Review

Therapeutic Delivery

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Nanocarriers: a versatile approach for mucosal vaccine delivery

Infectious agents generally use mucosal surfaces as entry port to the body thereby necessitating the need of development of mucosal vaccine as vaccination is important for disease avoidance and suppression. Vaccination through mucosal route is a promising strategy to elicit efficient immune response as parentally administered vaccines induce poor mucosal immunity in general. Safety, economy and stability are highly desired with vaccines and this can be achieved with use of delivery cargos. This review focuses on challenges related with mucosal vaccines and use of nanocarriers as suitable cargos to cater the antigen effectively to the desired site. The review also includes different factors which are to be considered regarding the performance of the nanocarriers and clinical status of these systems.

Mucosal immunity is central to immune homeostasis and protection against the majority of pathogens that attack through mucosal surfaces. Mucosal surface acts as the most important boundary between the external environment and body. Besides maintenance of normal physiology, mucosal surfaces also prevent the access of hazardous pathogens to body and therefore targeting/enhancing the mucosal immunity can greatly improve the health benefits and patient compliance. Innate and adaptive immunity comprises the two arms of the mucosal immunity. Innate immune response plays role in preventing early infections while adaptive immune responses perform the task of preventing infections from pathogens already present in the body. Conventional vaccines produce cellular immunity only but no or weak mucosal immunity and thus strengthen only the systemic immune response. A vaccine can work most efficiently if it can elicit both humoral and cellular immune responses; and this can be achieved by mucosal vaccination. Mucosal vaccination involves administration of an antigenic substance by oral, nasal, pulmonary, sublingual and urinogenital routes. Vaccination via these routes generally results in the generation of mucosal antibody and

10.4155/TDE.14.89 © 2015 Future Science Ltd

Nishi Mody1, Rajeev Sharma1, Udita Agrawal1 & Suresh P Vyas*,1 Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar, Madhya Pradesh 470003, India * Author for correspondence: [email protected] 1

cell mediated immune response [1,2] . This increased immune response paves the way for development of vaccines to be delivered to the mucosal site of interest thereby producing a strong protective effect vaccine. Further advantages of mucosal vaccination via oral or nasal delivery routes include ease of self administration and noninvasiveness. This improves overall patient compliance, especially in pediatric patients, and does not necessarily require trained personnel for administration, thereby reducing costs and facilitating use in mass immunization programs [3] . Mucosal vaccines: routes of administration Mucosal vaccines can be administered by oral, ocular, vaginal, rectal, pulmonary, nasal and sublingual routes. Oral route is the most convenient and captivating route for vaccine administration. Common mucosal immune system (CMIS) generates immune response in small intestine, descending colon and mammary and salivary glands [4] . With oral vaccination it is possible to administer particulate antigens and these particulate antigens are better taken up by the Peyer’s patches present in the Gut Associated Lymphoid Tissue (GALT) than the soluble

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Key terms Nanocarriers: Nano sized drug/antigen cargos.

• Economic.

Dendritic cells: Antigen-presenting cells, (also known as accessory cells) of the mammalian immune system.

With these advantages, mucosal vaccination serves as good alternative to parental vaccination [8] . When antigen is administered parentally IgG are produced which provide protection against systemic circulation only but mucosal vaccination produces both IgG and IgA and thus can trap the pathogen at entry level only and even then if some pathogen crosses the epithelium, they are acted upon by circulating IgG antibodies thereby eliciting the dual protection. Mucosal vaccination can also develop immunity at site other than that of administration because of the well connected network of surface IgA, also known as CMIS [2,6] .

M cells: Also known as microfold cells are cells found in the follicle-associated epithelium of the Peyer’s patch as well as in BALT (Bronchus-associated lymphoid tissue). CD8 + T cells: Cytotoxic T lymphocytes accompanied with CD8 glycoprotein. CD4 + cells: White blood cells that are essential part of immune system. Mucosal immunization: Immunization developed after exposure/presentation of antigen to the mucosal surfaces.

antigens. GALT is responsible for generation of surface IgA and adds to immunization but it is also associated with certain disadvantages like exposure to acidic environment and enzymatic degradation in Gastrointestinal tract (GIT) although these drawbacks can be overcome by encapsulating antigen within the nanocarriers. Another good alternative route of vaccination is nasal route where Nasal associated lymphoid tissue (NALT) is accountable for the immune response generation. NALT is made up of goblet cells, dendritic cells and M cells, the most abundant are the M cells which function in a manner similar to Peyer’s patch. Unlike the oral route, nasal route is more permeable with less hostile environment, retains long term memory and produces faster immunity as compared with oral one because of the presence of dendritic cells [5] . Apart from oral and nasal routes, sublingual, pulmonary, rectal and vaginal routes are also under investigation as good alternative vaccination strategy. Sublingual route is commonly used for vaccination in case of allergies, particularly for Type I allergies, rectal routes can be suitably used to elicit immune response at distant site of CMIS while the vaginal route serves as attractive route of vaccination against infections like HIV, HSV and HPV but rectal and vaginal routes are not preferred much because of lack of patient compliance and chances of expulsion associated with these routes [6,7] . Advantages of mucosal vaccination Mucosal vaccines possess certain advantages over the parenteral route which can be enlisted as: • With mucosal route, self administration is possible thereby eliminates the need for medical assistance; • Large dose of antigen as compared with that of parenteral route can be administered; • Provides dual immune response by generating both IgG and IgA;

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Organs of the immune system A number of organs and tissues are involved in the development of the immune responses. On the basis of their function they can be divided into primary, secondary and tertiary lymphoid organs (Figure 1) . Primary lymphoid organ consists of bone marrow and thymus. Thymus is the organ where development and maturation of T-cells occur that protects the body from infections. Bone marrow is responsible for the origin and development of B cells. Secondary lymphoid organ comprises spleen, lymph nodes and various mucosal-associated lymphoid tissue (MALT) and GALT. The function of secondary lymphoid organs is to trap antigen and provide site for the interaction of antigen and mature lymphocytes. Tertiary lymphoid tissues work in case of inflammations. Vigilance job performed by the mucosal surfaces involves antigen presenting cells (APCs), including dendritic cells (DCs), macrophages and B-cells. APCs, having specific functions and subsets, are crucial initiators of adaptive immune responses and vaccine-induced immunity. Microfold (M) cells are another type of cells specifically involved in the transport of substances across the epithelial surface after uptake and processing by DCs in order to initiate immune responses [9] which involves differentiation of T-cells to subsets (Th1, Th2, Th17 or T regulatory cells). T cells are directed toward the submucosal regions by mucosal homing markers in order to perform their effector functions. DCs and related T-cells also interact with B-cells and promote their differentiation along with the production of antibody at multiple mucosal sites [10] . Mechanism of mucosal vaccination Mucosal immune system has two sections viz., effecter sites and inductive sites. Inductive site, as the name suggests, is the site where induction of initial immune response occurs as a result of antigen sampling which

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Nanocarriers: a versatile approach for mucosal vaccine delivery 

leads to the activation of immune cells (Figure 2) . Effecter site is the site where antibody and component of immune system performs their specific task upon activation [11] . MALT and their adjacent lymph nodes are the main inductive site for mucosal immune response. MALT comprises the bronchus-associated lymphoid tissue, the NALT and the GALT, which includes isolated lymphoid follicles and Peyer’s patches [12] . M-cells are present abundantly in follicle associated epithelium of MALT which can be characterized by the presence of short irregular microvilli and transcytotic capabilities. These structures promote the contact and the selective uptake of antigen in lumen followed by subsequent delivery to the underlying lymphoid follicles [3] . CD8 + and CD4 + T lymphocytes are the major effector cells in the mucosal surfaces which collectively compose about 80% of the mucosal lymphoid cell population. CD4 + T helper cells get differentiated to memory or effector T cell in presence of professional APCs DC’s, B cells and macrophages whereas CD8 + cells can be activated to cytotoxic T-lymphocyte [3] . The nearness of a high number of APCs and immune cells to the mucosal inductive sites is the basis for an efficient response obtained by the mucosal immune system against potentially harmful foreign incursion. Mesenteric lymph nodes, intestinal Peyer’s patches and isolated lymphoid follicles are the part of inductive site at which the activation of antigen specific B- and T- cell takes place followed by clonal expansion and differentiation into T and B effector cells. After the differentiation process migration of T and B effector cells from inductive site to effector site occurs. Lamina propria serves as effector site where infected cells are lysed by the cytotoxic T-lymphocyte and B cells are differentiated to plasma cells which secret immunoglobulin A (IgA) in large quantities. IgA along with IgM serves as the predominant antibody isotype in intestinal secretions. IgA and IgM are transported across ECs through polymeric Ig receptors while neonatal Fc receptors are involved in the transport of IgG across ECs [13] . To summarize, mucosal vaccination can provide protection by any of the following means: • Mucosal vaccination stimulates the production of IgA antibody. IgA prevents the attachment and colonization of the noninvasive pathogen at mucosal surface. • Increased immunity prevents penetration and replication of the invasive pathogen (e.g., viruses and invasive bacteria) in mucosal cell. • IgG production provides systemic immunity. It prevents the attachment and penetration of

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Lymphoid organs

Primary lymphoid organ Thymus Bone marrow

Secondary lymphoid organ Lymph node Spleen

Tertiary lymphoid organ CALT

MALT

Figure 1. Organ of the immune system. CALT: Conjuctiva associated lymphoid tissue; MALT:Mucosal-associated lymphoid tissue.

pathogen that can reach to various tissues through the systemic circulation. • Mucosal immunization can activate cell mediated immune response and thus helps in eliminating intracellular pathogens. Challenges in mucosal vaccine design A wide number of factors like dilution of antigen by mucosal secretions, seizing in thick mucus gels, proteolytic degradation by proteases and nucleases, harsh gut milieu, mucosal tolerance and epithelial barriers represent the major challenge for development of mucosal vaccines such as (Figure 3) . Vaccine formulations containing constituents such as proteins, DNA and polysaccharides are highly labile and could be structurally degraded and lost of bioactivity while their transit through the gut or via mucosal layer due to meticulous chemical environment of stomach and rectal/vaginal mucosal layer, if not properly protected [14–16] . All these factors collectively pave way for the development of various delivery systems and devices that can efficiently deliver vaccine formulation with improved immune response, greater stability, cell selective targeting and immunomodulatory properties [17] , and nanotechnology has emerged out as boon in the field of mucosal vaccine development. Nanotechnology: a good alternative for mucosal immunization Nanotechnology has created its space in every field of science including vaccinology [18] and is under continuous exploration for rationally designed carriers to deliver therapeutic agents at mucosal surface. Solubility, stability and surface tailorability are a few properties of therapeutic agents that can be easily modified with nanotechnology and thus these nanotechnology-based bioactives are of great interest in present scenario. Tailored nanocarriers can safely cargo bioactives to specific mucosal site like eye, lung air way and the GI tract [19,20] . Because of the small size owned by nanopar-

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IgA

i M-cell

pIg receptor iv

IL-17

DC

Th17 v BAFF APRIL IgA+ iii

B

ii

Plasma cell

Homing to mucosal surfaces IFNγ+ T-bet

IL-4+ GATA3

IL-17+ RORγt

TGFβ+, IL-35+, IL-10+ Foxp3

Th1

Th2

Th17

Treg

Expansion of Th effector cells (Th1, Th2, Th17) and Treg cells

T0 Naive T cell

Lymph node

Figure 2. Role of mucosal surface in generating immune response. (A) Mucosal surfaces constitute the largest interface between the body and the external environment, including the respiratory (purple), GI (green) and genital (blue) tracts. (B) Mucosal immunity plays a crucial role in defense against invading pathogens at the epithelial cell surface, involving a complex network of innate and adaptive immune components. (i) Continuous pathogen surveillance is mediated by specialized antigen transport cells (M-cells) and antigen processing cells (DCs). (ii) Mucosal DCs are particularly important at initiating adaptive immune responses by migrating to the draining lymph node and mediating the expansion of antigen-specific native T-cells into T helper subsets, involving an upregulation of transcription factors (T-bet, GATA3, RORgt or Foxp3) and lineage-defining cytokines (INFg, IL-4, IL-17, TGFb, IL-35 and IL-10). (iii) Expanded T-cell subsets will come back to mucosal surfaces to perform their effector functions. (iv) Th17 cells and IL-17 expression can upregulate polymeric Ig (pIg) receptor expression and IgA class switching, enhancing IgA secretion. (v) In addition, soluble factors (BAFF, APRIL) secreted by DCs and epithelial cells can promote T-cell Independent (TI) IgA class switching. Increased IgA production and translocation through epithelial cells hinders pathogen invasion and promotes immunity at mucosal surfaces. Adapted with permission from [10] © Elsevier (2011). For color images please see online: www.future-science.com/doi/full/10.4155/TDE.14.89

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Nanocarriers: a versatile approach for mucosal vaccine delivery 

Review

Intranasal vaccine delivery • Complex geometry • Mucociliary clearance • Narrow nasal entrance C h a l l e n g e s

Oral vaccine delivery • Physical barriers • Enzymatic degradation • Low permeability • Acid-induced hydrolysis

Rectal/vaginal vaccine delivery • Expulsion of vaccine • Lack of patient compliance • Hormonal changes affects immune response

Figure 3. Various challenges of mucosal vaccine delivery.

ticles, they exhibit extraordinary properties like high surface area to volume ratio and high diffusion rate. Other advantages of nanocarrier-based delivery system over conventional system include [21] : • Localized drug delivery is possible with targeted nanocarriers; • Because of their size, these carriers are better taken up by the antigen presenting cells thereby facilitating the delivery of antigen to the antigen presenting cells; • Increased bioavailability with nanocarrier dependent therapeutics; • Apart from carriers, they also work as an adjuvant to provoke the immune response and thus produce lager response as compared with conventional vaccines; • Can maximize drug concentration at mucosal surfaces without causing harm to the body. With these advantages, many nanocarriers have been explored for the mucosal vaccine delivery which includes polymeric nanoparticles, nanocapsules, liposomes, noisome, bilosomes, micelles, dendrimers and nanotubes. Nanocarrier system can be given orally as oral administration allows uptake by intestinal epithelial cells in the mucosa of the GI tract,

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Peyer’s patches and M cells [22] . The material used for formulating nanocarriers should be nonreactive and preferable biocompatible. The release of the bioactive from nanocarriers follows any of the mechanism, alone or in combination, including erosion, degradation, diffusion or swelling of the matrix. Nanocarrier should provide optimum encapsulation, enough stability and necessary permeability to the antigen/drug [3] . Nanocarriers: suitable delivery vehicles for mucosal immunization The use of nanotechnology in mucosal immunology is a rapidly growing field in medicine and has generated significant interest over the past few years. Many parameters directly or indirectly govern the ability of the nanocarrier system to deliver the antigen to the APCs by modulating the rate of antigen release and/or nanocarriers uptake by M cells or APCs. The novel carriers (liposomes, micelles, nanoparticle, etc.) have been developed as a mucosal vaccine delivery system to protect vaccine candidate antigen from the hostile Key terms Liposome: Bilayered, lipidic and spherical structure enclosing aqueous compartment. Dendrimers: Hyperbranched tree-like structures with central core and branching.

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Review  Mody, Sharma, Agrawal & Vyas environment of mucosal lumen and increase their uptake and transcytosis via mucosa especially M cells. Particle size, stability, hydrophobicity/hydrophilicity, crystallinity of polymers, bioadhesiveness and the nature of additives are the factors governing the performance of nanocarrier system. Major features to be fulfilled by nanocarriers to be used for delivery of vaccine are antigen protection, transport across mucosal cell and delivery of antigen to APCs. The molecular designing of carriers together with the knowledge of generation of mucosal immunity mechanism, receptors involved in internalization of specific antigen and molecules involved in the process are taken into account to ensure effective generation of immune response against the antigen [6,23] . Since the novel carriers mimic the pathogenic size of particles, they are selectively taken up by the M cells and are transcytosed to underlaying lymphoid tissue. Their interaction and processing in the lymphoid tissue ultimately depends upon their nature and composition. Protection of antigen is chief prerequisite for nanocarriers as upon introduction to the harsh pH condition and enzymatic activity of GIT environment, most of the antigens (polysaccharides, DNA and/or proteins/ polypeptides,) may be degraded in biological system, hence they are inadequately transported across mucosal epithelial [24] . Nanocarriers should be competent to carry antigens to immune-competent cells such as dendritic cells and macrophages so that they are able to identify antigen and activate the immune system [25] . Factors influencing the performance of nanocarriers Performance of nanocarrier can be best assessed with their ability to release the antigen in the vicinity of antigen presenting cells. Different physicochemical and pharmaceutical factors can affect the interaction and behavior of the nanocarriers within the biology and these include particle size, glass transition temperature, nature of nanocarrier, crystallinity of polymers, bioadhesiveness, ratio of copolymers, transition temperature and nature of excipients present (Figure 4). The uptake of nanocarrier system is governed by the size of the carrier system as small sized system interacts better with mucosal surface. Relationship between immune response generated and particle size was studied and it was found that particles larger than 7 μm generated mucosal immunity while that of 4 μm or less produced humoral immunity. The uptake of nanocarriers also depends upon its lipophilic or hydrophilic nature. Although lipophilicity is required to cross the membranes but to interact with biological mileu, hydrophilicity is also required so an intermediate having balanced hydrophilic and lipophilic carrier will serve the best in terms of antigen load-

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ing, release, stability and interaction with the mucosal surfaces [6] . In case of polymeric carriers, glass transition temperature, adhesion strength of polymer, ratio of copolymer and polymer cyrstallinity are to be considered while formulating the vaccine. Amorphous content of the polymer and antigen solubility are inversely proportional. With increase in the amporphous nature, the release of antigen will also increase while reciprocate is true for crystallinity, in other words, increased crystallinity will decrease the rate of release of loaded bioactive [8] . Additive blended with polymers also affects the properties like particle size, hydrophobicity, zeta potential, polydispersity index, entrapment efficiency and release profile and thus selection of the additives as well as co-polymer should be critically observed [26] . Bioadhesive nature of the polymer influences its interaction with the mucosal surface in a direct proportion manner. Good is the bioadhesion, better will be the interaction of the carrier system and it will reside for a long time at the desired site of action thus increasing the efficiency of the system [6] . Nanocarriers explored so far for mucosal immunization Polymeric nanocarriers

Biodegradable polymeric nanocarriers have achieved significant consideration as drug delivery vehicles for the mucosal delivery of antigens. Polymeric materials can be modulated for the desirable physicochemical properties (e.g., surface charge for optimum interaction with mucosa), antigen loading, release pattern and biological performance of nanocarriers. The most explored polymers for mucosal vaccine delivery are poly(lacticco-glycolic acid) (PLGA), poly(ɛ-caprolactone) (PCL), poly(lactic acid) (PLA), poly(methacrylic acid) (PMAA), polyalkyl cyanoacrylates (PACA), polyacrylic acid (PAA) [27] , dextran, chitosan, alginate, etc. To augment the active targeting potential of a nanosystem to specific cells, a number of surface functionalization methods have been developed [28] . The surface modification strategies and/or the addition suitable stabilizer to the polymeric matrix can be included in the particle composition either by physical blending, surface adsorption or by covalent attachment. US FDA approved PLA and PLGA are the prime synthetic polymers exploited for the encapsulation of antigens. The biocompatibility, biodegradability and safety of these polymers, collectively with their capacity of controlled release are well documented, which led to their authorization for number of humans clinical applications, and also to a number of marketed products [5] . Results have revealed that these nanoparticles can also render immunogenic antigens that are otherwise not sufficiently effective when administered mucosally.

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Nanocarriers: a versatile approach for mucosal vaccine delivery 

Decrease in particle size increases absorption and thus immune response

It affects the release rate of the payload

Glass transition temperature For reaching to target cells, nanocarrier should possess stability in biologic milieu

Particle size

Lipophilic/ hydrophilic nature

Stability in biological milieu

Nature of additive

Additives can alter different pharmaceutical properties and hence the performance of nanocarriers

Review

Intermediate lipophilicity generates highest immune response

Ratio of copolymer

Bioadhesion

It affects the lipophilic/hydrophilic nature of the nanocarrier and so affects the release rate of the payload

Good bioadhesiveness of the polymers increases the interaction with the mucosal surface Figure 4. Factors affecting the performance of nanocarrier.

Antigen encapsulation into polymer leads to variation in veracity and immunogenicity of antigen may occur because of the revelation to high shearing stress, solvent and pH variation leading to polymer degradation. Adsorbing of the antigen on polymeric system surface rather than encapsulation within the nanoparticle can overcome the situation [29] . Polymer nanoparticles internalization at different mucosal sites has been reported to be extremely dependent on size. Chitosan nanocarriers due to their polycationic nature have mucoadhesive properties which make them efficient nanocarriers for transmucosal vaccine delivery  [30] . It has the capability to open the tight junctions to facilitate the cellular permeability of bioactives. Ye et al. designed chitosan-based nanoparticulate vaccines CPE30-CS-pVP1 which targeted M cells and significantly enhanced CVB3 specific mucosal IgA and T cell immune responses and concluded that these vaccines resulted in enhanced protection against CVB3-induced myocarditis [31] . The Poly ethylene glycol (PEG) modification on the surface of particles leads enhanced biological and physical stability. Steric stabilization attributed to the formation of a hydrophilic PEG shell around the

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particle core. In a study nasal administration of PLAPEG block co-polymers nanoparticles in rats showed enhanced mucosal transport, stability, which correlated well with the prolonged blood circulation time of a tetanus toxoid as compared with non-PEGylated PLA particles [32] . It was found that serum IgG and mucosal IgA antibodies were elicited in a prolonged fashion. It was also found that the antibody levels were appreciably advanced than those elicited by tetanus toxoid (TT) alone or TT encapsulated in plain PLA nanoparticles. Primard et al. prepared nanoparticles of PLGA coated with a muco-adhesive chitosan-derivate layer to transport an encapsulated antigen with a Toll-Like Receptor-7 agonist as immunostimulatory signal [33] . It was observed that the mice developed a high systemic immune response equivalent to mice injected subcutaneously when immunized intranasally. In a study by our group mucoadhesive alginate-coated chitosan microparticles were evaluated for their potential as oral vaccine against anthrax. The results showed that developed system was transported to the Peyer’s patch upon oral delivery and was competent to induce potent mucosal and systemic immune responses [34] .

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Review  Mody, Sharma, Agrawal & Vyas In a study by our lab, the targeting potential of Lectin (LTA)-anchored chitosan nanoparticles was explored for oral mucosal immunization against hepatitis B. The results assessed by estimating secretory IgA level in the different body secretions, and cytokine (IL-2 and IFN-γ) levels in the spleen homogenates, demonstrated that LTA-anchored nanoparticles showed high cellular and humoral responses [35] . Polymer can be also modulated/designed for intracellular trafficking to bias antigen presentation. For this stimuli-sensitive polymers that are responsive to changes in redox potential or pH can be exploited for intracellular antigen targeting. Nanoparticles based on pH-sensitive acrylic acid or ketal-containing polymers destabilize membranes in a pH dependent manner and take benefit of the acidification of endosomes [36,37] . Liposome as mucosal vaccine delivery system

Mucosal vaccine delivery with liposomes as carrier candidate provides better alternatives and opportunities for site specific vaccine delivery. Firstly, pioneers Gregory Gregoriadis and Allison informed about the application of liposomes as immunological adjuvants in 1974. Since that time, liposomes and related vesicular carriers have been established as robust systems for induction of humoral and cell mediated immunity to a broad spectrum of infectious diseases. Numerous researches have focused to improve antibody responses to antigens administered through mucosal route via antigen encapsulating liposomes. Liposomes are proficient carrier to improve the delivery of antigens beyond the mucosal membranes in mammalian cells and perform important roles in the stimulation of better adaptive immune responses [6,38] . Liposomes offer a wide range of choices for the design of vaccine candidates due to their structure and adjuvant property to enhance the considerable immune response triggered by weak antigens. Liposomal surface can be engineered through chemical conjugation of lectins, bioadhesives, mannose derivatives, etc. that have been used for their efficient delivery of encapsulated antigens (Figure 5) . Numerous approaches such as incorporation of Toll-like receptor ligands, Immunoglobulins (Ig), including lipopolysaccharide, Pam2 or Pam3 derivatives and CpG motif widely reported for stronger immune response stimulation [39–42] . Vyas and co-worker delivered antigen to mucosa associated lymphoid tissues by oral route and nasal associated lymphoid tissues by nasal route using liposomes/engineered liposomes as nanocarriers and it has been reported that liposomes showed stronger immune response when administered as vaccine adjuvant [13,43–45,]. Cationic liposomes have been investigated as a novel adjuvant in mucosal vaccine delivery system. Zhuang et al. reported that effect of PEGylation

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on lymph nodes targeting and the immunogenicity of cationic liposome formulated vaccines. The obtained data showed that incorporating a small amount of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine(polyethylene glycol)- DSPE-PEG2000 into DOTAP liposomes not only increased the passive lymph nodes targeting of DOTAP-formulated vaccines but also modulated their biodistribution in vivo, which consequently improved the efficiency of cationic liposomeformulated vaccines [46] . In another study, Senchi and coworkers reported the effectiveness of oligomannosecoated liposomes with the poly (I:C) adjuvant as an intranasal antigen-delivery system for the induction of viral specific mucosal and systematic immunity against HPIV3. On the other hand, no significant immune responses were found in mice immunized with oligomannose coated liposome loaded with hemagglutininneuraminidase (OML-HN) without the adjuvant [47] . Henderson et al. found that nasal immunization with cationic liposomes complexed with noncoding plasmid DNA adjuvant and data suggested that considerable potent mucosal IgA antibody responses generated, as well as systemic IgG responses in mice [48] . Wang et al. developed a galactose-modified liposome which can be specifically recognized by macrophage antigen presenting cells. After mucosal immunization suggested that ovalbumin (OVA)-encapsulated galactosylated liposome induced significantly higher mucosal IgA and systemic IgG antibody titers and is a potential antigen delivery carrier for further clinical studies [49] . Recently a research group synthesized Salmonella Enteritidis Agcontaining pH-sensitive fusogenic polymer (succinylated poly(glycidol) (SucPG) and 3-methylglutarylated poly(glycidol) [MGluPG]) - engineered liposome formulation. After nasal immunizationtion with OVAcontaining SucPG-modified liposomes, significant Agspecific protective immune response was evaluated in the serum and intestine [50] . De-Veer with his co-workers worked on liposomesin-oil adjuvant formulations and concluded that the immune response was prolonged after vaccination. They have encapsulated diphtheria toxoids along with poly (I:C) in a liposome composed of lecithin and cholesterol. When this formulation was given subcutaneously, persistant immune response was generated. This may be attributed to the reduction in antigen transport to the draining lymph nodes [51] . At present, as reported previously [38] , at least 8 antigen containg liposomebased adjuvant systems are endorsed for human use or are under clinical trials stage (Table 1) [52–64] . Niosomes

Nonionic surfactant-based vesicles (niosomes) are self-assemblages of nonionic amphiphiles into closed

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Nanocarriers: a versatile approach for mucosal vaccine delivery 

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Fusogenic liposomes Cationic liposomes

Ligand-decorated targeted liposomes Plain

Polymer-coated liposomes Antibody-anchored liposomes

Liposome with surface-associated antigens

Ligand Polymerized liposomes

Polymer Viral component Antigen

Figure 5. Various engineered liposomes for mucosal vaccine delivery.

bilayer structures. Niosomes-based mucosal immunization offers all the benefits of DNA vaccines, and in addition, overcomes the disadvantages of classical invasive methods of vaccination [65] . Niosomes seem to be similar in terms of their physical properties to liposomes, being prepared in same methods and under a kind of conditions developing unilamellar or multilamellar structures. Niosomal-based mucosal vaccine research is recently expanding because niosomes are able to overwhelm drawbacks associated with liposomes, and surfactants are easily derivatized and give a higher flexibility to the vesicular structure and besides they have lesser costs than phospholipids [65,66] . Niosomal mediated vaccine delivery overcomes the poor immunogenicity of antigen in the progressive development of new vaccine adjuvant, or carriers that enhance the efficiency of vaccines [67] . More recently, niosomes were reported for potential intranasal immunization; the vesicles comprised either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine-rich peptide (DTK) for the stimulation of defensive immunity against genital herpes infection in mice [68] . Moreover, Spans, cholesterol and cetyl trimethyl ammonium bromide containing cationic niosomal formulations were explored for the induction of the immune response against leish-

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maniasis in BALB/c mice [69] . Vyas and co-worker synthesized polysaccharide o-palmitoyl mannan engineered niosomes as nanocarriers for oral delivery of DNA vaccine for the induction of potent humoral, cellular as well as mucosal immunity against hepatitis B in Balb/c mice. The rationale of this approach is to defend the niosomes from bile salt which was liable for decomposition and also from enzymatic degradation in GIT [70] . Jain et al. synthesized TT loaded mannosylated niosomes by the reverse-phase evaporation method using sorbiton monostearate (Span 60), cholesterol and stearylamine. Prepared niosomes were administered through oral route and immune response was evaluated by estimating serum IgG titer, IgG2a/IgG1 ratio in serum and sIgA levels in albino rats. The obtained data were compared with alumadsorbed TT following oral and intramuscular immunization, and it was concluded that mannosylated niosomes elicited a significant mucosal immune response (sIgA levels in mucosal secretions) as compared with alum-adsorbed TT and plain uncoated niosomes [70] . Bilosomes

Conventional vesicles like liposomes and niosomes have their own limitations because susceptibility to bile salt caused decomposition and enzymatic degradation

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Table 1. Selected liposome and lipid-based vaccines approved for human use or in clinical trials. Name

Company

Disease

Description

Status

Inflexal® V

Crucell

Influenza

Virosomes – reconstituted influenza viral membranes (phospholipids, hemagglutinin and neuraminidase) supplemented with PC

Marketed

[52,53]

Ref.

Epaxal®

Crucell

Hepatitis A

Formalin-inactivated hepatitis A virus adsorbed to virosomes

Marketed

[54,55]

Stimuvax

Merck KGaA, Oncothyreon

Nonsmall cell lung cancer

BLP25 (palmitoylated MUC1), MPL, DPPC, DMPG, Chol

Phase III

[56,57]

RTS,S/AS01

GlaxoSmithKline

Malaria

Recombinant fusion of P. falciparum circumsporozoite protein and hepatitis B surface antigen, PC, Chol, MPL, QS21

Phase III

[58,59]

Vaxisome

NasVax

Influenza

Inactivated influenza vaccine, CCS, Chol

Phase II

[60,61]

JVRS-100

Juvaris BioTherapeutics

Influenza

Inactivated influenza vaccine, DOTIM, Chol, noncoding plasmid DNA

Phase II

[62]

Vaxfectin

Vical

Influenza

Plasmid DNA-encoded influenza proteins, GAP-DMORIE, DPyPE

Phase I

[27,63]

CAF01

CAF01 Statens Serum Institut

Tuberculosis

Subunit protein antigen Ag85B-ESAT, DDA, TDB

Phase I

[64]

Reproduced with permission from [38] © Elsevier (2012).

in the GI tract remains as the main obstacle for their effective oral delivery and led to the progression of new engineered versions of vesicles [71] . Mann et al. synthesized nonionic surfactant vesicle having liposome- like structures and became stable though incorporation of bile salts for the oral delivery of vaccines. These bile salt stabilized vesicles named as ‘Bilosomes’. Bilosomebased DNA vaccine/antigen delivery elicited better both cellular as well as humoral immune responses which was equivalent to immune response produced by subcutaneous route [72] . Oral immunization of tetanus toxoid entrapped in bilosomes is capable of inducing a stronger T helper type 2 (Th2) response characterized by systemic IgG1 [73] . Consequently, bilosomes have been demonstrated to induce significant IgG2a production as well as IgG1 against viral influenza antigen [74] . Arora et al. formulated recombinant hepatitis B surface (HBsAg) antigen loaded mannan surface anchored bilosomes. After oral immunization obtained that modified bilosomes have shown dual action, in other words, provided more stability in GI tract as well as acted as specific targeting for antigen processing cells such as macrophages and dendritic cells. Finally it was concluded that investigated system to be more immunogenic in comparison to plain bilosomes alone as considerable anti-HBsAg immune responses were obtained [75] . Premanand et al. investigated that recombinant baculovirus displaying VP1 (Bac-VP1) with bilosomes could be a promising GI delivery vaccine candidate against EV71 infections. Oral delivery of Bac-VP1 significantly induced VP1-specific humoral (IgG)

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and mucosal (IgA) immune responses [76] . A research group recently synthesized TT loaded glucomannanmodified bilosomes (GM-bilosomes) and investigated the stability and potential of eliciting immune response. On the basis of obtained results it was concluded that GM-bilosomes exhibited excellent stability in different simulated biological fluids with significant higher systemic immune response (serum IgG level) as compared with plain bilosomes, niosomes and alum adsorbed TT administered through oral route [77] . Virosome

Virus-like particles consisting unilamellar phospholipids bilayered vesicle with reconstructed virus envelopes (viral genetic material absent) depict as a novel vaccine delivery candidate having size 150–200 (nm) with greater affinity for mucosal surface receptors [78] . Virosomes reconstructed from influenza virus envelope, retain their specific receptor recognition and binding characteristics with membrane fusion activity of the native virus due to presence of viral haemagglutinin (HA). Huckriede et al. reported the comparison of immune response between virosomes mediated vaccine delivery and conventional subunit vaccine. Finally it is concluded that virosomes as adjuvant stimulate a more balanced T helper 1 versus T helper 2 response and elicit more potent T helper 1 immune response than conventional subunit vaccine [79] . A study reported first time, after intranasal administration of virosomal influenza vaccine with heat-labile toxin adjuvant, and found potent elicitation of humoral as well as cell-mediated

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Nanocarriers: a versatile approach for mucosal vaccine delivery 

responses and potential to express stronger protection from influenza virus infection [80] . Another research group also investigated that trivalent virosome-based subunit vaccine with heat-labile toxin adjuvant offers total prevention from Influenza infection with greater immunological protection against homologous virus challenge [81] . Morgane et al. compared the immune protection after intramuscular and intranasal immunization with gp41-subunit antigens grafted virosomes against HIV-1. On the basis of obtained data it is concluded that all protected animals showed gp41specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular cytotoxicity activities [82] .

Key term Virosome: Drug-or vaccine-delivery mechanism consisting of unilamellar phospholipid membrane vesicle incorporating virus derived proteins.

infection and thus in primates, it is a potential and useful approach for enabling mucosal vaccines targeting M cell [88] . Nanoemulsion

Dendrimers

Nanoemulsion droplets produced by the dispersion of two immiscible liquids as suspensions with the aid of emulsifier display colloidal stability and are able to deliver encapsulated antigen straight onto mucosal surfaces. The size range of nanoemulsion is appropriate for the uptake by M cells of mucosa and consequently offered to APCs [89] . Emulsion-based Freund’s vaccine adjuvant is used as popular standard in development of new vaccine adjuvants [90] . Baker et al. studied the recombinant Bacillus anthraces protective antigen (rPA) vaccine-based nanoemulsion intranasally [91] . It was concluded that immune response was elicited only with intranasally administrated nanoemulsion-based vaccine and not with vaccine containing rPA alone. Both anti-rPA IgG and anti-rPA IgA antibodies were induced by intranasal immunization. Makidon et al. delivered recombinant HbsAg in the emulsified form to mucosal effector sites intranasally. The system produced high immune response, and elevated titers of both IgA and IgG. The response indicates the potential use of nanoemulsion for NALT mucosal immunization and thus nanoemulsion can be exploited as novel delivery system for immunizing the mucosal immune system [92] .

Dendrimers are tree-like synthetic, hyper branched macromolecular polymer-based nanosystem that consists of an inner core attached in series of branches which can be either hydrophilic or hydrophobic in nature depending upon the polymer. Dendrimers exhibit significantly improved physical and chemical properties and antigens can be either incorporated depending upon their nature into the core or attached to periphery [87] . Misumi et al. evaluated the M cells’ targeting ability of dendrimer based on tetragalloyl-D-lysine (TGDK), in in vitro human M-like cell culture models and in vivo nonhuman primate. It was found that TGDK was proficiently carried from the lumen of the intestinal tract into rhesus Peyer’s patches by M cells and subsequently accumulated in germinal centers. Orally administrated TGDK conjugated rhesus CCR5-derived cyclopeptide in rhesus macaque resulted in a noteworthy amplification in stool IgA response against rhesus CCR5-derived cyclopeptide. TGDK-mediated vaccine delivery produced a neutralizing activity against SIV

Conclusion & future perspective Mucosal vaccination is a good alternate to parenteral immunization with several advantages including ease of administration and patient compliance. Alike parenteral immunization, mucosal immunization provides both cellular as well as humoral immunization and can attack the pathogens at the entry portal only thereby maximizing the efficiency of immunization. Nanocarrires are seeking much interest now a days as a good cargo to carry bioactive to the mucosal site because of the unique properties like size, better interaction and penetration, sustained release and prolonged therapeutic effect. Researchers are exploring the field of mucosal immunization with nanotechnological approach in order to find out an effective drug delivery system against pathogens. Till date liposomes and engineered nanoparticles have gained much attention among different nanocarriers used owing to their biocompatibility, ease of manufacturing and better in vitro performances. Further studies are being carried out to lead these

SLNs

Solid lipid nanoparticles (SLNs) are efficient colloidal carriers prepared from solid lipids (e.g., triglycerides, fatty acids, etc.) and can integrate either lipophilic or hydrophilic drugs [83] . SLN can be utilized for the sustain release of antigen which results in a extended lasting immune response with negligible toxicity as compared with polymeric nanoparticles. Moreover, SLN can be exploited as an effective nonviral transfection agent [84] . Zhang et al. reported that the oral administration of wheat germ agglutinin modified insulin loaded SLNs can enhance the hypoglycaemic effect in rats [85] . In a study it was observed that intranasal administration of lipid microparticles loaded with HBsAg to female albino rat results in significant mucosal as well as systemic immune responses [86] .

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Review  Mody, Sharma, Agrawal & Vyas laboratory scale developments to approved marketed formulations. Regardless of this, there are many hurdles to be overcome in future as very little is known about the effect of nanoparticles on the human system. Also during the penetration process these nanocarriers might alter the microstructure of the mucus barrier and even after crossing the barrier neither they nor their by-product should cause any kind of toxic effects. A deep understanding of the nanocarriers on the mucosal immune system at cellular and molecular level is now needed.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Background • Mucosal surface maintains the normal physiology and also prevents the access of hazardous pathogens to body. • Innate and adaptive immunity comprises two arms of the mucosal immunity.

Routes of administration • Mucosal vaccines can be administered by oral, ocular, vaginal, rectal, pulmonary, nasal and sublingual routes. • Oral route is the most convenient and fascinating route for vaccine administration as it is possible to administer particulate antigens and these particulate antigens are better taken up by the Peyer’s patches present in the Gut Associated Lymphoid Tissue than the soluble antigens. • Nasal route is more permeable with less hostile environment, retains long term memory and produces faster immunity as compared with oral one because of the presence of dendritic cells.

Mechanism of mucosal vaccination • Cellular immunity is cell mediated immunity that does not involve antibodies, but rather involves the activation of antigen-specific cytotoxic T-lymphocytes. • Humoral immunity or antibody mediated immunity is mediated by macromolecule in humorus or body fluid. • M cells (or microfold cells) are the cells found in the follicle-associated epithelium of the Peyer’s patch as well as in Bronchus-associated lymphoid tissue. • Dendritic cells are antigen-presenting cells (also known as accessory cells) of the mammalian immune system.

Nanotechnology: a good alternative for mucosal immunization • Nanocarriers such as polymeric nanoparticles, nanocapsules, liposomes, noisome, bilosomes, micelles, dendrimers and nanotubes have been explored for the mucosal vaccine delivery. • Nanocarriers not only increase the drug concentration at the site but also work as an adjuvant to provoke the immune response and thus produce lager response as compared with conventional vaccines.

Nanocarriers: suitable delivery vehicles for mucosal immunization • Particle size, stability, hydrophobicity/hydrophilicity, crystallinity of polymers, bioadhesiveness and the nature of additives are the factors governing the performance of nanocarrier system 5

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