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    Abstract

    The application of nanotechnology to the treatment of cancer or other diseases has been boosted during the last decades due to the possibility to precise deliver drugs where needed, enabling a decrease in the drug’s side effects. Nanocarriers are particularly valuable for potentiating the simultaneous co-delivery of multiple drugs in the same particle for the treatment of heavily burdening diseases like cancer. Immunotherapy represents a new concept in the treatment of cancer and has shown outstanding results in patients treated with check-point inhibitors. Thereby, researchers are applying nanotechnology to cancer immunotherapy toward the development of nanocarriers for delivery of cancer vaccines and chemo-immunotherapies. Cancer nanovaccines can be envisioned as nanocarriers co-delivering antigens and adjuvants, molecules often presenting different physicochemical properties, in cancer therapy. A wide range of nanocarriers (e.g., polymeric, lipid-based and inorganic) allow the co-formulation of these molecules, or the delivery of chemo- and immune-therapeutics in the same system. Finally, there is a trend toward the use of biologically inspired and derived nanocarriers. In this review, we present the recent developments in the field of immunotherapy, describing the different systems proposed by categories: polymeric nanoparticles, lipid-based nanosystems, metallic and inorganic nanosystems and, finally, biologically inspired and derived nanovaccines. WIREs Nanomed Nanobiotechnol 2017, 9:e1421. doi: 10.1002/wnan.1421

    For further resources related to this article, please visit the WIREs website.

    INTRODUCTION

    In the last decades of medical research, the need for effective drug delivery systems (DDS) has emerged as a major concern especially for the treatment of diseases like cancer, malaria, and HIV/AIDS, which require systemic administration of therapeutics. Despite all the advances in research and in therapy, cancer is still a major cause of death worldwide.[1, 2] Nanotechnology is emerging as a multidisciplinary area that is revolutionizing the treatment of diseases in the 21st century.[3, 4] The applications of nanotechnology in the delivery of therapeutics, either for the treatment of cancer or other diseases, have been boosted during the last decades, with the development of different nanocarriers for drug delivery applications.[2, 5] The effort to develop an effective DDS is directed toward the achievement of a greater fractional distribution of the therapeutic agent at the target site (or affected body part) and none or negligible distribution to the nontargeted sites in the body.[6, 7]

    The current treatment of cancer is based on the co-administration of multiple chemotherapeutics to overcome the complexity of the tumor microenvironment.[8] Many efforts have been devoted to develop nanocarriers that are able to co-deliver chemotherapeutics, cytokines, and antibodies.[9] Cancer immunotherapy represents a recent approach in the fight against tumors and applications of nanotechnology to the development of cancer vaccines and the delivery of immunogenic agents are currently being studied.[10, 11] The activation of the patient’s own immune system to help fighting the cancer was first proposed by Coley[12] over one century ago.

    In the immune system, antigen presenting cells (APC) continuously scavenge the body in search of exogenous (e.g., bacterial or viral) or endogenous (e.g., cancer and virus infected cells) antigens (Figure 1); they uptake and process the antigens to expose them onto the major histocompatibility complexes (MHC) I or II.[14, 15] APC prime naïve T-cells through a combination of three signals: (1) immunological synapsis between MHC complex and T-cell receptor; (2) presence of co-stimulatory factors (CD80 and 86 on APC binding to CD28 on T-cells); and (3) a suitable cytokine environment surrounding the T-cells.[16] T-cells can differentiate either in CD4+ (helper)-cells or in CD8+ (cytotoxic)-cells.[17] A CD8+cytotoxic response is necessary in the treatment of cancer with cancer vaccines because only cytotoxic T lymphocytes can directly kill another cell; moreover, tumor-associated antigens (TAA) are often intracellular elements, which prevent the recognition from circulating antibodies.[16, 18]

    Figure 1.

    Schematic of the leading characters in cancer immunotherapy and their relationship. The three different phases of immunization, T-cell activation, and immunosuppression in the tumor microenvironment are depicted. (Reprinted with permission from Ref [13]. Copyright 2011 Nature Publishing Group)

    In recent years, different strategies have being developed to achieve the stimulation of the patient immune system against the tumor: chimeric T-lymphocytes, checkpoint inhibitors, and cancer vaccines.[19] The use of nanocarriers for drug delivery and, in particular, the development of dual therapeutic DDS is sought in two of these strategies: the check-point inhibitors and the vaccines. The advantages derived from the administration of nanocarriers are (Table 1): the delivery of therapeutic compounds to a specific target (whether this is the tumor microenvironment or the cells of the immune system); in addition, the loading of immunostimulatory compounds within particulate carriers greatly improves their safety profile, allowing, in some cases, an increase in the dosage; finally, the nanocarriers themselves may work as adjuvant, reducing the need for the co-administration of adjuvants and antigens.[19, 51] Among others, inorganic materials like mesoporous silica and silicon, as well as polymers like acetalated dextran (AcDEX), have shown inherent adjuvant properties, activating the dendritic cells.[31, 32, 52, 53] In recent examples, polymers not displaying such properties were covalently modified with adjuvants and antigens, yielding self-assembling nanovaccines.[54] Furthermore, nanoparticles were covalently bound to the surface of therapeutic T-cells and act as cytokine reservoirs in adaptive T-cell therapy or to enhance the delivery of chemotherapeutics.[55, 56] Another enticing application of nanoparticles in the immunological field is the development of artificial APC, presenting antigen-loaded MHC I complexes and antibodies targeting CD28 for an optimal priming of naïve T-cells.[57, 58]

    Table 1. Advantages and Disadvantages of the Delivery Systems Presented in the Review
    Delivery System Advantages Disadvantages References
    Polymer-based Nanovaccines
    • Versatile in: size, morphology, surface functionalization
    • High loading of antigens and adjuvants
    • Form hydrogels
    • Induce stronger cellular response and longer lasting humoral response compared to liposomes
    • Degradation/inactivation of the payload in the preparation process
    • Cytocompatibility of the degradation products
    [19, 20]
    Lipid-based Nanovaccines
    • Biocompatible
    • Easy functionalization
    • Poorly immunogenic
    • Stability
    [20-26]
    Metallic Nanovaccines
    • Optical properties (combined chemo-immunotherapy)
    • Easy functionalization
    • Controllable size
    • Particular biodistribution among the cells of the immune system
    • Non-biodegradable
    [19, 27-30]
    Inorganic Nanovaccines
    • Self-adjuvant function
    • Easy preparation
    • Easy functionalization
    • Biocompatible and biodegradable
    • High porosity
    • High loading degree
    • Short circulation time (particles not functionalized)
    • Stability in basic solutions
    [31-44]
    Biologically-derived Nanovaccines
    • Biocompatible
    • Enhanced circulation time
    • Innovative antigenic source
    • Stability
    • Yield
    [45-50]

    Nanocarriers made from different materials (Table 1), such as polymeric nanoparticles, micelles, liposomes, inorganic nanoparticles and biomimicking nanoparticles (Figure 2), have all been proposed as cancer vaccines delivery systems.[11, 32, 64] The simultaneous delivery of an antigen (usually a protein or a small peptide) together with an adjuvant in the same nanovector can be envisioned as a dual therapeutic delivery. The TAA are usually poorly immunogenic due to their similarity with the self, thereby they require the coadministration of adjuvants to fully activate the APC.[16] Adjuvants are molecules presenting features similar to pathogens, which are recognized by the pattern recognition receptors and lead to the activation of the APC.[65] The co-administration of antigen and adjuvant in soluble form will not prime CD8+ T-cells because soluble antigens endocytosed from the APC are presented onto MHC II, leading to a CD4+ humoral response.[15] However, antigens uptaken by APC can be cross-presented: an exogenous antigen can be presented also on MHC I complexes and not only on MHC II.[66] The delivery of antigen with micro- or nano-sized particles greatly enhances the cross presentation, resulting in the priming of cytotoxic T-cells.[67, 68] The enhancement in the cross presentation with particulate systems is due to a different uptake mechanism by the dendritic cell, but can be further improved with the design of particles able to escape from the endosomes: among the different strategies studied, pH-sensitive particles increased the concentration of the antigen in the cytoplasm following a destabilization of the endosomal membrane,[69, 70] while a reducible (disulfide) bond between the antigen and the particle exploited the intracellular reductive environment to increase the antigen cross presentation when compared to a non-reducible bond.[71]

    Figure 2.

    The different systems presented in this review. Schematics and TEM pictures of the different categories of systems investigated for dual therapeutic delivery in cancer immunotherapy. (a), (c), (e), (g) scale bars 100 nm. (Reprinted with permission from: (a) Ref [49]. Copyright 2016 Elsevier; (b) Ref [59]. Copyright 2015 Elsevier; (c) Ref [46]. Copyright 2016 Wiley; (d) Ref [60]. Copyright 2005 Nature Publishing; (e) Ref [61]. Copyright 2015 Elsevier; (f) Ref [62]. Copyright 2014 Elsevier; (g) Ref [63]. Copyright 2016 ACS Publications)

    Most of the nanoparticulate vaccines developed so far have been developed for subcutaneous or intradermal use and are intended to be target to the resident population of dendritic cells in the skin.[72] An innovative delivery route and target for the nanovaccines are the lymphatic vessels and the lymph nodes: hydrophobic particles smaller than 200 nm can extravasate in the lymphatic vessels and reach the lymph nodes.[73] Several studies have investigated in vivo the characteristics of an ideal nanoparticulate system for the targeting of the dendritic cell population in the lymph nodes; according to Reddy et al. the particles presenting the highest retention in the lymph nodes and uptake by the resident APC population are the 25 nm particles, while 100 nm particles are less efficiently transported.[72, 74] The transport and retention in the lymph nodes are influenced also by the surface characteristics of the nanovaccine:[75] positively charged liposomes displayed a depot effect at the injection site, and did not elicit the priming of long-term memory cells, while the same liposomes modified with mannose, traveled to the lymph nodes and elicited long-term production of immunoglobulins.[76] Moreover, particles presenting positively charged surfaces are more efficiently uptaken by cells, both in periphery and in the lymph nodes.[77-79]

    Some chemotherapeutic agents have a positive immunogenic effect on the tumor microenvironment.[11] For example, some chemotherapeutic agents at low dose, such as 5-fluorouracil,[80] doxorubicin,[81] and paclitaxel,[82] can distinctly induce the immunogenic death of cancer cells. After the immunogenic cell death, the remains of the cancer cells themselves serve as a ‘vaccine.’ The nanocarriers may also be loaded both with an antigens and low-dosage chemotherapeutics to achieve a combined chemo-immunotherapy (Figure 3).[11]

    Figure 3.

    Envisioned future treatment of cancer. Nanoparticles are targeted to the cancer cells and are uptaken. The chemoterapeutics released from the particles kill the cancer cells, controlling the progression of the tumor. The immune system can then resume its fight against the cancer cells. A second set of nanoparticles, the actual vaccine, is delivered to dendritic cells (DCs), inducing the priming of cytotoxic T-lymphocytes against the antigens expressed by the tumor. Abbreviations: CTLs: cytotoxic T lymphocytes; DCs: dendritic cells; i.v.: intravenous; siRNA: small interfering RNA; TCV: therapeutic cancer vaccine; TLR: Toll-like receptor. (Reprinted with permission from Ref [11]. Copyright 2016 Elsevier)

    In this review, we describe how the nanocarriers are currently being adopted to improve contemporary cancer therapies, which are summarized in Table 2 and discussed in more detail below. The DDS proposed for the co-loading of antigens, adjuvants or chemotherapeutics will be classified based on the type of nanocarriers used.

    Table 2. Nanocarrier-based approaches to elicit anti-tumor immunity. Granulocyte-macrophage colony-stimulating factor, GMCSF; Transgenic adenocarcinoma mouse prostate, TRAMP; Glucopyranosyl lipid adjuvant, GLA; Toll-like receptor ligand, TLRL; intradermal, i.d.; Poly (ethylene glycol)-b-poly (l-lysine)-b-poly (l-leucine), PEG-PLL-PLLeu; Human basic fibroblast growth factor, bFGF; 3-methylglutarylated linear poly(glycidol), MGlu-LPG; 3-methylglutarylated hyperbranched poly(glycidol), MGlu-HPG; Major histocompatibility complex, MHC; Polyribocytidylic acid PIC; Pyruvate dehydrogenase E2 protein, PDE2.
    Nanocarriers Size Surface compound Payload 1 Payload 2 Payload 3 Tumor model Effect References
    PLGA <500 nm Naked OVA/gp 100 BSA Tumor lysate B16-F10 cells, s.c. C57BL/6 mice Animals vaccinated with DC exposed to particles containing the tumor lysate resulted in reduced tumor volume. [83]
    PLGA 90 nm and 248 nm Naked Anti-DEC205 antibody OVA α-GalCer / KRN7000 OVA-expressing EG7 (s.c.), C57BL/6 mice Co-delivery of α-GalCer and protein antigen to CD8α + DCs triggered antigen-specific antibody and cytotoxic CD8+ T cell responses, leading to a potent antitumor response. [84, 85]
    PLGA 244–293 nm Naked Paclitaxel Lipopolysaccharide derivatives B16-F10 cells, s.c., C57BL/6 mice Effectively induced infiltration of PACs in tumor microenvironment, activated these cells to an immune stimulatory state as well as facilitated infiltration of T cells. [82, 86]
    PLGA 248–604 nm Naked Pentaerythritol lipid A OVA OVA-expressing E.G7 cells, s.c., C57BL/6 mice Pentaerythritol lipid A promoted the secretion of immune potentiating cytokine, IL-12p70, and upregulated key costimulatory surface proteins, CD86 and CD40, in murine dendritic cells. [87]
    PLGA 350 and 450 nm Naked Monophosphoryl lipid A OVA Markedly increase in clonal expanded CD4+ T cells, which were capable of cytokine secretion and expressed an activation and memory surface phenotype (CD62L lo, CD11a hi, and CD44hi). [88]
    PLGA 350 and 410 nm Naked 7-Acyl lipid A TRP2 peptide180–188 B16-F10 cells, s.c. C57Bl/6 mice Efficient in inducing immunostimulatory mileu at the tumor microenvironment, as evidenced by decreased level of VEGF and elevated levels of IL-2, IL-6, IL-12, IFN-γ and TNF-α. [89]
    PLGA 80 nm Naked TRP2 peptide180–188 Monophosphoryl lipid A B16-F10 cells, s.c., B6 mice Down-regulation of MHC class I expression on tumor cells as an escape mechanism from antigen-specific T cells, and partial inhibition of tumor growth in immunized mice. [90]
    PLGA 350-450 nm Naked OVA / MUC 1 MPLA Increase in the expression of co-stimulatory signals by dendritic cells and in the activation of T-cells. [91]
    PLGA 2.66 µm and 212 nm Functionalized phospholipidic bilayer OVA bound to the pegylated lipids MPLA α-GalCer Low doses of the antigen and the adjuvants were able to induce a humoral response detectable after 150 days. Difference on-set of the antibody response (rapid for α-GalCer; long lasting for MPLA). [92]
    PLGA Microparticles Naked OVA CpG-ODN OVA-expressing B16 cells, s.c., C57BL/6 mice Triggered clonal expansion of primary and secondary antigen-specific CD4 and CD8 T cells. Exogenous antigen and TLR-based adjuvants to vaccinate for protective and therapeutic CD4 and CD8 T cell immunity. [93]
    PLGA 20–200 nm Naked Prostate tumor lysate CpG-ODN TRAMP model, C57BL/6 mice Immunization of mice yielded substantial cytotoxic T cell (CTL) responses and interfered with tumor growth in TRAMP mice, a pre-clinical transgenic mouse model of prostate carcinoma. [94]
    PLGA (SVP) Naked OVA Resiquimod/CpG Enhanced humoral and cellular immune response after immunization of mice, without the side effects of the adjuvants’ systemic administration. [95]
    PLGA <250 nm; see Table 1 in reference section PEGylated lipidic layer functionalized with targeting moieties OVA Resiquimod / Pam3Csk4 Poly IC OVA-expressing B16 cells, s.c., C57BL/6 mice Enhanced uptake from dendritic cells leading to augmented humoral and cellular immune response.

    Prolonged survival and control over the tumor growth in vivo.

    [96, 97]
    Poly (γ-glutamic acid) 2.2–3.4 µm Naked Paclitaxel Imiquimod B16-F10 cells, s.c., C57BL/6 mice The anti-tumor response had systemic memory response since the vaccinated mice significantly deferred secondary tumor development. [98]
    Acetalated dextran 40 nm Naked Lipopolysaccharide OVA Induced strong OVA peptide-specific CD4+ and CD8+ T cell proliferation, and a robust OVA-specific humoral immune response (IgG1 > IgG2a). [99]
    Chitosan hydrogel GMCSF OVA Increased in the number of OVA-specific CD4+ and CD8+ INF-γ + T cells, leading to enhanced humoral and cellular immunity. [100]
    Lipid nanocapsules 70 nm Chitosan Paclitaxel CpG GL261 cells orthotopic injection, C57BL/6J mice Potentiated both CpG immunopotency and PTX antitumor activity by enhancing its delivery into the tumor microenvironment. [101]
    Liposome 140 nm Naked TLR7 agonist, IMQ TLR4 ligand, GLA Significantly reduced IL-5 and enhanced interferon gamma production upon antigen-specific stimulation of cells from immunized mice [102]
    Liposome 150 nm PEGylation, maleimide Anti-CD40 CpG B16F10 cells, s.c., C57BL/6 mice Successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. [103]
    Liposome 690 nm Naked Hepa1-6 cell lysate PIC Heap1-6 cells , s.c., C57BL/6 mice Enhanced PIC uptake and consequential TLR3 signaling in bone-marrow dendritic cells, which in turn promoted DC maturation and type I IFN production. [104]
    Liposome 94–99 nm MGlu-HPG 3,5-didodecyloxybenzamidine OVA E.G7-OVA cells, s.c., C57BL/6 mice Promoted cytokine production, enhanced antigen presentation via both MHC I and II, induced antigen-specific antibody production and Th1-dominant immune responses. [21]
    Liposome 108–136 nm MGlu-LPG) or MGlu-HPG OVA E.G7-OVA cells, s.c., C57BL/6 mice Surface modification induced much more effective generation of OVA-specific cytotoxic T cells (CTL), and significantly reduced the tumor burden. [22]
    Liposome 96–150 nm 3-methylglutarylated dextran OVA E.G7-OVA cells, s.c., C57BL/6 mice The antigen-specific humoral and cellular immunity was induced more effectively by surface modification, and significantly suppressed tumor growth and extended the mice survival. [23]
    Liposome and lipoplex hybrid complex 158–1078 nm Methylglutarylated poly(glycidol) IFN-γ-encoding plasmid DNA OVA E.G7-OVA cells, s.c., C57BL/6 mice Immunization through the liposome–lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects. [24]
    Liposome 181–7821 nm PEGylation α-Galactosylceramide OVA-peptide Long TRP2-peptide B16.F10luc2 or B16.OVA cells, s.c., C57Bl/6 mice Surface PEGylation did not improve immune responses and enhanced production of IFN-γ, increased cytotoxic T-cell responses and tumor survival were observed. [105]
    Liposome 240–370 nm Naked Listeriolysin O OVA B16 or MO5 cells, (i.d.), C57BL/6J mice Engendered higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor frequency. IFN-γ production upon specific stimulation by MHC I-restricted peptide was also significantly stronger. [25]
    Liposome 100–173 nm Naked Monophosphoryl lipid A bFGF LL/2 cells, i.v., C57 mice Effectively induce humoral immunity through cross-reaction, mediate Th1 immune response preferentially and enhance antitumor activity in vivo. [106]
    Liposome 100 nm L-rhamnose, and MUC1 TLRL-MUC1- GalNAc-O-Thr Rhamnopyranoside-cholesterol BALB/c mice Successfully triggered both T-cell and humoral immunity enhanced by anti-L-rhamnose antibody-dependent antigen uptake. [26]
    Lipid microbubbles-avidin- biotin-lipoplexes Biotinylated lipoplexes OVA mRNA immunomodulating TriMix mRNA MO4 or E.G7-OVA tumor cells, s.c., C57BL/6 Effective induction of antigen-specific T cells resulted in specific lysis of antigen-expressing cells. DC sonoporation using microbubbles can elicit powerful immune responses in vivo. [107]
    Nanoscale liposomal polymeric gels 120 nm PEGylated interleukin-2 TGF-β inhibitor (SB505124) B16-F10 cells, s.c., C57BL/6J mice Delayed tumor growth, increased survival of tumor-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8+ T-cell infiltration. [108]
    PEG-PLL-PLLeu micelles 142 nm PEGylated Poly I:C (PIC, a TLR3 agonist) OVA STAT3 siRNA OVA-transfected B16 cells, s.c., C57BL/6J mice Co-delivery of immunopotentiator (PIC) and immunosuppressive gene silencer (STAT3 siRNA) improved the therapeutic efficacy of cancer vaccines by modulating TADCs and overcoming tumor immunosupression. [59]
    Oxidized multiwalled carbon nanotubes 10–40 nm in diameter CpG OVA B16-NY-ESO-1 or CT26-NY-ESO-1 cells, s.c., C57BL/6 mice Served as an intracellular antigen depot, favored the induction of strong CD4+ T as well as CD8+ T cell-mediated immune responses against the NY-ESO-1. The vaccination significantly delayed the tumor development and prolonged the mice survival. [109]
    Gold nanoparticles 23 nm CpG Red fluorescent protein (RFP) CpG RFP-transfected B16F10, s.c., C57Bl/6 mice Induced antibody production through a Th1-driven pathway and priming CTL responses in an antigen-specific manner, and exhibited significant antitumor efficacy. [110]
    Gold nanoparticles 16-176 nm Immune-polyelectrolyte multilayer Polyinosinic–polycytidylic acid OVA peptide and its derivative C57BL/6J mice Increase of DC activation and antigen presentation in draining lymph nodes promoted high levels of antigen-specific CD8+ T cells in peripheral blood. Also exhibited a potent recall response after boosting. [111]
    Gold nanorods 91.5 nm PEGylated Dox Y-shaped CpG ODNs H22 cells, s.c., BALB/c mice Y-shaped CpG ODNs are effective in inducing the cytokines, such as TNF-α and IL-6, Dox can efficiently intercalate into the Y-shaped CpG ODNs, which provide additional advantages for chemotherapy. [28]
    Gold nanoclusters 1.13 nm Naked CpG OVA Balb/C mice One-pot synthesis of fluorescent AuNCs by using OVA-CpG ODNs conjugates as the templates can enhance the immunological responses and immunostimulatory activity. [30, 112]
    Hollow CuS nanoparticles 85 nm Chitosan CpG EMT6-OVA, s.c., BALB/c mice Photothermal immunotherapy elicits more effective systemic immune responses, resulting in combined anticancer effects. [29]
    Mesoporous silica rods 88 × 4.5 µm Naked GMCSF CpG-ODN EG7.OVA cells, s.c., C57Bl/6J mice Formation of a 3D cellular microenvironment recruited substantial numbers of DCs, enhanced systemic TH1 and TH2 serum antibody and cytotoxic T cell levels. [113]
    Porous silicon nanoparticles 171 nm Anti-CD326 antibody Anti-CD326 antibody Sorafenib Anti-CD326 antibody enhanced the interaction of effector immune and cancer cells for subsequent phagocytosis and cytokine secretion. [43]
    Core/shell nanoparticles 110 nm B19-F10 cell membrane B19-F10 cell membrane antigens The full array of cancer cell membrane antigens, offer a robust platform with applicability toward multiple modes of anticancer therapy [45]
    Reduced cancer cells 400 or 500 nm B16-F10 cell membrane CpG DNA C57BL/6J mice Facilitated the induction of OVA-specific IgG1 and IgG2a antibody, and the induction of antigen-specific cellular and humoral immune responses. [46]
    Dendritic cell-derived membrane vesicle 30–100 nm TSG 101, MHC II B16 cell-derived exosomes LLC cell-derived exosomes B16 cells or LLC cells, s.c., C57BL/6 mice Resulted in cross-protection responses, and Induced specific cytotoxic T lymphocytes (CTL)-dependent tumor rejection and suppressed the growth of both types of tumor in mice [47]
    Plasma membrane vesicles 347 and 410 nm GPI-anchored antigen HER-2 GPI-anchored IL-12 GPI-anchored B7-1 D2F2/E2 cells, s.c., BALB/c mice Incorporation of IL-12 and B7-1 onto the PMVs enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. [48]
    Plasma membrane vesicles CD11c-ScFv and DEC-205-ScFv OVA, or OVA peptide antigen B16 melanoma

    antigens

    B16-OVA cells, i.v., C57BL/6 mice Stimulated strong B16-OVA-specific CTL responses in splenic T cells, a marked protection against tumor growth, and prolonged disease-free survival. [114]
    PDE2 protein nanoparticles 30.2 nm melanoma-associated gp100 epitope CpG B16-F10 melanoma cells, s.c., C57BL/6 mice Greatly increased in the frequency of melanoma epitope-specific CD8+ T cells, delayed the onset of tumor growth by ~5.5 days and increased animal survival time by approximately 40%. [49]
    PDE2 protein nanoparticles 30 nm CSIINFEKL peptide CpG Co-delivery of peptide epitopes and CpG activator in a viral mimic protein nanoparticle facilitated enhanced DC activation and cross-presentation. [50]

    POLYMER-BASED NANOVACCINES

    Most of the nanocarriers for drug delivery proposed in the literature are based upon polymeric particles, especially the ones made of poly (lactic-co-glycolic) acid (PLGA) or dextran as such or after acetalation (AcDEX). These particles present several advantages: they are versatile in size, morphology, and surface functionalization; moreover, depending on the preparation methods, they can display high loading of the therapeutics, form hydrogels or self-assembly into micelles. However, they still retain some disadvantages, like the production of proinflammatory molecules upon degradation of the polymer and the possible degradation and inactivation of the therapeutic pay load (antigens are usually small peptides) in the preparation process.[19, 69]

    Micellar-based Nanovaccines

    Micelles are nanosized particles formed by the self-assembly of amphiphilic polymers in a solution above their critical micelle concentration.[115] Nanosystems based on cationic self-assembly micelles made of polypeptides were employed for the co-loading of a model antigen (chicken ovalbumin, OVA), an adjuvant (poly I:C) and a siRNA (STAT3 inhibitor); the systems were targeted to the immunosuppressed dendritic cells in the tumor microenvironment and resulted in the activation of the dendritic cells, priming of cytotoxic T-lymphocytes and increased survival in a melanoma mouse model.[59]

    Amphiphilic diblock copolymers modified to introduce thiol-reactive groups were used by Wilson et al. to obtain pH-sensitive, cationic, and self-assembling micelles. The nanoparticles were then loaded with antigen (OVA by thiol bond) and with adjuvant (CpG oligonucleotide adsorbed on the micelle’s surface), producing a nanovaccine formulation able to induce a Th1 response with the priming of CD8+ T-lymphocytes, due to enhanced cross presentation of the antigen into DCs.[69]

    PLGA Particles

    PLGA is a biocompatible and biodegradable polymer approved by the Food and Drug Administration. It has been widely used in the pharmaceutic field for the preparation of micro- and nanoparticles, enabling the loading of all kinds of therapeutics, from small molecules to proteins.[116]

    The effect of the molecular weight of PLGA on the encapsulation efficiency and release profile of multiple antigens co-loaded in the same nanoparticle was evaluated by Solbrig et al.; the simultaneous loading of a known antigen (OVA; gp100) with bovine serum albumin (BSA) and tumor lysates, despite the lowest encapsulation efficiency, showed a sustained release of the antigen over 1 week, inducing the release of pro-inflammatory cytokines (IL-2 and IFN γ), and exerted a protective effect in vivoagainst a melanoma model when dendritic cells were exposed to the particles before injection in the animal.[83] In a recent study, PLGA nanoparticles (with dimensions between 200 and 600 nm), prepared by double emulsion technique, and encapsulating a model antigen, OVA, and a TLR-4 agonist (pentaerythritol, PET Lipid A) promoted the activation of APC in vitro, as shown by the both the increased expression of co-stimulatory signals (CD40 and CD86) on the cell membrane and by an increased secretion of pro-inflammatory cytokines (mainly IL12p70).[87] However, the same study failed to prove an advantage in the administration of the formulation loaded with both antigen and adjuvant over the controls in vivo. However, nanoparticles loaded with OVA and a different TLR-4 agonist (7-acyl lipid A) induced stimulation of immune cells both in vitro and in vivo, increasing the number of CD4+ and CD8+ lymphocytes.[88]

    The same TLR-4 agonist, 7-acyl lipid A, was co-loaded with the antigenic peptide Trp-2 into PLGA nanoparticles. In vivo, this vaccine achieved better control over a B16-melanoma mouse model compared to particles containing only the antigen. As argued by the authors, the development of novel vaccines should move toward the assessment of less antigenic peptides, like Trp-2, similar to the real tumor environment and not focus on model antigens, like OVA.[89] Hanlon et al. encapsulated tumor lysate derived from ovarian cancer cells in PLGA nanoparticles achieving the activation of dendritic cells and the priming of T-cells in vitro, while the administration of tumor lysate in solution lead to T-cell anergy.[117] Zhang et al. loaded PLGA with two different murine melanoma antigens (hgp100 and Trp-2), enhancing the uptake by dendritic cells in vitro and in vivo, and increasing the efficacy of the vaccine compared to the administration of the antigens together with Freund’s adjuvant, as recently discussed by Temizoz et al.[118] Moreover, they proved the efficacy of a prophylactic vaccination with nanoparticles co-loaded with Trp-2 and monophosphoryl lipid A (MPLA) against subcutaneously inoculated melanomas.[90]

    Elamanchili et al. developed PLGA nanoparticles encapsulating MPLA and the MUC1 antigen, and the nanovaccine avoided the tolerance toward the antigen in vitro, enhancing the expression of co-stimulatory signals on dendritic cells and the activation of T-lypmhocytes.[91] PLGA micro- and nano-particles were coated with a functionalized phospholipidic bilayer and the antigens were bound to the PEGylated moieties of the lipids and displayed on the surface of the particle, while the adjuvants (MPLA and α-galactosylceramide, α-GalCer) were loaded in the lipidic bilayer. These particles increased the production of antibodies in vivo at low doses of both antigens and adjuvants, with a different effect between the two adjuvants (long-lasting, up to 150 days for MPLA, rapid peak for α-GalCer).[92] PLGA microspheres were assessed for the endosomal co-delivery of CpG-oligonucleotide and OVA into dendritic cells in vitro and for the immunization effect against infective diseases and melanoma tumor models in vivo, with promising results against melanoma (four complete remissions over five animals treated with the particles).[93] The administration of PLGA microspheres loaded with tumor lysate as a source of antigens and CpG-oligonucleotide as adjuvant was able to reduce the tumor volume in TRAMP mice, a model of prostate cancer resistant to the therapy with dendritic cells. The antigens and the adjuvant were encapsulated in the spheres by spray-drying technique; moreover, they were sterilized with γ-rays and their efficacy was proven also after the sterilization.[94] Synthetic vaccines particles were developed by Ilyinskii et al. for the co-encapsulation of antigen and adjuvants: OVA was co-encapsulated with resiquimod (TLR-7/8 agonist) and CpG oligonucleotide, resulting in an enhanced humoral and cellular immune response, preventing the side effects and toxicity associated with a systemic administration of the powerful adjuvants.[95] In another study, OVA was co-encapsulated with resiquimod and poly I:C into PLGA nanovaccines that were further functionalized with a PEGylated lipidic coating and three different antibodies for the targeting to dendritic cells’ receptors (CD 11c, CD-40, and DEC-205).[96] The targeting enhanced the uptake of the particles from dendritic cells both in vitro and in vivo, leading to the activation of CD4+ and CD8+ T-cells in vitro. The same group developed PLGA nanoparticles loaded with poly I:C and Pam3CSK4 as adjuvants and OVA as antigen, targeted to the CD40 receptor on dendritic cells and assessed their efficacy in vivo over murine tumor models and the vaccination with the system increased the particles’ uptake in dendritic cells, resulting in priming of cytotoxic T-cells and prolonged survival of the animals with control over the tumor growth.[97]

    Nikitczuk et al. demonstrated that following the administration in the tumor microenvironment of a vaccine system based on PLGA vesicles prepared with the double emulsion method of an average size of 500 nm, there was a type 1 mediated (with secretion of interferon γ, IFNγ) response against the tumor in a murine model of T-cell lymphoma.[119]

    In order to promote an immune response resulting in the priming of cytotoxic T-lymphocytes, antigens need to be delivered in the cytoplasm of dendritic cells, thereby Bruno et al. developed PLGA microparticles loaded with OVA and functionalized with tetraphenyl chlorine disulphonate as a photosensitizer to trigger the release of the antigen in the cytosolic compartment. The immunization with these particles induced the production of CD8 T-lymphocytes and the prevention of tumor growth.[120]

    Important players in the immune population are the natural killer (NK)-cells: these cells, part of the innate immune system, kill tumor cells that do not express the MHC I on their surface (a frequent mutation in tumor cells, due to the selective pressure by cytotoxic T-lymphocytes) or stressed/tumoral cells that overexpress stress ligands recognized by activated NK-cell’s receptors.[121, 122] Dölen et al. screened the efficacy of cancer vaccines formulated by co-encapsulation of a model antigen (OVA) with different adjuvants (TLR-agonists or invariant-NKT cells agonists). Nanosystems loaded with α-galactosylceramide (α-GalCer, agonist of the invariant-NKT cells) involved NK cells in the immune response, produced a higher secretion of IFNγ, and allowed the development of a higher number of cytotoxic CD8+ T cells.[123] Macho Fernandez et al. used DEC205 antibodies to target PLGA nanoparticles loaded with OVA and α-GalCer to iNKT cells and the vaccination with the particles in tumor models resulted in a potent antitumor response.[84, 85]

    The immunostimulant effect promoted by traditional chemotherapeutics was recently proposed: in disagreement with previous theories, Zitvogel et al. proposed a model of positive interaction between the immune system and chemotherapeutic drugs administered at the therapeutic dosage.[124] This interaction was proved for several drugs and led to the development of systems combining traditional chemotherapeutics with immunotherapeuteutics.[98, 125-127] PLGA microparticles were used for in situ immunization against animal models of lymphomas and melanoma: the particulate systems were co-loaded with doxorubicin (Dox) and CpG. The authors reported that the intratumoral administration of low-dosage Dox together with CpG in combination with the systemic administration of a check-point inhibitor resulted in the complete eradication of one of the lymphoma and in reduced tumor volumes in the other two tumor models assessed.[128] PLGA polymer was used also for the co-encapsulation of paclitaxel and a TLR-4 agonist (a phthalic acid derivative of lipopolysaccharide, LPS) into nanoparticles prepared by an oil-in-water single emulsion evaporation method (Figure 4).[82]

    Figure 4.

    Schematic of the immune response induced after administration of PLGA nanoparticles co-loaded with paclitaxel and an adjuvant, a Toll-like Receptor (TLR) agonist 4. The administration of paclitaxel to the tumor cells will induce their death, with the releasing of tumor antigens. The antigens will be uptaken by APCs, mainly dendritic cells (DCs) and macrophages (MΦ). The simultaneous administration of the adjuvant will activate the antigen-presenting cells, stimulating the expression of activation markers (CD40, CD80, and CD86). The activated APCs will secrete cytokines like IL-12, inducing the activation of T-cells, both T-helper and cytotoxic T-lymphocytes directed against the cancer cells. This resulted in an improved control over the tumor volume in vivo. (Reprinted with permission from Ref [82]. Copyright 2013 ScienceDirect)

    Acetalated Dextran

    AcDEX is a highly biocompatible polysaccharide obtained from dextran in a single-step reaction.[53] This material shows interesting immunostimulant properties toward APC, is water insoluble and can be easily formulated into micro- and nano-particles (by single or double emulsion methods).[52, 53] Moreover, the polymer can be further functionalized with spermine, to yield a positively charged polymer to increase the uptake by the cells, or with mannose, to specifically target APC, thus enhancing the uptake of the antigen and the presentation on the MHC complexes.[129, 130] This biomaterial is highly versatile and was employed in the development of nanocomposites for the co-delivery of paclitaxel, sorafenib, and metothrexate.[61] Two different adjuvants, CpG oligonucleotide and polyinosinic:polycytidylic acid (Poly I:C) were successfully co-encapsulated into AcDEX microparticles and induced higher immunostimulation and cytokine secretion compared to PLGA particles or free antigens in solution in vitro.[131]

    Non-AcDEX did not show any immunostimulatory property per se and was used to co-encapsulate OVA and lipopolysaccharide into a nanovaccine formulation. It allowed, however, an enhanced uptake by APC in a mannose receptor-dependent manner, leading to an enhanced immunostimulation in vitroand in vivo.[99]

    Hydrogels

    Hydrogels made of polysaccharides represent an attractive vehicle for the delivery of antigens due to their intrinsic adjuvant properties.[132] The immunostimulant properties of antigen-loaded hydrogels are dependent on the interaction between the vaccine particles and dendritic cells and are ruled by the antigen encapsulated, while the type of immune response induced is ruled by the surface characteristics and functionalization of the nanosystems.[133] Jain et al. developed nanosized hydrogel by free radical polymerization and loaded them with OVA and CpG oligonucleotide for the co-delivery of antigen and adjuvant in the endosomal compartments of dendritic cells.[134]

    LIPID-BASED NANOVACCINES

    The main lipid-based systems investigated for cancer immunotherapy applications are liposomes. For example, lipid nanocapsules coated with chitosan were developed for the combinatorial chemo- and immuno-therapy with paclitaxel and CpG in the treatment of glioblastoma, with improvement in the overall survival of animal models.[101]

    Xu et al. proposed the co-administration of two different nanosystems: a lipid/calcium phosphate nanoparticle loaded with antigen and adjuvant (Trp-2 and CpG oligonucleotide) was functionalized with mannose to enhance the uptake from DC, while a liposome/protamine/hyaluronic acid system was loaded with siRNA against TGF-β to reduce the immunosuppression in the tumor microenvironment. The rationale between the co-administration of the two system is the lack of efficacy found by the authors in the vaccination with the lipid/calcium phosphate particles against advanced-stage melanoma models. The administration of the two particles in the late-stage melanoma models resulted in an enhanced inhibition of the tumor growth compare to a control treated only with the nanovaccines.[135]

    Liposomes

    Liposomes are nanosized vesicles delimited by one or more bilayer membranes made of phospholipids.[60] These systems have been proposed and studied as carriers for antigens and adjuvants and for the co-formulation, while investigating the effect of size, lamellarity, and surface charge.[136-138] Liposomes with an average size of 140 nm (suitable for sterile filtration), co-loaded with two adjuvants, TLR 4 and 7 agonists, glucopyranosyl lipid adjuvant and imiquimod, promoted the activation of APC with a synergistic effect compared to either adjuvant alone.[102] Moreover, targeting moieties (antibodies targeted against CD40) and antigens (CpG) can be covalently bound on the surface of PEGylated liposomes, showing similar results in tumor inhibition and immunostimulation compared to the soluble molecules, but with improved biocompatibility.[103]

    In particular, to enhance the immunostimulative potential of liposomal formulations, cationic liposomes loading adjuvant (poly I:C) were developed and assessed in vivo: they induced the priming of anti-tumoral CD8+ T-lymphocytes and the production of IFN-γ.[104] Cationic liposomes may also result from the addition of 3, 5-didodecyloxybenzamidine as cationic lipid, with the further modification of the liposomes with pH-sensitive moieties to promote the endosomal delivery of antigens.[21-23] Yuba et al. combined negatively charged, pH-sensitive liposomes with cationic liposomes in lipoplexes: the negative liposomes were loaded with OVA, while the positively charged ones encapsulated a DNA plasmid encoding IFN-γ gene.[24] Cationic liposomes were investigated also as carriers for the intravenous administration of a formulation composed by the Trp2 antigen (long peptide form) and α-galactosylceramide, adjuvant directed to iNKT: the intravenous administration of such nanosystems resulted in an increased IFN-γ production, priming of cytotoxic T-lymphocytes, and improved survival in melanoma mouse models.[105]

    Another way to improve the lack of immunogenicity displayed by liposomes is the co-loading of the antigen (OVA) with listeriolysin O (LLO), the purified virulent component of Listeria, bacterium able to escape from endosomes and live intracellularly. These liposomes promoted an increase in the production of IFN-γ and cytotoxic T-lymphocytes, leading to a prophylactic action of the vaccine against OVA expressing tumors in vivo; however, there was no difference in the anti-OVA antibody titer between the formulations with and without LLO, despite the shifting of the antigen presentation to MHC I complexes.[25] Miyabe et al. proposed the use of highly fusogenic liposomes loaded with dinucleotides as adjuvants, with promising immunostimulatory results in vitro.[139]

    Zhong et al. proposed a liposomic system co-loading a TLR 4 agonist (monophosphoryl lipid A, MPLA) together with basic fibroblast growth factor (bFGF, a proangiogenic factor) for the treatment of metastised lung carcinoma in vivo; the administration of their system resulted in an increase in the production of IFN-γ, together with anti-bFGF antibodies.[106]

    MUC 1 is a dysfunctional mucin produced by tumoral cells and represents an attractive target for cancer immunotherapy: Lakshminarayanan et al. developed a liposomal formulation loading a tripartite vaccine formed by the adjuvant (Pam3CysSK4), a T-helper epitope peptide and an abnormal mucin peptide, inducing the stimulation of cytotoxic lymphocytes and the production of antibodies against MUC 1.[140] An abnormal mucin peptide, conjugated to the adjuvant (Pam3CysSK4), was inserted in the phospholipidic bilayer; the liposome surface was further functionalized with l-Rhamnose, a xenoantigen which induces a rapid recognition of the liposomes from the immune system. The system was able to induce both cellular- and humoral-mediated immune responses against the mucin antigen.[26]

    An interesting formulation was also proposed by Dewitte et al., where they co-loaded cationic liposomes with antigen mRNA and TriMix mRNA (mix of three mRNA adjuvants, including CD40 and CD70); the liposomes were then bound to the surface of microbubbles via biotin-avidin bonds to achieve an ultrasound-mediated delivery, both in vitro and in vivo (as shown in Figure 5).[107, 141]

    Figure 5.

    Delivery of mRNA in dendritic cells with ultrasounds. (a) Mode of action of the system: when the ultrasound is applied, after the sonoporation of the cells, the liposomes deliver their cargo intracellularly. (b) Summary of the methods used to prepare the particles: the liposomes are mixed with the mRNAs; then the avidin molecules on the surface of the microbubbles react with the biotins on the surface of the liposome, binding them to the bubble. (Reprinted with permission from Ref [107]. Copyright 2014 Elsevier)

    Nanosized liposomes were used as outer template in the assembly of a complex multistage system. For example, the transforming growth factor-b (TGF-b) encapsulated cyclodextrins and a cytokine (IL2) were entrapped in a degradable hydrogel made of a central polyethylenglycole molecule flanked by poly lactic acid (PLA) monomers with two reactive acryl groups on the side. The acryl groups on one chain react with others from another chain upon UV-irradiation (Figure 6). The system delivered in a sustained fashion both the active compounds, leading to an improvement in the survival on a mouse melanoma model.[108]

    Figure 6.

    Preparation of the nanolipogels. Schematic of the production process. (a) Methacrylate-f-CD encapsulate the TGF-β inhibitor. (b) Then, the nanolipogels are produced from liposomes loaded with the biodegradable crosslinking polymer and the cytokine. The gel is formed after photoinduced polymerization. Abbreviations: CD, cyclodextrin; NHS, N-Hydroxysuccinimide. (Reprinted with permission from Ref [108]. Copyright 2012 Nature Publishing)

    METALLIC AND INORGANIC NANOVACCINES

    The most popular metallic particles investigated as nanovaccines are gold nanosystems. However, there is interest also in other inorganic particles, like mesoporous biomaterials (silica and silicon).

    An innovative nanosystem made of multi-walled carbon nanotubes co-loading antigens (OVA in the preliminary studies, then a well-known melanoma antigen, NY-ESO-1) and adjuvant (CpG) enhanced the immunostimulation, the production of IFN-γ, and improved the overall survival of animal models.[109]

    Gold-based Materials Acting as Nanovaccines

    Gold nanoparticles are suitable as carriers for the delivery of drugs, peptides, and nucleic acids.[142] The particles can assume different shapes based on the synthesis conditions and are suitable for further functionalization with targeting moieties.[143] The role of gold nanoparticles in immunotherapy was reviewed by Almeida et al.[62] Interestingly, the distribution of gold nanoparticles between the cells of the immune system is really varied: most of the particles are uptaken by B cells, followed by DC, granulocytes and T cells, with a small percentage (ca. 3%) of particles found in myeloid-derived suppressor cells.[27]

    Gold nanoparticles, presenting sizes suitable for targeting to lymph nodes, were formulated by covalently attaching a model antigen (red fluorescent protein, RFP) and an adjuvant (CpG) to the surface of the nanoparticle, inducing a potent immune response and resulting in delayed growth of tumor and prevention of lung metastases.[110]

    An adjuvant (poly I:C, negatively charged) and a peptidic antigen (OVA, or OVA modified with nona-arginine to increase the positive charge) were deposited layer-by-layer over gold nanoparticles resulting in a polyelectrolyte multilayer assembly (Figure 7). This system presented an intrinsic high loading degree of both adjuvant and antigen due to the production technique, and induced immunostimulation both in vitro and in vivo.[111]

    Figure 7.

    Layer-by-layer deposition of adjuvant and antigen on the surface of gold nanoparticles. The system is self-assembled due to the opposite charge between the elements (gold nanoparticles, adjuvant, and antigen). (Reprinted with permission from Ref [111]. Copyright 2015 ACS Publications)

    Tao et al. developed gold nanorods for combination chemo- and immune-therapy: the nanorods were co-loaded with doxorubicin and an adjuvant (CpG oligonucleotide in a particular, Y-like shape). These rods can also be used to induce hyperthermia in the tumor tissue, achieving a control over the progression of the subcutaneous tumors.[28] A combination of photothermal and immunotherapy, albeit using copper sulfide-based nanoparticles encapsulating CpG and coated with chitosan, was proposed by Guo et al.; the nanosystem showed increased efficacy against a breast cancer model compared to either therapy alone.[29]

    Gold nanoclusters are particles consisting of few atoms and they are characterized by photoluminescence, which can be adjusted to different wavelengths simply by changing the number of atoms in the core.[144] Such nanocluster can be synthesized in a ‘one-pot’ peptide biomineralization process, together with a OVA-CpG conjugate.[30] The same reaction can be employed in presence of a modified peptidic antigen (CYY-OVA) to shift the fluorescence to the red channel. These nanoclusters can be conjugated also with adjuvant molecules (CpG oligonucleotide) leading to a complete nanoparticulate vaccine presenting also properties useful for imaging.[112]

    Mesoporous Silica and Silicon Nanovaccines

    Mesoporous silica particles are produced with a bottom up approach in a sol-gel process, starting from organosilane precursors.[33] The surface of the particles can be further functionalized by introduction of reactive groups.[34] The potential role of mesoporous silica as a drug carrier in vaccine formulations was review by Mody et al.[32] Mesoporous silica nanoparticles loaded with CpG oligonucleotide were complexed with poly allylamine hydrochloride (PAH), demonstrating the attractive properties of the system as vaccine delivery system.[63]

    Mesoporous silica rods self-assembled into a macroporous structure upon subcutaneous injection in mice (Figure 8); moreover, the rods were co-loaded with an antigen (OVA), an adjuvant (CpG oligonucleotide), and a cytokine promoting the migration and maturation of dendritic cells (GM-CFS); they exhibited the sustained release of the three active compounds, the recruitment of immune cells (among them DC), the ability to induce a cellular- and humoral-mediated immune response against the model antigen.[113]

    Figure 8.

    Schematic of the proposed system. A dispersion of the mesoporous silica rods in buffer is injected into subcutaneous tissue of mice to form a pocket. After diffusion of the buffer from the pocket, there is the in situ spontaneous assembly of the rods with the formation of three-dimensional interparticle spaces where host immune cells can be recruited and educated by the payloads. Educated cells may then emigrate from the structure to interact with other immune cells. (Reprinted with permission from Ref [113]. Copyright 2015 Nature Publishing)

    Another interesting inorganic material is porous silicon (PSi).[35] This material can be easily prepared with a top-down process from silicon wafers, and can be further functionalized to display optimal properties (e.g., biocompatibility, high porosity, and possibility to tune the release of the therapeutics) for drug delivery.[36-41] Shahbazi et al. demonstrated the immunostimulatory properties of PSi; moreover, they were able to identify which of the surface functionalizations induced the highest immunostimulation.[31, 42]

    PSi nanoparticles were also functionalized with an antibody (anti CD326) against an antigen expressed by some cancer cell lines and were loaded with a model anticancer drug, sorafenib, as shown in Figure 9. The particles were selectively uptaken by the cancer cells expressing the antigen and were able to exert an antiproliferative effect toward those cells. Moreover, the authors investigated the antibody-dependent cell-mediated cytotoxicity of the system, showing cytotoxic activity by effector immune cells toward the cancer cells expressing the antigen and no effect was observed on the cells not expressing the antigen.[43]

    Figure 9.

    Schematic of the proposed system for chemoimmunotherapy. The PSi nanoparticle loads an anticancer drug and is conjugated to an antibody. When it will reach the tumor microenvironment, it will release the drug and interact with effector immune cells to create an antibody-dependent cell-mediated cytotoxicity. (Reprinted with permission from Ref [43]. Copyright 2015 Springer)

    BIOLOGICALLY DERIVED NANOVACCINES

    Cell Membrane-derived Systems

    In the last years there has been a shift toward biologically derived materials, like elements derived from the membrane of cancer cells, in the formulation of new cancer nanovaccines administered as such or used for the ex vivo pulsing of dendritic cells.[45-47, 145]

    A recent paper proposed the use of plasma membrane-derived vesicle as delivery systems for the co-delivery of adjuvant and antigens; the antigens were conjugated to glycosylphosphatidylinositol and inserted in the membrane, while the adjuvants (IL-12 and CD80) were incorporated in the membrane of the vesicle.[48] The administration of these systems in animal models lead to impressive improvements in the overall survival of the animals compared to the controls.

    Plasma membrane vesicles derived from B16-OVA murine cells were modified to attach OVA onto the surface, while the adjuvant (LPS, IFN-γ or GM-CSF) was encapsulated in the inner core; the vesicles were further decorated with antibodies targeted to DC (like DEC-205) and were shown to target dendritic cells both in vitro and in vivo.[114]

    Viral-Mimicking Nanovaccines

    Another innovative approach in the development of cancer vaccines mimics viral proteins. Nanoparticles prepared from the self-assembly of such proteins show significant advantages in size, passive targeting to the lymph nodes, and uptake by APC.[73]

    Particles made from the non-viral protein E2, modified to insert cysteine residues for further functionalization, were co-loaded with antigen (gp100, a melanoma TAA) and adjuvant (CpG) and promoted immunostimulation with the priming of CD8+ T-lymphocytes, increasing the overall survival time of murine melanoma models, as shown in Figure 10.[49, 50]

    Figure 10.

    Schematic representation of the production process of viral-mimicking particles. The E2 protein nanoparticle is covalently modified with CpG internally and with the antigenic peptide externally. The multifunctional system is then incubated with immature dendritic cells. The adjuvant is released, inducing the maturation of the dendritic cells and the subsequent priming of T-lymphocytes. (Reprinted with permission from Ref [50]. Copyright 2013 ACS Publications)

    CONCLUSION

    Cancer immunotherapy, particularly the systemic administration of check-point inhibitors, brought effective treatment in cancers with high mortality rates (melanoma, lung cancer). However, the side effects due to the systemic administration of such therapeutics are moderate in numerous patients. Thereby, the efforts of the researchers focus on the study and the development of targeted cancer vaccines. Nanotechnology is the source of exciting new possibilities in the field: particles made of innovative biomaterials can transport adjuvants and antigens, with a targeted delivery to the APC, improving the safety and the efficacy of the vaccines. The most studied systems are those based on polymeric nanoparticles, which provide a solid, reliable platform for the co-encapsulation of antigens and adjuvants, often biomolecules presenting different physicochemical properties. Liposomes are another popular choice as carriers, both in the traditional chemotherapy and in immunotherapy; however, they need further modifications, like the introduction of positively charged lipids or of elements derived from pathogens, directed to a general improvement of their antigenic properties. An enticing system is the one formed by gold nanoclusters, which add imaging features to the advantages of the targeted delivery of antigens and adjuvants. Inorganic porous material, like mesoporous silica and silicon, are characterized by intrinsic adjuvant properties, and are suitable carrier for a wide range of different payloads; moreover, these materials can be used to form self-assembled scaffolds able to recruit and prime APC over a longer period of time.

    The new trend in material science is the creative use of structures produced by nature, like particles made of viral proteins, or vesicles derived from the cell membrane: these systems present a degree of complexity impossible to recreate from scratch on a lab bench. Viral-derived particles present the appropriate danger signals to APC, without any risk derived from viral genetic material. Vesicles derived from the cells membrane can be used as source of neoantigens, if derived from the patient’s cancer cell, as biocompatible delivery system, and as biomimetic system, if derived from cells normally circulating in the blood (like red blood cells).

    Following the discovery that low dosages of various traditional chemotherapics can effectively boost the immune response against cancer cells, researchers are developing multifaceted particles, able to deliver the chemotherapic of choice to the tumor and, at the same time, induce activation and maturation of APC thanks to the co-loaded adjuvants.

    During the next years, several of these systems will advanced from the present preclinical studies to clinical studies and will eventually reach the market, revolutionizing the current protocols for the treatment of cancer. Moreover, these systems are highly versatile, and they may be tailored to each patient, with the creation of personalized treatments in the hospital pharmacy.

    ACKNOWLEDGMENTS

    Dr. H. A. Santos acknowledges financial support from the Academy of Finland (decision nos. 252215 and 281300), the University of Helsinki Research Funds, the Biocentrum Helsinki, the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013, Grant No. 310892), and the Sigrid Juselius Foundation.

     

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