nanoparticles: Particle design and potential vaccine delivery applications

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Polymeric micro/nanoparticles: Particle design and potential vaccine delivery applications

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Hua Yue a , Guanghui Ma a,b,∗ a

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National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China

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a r t i c l e

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Article history: Available online xxx

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Particle based adjuvant showed promising signs on delivering antigen to immune cells and acting as stimulators to elicit preventive or therapeutic response. Nevertheless, the wide size distribution of available polymeric particles has so far obscured the immunostimulative effects of particle adjuvant, and compromised the progress in pharmacological researches. To conquer this hurdle, our research group has carried out a series of researches regarding the particulate vaccine, by taking advantage of the successful fabrication of polymeric particles with uniform size. In this review, we highlight the insight and practical progress focused on the effects of physiochemical property (e.g. particle size, charge, hydrophobicity, surface chemical group, and particle shape) and antigen loading mode on the resultant biological/immunological outcome. The underlying mechanisms of how the particles-based vaccine functioned in the immune system are also discussed. Based on the knowledge, particles with high antigen payload and optimized attributes could be designed for expected adjuvant purpose, leading to the development of high efficient vaccine candidates. © 2015 Published by Elsevier Ltd.

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Keywords: Micro/nanoparticles Adjuvant Physiochemical property Preventive/therapeutic vaccine Antigen loading Immunological mechanism

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1. Introduction

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1.1. Adjuvants: avenue to the next-generation vaccine

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Vaccination is the most effective and economic way to prevent infection and severe outcomes caused by bacterial, viruses, or other pathogen. Owing to hundred kinds of vaccines, global death has decreased, along with cost for treatment for infectious diseases. The development of vaccine products was accompanied with rapid advancement of biotechnology, immunology, and biomaterials. The first generation of vaccine was dated from 1796, when Edward Jenner inoculated healthy people with attenuated live cowpox virus, opening the era of vaccination. By the 1940s, the vaccines were developed when passage in cell culture permitted the selection of attenuated mutants. Following that era, heat or chemical inactivation was applied to pathogens (e.g. typhoid, plague, and so on) to reduce the toxicity while maintaining the immunogenicity [1]. Although live or inactivated vaccines are capable of eliciting robust

∗ Corresponding author at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 Bei-Er-Tiao, Zhong Guan Cun, Beijing 100190, China. Tel.: +86 1082627072; fax: +86 1082627072. E-mail address: [email protected] (G. Ma).

immune response with the assist of endogenous adjuvant (nonspecific pathogenic component), the safety concerns has limited their clinic use, especially when extremely dangerous pathogens, such as human immunodeficiency virus (HIV) and hepatitis virus, were involved [2]. Therefore, the modern generation of genetically engineered vaccine or subunit vaccine, which have defined components, good stability and high safety, emerged from the recombinant hepatitis B virus vaccine in 1986. However, most of the new generation vaccines suffer from shortcoming of poor immunogenicity, and the exploration of high-performance adjuvant has become a rapidly expanding research area [3]. In addition to the progress of preventive vaccines for healthy people, vaccine-based treatments of chronic infected diseases and malignant tumors have attracted tremendous interests in the recent decades [4,5]. Compared with conventional chemotherapy or radiotherapy, vaccines strategy possesses relatively higher specificity and lower side effect. For example, the first Food and Drug Administration (FDA)-approved tumor vaccine (Provenge) could suppress metastatic castration-resistant prostate cancer by activating the power of the patient’s own immune system [6]. A crucial requirement for therapeutic vaccine is to elicit sufficient cellular immune responses against the target cells, thus not only the strength but also the polarization of the immune response should be considered in adjuvant design, making it even more challenging [7].

http://dx.doi.org/10.1016/j.vaccine.2015.07.100 0264-410X/© 2015 Published by Elsevier Ltd.

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Development of vaccines requires safe and efficient adjuvant or antigen delivery systems. Although being pursued, ideal adjuvants remain out of reach for now. Alum salt is the most commonly used adjuvant approved by FDA (in 1926), which can be found in diphtheria–tetanus–pertussis, human papillomavirus or hepatitis vaccines [8]. Alum salt induces the antibody related immune response by a depot effect and activation of antigen present cells (APCs) (via NLRP3 inflammasome). Nevertheless, the preventive effect was not satisfactory for some subunit vaccine (e.g. H5N1 split vaccine). Moreover, its incapacity in eliciting cellular response hindered its therapeutic application in treatment of infected or cancerous cells. In the following 80 years, adjuvants like water-in-oil emulsion (e.g. Freund’s adjuvant), microorganism component (lipopolysaccharide, LPS), and cytokines (Interleukine2) were developed to induce higher immune response. Albeit effective in animal models, these adjuvants were reported to induce adverse effects like fever, lethargy, and even shock. Their clinic applications were thus stagnated until MPL-A (Monophosphoryl Lipid A, a derivate of LPS) was adopted as an adjuvant in FDA approved cervical cancer vaccine in 2009. In this regard, there is still an unfulfilled need for a safe and potent adjuvant.

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1.2. Potential micro/nanoparticle-based adjuvant

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Compared with traditional adjuvant, the micro/nanoparticles (MP/NP) possess a particular advantage as they have similar size and structure as that of a pathogen, leading to a preferred interaction with antigen presenting cells (APCs). MP/NP, such as polymeric particles, liposomes and micelle, have shown promising signs owing to their attributes in bio-application [9–13]. Firstly, antigens that loaded in the particles can be protected from enzymatic degradation and rapid denaturation. Secondly, particles can improve the antigen uptake by APCs, promoting subsequent antigen process and cellular mediated response. Thirdly, the fine-tuned characteristics (e.g. size, surface charge or structure) of particles allow them to fulfill a prolonged antigen release for long-lasting humoral response, or targeted delivery and controllable release for specific cellular immunity [14–16]. Last but not least, the particulate vaccines can be administrated via alternative routes like oral, rectal, or nasal administration, rather than subcutaneously injection. The potential of polymeric particulate vaccine delivery systems has been widely recognized [17–19]. However, owing to the prevalent use of non-uniform sized particles, discrepant and even contradictory outcomes were often present in biological or immunological evaluations. This situation has kept compromising the progress in establishing reliable theoretical guidance for particle design [20]. Moreover, wide size distribution may lead to an uncontrollable tissue distribution of the adjuvant particles, magnifying the concern on safety. To resolve these problems, our research group has developed a special MP/NP fabrication method on the basis of microporous membrane emulsification and has achieved successes in preparing bio-degradable (e.g. polylactide PLA) and polysaccharide (e.g. chitosan) MP/NP with controllable and uniform size (Fig. 1) [21–24]. Particles at a range of 100 nm to 100 ␮m have been successfully prepared by choosing membrane with specific pore size. In addition, the scale-up equipment (40 L/h when pore size is 5.2 ␮m) has been established in our group, which can be applied for scaling-up test. Based on this kernel technique, MP/NP of different structure or physiochemical properties for specific characteristics and functions can be obtained (see our previous review [25]). The polymeric MP/NP with defined sizes and properties thus facilitated to exploring the relationship between the particle properties and immunomodulatory effects, providing enlightening paradigms in vaccine development. Herein, we provide a review on the correlation of the particle physiochemical properties and antigen loading mode with the

Fig. 1. Schematic diagram of membrane emulsification process (A) and the scanning electron microscope (SEM) images of as-prepared PLA microparticles (B) and chitosan microparticles (C).

resultant biological/immunological outcomes, mostly based on the study of using the aforementioned uniform MP/NP. The underlying mechanisms of how the particles-based vaccine functioned in the immune system were also discussed. On the basis of particle design concept, potential applications of polymeric MP/NP were implicated not only for prophylactic vaccine (against e.g. Influenza, Anthrax), but also for therapeutic vaccine (against e.g. chronically infected hepatitis and malignant tumor). 2. Particle design of uniform micro/nanoparticles for vaccine delivery 2.1. Particle property design for vaccine delivery The requirements of immune responses vary for specific antigen type and vaccination purpose. Antibody mediated humoral response is critical in the defense against extracellular pathogens for the preventive vaccine, while antigen specific cytotoxic T cells is mostly wanted for therapeutic vaccine. As APCs are pivots that translate the vaccine stimulation to immune system, their responses to MP/NP are very important for regulating the subsequent immune response. The major APCs responses include APCs uptake, intracellular trafficking pathway, and cytokine profile. For most vaccines, avidly taken up by APCs is preferred to provide sufficient antigen signal. Since lysosomes are correlated to the main degradation pathway of exogenous antigen for CD4 T cells mediated humoral response, lysosomal degradation is not a favored process in eliciting cellular mediated response. In contrast, lysosome escape allows the cross-presentation of exogenous antigen to activate CD8+ T cells (CTL), which is particularly desired for the therapeutic vaccine or cancer vaccine [26]. In addition, the type and level of cytokine secretion by APCs can also modulate the subsequent immune activation. Therefore, a deeper understanding of how the physical properties of particles regulate APCs mediated biological or immunological responses is highly required. These correlation results will benefit the design of a particulate vaccine with optimized delivery mode or stimulation efficacy as well as minimized potential hazards [27]. 2.1.1. Particle size Particle size is the primary attribute and plays a paramount role in the following fate of antigen when the antigen loaded

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particles encounter with APCs. In fact, some conflicting reports existed for a long term regarding the influence of particle size on immune responses. For example, a study suggested that 5 ␮m (PLA-glycolide) PLGA particles more efficiently enhanced immune responses, while another study showed that 1 ␮m particles were preferentially taken up by APCs and induced stronger immune responses than that of nanoparticles [28,29]. In contrast, polyacrylamide hydrogel particles of 35 nm and 3.5 m in size showed no difference in the magnitude of T-cell activation [30]. Such inconsistent results may originate from the intrinsic difference of the materials as well as the difficulty in preparing particles with controllable size and narrow size distribution. Uniform sized chitosan particles at different size (430 nm, 1.9 ␮m and 4.8 ␮m) (Fig. 2A–C) were prepared by utilizing the membrane emulsification technique, and particle size effect on biological response in macrophages (a professional APCs) was compared [31]. These chitosan particles showed bright autofluorescence, and the fluorescent intensity of cells was proved in direct proportion to the internalized particle volume. The particle number/surface area/volume of the internalized MP/NP was thus calculated according to the cellular fluorescence data, and corresponding antigen loading modes were suggested. 430 nm particles accumulated in the cells at a faster rate, and with a higher surface area (∼800 ␮m2 ) and particle number (330) (Fig. 2D), indicating their advantage for antigen adsorption and conjugation. In contrast, the internalized 1.9 ␮m microparticles displayed higher volume value (∼165 ␮m3 ), suggesting their superiority in encapsulating therapeutic molecules (Fig. 2E and F). Moreover, the nanoparticles preferred to promote the secretion of Th1-specific molecule signals (e.g. IFN, IL-12) rather than immune suppressors, showing the capability of NP to improve vaccine efficacy. On the basis of this design concept, PLA (FDA-approved material which undergone extensive clinic evaluations) was employed as the material to prepare MP/NP adjuvant for Hepatitis B vaccine [32]. In comparison with MP, NP promoted higher antigen internalization and diverse degradation in lysosomes as well as proteasomes (Fig. 3). This result indicated a possibility to cross present antigen to CTL, which was indispensable for the direct cytotoxic lysis against infected cells or tumor cells. In vivo experiments established that NP-antigen group indeed enhanced the hallmarks of cellular mediated response (e.g. CTL cytotoxicity and IFN-␥ cytokine). Particles size was also found to contribute to the vaccine function routes, as smaller particles (mostly <50 nm) were able to penetrate the compact barrier of lymphatic system [33]. Compared with 100 nm particles, 25 nm NP transported from the injection site directly to draining lymph nodes (LNs) and activated the vast amount of immune cells settled there. Nevertheless, particles with a very small size (especially less than 10 nm) were reported to cause potential cytotoxicity, which partially precluded their further application [34]. On account of the ultrahigh specific surface area of NP (1.4 nm), cell necrosis was triggered by oxidative stress and mitochondrial damage during the fierce interaction between NP and intracellular organelles. As particle size is a parameter that could be geared toward eliciting a desired immune type while minimizing toxicity, it needs to be seriously considered in vaccine design 2.1.2. Particle charge Surface charge is another feature that affects the in vivo distribution and cellular uptake of particle. As cell membrane is negatively charged, the internalizing process and thus the antigen delivery of particles may be influenced by the particles with different surface charge. Surface charge was found to play a very significant role in determining the type and amount of the particle coronas (absorbed protein from human plasma) [35]. For example, 100-nm carboxyl-modified (negatively charged) particles absorbed a larger number of immunoglobulins while less apolipoproteins than that

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for the positively charged particles or plain particles. However, different studies still show discrepancies in the correlation of particle charge and cell interaction. Cationic liposome-regulated immune responses were found to rely on the surface charge density, and low-charge particles failed to promote the antigen specific immune responses even at high concentrations [36]. In contrast, negatively charged [37] liposomes could also act as efficient adjuvants to induce cell-mediated immune response. To elucidate the role of particle charge on the APCs responses, three kinds of NP (∼215 nm) were compared, including negatively charged N-NP (carboxymethyl chitosan NP,  ∼ 45.8), neutrally charged M-NP (chitosan coated carboxymethyl chitosan NP,  ∼ 0.5), and positively charged P-NP (quaternized chitosan NP,  ∼ 39.2) (Fig. 4) [38]. As these particles were fabricated by using the membrane emulsification technique, uniform size as well as identical physicochemical properties could be assured, excluding influence of other factors except for surface charge. Both the cellular uptake rate and amount were found positively correlated with the surface charge in eight cell lines. Surface charge also decided the intracellular trafficking of different particles. P-NP escaped from lysosome and exhibited peri-nuclear localization, whereas the NNP and M-NP were highly colocalized with lysosome. Driven by these findings, we coated the PLA particles with cationic polymers (e.g. chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI)) to build positively charged surfaces and tested their adjuvanticity for therapeutic hepatitis B vaccine (Fig. 5) [39]. Owing to the merits of positive charge on improved antigen adsorption and lysosome-independent antigen processing, the cationic particlebased vaccine generated an enhanced antigen uptake, stimulation, and augmented Th1 polarized cytokines (e.g. IL-1␤, IL-12, for cellular response). In vivo testes showed a rapid and enhanced humoral immune response as well as a more efficient cell mediated immune responses, in comparison with aluminum-adsorbed vaccine and undecorated PLA particles. 2.1.3. Hydrophobicity Surface hydrophobicity also plays an integral role in the interaction between APCs and particles, since the cell membranes are formed by a lipid bilayer, exhibiting a hydrophobic interior. Particles prepared with hydrophobic and high-molecular weight polymers tended to be more efficiently and rapidly phagocytosed [40]. However, the understanding went short especially regarding the effect of hydrophobicity on biological response and antigen/drug delivery efficacy, as compared with that of size and charge. To understand the inherent relationship between the particle hydrophobicity and the APC activation, PLA were took as prototype materials, and the biological outcome of three particles (∼290 nm), including PLA, PLGA, and PEG-b-PLA (polyethylene glycol-PLA) were compared [41]. With the change of the molecular composition, the hydrophobicity increased gradually from PLA, PLGA, to PEG-b-PLA NPs, which dictated the decreased macrophage internalization and subsequent delivery performance. Compared with PEG-b-PLA particles, PLA and PLGA NPs were more preferentially internalized by macrophages. By using PLA, PLGA, and PEG-PLA particles, which were similar in size (∼1 ␮m) and morphology but different in surface hydrophobicity, their abilities in inducing immune responses were further evaluated [42]. Increased surface hydrophobicity of PLA-based particles greatly promoted the cellular internalization of recombinant Bacillus anthracis protective antigen (rPA) by dendritic cells (DCs a professional APCs). DCs treated with rPA-PLA (the most hydrophobic MP) exhibited a higher percentage of rPA priming cells (52.37%) than those treated by rPA-PLGA (50.33%) or rPA-PELA (44.17%). Meanwhile, the number of rPA priming cells increased more than 10-fold in both the axillary and the popliteal LNs in the PLA particle group as compared with the PLGA or PELA particle groups.

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Fig. 2. Internalization profiles of macrophages after 24 h incubation with 430 nm particles (A), 1.9 ␮m particles (B) and 4.8 ␮m particles (C). The internalization data were further expressed by means of particle number (D), surface area (E), and volume (F).

Fig. 3. The cellular response of APCs after incubation with PLA nanoparticle adjuvanted vaccine.

Fig. 4. Schematic image of charge effect on cellular uptake and intracellular trafficking.

Fig. 5. A strategy of using cationic polymer-coated PLA particles for therapeutic hepatitis B vaccine.

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Fig. 6. Measurement adhesion forces between different particles and live cells. (A) Schematic representation of the experimental setup for obtaining force curves on the cell surface with particle-coated tips. (B) SEM images of PLA, PLGA and PELA particles prepared for coating the cantilever tips (scale bar 1 mm). (C–E) Analysis of the adhesion-force distribution histogram for PLA (C), PLGA (D), and PELA (E) particles.

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Through coating the particles onto the automatic force microscope probes, the assay of relative adhesion forces exhibited that PLA MP exhibited the strongest interaction force with the cell membrane (2382.8 pN), followed by PLGA MP (1845.37 pN), and PELA MP (1631.36 pN) (Fig. 6). This mechanism investigation revealed that the increased surface hydrophobicity enhanced the physical interaction intensity between particles and cell membranes, which thus promoted the internalization of particles into cells. 2.1.4. Surface chemical group Most of the chemical groups on particles source from the original component of particle material. To fulfill the knowledge gap of intrinsic surface chemical group on biological and immunological outcome, we fabricated a novel kind of adjuvant (CS-NH2 MP, ∼1.0 ␮m) with abundant amino groups by membrane emulsification and layer-by-layer (LbL) assembly technology [43]. Compared with the amino-cross-linked MP (control MP), CS-NH2 MP with a high surface density of amino groups lead to improved vaccine efficacy. The level of IL-4 and IFN-␥, the secretion of rPA-specific IgG titers, and the antigen specific T cell proliferative in splenocytes were found significantly enhanced. In addition to intrinsic component, surface modification is also a routine strategy to modulate the performances of particulate adjuvants [44]. In one of the most typical examples, CD206 antibody was conjugated to the particles to guide their tracking down of DCs. Such an active targeting strategy was practical for exploring the particles as tumor vaccine adjuvant. In a recent study, a chitosan based NP adjuvant that modeled after granules of mast cells was endowed with attributes of LNs targeting as well as timed release of cytokines [45]. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. 2.1.5. Particle shape Shape is an essential property of natural bio-particles (e.g. versatile virus or bacterial), which is believed to be associated with their infection efficacy and the subsequent replication. However, investigation of shape effect has lagged far behind that of the size and charge, as the fabrications of particles with non-spherical shape are constricted upon limited material choice and restricted techniques.

This bottleneck was not broken until the emergency of some initial studies until recently, and shape was becoming a design parameter for micro- and nanoscale delivery carriers [46,47]. For example, shape effect of filaments particles (via polymer micelle assembly) was addressed, and worm shape particles were found with prolonged blood circulation and enhanced drug delivery efficacy than spherical particles [48]. In our study, we investigated the cellular behavior of nanospheres and nanorods in the interplay with different kinds of cells (including endothelia cells and macrophages) [49]. The rod-like particles possessed an appreciable advantage of being quickly internalized by cells, reminiscent of the marvelous cell infecting capacity of bacillus. Although particle internalization is a complex process, which is dependent on cell types and particle raw materials, macropinocytosis and phagocytosis were found be the predominant uptake mechanism for non-spherical particles [50,51]. Desimone group prepared cylindrical particles and pointed out that cells were readily to internalize particles with a diameter aspect ratio of 3 by using several different mechanisms of endocytosis. After optimizing the manufacture process of PRINT (Particle replication in non-wetting templates) technique, Liquidia Technologies drove the non-spherical PLGA-based particles (LIQ001, 80 × 80 × 320 nm) forward in the area of influenza vaccines [52]. These particles were safe and showed enhanced responses to influenza hemagglutinin in murine models. Recently, they have completed a Phase 1/2a clinical trial of LIQ001 + Fluzone involving both young and elderly healthy adults. In addition to the non-spherical particles, the two dimensional material graphene oxide (GO) has renewed interest in biomaterialbased application. We uncovered that the particle shape regulated both the intracellular distribution of particle and the cytokine profile response (Fig. 7) [53]. Intriguingly, the strong steric effect of flat micro-sized forced the micro-sized GO folding from 2 ␮m to 300 nm, and brought higher cytokine secretion of macrophage than that of nano-sized GO. This might be attributed to the continuous struggle of GO for a more stable state of GO, which betrayed its extrinsic properties inside cells and finally resulted in recognition and stronger response from APCs. With this pilot study, we constructed a high-performance tumor vaccine and preliminarily proved the concept of new dimensional material based delivery system.

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Fig. 7. Cellular response of macrophage after exposure with 350 nm and 2 ␮m GO. (A) Transmission electron microscopy images showing eligible cell interactions with different sized GO. (B) Cytokine profile showing that the cells stimulated with microsized GO secrete more immune activation-related cytokines.

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2.2. Antigen loading mode for vaccine delivery 2.2.1. Adsorption/encapsulation/mixture Efficient antigen loading is indispensable for subsequent delivery and immune stimulation. The major loading patterns are adsorption and encapsulation, which influence the bioactivity and release rate of antigen in a complex manner. On one hand, when antigens are microencapsulated with particle, inactivation of antigen may occur during this process and compromise the corresponding immunogenicity and even cause the adverse effect [54,55]. In contrast, antigens that formulated with particles by gentle adsorption process are expected to get a better chance to maintain their activities (the activity loss of adsorbed antigen on PLA NP is below 5%). On the other hand, the adsorbed antigen will be released relatively quickly, leaving a limited time window for APCs recruitment, whereas the encapsulated antigens show controlledrelease characteristics and lead to superior long-lasting antibody levels (more than 32 weeks) [56]. To achieve a better understanding of how the antigen loading way impact on the antigen exposure and the resultant antigen-specific immune responses, we compared three antigenNP formulations (Fig. 8) [57]. The combined formulation (composed of antigen encapsulated in nanoparticles and antigen absorbed on nanoparticles through simple mixture) induced more powerful antigen-specific immune responses than each single-component formulation (antigen encapsulated within nanoparticles, or soluble antigen mixed with blank nanoparticles). The potential of

combined formulation might be attributed to the antigen-depot effect at the injection site, effective supply of both adequate initial antigen exposure and long-term antigen persistence, and efficient induction of DC activation and follicular helper T cell differentiation in LNs. Therefore, understanding the effect of different antigen loading on the resultant immune responses has significant implications for rational vaccine design.

2.2.2. Other method: NP-based multi-adjuvant modality Besides the traditional antigen loading methods, a whole cell tumor antigen modality was constructed based on the particle adjuvants. This modality of whole cell tumor vaccine (WCTV) is especially suitable for the tumors whose specific antigens have not been identified. On account of the carrier function of CPP modified PLGA NP, two encapsulated stimulators (IL-2 and GM-CSF) in this particle were efficiently introduced, via the entry of PLA NP into tumor cell. In addition to the reinforced internalization of stimulators with the assist of NP, a lysosome-escape behavior of NP also contributed a lot to the significantly improved bioavailability of imported GM-CSF and IL-2. These adjuvant-primed tumor cells were further used as multi-adjuvant WCTV after inactivation. In vitro data demonstrated the programed promotions of multi-adjuvants on DC recruitment, antigen presentation, and T-cell activation. In vivo evaluations demonstrated the satisfactory effects on tumor growth suppression, metastasis inhibition, and recurrence prevention. In this aspect, the nanoparticles-based

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Fig. 8. Illustration of different antigen loading modes.

Fig. 9. Immunological mechanism of alum and MP/NP adjuvanted vaccine.

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multi-adjuvant served as a high-performance modality for therapeutic vaccine. 2.3. Immunological mechanism of the particle based adjuvant 2.3.1. Antigen reservoir In contrast to the rapid clearance of soluble antigen at injection site after administration of soluble antigen, the alum salt significantly prolonged antigen deposition. This “antigen reservoir” effect provided a longer time window for the recruitment of inflammation related cells (e.g. DCs, macrophage, and neutrophil) and promoted their subsequent interaction between these APCs and antigen. However, the antigen reservoir capability of particles varied with their components and the physiochemical properties. Whereas chitosan based particles deposited at the injection site for a longer period [39], PLA particles that were cleared (internalized by APCs) at a quick speed were favorable for further antigen process [58]. Moreover, in design of LN targeted antigen delivery, there’s a need for particles to directly migrate to the immune APC/T cells that settled in the draining lymph node, rather than stay at the injection sites. 2.3.2. APCs uptake and activation APCs are the leading actors in processing vaccine antigens, which receive the vaccine stimulation and motivate the other part of the immune system. These cells engulf the antigen and present the most characteristic peptide (major histocompatibility

complex MHC molecule, recognition signal) on cell surface. Based on specific MHC peptide signals and costimulators (stimulation signal), immune cells like CD4 and CD8 T cells will be activated to release their weapons (antibody or granule/perforin), exerting their functions in neutralizing the virus or attacking the infected cells, respectively. On the contrary, lacking of any MHC molecules (especially MHC I for CD8 T) and costimulators (CD80/86) or cytokines (communication signal), will lead to weak cellular response or immunity tolerance, thus conniving chronic diseases (chronic hepatitis B) or cancer progression. For instance, the incapacity of alum adjuvant in cellular response of alum adjuvant was found highly associated with downregulation of MHC I level expression in APCs [32]. Guided by this immunological process, most of the strategies (nano-sized, positively charged, rod-like) focused on increasing the chances of antigen to be massively and efficiently processed by APCs. The aforementioned vaccine adjuvants (chitosan NP, PEI modified PLA MP, non-spherical MP/NP) enjoyed the advantage of improving cellular uptake of antigen or activating the recognition or costimulator signals for T cells. Moreover, with the stimulation of exogenous particles, APCs secreted a range of signaling molecules or cytokines, which were vital to the following immunological regulation and activation. 2.3.3. Lysosome escape The internalized exogenous antigens were usually entrapped and processed by lysosome for CD4 T mediated antibody response,

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2.3.4. Complement activation Complement activation is capable of acquiring systematic immune response and is considered an alternative approach toward immunotherapy [67]. In the case of CS-NH2 MP, C3a (an index molecule for complement activation) was significantly enhanced upon the particle exposure, resulting in stronger antigen specific humoral response. Notwithstanding a similar cellular uptake level of the MP with different extends of amino group, the abundant amino groups on CS-NH2 MP could promote the covalent binding of C3b molecular and efficiently activate completion pathway. Considering possible acute inflammatory responses provoked by C3a, we carefully checked the pathological changes in the subcutis, and verified the fair cell infiltration and safety of CS-NH2 MP for using as a vaccine-delivery platform.

Fig. 10. Multiple mechanisms of immunity enhancement by the Gel MP based vaccine delivery system.

whereas the endogenous (self) antigens were processed via protease for CD8 T. A well-documented benefit of alum adjuvant is to elicit robust preventive humoral response. This can be partly 476 ascribed to the undivided antigen supply to lysosome-mediated 477 antigen processing, and the consequent MHC II signal upregulation 478 Q3 for efficient CD4 T cell activation (Fig. 9). However, the high fre479 quent antigenic variations among circulating virus (e.g. H1N1 and 480 H5N1) drove us to develop vaccines to elicit cross-protective CD8 T 481 cell response against both viruses [59]. Aiming to treat intracellular 482 infection and malignant cells, exogenous antigen also needs to be 483 cross-present through MHC I pathway to activate CD8 T cytotoxic 484 responses. Strategies to cross-present antigen include lysosome 485 escape of antigen, targeting Toll-like receptors with additive of dan486 ger signals (e.g. CpG) [60–62] and so on. In this review, we focused 487 on the lysosome escape way. 488 Antigen escape from lysosome was preferred when strong cel489 lular immune response was expected, and well-designed particle 490 adjuvants could trigger this process very efficiently. Considering 491 the advantage of a facile preparation condition, particles that bear 492 this merit but need no modification were intriguing candidates. 493 Under the “proton-sponge” effect, the positive charge of NPs (as 494 referred in P-NP) may trigger the influx of chloride ions to main495 tain charge neutrality, leading to osmotic swelling, physical rupture 496 of lysosomes, and the final antigen escape to cytoplasm [63,64]. 497 To further enhance the efficacy of lysosome escape, some stud498 ies attempted to modulate the intracellular trafficking of particles 499 by further functionalization or fine-tuning the particle component. 500 For example, we successfully developed a novel adjuvant candidate 501 (Gel MP) based on the thermal-gelation property and pH sensitive 502 properties of quaternized chitosan (Fig. 10) [65,66]. The majority 503 (close to 80%) of antigens (H5N1) in Gel MP/HA group were disso504 ciated from lysosomes, demonstrating an efficient antigen escape 505 from lysosome and release to the cytoplasm. A possible reason 506 for lysosome escape was the swelling and dissolution of pH sensi507 tive Gel MP under acidic microenvironment, which might result in 508 osmotic imbalance as proved by the improvement of erythrocyte 509 lysis. Another reason was the above-mentioned “proton-sponge” 510 effect in acidic endosome, which was related to high buffering 511 capacity (BC) of polymers. Compared with the positive PEI control, 512 a significantly higher BC was revealed for Gel MP in the pH range 513 for endosomal escape (pH 4.5–7), which was likely due to added 514 phosphate groups during NP preparation. 515 474 475

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3.1. Conclusions

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This article reviewed the recent insight and practical progress of MP/NP based adjuvant/antigen delivery system in our research group, emphasizing on the uniform-sized polymeric particles. In-depth understandings of particle–bio interactions make an important contribution to support the rapid expanding researches on particle adjuvants. However, it is not an easy task to draw an uncontested conclusion due to the complexity originated from the particle fabrication. By using a unique microporous membrane emulsification technique, we prepared particles with uniform and controllable size with good reproducibility, paving the way for further investigation on biological/immunological response. With these particles, the effect of a single property (e.g. size, charge, shape) can be clarified, and the influence of other factors is minimized to ensure reliable results. Taking advantage of successful exploration of particle-bio interaction, smaller size (raging from 20 nm to 1 ␮m), positive charge, rod shape, hydrophobic surface, specific chemical component were implicated in the active immune response. Moreover, the underlying versatile mechanisms of how the particles modulated in the immune system were also unveiled. Given the extensive knowledge, the tailored design of particle with high antigen payload and optimized attributes for expected purpose became feasible. 3.2. Perspectives In this article, we focused on constructing particles adjuvant under the knowledge of the influences of particle property on the immunological outcome. Intelligent micro/nanoparticles combining both antigen delivery and multi-stimulation are promising candidates for the new generation vaccines. Nevertheless, the capacity of adjuvant should still be optimized to cater for the vaccine demand of immune efficacy or longevity. With the thorough comprehension of the immunological effect of each property, more than one attribute (e.g. charge and shape) can be introduced into a single particle, via component control, ligand graft or surface modification, thus to achieve specific function (carrier or targeting) and activity (stimulation). In addition, the thorough theoretical progress of novel or traditional adjuvant addressed on molecular, cellular and animal level is highly appreciated [68], which will provide the design concept for maximizing the efficiency of the integrated multi-attributes while minimizing the potential hazard. Recalling the latest developments in preventive vaccine, particle-based vaccine promises compelling advantages even beyond adjuvant. Nevertheless, most of the studies are still in its research stage, and the clinical trial fairly lagged behind the overwhelming research work. Only scattered and few examples were

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translated from bench to the clinical Phase. The major handicap is associated with the quite limited material options due to the difficultly in developing material with good safety profile. To circumvent the problem, the well-proved materials (PLA/PLGA) with excellent biocompatibility are preferred for preventive vaccine, which is come-at-able for clinical trial. In the meantime, it is urgent to improve the quality of candidate materials (e.g. chitosan) base on the control of the preparation process and the comprehensive verification of the safety issue. Regarding the material that has not been fully approved but extensively proved, selecting a relative safe administration route, such as the transdermal (micro-needle), oral or mucosal delivery (e.g. chitosan based adjuvant) [69,70], is also an acceptable way to reduce the safety concern. As for both preventive and therapeutic vaccines, an overall challenge is the scale-up production of particles with uniform size and specific property. To achieve this goal, the corresponding manufacture apparatus should be developed to guarantee the repeatability of products, as well as provide enough samples for clinical trials. In our lab, pilot-scale equipment with automatic process control has been established, and a large amount of studies on fine-tuning the particles preparation conditions and process optimization are being performed. This progress may make an important contribution to the particle-based adjuvants, and is waiting to reach its full potential for further applications for humans. Author contribution Both authors have (1) screened relevant literature and discussed the review outline, (2) drafted the article or revised it critically for important intellectual content, and (3) approved the final version submitted. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments

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This work was supported by National Natural Science Foundation of China (51302265), National High Technology Research and Development Program of China (2014AA093604), and Major Project of the Ministry of Science and Technology of China (2014ZX09102045).

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