Elsevier

Vaccine

Volume 30, Issue 36, 3 August 2012, Pages 5341-5348
Vaccine

Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics

https://doi.org/10.1016/j.vaccine.2012.06.035Get rights and content

Abstract

Mucosal immunization offers various advantages over parenteral vaccination, but typically requires potent delivery systems and/or adjuvants to result in protective immunity. Here we report on the preparation of trimethylated chitosan (TMC) and chitosan (CHT) nanoparticles (NPs) loaded with hepatitis B surface antigen (HB), by a simple and scalable method. TMC:HB and CHT:HB NPs were prepared by direct coating of antigen by polymer. The impact of buffer, pH and tonicity of the dispersion medium on NPs’ polydispersity, zeta potential and association percentage of polymer with antigen was evaluated. Moreover, biological properties of both NPs were addressed in vitro by studying their effect on cell viability, transepithelial electrical resistance (TEER) and dendritic cell (DC) maturation. Finally, immunogenicity was assessed by evaluating IgG, IgG1, IgG2a, IgA titers and sIgA after both mucosal (nasal) as well intramuscular (i.m.) vaccination in a murine model. TMC:HB and CHT:HB NPs, prepared in acetate buffer pH 6.7 of three different tonicities, had comparable size, polydispersity, zeta potential and association percentage. TMC:HB NPs, but not CHT:HB NPs, had a mild negative effect on cell viability and TEER, and a considerable positive effect on DC maturation. After nasal and i.m. immunization, TMC:HB NPs in hypotonic medium and CHT:HB NPs in all media induced higher serum and nasal antibody titers compared with HB solution (P < 0.001). After i.m. injection, both TMC:HB and CHT:HB NPs induced higher IgG and IgG2a titers compared with alum adsorbed HB (P < 0.001). For CHT:HB NPs, the tonicity of the dispersion medium did not affect the mucosal and systemic immune responses. In conclusion, TMC NPs and CHT NPs are similarly potent mucosal immunoadjuvants for HB. Moreover, both polymers are potent immunoadjuvants for i.m. administered isotonic HB, resulting in higher IgG2a/IgG1 ratios compared with alum adjuvanted HB.

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.

References (47)

  • W. Boonyo et al.

    Chitosan and trimethyl chitosan chloride (TMC) as adjuvants for inducing immune responses to ovalbumin in mice following nasal administration

    J Control Release

    (2007)
  • A.M. Sadeghi et al.

    Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods

    Int J Pharm

    (2008)
  • M. Amidi et al.

    Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system

    J Control Release

    (2006)
  • N. Hagenaars et al.

    Role of trimethylated chitosan (TMC) in nasal residence time, local distribution and toxicity of an intranasal influenza vaccine

    J Control Release

    (2010)
  • C. Wischke et al.

    Increased sensitivity of chitosan determination by a dye binding method

    Carbohydr Res

    (2006)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

    J Immunol Methods

    (1983)
  • T. Jafari et al.

    Synthesis and cytotoxicity assessment of superparamagnetic iron–gold core–shell nanoparticles coated with polyglycerol

    J Colloid Interface Sci

    (2010)
  • B. Slütter et al.

    Mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination

    J Control Release

    (2009)
  • A. Hafner et al.

    Melatonin-loaded lecithin/chitosan nanoparticles: physicochemical characterisation and permeability through Caco-2 cell monolayers

    Int J Pharm

    (2009)
  • N. Nafee et al.

    Relevance of the colloidal stability of chitosan/PLGA nanoparticles on their cytotoxicity profile

    Int J Pharm

    (2009)
  • M. Amidi et al.

    N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model

    Vaccine

    (2007)
  • N. Hagenaars et al.

    Relationship between structure and adjuvanticity of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine

    J Control Release

    (2009)
  • B. Slütter et al.

    Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen

    Vaccine

    (2010)
  • Cited by (57)

    • Recent advances on chitosan as an adjuvant for vaccine delivery

      2022, International Journal of Biological Macromolecules
    • Nanodelivery system enhances the immunogenicity of dengue-2 nonstructural protein 1, DENV-2 NS1

      2020, Vaccine
      Citation Excerpt :

      Immunization with PA-TMC NPs, protective antigen (PA) from Bacillus anthracis, provokes an antibody response that effectively protects mice from bacterial challenge [45]. Among the systemic IgG response induced by TMC adjuvant, IgG1 subclass is indeed a major proportion, meaning that TMC preferentially enhances the Th-2 response [43,45]. In contrast, an oral vaccination of mice with TMC/Omp31 NPs or intranasal vaccination with r4M2e.HSP70 + TMC in mice result in a dominant IgG2a response [46,47].

    • Nanoparticles for mucosal vaccine delivery

      2020, Nanoengineered Biomaterials for Advanced Drug Delivery
    View all citing articles on Scopus
    View full text