TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

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Abstract

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283 ± 2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.

Introduction

The development of vaccine delivery systems remains a major challenge in the field of immunology. Commercially available vaccines are generally effective, however improvement in efficacy and safety, reduction in the number of administration, and ease of administration are still considered important issues to be solved over coming years (Plotkin, 2005).

The mucosal surface of the human body is the major site of entry for pathogens. Protective mucosal immune responses are most effectively induced by mucosal immunization through oral, nasal, rectal or vaginal routes (Neutra and Kozlowski, 2006). Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, reduced side effects, the possibility of self-administration without need for medical assistance and especially in developing countries, reduced risk of the unwanted spread of infectious agents via contaminated syringes. Furthermore, mucosally administered vaccines have the potential to initiate both local and systemic immune responses (Dietrich et al., 2003).

Although numerous protein antigens have been identified to generate immunity to infectious pathogens by mucosal routes, they have limited success due to delivery issues. Most antigens show no or little immunogenicity and are not sufficiently taken up following mucosal administration, thus they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles to evoke or increase immune responses (Freytag and Clements, 2005). In the mucosal milieu, encapsulation protects labile material from enzymatic and chemical destruction and improves uptake into M-cells. For nasal route, major delivery problems are; the mucociliary clearance of free antigens from the nasal cavity, poor absorption by nasal epithelial cells and generally low intrinsic immunogenicity (Slütter et al., 2008). The loss of encapsulated antigen by mucociliary clearance can be avoided by using bioadhesive polymers.

The successful development of mucosal vaccine delivery systems is mainly based on polymer and the properties it exerts like as mucoadhesion, penetration enhancing effect, ability to form different systems (Alpar et al., 2005). Mucosal vaccines are likely to be most effective when they mimic mucosal pathogens in key respects: they would ideally be multimeric and/or particulate, adhere to mucosal surfaces (or even better, adhere selectively to M cells), efficiently stimulate innate responses, and evoke adaptive immune responses that are appropriate for the target pathogen (Neutra and Kozlowski, 2006).

Chitosan is a cationic polysaccharide derived from chitin, which is a biocompatible, biodegradable and non-toxic material (Şenel and McClure, 2004). Mucoadhesive properties of chitosan make it an ideal candidate for the delivery of antigens to mucosal tissues. Furthermore, it was reported that chitosan could induce macrophage activation, as well as cytotoxic T lymphocytes (Tokura et al., 1999). The immunological adjuvant property of chitosan has been confirmed and extended to induce a wide range of antigens from bacteria, viruses and tumors (Nishimura et al., 1985, McNeela et al., 2001, McNeela et al., 2004). Chitosan derivatives which exert higher solubility compared to that of chitosan base, have been reported to be suitable for antigen delivery as well (van der Lubben et al., 2001, Amidi et al., 2007). Soluble N-trimethyl chitosan (TMC) and mono-N-carboxymethyl chitosan (MCC) have been shown to exert mucoadhesive properties and excellent absorption enhancing effects even at neutral pH (Thanou et al., 2001a, Hamman et al., 2002). TMC is a partially quaternized chitosan derivative and is freely soluble over a wide pH range as compared to other chitosan salts and derivatives. At all degrees of quaternization, TMC bears positive charges, independently of the environmental pH (Kotze et al., 1999). MCC is a polyampholytic polymer, able to form visco-elastic gels in aqueous environments or with anionic macromolecules at neutral pH values. MCC appears to be less potent compared to the quaternized derivative (Thanou et al., 2001b). The reader is referred to two recent papers which extensively review applications of chitosan and its derivatives in immunization (Sayın and Şenel, 2008a, Arca et al., 2009).

During encapsulation of the antigens or proteins, it is important to protect the biological nature of the entrapped material therefore, during preparation harmful organic solvents, heat or high shear forces should be avoided. In our previous study, nanoparticles prepared using TMC as well as MCC were shown to be promising for nasal immunization against tetanus toxoid (TT) (Sayın et al., 2008b). The objective of this study was to develop a novel delivery system by means of TMC and MCC complexation without using any crosslinker, and enhance the immunostimulant effect using two chitosan derivatives in the same formulation. Tetanus toxoid was used as the model antigen. The system was investigated in vivo for nasal immunization in mice model.

Section snippets

Materials

Purified tetanus toxoid (5000 Lf/ml) was kindly provided by Division of Bacteriology, NIBSC, UK. Low molecular weight (150 kDa) chitosan was supplied by Fluka, Norway (Deacetylation degree: >75–90%, mol. wt.: 150 kDa). All other chemicals were reagent grade chemicals.

Synthesis and characterization of TMC and MCC

TMC was synthesised from chitosan via a two-step methylation procedure described previously (Thanou et al., 2001b). After the first methylation step, TMC underwent a second methylation step and yielded TMC with a degree of

Results and discussion

The main goal of the present work was to investigate the potential of the nanoparticles developed by complexation of the oppositely charged chitosan derivatives without using a crosslinker as antigen delivery systems for nasal immunization. For this purpose positively charged TMC and negatively charged MCC was synthesised as previously described (Sayın et al., 2008b). 1H NMR spectra revealed the quaternization degree or methylation of TMC as 57% and carboxymethylation of MCC as 70%.

Conclusions

In this study, it was the first time to develop a TMC/MCC complex nanoparticulate system for mucosal immunization. Nanoparticles were prepared by ionic gelation method based on the complexation between oppositely charged chitosan derivatives (positively charged TMC and negatively charged MCC) without using any organic solvent and or any crosslinker. The loading of TT to TMC/MCC nanoparticles has been proven to be a very mild process resulting in a very high loading efficiency maintaining the

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