Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics
Highlights
► Hepatitis B surface antigen-containing chitosan and TMC nanoparticles were prepared. ► At several pHs/tonicities these particles have comparable physical properties. ► The nanoparticles, administered in hypotonic medium, are potent nasal adjuvants. ► At higher tonicities immunogenicity is maintained for chitosan nanoparticles only. ► Nanoparticles, administered i.m., are more immunogenic than alum adsorbed antigen.
Introduction
Most of the currently available vaccines are administered via the intramuscular (i.m.) or subcutaneous (s.c.) route. Although these vaccines induce strong and robust systemic immune responses, due to drawbacks associated with parenteral administration, like pain upon injection and the lack of mucosal immunity, development of non-invasive immunization strategies is intensified [1], [2], [3], [4], [5], [6], [7], [8]. Mucosal immunization routes are of particular interest among these non-invasive routes, as they generally lead to the induction of mucosal immunity [5], [9]. Among the mucosal routes, nasal administration is very attractive, as application of nasal vaccines is relatively simple (compared to pulmonary administration) and the nasal cavity offers a more gentile digestive environment than the gastro-intestinal tract.
Nonetheless, there are several challenges that must be overcome to induce systemic and mucosal immune responses via nasal administration. The residence time of antigens in the nasal cavity is limited due to mucociliary clearance. Moreover, the uptake of antigens through the nasal epithelium is restricted due to their large size and the tolerogenic nature of the mucosal epithelium can complicate a robust immune response. Encapsulating antigens within mucoadhesive polymeric nanoparticles is a popular method to deal with these challenges [10]. The mucoadhesive polymer helps to prolong the residence time of the antigen, the polymeric matrix may protect the antigen against enzymes [11], and the nanosized particles facilitate its uptake by dendritic cells (DCs) and help the antigen to cross the epithelial barrier [12]. It has been shown that specialized antigen-sampling cells (microfold cells) in the nasal associated lymphoid tissue (NALT) can take the particulate antigens up, promoting mucosal and systemic immune responses [13]. Multimerization of antigenic epitopes on the surface of the nanoparticles and co-encapsulation of antigen and adjuvants can also potentiate and modulate the immune responses [14].
Chitosan (CHT) is a known mucoadhesive, safe and biodegradable polymer which has been frequently studied for nasal vaccine delivery [4], [15], [16]. A drawback of CHT, however, is its limited aqueous solubility at neutral or alkaline pH. Therefore, CHT derivatives with improved solubility over a wide pH range have been developed. Among the CHT derivatives synthesized, N,N,N-trimethyl chitosan (TMC) is the most studied polymer for nasal delivery of antigens. Like CHT, TMC is mucoadhesive and biodegradable [12] and has been used successfully, usually in the form of nanoparticles (NPs), for nasal, pulmonary and oral delivery of antigens in mice [9], [17], [18]. As both CHT and TMC NPs can be prepared by simple and scalable methods like polyelectrolyte complexation and ionotropic gelation [19], [20] and possess limited toxicity, these delivery systems have great potential as adjuvants for nasal vaccination.
The hepatitis B vaccine is a major candidate to benefit from nasal administration as it may reduce the number of booster injections necessary to reach protective serum titers. Moreover, nasal administration may provide mucosal immunity in addition to systemic protection, which can prevent transmission of the virus via mucosal surfaces. Therefore, in the present study we investigated whether CHT and TMC NPs with encapsulated hepatitis B surface antigen (HB), prepared by polyelectrolyte complexation, are suitable antigen carrier systems for nasal administration. The impact of the formulation, i.e., buffering system, pH and tonicity, on the physical characteristics of both types of NPs and the resulting HB specific antibody response was studied. We found that, if optimal formulation parameters are used, both CHT and TMC NP greatly enhance systemic as well mucosal HB antibody responses in mice that are superior to i.m. administration of alum adjuvanted HB.
Section snippets
Materials
Hepatitis B surface antigen (protein content determined by BCA assay method: 1.8 mg/ml; formulated in 8 mM sodium phosphate buffer (PB) pH 7.2; particle size: 33.1 ± 1.5 nm; zeta potential: −17.4 ± 1.2 mV) was obtained from Serum Institute of India. TMC with a degree of quaternization of 23.8% was synthesized from 92% deacetylated (MW 120 kDa) CHT (Primex, Avaldsnes, Norway) and characterized by NMR, as described by Bal et al. [14]. All cell culture reagents were bought from Invitrogen (Breda, The
Formulation and characterization of TMC:HB and CHT:HB nanoparticles
Plain HB NPs were dispersed in PB with different pHs (4–10) to determine the optimal pH for obtaining NPs with the smallest mean size, narrowest size distribution (PDI) and the highest surface charge. The smallest particle size and PDI (33.1 ± 1.5 nm, 0.267 ± 0.038) was found at pH 7.2. At this pH the HB NPs showed a zeta potential of −17.4 ± 1.2 mV (data not shown).
TMC:HB and CHT:HB NPs were prepared by adding an equal volume of TMC or CHT solution to a HB dispersion in PB buffer pH 7.2. Different w/w
Discussion
Nanoparticles prepared from CHT and its trimethylated derivative, TMC, have been widely used as an adjuvant for nasal vaccination [15], [17], [25], [30], [31], [32]. Among the various methods used for preparation of these nanoparticles, the most frequently used one is ionic gelation [25], [30], [33], [34]. In this method, the polycationic polymer is incubated with a polyanionic molecule. For negatively charged nanoparticulate antigens like whole inactivated influenza virus, simple incubation
Acknowledgements
The authors acknowledge Mashhad University of Medical Sciences, Mashhad, Iran, for providing a Research Fellowship to MT for carrying out the research work. The authors thank Dr. Christophe Barnier for his kind assistance in cell culture and FACS analysis.
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