Nanotechnology based therapeutic modality to boost anti-tumor immunity and collapse tumor defense

Accepted Manuscript Nanotechnology based therapeutic modality to boost anti-tumor immunity and collapse tumor defense

Xiaomeng Hu, Tingting Wu, Yuling Bao, Zhiping Zhang PII: DOI: Reference:

S0168-3659(17)30545-X doi: 10.1016/j.jconrel.2017.04.026 COREL 8771

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

23 February 2017 15 April 2017 18 April 2017

Please cite this article as: Xiaomeng Hu, Tingting Wu, Yuling Bao, Zhiping Zhang , Nanotechnology based therapeutic modality to boost anti-tumor immunity and collapse tumor defense. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi: 10.1016/j.jconrel.2017.04.026

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ACCEPTED MANUSCRIPT Nanotechnology based therapeutic modality to boost anti-tumor immunity and collapse tumor defense Xiaomeng Hua, Tingting Wua, Yuling Baoa,b , Zhiping Zhanga,c.d,* a

Tongji School of Pharmcy

b c

Department of Pharmacy, Tongji Hospital

National Engineering Research Center for Nanomedicine

d

Hubei Engineering Research Center for Novel Drug Delivery System, HuaZhong University of Science and Technology, Wuhan, P.R. China, 430030

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*Corresponding author E-mail: [email protected]

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Abstract Cancer is still the leading cause of death. While traditional treatments such as surgery, chemotherapy and radiotherapy play dominating roles, recent breakthroughs in cancer immunotherapy indicate that the influence of immune system on cancer development is virtually beyond our expectation. Manipulating the immune system to fight against cancer has been thriving in recent years. Further understanding of tumor anatomy provides opportunities to put a brake on

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immunosuppression by overcoming tumor intrinsic resistance or modulating tumor microenvironment. Nanotechnology which provides versatile engineered approaches to enhance therapeutic effects may potentially contribute to the development of future cancer treatment

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modality. In this review, we will focus on the application of nanotechnology both in boosting

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anti-tumor immunity and collapsing tumor defense.

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Keyword: Nanotechnology, Immunotherapy, Tumor microenvironment, Dendritic Vaccination, Adoptive T cell therapy, Checkpoint therapy, Immunosuppression

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1. Introduction Over the past few years, immunotherapy has been developing vigorously and become the fourth modality pillar of cancer treatment after surgery, chemotherapy and radiotherapy [1]. Exploitation of the immune system in cancer therapy was proposed over a century ago. In 1894, the surgeon William Coley demonstrated that heat-killed bacterial products (Coley toxins) can be used to inhibit tumor growth [2]. This kind of crude vaccine ignited the interest in developing

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cancer immunotherapy. However, the serious side effects and subsequent achievements in radiotherapy rapidly shielded the silver lining from the cloud. Cancer immunotherapy wandered around the edge for almost a half century.

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Even so, scientists continuously made breakthroughs to illustrate the essential role of the

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immune system. It was presumed that tumor cells can be specifically recognized by immune cells. In 1943, being inspired by the work of Clowes and Baeslack [3], the virologist Ludwik Gross suggested the existence of tumor-specific antigens which were preferentially expressed on tumor cells [4]. While the process of specifically inducing anti-tumor effect via antigens was unclear

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until the dendritic cells (DCs) were found as the initiators of the immune system by the Nobelists Ralph Steinman and Zanvil A. Cohn in 1973 [5]. Lately, the discovery of interaction between T

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cell receptors (TCRs) and major histocompatibility complex (MHC) ultimately provided an integral interpretation about the processing of antigen [6]. During the same period, using interleukin-2 (IL-2) for lymphocytes activation ignited the motion of immunologist to investigate

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cytokine in various tumor types, such as breast cancer, renal cell cancer, glioblastoma, lymphoma, and melanoma [7-10]. Another cytokine, interferon-α (IFN-α), was approved by the United States Food and Drug Administration (FDA) for immunotherapy in hairy cell leukemia in 1986, and then IFN-α2 was approved as the adjuvant treatment in 1995 [11, 12]. In1998, IL-2 was approved by

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the FDA to treat metastatic melanoma. Generally, tumor cells are opportunistic to trigger T cell tolerance, which provides great convenience for tumor to escape from immune surveillance. Major progresses have been made on the immune checkpoint pathways to regulate the negative feedback of T cell. The FDA approved Ipilimumab, the anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody for advanced metastatic melanoma in 2011. In 2014, the first anti-programmed death

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receptor-1 (PD-1) antibody, Pembrolizumabanti was approved. Another anti-PD-1 immune checkpoint inhibitor, Nivolumab, was approved by FDA for treating patients with advanced squamous-cell non–small-cell lung cancer in 2015. Treatments based on checkpoint blockade have been prosperously investigated in many clinical trials [13-15] (Fig. 1).

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Figure 1. Timeline of important events in cancer immunotherapy. Adapted with permission

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from [1]. Copyright 2012 American Cancer Society, Inc.

Cancer is one of the top three killers of human. The ideal cancer immunotherapy not merely

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aims at strengthening anti-tumor immunity, but also possesses the ability to collapse tumor defense to the immune system. Fortunately, years of concentrated efforts in nanotechnology have given us numerous options to achieve this goal. Nanotechnology involves multidisciplinary fields, such as physics, chemistry, biology and engineering, to develop diverse devices in nanoscale. With

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a wide range of innovative nano-materials developed for medical application, nanotechnology can be regarded as excellent medium to promote interdisciplinary cooperation [16]. The application of nanotechnology can fulfill diverse requirements from pharmaceutical formulation, such as

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protecting payloads, delivering therapeutic agents to targeted area, extending the circulation in blood and so on. Especially in cancer immunotherapy, nanotechnology provides promising

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strategies for manipulating the immune system to fight against tumor. For vaccination, nanoparticles can be employed as delivery system to promote anti-tumor immunity of traditional

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vaccine, such as protecting payloads, enhancing cross-presentation and promoting DCs maturation and migration. In the field of T cell therapy, nano-engineering can be used to facilitate T cell expansion ex vivo or in vivo for effective anti-tumor immune response. In cooperation with nanotechnology, immunomodulatory therapy can effectively overturn the tumor immunosuppressive microenvironment and create more feasible conditions for the immune system to eliminate tumor cells. In this review, based on current development of immunotherapy, we will discuss the potential application of nanotechnology to enhance the anti-tumor immune response and overcome tumor immune resistance.

2. Strengthen the immune system 2.1 Dendritic cell immunotherapy As the most professional antigen-presenting cells (APCs), DCs can act as the initiator and modulator in the immune response. They can capture and process antigens to form MHC/peptide complexes, begin to mature accompany with the expression of co-stimulatory molecules, adhesion molecules and chemokine receptors, migrate to lymphoid organ and subsequently motivate naive T cells to become cytotoxic T lymphocytes (CTLs) or helper T cells [17, 18] (Fig. 2). Due to the 4

ACCEPTED MANUSCRIPT outstanding capacities in regulating and activating the immune system, DCs have been considered

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as the attractive target in several immunotherapeutic approaches for cancer treatment.

Figure 2. Attacks from the immune system and defense from the tumor. In peripheral tissues, antigens are captured and processed by distinct DCs to form MHC–peptide complexes. Due to antigen deposition and inflammation, these DCs enter mature state and successively migrate to lymphoid tissues to induce potent immune response. Lymph node resident DCs also conduct the stimulation after encountering with free antigens which are diffused to draining lymph nodes. After activation, immune effector cells are trafficked through blood vessel to battle with tumors. 5

ACCEPTED MANUSCRIPT In tumor site, tumors involve various mechanisms to create a feasible microenvironment, including undergoing mutation, changing physical condition, secreting cytokines (eg. TGF-β, IL-10) and recruiting suppressive immune cells (eg. Tregs, TAMs, MDSCs). In order to achieve the goal of eradicating tumor cells, not only the immune system needs to be strengthened, the immunosuppressive state created to defense immune attack should also be collapsed. TGF-β, IL-10, Tregs, TAMs, MDSCs and TILs represent transforming growth factor-β, interleukin-10, T regulatory cells, tumor associated macrophages, myeloid-derived suppressor cells and tumor

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infiltrating lymphocytes, respectively. Adapted with permission from [19]. Copyright 2014 Elsevier Inc. 2.1.1 Dendritic cell vaccine

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The antigen delivery ability of DCs has been utilized to develop cellular vaccination. The

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therapeutic process includes isolating DCs from peripheral blood by density gradients centrifugation, ex vivo pulsing with tumor antigens and transfusing back to the organism. This therapy was prosperously developed from the mid 1990s. The first generation of DC vaccine involved partially mature DCs which expressed co-stimulatory molecules at a suboptimal level

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and constituted weaker immunogens [1, 20]. To overcome the limitations of the first generation of DC vaccine, clinical trials undertook numerous approaches to obtain matured DCs for the second

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generation of DC vaccine [21, 22]. By using cytokine cocktail that involved IL-1a, tumor necrosis factor-α (TNF-α) and IL-6, it was able to induce the maturation of DCs with high expression of co-stimulatory molecules and chemokine receptors [23, 24]. Notably, the first DC vaccine,

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Provenge® (Sipuleucel-T) for metastatic castration-resistant prostate cancer was approved by FDA in 2010. DC vaccine can elicit CTLs activation and expansion. The results from Leonhartsberszger et al. [25] indicated that about 77% of patients with renal cell cancer were elicited with immune

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responses. It was also documented that the safety of DC based immunotherapy was expected to preserve the quality of life for cancer patients. In line with low toxicity, DC vaccines are capable to sufficiently improve the overall survival, which is generally regarded as the most objective measurement of therapeutic benefit. The median survival was improved to 4.1-month for patients who received treatment with Sipuleucel-T [26]. DC vaccine can elicit adaptive and innate

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anti-tumor immune responses coupled with the low occurrence of immune related adverse events [27]. Although hundreds of experiments were tested on DC vaccine, some drawbacks are still existed, which restrict the application. First of all, the poor migration to the lymph nodes and low occurrence in blood after injection of DC vaccine make a huge demand for antigen modified DCs, which are the direct cause for the other limited factors. Merely 5% transferred DCs can migrate to draining lymph nodes to activate T cells [28]. Secondly, the production is labor-intensive. All the processing steps described above, including cell isolating, antigen-loading and maturation are based on a complicated process and have high requirement for professional laboratory techniques [29]. Thirdly, the production cost hinders the applicability of DC vaccine. For each individual, the preparation of cell isolation, antigen-loading and maturation need to be specific, directly increasing the medical expenditure of patients. According to the advice from the UK National Institute for Health and Care Excellence, the cost of Provenge® is more than $73,000 for per course treatment [30]. Taking all these together, there is huge promotion room in therapeutic 6

ACCEPTED MANUSCRIPT effects, waiting for new therapies to reclaim. Due to the brilliant achievements in nanotechnology, it has sparked an interest to shift the field of DC vaccines generated in vitro to nano-based cancer vaccines, which can deliver antigen to DCs in vivo to realize in situ DC maturation and induce subsequent more efficient antigen-specific T cell response against cancer. 2.1.2 Application of nanotechnology in subunit vaccine In order to induce DCs activation in vivo, conventional cancer vaccines generally consist of

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attenuated or inactivated pathogens which can lead the indispensable problems such as infection risks, allergies and autoimmune responses [31]. It has prompted the development of safe subunit vaccines, which contain the minimal antigenic components necessary to induce appropriate immune responses. Subunit vaccines with specific components (recombinant antigen proteins,

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antigenic peptides, with or without adjuvants) are directly administered to patient to induce antigen-specific immune response. However, the efficacy of subunit vaccine is generally limited

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by the poor immunogenicity, weak stability and short in vivo half-lives. And most importantly, the anti-tumor immune response is influenced by the unsatisfied efficiency of antigen uptake, processing and presenting by endogenous DCs [31]. Nanotechnology, especially with the

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generation of innovative carriers, can provide a reasonable option to efficiently deliver antigens to DCs in situ and induce robust and long lasting immune response against cancer. Nanoparticles can be tuned to improve bioavailability of subunit vaccine, prolong half-lives, promote the interaction between innate and adaptive immune cells or even act as adjuvant by carefully controlling the size,

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shape, surface properties and payloads. The carry system based on nanotechnology applying in cancer immunotherapy can be categorized by the composition as following (shown in Table 1).

Structure

Size

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Compound

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Table 1. The application of nanotechnology in cancer therapy

Nanoparticle

50-400 nm

Characteristic

Efficient antigen

HGP100,

encapsulation and

siRNA,

surface

Paclitaxel,

modification

poly(I:C), CpG

Ovalbumin, 25-140 nm

micelle

Enhance antigen

siRNA, Poly

presentation

I:C, Trp2, CpG ODN

Enhance cell Dendrimer

Refs

Therapeutic carrier

and

adjuvant

[32], [36], [61], [149]

ODN

nanocarrier Polymeric

Medical application

Ovalbumin,

Polymers based

Payload

500 nm

uptake

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Therapeutic carrier and adjuvant

[33], [150]

Therapeutic Proteins

carrier

[34]

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GM -CSF, Nanogel

50-100 nm

Therapeutic

[35],

carrier and

[73],

adjuvant

[129]

paclitaxel,

Therapeutic

[37],

IL-12,

carrier and

[38],

c-di-GM P,

modification

[122]

Efficient antigen

gp-100,

encapsulation

Ovalbumin, LPA, CpG Ovalbumin,

Lipid based

Liposome

Surface

40-300 nm

modification

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nanocarrier

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CpG-ODN

Ovalbumin, Self-adjuvant, 5-200 nm

photothermal and imaging

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Nanoparticle

TRP2,

HGP100, Poly

50-150 nm

Inorganic nanocarrier 100-200 nm

Nanorod

40*10 nm

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nanoparticle

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Virus-like

45–50nm

30nm

particle

Vesicle

photothermal

encapsulation

CpG, Poly(I:C),

carrier

Efficient payload

Anti-CTLA-4

Antibody

encapsulation

antibody

carrier

Virus

[47-48]

and imaging Therapeutic

Self-adjuvant

[41],

Coated with

[215]

[216]

[49]

antigens

Accumulate to

Hsp70, Tumor

Therapeutic

[40],

specific site

lysates

carrier

[217]

Therapeutic Virus mimicry to

carrier and

[53],

passive target

immunotherapy

[184]

agent Immunogenic and reflect the

Biomimetic nano-carrier

adjuvant,

Hemagglutinin,

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Nanotube

carrier,

Efficient antigen

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Nanoshell

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I:C, siRNA

Therapeutic

100-400 nm

diversity and breadth of the

Therapeutic HER-2, ovalbumin, CpG, M PLA

tumor antigen

carrier and

[51],

immunotherapy

[218]

agent

repertoire Peptide based nanostructure

Human tolerated 70-90 nm

with multiple structure 8

Peptide

Therapeutic carrier

[219]

ACCEPTED MANUSCRIPT Polymer based nano-carriers With the good biocompatibility and the ability to undergo hydrolytic and biological degradation, polymers not only represent the essential role in conventional pharmaceutical formulations, but also commonly act as “building blocks” in engineering nano-materials based delivery systems. Polymers based nano-carriers mainly include polymeric nanoparticles, micelles, dendrimers and nanogels [32-35]. Generally, the widely used polymers contain poly(D,L-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA), poly(caprolactone) (PCL), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, polyethylenimine (PEI) and so on. And the varieties of encapsulation techniques make it possible to encapsulate the payload with different physicochemical properties. For example, the double-emuls ion method is efficient

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in encapsulation of proteins [32]. And the nanoprecipitation method is suitable to encapsulate both hydrophilic and hydrophobic therapeutics [36].

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Lipid based nano-carriers Owing to the excellent amphiphilic nature and good biocompatibility,

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lipid based nanostructures have been broadly used in protecting hydrophilic and lipophilic drugs and antigens. Liposomes have already been used in chemotherapeutic drug delivery to enhance therapeutic effect and reduce side effects. Until now, there are several drug-loaded liposomes ®

®

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approved in clinic, such as Lipusu (for paclitaxel) and Doxil (for doxorubicin). Lipid based nano-carries usually contain 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol [37, 38]. The rigidity and fluidity of nanostructures and the charge of the surfaces are determined by

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the component of lipids. Due to the convenience in modulating physical characters, lipid based nano-carriers are desirable for delivering therapeutics and further engineering [39].

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Inorganic nano-carriers Inorganic nano-carriers have superiorities in size control, morphology regulation and structural conformation. Furthermore, by introducing different inorganic materials, nano-carriers are endowed with some additional and useful properties such as iron based

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nanoparticles for magnetite application, gold based nano-carriers for photothermal therapy and imaging [40-44]. In addition, some inorganic nanoparticles are usually perceived to exhibit adjuvant effects [45]. Recently, nanoparticles based on aluminum [41], mesoporous silica

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nanoparticles [46, 47], zinc phosphate nanoparticles [48], gold nanoparticles [49] and graphene oxide nanosheets [50] have been regarded as potential immune adjuvants. Biomimetic nano-carriers Biomimetic nano-carriers have similar components with the donors, which could regulate many disease processes, notably in cancer and inflammation. Tumor derived subcellular vesicles have abilities to load adjuvant as well as reflect the diversity and breadth of the tumor antigen repertoire. It makes these membrane-enclosed subcellular particles as potential carriers to enhance payload uptake by DCs and promote activation of DCs [51]. Bacterial protoplast-derived nano-vesicles (PDNVs) possessed high productivity and safety and induced strong antigen-specific immune responses against bacterial infection [52]. Virus-like particles (VLPs), with the particular virus capsid and similar antigenicity with the parental virus, were successfully used as vaccines against infectious diseases and cancer [53-55]. Nanotechnology for antigen loading Nanoparticles can be used to encapsulate different hydrophilic/hydrophobic payloads by physical loading or through stimuli-responsive linkage with high entrapment efficiency [56]. The 9

ACCEPTED MANUSCRIPT antigen of subunit vaccine is easily degraded in vivo and nanoparticles can improve their stability after entrapment [48]. Xu and colleagues [57] formulated lipid-calcium-phosphate (LCP) nanoparticles with good encapsulating capacity of murine melanoma antigenic peptide TRP2 about 65%. The poor immunogenicity is another reason for the unsatisfied anti-tumor effect of subunit vaccine. Simply using s ingle peptide might be insufficient to generate efficient anti-tumor immune response because tumor cells have various mechanisms to escape immune surveillance. In our group, the lipid coated zinc phosphate hybrid nanoparticles (LZnP NPs) were formulated to co-deliver multiple antigenic peptides (TRP2 and HGP100) with high encapsulation efficiency, which benefited from the unique coordinative binding between peptides and materials of

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nanoparticles. The combination of peptides provides multiple epitopes for immune cells to recognize, making tumor cells more difficult to escape from the surveillance of immune system

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[48]. Nanoparticles with such sufficient room to accommodate payloads are the desirable

Nanotechnology for targeting delivery of antigen

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controlled delivery platform.

The potency of antigen-specific immune response is proportional to the efficient delivery of

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antigen and immune stimulus to DCs. To improve the delivery efficiency, nanoparticles can be designed to target DCs via modifying with specific targeting motifs such as C-type lectin receptors

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(Dec-205, mannose receptor), Fc receptor (FcR, the receptor for the IgG Fc fragment), TNF-α family receptor (CD40) and integrin receptor (CD11c) [58-60] (Figure 3). Dec-205 targeted nanoparticles may increase the access to the cytoplasmic MHC I loading machinery and facilitate

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the stimulation of anti-tumor CD8+ T cells [61]. Cruz et al. [62] demonstrated that gold nanoparticles conjugated with the Fc fragment were observed in a specific compartment in DCs , which showed an active uptake by interaction with the FcR and great capacity to induce lymphocyte proliferation. In addition, FcR-mediated uptake pathways were capable to lead

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efficient cross-presentation of antigen complexes [63]. Mannose targeted nanovaccine was formulated by incorporating DSPE-PEG-Mannose into erythrocyte-membrane coated polymeric nanoparticles. This targeted nanovaccine exhibited great potential to target resident DCs. Accompany with the superiority of DC uptake and activation in vitro and in vivo, the targeted nanoparticles significantly increased the vaccine-induced anti-tumor immune responses and

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enhanced the subsequent anti-tumor effects [64] (Figure 4a).

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Figure 3. Application of nanotechnology in the cancer immunity circle. The cancer immunity circle involves several steps for immune cells propagating through afferent and efferent limbs, which provides potential therapeutic targets for nanotechnology. For DC vaccine therapy and adoptive T cell therapy, the principle of immunotherapy is to broaden and amplify T cell responses. Nanotechnology can be applied in DC vaccine therapy to deliver payloads, enhance cross-presentation and promote DCs maturation and migration, leading potent T cell responses. In adoptive T cell therapy, the application of nanotechnology can facilitate T cell expansion ex vivo or in vivo. Immunomodulatory therapy can overcome the immunosuppressive mechanism conducted by tumors, serving as effective ways to collapse tumor defense. Nanotechnology can be applied in delivery of immunomodulators, immunochemotherapy and checkpoint therapy. Adapted with permission from [65]. Copyright 2013, Elsevier Inc.

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Figure 4. (a) Schematic illustration of the preparation of Man-RBC membrane-coated PLGA-SS-hgp100 nanoparticles (Man-RBC NPhgp ) and induction of anti-tumor immunity. (b) Expected mechanism of antigen presentation mediated by pH-sensitive polymer-modified liposomes. When trapped in endosomes, the polymer-modified liposomes are sensitive to the acidic environment and disrupt endosomes and release antigens into cytosol. This process enhances cross-presentation and the induction of the antigen-specific CTL. (c) The size dependent drainage of nanoparticles into lymph node. Fluorescent nanoparticles were injected into the hind footpads to image the traffic of nanoparticles to the lymph nodes at the different time points. Arrowheads represent popliteal lymph nodes and arrows represent with the injection sites. A reprinted with permission from [64]. Copyright 2015, American Chemical Society. B reprinted with permission from [66]. Copyright 2012, Elsevier Ltd. C reprinted with permission from [67]. Copyright 2008, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 12

ACCEPTED MANUSCRIPT Nanotechnology for cross-presentation The anti-tumor response of CD8+ T cell plays an essential role in immunotherapy. Generally, +

in DCs, peptide fragments need to bind to MHC class I molecules to induce CD8 T cell response. However, antigens from the extracellular environment are typically degraded into short peptides +

and loaded onto MHC class II molecules that are presented to CD4 T cells [68]. The biogenesis of MHC class I and II have a strict compartmentalization which leads the different ways to process

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antigens. In the endoplasmic reticulum pathway, endogenous but not exogenous antigens are selectively loaded on MHC class I molecules. In the endocytic pathway, both exogenous and endogenous antigens are loaded on MHC class II molecules [69]. It was identified by Kovacsovics-Bankowski et al. [70] that internalized antigens can undergo cross-presentation

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which provided exogenous antigens opportunities to form MHC class I complex. As mentioned, antigens enter DCs in the endocytic pathway and the loading process is sensitive to the lysosome

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function. The disruption of lysosome permits endocytic degraded peptides to bind with MHC class I molecules [69]. In order to enhance antigen cross-presentation, antigens need to escape from lysosome and access cytosol to bind to MHC class I molecules. The protein-loaded PLGA

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nanoparticles were demonstrated to help the protein escape from endosomes into the cytoplasm, which made it convenient for antigen accessing to the MHC class I loading pathway [71]. It was exemplified by Yuba et al. [66] that pH-sensitive polymer-modified liposomes disrupted endosomes and efficiently released antigens into cytosol after encountering with acidic

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environment in endosomes. This kind of pH-sensitive nanoparticles can sufficiently process antigens to form MHC class I-peptide complexes and activate antigen-specific CTLs (Figure 4b). Photosensitization can also be employed to facilitate cross-presentation. Antigens and

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photosensitizer were co-loaded in nanoparticles. After triggering by light, the antigen-containing endosomes were disrupted, which promoted MHC class I cross-presentation and induction of +

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CD8 T cell responses [72]. Nanoparticles, which can be decomposed in response to particular factors such as the light and weak acidic situation, are ideal candidates to facilitate antigens escape from the endo-lysosome to enhance cross-presentation [73-75]. Nanotechnology for DC maturation

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The “danger signal” is essential for triggering immune response, which may be lost in subunit vaccines accompany with removing unnecessary components of classic vaccines [31]. Therefore, besides antigenic payloads, subunit vaccines generally require adjuvant to enhance vaccine efficacy. With the help of nanoparticles, co-encapsulation of antigen and adjuvant has exhibited superiority to trigger T cell and B cell responses compared with simple admixture of antigen with adjuvant. Toll like receptors (TLRs) are a large family of surface receptors on DCs which can recognize most of pathogenic molecules, such as bacterial wall compounds (lipopolysaccharide-LPS) and CpG. With the ability to induce maturation of primed DCs, a prerequisite for effective immune response, TLRs agonists have been widely used as adjuvants to promote antigen presentation [38, 76, 77]. For instance, the TLR9 agonist CpG was conjugated on biomimetic protein nanoparticles with an antigenic peptide via acid-labile hydrazone bonds. It enhanced the release of CpG in the endosome compartment, leading to more robust activation of antigen-specific T cells [78]. In the work from Zhang et al. [79], the TLR4 agonist 13

ACCEPTED MANUSCRIPT monophosphoryl lipid A (MPLA) was co-loaded with antigenic peptides in PLGA nanoparticles. PLGA nanoparticles incorporating MPLA exhibited significant DC maturation in vitro and secretion of inflammatory cytokines. Compared with single peptide nanoparticles formulation, nanoparticles accompany with MPLA induced much stronger T cell response and tumor growth inhibition in a prophylactic setting. Similar results were also demonstrated in multiple peptide-loaded lipid-enveloped PLGA nanoparticles [56]. Nanoparticles, especially inorganic nanoparticles and cationic nanoparticles have been shown with some adjuvant effects. Gold based nanoparticles showed 15-fold greater increase in amount of secreted TNF-α, suggesting that nanoparticles without adjuvant can efficiently induce host immune responses than free antigen

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[80]. And aluminum oxyhydroxide nanorod can boost antigen-specific immune response based on the adjuvant effects of nanoparticles engaging with the NLRP3 inflammasome and the IL-1β

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production [41]. PEGylated graphene oxide nanosheets were demonstrated to induce potent cytokine responses by enhancing integrin β8 -related signaling pathways [50].

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Nanotechnology for drainage into lymph node

The diverse subpopulations of DCs vary from residence and function. In this review, we

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focus on two categories in cancer vaccine, the peripheral DC, a subset migratory to the draining lymph node, and the lymphoid tissue-resident DCs which express CD8 surface marker and are

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professional in cross-presentation [81]. Since the lymphoid organs play an essential role in adaptive immune response, transporting antigen to the draining lymph node is a key factor in inducing potent antigen-specific immunity.

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Nanoparticles with the controllable size are more appealing to lymph nodes. It was reported that smaller nanoparticles can be more efficiently transported to the draining lymph nodes via convective force and interstitial diffusion. With the size increasing, it becomes hard for nanoparticles to be directly transported to draining lymph node because of hindrance from the

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extracellular matrix. Generally, larger nanoparticles migrate to the lymph nodes mainly depends on internalization by peripheral DCs [82]. Manolova et al. [67] evaluated the trafficking of particulate to the draining lymph node by subcutaneously administrating polystyrene particles with the size between 20 and 2000 nm. It was demonstrated that particles with a size around 20 nm rapidly and efficiently arrived in the draining lymph node 2 h post-injection while 1000 nm

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nanoparticles were hardly detectable in the draining lymph node until 24 h post-injection. Furthermore, after depletion of DCs, the trafficking of 500 nm particles to the draining lymph node was abrogated but the trafficking of 20 nm particles was rarely affected (Figure 4c). In addition, nanoparticles with the hydrodynamic diameter around 5–6 nm are rapidly eliminated by renal filtration and urinary excretion and nanoparticles with the hydrodynamic diameter in sub-micrometer grade are usually cleared by phagocytes reticuloendothelial system [83, 84]. Nanoparticles with a size between 20 and 200 nm may be the appropriate choice both for promoting encapsulated antigens to be captured by APCs and protecting antigen loaded nanoparticles from clearance [85]. 2.2 T cell therapy The major principle of immunotherapy is to induce potent and lasting T cell response. In lymph nodes, primary T cell response requires T cells to recognize the processed and presented 14

ACCEPTED MANUSCRIPT antigen fragments through TCRs. Intracellular antigens can be cut into peptides in the cytosol of APCs and presented by MHC class I molecules to interact with CTLs which possess the ability to directly kill target cells. Extracellular antigens, which enter the APCs by the endocytic pathway, can bind to MHC class II molecules and be presented to T helper cells with immune regulatory effects. Generally, naive T cells continually recirculate between the blood and lymph nodes to search for antigens [86]. When encountering with activated DCs, the naive T cells are primed, following with activation, cytokine secretion and proliferation [87]. Among this process, antigen-specific T cells will be activated to attack tumor cells accompany with differentiation of long-lived memory cells [81]. Acting as the primary executor of immune system, T lymphocytes

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have attracted the attention of researchers to exploit the T cell therapy in cancer treatment.

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2.2.1 Adoptive T cell therapy

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Therapeutic or prophylactic vaccination can induce immunization actively. Passive immunization is another fundamental strategy to stimulate anti-tumor immunity which has entered the realm of clinical immunotherapy. Herein, passive immunization refers to adoptive T cell therapy that is the infusion of autologous or allogeneic T cells into patients [88]. In 1955, the

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adoptive transfer of lymphocytes was carried out in tumor bearing mice [89]. In 1970s, by co-transferring autologous leukocytes, Southam et al. [90] showed specific inhibitory effect on the

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growth of tumor in about half of patients with advanced tumor. In 1988, Rosenberg et al. [91] found that lymphocytes extracted from freshly resected melanoma can be expanded in vitro and possessed the ability to mediate specific lysis of tumor cells. By means of adoptively transferring

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these tumor infiltrating lymphocytes and IL-2 back into the patient, the objective regression of metastatic melanoma was witnessed in 60% of patients. Further understanding of T cells biology makes it possible to explore the potential of adoptive immunotherapy. Recent applications of adoptive T cell therapy include TIL therapy, CTL therapy and genetically engineered T cell

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therapy.

TIL therapy TIL therapy involves T cells isolated and enriched from tumor, which has been

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considered as promising treatment for metastatic melanoma. The study conducted with 93 patients with metastatic melanoma had shown that transfusion of autologous TILs can mediate durable complete tumor regression [92]. However, the efficacy of TILs appeared to be restricted to melanoma. Although other types of tumors also contain T cells, there are few clues about the specificities and functions of these T cells [93]. So in this review, we will focus on T cell therapy suited to be applied in various cancers. CTL therapy For CTL therapy, T cells were generated by in vitro stimulating purified CD8+ peripheral blood lymphocytes with antigen [94]. A phase I study for adoptive CTLs therapy has been permitted and about 80% of patients were induced with antigen-specific immune response [95]. Genetically engineered T cell therapy Accompany with the rapid development of genetic engineering in the past few years, it provides opportunities to genetically engineer normal lymphocytes with attractive functions, especially recognition of tumor associated antigens. Rosenberg’ group [96] used anti-CD19-chimeric-antigen-receptor (CARs) transduced T cells and IL-2 to treat patients with progressive B-cell malignancies. The effective application of engineered 15

ACCEPTED MANUSCRIPT T cell therapy provides opportunities for extending the adoptive T cell therapy approach to various cancers [97]. 2.2.2 Application of nanotechnology in adoptive T cell therapy Nanotechnology for CTL therapy Despite the achievements in clinical trials, the primary requirement for CTL therapy is to expand T cells ex vivo efficiently and economically. Traditional approach for T cells expansion

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uses autologous APCs to interact with T cells. However, leaving out the cost and cumbersome work, the involvement of autologous APCs is unable to guarantee the stable quality of T cell products on account of the affection from patient’s disease state [98]. Another way for expanding CTLs is usually done by employing synthetic anti-CD3/CD28 beads. CTLs produced in this way

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still suffer from variability in quantity, viability and functionality and even lose the antigen-specificity [99]. The lack of stable APCs is the current limitation for T cells expansion. It may be overcome by synthetic artificial APCs (aAPCs). With appropriate control of size, surface area and morphology, particle based on nanotechnology shows superiority in engineering aAPCs.

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During the activation of T cells, it will persistently induce a state of nanoscale TCR clustering [100, 101], which may be more accessible for particles in nano-size to trigger receptor than that of other larger particles. To identify the interaction between TCR clustering with

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nanoparticles, Perica et al. [40] synthesized a nano-aAPCs by coupling chimeric MHC-Ig dimer and anti-CD28 antibody to 50-100 nm paramagnetic iron-dextran nanoparticles. And external magnetic field served as a powerful tool to control particle behavior. Nano-aAPCs driven by an external magnetic field were inclined to bind activated T cells which have more available TCR

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clustering than naive T cells (Figure 5a). It is consistent with the former hypothesis that nano-aAPCs are suitable to interact with nanoclusters of TCR. The sensibility to TCR clustering of nanoparticles resulted in a lower threshold for T cells activation which efficiently increased the expansion of T cell ex vivo. The stimulation of T cells can also be promoted by enhancing the intensity of T cell stimulus [102]. Nanoparticles can meet the requirement by providing adequate

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surface area to present high density of T cell stimulus. Single walled carbon nanotubes (SWNTs) were used as artificial antigen-presenting construct. SWNTs can induce potent activation of T cells by presenting with high local concentration of stimulating antibodies such as anti-CD3. Owing to the large active surface area, SWNTs with high density of stimulus can be exploited as effective platform for T cell stimulation and expansion [103] (Figure 5b and c). In addition, the morphology of nanoparticles may influence the expansion of T cells. Results from Meyer et al. [104] demonstrated that non-spherical nano-aAPCs had superiority in T cell activation. It showed that ellipsoidal nano-aAPCs significantly mediated 15-fold expansion of T cells while traditional spherical particles only induced 3-fold expansion. With similar synthesis conditions and surface protein content of particles, non-spherical nano-aAPCs showed superiority in the induction of antigen-specific CTLs than spherical nano-aAPCs (Figure 5d and e). Cytokines play an important role in long-term T cell expansion [105]. IL-2 is frequently employed for T cell expansion but the efficacy is limited by the free form. Nanoparticles may act as excellent candidates for protection and controlled release of cytokines to promote T cell expansion. In the work from Fahmy and colleagues [106], they firstly constructed carbon nanotube (CNTs) binding neutravidin to present peptide-loaded MHC-I and the co-stimulatory 16

ACCEPTED MANUSCRIPT ligand anti-CD28. Then IL-2 and magnetite were co-encapsulated into PLGA nanoparticles to bind with CNTs to form carbon nanotube–polymer composite (CNP) (Figure 5f). The IL-2 requirement for CNP to match the same effect was about 1000-fold less than that of soluble form. With the assistance of controlled release of IL-2, CNP significantly improved T cell expansion and function compared with CNTs with exogenous IL-2. The anti-tumor efficiency of CNP-activated T cells was evaluated by transferring the activated cells to murine melanoma model. Significant delay in tumor growth was observed in the group treated with adoptively transferred

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CNP-activated T cells. As a favorable prognostic biomarker, TILs harvested from CNP treated mice were at least ten times higher than control.

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ACCEPTED MANUSCRIPT Figure 5. (a) Schematic of magnet-induced clustering by nano-aAPC. By applying magnetic field, the irondextran nanopartic les coupled with MHC-Ig dimers and co-stimulatory anti-CD28 will result in TCR cluster. (b) Schematic of anti-CD3-adsorbed SWNT scaffolds inducing T cell stimulation (c) Fluorescence microscopy of T cells (Cells were stained with CFDA-SE staining solution), T cells + SWNT (cells incubated with blank SWNT bundle) and T cells + SWNT + anti-CD3 (cells incubated with anti-CD3-adsorbed SWNT). (d) Schematic of non-spherical and spherical nanodimensional aAPC synthesis by conjugating MHC-Ig Dimer and anti CD-28 to PLGA nanoparticles. (e) The percent of CD8+ T cells that were also Thy 1.1 positive on blood was analyzed on day 6, 8, and 10 post injection. Ellipsoidal and spherical aAPCs were injected

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intravenously into irradiated mice accompanied by 106 antigen-specific CD8+ T cells bearing the marker Thy 1.1. “No treatment” groups received T cells only. (f) Schematic of the engineered CNP

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platform, including paracrine release of IL-2, multivalent antigen presentation and magnetic

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separation of CNPs from T cells. (g) Schematic of maleimide-based conjugation to nanoparticles to the surfaces of T cells via cell-surface thiols. A reprinted with permission from [40]. Copyright 2014, American Chemical Society. B and c reprinted with permission from [103]. Copyright 2008, American Chemical Society. D and e reprinted with permission from [104]. Copyright 2015,

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WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. F reprinted with permission from [106]. Copyright, 2014 Nature Publishing Group. G reprinted with permission from [107]. Copyright, 2010 Nature Publishing Group.

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Nanotechnology for genetically engineered T cells therapy

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Genetically engineering strategies provide opportunities to redirect tumor-specific T cells without affecting their initial specificity. However, the viability and function of these engineered T cells suffer from a rapid decline after transplanting back to hosts. With deliberate designation, nanoparticles may provide some solutions to reverse these unsatisfied situations. Irvine’s group

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[107] firstly fabricated drug-loaded liposomes and liposome-like nanoparticles with lipid bilayer including thiol-reactive maleimide headgroups. Then, taking use of substantial amounts of free thiols on the surfaces of T cells, the synthetic nanoparticles were stably conjugated to the plasma membrane of T cells (Figure 5g). By evaluating the tumor homing properties of T cells with surface-conjugated nanoparticles, it showed that in vivo tumor homing of adoptive transfusing T

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cells was not affected by nanoparticle conjugation. Actually, nanoparticle-decorated T cells can efficiently carry surface-tethered nanoparticles into antigen-expressing tumors. According to the results from in vivo T cell expans ion, cell-bound cytokine-loaded nanoparticles markedly elicited T cells proliferation about 81-fold higher than unmodified T cells. By this strategy, it is convenient to deliver a range of therapeutics such as adjuvant, protein and siRNA to collaborate with T cells to induce potent anti-tumor effects [108]. 2.2.3 Immune checkpoint therapy on T cells During the course of tumor invasion, there are complicated cell-intrins ic pathways participating in the immune response. Especially, CTLA-4 and programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway can inhibit T cell activation. Immune checkpoint CTLA-4 and PD-1 are often utilized as targets to block these inhibitory pathways [109] (Figure 6).

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Figure 6. Mechanisms of activating cytotoxic T Cells by immune checkpoint therapy. Reprinted with permission from [109]. Copyright, 2015 Elsevier Inc. The activation and subsequent differentiation of T lymphocytes are mainly depended on

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efficient interaction between APCs and T cells. Two stimuli are indispensible to this process. The one is frequently mentioned antigenic peptide MHC molecule, ligating to TCR. The other one is

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co-stimulatory signal mediated by the interaction between CD28 expressed on T cells and B7-1/2 (CD80/CD86) expressed on APCs [110]. Following the activation, not only proliferation and

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functional differentiation are induced but an incidental inhibitory program is also mediated, which will ultimately cease T cell proliferation. Overreaction of activated T cells will trigger negative feedback regulatory loops. CTLA-4, a competitor of CD28, has much greater avidity to bind with B7-1/2 than that of CD28. It can mediate a parallel program to eventually attenuate or prevent

CD28 co-stimulation [111]. Ipilimumab, the anti-CTLA-4 antibody for advanced metastatic melanoma, is the first immune checkpoint agent approved by FDA in 2011. Around the same period, a new inhibitory pathway, the PD-1/PD-L1 pathway was introduced. PD-1 is an inhibitory receptor mainly expressed on T cells. PD-L1, a functional partner of PD-1, is seldom expressed on normal tissue but abundantly expressed on the surface of various tumor cells [112]. The activation of PD-1/PD-L1 pathway can turn activated T cells into an anergic state, facilitate the apoptosis of activated T cells and secretion of immunosuppressive cytokines [113-115]. The blockade of PD-1 is able to modulate the immune suppression from tumor, restore the function of anergic T cells and induce a long term memory response [116-118]. Recently two PD-1 mAbs, Nivolumab from Bristol-Myers Squibb and Pembrolizumab (or Lambrolizumab) from Merck, were approved by FDA for cancer treatment [112]. Recent achievements of checkpoint immunotherapy are mainly based on T cells because of following compelling advantages. First of all, immune checkpoint blockade on T cells could efficiently enhance T cell response or reverse T cell exhaustion [119]. Secondly, T cell can provide 19

ACCEPTED MANUSCRIPT a stable target. Since genetic mutation frequently occurs in tumor, it is suboptimal to choose tumor cells as target. In addition, this therapeutic strategy makes it feasible to be applied in many different histologic tumors [111]. 2.2.4 Application of nanotechnology in immune checkpoint therapy on T cells Nanoparticles based delivery system, as the potential loading strategy, has already been applied in immune checkpoint therapy. First of all, involving nanotechnology is able to control the release of antibodies. It is available to exhibit more potent anti-tumor efficacy, reduce the cost of

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treatment and minimize the side effects from dose-dependent autoimmune disorders. In the work from Wang et al. [120], an innovative self-degradable microneedle was constructed by biocompatible hyaluronic acid and pH-sensitive dextran nanoparticles. Anti-PD1 antibody was

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encapsulated in the nanoparticles accompany with glucose oxidase that can turn blood glucose

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into gluconic acid, leading gradual self-dissociation of nanoparticles and sustained release of payload. It showed that the release of anti-PD1 antibody can last for three-day after the first administration. Microneedle patch treated mice exhibited more robust immune responses than that of free antibody. In addition, the functionalized structure of nanoparticles can also be used to

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increase protein activity and loading capability. Lei et al. [121] took advantage of nanoporous in functionalized mesoporous silica to trap therapeutic antibodies to blockade the immunoregulatory

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molecule CT LA-4. Nanopore geometry of this nanoparticle can load high amount of anti-CTLA-4 mAb and provide long-lasting local release, which showed stronger therapeutic effects than systemic administration of free antibody. With the development of genetic immunotherapy, these

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negative feedback pathways can be blocked by regulatory agents such as siRNA. Nanoparticles can efficiently deliver siRNA to the target cells and modulate negative feedback loops. Li et al. [122] fabricated CTLA-4 siRNA-loaded nanoparticles to restore the anti-tumor functions of T cells. The nanoparticles enhanced the activation and proliferation of T cell in vitro. In a melanoma model, this platform efficiently delivered CTLA-4 siRNA to CD4+ and CD8+ T cells in the tumor,

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increasing the percentage of CD8 T cells while decreasing the ratio of inhibitory T regulatory cells (Tregs). The knockdown strategy based on PD-1 ligand siRNA can also be used to improve the T cells response in DC vaccine. In the work from Hobo et al. [123], the PD-L1 and PD-L2 siRNA were co-delivered by lipid nanopartic les to specifically silence the PD-L expression on

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human monocyte-derived DCs. The ex vivo experiments indicated that the PD-L-silenced antigen-loaded DCs superiorly enhanced antigen-specific response of CD8+ T cells obtained from transplanted patients.

2.3 Application of nanotechnology for other immune cells The immune system also involves other functional immune cells to protect hosts from pathogens, such as natural killer (NK) cells, macrophages and neutrophils. NK cells, a type of lymphocytes, possess the ability to immediately and directly defense against pathogens without pre-exposure to antigens. Tumor may escape from immunosurveillance via the loss or down-regulation of MHC-I expression. The NK cell is insusceptible to these variations, which makes it as an ideal candidate [124]. The work from Nakamura et al. [125] showed that cyclic di-GMP encapsulated within liposomes efficiently activated NK cells via activating the stimulator of interferon genes (STING) pathway and induced anti-tumor effect against malignant melanoma. 20

ACCEPTED MANUSCRIPT Clinical studies demonstrated that the increased number of tumor infiltrated NK cells was correlated with improved prognosis [126]. However, the trafficking process of NK cells to the tumor may be confronted with direct immune evasion mechanisms of cancer cells [127]. Jang et al. [128] used magnetic nanoparticles to control the movement of NK cells. Under the help of an external magnetic field, the ratio of nanoparticles loaded NK cells infiltrated into the target tumor site was enhanced about 17-fold and the killing activity was still maintained. Macrophages can efficiently capture, process and present antigens. Recent findings indicated that macrophages localized in lymph nodes may play an important role in cross-presentation of antigen [129]. Antigen encapsulated in nanocarriers can be efficiently transported to the draining

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lymph node, and preferentially engulfed by medullary macrophages. Then these macrophages effectively primed CD8+ T cells, resulting in significant tumor growth inhibition [130]. Moreover,

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macrophages could induce naive CD4+ T cells to differentiate into either Th1 or Th2 cells [131].

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Jones et al. [58] synthesized mannosylated-poly(beta-amino esters) nanoparticles to deliver antigen encoded DNA to macrophages. The results indicated that nanoparticles without the help of adjuvant were able to improve standardized antibody titers and induce significant immune responses.

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Neutrophils are the sentinels to clean extracellular pathogens and induce inflammatory response [132]. Studies indicated that the phenotypic plasticity of tumor-associated neutrophils (TANs) was analogous to the M1/M2 phenotype of macrophages. Resident TANs can develop into anti-tumor phenotype (the “N1” TANs) and pro-tumor phenotype (the “N2” neutrophils) [133]. N1 neutrophils was considered as potent anti-tumor effector cells to coordinate adaptive

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immune response, produce pro-inflammatory cytokines, recruit other immune effector cells and directly mediate cytotoxicity to tumor. Lizotte et al. [53] constructed the self-assembled virus-like nanoparticles derived from cowpea mosaic virus (CPMV). With the inherent immunogenicity, CMPV nanopartic les were able to activate neutrophils in the tumor microenvironment to induce

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anti-tumor immune response. It was the first report that virus-like nanoparticles can be directly used in cancer immunotherapy rather than as a simple delivery system of therapeutic agents. They also exploited the treatment efficacy and systemic anti-tumor immunity of the virus-like nanoparticles in a wide range of tumor models including ovarian, colon, and breast cancers. With

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stable and nontoxic properties, drugs and antigens loading capacity, high scalable nano-manufacture and the inherent immunogenicity, CPMV derived virus-like nanoparticles could be regarded as an attractive vehicle in future cancer treatment.

3. Collapse tumor defense to immune system In current cancer immunotherapy, multiple approaches have been applied to recruit activated immune cells to specifically recognize and eliminate tumor. However, it is difficult to meet the therapeutic expectations due to the tumor-induced suppression. Since tumor cells are able to craftily manipulate the variation of living condition and find a way out among the activated immune cells, the immune system is confronted with the following hurdles. The developing tumor cells intrins ically undergo a series of changes including the immunogenic sculpting, activation of signaling pathways and expression of anti-apoptotic signals etc., directly resisting the tumor inhibitory actions from the immune system [134, 135]. Besides, the extrinsic microenvironment will develop into a feasible condition. The mutual interactions between physical condition and tumor

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ACCEPTED MANUSCRIPT Immunosuppressive cells and cytokines will be elaborately involved to indirectly impede the anti-tumor immune responses [124]. Effective therapeutic agents are just like sharp arrows. It needs a strong bow to straightly shot them to the tumor site, overcome obstacles and leave no space for immune escape. Fortunately, it can be realized by nanotechnology based delivery system. The delivery of varied therapeutic agents to tumor microenvironment via nano-carriers can improve efficiency of the immune system and modulate microenvironment at the same time. 3.1 Overcoming tumor intrinsic resistance

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To resist extrinsic tumor suppressive actions, tumors undergo crafty changes to escape from immune surveillance. One of these alterations is to evade tumor recognition by immune effector cells, usually including los ing antigen and MHC components and shedding NKG2D ligands. The

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other one of the changes is to trigger certain mechanisms of tumor to disturb immune destruction,

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such as inhibiting death-receptor signaling pathways and expressing anti-apoptotic signals. 3.1.1 Application of nanotechnology in enhancing recognition

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Through loss/down-regulation of tumor associated antigens and MHC components, tumor creates an “invisible” state for effector T cells [137]. Moreover, confronting with the countless

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attack from the immune system, tumor cells shed NKG2D ligands, depriving the induction of NK cells and the function of CTLs [138, 139]. One appropriate way to reverse the “invisible” state is to exploit available antigen-specific epitopes for T cell recognition. Lee et al. [140] found that the tolerance of self-antigenic epitopes may cause tumor survival. By introducing foreign antigenic epitope in tumor cells, T cells

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recognized these modified tumor cells as foreigners. This strategy efficiently facilitated the presentation of foreign antigen fragments and allowed antigen-specific CTLs to eradicate the

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tumor. In this work, they utilized hyaluronic acid based nano-carriers for targeting delivery of foreign antigens to tumor cells, leading preferential accumulation in tumor tissue and effective uptake by tumor cells (Figure 7a). The in vivo anti-tumor experiment indicated that mice treated with foreign antigens loaded hyaluronic acid based nano-carriers had almost 10-fold higher number of antigen-specific CD8+ T cells than no treatment mice (Figure 7b). NKG2D, which is +

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widely expressed by NK, CD8 T and NKT cells, represents a distinct receptor involving in triggering immune effector cells. Through shedding the binding ligand, tumor could successfully escape from immune surveillance. Gong et al. [141] prepared chitosan-based nanoparticles encapsulated with a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid to produce the dsNKG2D-IL-15 protein. The generated protein can bind to NKG2D ligand and efficiently activate NK or CD8+ T cells via the IL-15 moiety, thereby mediating anti-tumor immunity and retarding tumor growth.

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Figure 7. (a) Schematic illustration of self-tolerance conducted by tumors and the process of targeted delivery of foreign antigens into tumor cells to induce immunologic rejection of foreignized tumor cells. (b) Quantification of OVA-specific CD8+ T cells in splenocytes derived

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from TC-1 tumor-bearing mice. (c) Proposed mechanism of QD induced apoptotic cell death by activation of Fas/FasL signaling pathway. (d) Fas expression on the cell-surface of neuroblastomas by exposure to QDs. (e) The dose-dependent knockdown in STAT3 and reduction in VEGF levels by siRNA complexes in vitro. (f) Anti-tumor effect of siRNA complexes in vivo. A and b reprinted with permission from [140]. Copyright 2014, Elsevier B. V. C and d reprinted with permission from [142]. Copyright 2007, BioMed Central Ltd. E and f reprinted with permission from [143]. Copyright 2009, Elsevier Ltd. 3.1.2 Application of nanotechnology in inhibiting adverse pathway Besides tumors undergo intrinsic variation to escape from immune surveillance, tumor cells can develop the defect in receptors or signals to resist immune destruction and facilitate tumor cell survival and proliferation. Fas/FasL signaling pathway, a well-known cell death receptor signaling pathways, can mediate tumor cell apoptos is under the proper conditions [144]. Choi et al. [142] demonstrated that tumor cells treated with quantum dots can significantly up-regulate Fas expression than untreated control cells (Figure 7c and d). In the work from McCarron et al. [145], 23

ACCEPTED MANUSCRIPT Fas receptor antibody was covalently attached to camptothecin-loaded PLGA nanoparticles. Camptothecin possesses potent anti-tumor activity and can up-regulate the Fas expression on the surface of tumor cells [146]. The camptothecin and anti-Fas antibody showed the synergistic cytotoxic effect of neuroblastoma cells. For the other death receptors, such as TNF receptors, and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) [124], nanotechnology can also be employed to activate death signaling, such as loading TRAIL in nanoparticles to enhance its biological activity [147]. Not only can tumor cells involve deficiency of death-receptor signaling pathways to inhibit effective anti-tumor immune response, but also can induce anti-apoptotic signals to escape

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immune destruction. Signal transducer and activator of transcription 3 (STAT3) acts as a convergence for oncogenic signaling pathways [148]. The constitutive activation of STAT3 plays a

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key role to mediate immunosuppression for oncogenic development. Through secreting

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immunosuppressive factors such as IL-10 and vascular endothelial growth factor (VEGF), the activation of STAT3 can be rapidly propagated in an efficient way by mediating a crosstalk between tumor cells and diverse immune cells, which in turn generates immunosuppression to sustain tumor growth [149]. It is convenient to specifically deliver STAT3 inhibitor to tumor by

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nanoparticles. The stearic acid substituted polymeric nanoparticles were developed to deliver siRNA for efficient down-regulation of STAT3 and reduction of VEGF expression in melanoma cells, which significantly suppressed tumor growth [143] (Figure 7e and f). Immune cells in microenvironment also act as appropriate targets. According to the results from Lavasanifar and coworkers [150], STAT3 siRNA encapsulated PLGA nanoparticles efficiently mediated specific

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STAT3 silence in tumor-exposed DCs. The results indicated that this approach can restore DC maturation and functionality by up-regulating of CD86 expression, secreting high amount of TNF-α and significantly promoting T cell proliferation. The micelles co-delivering STAT3 siRNA, imiquimod and ovalbumin synergistically enhanced DC cross-presentation and CTL response

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[151]. In the work of Luo et al. [152], Poly I:C and STAT3 siRNA co-loaded micelles robustly elicited anti-tumor immune responses through modulating tumor-associated DCs in vivo. Another anti-apoptosis signal is PD-L1. While the PD-L1 mRNA was broadly found in various normal tissues, seldom evidences show ed PD-L1 expression on normal cells under normal

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physiological conditions. By comparison, in various tumor lesions, PD-L1 is a remarkable label and abundantly expressed on the surface of tumor cells and tumor associated APCs [113, 153]. This selective expression makes PD-L1 as the potential treatment checkpoint for a broad spectrum of advanced cancers [112]. As mentioned above, the immune checkpoint therapy based on PD-1 can enhance anti-tumor immunity. Anti-PD-L1 to block PD-1/PD-L1 pathway can also inhibit dysfunction of T cells and induce potent immune responses. Powles et al. [154] engineered an anti–PD-L1 IgG1 mAb (MPDL3280A, Genentech/Roche) which blockaded the interactions between PD-L1 and PD-1. MPDL3280A was identif ied as an efficient therapeutic agent with mild side effects in metastatic urothelial bladder cancer. The development of checkpoint therapy provides opportunities for application of nanotechnology in immunotherapy. With folic acid (FA)–functionalization, polyethylenimine nanoparticles successfully delivered PD-L1 siRNA to epithelial ovarian cancer cells which overexpressed folate receptors. The targeting nanoparticles increased siRNA uptake and led to 40-50% PD-L1 protein knockdown. Compared to scrambled siRNA treated controls, epithelial ovarian cancer cells treated with PEI–FA siRNA nanoparticles exhibited up to 2-fold more sensitive to T cell killing [155]. 24

ACCEPTED MANUSCRIPT 3.2 Modulating tumor microenvironment Accompany with the immune system to attack and kill tumor in tumor microenvironment, not only the defenses directly from tumor should be reversed but the ramparts built by tumor for indirectly resisting should be collapsed. In order to support tumor growth, tumor prefers to create a favorable peripheral condition including neovascularization, oxygen deficit, low pH and matrix metalloproteinases (MMP) production. Besides, the recruitment of immunosuppressive cells and

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secretion of immunosuppressive cytokines assist tumor cells to survive from immune attack. Although the sophisticated condition gradually becomes more desirable for tumor growth, it also gives new hints for further strategies of microenvironment modulation. In the majority of tumors, the vascular pore cutoffs size around 380 and 780 nm allow s larger molecules and nanoparticles to

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enter and retain in tumor tissue [156]. Hence, it is favorable to design therapeutics-loaded nanoparticles in appropriate size between 10 nm and 400 nm, which can pass through aberrant

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neoangiogenic tumor vasculature and target tumor site. Taking advantage of this property, diversified nanoparticles were applied in selectively delivering and releasing payloads [157-159]. However, it should be noted that the enhanced permeation and retention (EPR) effect of

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nanoparticles is not sufficient to access more distant and dense region of tumor. To help immune cells regain the control of tumor microenvironment, other specific conditions in tumor microenvironment should be exploited.

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3.2.1 Application of nanotechnology in peripheral condition

In the expansion stage, neoplastic tissue growth needs rapid angiogenesis to meet the demand for a large amount of nourishment. Tumor cells mediate these changes by secreting VEGF to

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stimulate vascular growth. However, newly formed vessels generally possess heterogeneous vascularization resulting in insufficient perfusion [160, 161]. It becomes difficult for tumor stroma to maintain enough oxygen to keep up with the requirement for rapid expansion, which leads to a local hypoxic condition in tumor regions. Owing to the lack of available oxygen, tumor produces energy through glycolytic activity. The accumulation of byproduct in this process, lactic acid,

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results in acidic tumor microenvironment (pH 6.6-7.0) [136]. Tumor cells also produce MMPs to degrade the extracellular matrix, creating a favorable environment for tumor growth and invasion [162]. Nutrition supply Rapid angiogenes is always serves as an important role in tumor development. Vast vasculature transports oxygen and nutrient to sustain abnormal expansion. To some extent, the anfractuous morphology of vasculature may hinder the attack from immune effector cells. Anti-angiogenic agents are capable to cut down the source of nourishment and break the barriers in the meantime. During angiogenesis, there are certain markers like the VEGF receptors (VEGF-R) which can be used as target. Previously vascular-targeting nanoparticles were used in chemotherapy to selectively kill tumor cells [163-165]. They can also be addressed with different purposes in modulating tumor microenvironment. For instance, Zhang et al. [166] co-formulated VEGF siRNA and gemcitabine monophosphate (GMP) into cell-specific targeted nanoparticles. Compared with respective therapy, the combined therapy was observed with a significant decrease of tumor microvessel density, 30–40% induction of tumor cell apoptosis and 8-fold reduction of tumor cell proliferation. 25

ACCEPTED MANUSCRIPT Oxygen Due to insufficient perfusion of oxygen during angiogenesis, the hypoxia condition directly affects the utilization of glucose and even attributes to tumor survival [167]. Taking advantage of diverse oxygen-sensitive modification, nanoparticles can be selectively degraded under hypoxic tumor microenvironment, resulting in the enhanced cell uptake and targeted release of therapeutics in tumor site. Perche et al. [168] constructed a hypoxia sensitive nanoparticles by utilizing azobenzene. The nanoparticles showed 3.2-fold higher cellular internalization under hypoxia than that of under normoxia. Park and his colleagues [169] demonstrated that hypoxia-responsive polymeric nanoparticles were selectively accumulated at the hypoxic tumor tissues. Through introduction of modified nanocarriers to regulate the level of oxygen, the fertility

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of tumor may be transformed into inaptitude area. Hypoxia-inducible factor-1α (HIF-1α) is a key transcription factor during hypoxia and can serve as a target in cancer therapy. Wang’s group [170]

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employed the mixed micelles to deliver HIF-1α siRNA aiming at the hypoxia environment. In

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both in vitro hypoxic mimicking culture and in vivo hypoxic tumor model, HIF-1α siRNA loaded micelles showed effectively knocking down the expression of HIF-1α, which remarkably inhibited cell proliferation, migration and angiogenesis. In addition, it was reported that hypoxia contributed to a tolerogenic phenotype of DCs, which significantly reduced capture of antigens and changed

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chemokine expression profile [171]. This phenomenon may provide possibilities for nanoparticles to relief tumor resistance by supplying appropriate amount of oxygen. pH On account of the pH difference between normal tissues (pH 7.4) and tumor

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microenvironment (generally around pH 6.6-7.0), it is appropriate to design nanopartic les to respond to the slight difference in threshold value of pH and release payloads. In the work from Wang group [172], acidic tumor microenvironment-responsive polymer of PEG-Dlink m-PDLLA

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was synthesized with an acid-degradable amide bond and applied in drug delivery. The nanoparticles can dissociate PEG corona and increase the zeta potential as the cleavage character

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of amide bond under tumor acidity which facilitated the cellular uptake of nanoparticles. The pH ultra-sensitive polymeric nanoparticles were recently developed for tumor penetration and

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effective drug delivery in tumor microenvironment. The amphiphilic polymer contained ionizable tertiary amine groups and the formulated nanoparticles can undergo a dramatic and sharp size transition in the slight difference of pH and dissociate small particles (less than 10 nm) for tumor penetration [173]. This kind of size-switchable nanoparticles may be prospected for releasing therapeutics in tumor microenvironment. The decreased pH can be adjusted accompany with reversing hypoxia. Prasad et al. [174] engineered polyelectrolyte albumin complex and MnO2 to produce O2 by reactivity of MnO2 toward peroxides and simultaneously increase the local pH + from 6.7 to 7.2 through consuming intratumoral H2 O2 and H . This strategy suggested a potential role of nanoparticles to regulate the peripheral condition of tumor microenvironment. MMP High concentrations of MMPs are detectable in tumor microenvironment. They cleave away the extracellular matrix to create space for invasion and metastasis [175]. With the benefit from EPR effects, enzyme-responsive nanoparticles can be rapidly delivered into tumor and release payloads in response to the increased levels of MMPs [176]. Huang et al. [177] modified nanoparticles with activatable cell-penetrating peptide. This kind of masking peptide can promote nanoparticles uptake by intratumoral cells after encountering high concentration of MMP and low pH in tumor microenvironment. This innovative delivery system utilized the enzyme sensitive property to provide opportunities for tumor targeting. 26

ACCEPTED MANUSCRIPT 3.2.2 Application of nanotechnology in cytokine modulation Recent years have witnessed that a couple of immunostimulatory cytokines IL-2 and IFN-α achieved FDA approval for cancer treatment. A number of cytokines, including granulocyte macrophage colony-stimulating factor (GM-CSF), IL-7, IL-12, IL-15, IL-18 and IL-21, have entered clinical trials for cancers [178]. In tumor microenvironment, the gaming arena of the immune system and tumor, cytokines play an intricate role. Besides cytokines, like IFN-γ, TNF-α,

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IL-2 and IL-12 protect host against cancer, there are a group of suppressive cytokines released by tumor and immunosuppressive cells, including macrophage colony-stimulating factor, TGF-β， IL-4，IL-6, IL-10, attributing to the inhibition of anti-tumor immunity. Both the achievements in the durable role of

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microenvironment have inspired researchers to explore the potential of nanotechnology to modulate cytokines in tumor microenvironment. TGF-β, as a pleiotropic cytokine, is highly

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produced by variety cancers, plays an important role in tumor growth and differentiation. It is able to maintain an appropriate environment for tumor cell to escape from immune attack, which renders it as a common target in tumor environment [179]. According to the work from Kano et al.

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[180], nano-carriers encapsulating TGF-β inhibitor exhibited potent inhibition on solid tumor. The assistance of nano-carriers may significantly contribute to the modulation of cytokine in clinical and practical cancer treatment. In addition, the inhibition of immunosuppressive cytokine may have the synergistic effect with immunostimulator. Park et al. [181] chose IL-2 to explore the

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potential synergic effect with TGF-β inhibitor. Allowing for the high-dose-related toxicity of cytokines can hinder the therapeutic effects in traditional administration [182], it is appropriate to apply nano-carriers for reducing the dose of cytokine via increasing the half-life in circulation.

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They introduced nanolipogels to encapsulate the hydrophobic TGF-β inhibitor and water-soluble cytokine IL-2. With the ability of sustained release, the polymeric nanoparticles significantly +

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promoted the activity of NK cells and CD8 T cells and prolonged the survival of tumor-bearing mice (Figure 8 a, b, c and d). Moreover, gene delivery based on nanotechnology can also serve

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as an alternative way to modulate cytokine. Recombinant murine IL-12 plasmid loaded polymersomes successfully suppressed tumor growth. It may relate to efficient infiltration of lymphocytes conducted by IL-12 [183]. Xu et al. [184] formulated liposome-protamine-hyaluronic acid nanoparticles with targeted modification to selectively deliver TGF-β siRNA to tumor microenvironment. Collaborating with the antigen-specific vaccine, this strategy resulted in about 50% knockdown of TGF-β, increased the infiltration of CD8+ T cells and reduced Tregs in the late stage of tumor. Different from traditional cytokine therapy, the application of nanotechnology makes it possible to combine various therapeutic agents to modulate cytokine in tumor environment.

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Figure 8. (a) Schematic illustration of nanolipogels particle system. (b) Survival plot of mice with

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aggressive B16 lung metastases after systemic therapy. (c) Quantification of number of NK cells present in tumors from tumor-bearing mice seven days after treatment. (d) The ratio of activated CD8+ T cells to Tregs in TILs from tumor-bearing mice after receiving sustained administration. (e) The production of IFN-γ in tumors six hours following the last PapMV treatment after receiving systematic administration of PapMV on days 7, 12, and 17 post implantation. (f) Quantification of proportions of infiltrated CD45+ cells in tumor 3 days following the second PapMV administration on day 15 post tumor implantation. (g) Quantification of proportions of MDSC within the CD45+ cell population. A, b, c and d reprinted with permission from [181]. Copyright 2012, Nature Publishing Group. E, f Chemical Society.

and g reprinted with permission from [185]. Copyright 2016, American

3.2.3 Application of nanotechnology in modulating immunosuppressive cells There is a delicate balance between effector and regulatory T cells. The ratio of these two kinds of cells has been considered as a critical indicator for the rejection or progression of tumor. The self-tolerance mediated by Tregs is an important reason for unsatisfied anti-tumor immunity 28

ACCEPTED MANUSCRIPT [186]. The immunosuppressive mediators such as TGF-β, IL-10 and IL-35 which are secreted by Tregs can suppress effector T cell expansion [187]. In tumor microenvironment, inhibiting the functions of Treg can be a desirable approach to promote the immune response. Adjuvant like CpG-ODN or Poly(I:C) can preferentially amplify antigen-specific effector T cells (Teff) over Treg, increasing the ratio of antigen-specific Teff to Treg. The pro-inflammatory type I cytokines were selectively produced after treated with therapeutic formulations contained CpG-ODN or +

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Poly(I:C), which significantly promoted the expansion of CD8 and CD4 T cells rather than Tregs. The balance of Teff and Treg was tipped to favor effector cells, indicating the influence of adjuvants on T cell populations [188]. Additionally, adaptive Tregs can suppress effector T cells

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by expressing cyclooxygenase 2 (COX-2) and producing PGE2 . COX-2 inhibitor can be used to reverse the favored infiltrating state of Tregs, which can inhibit the development of tumor [189].

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As a stromal component in tumor microenvironment, the heterogeneous population of

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macrophages which are termed as TAMs, closely resembles the M2-polarized macrophages. The conversion of macrophage to M2 macrophage can be mediated by cytokines such as IL-4, IL-13, TGF-β and IL-10 [190]. Contrary from the M1 macrophages involving in the inflammatory response and anti-tumor immunity, the M2 macrophages play a crucial role in promoting tumor

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growth. The suppressions conducted by M2 macrophages in tumor microenvironment are mainly involved in attracting Tregs and inducing CTL apoptosis, generally accompany with producing several suppressive cytokines such as IL-1β, IL-6, IL-10 and TGF-β [191]. To deal with the suppression from TAM, one choice is to block tumor supporting effects from M2 macrophages by depleting these cells in tumor microenvironment [192]. Treatment with liposomes encapsulating

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bisphosphonate clodronate efficiently mediated TAM depletion, resulting in remarkable tumor growth inhibition [193]. Doxorubicin loaded PLGA nanoparticles selectively depleted TAMs in tumor microenvironment after modified with mannose. The modified PLGA nanoparticles significantly increased the uptake of doxorubicin by TAMs and exhibited more effective control of

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tumor growth [194]. In current research, the most prominent way is to make M2 macrophage shift into M1 macrophage to inhibit immunosuppressive effects. Cationic polymer exhibited the immunological activity mediated by TLR4, which can reverse the polarization of TAMs, promote the Th1 and NK cells infiltration and eventually exhib it significant anti-tumor efficacy [195]. The

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cationic polymers can be further applied in drug delivery system with immunological activity especially on TAMs. Another negative regulation of the immune system is caused by MDSCs, which are described to be a potent suppressor of T cell response. There are several different factors, particularly two main groups which include factors such as VEGF, COX-2, M-CSF, IL-6 and GM-CSF for promoting the expansion of MDSCs, and factors of IFN-γ, ligands for TLRs, IL-4, IL-13 and TGF-β for directly activating MDSCs [196]. The suppressive activity of MDSCs could exhibit different types of immunosuppression in peripheral lymphoid organs and tumor site, respectively. In the peripheral lymphoid organs, MDSCs express high levels of enzymes, arginase 1 and iNOS, leading antigen-specific suppression of T-cell proliferation and function [197-199]. In tumor site, the suppression conducted by MDSCs is non-specific, including rapidly differentiation of TAMs, expansion of Tregs and inhibition of T cell function [196]. The most appropriate way to tackle the suppressive effects is to transform the expansion and activation of MDSCs into differentiation and maturation of macrophage or even functional APCs [200]. It has been identified that all trans retinoic acid (ATRA) could induce the differentiation of MDSCs into DCs and macrophages both 29

ACCEPTED MANUSCRIPT in vitro and in vivo [201]. However, the non-specific toxicity and low drug solubility limit the effectiveness of traditional ATRA treatment. In comparison, incorporating it into liposomes can increase the stability and reduce the toxicity of ATRA [202]. Liposome drug delivery system may be used for targeted delivery of ATRA to granuloc ytic myeloid derived suppressor cells for restoring the immunostimulatory phenotype of MDSCs [203]. Another promising approach to overturn the suppression of MDSCs is to prevent MDSCs formation and inhibit the suppressive functions. Recent studies indicated that IL-12 is able to reprogram MDSCs and force them to support CD8+ T cell attack by up-regulating co-stimulatory markers such as CD80, CD86, and MHC-II [204, 205]. IL-12 encoded plasmid was used as a therapeutic gene to be co-delivered with

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paclitaxel in single nanoparticles. Compared to the delivery of either paclitaxel or the plasmid alone, the combinational nanoparticles significantly suppressed tumor growth. IL-12 may

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overcome T cell suppression mediated by paclitaxel [206]. Another cytokine, IFN-α potently reduces T cell suppressive activity of MDSCs and promotes anti-tumor immunity [207]. Lebel et al. [185] showed that papaya mosaic virus (PapMV) nanoparticles possessed multiple desirable

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properties, especially the production of IFN-α, which can be favorable for cancer immunotherapy. PapMV treatment dramatically decreased the proportion of MDSCs resulting from systemic IFN-α

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production (Figure 8 e, f, and g). Abrogating suppressive ability of MDSCs can also be achieved by DCs produced IFN-α upon CpG ODN stimulation [208]. CpG ODN delivered by AuNPs was shown to reduce the suppressive activity of MDSCs and potentially induce DCs infiltration in tumor and significant tumor growth inhibition [209]. The intricate functions of immunosuppressive cells which directly influence cytokine and immune effector cells encourage the further exploration on nanotechnology to inhibit immunosuppressive responses.

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5. Conclusions

Over the past few years, the tendency of cancer treatment has gradually shifted from

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aggressively eliminating tumor by conventional treatment to adaptively mobilizing the immune system to fight against cancer. DC based vaccine, subunit vaccine, adoptive T cell therapy, checkpoint therapy or even modulation of tumor microenvironment are virtually based on the principle, inducing specific anti-tumor efficacy toward tumor and reducing the damage to normal tissues. Even a variety of successes have been achieved in recent research, it is hardly to say that

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these therapies sufficiently take advantages of the immune system. Besides destroying tumor development and restraining the expans ion, the immune system can also function as a promoter to facilitate tumor growth and transformation as well as sculpt immunogenic ity of tumor cell [134, 210]. The checkpoint immunotherapy which has been considered as the unprecedented clinical treatment for a wide range of cancers, recently was demonstrated that the anti-tumor efficacy was threatened by acquired resistance related to mutation defects [211]. In order to achieve better therapeutic outcome, it is important to overcome drawbacks of current therapies, including insufficient elicitation of the immune response, suboptimal anti-tumor effect, immunosuppression mediated by tumor and complex environment around tumor. These undesirable situations can be masterly evaded by the application of nanotechnology in immunotherapy, including targeted delivery of antigens, enhanced antigen presentation, increased T cells proliferation and effective modulation of tumor microenvironment. However, to achieve better therapeutic effects, it still needs further development of nanotechnology to improve the efficacy of immunotherapy. Nano-device may be taken up by other 30

ACCEPTED MANUSCRIPT tissues in some circumstances, limiting the ability to access the target site [212]. The unsatisfied controlled release of payloads and insufficient tumor penetration still are obstacles in application of nanotechnology. Although the anti-tumor effects of cancer immunotherapy have been benefited greatly from nanotechnology in animal tumor models with good safety and minimal side effects, clinical translations are confronted with some inevitable problems. Due to the physiological difference between human and animal, the advantages of nanotechnology may be dimed [213]. To promote clinical translation, the mainstream of research on nanotechnology based immunotherapy needs to be shifted from emphatic evaluation of anti-tumor efficacy to particular investigation of nano-bio interaction in blood, organs, biological barriers, cell membranes, intercellular

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environment etc. [214]. The tendency of nanotechnology applied in cancer immunotherapy in the future should involve multiple disciplines to cooperate, including genetic engineering, tumor

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histology, molecular biology, material science and so on. Under the help of nanotechnology

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cooperated with diverse fields, it is possible to boost the immune system and tackle the defense at the same time for expected cancer treatment.

Acknowledgement

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This work was supported by the National Natural Science Foundation of China (81373360 and 81673374); Fundamental Research Funds for the Central Universities (2015ZDTD048).

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