three-dimensional multi-channel image processing software for CLSM images.
2.7 ELISA of IL-8 and TNF-α
Following 24h of particle incubation, the cell culture media of the apical chamber of
triple cell co-cultures were collected separately and stored at −80°C until further use.
After centrifugation, TNF-α and IL-8 concentrations were quantified by a DuoSet ELISA
Development kit (DY 210 and DY 208, R&D Systems, UK) performed according to the
151
manufacturer's recommendations. The assay was performed in triplicates. An aliquot of
100 µl of the diluted capture antibody (mouse anti-human TNF-α or IL-8, concentration
of 4 µg/ml PBS) was incubated overnight in a 96-well immunoassay plate (NUNC,
MaxiSorp, Switzerland) at room temperature. Differing from the producer's protocol, the
plate was blocked with PBS supplemented with 1 % bovine serum albumin (BSA) and
0.05 % NaN3 for 1 h at room temperature. After washing with buffer, supernatants from
samples and the standards (0–10 ng/mL of recombinant human TNF-α and 0–2 ng/mL
of recombinant human IL-8) were pipetted into the wells and incubated at room
temperature for 2 h. After washing, the detection antibody (biotinylated goat antihuman TNF-α or IL-8, respectively) diluted in reagent diluent was added. The plate was
covered with an adhesive strip and incubated again for 2 h. Washing was followed by the
addition of horseradish peroxidase-conjugated streptavidin to the plates and incubation
for 20 min at room temperature in the dark. Finally, the substrate solution
(tetramethylbenzidine/H2O2; DY 999, R&D Systems, UK) was added. After 20 min in
darkness, the color development was stopped by adding 50 μL of 1 M H2SO4 and the
plate was put on a shaker (differing from the protocol) for 2 minutes. The absorbance
was then read at 450 nm using an ELISA reader (Benchmark Plus Microplate
Spectrophotometer, Switzerland). The concentration of the cytokine or chemokine was
determined by comparing the absorbance of the samples with standard samples using
log log regressions.
2.8 Statistics
Data from ELISA experiments were expressed as the mean ± standard error of the mean
and were compared by one-way ANOVA using Origin 7.01 software. Differences were
considered significant at p<0.05.
152
3. Results and discussion
3.1 Plasmid DNA NP enter human macrophages and dendritic cells
Regarding vaccination, uptake of antigenic materials into antigen-presenting cells (APC)
is the very first and mandatory step towards the induction of a specific immune
response. In general, there are three types of APC: dendritic cells (DC), macrophages (M)
and B-cells. Among these three cell types, DC play the most prominent role. In contrast
to lymphocytes, DC have during evolution maintained many of the so-called pattern
recognition receptors (PRR) and thus possess the ability of sensing invasion of bacterial
and viral pathogens (Lambrecht et al., 2001; Foged et al., 2002). After encountering such
antigens, immature DC are activated, initiate antigen internalization and transform into
a mature state with high T cell stimulating capability. Thereafter, mature DC can migrate
to local lymph nodes and present antigen(s) to resident T cells (Randall, 2010). After
successful antigen presentation, T cells become activated and migrate back to the site of
antigen exposure within the lung and eliminate infected cells.
In that interplay, pulmonary DC were shown to be imperative for the maintenance of T
cell activity as well as for a constant stimulation of T cells, even after their migration to
infected lung tissues (McGill et al., 2008). In view of pulmonary vaccination, human DC
are difficult to study, because they are sparse and make up only a small percentage of
the pulmonary cell population (Holt et al., 2005).
In order to study the uptake of particulate matter into pulmonary DC, as well as to follow
their interplay with macrophages and epithelial cells in vitro, a triple cell culture model
of the airway barrier was developed over recent years (Rothen-Rutishauser et al., 2005).
Within the scope of our study, we applied a potential NP carrier system for DNA
vaccines onto mentioned triple cell culture model of the respiratory tract and analyzed
by means of CLSM and ELISA of immunogenicity related cytokines (IL-8 and TNF-α).
153
In more detail, we involved three different categories of chitosan-based NP: 1) empty
CM25-TMC35 NP, 2) pGFP-loaded CM25-TMC35 NP and 3) pGFP-loaded CM25-TMC35-gPEG-Pam3Cys NP. Properties of NP can be found in table 1 and reflect previous findings
in our laboratory (Heuking et al., 2009b)
Table 1: Characteristics of chitosan derivatives: molecular weight (MW) and NP:
particle size, ζ potential (ZP), polydispersity index (PDI) and loading efficiency (LE) of
chitosan-based DNA preparations used in this study.
Formulation
Empty CM25-TMC35 NP
pGFP-loaded CM25-TMC35 NP
pGFP-loaded CM25-TMC35-gPEG-Pam3Cys NP
aanalyses
PDIa
MW
Size
ZP
(g/mol)
(nm)a
(mV)a
98,330
378.8 ±
10.8 ±
0.341 ±
52.1
1.2
0.028
404.7 ±
17.5 ±
0.522 ±
92.5 ±
164.1
1.6
0.041
2.5
421.1 ±
15.8 ±
0.576 ±
89.4 ±
173.0
1.6
0.059
3.8
98,330
259,000
LE
(%)a
n/a
were performed in triplicates; n/a, not applicable
In the following, we sprayed these three different NP suspensions onto the described
triple cell culture model by using a microsprayer device, which is commonly used for
intratracheal administrations. This microsprayer creates a fine plume of aerosolized
liquid (Bivas-Benita et al., 2005) and gives therefore a more realistic way of NP
administration than the simple addition of NP suspensions into the cell culture medium.
After exposure, we were able to visualize uptake of NP in all cell types of the co-culture
system by using CLSM techniques. Regarding the overall assessment of NP capture into
154
the particular cell types, we were able to estimate the percentage of internalized NP with
the help of XY- and XZ-projections as well as IMARIS reconstruction images. As a result,
we found that MDM (at the apical side of the culture model) phagocytosed most of the
NP (60-65%). MDDC located at the basolateral side were found to have internalized 1015% of NP, and epithelial cells (HBE) ingested only a minor percentage of NP of 5-10%.
Moreover, the extent of NP uptake was irrespective of pGFP-loading or TLR-2 agonist
functionalization, because all NP types gave similar percentages. In previous
investigations involving virosomes (0.1 - 0.2 μm in size) and polystyrene particles (0.2
μm and 1.0 μm in size), similar uptake patterns were observed (Blank et al., 2007; Hofer
et al., 2009).
Empty CM25-TMC35 NP
CM25-TMC35 pGFP NP
Conjugate pGFP NP
MDM
MDM
MDM
20 μm
20 μm
20 μm
Figure 1: CLSM micrographs of phagocytosed NP (green) by MDM (blue). XY and XYprojections clearly indicate that NP are mostly taken up by MDM (lozenge). Extracellular
NP are marked with an arrowhead. The horizontal bar represents 20 μm (Conjugate,
CM25-TMC35-g-PEG-Pam3Cys).
Considering different functions of each cell type in this triple in vitro model, MDM
constitute clearly the first line of defence and are constantly exposed to the entry of
155
antigenic materials into the human lung. MDM have also the ability to inhibit virus
growth and eliminate infected cells. Moreover, activated macrophages can produce
antiviral factors, (e.g., TNF-α and IFN-α/β) and chemokines, which are able to activate
additional cell types in the fight against infections (Murphy et al., 2004). Although MDM
possess only a minor antigen-presenting capacity, a direct cross-talk by exchanging NP
with MDDC was proposed (Blank et al., 2007). In line with our study, CLSM images of
human CD14-positive MDM demonstrate that a pronounced phagocytosis of NP took
place (lozenge-marked, figure 1). XY- and XZ-projections also allowed for the
recognition of non-phagocytosed NP for comparative purposes (arrowheads, figure 1).
Furthermore, we reconstructed the apical part of the co-culture by imaging software
(figure 2), where NP internalization was detected in all three experimental groups.
Secondly, considering the important function of DC as APC, we investigated the
capturing of NP by MDDC (located in and beneath the epithelium). Generally, it was
proposed that MDDC extend pseudopodia even in the absence of apical particles through
the epithelium towards the luminal side (Blank et al., 2007). Once particles are
deposited, MDDC can rapidly induce internalization (within minutes) followed by
transport to the apical side of the epithelium (Blank et al., 2007). In our study, we
observed clearly the uptake of all chitosan-based pGFP NP into MDDC after 24h by CLSM
(figure 3).
156
A
B
C
Figure 2: Visualization of NP uptake in the apical part of the triple cell culture model
(actin, red; macrophages, blue; nanoparticles, green), with A) empty CM25-TMC35 NP, B)
CM25-TMC35 pGFP NP and C) CM25-TMC35-g-PEG-Pam3Cys pGFP NP. Lozenge marks
indicate internalized NP into MDM; extracellular NP are marked with an arrowhead.
White bar (lower left corner) represents a 20 μm scale.
157
In addition, software reconstruction of the basal part of the co-culture model was
performed (figure 4).
Empty CM25-TMC35 NP
CM25-TMC35 pGFP NP
Conjugate pGFP NP
MDDC
MDDC
20 μm
MDDC
20 μm
20 μm
Figure 3: CLSM micrographs of phagocytosed NP (green) by MDDC (blue). XY and XYprojections clearly indicate that NP are internalized by MDDC (lozenge). Horizontal bar
represents 20 μm (Conjugate, CM25-TMC35-g-PEG-Pam3Cys).
From these images it becomes clear that MDDC were capable of taking up all three types
of chitosan-based NP. Interestingly, Blank et al. (2007) proposed different mechanisms
of particle transport through the epithelium by MDDC: i) uptake of particles through
cellular extensions of MDDC across the epithelium, ii) crossing of MDDC through the
entire epithelium followed by particles uptake, iii) particle exchange between MDDC, iv)
transfer of particles from MDM to MDDC via cell–cell contacts and v) transfer of particles
through interactions of MDM with MDDC located in or at the basolateral side of the
epithelium.
158
A
B
C
Figure 4: Visualization of different NP in the basal compartment of triple cell culture
model (actin, red; macrophages, blue; nanoparticles, green) with A) empty CM25-TMC35
NP, B) CM25-TMC35 pGFP NP and C) CM25-TMC35-g-PEG-Pam3Cys pGFP NP. Lozenge
marks internalized NP into MDM; extracellular NP are marked with an arrowhead.
White bar represents a 20 μm scale (lower left corner).
159
Although we were not able to reveal the exact way of migration of NP towards MDDC
followed by their uptake, we found a certain percentage of NP (10-15%) internalized by
MDDC after 24h of exposure. It is highly possible that during the exposure to NP, MDDC,
but also MDM, were stimulated by our chitosan-based pGFP NP. We therefore analyzed
for the secretion of two different immune mediators, IL-8 and TNF-α.
3.2 TLR-2 agonist functionalization of NP augments the immune response
In order to investigate a potential higher immunogenicity of CM25-TMC35-g-PEGPam3Cys pGFP NP due to the inclusion of an adjuvant moiety, the TLR-2 agonist Pam3Cys,
we studied the expression of IL-8 and TNF-α in the basal compartment of the co-culture
system. In general, IL-8 has a role as an essential regulator for the recruitment of
leukocytes and their successive trafficking to the mucosal site of infection. In our study,
we remarked a significantly (p<0.05) higher release of IL-8 owing to the exposure of
pGFP-loaded CM25-TMC35 and CM25-TMC35-g-PEG-Pam3Cys NP (9.21 ± 0.36 pg/mL and
13.72 ± 0.76 ng/mL, respectively), when compared to the use of medium alone (5.17 ±
0.54 ng/mL; figure 5). Hereby, CM25-TMC35-g-PEG-Pam3Cys pGFP NP elicited a
significantly (p<0.05) higher IL-8 related immune response than CM25-TMC35 pGFP NP.
Spohn et al. (2004) and Sadik et al. (2008) already described the IL-8 eliciting capacity of
the Pam3Cys moiety on stably TLR-2 transfected HEK cells and differentiated THP-1
macrophages, respectively. Interestingly, unloaded CM25-TMC35 NP had a weak ability
to trigger IL-8 levels (8.68 ± 1.06 ng/mL), although the difference was not significant in
comparison to the control. An IL-8 inducing property of chitosan was already mentioned
by Park et al. (2009). The general trend of this IL-8 ELISA goes in line with our previous
report (Heuking et al., 2009b), in which we noted a ten-fold higher induction of IL-8
chemokine from differentiated human-like macrophages (THP-1) by TLR-2 agonist
160
functionalization (CM25-TMC35-g-PEG-Pam3Cys pGFP NP versus CM25-TMC35 pGFP NP).
20,0
*
18,0
16,0
*
IL-8 [ng/ml]
14,0
12,0
NS
*
CM25-TMC35 NP
CM25-TMC35 pGFP NP
10,0
8,0
6,0
4,0
2,0
0,0
Medium control
Conjugate pGFP NP
Figure 5: IL-8 release in the apical compartment from co-culture model due to exposure
to empty CM25-TMC35 NP, CM25-TMC35 pGFP NP and Conjugate (CM25-TMC35-g-PEGPam3Cys) pGFP NP. Presented data are the mean ± standard error of the mean of three
independent experiments. Differences were considered significant for * (p<0.05), in
comparison to cells treated with culture medium; NS, not significant.
As a second indicator for an immune response, we examined the release of TNF-α from
the co-culture model. Generally speaking, TNF-α is involved in the regulation of
apoptotic cell death and tumorigenesis, but its main role consists in the regulation of
immune cells. In our study, we found in response to NP exposure TNF-α responses with
the ranking of CM25-TMC35-g-PEG-Pam3Cys pGFP NP > CM-25TMC35 pGFP NP >
unloaded CM25-TMC35 NP > medium control (figure 6). It appears that again the
functionalization with the TLR-2 agonistic moiety Pam3Cys was able to increase
161
significantly (p<0.05) a TNF-α specific immune response (2.30 ± 0.23 ng/mL), when
compared to unmodified CM25-TMC35 pGFP NP (1.62 ± 0.16 ng/mL) and control
medium (0.84 ± 0.15 ng/mL). Schjetne et al. (2003) reported that the Pam3Cys moiety
stimulates CD14-positive monocytes as well as immature DC in a TLR-2 specific manner
to produce high levels of TNF-α (around 1.5 – 2.0 ng/mL), which were comparable to
those induced by bacterial LPS.
TNF-alpha [ng/m l]
4,0
*
3,0
*
NS
2,0
NS
1,0
0,0
Medium control
CM25-TMC35 NP
CM25-TMC35 pGFP Conjugate pGFP NP
NP
Figure 6: TNF-α release in the apical compartment from co-culture model due to
exposure of empty CM-25TMC35 NP, CM25-TMC35 pGFP NP and Conjugate (CM25TMC35-g-PEG-Pam3Cys) pGFP NP. Presented data are mean ± standard error of the mean
of three independent experiments. Differences were considered significant for * (p<
0.05), in comparison to cells treated with culture medium (control); NS, not significant.
Similarly to the IL-8 results, empty CM25-TMC35 NP were also able to trigger some
release of TNF-α, although the difference was again not significant. Otterlei et al. (1994)
demonstrated that chitosan (depending on its molecular weight and degree of
162
deacetylation) can induce TNF-α production from human monocytes in a CD14dependent manner.
In conclusion, we were able to detect MDDC transferred pGFP NP being located at the
abluminal side of the HBE epithelium by CLSM. Although no significant difference in
uptake pattern was observed for all three NP systems studied, ELISA of IL-8 and TNF-α
demonstrated clearly that TLR-2 agonist functionalization facilitates an overall higher
immune response with the ranking of CM25-TMC35-g-PEG-Pam3Cys pGFP NP > CM25TMC35 pGFP NP > CM25-TMC35 NP. Hence, the novel TLR-2 agonist functionalized DNA
carrier merits further investigation as novel material for DNA vaccination.
Acknowledgments
The authors would like to thank B. Tschirren, A. Stokes, D. Raemy, A. Lehmann, L. Müller
and C. Brandenberger from the Institute of Anatomy in Bern for their technical support
in cell culture and immune analyses.
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167
Chapter 7
Preliminary in vivo
immunogenicity of TLR-2 agonist
decorated chitosan nanoparticles
encapsulating a plasmid DNA
vaccine against
Mycobacterium tuberculosis
S. Heuking1,2,3, D. Hedhli3, K. Huygen3 and G. Borchard1,2
1School
of Pharmaceutical Sciences, University of Geneva, Switzerland
2Centre
Pharmapeptides, Archamps, France
3WIV-Pasteur
Institut, Mycobacterial Immunology, Brussels, Belgium
168
Abstract
Within the scope of our study, we prepared a water-soluble and TLR-2 agonist
functionalized chitosan derivative (CM20-TMC20-60-g3.5%-PEG-Pam3Cys) of minor
toxicity (IC50 of 919 μg/mL). This novel co-polymer was then utilized for the
encapsulation of a plasmid DNA vaccine (pAg85A, encoding for the antigen 85A of
Mycobacterium tuberculosis), resulting in nano-sized particles. The immunogenicity
(measured as IL-2 and IFN-γ secretion) of pAg85A chitosan nanoparticles was then
investigated after intramuscular (IM) and intranasal (IN) administration. Reliable and
significant results were obtained for IM vaccinations only for pAg85A (without carrier)
immunizations in view of IL-2 (spleen) and IFN-γ (spleen) secretions. With regard to IN
immunizations, significant IFN-γ production was seen with the ranking pAg85A
(without carrier) > TMC20-60 pAg85A nanoparticles (NP). In addition, Ag85A-specific
antibodies were only detected by IM pAg85A (without carrier) vaccination. In a nutshell,
pAg85A chitosan nanoparticles, with or without TLR-2 agonist functionalization, did not
demonstrate a benefit over IM pAg85A vaccination. This observation might be related to
low in vitro and in vivo transfection efficiency of the particles.
1. Introduction
In general, a plasmid DNA (pDNA) vaccine can be defined as a bacterial plasmid vector
into which a gene of interest is inserted encoding for one (or more) antigenic protein(s).
Plasmid DNA vaccines are produced in bacteria (such as Escherichia coli) and after
purification injected into the host (Huygen, 2005). Overall, DNA vaccination might be an
answer for preventing infections caused by intracellular pathogens (such as
tuberculosis, influenza, hepatitis or HIV/AIDS) due to its distinct ability of inducing a
strong cytotoxic T lymphocyte (CTL) response in orchestration with CD4+ T helper cells
169
(cellular immunity) as well as the generation of antibodies (humoral immunity) (Kutzler
and Weiner, 2008).
Regarding the immunization against Mycobacterium tuberculosis (Mtb), several pDNA
vaccines encoding, e.g., for the antigen 85A (Ag85A), Ag85B, 65 kDa heat-shock-protein,
Mtb72F and others were examined at preclinical level and revealed different
magnitudes of immunogenicity and protection (Huygen, 2005). However, a major
drawback of pDNA vaccines is their low transfection efficacy and consequently only
minor quantities of antigenic protein(s) are produced in situ leading to a weaker
immune response. Moreover, at present, no pDNA vaccine causes protection superior to
the common BCG vaccine.
Therefore, improvements on pDNA immunization are indispensable and might be
achieved by plasmid optimization, co-formulation with adjuvants or changing the route
of administration, for instance to pulmonary vaccination (Kutzler and Weiner, 2008).
Considering pulmonary vaccine delivery, it is worth mentioning that airborne bacterial
and viral infections in the respiratory tract (especially pulmonary tuberculosis) are
causing a high rate of deaths per year (Woodland et al., 2004). Consequently, pulmonary
deposition of vaccines mimicking the natural way of infection might therefore be an
appropriate way for their prevention. Promise of pulmonary vaccination as a feasible
concept was indicated by several clinical trials using measles aerosol vaccines (Dilraj et
al., 2000; Bennett et al., 2002). Interestingly, nasal vaccination with volumes > 10 μL in
mice under anaesthesia was shown to facilitate effective delivery also to the lung (Alpar,
et al., 2005). Secondly, nasal administration can be easily realized by simply pipetting
small volumes (e.g., 50 μL) into nasal cavities.
Within the scope of nasal/pulmonary DNA vaccination, we selected a pDNA vaccine
encoding for the mycolyl-transferase protein Ag85A of Mtb (abbreviated pAg85A). The
170
plasmid pAg85A is among the most investigated pDNA vaccines against Mtb and
demonstrated promise due to its strong Th1 response in vivo (Huygen et al., 1996).
In order to protect pAg85A against nuclease degradation upon deposition at mucosal
epithelia, we encapsulated pAg85A into chitosan-based nanoparticles (NP). Moreover,
we equipped these pAg85A NP with an adjuvant moiety, the TLR-2 agonist Pam3Cys, as
described earlier (Heuking et al., 2009a).
In turn, Pam3Cys functionalized pAg85A nanoparticles were applied intranasally and
intramuscularly into C57BL/6 mice. The proliferative capacity of leukocytes in spleen,
lungs and cervical lymph nodes (intranasal) or solely spleen (intramuscular) was
evaluated by measuring secretions of T-cell helper type 1 (Th1) cytokines (IL-2 and IFNγ) in response to antigen exposure in vitro. Finally, titration of Ag85A specific total
antibodies in sera was performed by ELISA.
2. Materials and methods
2.1 Animals
C57BL/6 mice (n=120) were provided from the breeding facilities of WIV-Pasteur Site in
Ukkel (Belgium). Only female mice were used at an age of 6-8 weeks old at the start of
vaccination.
2.2 Plasmid DNA construction
Plasmid DNA encoding Ag85A (pAg85A) was prepared as previously reported (D’Souza
et al., 2006) and propagated in Escherichia coli DH5α followed by large scale purification
by GigaKit (Invitrogen, France). Correct propagation of pAg85A was controlled by
incubation with the BgLII restriction enzyme (Invitrogen, France) followed by gel
electrophoresis. Final concentration of pAg85A was 3.2 µg/µL in endotoxin-free TBE
171
buffer (Invitrogen, France).
2.3 Synthesis of and characterization of CM-TMC-g-PEG-Pam3Cys
The water-soluble chitosan derivative CM-TMC-g-PEG-Pam3Cys was prepared as
previously reported (Heuking et al., 2009b). Briefly, a N,N,N-trimethyl chitosan (TMC)
with a degree of trimethylation (%DTM) of 20% and degree of dimethylation (%DDM)
of 60% was synthesized. After successive carboxymethylation (CM-TMC), grafting of
NH2-PEG-Pam3Cys to CM-TMC was performed aiming at a grafting success of 4.0 %
(Heuking et al., 2009). The DTM, DDM, degree of 6-0-carboxymethylation (%DCM) and
the degree of grafting (%DG) were determined as described earlier (Heuking et al.,
2009b) by 1H NMR spectroscopy on a Varian VXR 300 MHz spectrometer (Varian,
Switzerland). Chitosan derivatives were dissolved in D2O and all other compounds in
CDCl3.
2.4 Nanoparticle preparation
Nanoparticles encapsulating pAg85A were formulated according to published methods
(Heuking et al., 2009a). More precisely, CM-TMC-g-PEG-Pam3Cys (at 7.49 mg/mL) or
TMC (at 4.4 mg/mL) were dissolved in Minniversol water. Separately, the plasmid
pAg85A (1 μg of pAg85A being equal to 3.1 nmol of phosphate groups) was dissolved in
5 mM aqueous Na2SO4 at appropriate concentrations in order to yield an N/P molar
ratio of 3:1. Both solutions were heated for 5 minutes at 55°C and the polymer solution
was then slowly added (approximately 1 drop per second) to the pAg85A solution and
subsequently vortexed at low speed for 30 seconds. Attention was paid to keep the final
volume ≤ 400 μL. Moreover, empty TMC nanoparticles (control group) were prepared by
adding the cross-linking agent pentasodium tripolyphosphate (TPP) at a molar ratio of
172
3:1 (positive amines N+(CH3)3 to TPP molecules) at room temperature via drop-wise
addition of TPP solution (0.58 mg/mL) to slowly vortexed polymer solution. The final
volume was similarly kept at a volume of ≤ 400 μL. Thereafter, particles were kept at
room temperature for at least one hour prior to further use.
2.5 Isotonicity
The isotonicity of nanoparticle dispersions as well as pAg85A solution was adjusted to
300 mOsmmol/kg by using appropriate small volumes (< 20 μL) of NaCl solution (0.16
mg/mL). Isotonicity was controlled by a MicroOsmometer (Knauer, Germany). All
measurements were done in triplicates.
2.6 Size and zeta potential
Hydrodynamic diameters were measured by Photon Correlation Spectroscopy
(ZetaSizer 3000 HS, Malvern, Switzerland). For measurements, 400 μL of isotonized
nanoparticle suspensions were diluted with RPMI1640 plain medium to 1.4 mL. Size
distribution data were obtained by the number-averaged value of three independent
groups of ten measurements. In addition, zeta potential was measured in triplicate via
micro-electrophoresis by using an aqueous dip cell (ZetaSizer 3000 HS, Malvern,
Switzerland).
2.7 Vaccination
All animal experiments were performed according to the regulations enforced by the
local ethical committee. C57BL/6 mice (n=120) were anaesthetized by intraperitoneal
injection of ketamine-xylazine and immunized three times intramuscularly (n=60) at
three weeks intervals with 100 µL of formulations I, II or III containing 25 µg of pAg85A
173
(see table 1) or with the control formulation IV (no pAg85A). Moreover, further mice
(n=60) were immunized intranasally with 50 µL of formulations I, II or III (containing
12.5 µg of pAg85A, respectively) or control formulation IV (no pAg85A).
Table 1 – pAg85A formulations used for this study (conjugate = CM-TMC-g-PEGPam3Cys).
Formulation
Delivery form
Content
I
Solution
pAg85A
II
Suspension
TMC20-60 pAg85A nanoparticles
III
Suspension
Conjugate pAg85A nanoparticles
IV
Suspension
TMC20-60 TPP nanoparticles
2.8 Cytokine measurements
Three weeks after the last immunization mice were sacrificed by cervical dislocation.
Spleens (intramuscular groups) or spleens, lungs and cervical lymph nodes (intranasal
groups) were removed aseptically, crushed and cells were passed over a nylon wool
column in order to eliminate debris. Leucocytes (4 × 106 cells/ml) from five mice per
group and per delivery route were tested individually (spleen) or pooled (lungs and
lymph nodes) for cytokine response to recombinant Ag85A (5 μg/ml), purified protein
derivative (PPD) (10 μg/mL), Ag85A240-260 (10 μg/mL), Ag85A260-280 (10 μg/mL) and
pokeweed mitogen (PWM, 5 μg/mL) as positive control. Non-stimulated cells were used
as negative control. Next, supernatants were harvested after 24h or 48h for IL-2 analysis
and after 72h for IFN-γ analysis as described earlier (D’Souza et al., 2003). IL-2
concentrations were determined by ELISA (antibodies were obtained from Bioscience,
Belgium) and expressed in pg/mL.
174
Similarly, IFN-γ activity was quantified by sandwich ELISA as reported (Romana et al.,
2006) and expressed in pg/mL. Data were regressed using log log regression with R >
0.95. The assay sensitivity for ELISA of IL-2 and IFN-γ was 3.1-39.1 pg/ml, depending on
the standard concentrations used.
2.9 Antibody measurement
Five mice per group were bled before the 1st, 2nd and 3rd immunization and sera were
isolated from the clot and stored at 4 °C for antibody detection. The antibody responses
to Ag85A was detected with an ELISA as specified earlier (Tanghe et al., 2000; Gartner et
al., 2008).
2.10 Transfection of HEK293T cells and Western Blot
Human embryonic kidney cells (HEK293T cells) were used for studying the transfection
of pAg85A chitosan nanoparticles (Gartner et al., 2007). HEK293T cells were grown in
Dulbecco's modified Eagle medium (DMEM) containing penicillin (100 U/ml),
streptomycin (100 μg/ml) and 10% foetal bovine serum. One day before transfection,
2.5 × 105 cells per well were seeded in six-well plates. Per well and per transfection, 1 μg
of pAg85A were applied with the following organization of controls and samples: 1)
lipofectamine TransIT-LT1TM (Mirus, France) for pAg85A transfection (positive control);
2) TransIT-LT1TM was also applied for transfection of a control plasmid (without Ag85A
sequence; negative control); 3) pAg85A alone; 4) TMC pAg85A nanoparticles; 5) CM20TMC20-60-g3.5%-PEG-Pam3Cys pAg85A nanoparticles. After 24 h of transfection cells
were harvested by pipetting, washed with cold PBS and lysed on ice during 2 min in a
lysis buffer containing 50 mM Tris–HCl pH 7.4, 250 mM sucrose, 1% (v/v) nonidet P-40
(NP-40) and complete protease inhibitor cocktail (Roche, Switzerland).
175
Protein concentration of each lysate was determined by Bradford assay (BioRad,
France) and diluted accordingly. Next, lysates were boiled in loading dye under reducing
conditions with 500 mM DTT for successive sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS-PAGE). Samples (25 μl per well) and the protein ladder
Precision Plus KaleidoscopeTM (BioRad, France) were separated by SDS-PAGE using 12%
gels. In the following, the mouse monoclonal Ag85-specific antibody TD17 (Drowart et
al., 1992; Huygen et al., 1994) was used for revealing Ag85A expression followed by
treatment with horseradish peroxidase coupled rabbit anti-mouse immunoglobulin (Ig)
as secondary antibody (Sigma, Switzerland). Blots were analyzed by using the
ChemiLuminescence Detection Kit (Applichem, Germany), Fuji Super RX films (Fuji,
Switzerland) and a Fujifilm FPM-100A film developer (Fuji, Switzerland).
2.11 Data analysis and statistics
Obtained data were expressed as the mean ± standard error of the mean and compared
by a one-way ANOVA analysis using Origin 7.03 software. Differences were considered
significant at p<0.05.
3. Results
3.1 CM-TMC-g-PEG-Pam3Cys synthesis and characterization
In order to develop a water-soluble chitosan derivative for vaccine delivery, we opted
for the synthesis of a TMC with 20% as DTM and 60% as DDM (named TMC20-60).
Jantapinkit et al. (2008) reported that such a TMC20-60 polymer exhibits the least
cytotoxicity on L929 fibroblasts (IC50 > 1000 µg/mL) in comparison to TMCs with higher
DTMs or lower DDMs (having IC50 values of 10 µg/mL). Accordingly, we synthesized a
TMC20-60, carboxymethylated the polymer (giving CM-TMC) and grafted then the TLR-2
176
agonistic moiety NH2-PEG-Pam3Cys to CM-TMC as already reported (Heuking et al.,
2009). 1H NMR spectroscopy confirmed a DTM of 18.5 %, DDM of 55.3 %, DCM of 19.2 %
and DS of 3.5 % for the finalized co-polymer, which will be abbreviated CM20-TMC20-60g3.5%-PEG-Pam3Cys (data not shown). In our hands, CM20-TMC20-60-g3.5%-PEG-Pam3Cys
displayed a much lower cytotoxicity on alveolar A549 cells by WST assay (IC50 of 919
µg/mL) when compared to our previously investigated co-polymer (IC50 of < 20 µg/mL;
Heuking et al., 2009). Moreover, we demonstrated (Heuking et al., 2009) that the
formation of TMC and CM-TMC-g-PEG-Pam3Cys DNA nanoparticles decreased strongly
the cytotoxicity by a factor of 50, so that we did not expect any cytotoxicity of CM20TMC20-60-g3.5%-PEG-Pam3Cys pAg85A nanoparticles (final concentration of CM20TMC20-60-g3.5%-PEG-Pam3Cys was 3.75 mg/mL). Consequently, we included CM20TMC20-60-g3.5%-PEG-Pam3Cys in further studies.
3.2 Characterization of pDNA nanoparticles
Nanoaggregation of TMC20-60 as well as CM20-TMC20-60-g3.5%-PEG-Pam3Cys with
pAg85A resulted in positively charged and nano-sized particles (see table 2). In
comparison to empty TMC20-60 TPP nanoparticles, pAg85A loaded nanoparticles
exhibited slightly higher polydispersities (see table 2), which might be explained by
differences in molecular weight between TPP (MW 367.9 Da) and plasmid DNA (MW
3575 kDa), allowing a potentially more controlled and homogenous formation of
TMC20-60 TPP nanoparticles. Interestingly, Csaba and colleagues (2010) noticed
likewise that the explicit use of TPP for the preparation of chitosan DNA nanoparticles
resulted in a more homogenous morphology of particles.
177
TABLE 2 Size distribution and zeta potential (ZE) values of pAg85A nanoparticles (Gr. =
formulation group; conjugate = CM20-TMC20-60-g3.5%-PEG-Pam3Cys; n/a = non
applicable; * = nanoparticles were prepared at a molar ratio of 3:1, +N(CH3)3:TPP).
Gr.
Chitosan derivative
N/P
Size
ZE
Poly-
applied
ratio
[nm]
[mV]
dispersity
I
None
n/a
n/a
n/a
n/a
II
TMC20-60
3:1
429.5 ± 170.2
18.9 ± 2.3
0.534
III
Conjugate
3:1
511.4 ± 166.5
15.8 ± 1.6
0.522
IV
TMC20-60
n/a*
438.8 ± 82.1
10.8 ± 1.2
0.341
After the first vaccination step in mice and successive storage at 4-5°C, inconvenient
precipitation of nanoparticles was detected by sight. The reason for this phenomenon
might be the addition of concentrated sodium chloride solution in order to adjust the
isotonicity to 300 mOsm/kg. Similar observations for pDNA containing chitosan
nanoparticles were already reported by others (Medburry et al., 2004; Strand et al.,
2008). Consequently, in an attempt to restore the initial nanoparticulate size, we applied
soft ultrasonication (Branson ultrasonic bath, 5510, 40kHz) for disaggregation purposes.
However, ultrasonication was shown to cause degradation of pDNA depending on time
and frequency applied (Walter et al., 1999; Kuo et al., 2003). Thus, we controlled the
integrity of plasmid pAg85A after ultrasonication treatment by gel electrophoresis. After
60 minutes of ultrasonication at room temperature, initial size and zeta potential values
were maintained (data not shown) and even after 75 minutes of ultrasonication, no
degradation of pAg85A was detected (see figure 1). Therefore, we set the time of
ultrasonication of formulations II, III and IV to one hour at room temperature prior to
second and third immunizations.
178
x
1
2
x
3
4
5
6
x
x
x
x
7
8
9
10
Figure 1: Gel electrophoresis after 75 minutes of ultrasonic treatment (47 kHz, room
temperature) with lane 1-2 CM20-TMC20-60-g3.5%-PEG-Pam3Cys pAg85A nanoparticles;
lane 3-4 TMC20-60 pAg85A nanoparticles; lane 5-8 pAg85A alone; lane 9-10: 1kpb DNA
ladder; x = treatment by ultrasonication.
3.3 Vaccination
3.3.1 Intramuscular immunizations
As positive control to intranasal vaccination, we injected three times all four pAg85A
formulations (group I, II, III and IV) into C57BL/6 mice intramuscularly (IM) and
analyzed for antigen specific immune responses after a total of 12 weeks. Splenocytes
from mice were challenged in vitro with the recombinant Ag85A protein (recAg85A),
purified protein derivative from Mycobacterium tuberculosis (PPD), two selected CD4+
antigenic peptide sequences (Ag 85A240-260, Ag 85A260-280, Huygen et al., 1994) and
pokeweed mitogen (PWM) as positive control. Subsequently, Th1-cytokines IL-2 and
IFN-γ were measured.
179
Relating to IL-2 levels, we observed that only IM injection of plasmid pAg85A (group I,
see figure 2a) triggered ascertainable, but weak IL-2 secretions. None of the chitosanbased pAg85A delivery systems (group II and III) induced any significant levels of IL-2.
Spleen: IL-2 (24h)
600,0
NS
IL-2 (pg/ml)
500,0
400,0
300,0
NS
200,0
100,0
*
P25
P27
PPD
CS
II
Ag85A
P27
II
CS
P25
II
PPD
Ag85A
I
P27
CS
I
P25
PPD
I
Ag85A
P27
I
CS
P25
I
PPD
Ag85A
0,0
II
II
III
III
III
III
III
IV
IV
IV
IV
IV
antigen/group
Figure 2a: IL-2 secretions in supernatant of spleen cells after 24h exposure to the
recombinant antigen 85A (recAg85A), purified protein derivative (PPD) Ag 85A240-260
(P25), Ag85A260-280 (P27) and non-stimulated cells (CS) (n=5 per group); bar = detection
limit of 3.1 pg/mL; ANOVA analysis were performed via comparison to non-stimulated
cells: *, p<0.05; NS, not significant.
180
1800,00
1600,00
IL-2 (pg/mL)
1400,00
1200,00
1000,00
800,00
600,00
400,00
200,00
0,00
PWM
PWM
PWM
PWM
I
II
III
IV
PWM/group
Figure 2b: IL-2 secretions in supernatant of spleen cells after 24h exposure to pokeweed
mitogen (PWM) (n=5 per group).
Exposure to pokeweed mitogen (PWM) as positive control demonstrated a different
onset of IFN-γ production (figure 2b), which renders a direct comparison of IL-2 results
difficult as not the same proliferative capacity of leukocytes was given. In contrast to
that, we observed the generation of IL-2 cytokines when splenocytes were exposed to
the purified protein derivative (PPD). PPD is the total protein extract of Mycobacterium
tuberculosis and contains, next to small amounts of Ag85A, all secreted, cytoplastic and
membrane-based proteins. In our hands, we noticed PPD-specific IL-2 secretions with
the tendency of CM20-TMC20-60-g3.5%-PEG-Pam3Cys pAg85A nanoparticles > TMC20-60
pAg85A nanoparticles (although obtained values were not significant). IL-2 levels for
group I (pAg85A alone) and group IV (unloaded TMC20-60 nanoparticles) were below
the limit of detection (see figure 2). Regarding the second Th1-cytokine, IFN-γ, only
splenocytes from mice vaccinated with pAg85A alone (group I) demonstrated Ag85A181
specific IFN-γ levels (see figure 3). Again in contrast to that, PPD challenged splenocytes
elicited IFN-γ secretions with the trend (although not significant) with CM20-TMC20-60g3.5%-PEG-Pam3Cys pAg85A NP > unloaded TMC20-60 NP > pAg85A in solution (see
figure 3). To our surprise, unloaded TMC20-60 NP (as control group) enabled PPDrelated IFN-γ production, although not statistically significant, this results questions the
validity of this particular IFN-γ assay.
Spleen: IFN-g (72h)
900,0
800,0
*
700,0
IFN-g (pg/ml)
600,0
NS
NS
500,0
400,0
NS
NS
300,0
200,0
100,0
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
0,0
I
I
I
I
I
II
II
II
II
II
III
III
III
III
III
IV
IV
IV
IV
IV
antigen/group
Figure 3: IFN-γ secretions in supernatant of splenocytes after 72h exposure to the
antigen 85A (recAg85A), purified protein derivative (PPD), Ag85A240-260 (P25),
Ag85A260-280 (P27) and non-stimulated cells (CS) (n=5 per group); bar = detection limit
of 39.1 pg/mL; ANOVA analysis were performed via comparison to non-stimulated cells:
*, p<0.05; NS, not significant.
182
3.3.2 Intranasal immunizations
Intranasal (IN) vaccination of C57BL/6 mice was performed by using 12.5 μg of pAg85A
per vaccination (in contrast to 25 μg of pAg85A for IM vaccinations) and was analyzed
for IL-2 (lung, lymph node and spleen) and IFN-γ secretions (spleen) in similar manner
as reported for IM immunizations.
Concerning the immune response in the lung, no proliferative IL-2 responses were
detected at all from lung cells after antigen exposure in vitro (data not shown). However,
we noted IL-2 secretions of leukocytes from cervical lymph nodes after antigen
challenge (see figure 4).
Lymph nodes: IL-2 (48h)
14,0
12,0
IL-2 (pg/ml)
10,0
8,0
6,0
4,0
2,0
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
0,0
I
I
I
I
I
II
II
II
II
II
III
III
III
III
III
IV
IV
IV
IV
IV
antigen/group
Figure 4: IL-2 response of pooled lymph node cells due to 48h exposure to the following
antigens: antigen 85A (Ag85A), purified protein derivative (PPD), Ag85A240-260 (P25),
Ag85A260-280 (P27) and non-stimulated cells (CS) (n=5 per group); bar = detection limit
of 3.1 pg/mL.
183
Above the detection limit, TLR-2 decorated NP harbouring pAg85A (group III) mediated
the strongest production of IL-2, which was specific to the antigen 85A, but not to PPD.
No Ag85A-specific IL-2 was found for group I (pAg85A alone), however, to our
astonishment, unloaded TMC20-60 nanoparticles (containig no pAg85A, control group
IV) showed an Ag85A-specific IL-2 response, which is comparable to the IFN-γ assay for
IM vaccinations questioning the assay´s validity (see figure 4). In addition, Ag85A- and
PPD-related IL-2 response of spleen cells were very weak (<5 pg/mL; see figure 5) and
only splenocytes of group III (TLR-2 agonist decorated pAg85A NP) vaccinated mice
exhibited modest and not significant PPD-specific IL-2 secretions.
Spleen: IL-2 (24h)
40,0
35,0
NS
IL-2 (pg/ml)
30,0
25,0
20,0
15,0
10,0
5,0
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
0,0
I
I
I
I
I
II
II
II
II
II
III
III
III
III
III
IV
IV
IV
IV
IV
antigen/group
Figure 5: IL-2 response of splenocytes in response to 24h exposure to various antigens:
antigen 85A (Ag85A), purified protein derivative (PPD), Ag85A240-260 (P25), Ag85A260-280
(P27) and non-stimulated cells (CS) (n=5 per group); bar = detection limit of 3.1 pg/mL;
ANOVA analysis were performed via comparison to unstimulated cells: *, p<0.05; NS, not
significant.
184
Regarding IFN-γ responses of splenocytes (see figure 6a), Ag85A exposure enabled IFNγ production with the trend of pAg85A alone (group I) > TMC20-60 pAg84A NP (group
II) > CM20-TMC20-60-g3.5%-PEG-Pam3Cys pAg85A NP (group III, not significant).
Unloaded TMC20-60 NP (group IV) as control showed no IFN-γ induction.
Spleen: IFN-g (72h)
90,0
80,0
*
70,0
*
IFN-g (pg/ml)
60,0
50,0
NS
40,0
NS
30,0
20,0
10,0
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
Ag85A
P25
P27
PPD
CS
0,0
I
I
I
I
I
II
II
II
II
II
III
III
III
III
III
IV
IV
IV
IV
IV
antigen/group
Figure 6a : IFN-γ secretions of splenocytes in response to 72h exposure to various
antigens: antigen 85A (Ag85A), purified protein derivative (PPD), Ag85A240-260 (P25),
Ag85A260-280 (P27) and non-stimulated cells (CS) (n=5 per group); bar = detection limit
of 15.1 pg/mL. ANOVA analysis were performed via comparison to non-stimulated cells:
*, p <0.05; NS, not significant.
185
Exposure to pokeweed mitogen (positive control) showed different magnitude (figure
6b) of IFN-γ production, which renders a comparison of results difficult as not the same
proliferative function of leukocytes was given.
3000,00
IFN-g (pg/mL)
2500,00
2000,00
1500,00
1000,00
500,00
0,00
PWM
I
PWM
PWM
PWM
II
III
IV
PWM/group
Figure 6b: IFN-γ secretions of splenocytes in response to 72h exposure to pokeweed
mitogen (PWM) (n=5 per group); bar = detection limit of 15.1 pg/mL.
3.3.3 Ag85A-specific antibodies
In our hands, only mice vaccinated with pAg85A alone (group I) demonstrated
significant Ag85A-specific IgG antibodies at 1:100 sera dilution (OD value of 0.69 ± 0.22,
one-way ANOVA, p < 0.05) and were lower when compared to previous findings (1:2560
dilution, OD value of 0.611 ± 0.246, Romano et al., 2006). Chitosan-based pAg85A
nanoparticles (group II and III) did not elicit significant Ag85A-specific immune
responses (data not presented). Therefore, we questioned the polymer´s ability of
transfection, which would impair the production of the antigenic protein 85A in vivo and
subsequently Ag85A-specific immune responses.
186
pAg85A IM vaccination
1,2
1
Mice 1
Mice 2
DO
0,8
Mice 3
Mice 4
0,6
Mice 5
Mice 6
0,4
NC
NC
0,2
0
100
200
400
800
1600
3200
6400
12800 25600
dilution steps
Figure 7: Detection of anti-Ag85A IgG antibodies in sera of pAg85A-vaccinated mice
(n=6 , group I); NC, negative control.
3.4 pAg85A transfection in HEK cells
In order to investigate the transfection capacity of our chitosan derivatives, we aimed at
transfecting human embryonic kidney 293T (HEK293T) cells with pAg85A by applying
both chitosan derivative polymers, respectively. For comparison purpose, we also
involved the commercially available transfection agent TransITTM. In figure 8 is shown
that all negative controls (lanes 2 and 3) gave no protein signal. Lipofectamine
TransITTM mediated pAg85A transfection resulted in a protein (lane 4) with a molecular
weight of around 32 kDa, similar to the recombinant protein Ag85A as positive control
(lane 1).
187
1
2
3
4
5
6
7
50 kDa
37 kDa
25 kDa
Figure 8: Western blot detection of equal amounts of HEK cell lysates under reducing
conditions: lane 1, recombinant protein Ag85A; lane 2, pAg85A alone; lane 3, control
plasmid delivered by lipofectamine TransITTM; lane 4, pAg85A deliverd by TransITTM;
lane 5, TMC20-60 pAg85A NP; lane 6: CM20-TMC20-60-g3.5%-PEG-Pam3Cys pAg85A NP;
lane 7: cell culture medium.
With regard to polymeric chitosan effectuated pAg85A delivery, both delivery systems
gave a weak protein signal at a higher molecular weight of around 40 kDa. An
explanation for that might be N-glycosylation of the protein Ag85A in eukaryotic
HEK293T cells, what would increase in turn the molecular weight. However, the same
phenomenon would occur for the pAg85A transfection using TransITTM lipofectamine
and that was not observed (lane 4). In addition, we investigated the GFP transfection in
alveolar A549 cells and found a relatively low transfection efficiency mediated by both
polymer systems (TMC20-60: 6.3 ± 2.8 and CM20-TMC20-60-g3.5%-PEG-Pam3Cys: 6.4 ±
3.6) when fluoresence microscopy was used for an approximative quantification.
188
4. Discussion of results
In order to elicit protective immunity against Mtb infection, Th1 associated cytokines
(such as IL-2, IFN-γ or IL-12) are considered to play an important role. In that way, it is
worth mentioning that IM Ag85A vaccinated IFN-γ knockout mice were able to produce
significant amounts of Ag85A-specific antibodies and IL-2, but died quickly upon
infection challenge with Mtb (O’Souza et al., 2000). It was consequently stated that the
Th1 cytokine IFN-γ mediates the protective function of the pAg85A vaccine and IFN-γ´s
critical role in controlling MtB infections is generally accepted (Giri, 2008). CD4+ and
CD8+ T lymphocytes are the producers of IFN-γ, which in turn is strongly involved in the
activation of macrophages followed by stimulation of microbicidal mechanisms. In
addition to IFN-γ, induction of IL-4, a balanced Th1 versus Th2 response, local IgA
antibodies, suppression of Treg cells and stimulation of a Th17 response might be
helpful for achieving immunity against Mtb infection (Giri, 2008).
Within the scope of our study, we analysed for IFN-γ and IL-2 secretions of leukocytes
from spleen (IM vaccination) or spleen, lungs and lymph nodes (IN vaccination) in
response to Mtb-related antigens. Exposure of leukocytes to the PWM antigen (positive
control) showed comparable proliferative capacities (no intrinsic defect in cytokine
production) within each cytokine assay for all formulations studied (IM and IN
vaccinations). Exceptions were found for IN-splenocyte IFN-γ and IM-splenocyte IL-2
bioassays. Noteworthy, PPD and 85A antigen induced cytokine levels were in all cases
below PWM triggered responses (positive control). Most measured IL-2 and IFN-γ
concentrations were lower than the limit of detection or not significant in comparison to
non-stimulated cells (one-way ANOVA).
Moreover, leukocytes from the cervical lymph nodes produced Ag85A-specific IL-2
cytokines when exposed to unloaded TMC20-60 TPP NP (containing no pAg85A, control
189
group IV), which is questioning the informative value of this particular experiment.
After all these exclusions, reliable and significant results were obtained for IM
vaccinations only for pAg85A (formulation group I) immunizations in view of IL-2
(spleen) and IFN-γ (spleen) secretions. With regard to IN immunizations, significant
IFN-γ production was seen with the ranking of pAg85A (group I) > TMC20-60 pAg85A
NP. In addition, Ag85A-specific antibodies were only detected after IM pAg85A
vaccination.
In a nutshell, pAg85A chitosan nanoparticles, with or without TLR-2 agonist
functionalization did not demonstrate a higher immunogenicity and benefit over IM
pAg85A vaccination. A reason for this is probably the low transfection efficiency of
chitosan DNA nanoparticles in vivo as indicated by Western blot experiment and GFP
transfection studies in vitro. In terms of further vaccination studies, optimizations
enabling a clear benefit of chitosan-based DNA vaccine delivery have to be
demonstrated. Valuable concepts might be:
i) the absorption of DNA vaccine onto unloaded TPP chitosan nanoparticles;
ii) optimization of DNA transfection efficiency by modifying molecular weight and
degree of acetylation;
iii) modification of chitosan nanoparticles with targeting moieties that enhance DNA
transfection in vivo;
iv) the inclusion of highly DNA transfective polymers into chitosan nanoparticles.
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194
195
Chapter 8
Discussion
and future perspectives
196
Pulmonary vaccination
Most respiratory pathogens invade the human body through mucosal epithelia and
cause a high rate of deaths per annum (Woodland et al., 2009). Fighting against lung
infections, pulmonary delivery of vaccines represents an attractive alternative by
mimicking the natural way of infection and therefore eliciting a local immunity.
This immunity constitutes a first line of defense and prevent further entrance of
pathogens. In animal models, aerosol delivery involves intratracheal instillation and
insufflation or the use of exposure chambers, whereas for clinical trials a delivery device
is required. Microsized particles (1-5 μm in size) are generated by dry-powder inhalers
and aerosols from liquid suspended particles by nebulizers before being delivered into
the respiratory tract (Lu and Hickey, 2009). In history, aerosol vaccination was applied
in human subjects for more than a century and includes aerosol vaccines against
anthrax, plaque, tularemia, smallpox, tetanus and botulism (Giudice et al., 2006). When
compared to common parenteral immunization, the following benefits of aerosolized
vaccines are being discussed: i) delivery of vaccines into the respiratory tract can trigger
the secretion of local IgA antibodies being capable of crossing mucosal epithelia and
preventing further entrance of pathogens; ii) the particular non-invasive nature of
antigen delivery into the lungs circumvents the common use of needles and by these
means the main reason for unsafe injections. According to WHO reports (World Global
Burden of Disease Study, 2002), these unsafe injections cause 21,000,000 cases of
hepatitis B, 2,000,000 cases of hepatitis C and 260,000 cases of HIV/AIDS around the
world; iii) in contrast to conventional vaccines, the application of pulmonary dry powder
vaccines could stop the common imperative of an intact cold chain for storage; iv) for
the administration of vaccines via inhalers, no specially trained medical personnel
would be required.
197
In addition, a recent systematic review about measles immunization in children (10
months and older) reported that aerosol measles vaccination was more immunogenic in
comparison to subcutaneous vaccination (Low et al., 2008). A current phase II/III
clinical trial with 2000 healthy infants (9 to 12 months old) initiated by the WHO will
further investigate the safety and efficacy of aerosol measles vaccination (Simon et al.,
2010).
Aerosol delivery of plasmid DNA vaccines
In order to embrace presented advantages of pulmonary immunization with DNA
vaccines (see part I, general), pulmonary plasmid DNA vaccination was considered to be
a promising concept of vaccination and might be applied in the future against
intracellular pulmonary pathogens, such as Mycobacterium tuberculosis, respiratory
syncytical virus (RSV) and severe acute respiratory syndrome coronavirus (SARS)
(Bivas-Benita et al., 2005). Until now, most in vivo studies have focused primarily on
gene therapy in the lung (Aneja et al., 2009) and raised expectations that the most
efficient and safest pulmonary gene delivery systems will find their application also for
the transport of DNA vaccines. In addition to common barriers in gene therapy (see part
I, general), the delivery of plasmid DNA into the lung has to overcome further
extracellular obstacles, e.g., withstanding shear forces during aerosolization and
crossing the respiratory mucus layer, which is covering conducting airways or the liquid
layer in the alveoli (Sanders et al., 2009). So far, pulmonary delivery of plasmid DNA
vaccines was only reported for very few antigens (table 1).
Until now, the only study of pulmonary DNA vaccination demonstrating protective
immunity was reported by Orson et al. (2006). An influenza antigen hemagglutinin (HA;
from viral strain A/PR8/34) expressing DNA (pHA) was incorporated into PEI particles
198
and aerosolized into mice. When compared to intravenous delivery of the same HA
plasmid in macroaggregated albumin (MAA)-PEI particles, inferior virus neutralization
antibodies were found two weeks post immunization. However, once adjuvant plasmids
encoding the cytokines IL-12 and granulocyte-macrophage colony stimulating factor
(GM-CSF) were co-aerosolized in the same PEI formulation, a significant increase in
neutralizing antibody titers was detected together with protection against subsequent
influenza challenge (Orson et al., 2006).
Table 1: Overview of pulmonary DNA vaccination studies (Mycobacterium tuberculosis =
MTb; poly(D,L-lactide-co-gylcolide = PLGA; polyethylenimine = PEI; NP = nanoparticles;
RSV = respiratory syncytical virus).
Encoded protein
Delivery
(pathogen)
route
Delivery system
References
Hepatitis B surface
Intratracheal
None
Lombry et al.,
(hepatitis)
(instillation)
Eight epitopes
Intratracheal
(MTb)
(microsprayer)
Hemagglutinin
Aerosol
(influenza)
(nebulizer)
Rv1733c
Intratracheal
(MTb)
(microsprayer)
2004
Chitosan NP
Bivas-Benita et al,
2005
PEI NP
Orson et al.,
2006
PGLA-PEI NP
Bivas-Benita et al.,
2009
This result also reflects previous findings (O`Hagan et al., 2006; Schlosser et al., 2008;
Heit et al., 2008), suggesting that the adjuvant has to be co-delivered within the same
199
particulate vector as the antigen in order to maximize a homogenous stimulation of
antigen-presenting cells (APC) in vivo.
Functionalization of chitosan DNA nanoparticles with TLR agonists for pulmonary
vaccination
The present strategy of finding a safe and potent APC stimulating adjuvant correlates
with an understanding of the human innate immune system. Hereby, so-called Toll-like
receptor (TLR) agonists have been lately considered as very auspicious due to their
ability to elicit a potent innate immune response which, in turn, affects strongly the
initiation of adaptive immunity (Iwasaki and Medzhitov, 2004). TLRs are a family of at
least ten receptors able to recognize and discriminate highly conserved microbial
structural motives of bacteria, viruses, fungi and protozoae. Their activation results in an
immune response accompanied by increased levels of pro-inflammatory and immunerelated cytokines. Successively, maturation of dendritic cells and migration to regional
lymph nodes followed by a facilitated presentation of antigens to T lymphocytes was
described (Iwasaki and Medzhitov 2004).
In order to ensure the ideal combination of adjuvant (e.g., Toll-like receptor agonist) and
antigen or antigen producing unit (plasmid DNA vaccine) in the same vaccine
formulation, we investigated the new strategy of coupling the adjuvant to the delivery
system followed by particle preparation incorporating the plasmid DNA. As delivery
system, we selected the polysaccharide chitosan (molecular weight ≥ 100,000 Da),
which demonstrated promise for pulmonary DNA vaccination (Bivas-Benita et al, 2005).
Starting from chitosan, we developed a new water-soluble polymeric chitosan
derivative, 6-O-carboxymethyl-N,N,N-trimethyl chitosan (CM-TMC) possessing a distinct
carboxylic moiety for further coupling/grafting of amine-based targeting ligands.
200
At first, we opted for an adjuvant with the Pam3Cys moiety (TLR-2 agonist), which
demonstrated promise in several preclinical studies (see chapter 4). We established a
new chemical synthesis for the grafting of Pam3Cys through a PEG spacer to CM-TMC
(chapter 3). The successful grafting in CM-TMC-g-PEG-Pam3Cys was confirmed by using
spectroscopic (1H and
13C
NMR, mass, UV/VIS and FTIR) and chromatographic (SEC-
MALLS) methods. Successively, Pam3Cys functionalized nanoparticles (NP) were
prepared by complex coacervation with plasmid DNA (pDNA) expressing green
fluorescence protein (GFP). pDNA NP were of around 400 nm in size, and displayed a
positive zeta potential of 27.9 ± 1.6 mV. Furthermore, NP had the ability to protect
against DNAse I enzymatic degradation and to transfect A549 cells. Regarding
immunogenicity, in vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated
human macrophage-like THP-1 (mTHP-1) cells demonstrated that functionalization of
pDNA NP induced IL-8 secretion by mTHP-1 macrophages, which was increased by 10fold compared to non-functionalized carriers.
In the course of our studies, we subjected a further potential adjuvant to examination of
adjuvant properties in a chitosan based vaccine delivery system: 9-benzyl-8hydroxyadenine (8HA), a TLR-7 agonistic moiety (see chapter 5). In analogy to our
precedent study, we grafted 8HA by using a PEG spacer to carboxy functionalized CMTMC. The DNA carrier system CM-TMC-g-PEG-8HA was shown to exhibit comparable
characteristics to CM-TMC-g-PEG-Pam3Cys in terms of size, charge, DNase I protection
and transfection of A549 cells. With regard to immune stimulation, the CM-TMC-g-PEG8HA co-polymer and successive pDNA NP formulation were shown to elicit superior IL-8
and IL-12 related immune responses using mTHP-1 macrophages, when compared to
their non-functionalized counterparts. As a summary of part II of this thesis, we have
clearly demonstrated that functionalization of a vaccine delivery device (polymeric
201
chitosan) with TLR agonistic moieties is a chemically feasible procedure. In addition,
pDNA NP based on these co-polymers have the ability to transfect A549 cells and to
elicit a superior immune response in vitro.
In part III of this thesis, we aimed at first to investigate in a more systematic manner the
uptake of NP uptake upon deposition in the respiratory tract (chapter 6). Hereby, we
applied a triple cell culture model being composed of bronchial epithelial cells,
monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC).
First, we focused on elucidating whether TLR-2 functionalized NP can reach MDDC being
located beneath the epithelium. In our experiment, pDNA loaded NP were administered
via microsprayer onto the in vitro model and cellular uptake of NP was visualized after
24h by staining of cell inserts followed by confocal laser scanning microscopy (CLSM).
Interestingly, we noticed pDNA NP transfer into MDDCs independent of modification
with the TLR-2 agonist Pam3Cys. On the other side, the functionalization of pDNA NP
with the TLR-2 agonist Pam3Cys correlated with an enhanced induction of two proinflammatory cytokines, IL-8 and TNF-α, respectively. Hence, we can conclude that
chitosan-based pDNA delivery systems had the ability to reach MDDC and that the
inclusion of the Pam3Cys moiety enabled a higher immunogenicity being advantageous
for pulmonary immunization.
In order to study the potential of the CM-TMC-g-PEG-Pam3Cys co-polymer for pulmonary
pDNA vaccination, we involved a pDNA vaccine encoding for the mycolyl-transferase
protein Ag85A (abbreviated pAg85A) of Mycobacterium Tuberculosis (MTb). The
plasmid pAg85A vaccine is among the most studied pDNA vaccines against MTb and
showed promise due to its strong Th1 response in vivo (see chapter 7).
In our study, Pam3Cys functionalized pAg85A NP were applied intranasally (IN) and
intramuscularly (IM) into C57BL/6 mice. For IM immunizations, significant immune
202
responses were only obtained for IM vaccinations using pAg85A without chitosan
formulation when IL-2 (spleen) and IFN-γ (spleen) secretions were analysed.
With regard to IN immunizations, significant IFN-γ productions were seen with the
ranking of pAg85A alone > TMC pAg85A NP. Ag85A-specific antibodies were only
remarked by IM pAg85A vaccination. In a nutshell, pAg85A chitosan NP, with or without
TLR-2 agonist functionalization, did not demonstrate superior immunogenicity over IM
pAg85A vaccination. A reason for that is probable the relatively low transfection
efficiency of chitosan DNA nanoparticles in vivo being indicated by Western Blot
experiment and GFP transfection studies in A549 cells.
In order to approach this problematic issue and improve DNA delivery, several factors
affecting chitosan’s gene delivery efficiency were described. At first, in chapter 5 was
revealed that the molecular weight influences DNA transfection in vitro. Mao et al.
(2010) presented further decisive parameters affecting the DNA transfection in vitro and
in vivo, such as chitosan’s degree of deacetylation, N/P ratio between positively charged
polymer and negatively charged pDNA and pH of culture medium.
Within the scope of further DNA vaccination studies, the following concepts might be
helpful for the optimization of chitosan-based DNA delivery: i) the absorption of pDNA
vaccine onto unloaded TPP-crosslinked chitosan nanoparticles, ii) optimization of DNA
transfection efficiency by varying the molecular weight and degree of acetylation, iii)
modification of chitosan nanoparticles with targeting moieties, that enhance DNA
transfection in vivo, iv) the inclusion of highly DNA transfective polymers into chitosan
nanoparticles, v) the use of chitosan or chitosan derivatives as coating material for other
polymers (e.g., PLGA) in order to provide a versatile particle technology for DNA
delivery.
203
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Expert Opin. Drug Deliv. (2009) 567-583.
Bivas-Benita, M., van Meijgaarden, K.E., Franken, K.L., Junginger, H.E., Borchard, G.,
Ottenhoff, T.H. and Geluk, A. Pulmonary delivery of chitosan-DNA nanoparticles
enhances the immunogenicity of a DNA vaccine encoding HLA-A*0201-restricted
T-cell epitopes of Mycobacterium tuberculosis, Vaccine. (2004) 22, 1609-1615.
Bivas-Benita, M., Ottenhoff, T.H., Junginger, H.E. and Borchard, G. Pulmonary DNA
vaccination: concepts, possibilities and perspectives, J. Control. Rel. (2005) 107,
1-29.
Bivas-Benita, M., Lin, M.Y., Bal, S.M., van Meijgaarden, K.E., Franken, K.L., Friggen, A.H.,
Junginger, H.E., Borchard, G., Klein, M.R. and Ottenhoff, T.H. Pulmonary delivery of
NA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to
PLGA-PEI nanoparticles enhances T cell responses in a DNA prime/protein boost
vaccination regimen in mice, Vaccine (2009) 27, 4010-4017.
Giudice, E.L. and Campbell, J.D. Needle-free vaccine delivery, Adv. Drug Deliv. Rev. 58
(2006) 68-89.
O´Hagan, D.T., Singh, M. and Ulmer, J.B. Microparticle-based technology for vaccines.
Methods (2006) 40, 10-19.
Heit, A., Busch, D.H., Wagner, H. and Schmitz, F. Vaccine protocols for enhanced
immunogenicity of exogenous antigens. Int. J. Med. Microbiol. (2008) 298, 27–32.
Iwasaki, A. and Medzhitov, R. Toll-like receptor control of the adaptive immune
responses. Nat. Immunol. (2005) 5, 987-995.
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Lombry, C., Marteleur, A., Arras, M., Lison, D., Louahed, J., Renauld, J.C., Préatand, V. and
Vanbever, R. Local and systemic immune responses to intratracheal instillation of
antigen and DNA vaccines in mice, Pharm. Res. (2004) 21, 127-135.
Low, N., Kraemer, S., Schneider, M. and Restrepo, A.M. Immunogenicity and safety of
aerosolized measles vaccine: systematic review and meta-analysis. Vaccine
(2008) 26, 383-398.
Lu, D. and Hickey, A.J. Pulmonary vaccine delivery, Expert Rev. Vaccines 6 (2007), 213226.
Mao, S., Sun, W. and Kissel, T. Chitosan-based formulations for delivery of DNA and
siRNA. Adv. Drug Deliv. Rev. (2010) 62, 12-27.
Miller, M.A. and Pisani, E. The cost of unsafe injections, Bull. World Health Organ. 77
(1999) 808-811.
Orson, F.M., Kinsey, B.M., Densmore, C.L., Nguyen, T., Wu, Y., Mbawuike, I.N. and Wyde,
P.R. Protection against influenza infection by cytokine-enhanced aerosol genetic
immunization, J. Gene Med. (2006) 8, 488-497.
Sanders, N., Rudolph, C., Braeckmans, K., De Smedt, S.C. and Demeester, J. Extracellular
barriers in respiratory gene therapy, Adv. Drug Deliv. Rev. (2009) 61, 115-27.
Schlosser, E., Mueller, M., Fischer, S., Basta, S., Busch, D.H., Gander, B., Groettrup, M. TLR
ligands and antigen need to be coencapsulated into the same biodegradable
microsphere for the generation of potent cytotoxic T lymphocyte responses.
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needle-free injection devices. New Generation Vaccines (2010) 4th edition, 405414.
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Woodland, D.L and Randall, T.D. Anatomical features of anti-viral immunity in the
respiratory tract, Semin. Immunol. 16 (2004), 163-170.
206
Chapter 9
Summary of thesis
207
The investigations described in this thesis aimed at the evaluation of novel adjuvant
functionalized DNA carrier systems for pulmonary vaccination.
Plasmid DNA vaccination confers the induction of a potent cytotoxic T lymphocyte
response being required for the prevention of intracellular pathogens (e.g.,
Mycobacterium tuberculosis). Despite promising results of plasmid DNA immunizations
in pre-clinical trials, studies in non-human primates and humans have failed so far in
achieving protective immunity. The main reason for this drawback was related to the
low transfection efficacy of plasmid DNA vaccines. Therefore amelioration strategies (in
dependence of the targeted infectious diseases) were undertaken, ranging from plasmid
DNA optimization, formulation into particles, inclusion of adjuvants to changing the
route of administration, to e.g., aerosol vaccination.
Aerosol delivery of vaccines is considered a promising route of immunization. The
unique architecture of the human lung enables the induction of both, local and systemic
immunity against pulmonary invading pathogens due to the presence of respiratory APC
(macrophages and dendritic cells) and the bronchus-associated lymphoid tissue (BALT).
A current clinical phase II/III trial involving an aerosol device for the administration of a
measles vaccine into 2000 human objects underlines the potential of vaccination to the
lung.
Moreover, the common strategy to improve the effectiveness of vaccines is to formulate
the antigen or antigen producing plasmid into particles together with the inclusion of a
potent stimulator of the innate immune system (adjuvant) leading to an increased
immunogenicity and adaptive immunity against subsequent infection challenge.
We addressed the need of such a modified DNA delivery system and synthesized novel
stimulators of innate immune receptors (Toll-like receport (TLR) agonists).
208
Successively, we used these adjuvants for the functionalization of DNA nanoparticles
based on a chitosan polymer.
In part II of this thesis, we synthesized at first different water-soluble TLR agonists: NH2PEG-Pam3Cys targeting the TLR-2 (chapter 3) and NH2-PEG-8HA targeting the TLR-7
(chapter 5). TLR agonisitic moieties we then grafted onto a novel water-soluble chitosan
derivative, CM-TMC, respectively. The successful grafting in CM-TMC-g-PEG-Pam3Cys and
CM-TMC-g-PEG-8HA was confirmed by using spectroscopic (NMR, mass, UV/VIS and
FTIR) and chromatographic (SEC-MALLS) methods. Separately, these co-polymers were
used for the preparation of DNA NP by complex coacervation. Both DNA carrier systems
demonstrated the following physico-chemical and biological properties:
i) particle size was around 400 nm; ii) positive surface charge; iii) ability to protect the
DNA against DNAse I enzymatic degradation; iv) relative low cytotoxicity at
concentrations employed for biological assays and v) transfection capability (5-10%) in
A549 cells. Regarding immunogenicity of the CM-TMC-g-PEG-Pam3Cys DNA carrier,
further in vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated human
macrophage-like THP-1 (mTHP-1) cells demonstrated that functionalization of DNA NP
induced IL-8 secretion by mTHP-1 macrophages, which was increased by ten-fold
compared to non-functionalized carriers (chapter 4).
In addition, the inclusion of the TLR-7 agonistic moiety in CM-TMC-g-PEG-8HA DNA NP
demonstrated gain in immune stimulation owing to superior IL-8 (two-fold) and IL-12
(two-fold) cytokine secretions from mTHP-1 macrophages, when compared to their
unfunctionalized counterparts (chapter 5).
In part III of this thesis, we investigated in a more systematic manner the uptake of CMTMC-g-PEG-Pam3Cys pDNA NP uptake by using a triple cell culture model of the human
respiratory tract (chapter 6). Interestingly, we were able to detect internalization of
209
chitosan-based pDNA NP into monocyte-derived dendritic cells (MDDCs) irrespective of
the functionalization with the TLR-2 agonist Pam3Cys. On the other side, the
functionalization of pDNA NP with the TLR-2 agonist Pam3Cys correlated with an
increased induction of two pro-inflammatory cytokines, IL-8 and TNF-α, respectively.
This study allows the conclusion that chitosan-based pDNA carrier systems had the
ability to reach MDDC and that the inclusion of the Pam3Cys moiety elicited a superior
immunogenicity being advantageous for immunization.
In addition, we examined the potential of the CM-TMC-g-PEG-Pam3Cys co-polymer for
intranasal/pulmonary DNA vaccination in vivo (chapter 7). Hereby, we involved a pDNA
vaccine (pAg85A) encoding for the mycolyl-transferase protein Ag85A of Mycobacterium
Tuberculosis (MTb). We formulated CM-TMC-g-PEG-Pam3Cys pAg85A NP and applied
them intranasally (IN) and intramuscularly (IM) into C57BL/6 mice. In view of cytokine
secretions (IL-2, IFN-γ) from immune organs (spleen, lymph nodes) as well as titer of
specific antibodies, we did not observe a superior immunogenicity of i) IN vaccinations
and ii) TLR-2 agonist functionalization of pAg85A NP, when compared to IM pAg85A
vaccination. An explanation might be the relative low transfection efficiency of chitosan
DNA nanoparticles in vivo. Related to that, several strategies were proposed in order to
solve this problem and to enhance the DNA transfection efficiency (chapter 8).
210
Chapter 10
Résumé de these
211
Ce travail de thèse vise à l´évaluation de nouvelles nanoparticules (NP) vectorisant un
antigène sous forme d’acide nucléique, fonctionnalisées par de puissants adjuvants pour
une vaccination par voie respiratoire.
La vaccination par ADN est une nouvelle approche vaccinale pour induire une réponse
immunitaire spécifique contre un agent pathogène. Suite à son administration, un vaccin
à ADN permet la synthèse in vivo de l´antigène, puis sa présentation sous forme de
peptides antigéniques par les molécules du complexe majeur d´histocompatibilité
(CMH) de classe I. Par conséquent, les lymphocytes T CD8+ sont stimulés, et déclenchent
une réponse cytotoxique dirigée contre l´antigène .
Malgré des résultats pré-cliniques prometteurs de vaccination par ADN, les essais
réalisés chez le primate ou chez l’Homme n´ont pas permis l’induction d’une réponse
immunitaire protectrice. A cause de cela, plusieurs stratégies ont été proposées, comme
le changement de la voie d’administration, la complexation de l’ADN avec un polymère
sous forme (nano)particulaire ou l´ajout d´un adjuvant.
La vaccination par la voie respiratoire est considérée comme prometteuse en vertu de
plusieurs études portant sur des vaccins contre la rougeole, au niveau clinique.
Actuellement, un vaccin contre la rougeole est en cours d´essai clinique (phase II/III)
initié par l´OMS, qui va examiner l´efficacité et la sécurité de cette voie vaccinale.
De plus, la stratégie prédominante pour améliorer l´efficacité des vaccins consiste en la
formulation de l´antigène et de l´adjuvant dans le même vecteur de vaccination.
Dans le cadre de nos études, deux adjuvants ont été choisis et synthétisés : un agoniste
de Toll-like receptor-2 NH2-PEG-Pam3Cys (chapitre 3) et un agoniste de Toll-like
receptor-7 NH2-PEG-8HA (chapitre 5). Nous avons ensuite greffé séparément ces deux
molécules TLR agonistes sur un nouveau dérivé chimique de polymère chitosan (CMTMC).
212
La réussite de la greffe a été vérifiée avec des méthodes spectroscopiques (RMN, masse,
UV/VIS, IR) et chromatogéniques (SEC-MALLS).
Ce nouveau co-polymère est capable de former des NP par complexation avec l´ADN en
donnant les propriétés suivantes : i) une taille de 400 nm environ ; ii) une charge de
surface positive ; iii) la capacité de protéger l´ADN contre les dégradations enzymatiques
par la DNAse I ; iv) une cytotoxicité relativement faible au vu des concentrations
utilisées pour les essais biologiques et v) la capacité de transfection (5-10%) dans des
cellules A549. Concernant l´immunogenicité de CM-TMC-g-PEG-Pam3Cys ADN NP, une
étude in vitro utilisant des cellules THP-1, lignée de macrophages humains, a montré que
la fonctionnalisation de l´ADN sous forme particulaire avec l´adjuvant Pam3Cys (TLR-2
agoniste) induisait une sécrétion d´IL-8 dix fois plus importante que celle induite par
l´ADN vecteur non-modifié (chapitre 4). De la même façon, la fonctionnalisation de
l´ADN NP avec le TLR-7 agoniste (8HA) a montré une immunogénicité supérieure en
analysant la sécrétion de deux cytokines (IL-8, IL-12) par les cellules THP-1 (chapitre 5).
Dans la deuxième partie expérimentale (partie III) de cette thèse, nous avons étudié
l´internalisation de CM-TMC-g-PEG-Pam3Cys ADN NP dans un modèle in vitro de
muqueuse bronchique reconstruite. Ce modèle « à trois étages » est composé de cellules
épithéliales bronchiques, couvrant des cellules dendritiques dérivées de monocytes
(CDDM) et recouvertes par des macrophages dérivés de monocytes (MDM, chapitre 6).
Nous avons détecté l´internalisation de l´ADN NP dans des CDDM indépendamment de
sa fonctionnalisation par le TLR-2 agoniste. En revanche, la fonctionnalisation de NP par
le Pam3Cys est en corrélation avec une immunogenicité supérieure, due à des
concentrations élevées de deux cytokines pro-inflammatoires, IL-8 and TNF-α.
Enfin, nous avons examiné le potentiel adjuvant du co-polymère CM-TMC-g-PEGPam3Cys pour la vaccination à ADN par la voie intranasale/pulmonaire in vivo (chapitre
213
7), en utilisant le vaccin à ADN pAg85A, exprimant l´agent antigène 85A de
Mycobacterium Tuberculosis (MTb). Le plasmide pAg85A a été vectorisé avec CM-TMC-gPEG-Pam3Cys sous forme nanoparticulaire, administré par voie intranasale (IN) et
intramusculaire (IM) chez la souris C57BL/6. En analysant la sécrétion de cytokines (IL2, IFN-γ) par les cellules des organes immunitaires (rates, ganglions) et les titres
d’anticorps spécifiques de l’antigène, aucun avantage immunogénique n’a été observé
concernant i) la vaccination par la voie intranasale par rapport à la voir IM et ii) la
fonctionnalisation ou non de pAg85A NP avec l´agoniste de TLR-2 (Pam3Cys).
Une explication plausible pourrait être la relativement faible capacité des vecteurs
dérivés de chitosan de transfecter des cellules in vivo. Plusieurs stratégies pour aborder
ce problème et pour améliorer l´efficacité de la transfection sont présentées dans le
chapitre 8.
214
List of Abbreviations
AIDS
acquired immunodeficiency syndrome
APC
antigen-presenting cells
BALT
broncho-alveolar lymphoid tissue
CLSM
confocal scanning laser microscopy
CM-TMC
6-0-carboxymethyl-N,N,N-trimethylchitosan
CO2
carbon dioxide
CTL
cytotoxic T lymphocyte
EDC
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
ELISA
enzyme-linked immunosorbent assay
FCS
foetal calf serum
GFP
green fluorescence protein
GM-CSF
granulocyte/macrophage colony-stimulating factor
8HA
2-chloro-9-benzyl-8-hydroxyadenine
HBE
bronchial 16HBe14o- cells
HIV
human immunodeficiency virus
IFN
interferon
IL
interleukin
LE
loading efficiency
LMC
low molecular weight chitosan
LPS
lipopolysaccharide
MDDC
monocyte-derived dendritic cells
MDM
monocyte-derived macrophages
mTHP-1
macrophage-like THP-1 cells
215
NHS
N-hydroxysuccinimide
NH2-PEG-Pam3Cys
amido-[Nα-palmitoyl-oxy-S-[2,3-bis(palmitoyl-oxy)-(2R)-propyl][R]–cysteinyl]-α-amino poly(ethylene glycol)
NP
nanoparticles
mAB
monoclonal antibody
MHC
major histocompatibility complex
MTb
Mycobacterium tuberculosis
PAMP
pathogen-associated molecular pattern
PBS
phosphate buffered saline
pDNA
plasmid deoxyribonucleic acid
PEG
poly(ethylene) glycol
PEI
polyethylene imine
PLGA
poly(D,L-lactide-co-glycolide)
PMA
phorbol 12-myristyl 13-acetate
PN
passage number
PRR
pattern-recognition receptor
SARS
severe acute respiratory syndrom
rAg85A
recombinant protein antigen 85A
TEM
transmission electron microscopy
Th
T helper cell
TLR
toll-like receptor
TNF-α
tumor necrosis factor alpha
WHO
World Health Organization
216
Publications
1. Esmaeili, F., Heuking, S., Junginger, H.E. and Borchard, G. Progress in chitosan-based
vaccine delivery systems, Journal of Drug Delivery and Science Technology (2010) 20,
53-61.
2. Heuking, S., Iannitelli, A., Di Stefano, A. and Borchard, G. Toll-like receptor-2 agonist
functionalized
biopolymer
for
mucosal
vaccination.
International
Journal
of
Pharmaceutics (2009) 381, 97-105.
3. Heuking, S., Adam-Malpel, S., Sublet, E., Iannitelli, A., di Stefano, A. and Borchard, G.
Stimulation of macrophages using Toll-like receptor-2 (TLR-2) agonist decorated
nanocarriers. Journal of Drug Targeting (2009) 17, 662-670.
4. Heuking, S. and Borchard, G. Functionalization with a TLR-7 agonist enhances the
immunogenicity of chitosan DNA nanoparticles in human THP-1 macrophages.
To be submitted
5. Heuking, S. ,Rothen-Rutishauser, B., Gehr, P. and Borchard, G. Fate of TLR-2 agonist
functionalized pDNA nanocarriers upon deposition at the bronchial epithelium in vitro.
To be submitted
Oral Presentations
1. Heuking, S., Adam-Malpel, S., Sublet, E., Iannitelli, A., di Stefano, A. and Borchard, G.
“Targeting of macrophages using Toll-like receptor-2 (TLR-2) agonist decorated
nanocarriers”, June 23, 2009, PhD Day of the School of Pharmacy, Hermance,
Switzerland.
2. Heuking, S., Adam-Malpel, S., Sublet, E., Iannitelli, A., di Stefano, A. and Borchard, G.
“TLR agonists as adjuvants for the delivery of DNA vaccines into the lung”, July 24, 2009,
WIV-Pasteur Institut, Mycobacterial Immunology, Brussels, Belgium.
217
3. Heuking, S., Adam-Malpel, S., Sublet, E., Iannitelli, A., di Stefano, A. and Borchard, G.
“Functionalization of chitosan DNA nanoparticles with Toll-like receptor agonists for
pulmonary vaccination”, November 3-5, 2009, Optimization of Inhaled Tuberculosis
Therapies and Implications for Host-Pathogen Interactions, New Delhi, India.
Awards
Heuking, S., Adam-Malpel, S., Sublet, E., Iannitelli, A., di Stefano, A. and Borchard, G. “In
vitro Evaluation of Toll-like Receptor-2 Agonist Functionalized Nanocarriers”, Swiss
Pharma Science Day, Swiss Society of Pharmaceutical Sciences, September 2, 2009, Bern,
Switzerland. 2nd poster price
218
Curriculum Vitae
Simon Heuking
Rue des Mouettes 9
CH-1227 Les Acacias
Switzerland
Phone (+41) 22 – 310 16 75
Fax: (+41) 22 – 379 65 67
Date of birth: 11/09/1978
Nationality: German
WORK EXPERIENCE
University of Geneva
Geneva, Switzerland
January 2006 –
present
Kreuz-Apotheke (Public Pharmacy)
Dinslaken, Germany
June – December
2005
Fontane-Apotheke (Public Pharmacy)
Berlin, Germany
November 2004 –
April 2005
Pfizer
Department of Pharmaceutical Research and Development,
Sittingbourne, United Kingdom
Project: Scale-down of small-scale coating machines.
May – October
2004
Bayer AG
Department of Pharmaceutical Analytics, Leverkusen, Germany
Project: Release study of different pre-clinical compounds.
August 2001
Klinikumsapotheke (Hospital Pharmacy)
Duisburg, Germany
March 2001
EDUCATION
Study of Pharmacy
University of Paris XI, Châtenay-Malabry, France
(Erasmus Exchange Programme)
February – July
2003
Study of Pharmacy
Philipps – University of Marburg, Germany
April 2000 –
June 2005
Languages
German
English
French
Spanish
Japanese
Swedish
native language
fluent in speaking and writing (B2 level)
fluent in speaking and writing (B2 level)
intermediate knowledge (B1 level)
basic knowledge (JLPT 4 exam)
basic knowledge (A2 level)
219
Acknowledgements
First and above all, my thanks go to Gerrit, who made this entire work possible. I
remember well my very first days in Archamps in January 2006. As your first male PhD
student in Archamps/Geneva, we had to create a lot of kinetic energy in order to set-up a
working laboratory. There is a lot I would like to thank you for. Most important, I could
always rely on your guidance and strong support throughout all these years we worked
together. I learned a lot about science and how to organize myself due to many
discussions and shared work. I was always appreciating your scientific way of thinking
as well as your dedication for science and work. You are a great scientist and supervisor.
Thank you a lot for all I could learn from you. I am especially grateful that you did not
lose your faith in our projects, although primarily it took me quite some time for the
synthesis and characterization of chitosan derivatives. In addition, I am exceedingly
thankful for enabling the visit of international conferences (ITT, CRS, APV, GPEN,
EUCHIS, …) and excellent laboratories (Imperial College, London; Pasteur-Institute,
Brussels; Institute of Anatomy, Bern). These were enriching experiences giving me many
insights about science from different perspectives. Besides work, I am very grateful for
all your invitations to many great get-togethers. At this point, my warm thanks go as
well to Christiane. She made every BBQ, dinner and get together to a remarkably
delicious and exquisite event. I remember the many relaxed moments we laughed
together and shared a beer either at Archamps or at your place.
Life would not have been the same without my dear colleagues in Archamps. First, many
thanks to Claudia for friendship and for your help, when I was confronting you with my
crazy chemical and biological questions. We spent a lot of lab time together and I will be
looking back to these days with a smile. It is an un-spoken secret that the best part of
one´s thesis is done by technicians. My thesis was there no exeption and thus un grand
220
merci beaucoup to Emmanuelle for helping me with cell culture and cell biology. Also
thanks to Sarah for setting up thoroughly our cell culture and cell biology labs. In
addition, it is a further un-spoken secret in universities (probably also in other working
structures) that the secretary is the unique heart of the institution. Merci beaucoup
Valerie for welcoming me and your introduction into the french grammar, bonne vie
pour toi. In Archamps, there was always time for a café outside, hence, motsahkern to
Farnaz and tuck så mycket to Annasara for establishing savoir-vivre in Archamps.
Namaste and dhanyawad to Akash for teaching me some hindi. A big merci to Charlotte
for the edition of french summary; you are most welcome in Archamps. Many
thanks/mille grazie to Antonio I. & Antonio di S. for your incredible help with all the
very challenging chemical part of my PhD thesis. Thanks to all my bachelor and master
students, I learned from you how in order to move our common projects forward.
In Geneva (ScII), merci beaucoup to Olivier for you help with SEC-MALLS. Thanks to all
members of the research groups “Pharmacie Galénique”
and “Technologie
Pharmaceutique” for welcoming me and philosophical discussions, e.g, about “bosón de
higgs y sobre el sentido y la referencia” (¡gracias a Martha y Iván!). There is not enough
space to name you all, however, I will express my gratitude as always with a fine
confection of swiss chocolate. The neighbor lab of pharmaceutical biochemistry of Prof.
Leonardo Scapozza was a second home for me. Grazie mille Leonardo S. for organizing
our excellent PhD school. E´stato un vero piacere incontrarti. Herzlichen Dank/mille
grazie to Remo, Ralitza, Leonardo L. and Patricia for your support for my DNA
preparations and Western blots. Merci vielmal to Yvonne for your friendship; one day I
will say Alegra and visit your Graubünden. Andrea, one day we have to play the “Kanon”
de Johann Pachelbel. Shokran and hamdo illah to Majdeline. In addition, thanks to
Elisabeth Rivara-Minten for your support with NMR spectroscopy.
221
Most interesting parts of PhD thesis consisted of visits of other excellent research labs.
My stay at the Department of Mycobacterial Immunology (Pasteur-Institute, Brussels)
was an eye-opener. My deepest gratitude goes to Kris Huygen. She has a great scientific
mind. Shokran to Dorsaf for showing me how to manipulate mice and to perform
successive immune analysis. I enjoyed the existentialist discussions with Olivier, Marta
and Boussa and especially the guided tour through the mostly undiscovered second
tower of the Pasteur-Institute. The Department of Histology (Instiute of Anatomy, Bern)
was the second formidable event during my thesis. Special thanks go to Peter and
Barbara B., who offered my the possibility to work in their labs. I felt at home and
enjoyed plenty of support from this excellent research group. Herzliches Dankeschön to
Barbara T. (cell culture), Andrea (staining), David R. (ELISA), Michael, David S.
(canadian-swiss insights), Andrea (CLSM), Loretta, Christina (microsprayer), Dagmar,
Mohammed (shokran) and Martin&Kirsten (scottish insights).
From work to private, I am especially thankful for my friendship with Alex, who made
my Genevian settling in much easier. I am looking forward to spend more time with you
and your young wonderful familiy (Aurélie and Joanna). To me, the most important
meaning in life are my families. To my parents in Germany, Werner and Hildegard, my
deepest gratitude comes to you. I am also very fortunate to have two unique siblings,
Lukas and Angela with family (Markus, Benjamin, Johannes and Moritz). Family
moments are the most precious in my life and I am therefore very happy to have now a
second family in Sweden: Eva, Rainer and Inga. Hjärtliga hälsningar och kramar och tack
så mycket for your support and I am looking to move much closer to you. My beloved
Pernilla, words are not enough for saying thank you and fortunately there will be plenty
of other means to do that. I am full of joy for our own family with Emma Sophie.
We have a life together.
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