Chitosan, a linear amino polysaccharide consisting of β1,4-linked monomers of d
-glucosamine and N
-glucosamine, is obtained by partial deacetylation of chitin, a long-chain polymer of N
-acetylglucosamine. Hence the degree of deacetylation directly affect the physical properties (molecular weight, solubility and biodegradability) and biological properties (cytotoxicity, antigen binding capacity and adjuvant activity) of chitosan [86
]. Because chitosan is insoluble in water at neutral pH, many soluble chitosan derivatives have been produced by substituting amine and/or hydroxyl functional groups of chitosan with hydrophilic groups such as thiol [89
], sulphate [91
] hydroxyalkyl [92
], carboxyalkyl [94
] and succinyl [95
]. Other methods involve the use of poly(ethylene glycol) (PEG) [96
] and Poloxamer [97
] to enhance the solubility of chitosan. Among the derivatives, quaternized N
-alkyl chitosan derivatives behave as cationic polyelectrolytes which are highly soluble in water [98
]. They also have mucoadhesive and penetration-enhancing properties which, however, depend on the degree of substitution or quaternization in chitosan.
Apparently, the mucoadhesive property of chitosan in mucosal route emerges from the electrostatic interaction between the cationic charge of chitosan and negatively charged mucins. Thus, chitosan particles can reside longer in the mucosal surfaces leading to a higher possibility of particles to enter through M cells and/or epithelial cells in the mucosal routes [52
]. Due to these consequences, chitosan derivatives have been developed as antigen delivery carriers which are supposed to assist the transportation of antigens to mucosal immune compartments, prolong the interaction time between antigen and immune cells and initiate the immune reactions from local to distal sites. The other advantage of chitosan carrier systems is the ease of tuning the release of antigens in the mucosal sites because different physicochemical properties such as solubility, surface charge and hydrodynamic size of the chitosan particles can vary the release pattern of antigens from the chitosan particulate systems. Hence, the delivery of antigens in polymeric particulates, either as microparticles or nanoparticles, has been employed as a successful approach for mucosal delivery [99
4.1. Formulations of Chitosan Particles
Chitosan-based particulates for needle-free vaccine delivery can be formulated by ionic interaction between cationic chitosan derivatives and anionic crosslinking substrates such as tripolyphosphate (TPP) or sodium sulfate. The spontaneous self-assembly of oppositely charged particles gives rise to microparticles or nanoparticles depending upon the molecular weights of chitosan derivatives and crosslinkers. Usually, protein-loaded chitosan particulates have been formed by ionotropic gelation of chitosan with TPP. In this process, an aqueous solution of antigens with TPP is stirred with an aqueous solution of chitosan to form nanoparticles at room temperature. The process, therefore, avoids organic solvents and high temperature and hence protects the protein or antigen integrity [101
]. For example, chitosan nanoparticles loaded with TT through ionotropic gelation method was investigated as vaccine delivery vehicles through nasal route [104
]. Similarly, chitosan nanoparticles were developed as a carrier system for nasal delivery of an influenza subunit vaccine by stirring a solution containing influenza A subunit H3N2 with TPP in a solution of N
-trimethyl chitosan (pH 7.4) at room temperature. Intranasal immunization with influenza subunit vaccine-loaded chitosan nanoparticles induced strong IgG and IgA levels [105
]. Alternatively, ionic crosslinking of sodium sulfate with an aqueous solution of chitosan could produce chitosan particulates with porous structures [106
]. As a result, a large number of antigens could be richly loaded by entrapping within the porous particles or protruding on the outer surfaces of chitosan particulates.
Another promising formulation of needle-free vaccine delivery involves the use of antigen conjugates with chitosan derivatives. The chemical crosslinking of trimethyl chitosan with OVA antigens has ameliorated the immunogenicity of the antigen [107
]. Besides, nasal immunization of trimethyl chitosan-ovalbumin conjugates has generated high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG in mice [108
]. However, the aggregation behavior of chitosan particles has hindered their efficient use in vaccine delivery. To minimize the aggregation problems, either chitosan or antigen can be conjugated with PEG that offers improved stability and water solubility to conjugated substances. In a typical example, chitosan was covalently conjugated to PEG, stirred with TTP to prepare stable microspheres and further loaded with Bordetella bronchiseptica
dermonecrotoxin (BBD) antigens to develop an effective nasal vaccine against atrophic rhinitis in animals [97
]. Thus, BBD-loaded pegylated chitosan microspheres, owing to their efficient release of antigens and subsequent stimulation of cytokines from macrophage cells in vitro, has represented as a promising vaccine delivery system for nasal immunization.
Apart from PEG, Poloxamer or Pluronic has been utilized in many vaccine formulations for needle-free immunizations. In an attempt of mucosal delivery of TT antigen, a mixture of chitosan and Pluronic F-127 boosted through nasal route, following the first immunization of TT antigen through intraperitoneal injection, showed a significant improvement in the systemic antigen-specific IgG response and mucosal IgA response in the nasal and lung washes in mice [109
]. The results indicated that Pluronic may act as an adjuvant to induce an additive or synergistic effect on mucosal immunization of vaccines.
In another study, chitosan microspheres, prepared with BBD antigens in the presence of Pluronic F-127, showed a higher amount of antigen release and stimulated a higher level of immune responses in mouse alveolar macrophage cells in vitro [96
]. Further, nasal immunization of BBD-loaded Pluronic F-127/chitosan microspheres induced higher BBD-specific IgA responses in nasal secretions in the mice. Moreover, these immunized mice, when challenged with B. bronchiseptica
through nasal route, survived longer than the control groups, demonstrating the potential of chitosan particulates for nasal delivery of vaccines.
4.2. Mucoadhesive Chitosan Particles
Mucoadhesive chitosan microspheres can be simply produced by coating chitosan microspheres with a mucoadhesive polymer to enhance the stability and efficiency of microspheres for mucosal delivery of vaccines. Accordingly, a mucoadhesive and pH-sensitive polymer, Eudragit, was coated on chitosan microparticles to deliver OVA antigen in mice [110
]. Oral immunization of OVA by Eudragit-coated chitosan microparticles induced high levels of OVA-specific IgA and IgG in fecal and plasma, respectively. Similarly, the coating of thiolated Eudragit on chitosan microspheres containing bovine serum albumin (BSA) antigen not only protected the release of BSA from the microspheres at gastric pH but also increased the mucoadhesive potential of the microspheres in mucosal tissues in vitro and in vivo [111
]. In the same way, another pH-sensitive polymer, hydroxypropyl methylcellulose phthalate (HPMCP), was mixed with trimethyl chitosan in the presence of hepatitis B surface antigen (HBsAg) to form nanoparticles by self-assembly [112
]. Further experiments showed that HPMCP protected the release of HBsAg from chitosan nanoparticles at gastric pH, suggesting that HPMCP-coated chitosan nanoparticles could be harnessed for HBsAg delivery by the oral route. In another study, the efficacy of glycol chitosan as a mucoadhesive coating material to deliver HBsAg through nasal route was investigated [113
]. Owing to higher mucoadhesive potential, glycol chitosan nanoparticles showed low nasal clearance thus increasing the mucosal uptake of antigen which eventually enhanced humoral and mucosal immunity. To demonstrate the effect of mucoadhesive coating on the delivery carriers for mucosal vaccines, glycol chitosan was coated on HBsAg-loaded PLGA nanoparticles and delivered through nasal route [114
]. The results showed that the lower nasal clearance of glycol chitosan coated-PLGA nanoparticles, compared to uncoated PLGA nanoparticles, resulted in higher uptake of antigens through local or systemic circulation, to produce higher mucosal and systemic immune responses.
4.3. Targeted Chitosan Particles
Undoubtedly, chitosan particles are promising carriers for needle-free vaccine delivery. However, the antigen delivery or targeting efficiency of chitosan particles varies with the particular cells or tissues. This issue can be solved by selective targeting of cells through ligand-receptor interaction. Thus, chitosan particles or antigens can be conjugated with various ligands or antibodies which can specifically bind to the specific cells or their receptors. APCs and M cells have been selected as two common targeted cells for vaccine delivery by chitosan particles. Because mannose receptors are surrounded on the surfaces of APCs such as macrophages [115
], mannose as a specific ligand was conjugated with chitosan microspheres and used them for nasal delivery of vaccines [116
]. Accordingly, mannosylated chitosan microspheres (MCMs), loaded with BBD antigens, exhibited high binding affinity with macrophage cells. Nasal immunization of BBD antigen with MCMs showed higher antigen-specific IgA responses in saliva and serum of mice than nasal immunization of BBD antigen with chitosan microspheres only (Figure 6
). The results concluded that the high level of immune responses, although partly contributed by adjuvant property of chitosan, was majorly due to higher uptake of MCMs in macrophage cells through ligand-receptor interaction.
As stated earlier, conjugation of receptor-specific antibodies to antigens is an alternative approach of targeting APCs for selective delivery of antigens [117
]. It is now well-documented that the differences in levels of cellular and humoral immune responses primarily depend upon the type of interactions between antibodies and receptors on APCs [119
]. Therefore, many studies have focused to find novel ligands which can strongly bind to the receptors of APCs to stimulate appropriate immune responses. In an attempt to find an effective ligand for dendritic cells, a novel peptide, TPAFRYS (TP) was isolated by phage display technique. Elegantly, TP-conjugated chitosan nanoparticles had stronger binding specificity towards dendritic cells than myoblasts or macrophages [121
]. Further, subcutaneous immunization of TP-conjugated chitosan nanoparticles with OVA antigen exhibited high production of OVA-specific serum IgG in mice demonstrating the efficacy of targeted delivery of vaccines in APCs.
Because M cells play a key role as the entry point for particles or antigens into lymphoid tissues to induce immune reactions of mucosal vaccination [7
], various M cell targeting ligands have been entertained for mucosal vaccines [122
]. Lectins are one of the natural ligands that have high affinity with sugar moieties on intestinal cells [123
]. For example, Ulex europaeus agglutinin I (UEA1), an α-l
-fucose-specific lectin, owing to its specificity with M cells has been frequently linked with delivery carriers of mucosal vaccines [124
]. Hence, the efficacy of UEA-1 to target murine M cells and subsequent induction of immune reactions was evaluated by comparing the efficiency of antibodies production between oral immunization of UEA-1-coated microparticles with HBsAg and intramuscular immunization of alum with HBsAg [125
]. While both routes of immunization produced similar levels of HBsAg-specific antibodies, oral immunization also elicited sIgA responses in vaginal, intestinal and salivary secretions [125
]. In the further comparative study, BSA-loaded chitosan nanoparticles, coated with UEA-1-alginate, were administered through oral route while BSA-loaded chitosan nanoparticles with alum were immunized in mice through parenteral route [126
]. The results revealed that oral immunization of antigen by UEA1-alginate-coated chitosan nanoparticles elicited superior systemic response plus dominating mucosal response than the antigen delivered by parenteral immunization. These data show the potential of chitosan nanoparticles combined with targeting ligand as an effective delivery system for oral immunization.
Some peptides that have specific binding capacity with M cells have been identified and exploited for M cell targeting vaccine delivery. These peptides are usually discovered by both in vitro and in vivo phage display screening technology. For example, VPPHPMTYSCQY (P25) and LETTCASLCYPS (P8) peptides, identified by in vivo screening method, have shown strong binding on the surfaces of intestinal tissues while another peptide, YQCSYTMPHPPV has demonstrated to assist the efficient delivery of polystyrene particles to M cells in a mouse model [127
]. In another study, CTGKSC and LRVC peptides, identified by in vitro phage display method, have enhanced the transportation of polycaprolactone-PEG nanoparticles into the FAE through M cells in the intestine [128
]. Another peptide, SFHQLPARSPLP (Co1), selected by screening phage display library, was fused with enhanced green fluorescent protein (EGFP) antigen to determine the ability of Co1 to deliver antigen by targeting M cells [129
]. Compared to EGFP alone, the fused EGFP-Co1 showed higher binding affinity to M cells, entered easily in Peyer’s patches and subsequently elevated the production of fecal IgA and serum IgG, signifying Co1 as a promising ligand for M cell-targeted antigen delivery.
Similarly, an M cell-homing peptide, CKSTHPLSC (CKS9), selected by the phage display technique, was chemically conjugated to chitosan nanoparticles (CKS9-CNs) and determined the targeting ability of CKS9-CNs to M cells [130
]. Compared to chitosan nanoparticles, CKS9-CNs were significantly accumulated into Peyer’s patches in small intestines of rat. These results indicate that CKS9, owing to its enhanced targeting and transcytosis ability of particles, could be used for efficient delivery of mucosal vaccines. Accordingly, a vaccine was developed by loading a model antigen, Brachyspira hyodysenteriae
membrane protein B (BmpB) into PLGA microparticles, which were further coated with CKS9-conjugated chitosan and administered in mice through oral route [131
]. Oral immunization of CKS9-chitosan-PLGA microparticles with BmpB vaccine exhibited enhanced levels of IgG in serum and sIgA in both intestinal and fecal secretions (Figure 7
). These data indicated that the use of M cell targeting peptide could improve the effectiveness of oral immunization of particulate vaccines.
4.4. Adjuvant Activity of Chitosan Particles
An immunologic adjuvant is defined as any substance that tends to accelerate, enhance or prolong antigen-specific immune responses of vaccine antigens [132
]. The intrinsic activity of polymers to stimulate various cellular functions of cells and immune cells gives rise to the adjuvant property to polymers [133
]. Numerous studies have evaluated the potential of biodegradable polymers as adjuvants including PLGA and chitosan. Chitosan and its derivatives have been studied in numerous vaccine formulations and shown the adjuvant activity when given in combination with the vaccines [134
]. Chitosan-based intranasal vaccine against hepatitis B was formulated to study the adjuvant effect of the chitosan and its underlying mechanism of action. In vivo and in vitro experiments exhibited increased residence time of HBsAg encapsulated chitosan particles in the nasal cavity due to the interaction of positively charged particles and the negatively charged mucosa of the nasal cavity. In addition, the decrease in transepithelial electrical resistance (TEER) values with a consequently increased transport of HBsAg across the monolayered human colonic carcinoma-derived Caco-2 cells was observed in the presence of chitosan but not in the group without chitosan, indicating that chitosan is capable of opening tight junctions. It further showed that chitosan increased the uptake of antigens by dendritic cells and promoted their maturation. Mice immunized with HBsAg plus chitosan showed the significantly increased level of anti-HBs sIgA, IFN-γ- and IL-2 than the mice immunized with alum-based vaccine and plain HBsAg [135
Another study evaluated the effects of alginate modification on absorption properties of fluorescein isothiocyanate (FITC)-BSA loaded N
-trimethyl chitosan chloride (TMC) nanoparticles. In addition, the feasibility of applying TMC nanoparticles loaded with a model vaccine urease, a vaccine protein against Helicobacter pylori
infection, in oral vaccination was studied. TMC nanoparticles modified with alginate showed higher FITC-BSA permeation efficiency as compared to non-modified TMC nanoparticles. Further, in vivo studies showed that orally administrated urease loaded TMC nanoparticles were able to induce a significantly higher titers of both IgG and IgA as compared with those mice who were immunized with either urease solution or urease co-administered with TMC solution, indicating that TMC nanoparticles are potential carriers for oral protein as well as vaccine delivery [136
Recently, a study reported that intranasal administration of chitosan alone could completely protect BALB/c mice from lethal infection by H7N9 virus, a highly pathogenic virus, by the stimulation of the innate immune system. In vivo experiments showed that mice challenged with lethal dose of H7N9 (10 × LD50
) could be protected even after ten days of the intranasal chitosan administration. It further demonstrated that the infiltration of leukocytes in the bronchoalveolar lavage and proinflammatory cytokines were significantly enhanced in the lungs of mice treated with chitosan as compared with untreated groups, indicating the potent activation of mucosal immune responses by intranasally delivered chitosan [137
Similarly, chitosan has exhibited to induce various cytokines, interleukin (IL)-1 and colony-stimulating factor (CSF) in macrophages in vitro [138
]. Moreover, chitosan derivatives have shown their adjuvant activities such as activation of peritoneal macrophages, suppression of tumor growth and protection of the host against bacterial infection in mice and guinea-pigs [87
]. Similarly, chitosan has significantly elevated the number of T cells, dendritic cells and natural killer cells in herpes simplex virus (HSV) infected mice [139
]. Further, the adjuvant function of chitosan was compared with a standard vaccine adjuvant, cholera toxin (CT), in a study of vaccine delivery in mice infected with Helicobacter pylori
]. Oral immunization of the vaccine with chitosan had higher or comparable humoral immune response, Th1/Th2 cell immune reaction and H. pylori
elimination rate than the same vaccine administered with CT. In another study, mice were challenged with a lethal dose of various influenza viruses following the nasal immunization of a vaccine containing a matrix protein 1 in combination with chitosan to mice [141
]. Nasal immunization with chitosan effectively protected the mice against the challenge of both homologous and heterologous influenza viruses to various extents.
An informative study was designed to gain insight into the immunogenicity of N
-trimethyl chitosan, either in solution or nanoparticle formulation, by delivering diphtheria toxoid (DT) through transcutaneous route in combination with microneedles [142
]. Microneedle-based transcutaneous immunization of DT with or without chitosan solution formulation elicited 8-fold higher IgG titers in the latter than the former. However, neither topically applied DT-loaded chitosan nanoparticles were able to enhance the IgG titers compared to DT alone nor microneedle application could improve the immunogenicity of the DT-loaded chitosan nanoparticles. The consequence was due to the limited transport of the chitosan nanoparticles into the skin using the microneedle conduits compared to chitosan solution. It means the combination of microneedles and adjuvant can assist the transport of antigens into the skin and the activation of the APCs for successful transcutaneous immunization. The study revealed that chitosan in solution offers great potential as an adjuvant for transcutaneous immunization with microneedles but not when formulated in nanoparticles.
To enhance immunogenicity of antigens with low-dose immunization through sustained intradermal delivery, a system comprising OVA-loaded chitosan microneedles with dissolving patches was developed and tested in rats. The immunization results showed that rats administered with chitosan microneedles containing low-dose OVA (200 μg) had significantly higher antibody levels than those administered with intramuscular injection of full-dose OVA (500 μg) [143
]. Moreover, the delivery of OVA with chitosan microneedles induced significantly stronger immune responses compared with the delivery of same dose of OVA and chitosan by intramuscular injection indicating chitosan microneedles as an efficient vaccine delivery system with enhancing adjuvant activity.