Nanomaterials for enhanced immunity as an innovative paradigm in nanomedicine.

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Nanomaterials for enhanced immunity as an innovative paradigm in nanomedicine

Since the advent of nanoparticle technology, novel and versatile properties of nanomaterials have been introduced, which has constantly expanded their applications in therapeutics. Introduction of nanomaterials for immunomodulation has opened up new avenues with tremendous potential. Interesting properties of nanoparticles, such as adjuvanticity, capability to enhance cross-presentation, polyvalent presentation, siRNA delivery for silencing of immunesuppressive gene, targeting and imaging of immune cells have been known to have immense utility in vaccination and immunotherapy. A thorough understanding of the merits associated with nanomaterials is crucial for designing of modular and versatile nanovaccines, for improved immune response. With the emerging prerequisites of vaccination, nanomaterial-based immune stimulation, seems to be capable of taking the field of immunization to a next higher level. Keywords: adjuvant • antigen • antigen presenting cells • cross-presentation • immune response • nanoparticle • vaccine

Immune response & nanomaterials Immune reaction initiates with recognition of pathogen followed by triggering of a series of nonspecific innate or specific adaptive immune response. Dendritic cells (DCs) are one of the specialized antigen-presenting cells (APCs), which present antigen to their cognate naive T-cell partners and educate them what to do with it. When DCs meet pathogen (as endogenous danger signals) or adjuvants (nanomaterials as engineered danger signals), they take up the antigenic sources, begin to mature and express the chemokine receptor CCR7, which allows them to migrate toward the nearest draining lymphatic vessels and lymph node. In the lymph node, DCs present processed antigenic peptides with costimulatory molecules to T cells to initiate an adaptive antigen-specific immune response. During the antigen processing and presentation, DCs use receptor pattern-recognition receptors (PRRs), which can sense either pathogen-derived or endogenous danger signals. By several PRRs,

10.2217/NNM.14.200 © 2015 Future Medicine Ltd

a pathogen may be recognized and the quality of the induced adaptive immunity is determined by the danger signals to which the DC is exposed. PRRs largely recognize pathogen-derived biomolecules referred to as pathogen-associated molecular patterns such as lipopolysaccharide (LPS) and viral double-stranded RNA. One of the major PRR classes is the Toll-like receptor (TLR) family, members of which recognize a large number of pathogen-derived ligands and a smaller number of endogenously derived ligands. For example, TLR4 expressed on the plasma membrane recognize LPS, while TLR7, TLR8 and TLR9, which are present in the endosomal compartment, recognize bacterial RNA or DNA. Other PRRs include cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which activate the DCs inflammasome in response to bacterial and endogenous danger [1] . Small molecules, TLR agonists and NLR agonists, are present in the form of patterns on the surface of pathogens that are recog-

Nanomedicine (Lond.) (2015) 10(6), 959–975

Anushree Seth1, Doo-Byoung Oh2 & Yong Taik Lim*,3 Graduate School of Analytical Science & Technology, Chungnam National University, Daejeon 305–764, South Korea 2 Biochemicals & Synthetic Biology Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon 305–806, South Korea 3 SKKU Advanced Institute of Nanotechnology (SAINT), School of Chemical Engineering, Sungkyunkwan University, Suwon 440–746, South Korea *Author for correspondence: Tel.: +82 312 994 172 Fax: +82 312 994 119 yongtaik@ skku.edu 1

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Review Seth, Oh & Lim nized by the TLRs, NLRs and other PRRs presented by the immune cells enabling them to distinguish between self and nonself [2–5] . Nanomaterials provide a platform for generation of such patterns on its surface that can be efficiently recognized by the immune cells. Despite the fact that the human body has a wellestablished immune system, in most of the cases, the immune cells are rendered inactive or tolerant by the pathogen or tumor cells [6,7] . The immune cells are either not able to recognize the pathogen because of the presence of a protective capsule or they are suppressed and disabled by released toxins [8–10] . In order to avert this situation, the immune cells can be trained ahead of time using prophylactic vaccination where specific antigen and adjuvant prepares the immune system to develop a robust immune response upon any future host-pathogen encounter. More specifically, prophylactic vaccines can induce the activation of T cells and B cells of the adaptive immune system, eliciting the differentiation of these lymphocytes into long-lived memory cells that will rapidly respond if the pathogen is encountered in the future. In other instances, where prophylactic vaccination seems to be impractical, therapeutic vaccination is preferred where immune stimulation helps suppressed immune cells in combating the disease. In yet another approach, the therapeutic immune cells which are activated and programed against specific disease are administered inside the body. In all the cases, the antigens and adjuvants that are defined as components capable of enhancing and/or shaping antigen-specific immune responses act as a representation of real pathogenic organism, so that the immune cells generate specificity for that particular pathogen. A closer look reveals that the structure of these pathogens resembles submicron or nm size range particles. Therefore, intuitively, if antigen and adjuvants are associated with nanoparticle-based systems or delivering agents such as nanoparticles, virus-like particles, nanoemulsions, dendrimers and liposomes, they are expected to be closer to real pathogens as compared with free soluble form (Table 1) . Lately, various interesting properties of nanomaterials have been exploited and engineered to create advanced materials for triggering both the arms of immune response (Figure 1) . Recent progress in the synthesis of multifunctional biodegradable/biocompatible nanomaterials has provided new momentum to translate discoveries from basic immunology into novel therapies and diagnostics for numerous diseases, including cancer, infectious diseases and autoimmunity. Novel synthetic nano- and microparticles have shown promise as potent adjuvant for vaccines, drug carriers for cancer immunotherapy [48,49] . These nanoagents could serve more than one

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purpose for more enhanced and effective immunization and immunotherapy. These remarkable features of nanomaterials are extensively discussed in this review article. Nanomaterials as vaccine delivering agents One of the most common utility of nanomaterials is in the form of delivering vehicle for therapeutic agents. Nanomaterials are used in vaccine delivery for imparting enhanced stability, encapsulation efficiency of unstable and small molecules and enabling controlled delivery. It has been found that nanoparticle-based vaccination results in the production of higher antibody titer as compared with free protein irrespective of the route of administration (oral, intranasal or subcutaneous) used [50] . Nanomaterials are also advantageous when delivery of an antigen in a tissue-specific manner (gastrointestinal, pulmonary or vaginal) is required to generate a robust localized, as well as systemic, immune response. Oral & other mucosaldelivery

Most vaccines not only require refrigeration or proper storage in order to maintain their activity and stability before administration but also need appropriate protection in vivo for maximum efficacy. Ensuring stability is particularly more crucial when labile biomolecules such as subunit vaccines, conjugate vaccines, cytokine, siRNA, DNA vaccines or recombinant vectors are used for immunization. A majority of antigens or adjuvants used for vaccination are biomolecules such as proteins, peptides, polysaccharides or oligonucleotides which are susceptible to degradation and inactivation inside the body. Nanomaterials are known to impart protection by encapsulating biomolecules and averting the detrimental effects of pH and enzymes when administered orally. The orally administered nanoparticle vaccines are capable of eliciting localized immune response in colon and vagina by coating them with specific combination of pH responsive polymer Eudragit® (Evonik Industries, Germany) [51] . DNA molecules can also be successfully delivered orally using nanoparticles [52] . Orally administered chitosan nanoparticle encapsulating DNA for a food allergen was able to induce production secretory IgA and serum IgG, capable of providing prophylactic protection against food allergy [53] . Nanoparticles aid in enhancement of uptake of antigen by the M cells in Payer’s patch and gut associated lymphoid tissue and thereby contributing to enhanced immunity [16,54–55] . Active targeting of M cells is achieved using nanoparticle coated with specific ligands such as ulex europaeus 1 (UEA-1) lectin or arginylglycylaspartic acid (RGD peptide) for effective vaccine delivery [11,17,56–57] .

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Table 1. List of nanoparticulate systems used for immune stimulation. Nanoparticle systems

Components

Ref.

Liposomes

Phospholipids

[11–15]

Polymeric nanoparticle

PLGA, chitosan, poly-anhydride, tri-polyphosphate, poly-propylene sulphide

[16–28]

Metal nanoparticle

Gold, iron oxide, manganese oxide

[29–35]

Nanoemulsion

Squalene, MF59, addvax, incomplete Fruend’s adjuvant

[36,37]

Virus-like particles

Noninfectious virus without genetic material

Self-assembled proteins

Ferritin

[38–40] [41]

Polymer hydrogel/nanogel Polymethylmethacrylate, poly-γ-glutamic acid, pullulan

[42–47]

PLGA: Poly lactic-co-glycolic acid.

Mucosal delivery of antigen is one of the critical issues in vaccination because mucus layer can act as a biological barrier to exogenous antigen, it will be important to devise ways to deliver antigens efficiently through mucus layer and induce strong mucosal immunity. Since most of the pathogen gain access inside the body of host by mucosal routes, administration of vaccine for adequate localized and systemic immune response is crucial [58,59] . For mucosal delivery, nanoparticles prepared with mucoadhesive materials such as chitosan or poly(gamma-glutamic acid) (γ-PGA) establish close contact with the mucosal tissues and enhance the residence time, mucosal uptake, thereby increase the bioavailability of the antigen [18,60–61] . Polyanhydride nanoparticles delivered intranasally are capable of inducing protective immune response against respiratory infectious disease in a single administration [62] . Moreover, nanoparticles can also efficiently deliver DNA vaccines at nasopharyngeal and pulmonary sites and augment the immunogenicity [63–67] . Other mucosal delivery routes, intranasal and sublingual routes, have been suggested, vaccine stability and possible brain toxicity issues should be addressed. In future studies, vaccine delivery systems that can transport antigen through mucus layer and induce effective immune responses with minimum antigen and adjuvant dose, shall be highly required. Mucosal delivery of antigen can be improved using mucus penetrating nanoparticles for local as well as systemic immune response [68] . Delivery to lymphatic system

Lymphatic vessels and lymph nodes are crucial targets for vaccine delivery as DCs after uptake of the antigen migrate to the lymph node where DCs prime other lymphocytes [69,70] . Size is an important parameter for controlling the kinetics of nanoparticles in lymph node. It was found that the 25 nm nanoparticle illustrated optimum adjuvanticity by traversing the lymphatics and interacting with the lymph


node residing DCs as compared with larger 100 nm nanoparticles. Interestingly, complement activation was observed by modulating the surface of these nanoparticles [71] . Lipid-based nanoparticles have been found to traverse through the lymphatics, thus, emerging as a promising nanomaterial for ensuing powerful immune response [72–74] . The significance of lymphatic delivery in tumor immunotherapy is emphasized in a recent report illustrating anti-tumor immune response generated by adjuvanted nanoparticles directed toward tumor draining lymph node [75] . Recently, it has been shown that amphiphilic vaccines (5–20 nm micelles) consisting of albumin binding domains drastically enhances the lymph node accumulation of subunit vaccines [76] . Interestingly, this approach harnesses the lymph node-targeting capability of an albumin bound nanomicelle. In another innovative approach nanoparticle made up of heparin and chitosan was used for delivery of cytokine (TNFα or IL-12) to the lymphatics, mimicking the mechanism of naturally occurring mast cell granules for immune modulation [77,78] . Other benefits of using nanomaterials as a delivery agent

Exploiting the presence of large number of Langerhans cell or immature DCs beneath the epidermal layer of skin has led to the development of intradermal, subcutaneous and transcutaneous vaccine delivery strategies [79,80] . Nanomaterials are known to deliver vaccines by these routes of administration and are successfully taken up by the residing DCs for amplified immune stimulation [42,81–82] . Simultaneous delivery of antigen and adjuvant is also crucial for efficient immune response, which could be achieved by coencapsulation of antigen and adjuvant in a nanoparticle. Codelivery of antigen and adjuvant using a 500 nm hydrogel particle in an APCs drives enhanced activation as compared with soluble protein [83] . In case of cell-based therapy, nanoparticles

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Polyvalent presentation for B-cell activation

Cancer cells Bacteria

B cells

Antibody production B cells

Prophylactic vaccine

Virus CD4 T

BCR cross-linking

Humoral immunity

MHC II MHC I

Immature DCs

CD8 T

Activated DCs

Nanoparticles for programming of DCs

CTL

Virus clearance, cancer therapy

Therapeutic vaccine

Cellular immunity

Nanoparticles for cross-presentation

Figure 1. Triggering and modulation of immune responses. Immature DCs were matured and activated by the cancer cells or pathogens (virus, bacteria). Activated DCs present antigens (through MHC I and MHC II pathway) to naive T cells and two types of helper T cells (CD4 and CD8) subsequently activate B cells and CTLs, respectively. Finally, humoral immunity which is related to antibody generation (prophylactic vaccine) and cellular immunity to viral clearance or cancer immunotherapy (therapeutic vaccine) are induced. Nanoparticles can be used to modulate (i.e., programing [activation and maturation] of DCs, induction of MHC I pathway and efficient B cell receptor [BCR] crosslinking) the immune response. CTL: Cytotoxic T lymphocyte; DC: Dendritic cell.

where the cells are manipulated ex vivo, efficient delivery of antigen and adjuvant inside the cell is important. Nanoparticles are readily phagocytosed by APCs and thus aid in desired modulation of APCs [84] . Another beneficial outcome of using nanoparticle for vaccines delivery is significant reduction in antigen dose required for protective immunity [43] . Nanoparticle carriers are known to stably deliver DNA encoding for specific ligands in combination with TLR agonist for dramatic anti-tumor immune response [44] . Self-assembled nanoparticles have also emerged a novel platform for delivering of labile protein antigen in APCs [12,36,45,85] . Various reports demonstrate that antigen encapsulated in nanomaterials illicit enhanced cellular uptake as compared with free antigen. Interestingly, nanoemulsions enable DCs to engulf antigen-primed epithelial cells, thereby augmenting the overall antigen uptake by DCs [13] . A wide range of materials are being explored for designing nanocarriers for vaccine delivery. Liposomes prepared from polar lipids derived from nonpathogenic bacteria have been known to efficiently fuse with APCs, leading to potent immune response [86] . Liposomal formulation for localized delivery of monoclonal antibody anti-CD137 and cytokine IL-2 in tumor significantly reduced the inflammatory toxicity [87] . Using nanoparticles not only enhances the potency of the immune

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response generated but is also capable of producing long lasting B-cell-mediated immune response due to formation of germinal center in the lymph node [88] . Nanoparticle-based systems for delivery of TLR agonists reduce the systemic exposure of TLR agonists and minimize the undesirable systemic cytokine secretion and associated toxicity [89] . The release rate of antigen has a significant role in the intensity and nature of immune response generated. The depot effect due to emulsion-based vaccines or polymer matrix results in sustained release of antigen and adjuvant resulting in recruitment and programing of APCs [19,20] . However, too slow release of antigen and prolonged persistence of antigen can lead to T-cell apoptosis leading to insufficient immune response [20] . Therefore, controlling the rate of antigen release is an important consideration for vaccine formulation development which can be readily modulated using nanoparticles of different size and material. Nanomaterials as adjuvants Physicochemical properties of nanoparticles & immune response

The presence of nanomaterial without any adjuvant can in itself contribute to immune stimulation. Various polymeric materials that are used to synthesize nanoparticles have been known to possess adjuvant



properties. Biodegradable nanoparticles made up of a variety of polymers such as poly lactic-co-glycolic acid (PLGA), N-trimethyl chitosan and tripolyphosphate, contribute differentially to immune modulation [90,91] . Cross-linked polymethylmethacrylate hydrogel nanostructure is shown to be capable of causing in vitro and in vivo activation of CD4 T cells and CD8 T cells [45] . Interaction of chitosan nanoparticles with lymphocytes causes cytokine production, lymphocyte proliferation [92] . In another study, N-trimethyl chitosan nanoparticles elicited robust antibody response against influenza antigen after intranasal administration [18] . Chitosan is a generally regarded as safe (GRAS) material and chitosan nanomaterials have been explored for immunomodulation since long time. However, the material is not approved by US FDA possibly due to variability in purity, dosage-dependent toxicity and differential biodistribution and toxicity profiles [93] . Despite promising results in animal experiments, chitosan nanomaterials need to be extensively verified by the regulatory agency for clinical translation [45] . Various properties of nanomaterials such as size, hydrophobicity, molecular weight, surface charge, degradation profile and source or origin biomaterial, are known to play a role in adjuvanticity. Hydrophobic materials released from dying cells are considered to be conventional damage-associated molecular patterns which trigger the immune response [21] . A systematic study using gold nanoparticles with varying surface hydrophobicity revealed a direct correlation between surface hydrophobicity and cytokine mRNA expression levels as a measure of immunogenicity [22] . In another report, nanoparticles made up of amphiphilic polymeric material (hydrophobically modified γ-PGA) acted as an adjuvant for enhanced cellular immune response against (HIV)-1 gp120 [12] . In addition to hydrophobicity, surface chemical moieties such as carbonyl groups, hydroxyl groups and alkyl ethers are known to contribute to pathogen-mimicking characteristics of nanoparticles. Bioinformatics analysis revealed a complex interplay of surface properties led to the formation of distinct molecular pattern which causes immune stimulation in APCs [94] . The key idea is to functionalize the nanoparticle in such a way that it resembles a pathogen as closely as possible. Some of such pathogen-mimicking approaches includes coating of nanoparticle with carbohydrates such as galactose, and mannose which are found on the surface of pathogen. Such functionalized nanoparticles have been used for targeting C-type lectin receptors present on the surface of APCs [23,95–96] . Nanoparticles coated with specific danger signals or functionalities are known to activate NOD-like receptor pyrin 3-containing domain inflammasome contributing to adjuvanticity of nanoparticles [97–99] .


Review

In addition to surface chemistry, the overall geometry of the nanoparticle also contributes to adjuvant effect of nanomaterials. It has been found that polymers have higher level of adjuvanticity in the form of nanoparticles as compared with polymeric films [100] . Also, the size of the nanoparticle used for vaccination has major role to play in terms of adjuvanticity [101,102] . Since the different formulations of the same immunostimulant adjuvant materials are capable of generating varying immune responses, care should be taken in the preparation of adjuvant materials. In future studies, rational selections of suitable adjuvant and administration route that will optimally activate immune cells for desired immune response shall be crucial factor in the success of vaccine adjuvant. Immunomodulation by nanoparticles containing siRNA

Immunomodulation of DCs by delivering siRNA is an emerging strategy for immune modulation by nanoparticles. Silencing of undesirable genes can be achieved by using RNA interference or siRNA [103] . In a recent study, immunosuppressive gene in DCs SOCS1 (suppressor of cytokine signaling 1) was silenced using siRNA and combination of siRNA with tumor antigen and adjuvant using PLGA nanoparticles lead to dramatic anti-tumor immune response [104,105] . The strategy seems to have immense potential in DCs based immunotherapy for cancer. Another target for siRNA is signal transducer and activator of transcription 3 (STAT3) which is known to inhibit production of anti-tumor immune response by immune cells in the presence of tumor cells [24] . Targeting STAT3 in DCs using siRNA-nanoparticle has illustrated encouraging anti-tumor immune response [106,107] . TGF-β is another regulatory cytokine known to generate an immunesuppressive environment in tumor [108,109] . Silencing TGF-β gene using nanoparticle based siRNA technology, leads to reduction in number of regulatory T cells in tumor and significant enhancement in efficacy of vaccination [110] . Expression of programed death ligand 1 (PD-L1) on the surface of immune cells results in negative regulation of the immune system [111] . siRNA lipid nanoparticles have been shown to be capable of silencing PD-1 ligands and improving the immunogenicity of mRNA vaccine [29] . Since the phenomenon of RNA interference occurs in the cytosol, for effective gene silencing, delivery of the target siRNA in the cytosol is essential. Programed nanoparticles modified with pH dependent fusogenic peptide which were capable of efficiently escaping from endosome have demonstrated superior silencing of gene in DCs as compared with uncoated nanoparticle [30] .


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Review Seth, Oh & Lim Although, siRNA treatment is associated with limitations such as in vivo stability and bioavailability, using siRNA nanoparticle seems to have immense potential for opposing the immunesuppressive milieu in diseased condition. Nanomaterials for polyvalent presentation of antigen & adjuvant Any macroscopic material when transformed to a nanoscale dimension is accompanied by remarkable increase in the total surface area of that material. The large surface area of nanoparticle can be exploited for the conjugation and presentation of various antigens and adjuvants. The polyvalent presentation of antigen and adjuvant assists immune cells by unique recognition and triggering of immune response as compared with free soluble form. Recently, it has been shown that CpG-ODN (cytosine triphosphate deoxynucleotide [‘C’] followed by a guanine triphosphate deoxynucleotide [‘G’]-oligodeoxynucleotide) which is a TLR9 agonist when conjugated on the surface of nanoparticle resulted in enhanced immune stimulation in macrophages as compared with free CpG-ODN [112,113] . The concept of polyvalent nanoparticle leading to enhanced immune response is inspired by biological entities present in nature where polyvalency enhances the affinity and strength of interaction [114] . This concept is of particular importance in boosting humoral immunity as interaction of multiple antigens in close vicinity with the B-cell receptor is known to cause receptor cross-linking, which subsequently leads to alteration in B-cell proliferation, intracellular signaling and activation [14,31,115] . The use of nanoparticles for the presentation of antigen in a polyvalent manner has shown to augment humoral immune response and produce a protective immune response against both viral and tumor challenge [32–33,116–117] . The size of the nanoparticle used for display of antigen or adjuvant affects the intensity of immune response generated. In a size-dependent study, it was found that the gold nanoparticle of size range 8–17 nm facilitated maximum antibody binding [118] . In other studies, 15 nm gold nanoparticles conjugated to CpG ODN illustrated optimum immune stimulation [41,112] . For eliciting powerful immune response, it is crucial that the immune cells recognize the epitope on antigen and produce antigen-specific antibody. It has been known that antibody against conserved regions of particular protein antigen has the potential to provide protection against a broad spectrum of pathogens, particularly viruses which are prone to frequent mutations. In case of influenza virus the conserved regions of hemagglutinin (HA) protein is capable of eliciting immune response across different

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strains of influenza virus [38] . Nanomaterials provide a platform for controlling the epitope of the protein antigen exposed to the immune cells by modulating the conformation of the proteins during conjugation. Single epitope presenting nanoparticles could also be generated by using self-assembled proteins such as ferritin which was capable of presenting conserved regions of HA in trimeric form [39] . In a yet another approach, polyvalent presentation of antigen could also be achieved by using bacteriophage nanoparticles and virus-like particles [25,40,119] . Although polyvalent presentation of antigen and immunostimulatory components is very promising concept to induce strong immune response, the synthesis or fabrication of vaccine system having repetitive and ordered nanoscale pattern (for example, virosome) requires multiple preparation steps. Antigen presenting nanoparticle combined with a danger signal is expected to be acting in a pathogen-mimicking manner [120,121] . All the features of a prospective pathogen such as nanoscale size range, antigen and adjuvants displaying as molecular patterns on the surface, lipid bilayer and danger signal can be incorporated in a single nanoparticulate system to elicit a robust immune response. Future studies are needed to device an easy and robust chemical strategy to realize such polyvalent nanoscale repetitiveness on nanoadjuvant materials. Nanomaterials enabling endosome escape for cytotoxic T lymphocytes response Th1 response is crucial for triggering cytotoxic T lymphocytes which have the ability to kill the virus infected cells and tumor cells directly. T-cell receptor is specific for recognition of antigen peptide associated with MHC I molecule on APCs. For cross-presentation or an exogenous antigen to be associated with MHC I, cytosolic delivery of antigen in the APCs is crucial. While most of the nanoparticles after endocytosis are either trapped or undergo degradation inside the endosome; delivery of antigen in the cytosol could be facilitated by imparting nanoparticles with unique properties to escape the endosome. At the acidic pH of endosome, amphiphilic nanoparticles demonstrated membrane-disruptive properties, indicating their potential for cytosolic delivery of antigen [122] . Using biodegradable PLGA nanoparticle for encapsulation of model antigen led to enhanced and sustained crosspresentation in bone marrow derived DCs [123] . Adoptive transfer of T cells which were activated by DCs treated with PLGA nanoparticle encapsulating antigen, exhibited effective anti-tumor response [124] . The endosomal escape of the PLGA nanoparticle is expected to be due to surface charge reversal at the endosomal



pH followed by interaction with the endosomal membrane [125] . PLGA particle coated with endoplasmic reticulum-targeting peptide also demonstrated endosomal escape and cross-presentation in vitro [126] . Poly (γ-glutamic acid) nanoparticles were shown to induce endoplasmic reticulum and endosomal fusion mimicking the classical presentation of antigen to MHC I [37] . Nanoparticles and micelles are modified using cationic polymers such as polyethylene imine, protamine sulphate, poly-l-lysine to enable efficient interaction with negatively charged cell membrane and allow cross-presentation [34,127–130] . Lately, poly(propylene sulphide) has emerged as a novel material for nanocarrier design capable of eliciting cross-presentation and cytotoxic T-cell response [35,131] . In yet another approach, 100-fold enhancement in cross-presentation was observed by using alpha-alumina nanoparticles which facilitated the breakdown of cellular components or autophagy in DCs [132] . Nanoemulsions are also capable of eliciting delivery of antigen in the cytoplasm and subsequent cross-presentation [133] . In nature, the surface proteins of viruses undergo conformational change in acidic pH, allowing them to fuse with endosomal membrane and subsequently escape into the cytoplasm [134–136] . Inspired by this concept, several researchers have used fusogenic peptides for facilitating endosomal escape of nanoparticles [26,137] . Nanoparticles demonstrating pH responsive properties have shown to be beneficial for cross-presentation and cytotoxic T lymphocytes response [46] . Since most of nanoparticle carriers for cytosolic delivery include membrane-disrupting properties, parallel systemic immunotoxicity studies need to be conducted when some nanomaterial is adopted for cross-presentation. Nanomaterials for targeting of immune cells Immune cells express specific surface markers which can be targeted by using specific targeting ligands such as small molecules or antibodies thereby, enabling the nanoparticles to specifically interact with desired cells and enhance therapeutic efficacy [138,139] . For passive targeting of DCs in vivo, 20–45 nm size nanoparticles were found to be optimum for traversing the lymphatic and nonspecifically phagocytosed by DCs without any targeting ligand [47] . DCs can also be targeted actively by using nanoparticles conjugated to targeting ligands which are specific to DCs. DC immunoreceptor, Fc gamma RII and DEC205 have been identified as DC-specific receptors which help in uptake and cross-presentation of antigens [27,140–142] . Careful selection of target receptor on DCs is crucial for desirable immune response. In a study, it was found


Review

that C-type lectins DEC-205, DC immunoreceptor, blood DC Ag-2 or the FcR CD32 are favorable selections for in vivo DC targeting [143] . It has been shown that nanoparticles are more effective in targeting DCs as compared with larger microparticles, when coated with ligand specific for C-type lectic DC-sign present on the DCs [144] . B cell targeting using nanoparticle conjugated to monoclonal antibody CD20 Ab (rituximab) and antisense G3139 is found to be advantageous for overcoming the nonspecific immunostimulatory effects of CpG [145] . Liposomal formulation containing antibodies against CD19, CD20 and CD37 was found to have improved pharmacokinetic profile, B cell targeting and therapeutic effect in leukemic mice [146] . Nanoparticlebased selective targeting of T cell is also achieved by conjugate peptide MHC complex on the surface of nanoparticles [147] . Liposome-mediated targeting of adoptively transferred T cells demonstrated enhanced effector T-cell function and efficient anti-tumor immunotherapy [148] . The importance of targeting was accentuated in a study in which it was found that the lineage of T cells can be controlled using targeted nanoparticle conjugated with antibodies against CD4 for delivery of cytokines [149] . Future studies are required to target specific DCs, the most potent professional APCs of the immune system. Because different DCs subsets have been reported with varying expression of TLRs and capacities to induce different types of adaptive immune responses, the specific targeting of DCs by specific targeting ligand and rational selection of administration route could lead to generation of tailored immune response. Nanomaterials for imaging of immune cells Various imaging modalities such as fluorescently labeled nanoparticle, nanoparticles loaded with quantum dots, magnetic nanoparticle by MRI and gold nanoparticle by CT imaging, can be used for the labeling and tracking of therapeutic immune cells [106,117,150] . Lymph nodes are specialized immune organs where majority of the antigen carrying APCs migrate and prime the effector B cells and T cells. Simultaneous imaging and tracking of nanoparticle carrying therapeutic cells assists in ensuring their movement to lymph nodes which helps in evaluating the success of immunotherapy [117,151] . Magnetic nanoparticles are one of the conventionally used MRI agents for imaging of APCs [28,152–153] . Magnetic nanoparticles are endowed with functionalities which enable them to image-specific intracellular regions [154] . 19F MRI based nanoparticles and nanoemulsions have been successfully used for imag-


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Pathogen mimicking

Virus (20–200 nm)

Bacteria (500–5000 nm)

~100 nm Adjuvanticity ~25 nm Size of nanomaterial

Lymph node trafficking

Figure 2. Importance of size of nanoparticle for immunomodulation. Pathogen mimicking: size of nanomaterial falls in the same range as that of pathogen (virus and bacteria). Adjuvanticity: smaller size (∼25 nm) nanoparticles are known to traverse through the lymphatics to reach the lymph node unlike the larger size (∼100 nm) nanoparticle. Lymph node is a crucial site for antigen uptake by APCs and priming of immune cells (T cells and B cells). Size of nanoparticle affects the cellular uptake of nanoparticle by APCs, thereby influencing the adjuvanticity of the nanoparticle.

ing of therapeutic immune cells [155,156] . In order to overcome the limitations associated with individual modalities and to increase the sensitivity and resolution of the imaging probes, nanoparticle-based multimodal imaging modalities are being developed. Successful tracking and labeling of therapeutic immune cells was demonstrated by combining various imaging modalities such as optical, near infrared and MRI using nanomaterials [157–160] . Imageable nanoparticles are also used to study the trafficking and biodistribution of DCs, macrophages and specific T lymphocytes in vivo, homing to lymph node and spleen and migration toward tumor [15,161] . The kinetics of movement of T lymphocytes helps in generating a better understanding of cytotoxic T-cell response in tumors [162] . Moreover, codelivery of therapeutic agent along with imaging of T cells could efficiently be performed by using nanosystems such as biodegradable nanoparticles, liposomes or dendrimers [163] . In cell-based therapy for tumors, various aspects such as priming, activation and imaging can be pooled in a single nanoparticle system for imparting enhanced functionality to therapeutic cells [104,164] . Future studies are needed to assess the final fate of

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nanomaterials used for the labeling and trafficking of target immune cells. Conclusion & future perspective This review summarized how synthetic and engineered nanomaterials have been used as delivery systems or adjuvants for regulation, amplification and qualitatively alteration of vaccine immune responses (Figure 2) . The development in the synthesis of multifunctional nanoparticles has led to emergence of novel concept of immunotherapy for the treatment of wide variety of diseases such as cancer, infectious disease, autoimmune diseases and cardiac diseases. Recently, erythrocyte membrane-coated nanoparticles and staphylococcal α-hemolysin were introduced as an effective nanomaterial for capturing of virulent toxins [165] . However, this rapidly moving field is still in very early stage and several aspects should be considered for the generation of nanomaterials having clinical relevance. In the design of nanomaterials used for immunotherapeutics, the design principles should vary in accordance with the final purpose or application. For instance,



complicated nanostructured materials may be contemplated and cost does not present a major consideration in therapeutic vaccines against cancer. However, several aspects such as wet formulations, unrefrigerated storage, needle-free administration routes, easy formulation and minimal number of biological molecules at low cost, should be crucial factors that must be taken into account while designing nanomaterials for the treatment and prevention of infectious disease, which mostly requires mass immunization. Although early developments in cancer vaccine (i.e., Provenge [Dendreon]) and a few nanomaterialsbased vaccine adjuvants (Alum; MF59) have shown some promising prospects for the clinical translation,

most of the immunomodulatory strategies often target complex pathways involved in regulating both immunity and tolerance, which suggest that the path for clinical translation may be substantially longer than for small-molecule drugs with simpler mechanisms of action. Therefore, several challenging issues should be addressed before immunomodulation by nanomaterials becomes a reality. The biggest challenge that nanomaterials face at present is meeting all the safety guidelines required for gaining clinical acceptance, particularly those required by the FDA and other regulatory guidelines. Over the past decade, various nanoparticle/nanomedicine platforms have been screened and studied in terms of their size, shape and surface properties

Nanomaterial as a vaccine-delivering agent Degradation/ inactivation Free antigen/adjuvant

Nanomaterial for immunostimulation Enzyme and acid

Hydrophobicity/surface chemistry

Oral delivery Controlled release/ depot effect

Review

Immunomodulation Size Nanoparticle

Antigen uptake

I Polyvalent presentation

Codelivery

Mucosal delivery

MHC I TCR J Endosome escape and cross-presentation

Figure 3. Schematic depicting promising features of nanomaterials for immunomodulation. (A) Nanoparticle imparts protection from degradation to labile biomolecules in physiological environment inside the host body. Free or soluble form of antigen or adjuvant is more susceptible to degradation or inactivation due to lack of any protective shield. (B) The protective ability of nanoparticle makes it a promising platform for oral delivery of vaccines. (C) Nanoparticle-based vaccines release the vaccine in a controlled or sustained manner resulting in depot effect for enhanced and prolonged immunization. (D) While intracellular delivery of free antigen or adjuvant into the cell is difficult to achieve, nanoparticle-based formulations lead to dramatic increase in antigen uptake. (E) Nanoparticle-based formulations ensure codelivery of the components encapsulated (antigen and adjuvant) in a predetermined ratio inside the cell as compared with differential delivery of antigen and adjuvants in case of soluble counterparts. (F) Mucoadhesive and mucus-penetrating nanoparticles enable mucosal delivery of vaccines while free form of vaccine is at risk of being washed away without penetration. (G & H) Parameters such as nanoparticle hydrophobicity, surface chemistry and size, are amenable to modulation for controlling the intensity and type of immune response. (I) Nanoparticles provide a platform for polyvalent presentation of antigen and adjuvant leading to improved immunogenicity. (J) Nanoparticles are capable of escaping the endosome thereby, delivering the antigen in cytosol which is essential for cross-presentation.



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Review Seth, Oh & Lim to meet these guidelines and market request. Clinical translation of nanomaterials for immunomodulation requires a complete understanding of particle size, composition, formulation, internal and external structure, chemical reactivity and stability and their interaction with the human body, such as biodistribution, toxicity and biocompatibility. In fact, nanomaterials can also be functionalized with biomolecules, enabling them to target specific organelles within certain tissues or even the entire cells for localization in the targeted area. Due to the nanosize, they are normally composed of thousands of atoms with a high surface area so that a higher and multifunctional therapeutic payload (antigens and immunostimulatory components) can be carried to or encapsulated in the nanostructure. Because such nanostructured materials are often multimodular, thorough and systematic investigations are necessary to examine their mechanism(s) of action and impact on the immune system. In fact, previous studies strongly suggest that nanomaterials do interact with the immune systems, there has been limited guidelines for assessing their possible immunotoxicity. To increase clinical relevance of nanomaterials-based immunomodulation technology, standard immunotoxicological methods to investigate various aspects of immunotoxicity of nanomaterials should be addressed. Toxicity associated with nanomaterials such as generation of reactive oxygen species by metal nanoparticles, allergic sensitization, hypersensitivity and induction or augmentation of autoimmune disease must be considered before clinical application of nanovaccines [166,167] . Concentrations of cytokines in the systemic blood circulation are considered as one the biomarkers to test the efficacy of nanovaccines [168] . In addition to the safety issues, the process of scaling up and manufacturability should not be ignored. Although the incorporation of various components into a single nanoparticle vaccine provides versatility in clinical applications, it also requires the need for sophistication in the production process. Large-scale production and distribution of vaccines will require nanoparticle platforms that are robust and reproducible. While several approaches using lithography and microfluidics are encouraging, they are at early stages of development and need further optimization. The requirement for multiple chemical and immunological components with layers of synthetic chemistry process technology could lead to substantial production costs. In this regard, a careful cost–benefit analysis should be taken into account for the real clinical application of nanomaterials for immunomodulation.

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Designing long-circulating particles conjugated with monoclonal antibodies or other targeting agents that can successfully target critical APCs residing in the spleen, lymph nodes and bone marrow, seems to be a promising area in nanomaterial-based immunomodulation. Monoclonal antibody technology conjugated to nanomaterials for vaccination is relatively unexplored and seems to have tremendous potential for developing specialized vaccination strategies by targeting specific immune cells. Especially, leukocytes can be attractive targets for targeted particle therapies as they are present in large numbers in lymphoid organs. Deriving inspiration from nature, nanomaterial design should evolve in such a way that it is able to imitate the disease causing pathogen required for directed and pathogen specific stimulation of immune response. Since the nanomaterials for immunomodulation can have many different shapes and chemical compositions, the effects of nanoparticle properties on the immune system should be systematically explored in order to find out whether it stimulates the immune system or induces undesirable side effects Finally, the scientific gaps between engineered nanomaterials and their immune responses should be systematically studied. For example, more work must be done in understanding the relationship between the endocytosis and intracellular trafficking of nanoparticle systems and resultant immune responses. By the rationally design nanomaterials with specific size, shape, morphology and surface functionality, tailored immune responses such as the balance of cellular and humoral immunity, the ratio of effector to memory T cells, the functional properties of activated T cells, can be generated (Figure 3) . Although, the field of materials-based immunomodulation for the cancer and infectious disease is still in an early stage and significant advances are yet to be made, potent prophylactic and therapeutic vaccines can be generated by facilitating closer collaborations between scientists in immunology and engineers in materials. Financial & competing interests disclosure This research has been supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST; no. 2012M3A9C6050070 and 2013M3A9B6075888), the Korea Health Technology R&D Project, Ministry of Health & Welfare (no. A111918). The authors have no other 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 apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary • Nanoparticle technology has demonstrated capability to enhance innate and adaptive immunity, for both therapeutic and prophylactic immunization. Exploiting the fact that nanoparticles fall in the same order of size range as that of disease causing pathogen, nanoparticles seem to possess unique characteristics for augmenting immune response. • Nanoparticles as a vaccine delivery vehicle for imparting stability, controlled delivery, enhanced vaccine uptake, reduction of vaccine dose, co-delivery of antigen and adjuvant. Nanomaterials are desirable carriers for vaccine delivery through oral, mucosal, subcutaneous and intradermal routes. • Nanoparticles possess adjuvanticity by modulating the physiochemical properties such as material, size, surface chemistry, hydrophobicity for modulation of type and intensity of immune response. Recently, delivering siRNA using nanomaterials for silencing of immunesuppressive genes such as SOCS1, STAT3, TGF-β, PD-L1 has emerged as innovative approach for immunestimulation. • Polyvalent presentation of antigen and adjuvant enables B cell receptor cross-linking for eliciting improved humoral response. Harnessing the properties of nanoparticles for designing of patterns of danger signals or antigens in a defined conformation is intended to mimic pathogen-associated molecular patterns present on pathogen. • Nanoparticles for simultaneous imaging, priming and tracking of therapeutic immune cells, help in confirming the success or failure of immunotherapy. Moreover, with the help of nanomaterials other immune cells such as T cells biodistribution and kinetics of movement in the tumor helps in getting a deeper insight in cyto-toxic T-cell response. • Nanomaterials aid in endosomal escape by using pH responsive materials, fusogenic peptides, endoplasmic reticulum targeting or materials that enable surface charge reversal at acidic pH or autophagy. These nanoparticles mediated mechanisms help in augmenting T-cell-mediated immune response which is crucial for combating tumors and viruses. • For increasing the specificity of vaccine carrier active and passive targeting of immune cells (APCs, B cells, T cells) is beneficial. DC immunoreceptor, Fc gamma RII, DEC 205, DC Ag-2 and FcR CD32 are known potential targeting ligands for active targeting of dendritic cells. • In conclusion, nanomaterials have emerged as versatile, accessible and amenable platform for improved and effective vaccination. Systematic analysis of nanoparticle-based immune modulation and immune-toxicity is required for realizing clinical translation of nanovaccines.

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