Chitosan-based particulate systems for the delivery of mucosal vaccines against infectious diseases

G Model

ARTICLE IN PRESS

BIOMAC-8396; No. of Pages 11

International Journal of Biological Macromolecules xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Chitosan-based particulate systems for the delivery of mucosal vaccines against infectious diseases Bijay Singh a , Sushila Maharjan a , Ki-Hyun Cho b , LianHua Cui c , In-Kyu Park d , Yun-Jaie Choi b , Chong-Su Cho b,∗ a

Research Institute for Bioscience and Biotechnology, Kathmandu 44600, Nepal Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea c Department of Animal Science, College of Agriculture Science, Yanbian U niversity, Yanji, JiLin, 133002, China d Department of Biomedical Sciences, BK21 PLUS Center for Creative Biomedical Scientists at Chonnam National University, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 61469, Korea b

a r t i c l e

i n f o

Article history: Received 30 July 2017 Received in revised form 23 September 2017 Accepted 11 October 2017 Available online xxx Keywords: Chitosan Mucosal vaccine Antigen delivery

a b s t r a c t Given that most pathogens enter the body at mucosal surfaces for infection and mucosal immune responses act as the first line of defense against the invading pathogens, mucosal vaccination is the most effective method to prevent infectious diseases. However, the development of mucosal vaccines requires an efficient antigen delivery system which should protect the antigens from physical elimination and enzymatic degradation, target mucosal inductive sites, and appropriately stimulate the mucosal and systemic immunity. Accordingly, polymeric particles have garnered much attention because the physicochemical properties of polymers can be adjusted to resolve the issues associated with mucosal vaccine delivery. Particularly, chitosan-based polymeric carriers are the most promising vehicles for mucosal vaccine delivery because chitosan is biodegradable, biocompatible and mucoadhesive in nature. Similarly, chitosan can be modified with chemical and biological molecules to develop delivery carriers for controlled or targeted therapy. Moreover, they can be converted to various formulations, such as solid, liquid and gel, with a wide range of particle sizes. In this review, we highlight and discuss advances in the development of chitosan-based particulate systems, specifically for the delivery of mucosal vaccines against infections. © 2017 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mucosal immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mucosal vaccine delivery by particulate systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Chitosan-based particulate systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Preparation of chitosan particulates formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Ionic crosslinking methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.2. Chemical crosslinking methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Spray drying methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Modified chitosan particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Mucoadhesive chitosan particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Targeting delivery using chitosan particulates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8.1. Targeting antigen presenting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8.2. Targeting M-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8.2.1. Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8.2.2. Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. E-mail address: [email protected] (C.-S. Cho). https://doi.org/10.1016/j.ijbiomac.2017.10.101 0141-8130/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

2

9. Adjuvant activity of chitosan particulates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 10. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Mucosal surfaces (nasal, respiratory, oropharyngeal, gastrointestinal and urinogenital) are the most common routes of entry of many pathogens (viruses and bacteria) for infection. It is now increasingly evident that local mucosal immune responses play a pivotal role in protecting against these invaders. Vaccination is the simple way of boosting the immunity to fight off the infections. Unlike parenteral vaccination, mucosal vaccination not only provides humoral and cell-mediated immune protection at mucosal sites but also confers systemic immunity [1]. Besides, mucosal vaccines have garnered much attention due to their ease of administration, high patient compliance and feasibility of mass vaccination. Although several mucosal routes (nasal, pulmonary, oral, vaginal and rectal) are available for vaccine administration, the nasal and oral routes are more effective to induce immunity at distant mucosal sites in addition to the local site of vaccine delivery [2,3]. However, both routes of vaccine delivery have their own pros and cons. While oral route has the largest mucosal surface area and specialized epithelial cells termed microfold cells (M cells) for the higher possibility of antigen uptake, the route is compromised by harsh gastric pH and enzymes for antigen degradation thereby necessitating protective formulations to overcome the barriers of the oral delivery route. On contrary to oral route, antigens delivered through nasal route do not confront with pH and enzyme barriers requiring only a low dose of antigen for delivery. However, vaccine delivery through nasal route is compromised by rapid mucociliary clearance, inefficient antigen uptake, and poor patient acceptability. A number of formulations have been developed to improve the efficacy of mucosal delivery. These formulations, usually prepared from polymers, are in use as delivery systems because they protect the antigens from degradation, increase the residence time of antigens at mucosal surfaces, release the antigens at specific sites and target M cells to enter into immune compartments. Although a vast array of polymers, both natural and synthetic, have been recognized for their potential to deliver mucosal vaccines efficiently, chitosan holds a special position due to its unique properties. Chitosan is a natural polysaccharide and hence it is biodegradable and biocompatible. It is cationic, non-toxic and mucoadhesive in nature. Chitosan can be modified with chemical and/or biological molecules and they can be converted to various formulations, such as solid, liquid and gel, with a wide range of particle sizes. 2. Mucosal immunity Mucosal surfaces represent a major portal of entry for many pathogens and the immunological activity occurred at these surfaces by mucosal immune system plays a key role, as the first line of defense, to protect these surfaces from the external invaders. The mucosal immune system consists of an integrated network of tissues, lymphoid and constitutive cells, and effector molecules (antibodies, cytokines, and chemokines) [4]. These factors respond to pathogens (or mucosal vaccines) through a complex orchestration of cellular processes stimulating innate and adaptive immune responses to confer protection (Fig. 1). Mucosal immune responses are initiated in organized mucosaassociated lymphoid tissues (MALT), namely gut-associated

lymphoid tissue (GALT) or Peyer’s patches in the small intestine and bronchus-associated lymphoid tissues (BALT) in respiratory tracts. The MALT is a highly compartmentalized immunological system that functions independently from the systemic immune system, and it consists of large populations of antigen presenting cells (dendritic cells, T lymphocytes) and plasma cells (B lymphocytes). In the presence of signals, antigen-specific antibodies produced by the plasma cells are secreted to mucosal surfaces as secretory IgA (sIgA) which can bind, neutralize and eliminate the pathogens from the body. As sIgA is the very first line of defense against the invading pathogens, induction of potent IgA responses is a pre-requisite for successful mucosal vaccination. Above all, the critical barrier to successful antigen delivery is to pass pathogens or antigens from mucosal tissues to immune compartments through M cells. The M cells are specialized epithelial cells, specifically located on the follicle-associated epithelium (FAE) in mucosal tissues, that capture and transport foreign particles across the epithelial barrier to underlying lymphoid cells [5]. The transcytosed particles are then passed either to B cells to activate the secretion of sIgA (mucosal response) or to dendritic cells which present antigens to T cells to initiate the production of IgG (humoral response). Hence, considering the key role of M cells in antigen delivery across the epithelial barrier, M cells are the most amenable targets for antigen delivery in the mucosal route to induce mucosal immunity. 3. Mucosal vaccine delivery by particulate systems The use of polymers as carrier systems has flourished in mucosal vaccine delivery as they offer an advantage of delivering antigens to a specific target site. The other benefit of polymeric carrier systems is the way they control the release of antigens, slow or burst, from their grip in the mucosal sites. In the case of oral delivery, the antigens should be protected from harsh gastric pH, bile juices and digestive enzymes in the gastrointestinal (GI) tract. To overcome these barriers in GI tract, the encapsulation of antigens in polymeric particulates such as microparticles and nanoparticles has emerged as a promising approach for mucosal delivery [6,7]. The choice of polymeric particulate systems for antigen delivery arises from their ease of modifications to tune up physicochemical properties such as surface charge, hydrodynamic size, and solubility of the particles. Besides, polymeric particles have the capability to enhance the immune responses to mucosally delivered antigens. In many cases, particulate antigens have greater access to immune compartments in MALT through M cells compared to soluble antigens [6]. Additionally, particulate antigens also find their way through paracellular route to enter to underlying lymphoid cells from FAE in mucosal tissues [8]. Naturally, innate immune system interacts with invading pathogens through a ligand-receptor interaction between pathogen associated molecular patterns (PAMPs) of pathogens [9] and pattern recognition receptors (PRRs) of the cells of immune system [10]. Various cells, including neutrophils, macrophages and dendritic cells express PRRs on their cell surfaces which recognize a pathogen as an extracellular particulate antigen. Upon detection of PAMPs on pathogens, PRRs trigger inflammatory responses that not only destroy the invading pathogens but also activate the adaptive immune system. In the same way, polymeric particles with antigens mimic natural pathogens and efficiently present to

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

3

Fig. 1. Schematic diagram of various immune responses induced by particulate vaccine system. Upon encounter with an antigen, B cells convert themselves to antibody secreting plasma cells that produce antibodies for excreting the pathogens to mucosal surfaces (mucosal response) whereas dendritic cells (DCs) present the antigen via major histocompatibility complex (MHC) class I and class II molecules to CD8+ and CD4+ T-cells. Activation pathway of CD8+ T cells and CD4+ Th1 cells produces cytotoxic T lymphocytes (CTL) and activated macrophages that kill intracellular pathogens or infected cells (cellular response) while activation pathway of CD4+ Th2 cells produces activated B lymphocytes that secrete antibodies for neutralization of extracellular pathogens (humoral response).

antigen presenting cells leading to higher immune responses [11]. Because the size of particulate vaccines is comparable to those of common pathogens and the immune system has evolved to react against these pathogens, the particulate vaccines are readily taken up by antigen presenting cells. It is to be noted that particles larger than 0.5 ␮m are internalized in antigen presenting cells via phagocytosis while soluble antigens or smaller particles are mainly internalized via endocytosis [12]. Importantly, internalization of particulate vaccines through phagocytosis into phagosomes has key consequences because phagosomes are considered as competent organelles for cross presentation of antigens [13–15]. In contrast to soluble antigens which are preferentially presented by the MHC class II pathway, particulate antigens internalized in phagosomes can enter both MHC class I and MHC class II pathways [16] thereby allowing the induction of cytotoxic T cell responses, a feature that is not attainable by endocytosis of soluble antigens. These cellular immune responses have major role to prevent infections caused by intracellular bacteria and viruses. Another salient feature of particulate carriers is the possibility to deliver relatively large quantities of particulate antigens inside the antigen presenting cells, or the prolonged release of antigens into antigen presenting cells leading to extended antigen presentation compared with soluble antigen [14,17,18]. The other benefit of particulate carriers is the concomitant delivery of antigen and adjuvants to the antigen presenting cells to switch the desired cellular or humoral immune response pathway [19–21]. As a result, particulate vaccines are able to induce strong immune responses

that are critical for immunity to many pathogens including most viral infections. 4. Chitosan-based particulate systems Considering the optimum properties of particulate carriers and the conditions required for the delivery of antigens via mucosal routes, chitosan and its derivatives have garnered great attention to develop as particulate carriers for antigen delivery. The favorable properties of chitosan-based polymers that make them suitable for antigen carriers are concisely described here. Basically, chitosan is a linear amino polysaccharide composed of ␤1,4-linked monomers of d-glucosamine and N-acetyl-dglucosamine. Generally, chitosan is obtained by partial deacetylation of chitin, and hence the physical and biological properties of chitosan depend upon the degree of deacetylation and molecular weight [22]. Since the amount and distribution of acetyl groups in chitosan affect biodegradability and cytotoxicity, the degree of chitosan deacetylation directly influences on the efficiency of antigen delivery. Moreover, the adjuvant activities of chitosan also depend upon the degree of deacetylation [23,24]. Similarly, the pKa of the primary amine group of chitosan is around 6.5, the polymer at neutral pH carries no charge, and hence chitosan is insoluble in water. To make chitosan compatible to deliver antigens that are soluble and stable at neutral pH, chitosan derivatives have been made that are soluble at neutral pH retaining their activity and cationic property. Because chemical modifications are possible

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

4

to substitute both amine and hydroxyl functional groups present at chitosan, various chitosan derivatives have been produced by introducing hydrophilic groups such as hydroxyalkyl [25,26], carboxyalkyl [27], succinyl [28], thiol [29,30] and sulphate [31] or by grafting solubility enhancer polymers such as poly(ethylene glycol) (PEG) [32] and Poloxamer [33]. Among quaternary chitosan derivatives, N-trimethyl chitosan is of particular interest because it has permanent cationic charge and it is soluble over a wide range of pH. It also demonstrates high mucoadhesive and penetration-enhancing ability even at neutral pH. However, the mucoadhesive and penetration-enhancing properties of chitosan derivatives depend on the degree of substitution or quaternization in chitosan. It appears that the mucoadhesive property of chitosan is primarily due to the electrostatic interaction between the positively charged polymer and negatively charged cell surfaces and mucus. The adherence of chitosan with mucus in the body is largely attributed to mucins that contain significant proportions of negatively charged sialic acid at physiological pH. The longer the mucoadhesion of chitosan particulates with mucus, the greater the residence time of particles in mucosal routes. That leads to the greater possibility of particles to reach M cells, penetrate epithelial cells, or release antigens resulting in effective interaction with immune cells to generate immune responses. Because of the significant role of the mucoadhesive property of particles in antigen delivery, chitosan has been developed as an antigen carrier. The another feature of chitosan, that makes it suitable for antigen delivery, is its penetration-enhancing ability to transport antigens to immune compartments through paracellular route. It has been reported that chitosan can open the tight junctions between the epithelial cells through the alterations in the distribution of F-actin filaments [34]. 5. Preparation of chitosan particulates formulations Chitosan-based particulates for antigen delivery can be prepared by both physical and chemical methods. Although both methods have pros and cons, physical methods to yield particles are preferred over the chemical crosslinking methods because the proteins are chemically modified with crosslinking agents, the proteins are degraded in organic solvents, and the unreacted crosslinkers are difficult to remove from the formulations. Here, various techniques of antigen formulations are discussed. 5.1. Ionic crosslinking methods Chitosan-based particulates can be prepared by ionic crosslinking between cationic chitosan derivatives and anionic molecules or crosslinkers, such as tripolyphosphate (TPP), sodium sulphate or cyclodextrin. Ionic interactions between oppositely charged particles result in spontaneous self-assembly to microparticles or nanoparticles depending upon the molecular weights of polymers and crosslinkers. The ionic crosslinking method is of particular interest in protein/antigen formulations because the method does not use chemical crosslinkers, organic solvents and high temperature which may degrade or modify the protein/antigen structures [35–37]. Particularly, protein/antigen-loaded chitosan particulates have been typically made by ionotropic gelation of chitosan with TPP. The gelation process spontaneously forms nanoparticles upon addition of an aqueous solution of TPP incorporated with proteins to an aqueous solution of chitosan, under stirring at room temperature. Following the process, chitosan nanoparticles have been loaded with insulin [38] or tetanus toxoid (TT) [39] and investigated as nasal delivery vehicles. In a similar study, N-trimethyl

chitosan nanoparticles were investigated as a carrier system for the nasal delivery of an influenza subunit vaccine. The antigen-loaded nanoparticles of 850 nm in size with zeta-potential (+13 mV) were prepared by mixing a solution containing N-trimethyl chitosan and influenza A subunit H3N2 with TPP solution while stirring at room temperature and pH 7.4. Intranasal immunization with the antigenloaded nanoparticles resulted in strong IgG and IgA levels [40]. Precipitation or coacervation is another known method of preparing chitosan particulates in which sodium sulphate is gradually added to aqueous solution of chitosan containing 2% acetic acid to form ionically crosslinked particles. Using this method, positively charged chitosan microparticles of 4.3 ␮m in size and zeta-potential (+20 mV) with porous structures could be produced [41]. The antigens could also be abundantly loaded within the porous microparticles because the antigens not only adsorbed on the outer surfaces of chitosan microparticles but also entrapped within the microparticles. Remarkably, in vitro studies displayed a very low release of ovalbumin within 4 h from the microparticles, which is a favorable feature for antigen delivery as the considerable amount of remaining entrapped ovalbumin would be released by intracellular digestion after the uptake in the Peyer’s patches. Further in vivo studies demonstrated that microparticles smaller than 10 ␮m could be predominantly taken up by M-cells of Peyer’s patches [42]. Hence, the size of particles produced by this method adds an extra advantage to the carrier system. According to precipitation methods, both nanoparticles and microparticles were produced depending upon the molecular weights of chitosan used [43]. The particles were further incubated with antigens at room temperature stirring overnight to make antigen-loaded chitosan particulates. Thus, the adsorptive loading of ovalbumin on chitosan particles of various sizes (0.4, 1 and 3 ␮m) with similar zeta-potential (+25 mV) was prepared and the effects of sizes of chitosan particles on the immune response were evaluated by intranasal vaccine delivery. The experiment demonstrated that the smaller the chitosan particulates (≤0.5 ␮m), the higher the induction of IgG and IgA responses. 5.2. Chemical crosslinking methods Chemical crosslinking of antigens with chitosan derivatives is another promising approach of antigen conjugates for mucosal delivery. In this method, chitosan and antigen are individually functionalized with a bifunctional crosslinker N-succinimidyl-3(2-pyridyldithio) propionate to accommodate a disulfide bond between the conjugate. After 1 h of shaking at room temperature, the functionalized chitosan is treated with dithiothreitol for 30 min at room temperature to obtain thiolated chitosan. The latter is further mixed with functionalized antigen overnight allowing a disulfide bond formation to produce chitosan-antigen conjugates. Following the protocols, it is reported that conjugation of ovalbumin to trimethyl chitosan improves the immunogenicity of the antigen [44]. Moreover, nasal immunization of mice with the trimethyl chitosan-ovalbumin conjugates produced high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG [45]. 5.3. Spray drying methods Crosslinked and non-crosslinked chitosan microspheres can be prepared by spray drying method. The common cross-linking agents are formaldehyde (FA), glutaraldehyde (GA) and TPP and they influence on the physical properties of the resulting microspheres. For example, when a crosslinking agent (either FA or GA) was used, the resulting microspheres were smooth, spherical, and positively charged with zeta potential (+14–18 mV), and their particle size ranged from 2 to 10 ␮m [46]. In a separate study,

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

the influence of cross-linking agents on the properties of spray dried chitosan microspheres was extensively investigated. The outcomes of the study demonstrated that the zeta potential, surface morphology, and releasing property of the spray dried chitosan microspheres were remarkably influenced by the type (ionic or chemical) and extent of cross-linking agents [47]. Another important factor affecting the properties of the particles formed are the compositions of formulation and the processing parameters of spray drying method. In a typical example, chitosan solution of different concentrations in the presence or absence of a model antigen, bovine serum albumin (BSA) were used to prepare chitosan microparticles under different conditions. The size of microparticles was mainly influenced by formulation variables, while particle morphology was influenced by both formulation and process variables [48]. The microparticles were positively charged with zeta potential (+15–19 mV) and their size ranged between 3 and 8 ␮m. Apparently, BSA-loaded microparticles were larger in size with more distorted surface than their corresponding blank microparticles. Further, the observation of the loaded BSA retaining its integrity after the preparation process suggested that the chitosan microparticles prepared by spray drying method could be efficiently used for antigen delivery.

6. Modified chitosan particles Although chitosan particulates have been widely used in vaccine delivery, the aggregation behavior of chitosan particles is one of the problems associated with their long term stability, storage, and use. Such kind of problems with polymers or proteins is usually minimized by conjugation of PEG through a process called PEGylation that offers improved water solubility and stability of the conjugated substances. Accordingly, chitosan was covalently conjugated to activated PEG using N-hydroxy succinimide and 1ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the resulting PEGylated chitosan was stirred with TTP and sonicated to prepare microspheres [33]. The PEGylated chitosan microspheres of 3 ␮m in size and with zeta potential (+8 mV) were further loaded with Bordetella bronchiseptica dermonecrotoxin (BBD) antigens and evaluated them for nasal vaccination. BBD-loaded PEGylated chitosan microspheres were physically more stable, released more antigens, and stimulated more cytokines from macrophage cells in vitro as compared to BBD-loaded chitosan microspheres suggesting that PEGylated chitosan could be developed as a promising vaccine delivery system. Pluronic or Poloxamer is a triblock copolymer consisting of a central hydrophobic block of poly(propylene oxide) flanked by two hydrophilic blocks of PEG. Pluronic is a typical multipurpose excipient that has been widely used in a variety of pharmaceutical formulations due to its capability of increasing aqueous solubility and drug stability. Hence, many vaccine formulations have been made with Pluronic F-127 as nasal delivery vehicles of vaccines. In a study, to examine the effect of mucosal delivery of TT with Pluronic F127 and chitosan on immune responses, a number of mice first immunized intraperitoneally with TT and boosted intranasally with TT in Pluronic F127/chitosan at week 4, demonstrated a significant enhancement in the systemic antigen specific IgG response and mucosal IgA response in the nasal and lung washes [49]. The results suggest that the two component system of Pluronic F127 and chitosan appear to induce an additive or synergistic effect on the immune responses representing an attractive avenue for the mucosal delivery of vaccines due to the protection of the antigens by Pluronic F127. Another effective nasal delivery of vaccine with chitosan microspheres with Pluronic F-127 was demonstrated where chitosan microspheres were prepared through an ionic gelation process

5

with TPP to load BBD antigens in the presence of Pluronic F-127 [32]. Importantly, BBD-loaded Pluronic F127/chitosan microparticles showed a greater amount of BBD release than BBD-loaded chitosan microparticles due to hydrophilic property of Pluronic. The former microparticles also stimulated higher immune activities of mouse alveolar macrophage cells in vitro. Following nasal delivery of these microparticles to mice, there were higher BBDspecific IgA responses in nasal secretions in the mice immunized with BBD-loaded Pluronic F127/chitosan microparticles than controls. Further, these immunized mice were challenged with B. bronchiseptica via the nasal cavity to observe the protective immunity. The survival rate of the mice immunized with BBD-loaded Pluronic F127/chitosan microparticles was higher than those of other groups, demonstrating a successful vaccine delivery system.

7. Mucoadhesive chitosan particles Particulate carrier systems have advanced in many forms such as nano-/microspheres, emulsions, liposomes, virosomes, immune stimulating complex and virus like particles for vaccines delivery. Among them, microspheres accommodate several distinctive features of vaccine delivery system to stimulate host immune system. First, the microspheres can be prepared with an optimum size that is appropriate for particle trafficking into the body and subsequent uptake by antigen presenting cells [50]. Second, the microspheres can be easily functionalized with various ligands or peptides for targeting delivery of vaccines [51]. Third, the microspheres can be formulated to control the release of antigens at specific sites of the mucosal surfaces [52]. Mucoadhesive chitosan microspheres have been extensively used for nasal or oral vaccination. In a study, BSA loaded in mucoadhesive chitosan microspheres with an average size of 5 ␮m, prepared by a spray drying method, when delivered through nasal route, enhanced systemic immune response and the response was dependent on the nature of chitosan used in the preparation of microspheres [53]. In another study, nasal delivery of BBD in chitosan microspheres with an average size of 5 ␮m and zeta potential (+18 mV), prepared by ionotropic gelation of chitosan with TPP, increased BBD-specific IgA titers in the nasal cavity of mice in a time- and dose-dependent manner [54]. Due to the successful induction of mucosal immune responses in mice, a challenging experiment was performed in pigs, as a part of vaccine development, to observe the level of protection from the infection. First, a group of pigs were immunized with BBD-loaded chitosan microspheres through intranasal route and further challenged with a field isolate of B. bronchiseptica [55]. The results of these experiments demonstrated a significant increase in BBD-specific IgA and IgG titers in the nasal wash and serum of the immunized pigs. Compared to control pigs, the clinical signs of infection were about 6-fold lower in the immunized pigs. A strategy of coating chitosan microspheres with a pH-sensitive and/or mucoadhesive polymer was put forward in order to enhance the efficiency of microspheres for oral delivery. Accordingly, in order to evaluate as an oral immune delivery system, Eudragit-coated chitosan microparticles containing ovalbumin (OVA), prepared by both ionic gelation and emulsification–solvent evaporation method, were administered to Balb/C mice [56]. When OVA-specific antibodies were measured, there were the high induction of OVA-specific IgG in plasma and OVA-specific IgA in fecal. Similarly, the coating of thiolated Eudragit, by simply mixing, on BSA-loaded chitosan microspheres prepared by ionic gelation, lowered the release of BSA from the microspheres at gastric pH but enhanced the mucoadhesive property of these microspheres both in vitro and in vivo [57].

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11 6

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

One of the convenient methods used in the development of oral vaccine delivery system is the entrapment of antigens in polymeric nanoparticles formed by ionic interaction between cationic and anionic polymers. For instance, a cationic trimethyl chitosan was mixed with hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive anionic polymer, to entrap hepatitis B surface antigen (HBsAg) for oral delivery [58]. HPMCP not only improved the acid stability of the chitosan nanoparticles but also protected the loaded HBsAg from gastric destruction, suggesting that HPMCP/chitosan nanoparticles could be used in the oral delivery of HBsAg vaccine. Mucoadhesive glycol chitosan nanoparticles were evaluated to obtain systemic and mucosal immune responses against nasally administered HBsAg [59]. The results demonstrated that glycol chitosan nanoparticles, with lower nasal clearance rate as well as better mucosal uptake, induced strong humoral and mucosal immunity when compared to chitosan nanoparticles. This study demonstrated that this newly developed system had great potential for mucosal administration of vaccines. In a similar study, the efficacy of chitosan and glycol chitosan as a mucoadhesive coating material for PLGA encapsulated HBsAg nanoparticles in nasal vaccine delivery was investigated [60]. The glycol chitosan coated-PLGA nanoparticles demonstrated lower nasal clearance, better local or systemic uptake, and higher systemic and mucosal immune response compared to chitosan coated and uncoated PLGA nanoparticles. 8. Targeting delivery using chitosan particulates Although chitosan particulates have demonstrated as promising carriers for antigen delivery, their low targeting efficiency to specific cells limits their use in broad applications. The low efficiency of

chitosan particulates can be overcome by selective targeting of cells for antigen delivery. The targeting strategy offers a great advantage to induce high humoral and cellular responses even with lose dose of antigen delivery. Depending upon the cells and receptors, particulates can be decorated on their surface with carbohydrate ligands or antibodies that bind specifically to cell receptors. Here, we described two specific cells, namely antigen presenting cells and M cells, which are targeted for antigen delivery by various chitosan particulates. 8.1. Targeting antigen presenting cells Vaccine efficacy is improved upon specific delivery to antigen presenting cells, such as dendritic cells and macrophages. Usually, mannose is used as a specific ligand in selective targeting of particulate carriers for vaccine delivery to these antigen presenting cells [61]. Accordingly, chitosan microspheres with their surface decorated with mannose moieties were used as carriers for nasal delivery of vaccines [62]. In the study, mannosylated chitosan microspheres (MCMs) were prepared by ionic gelation method with TPP, and BBD antigens were loaded in the MCMs with continuous shaking condition at 37 ◦ C overnight. These MCMs showed high binding affinity with mannose receptors on macrophage cells. Consequently, the mice immunized with BBD antigen with MCMs through nasal route exhibited higher antigen-specific IgA responses in their saliva and serum than the mice immunized with BBD antigen with chitosan microspheres without ligand through the same route (Fig. 2). Although the high immune responses are considered as a direct result of enhanced uptake of MCMs in macrophage cells through receptor-mediated endocytosis by ligand-receptor interaction, the consequence of high immune responses appeared to be

Fig. 2. Anti-BBD IgA levels in (A) nasal wash; (B) saliva; (C) serum and (D) anti-BBD IgG levels in serum (data are means ± standard deviations, n = 3). Significant differences between untreated and immunizedgroups was expressed as *p < 0.001 and **p < 0.05 and between BBD-CMs and BBD-MCMs groups as ## p < 0.05.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

7

Fig. 3. In vivo localization of Alexa 488-labeled chitosan nanoparticles (a) compared with Alexa 488-labeled CKS9-conjugated chitosan nanoparticles (b) on rat Peyer’s patch region at 1 h after injection into closed ileal loops.

partly contributed by inherited property of chitosan which activates the functions of macrophages for immune reactions. Over the past decades, many studies have explored the conjugation of antigens with receptor-specific antibodies to target different receptors on dendritic cells for selective delivery of antigens [63,64]. Depending upon the degree of interactions between various antibodies and receptors on dendritic cells, they would elicit markedly different cellular and humoral immune responses [65,66]. It is therefore important to identify new candidate ligands that can specifically bind to receptors of dendritic cells to induce more effective and longer lasting immune responses. In pursuit of ligands, a novel dendritic cell-targeting peptide, TPAFRYS (TP) was identified by phage display technique and conjugated to chitosan in order to develop an efficient dendritic cell-targeting vaccine delivery carrier [67]. TP-conjugated chitosan nanoparticles exhibited higher targeting specificity in dendritic cells than macrophages or myoblasts. Furthermore, immunization of mice with OVA-loaded TP-conjugated chitosan nanoparticles demonstrated high production of OVA-specific serum IgG. Thus, the study exemplified dendritic cell-targeting strategy as a potential approach to enhance the efficacy of vaccines. 8.2. Targeting M-cells The uptake of particulate antigens by specialized M cells in Peyer’s patches of MALT is the determining step in inducing efficient immune responses of mucosal vaccination [68]. Several M cell targeting ligands, such as proteins, peptides or antibodies, have been identified and exploited for mucosal delivery of vaccines [69]. Here, we will describe several ligands of M cells for targeted delivery of vaccines. 8.2.1. Lectins Lectins are naturally occurring proteins that have high affinity with sugar moieties. Various lectins bind specifically to oligosaccharides on intestinal cells [70]. Especially, an ␣-l-fucose-specific lectin, Ulex europaeus agglutinin I (UEA1), is almost exclusively M-cell specific [71]. Due to this specificity, UEA1 has been used as a ligand for targeted oral vaccine delivery to M cells. As a proofof-concept, oral delivery of HBsAg loaded microparticles coated with UEA-1 to target murine M cells not only yielded comparable

HBsAg-specific antibodies to intramuscular vaccines with alumHBsAg but also elicited sIgA production in intestinal, vaginal and salivary secretions [72]. Lectin-mediated targeted delivery of vaccine is prominently demonstrated in an investigation using surface engineered polymeric nanoparticles with UEA1 for oral immunization [73]. Chitosan nanoparticles, prepared by ionic gelation and loaded with BSA antigen by adsorption, were further coated by UEA-1 conjugated alginate. To compare the capacity of exerting immune responses in vivo by these resulting chitosan nanoparticles against the aluminum hydroxide gel-based conventional vaccine, they were separately delivered in mice and their efficiencies to induce antigen-specific IgA in mucosal secretions and IgG in serum were measured. Enzyme-linked immunosorbent assay (ELISA) results indicated that immunization with UEA1 conjugated alginatecoated chitosan particles induced comparable systemic immune response but dominated mucosal immune responses compared to the conventional vaccine exhibiting the potential of UEA1 as a ligand for effective oral delivery of vaccines.

8.2.2. Peptides Peptides for M cell targeting have been of particular interest because of their easy synthesis and conjugation with carriers. Their applicability for vaccine delivery to M cells has been elucidated by several studies. Generally, in vivo phage display screening technology is used to identify the peptides that target delivery to M cells. Using the in vivo screening method, several targeting peptides, including LETTCASLCYPS (P8) and VPPHPMTYSCQY (P25) were identified to bind to receptors on the surface of intestinal tissues [74]. One of the analogs of peptide P8, YQCSYTMPHPPV demonstrated the enhanced delivery of polystyrene particles to M cells in a mouse model. In another phage display method using an in vitro model of the human FAE, two peptide sequences, CTGKSC and LRVC, were identified, and these peptides, when individually coupled to polycaprolactone-PEG (PCL-PEG) nanoparticles, enhanced their transportation into the FAE than PCL-PEG themselves [75]. An M cell-targeting ligand, SFHQLPARSPLP (Co1), selected by using a screening phage display library against an in vitro M-like cell culture model, was fused with a model antigen, enhanced green fluorescent protein (EGFP), to study the efficacy of ligand-mediated antigen delivery [76]. The resulting EGFP-Co1 showed the greater

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11 8

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

binding affinity to M cells and higher uptake in the immunogenic compartments of Peyer’s patches. Consequently, there were elevated immune responses of serum IgG and fecal IgA after oral delivery of EGFP-Co1 compared to EGFP alone, suggesting Co1 as a promising ligand for targeted delivery of mucosal vaccines. Similarly, a new peptide, CKSTHPLSC (CKS9), selected by the phase display technique, was conjugated to chitosan nanoparticles to evaluate CKS9-mediated transcytosis of nanoparticles through M cells to underlying immune compartments in Peyer’s patches [77]. As illustrated in Fig. 3, CKS9-conjugated chitosan nanoparticles entered and accumulated more specifically into PP regions than chitosan nanoparticles. To further explore CKS9-mediated vaccine delivery, a model antigen, Brachyspira hyodysenteriae membrane protein B (BmpB) was loaded into porous PLGA microparticles, coated with CKS9-coupled chitosan, and delivered in mice through oral route [78]. As shown in Fig. 4, oral immunization of BmpB vaccine with CKS9-chitosan-PLGA microparticles demonstrated high levels of IgG in serum and sIgA in both intestine and excreta. These results suggest that particulate vaccine with M cell targeting peptide could be a promising tool for targeted delivery of oral vaccine.

9. Adjuvant activity of chitosan particulates The ability of biodegradable particles to promote vaccinespecific immunity, usually termed as adjuvant activity, has been demonstrated in many studies. Often, these particles exhibit adjuvant effects in parenteral vaccine delivery probably because they are readily taken up by M cells in MALT, and subsequently engulfed by antigen presenting cells initiating immunological responses. Although the mechanism of action of particulate vaccine adjuvants is not fully understood, the common postulates are that polymeric particles induce cytokine release by epithelial cells, activate macrophages and natural killer cells (NK), increase cytotoxicity and induce mitosis in cells producing interleukins and interferons, or simply increase absorption of antigens [79]. As stated earlier, while chitosan showed induction of cytokines, interleukin (IL)-1 and colony-stimulating factor (CSF) in macrophages in vitro [80], the adjuvant activity of chitosan and its derivatives was also shown in vivo using mice and guinea-pigs with the induction of cytotoxic macrophages, the activity of natural killer cells, and cell-based immunity against bacterial infection [23,24]. Recently, a study has verified the function of chitosan as

Fig. 4. BmpB-specific immune response detection by ELISA after oral administration. Anti-BmpB IgA levels in feces (A) and intestine (B), anti-BmpB IgG levels in serum (C) and anti-BmpB IgG1 and IgG2a (D) levels.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

an immunomodulating adjuvant on T cells and antigen presenting cells in herpes simplex virus (HSV) infection [81]. In HSV infected mice, chitosan treatment significantly increased the frequencies of CD4+ T-cells, dendritic cells and natural killer cells compared to those in the control group. Besides, there was a marked reduction of anti-HSV IgG antibody in chitosan treated mice. Remarkably, a solid comparison between cholera toxin (CT) and chitosan as an adjuvant was exemplified in a study with Helicobacter pylori vaccine delivery in mice infected with H. pylori [82]. The present findings demonstrated that an oral delivery of the H. pylori vaccine with chitosan as an adjuvant had greater or similar immunotherapeutic effect in terms of H. pylori elimination rate, the humoral immune response and the Th1/Th2 cell immune reaction when compared to the same vaccine delivered with CT as an adjuvant. In another report, a matrix protein 1 (M1) in combination with chitosan as an adjuvant was administered intranasally to mice [83]. Later, the mice were challenged with a lethal dose of various influenza viruses. Because M1 protein is conserved in all influenza A strains, nasal delivery of the vaccine with chitosan not only protected the mice effectively against the challenge of the homologous influenza virus but also protected from heterologous viruses at different capacities. Thus, an intranasal delivery of influenza M1 vaccine using chitosan as an adjuvant induced crossprotection against various influenza virus infections in mice. These results suggest that chitosan is a potential modulator or immune stimulator as an adjuvant in vaccine delivery.

10. Conclusions and perspectives Theoretically, mucosal administration of antigens may result in the induction of protective mucosal immune responses that initiate in organized lymphoid tissues at mucosal surfaces, the major portal of entry of pathogens for infections. In practice, however, rapid mucociliary clearance and enzymatic digestion of antigens in mucosal surfaces lower the bioavailability of antigens to enter mucosal inductive sites and fail to generate adequate local mucosal immune responses against the mucosal vaccines. Among the alternative ways of delivery of vaccines investigated, particulate vaccines have several advantages for mucosal delivery. M cells are particularly accessible to particulate vaccines, but not to vaccines alone, and actively transport the particles across the epithelial barrier to underlying lymphoid tissues to initiate the immune reactions [84]. In addition, particulate vaccines, mimicking natural pathogens, are readily taken up by mucosal dendritic cells in lymphoid tissues to produce effective immune responses [85]. Chitosan-based particulate systems appear to be the most promising carriers among the polymeric delivery systems for the delivery of mucosal vaccines. The preference of chitosan derivatives over other polymers is simply because they can be modified in a number of ways to enhance the efficacy of mucosally administered vaccines; they can protect antigens from degradation, increase the residence time of antigens at mucosal surfaces, target antigens to M cells through ligand-receptor interaction, deliver antigens abundantly at the immune compartments, and effectively induce both systemic and mucosal immunity [86]. Despite these advantages, very little is known how chitosan particles work and how different types of immune responses are induced. In general, the potency and type of immune responses are largely dependent on the physicochemical properties of the vaccine formulations. While mucoadhesive property, colloidal stability, and antigen releasing ability of particulate vaccines hold a significant role in antigen delivery at the mucosal surfaces, the size, shape, and charge of the particles determine the endocytosis pathway of particles into antigen presenting cells which subsequently influ-

9

ence in the way of presentation of antigenic peptides to immune cells. Hence, more studies at molecular levels are needed to elucidate the mechanism of action of these particulate vaccines to engineer the physicochemical properties of the chitosan particles for effective vaccine delivery resulting in elevated immune responses. Moreover, the efficiency of vaccine delivery is predominantly dependent on the immunogenic properties of the antigens. It is therefore essential to optimize the physical and biological properties of particles and antigens, respectively, and the particulate vaccine formulations require to maintain these optimized properties in vitro and in vivo to achieve clinical significance of chitosan-based systems for vaccination. Acknowledgement This work was supported by the Ministry of Science, ICT and Future Planning (Project No. 2016R1D1A1B03933491). This work was also supported by the Technological Innovation R&D Program (S2273990) funded by the Small and Medium Business Administration (SMBA, Korea). References [1] J. Holmgren, C. Czerkinsky, Mucosal immunity and vaccines, Nat. Med. 11 (Suppl. 4) (2005) S45–53. [2] J. Mestecky, The common mucosal immune system and current strategies for induction of immune responses in external secretions, J. Clin. Immunol. 7 (4) (1987) 265–276. [3] M.R. Neutra, P.A. Kozlowski, Mucosal vaccines: the promise and the challenge, Nat. Rev. Immunol. 6 (2) (2006) 148–158. [4] C. Czerkinsky, F. Anjuere, J.R. McGhee, A. George-Chandy, J. Holmgren, M.P. Kieny, K. Fujiyashi, J.F. Mestecky, V. Pierrefite-Carle, C. Rask, J.B. Sun, Mucosal immunity and tolerance: relevance to vaccine development, Immunol. Rev. 170 (1999) 197–222. [5] K.A. Woodrow, K.M. Bennett, D.D. Lo, Mucosal vaccine design and delivery, Annu. Rev. Biomed. Eng. 14 (2012) 17–46. [6] A.K. Andrianov, L.G. Payne, Polymeric carriers for oral uptake of microparticulates, Adv. Drug Deliv. Rev. 34 (2–3) (1998) 155–170. [7] A. des Rieux, V. Fievez, M. Garinot, Y.J. Schneider, V. Preat, Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach, J. Control. Release 116 (1) (2006) 1–27. [8] M. Amidi, E. Mastrobattista, W. Jiskoot, W.E. Hennink, Chitosan-based delivery systems for protein therapeutics and antigens, Adv. Drug Deliv. Rev. 62 (1) (2010) 59–82. [9] Y. Vanloubbeeck, J. Hostetter, D.E. Jones, The biology of dendritic cells and their potential use in veterinary medicine, Anim. Health Res. Rev. 4 (2) (2003) 131–142. [10] S. Akira, TLR signaling, Curr. Top. Microbiol. Immunol. 311 (2006) 1–16. [11] I. Jabbal-Gill, P. Watts, A. Smith, Chitosan-based delivery systems for mucosal vaccines, Expert Opin. Drug Deliv. 9 (9) (2012) 1051–1067. [12] S. Burgdorf, C. Kurts, Endocytosis mechanisms and the cell biology of antigen presentation, Curr. Opin. Immunol. 20 (1) (2008) 89–95. [13] M.-L. De Temmerman, J. Rejman, J. Demeester, D.J. Irvine, B. Gander, S.C. De Smedt, Particulate vaccines: on the quest for optimal delivery and immune response, Drug Discov. Today 16 (13) (2011) 569–582. [14] H. Shen, A.L. Ackerman, V. Cody, A. Giodini, E.R. Hinson, P. Cresswell, R.L. Edelson, W.M. Saltzman, D.J. Hanlon, Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles, Immunology 117 (1) (2006) 78–88. [15] S. Chadwick, C. Kriegel, M. Amiji, Nanotechnology solutions for mucosal immunization, Adv. Drug Deliv. Rev. 62 (4–5) (2010) 394–407. [16] S. Burgdorf, C. Kurts, Endocytosis mechanisms and the cell biology of antigen presentation, Curr. Opin. Immunol. 20 (1) (2008) 89–95. [17] R. Audran, K. Peter, J. Dannull, Y. Men, E. Scandella, M. Groettrup, B. Gander, G. Corradin, Encapsulation of peptides in biodegradable microspheres prolongs their MHC class-I presentation by dendritic cells and macrophages in vitro, Vaccine 21 (11–12) (2003) 1250–1255. [18] M. Singh, X.M. Li, J.P. McGee, T. Zamb, W. Koff, C.Y. Wang, D.T. O’Hagan, Controlled release microparticles as a single dose hepatitis B vaccine: evaluation of immunogenicity in mice, Vaccine 15 (5) (1997) 475–481. [19] E. Schlosser, M. Mueller, S. Fischer, S. Basta, D.H. Busch, B. Gander, M. Groettrup, TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses, Vaccine 26 (13) (2008) 1626–1637. [20] S. Fischer, E. Schlosser, M. Mueller, N. Csaba, H.P. Merkle, M. Groettrup, B. Gander, Concomitant delivery of a CTL-restricted peptide antigen and CpG ODN by PLGA microparticles induces cellular immune response, J. Drug Target. 17 (8) (2009) 652–661.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11 10

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

[21] M.R. Tafaghodi, S.A. Jaafari, Nasal immunization studies using liposomes loaded with tetanus toxoid and CpG-ODN, Eur. J. Pharm. Biopharm. 64 (2) (2006) 138–145. [22] A.K. Singla, M. Chawla, Chitosan: some pharmaceutical and biological aspects – an update, J. Pharm. Pharmacol. 53 (8) (2001) 1047–1067. [23] K. Nishimura, S. Nishimura, N. Nishi, I. Saiki, S. Tokura, I. Azuma, Immunological activity of chitin and its derivatives, Vaccine 2 (1) (1984) 93–99. [24] K. Nishimura, S. Nishimura, N. Nishi, F. Numata, Y. Tone, S. Tokura, I. Azuma, Adjuvant activity of chitin derivatives in mice and guinea-pigs, Vaccine 3 (5) (1985) 379–384. [25] K. Kurita, Chitin and chitosan: functional biopolymers from marine crustaceans, Mar. Biotechnol. (NY) 8 (3) (2006) 26–203. [26] H. Sashiwa, N. Kawasaki, A. Nakayama, E. Muraki, H. Yajima, N. Yamamori, Y. Ichinose, J. Sunamoto, S. Aiba, Chemical modification of chitosan. Part 15: synthesis of novel chitosan derivatives by substitution of hydrophilic amine using N-carboxyethylchitosan ethyl ester as an intermediate, Carbohydr. Res. 338 (6) (2003) 557–561. [27] F.R.d. Abreu, S.P. Campana-Filho, Preparation and characterization of carboxymethylchitosan, Polímeros 15 (2005) 79–83. [28] Y. Kato, H. Onishi, Y. Machida, N-succinyl-chitosan as a drug carrier: water-insoluble and water-soluble conjugates, Biomaterials 25 (5) (2004) 907–915. [29] A. Bernkop-Schnurch, D. Guggi, Y. Pinter, Thiolated chitosans: development and in vitro evaluation of a mucoadhesive, permeation enhancing oral drug delivery system, J. Control. Release 94 (1) (2004) 86–117. [30] M. Roldo, P. Hornof, A. Caliceti, Mucoadhesive thiolated chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation, Eur. J. Pharm. Biopharm. 57 (1) (2004) 115–121. [31] K.R. Holme, A.S. Perlin, Chitosan N-sulfate, A water-soluble polyelectrolyte, Carbohydr. Res. 302 (1–2) (1997) 7–12. [32] M.L. Kang, H.L. Jiang, S.G. Kang, D.D. Guo, D.Y. Lee, C.S. Cho, H.S. Yoo, Pluronic F127 enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of Bordetella bronchiseptica antigens containing dermonecrotoxin, Vaccine 25 (23) (2007) 4602–4610. [33] H.-L. Jiang, I.-K. Park, M.-L. Kang, H.-S. Yoo, Y.-J. Choi, T. Akaike, C.-S. Cho, Immune stimulating activity of an atrophic rhinitis vaccine associated to pegylated chitosan microspheres in vitro, Polym. Adv. Technol. 18 (3) (2007) 220–225. [34] L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A.N. Fisher, S.S. Davis, Chitosan as a novel nasal delivery system for vaccines, Adv. Drug Deliv. Rev. 51 (1–3) (2001) 81–96. [35] S. Mao, U. Bakowsky, A. Jintapattanakit, T. Kissel, Self-assembled polyelectrolyte nanocomplexes between chitosan derivatives and insulin, J. Pharm. Sci. 95 (5) (2006) 1035–1048. [36] Y. Xu, Y. Du, R. Huang, L. Gao, Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier, Biomaterials 24 (27) (2003) 5015–5022. [37] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M. Alonso, Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers, J. Appl. Polym. Sci. 63 (1) (1997) 125–132. [38] R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (10) (1999) 1576–1581. [39] A. Vila, K. Sanchez, I. Janes, T. Behrens, J.L. Kissel, M.J. Vila Jato, Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice, Eur. J. Pharm. Biopharm. 57 (1) (2004) 123–131. [40] M. Amidi, S.G. Romeijn, J.C. Verhoef, H.E. Junginger, L. Bungener, A. Huckriede, D.J.A. Crommelin, W. Jiskoot, N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model, Vaccine 25 (1) (2007) 144–153. [41] I.M. van der Lubben, J.C. Verhoef, A.C. van Aelst, G. Borchard, H.E. Junginger, Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer’s patches, Biomaterials 22 (7) (2001) 94–687. [42] I. Van Der Lubben, F. Konings, G. Borchard, J. Verhoef, H. Junginger, In vivo uptake of chitosan microparticles by murine Peyer’s patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry, J. Drug Target. 9 (1) (2001) 39–47. [43] T. Nagamoto, Y. Hattori, K. Takayama, Y. Maitani, Novel chitosan particles and chitosan-coated emulsions inducing immune response via intranasal vaccine delivery, Pharm. Res. 21 (4) (2004) 671–674. [44] B. Slutter, P.C. Soema, Z. Ding, R. Verheul, W. Hennink, W. Jiskoot, Conjugation of ovalbumin to trimethyl chitosan improves immunogenicity of the antigen, J. Control. Release 143 (2) (2010) 207–214. [45] B. Slutter, S.M. Bal, I. Que, E. Kaijzel, C. Lowik, J. Bouwstra, W. Jiskoot, Antigen-adjuvant nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles? Mol. Pharm. 7 (6) (2010) 2207–2215. [46] P. He, S.S. Davis, L. Illum, Chitosan microspheres prepared by spray drying, Int. J. Pharm. 187 (1) (1999) 53–65. [47] K.G.H. Desai, H.J. Park, Preparation of cross-linked chitosan microspheres by spray drying: effect of cross-linking agent on the properties of spray dried microspheres, J. Microencapsulation 22 (4) (2005) 377–395.

[48] C. Kusonwiriyawong, W. Pichayakorn, V. Lipipun, G.C. Ritthidej, Retained integrity of protein encapsulated in spray-dried chitosan microparticles, J. Microencapsulation 26 (2) (2009) 111–121. [49] M.A. Westerink, S.L. Smithson, N. Srivastava, J. Blonder, C. Coeshott, G.J. Rosenthal, ProJuvant (Pluronic F127/chitosan) enhances the immune response to intranasally administered tetanus toxoid, Vaccine 20 (5–6) (2001) 23–711. [50] M.L. Kang, C.S. Cho, H.S. Yoo, Application of chitosan microspheres for nasal delivery of vaccines, Biotechnol. Adv. 27 (6) (2009) 857–865. [51] M.A. Islam, J. Firdous, Y.J. Choi, C.H. Yun, C.S. Cho, Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review, Int J Nanomedicine 7 (2012) 6077–6093. [52] B. Singh, S. Maharjan, T. Jiang, S.K. Kang, Y.J. Choi, C.S. Cho, Attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum, Biomaterials 59 (2015) 144–159. [53] S. Somavarapu, P. He, Y. Ozsoy, H.O. Alpar, Chitosan microspheres for nasal delivery of model antigen bovine serum albumin, J. Pharm. Pharmacol. 50 (S9) (1998) 166. [54] M.L. Kang, S.G. Kang, H.-L. Jiang, S.W. Shin, D.Y. Lee, J.-M. Ahn, N. Rayamahji, I.-K. Park, S.J. Shin, C.-S. Cho, H.S. Yoo, In vivo induction of mucosal immune responses by intranasal administration of chitosan microspheres containing Bordetella bronchiseptica DNT, Eur. J. Pharm. Biopharm. 63 (2) (2006) 215–220. [55] M.L. Kang, S.G. Kang, H.L. Jiang, D.D. Guo, D.Y. Lee, N. Rayamahji, Y.S. Seo, C.S. Cho, H.S. Yoo, Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs, J. Microbiol. Biotechnol. 18 (6) (2008) 1179–1185. [56] M. Hori, H. Onishi, Y. Machida, Evaluation of eudragit-coated chitosan microparticles as an oral immune delivery system, Int. J. Pharm. 297 (1–2) (2005) 223–234. [57] J.S. Quan, H.L. Jiang, E.M. Kim, H.J. Jeong, Y.J. Choi, D.D. Guo, M.K. Yoo, H.G. Lee, C.S. Cho, pH-sensitive and mucoadhesive thiolated eudragit-coated chitosan microspheres, Int. J. Pharm. 359 (1–2) (2008) 10–205. [58] A. Farhadian, N.M. Dounighi, M. Avadi, Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery, Hum. Vaccin. Immunother. 11 (12) (2015) 2811–2818. [59] D. Pawar, K.S. Jaganathan, Mucoadhesive glycol chitosan nanoparticles for intranasal delivery of hepatitis B vaccine: enhancement of mucosal and systemic immune response, Drug Deliv. 23 (1) (2016) 185–194. [60] D. Pawar, S. Mangal, R. Goswami, K.S. Jaganathan, Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity, Eur. J. Pharm. Biopharm. (2013) 550–559. [61] Y. Phanse, B.R. Carrillo-Conde, A.E. Ramer-Tait, R. Roychoudhury, N.L. Pohl, B. Narasimhan, M.J. Wannemuehler, B.H. Bellaire, Functionalization of polyanhydride microparticles with di-mannose influences uptake by and intracellular fate within dendritic cells, Acta Biomater. 9 (11) (2013) 8902–8909. [62] H.L. Jiang, M.L. Kang, J.S. Quan, S.G. Kang, T. Akaike, H.S. Yoo, C.S. Cho, The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization, Biomaterials 29 (12) (2008) 1931–1939. [63] L.J. Cruz, P.J. Tacken, J.M. Pots, R. Torensma, S.I. Buschow, C.G. Figdor, Comparison of antibodies and carbohydrates to target vaccines to human dendritic cells via DC-SIGN, Biomaterials 33 (16) (2012) 4229–4239. [64] L.J. Cruz, P.J. Tacken, R. Fokkink, C.G. Figdor, The influence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells, Biomaterials 32 (28) (2011) 6791–6803. [65] V. Apostolopoulos, T. Thalhammer, A.G. Tzakos, L. Stojanovska, Targeting antigens to dendritic cell receptors for vaccine development, J. Drug Deliv. 2013 (2013) 869718. [66] C. Macri, C. Dumont, A.P. Johnston, J.D. Mintern, Targeting dendritic cells: a promising strategy to improve vaccine effectiveness, Clin. Transl. Immunol. 5 (3) (2016) e66. [67] S.N. Jung, S.K. Kang, G.H. Yeo, H.Y. Li, T. Jiang, J.W. Nah, J.D. Bok, C.S. Cho, Y.J. Choi, Targeted delivery of vaccine to dendritic cells by chitosan nanoparticles conjugated with a targeting peptide ligand selected by phage display technique, Macromol. Biosci. 15 (3) (2015) 395–404. [68] B. Singh, S. Maharjan, T. Jiang, S.K. Kang, Y.J. Choi, C.S. Cho, Combinatorial approach of antigen delivery using M cell-Homing peptide and mucoadhesive vehicle to enhance the efficacy of oral vaccine, Mol. Pharm. 12 (11) (2015) 3816–3828. [69] T.-E. Park, B. Singh, S. Maharjan, T. Jiang, S.-Y. Yoon, S.-K. Kang, J.-D. Bok, Y.-J. Choi, C.-S. Cho, Mucosal delivery of vaccine by M cell targeting strategies, Curr. Drug Ther. 9 (1) (2014) 9–20. [70] I. Lambkin, C. Pinilla, C. Hamashin, L. Spindler, S. Russell, A. Schink, R. Moya-Castro, G. Allicotti, L. Higgins, M. Smith, J. Dee, C. Wilson, R. Houghten, D. O’Mahony, Toward targeted oral vaccine delivery systems: selection of lectin mimetics from combinatorial libraries, Pharm. Res. 20 (8) (2003) 1258–1266. [71] M.A. Clark, M.A. Jepson, N.L. Simmons, T.A. Booth, B.H. Hirst, Differential expression of lectin-binding sites defines mouse intestinal M-cells, J. Histochem. Cytochem. 41 (11) (1993) 1679–1687. [72] P.N. Gupta, K. Khatri, A.K. Goyal, N. Mishra, S.P. Vyas, M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B, J. Drug Target. 15 (10) (2007) 701–713.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101

G Model BIOMAC-8396; No. of Pages 11

ARTICLE IN PRESS B. Singh et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx

[73] B. Malik, A.K. Goyal, T.S. Markandeywar, G. Rath, F. Zakir, S.P. Vyas, Microfold-cell targeted surface engineered polymeric nanoparticles for oral immunization, J. Drug Target. 20 (1) (2012) 76–84. [74] L.M. Higgins, I. Lambkin, G. Donnelly, D. Byrne, C. Wilson, J. Dee, M. Smith, D.J. O’Mahony, In vivo phage display to identify M cell-targeting ligands, Pharm. Res. 21 (4) (2004) 695–705. [75] L. Fievez, C. Plapied, D. Plaideau, A. Legendre, V. des Rieux, H. Pourcelle, C. Freichels, J. Jerome, V. Marchand, Y.J. Preat, In vitro identification of targeting ligands of human M cells by phage display, Int. J. Pharm. 394 (1–2) (2010) 35–42. [76] S.H. Kim, K.W. Seo, J. Kim, K.Y. Lee, Y.S. Jang, The M cell-targeting ligand promotes antigen delivery and induces antigen-specific immune responses in mucosal vaccination, J. Immunol. 185 (10) (2010) 95–5787. [77] M.K. Yoo, S.K. Kang, J.H. Choi, I.K. Park, H.S. Na, H.C. Lee, E.B. Kim, N.K. Lee, J.W. Nah, Y.J. Choi, C.S. Cho, Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cell-homing peptide selected by phage display technique, Biomaterials 31 (30) (2010) 7738–7747. [78] T. Jiang, B. Singh, H.S. Li, Y.K. Kim, S.K. Kang, J.W. Nah, Y.J. Choi, C.S. Cho, Targeted oral delivery of BmpB vaccine using porous PLGA microparticles coated with M cell homing peptide-coupled chitosan, Biomaterials 35 (7) (2014) 2365–2373.

11

[79] I.M. van der Lubben, J.C. Verhoef, G. Borchard, H.E. Junginger, Chitosan and its derivatives in mucosal drug and vaccine delivery, Eur. J. Pharm. Sci. 14 (3) (2001) 201–207. [80] K. Nishimura, C. Ishihara, S. Ukei, S. Tokura, I. Azuma, Stimulation of cytokine production in mice using deacetylated chitin, Vaccine 4 (3) (1986) 151–156. [81] B. Choi, D.H. Jo, A.K. Anower, S.M. Islam, S. Sohn, Chitosan as an immunomodulating adjuvant on T-Cells and antigen-Presenting cells in herpes simplex virus type 1 infection, Mediators Inflamm. 2016 (2016) 4374375. [82] Y. Gong, L. Tao, F. Wang, W. Liu, L. Jing, D. Liu, S. Hu, Y. Xie, N. Zhou, Chitosan as an adjuvant for a Helicobacter pylori therapeutic vaccine, Mol. Med. Rep. 12 (3) (2015) 4123–4132. [83] Z. Sui, Q. Chen, F. Fang, M. Zheng, Z. Chen, Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant, Vaccine 28 (48) (2010) 7690–7698. [84] N.A. Mabbott, D.S. Donaldson, H. Ohno, I.R. Williams, A. Mahajan, Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium, Mucosal Immunol. 6 (4) (2013) 666–677. [85] D.J. Irvine, M.A. Swartz, G.L. Szeto, Engineering synthetic vaccines using cues from natural immunity, Nat. Mater. 12 (11) (2013) 978–990. [86] H.C¸. Arca, M. Günbeyaz, S. S¸enel, Chitosan-based systems for the delivery of vaccine antigens, Expert Rev. Vaccines 8 (7) (2009) 937–953.

Please cite this article in press as: B. Singh, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.101