Research paper
Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice

https://doi.org/10.1016/j.ejpb.2003.09.006Get rights and content

Abstract

High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50–60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70–200 μg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.

Introduction

Over the last decade, chitosan (CS) has been attracting increasing attention as a biomaterial and as a pharmaceutical excipient for drug delivery because of its favorable biological properties [1]. Besides its low toxicity and susceptibility to biodegradation, CS has shown mucoadhesive properties as well as an important drug penetration enhancement capacity across mucosal barriers [2], [3], [4], [5], [6]. Specific reports on the nasal administration of high molecular weight (Mw) CS solutions have shown that CS enhances the nasal absorption of peptide drugs and also antigens [7], [8], [9], [10].

As a new mucosal delivery vehicle, as an alternative to high Mw CS solutions, we have recently developed CS nanoparticles. These nanoparticles can be made of a wide range of CS Mws using a very mild and friendly ionic gelation technique [11]. Their size, surface characteristics and in vitro release properties can be controlled by adjusting the formulation conditions and by the incorporation of additional polymer, e.g. poloxamers [12]. A variety of drugs, going from low to high Mw, hydrophilic and lipophilic drugs, have been efficiently incorporated into these particles [13]. Furthermore, the ability of these nanoparticles to facilitate the nasal transport of macromolecules has been clearly shown using insulin as a model peptide. More specifically, the plasma glucose levels following nasal administration of insulin-loaded nanoparticles to conscious rabbits were significantly lower than those corresponding to the same dose of CS solutions [14]. These studies led us to accept that the mechanism of action of these CS nanoparticles may be different as compared to that of CS solutions. An additional observation is that the positive effect of CS solutions in promoting drug transport has only been shown for relatively high Mw CS (higher than 70 kDa). A step forward on the evidence of the potential of CS nanoparticles as nasal carriers for macromolecules was very recently shown using tetanus toxoid (TT) as a model antigen. In this study we found that CS nanoparticles (Mw 70 kDa) were able to elicit high and long-lasting IgG immune responses following nasal administration to conscious mice [15]. Consequently, the results suggested that CS nanoparticles were not simply able to facilitate the transport of TT through the nasal epithelium but also to improve its delivery to the immunocompetent cells. However, the mechanism by which this antigen was delivered to the nasal mucosa and the role of the particle's characteristics have not been elucidated until now.

The nasal route holds great promise from the perspective of vaccination due to the particular organization of the nasal mucosa [16]. The nasal mucosa is the first site of contact with inhaled antigens, and the nasal-associated lymphoid tissue (NALT) at the base of the nasal cavity (Waldeyer's ring in humans) is important in the defense of mucosal surfaces. Additionally, the nasal epithelium is leaky and there are underlying blood vessels, cervical lymph nodes and lymphoid cells to which the antigen may have direct access if it can be adequately transported across the epithelium. However, in spite of this attractive configuration, so far the poor transport characteristics of antigens across the nasal barrier have made this modality of vaccination non-viable.

An interesting approach that has been explored in order to improve the transport of antigens across the nasal mucosa has been their encapsulation into polymeric particles [17]. Polymers most frequently used for this application are PLA (poly (lactic acid)) and PLGA (poly (lactic-co-glycolic acid)) [18], [19], [20], [21], [22]. These studies showed that, following nasal administration, antigens encapsulated in PLGA microparticles elicit a higher immune response than fluid vaccines. To explain this positive response it was hypothesized that encapsulated antigens are sampled by specialized cells, similar to the microfold (M) cells which overlie the NALT, and then transported to the underlying antigen presenting cells (APCs). More recent work carried out by our group has revealed that the size and surface composition play a significant role in the ability of these biodegradable particles to target the antigen to the APCs. More specifically, we observed that a hydrophilic PEG coating around PLA nanoparticles has a very positive effect on the immune response of the encapsulated antigen [23]. This was justified by the improved stability of the particles in contact with the mucus layer, followed by a greater transport of the antigen encapsulated in PEG-PLA nanoparticles as compared to that of antigen-loaded PLA nanoparticles [15], [24].

Based on this information, the aim of the present work was to explore further the potential of CS nanoparticles as vehicles for the nasal administration of vaccines. For this purpose, TT was chosen as a model antigen. We first studied the effect of CS dose on the efficacy of CS nanoparticles at eliciting long-lasting immune responses. Then, we investigated the mechanism of interaction of the nanoparticles with the nasal mucosa. Finally, we studied the effect of CS Mw (23, 38 and 70 kDa) on the ability of these nanoparticles to elicit significant immune responses.

Section snippets

Chemicals and animals

CS, in the form of hydrochloride salt (Protasan® 110 Cl, Mn >50 kDa, deacetylation degree: 87%), was purchased from Pronova Biopolymer, S.A. (Norway). Pentasodium tripolyphosphate (TPP) and trehalose were supplied by Sigma (USA). Glucose was provided by Merck (Darmstadt, Germany) and sucrose by Probus S.A. (Barcelona, Spain). Ultrapure water (MilliQ Plus, Millipore Iberica S.A., Spain) was used throughout. Purified TT (Mw 150 kDa, 85–95% monomeric) dissolved in phosphate buffer saline (pH 7.4)

Results and discussion

The main goal of the present work was to investigate the potential interest of low Mw CS nanoparticles as antigen delivery carriers for nasal administration. Using TT as a model antigen, we studied the effect of the CS dose and CS Mw on the efficacy of nanoparticles at eliciting enhanced and long-lasting immune responses. Besides this primary goal, we also expected to obtain information regarding the suitability of the nanoparticles' formation process for the preservation of TT immunogenicity.

Conclusions

In light of these results, low Mw CS nanoparticles could be considered as promising new nasal vaccine delivery systems able to provide high and long-lasting mucosal and humoral immune responses.

Acknowledgements

This work was supported by grants from the Commission of Science and Technology (CICYT-SAF 97-0169, UE-FEDER/DGESIC 1FD97-2363). The authors wish to thank the WHO and the NIBSC for the donation of TT and ELISA reagents. The advice from Professor Teresa Criado, Professor Carlos Ferreirós, Professor Ramón Anadón and Professor Florencio Martı́nez of the University of Santiago de Compostela is greatly appreciated.

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