Synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity

Accepted Manuscript Synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity Sun-Young Kim, Young-Woock Noh, Tae Heung Kang, Jung-Eun Kim, Sohyun Kim, Soong Ho Um, Doo-Byoung Oh, Yeong-Min Park, Yong Taik Lim PII:

S0142-9612(17)30183-7

DOI:

10.1016/j.biomaterials.2017.03.034

Reference:

JBMT 18004

To appear in:

Biomaterials

Received Date: 14 December 2016 Revised Date:

20 March 2017

Accepted Date: 23 March 2017

Please cite this article as: Kim S-Y, Noh Y-W, Kang TH, Kim J-E, Kim S, Um SH, Oh D-B, Park Y-M, Lim YT, Synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.03.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Synthetic Vaccine Nanoparticles Target to Lymph Node Triggering Enhanced Innate and Adaptive Antitumor Immunity Sun-Young Kim‡,1, Young-Woock Noh‡,1, Tae Heung Kang‡,2, Jung-Eun Kim1, Sohyun Kim1,

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Soong Ho Um1, Doo-Byoung Oh3,*, Yeong-Min Park2,* and Yong Taik Lim1,*

SKKU Advanced Institute of Nanotechnology (SAINT) and School of Chemical

440-746, Republic of Korea 2

Department of Immunology, School of Medicine, Konkuk University 268, Chungwondaero

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Chungju-si, Chungcheongbuk-do, Republic of Korea 3

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Engineering, Sungkyunkwan University 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do

Synthetic Biology and Bioengineering Research Center, Korea Research Institute of

Bioscience and Biotechnology (KRIBB) 125, Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea

These authors contributed equally

*

To whom all correspondence should be addressed

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‡

Address correspondence to: [email protected] (Y.T. Lim), [email protected] (Y.-M.

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Park), [email protected] (D.-B. Oh)

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Keywords: vaccine, lymph node, delivery, adjuvant, cancer immunotherapy

Abstract

In this study, synthetic vaccine nanoparticles (SVNPs) that efficiently targeted lymph nodes, where immune responses against foreign antigens are primed, were developed to enhance antitumor immunity. The size (20-70 nm) and surface character (amination) of poly(γglutamic acid)-based SVNPs were selected for effective loading and delivery (i.e., migration and retention) of model tumor antigen (OVA) and toll-like receptor 3 agonist (poly (I:C)) to immune cells in lymph nodes. Antigen-presenting cells treated with SVNP-OVA and SVNP1

ACCEPTED MANUSCRIPT IC showed higher uptake of OVA and poly (I:C) and higher secretion of inflammatory cytokines (TNF-α, IL-6) and type I interferon (IFN-α, IFN-β) than those treated with OVA and poly (I:C) alone. In vivo analysis revealed higher levels of activation markers, inflammatory cytokines, and type I IFNs in the lymph nodes of mice immunized with SVNPIC compared to those of mice in other groups. SVNP-IC-treated mice showed significantly

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greater in vivo natural killer cell expansion/activation (NK1.1+ cells) and CD8+ T cell response (CD8+ INF-γ+ cells) in innate and adaptive immunity, respectively. Both preventive and therapeutic vaccination of EG7-OVA tumor-bearing mice using the simultaneous injection of both SVNP-OVA and SVNP-IC induced higher antitumor immunity and

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inhibited tumor growth.

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1. Introduction

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Graphical abstract

The tuning of physicochemical properties of nanoparticles (NPs) to modulate the

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immune response has received considerable research impetus [1-3]. Size and shape dictate the interaction of NPs with antigen-presenting cells (APCs), and as such, the rational design and synthesis of NPs are prerequisites for modulating the immune response against a target disease [4-9]. On injection of NPs containing antigens and immunomodulatory compounds, APCs surrounding the injection point are recruited and programmed, followed by their migration into lymph nodes to deliver antigenic information to T cells and B cells and consequent induction of an adaptive immune response [10-12]. The generation of an adaptive immune response relies on the efficient drainage or trafficking of the antigens to lymph nodes for the subsequent processing and presentation of foreign molecules to T and B lymphocytes. Lymphatic vessels have evolved to drain pathogens into lymph nodes, thus enabling the 2

ACCEPTED MANUSCRIPT immune system to rapidly mount a response. Lymph nodes have thus become critical targets for the delivery of vaccines and immunotherapeutic agents, because the direct delivery of vaccine components into APCs residing in lymph nodes can induce another arms of adaptive immune response, in addition to that induced by migrated APCs. However, most of the commercialized vaccination strategies resulted in the inefficient and transient delivery of

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antigen and adjuvants to the lymphoid tissue, because the delivery carriers in vaccine were not optimized for direct migration through lymphatic vessels [13-15]. Thus, new approaches to improve the targeting and retention of vaccine components in the lymph nodes are envisaged to have significant impact on the potency and efficiency of new vaccines. Three

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major approaches have been proposed to improve the vaccine targeting efficiency: size tuning, hitch-hiking on albumin, and PEGylation. Previous studies have shown that the kinetics of

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NP migration through lymphatic vessels is highly dependent on NP size [13-15]. NPs of less than 5-nm diameter can easily enter the bloodstream, whereas those of over 100-nm diameter remain at the injection site and do not move into lymphatic vessels. Typically, larger particles with diameters in the range of 500–2000 nm are carried into lymph nodes by dendritic cells (DCs). It has been reported that 15-70-nm diameter NPs are optimal for rapid entry into lymphatic vessels and migration into lymph nodes [13, 14]. As a novel strategy for

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developing lymph node-targeting vaccines, liposomes containing synthetic peptide antigens and immunostimulatory adjuvants were designed and fabricated to hitch-hike on albumin proteins, which migrate into lymph nodes [16]. In addition, PEGylated hydrogel

that can

dramatically improve in vivo lymphatic drainage and efficiently target multiple immune cell

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subsets have been proposed [17]. Concerning the effect of vaccine retention on the immune response, rapid clearance of vaccines would limit the quality and duration of the generated

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immune memory because the adaptive immune response can be maximized by sustained antigen/inflammation over several days [18]. In this study, we designed and developed synthetic vaccine NPs (SVNPs) that are

expected to improve the targeting and retention of cancer vaccines (i.e., tumor antigens and immunostimulatory adjuvants) in lymph nodes and are expected to induce a strong and effective immune response for enhanced cancer immunotherapy. The size and surface characteristics of SVNPs were tuned for the effective loading and delivery of antigens and immunostimulatory materials into lymph nodes and for the induction of efficient migration through lymphatic vessels. To fabricate SVNPs (20-70 nm in diameter) and facilitate loading of negatively charged protein tumor antigen (OVA) and toll-like receptor 3 (TLR3) agonist 3

ACCEPTED MANUSCRIPT (polyinosinic-polycytidylic acid, poly (I:C)), the carboxyl-terminated surfaces of poly(γglutamic acid) (γ-PGA) NPs were further modified with amine moieties (Fig. 1A). Poly (I:C) is a synthetic analog of double-stranded RNA and is known to interact with TLR3 expressed on the membrane of B-cells, macrophages, and DCs [19, 20]. The poly (I:C)-loaded SVNPs

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are expected to stimulate innate immune system, which senses invading viral pathogens and initiates signaling pathways and induces protective genes, including those encoding type I IFNs and pro-inflammatory cytokines that directly limit viral replication and help direct subsequent adaptive immune responses [21]. The lymph node-targeting SVNPs were

2. Materials and Methods

2.1. Materials

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and adaptive antitumor immunity (Fig. 1B).

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developed to trigger the protective mechanism in lymph nodes and thus enhance both innate

Poly(γ-glutamic acid) (γ-PGA, Mw = 50 kDa) was provided by BioLeaders Corporation (Daejeon, Korea). Cholesteryl chloroformate (97%), 1,1′-carbonylbis-1H-imidazole, 1,2-

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ethanediamine, polyinosinic-polycytidylic acid sodium salt (low molecular weight poly (I:C)), and chicken egg ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The endotoxin content was analyzed with the Limulus amoebocyte lysate assay (QCL-1000;

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Lonza, Walkersville, MD, USA) as per the manufacturer’s protocols and was found to be 0.6 endotoxin units/mg of soluble OVA [22]. Fluorescein isothiocyanate-conjugated ovalbumin

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(FITC-OVA) was obtained from Thermo Scientific (Waltham, MA, USA). IRDye800-labeled OVA was prepared using the IRDye800 NHS ester (Li-COR, Lincoln, NE, USA). Rhodamine-labeled poly (I:C) was prepared using the 5′ EndTagTM Nucleic Acid Labeling System (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions.

2.2. Preparation of SVNPs Cholesteryl chloroformate (2.25 g, 5 mmol) was slowly added to a solution of 1,2ethanediamine (16.7 mL, 250 mmol) in toluene (anhydrous, 250 mL) in an ice bath. The reaction was carried out at 25°C for 16 h, followed by washing with deionized water, drying 4

ACCEPTED MANUSCRIPT over anhydrous magnesium sulfate, and evaporation in a rotary evaporator. The residue was dissolved in dichloromethane (20 mL) and then added to methanol (20 mL). The resulting suspension

was

filtered

to

remove

biscarbamate

by

syringe-filtration

(1

µm,

polytetrafluoroethylene). The filtrate was evaporated in a rotary evaporator to obtain a white solid. To synthesize γ-PGA-CH, 500 mg γ-PGA (3.876 mmol) in DMSO (10 mL) and 46 mg

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cholesterol-amine (0.097 mmol) in tetrahydrofuran (10 mL) were reacted in the presence of 1,1′-carbonyldiimidazole (63 mg, 0.3876 mmol) at 40°C for 18 h. The reaction mixture was evaporated in a rotary evaporator to remove tetrahydrofuran. The reaction mixture was then

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poured into acetone. After centrifugation, the precipitate was collected, dried, and mixed with NaHCO3 solution and stirred for 4 h. Amberlite IR-120H beads were treated by ion exchange. After filtration with the beads, the reaction mixture was dialyzed through a 10-12 kDa

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molecular weight cut-off membrane against deionized water for 2 days. The solution was freeze-dried. Next, the γ-PGA-CH conjugate was aminated in the presence of ethylene diamine in DMSO. For amination, γ-PGA-CH (100 mg, 0.773 mmol) and ethylene diamine (10.3 mL, 155 mmol) were slowly mixed in DMSO (20 mL) containing 1,1′carbonyldiimidazole (752.8 mg, 4.63 mmol) under constant stirring for 12 h. The reaction

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mixture was poured into acetone. After centrifugation, the precipitate was collected, dried, and mixed with NaHCO3 solution and stirred for 4 h. The reaction mixture was dialyzed through a 10-12 kDa molecular weight cut-off membrane against deionized water for 2 days. The solution was freeze-dried. Elemental analysis of γ-PGA-CH-NH2 was performed to

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determine the ratio of C, N, and H (Table 1). To generate the complexes of γ-PGA-CH-NH2OVA (SVNP-OVA) and γ-PGA-CH-NH2-poly (I:C) (SVNP-IC), γ-PGA-CH-NH2 NP

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solution was added to the solution of poly (I:C) and OVA at a specified mass ratio of 0.5:1 (γ-PGA-CH-NH2 : OVA) and 1:2 (γ-PGA-CH-NH2 : poly (I:C)) and reacted for 2 h to form a stable complex.

2.3. Characterization of SVNPs The size distribution and zeta potential of γ-PGA-CH and γ-PGA-CH-NH2 NPs in aqueous phase were analyzed by dynamic light scattering (DLS) using an electrophoretic light scattering photometer (ELS-Z, Otsuka Electronics, Osaka, Japan). The surface morphology and size of the SVNPs were analyzed using a high-resolution transmission electron 5

ACCEPTED MANUSCRIPT microscope (JEOL Ltd., Japan). The samples were stained with 2% uranyl acetate (SigmaAldrich).

2.4. Mice and cell lines C57BL/6 mice (female, 6–8 weeks old) were purchased from Orient Bio (Seongnam, Korea)

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and maintained under pathogen-free conditions. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University School of Medicine, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and abides

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by the Institute of Laboratory Animal Resources (ILAR) guide. EG7-OVA (EL-4 thymoma cells transfected with chicken albumin cDNA, American Type Culture Collection, ATCC,

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Manassas, VA, USA) cells were cultured in RPMI medium (Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Scientific), 5 × 10-5 M 2-mercaptoethanol (Sigma-Aldrich), 50 IU/mL penicillin, and 50 µg/mL streptomycin (Thermo Scientific).

2.5. Generation of BMDCs and BMMΦs from mice

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Bone marrow-derived DCs (BMDCs) and bone marrow-derived macrophages (BMMΦs) were generated from the bone marrow of C57BL/6 mice. Briefly, the bone marrow was collected from the tibias and femurs of these mice. Red blood cells were depleted using red blood cell lysing buffer (Sigma-Aldrich). Bone marrow cells (2 × 106 cells) were collected

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and cultured in 100-mm Petri dishes containing 10 mL RPMI medium, supplemented with 10% of heat-inactivated FBS and 20 ng/mL of recombinant mouse granulocyte macrophage

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colony-stimulating factor (Peprotech, Rocky Hill, NJ, USA) for BMDCs or macrophage colony-stimulating factor (Peprotech) for BMMΦs. After 7 days, differentiated cells were harvested, washed, and used in further experiments.

2.6. Analysis of cellular uptake BMDCs or BMMΦs were incubated with SVNP-OVA-FITC (5 µg/mL) and SVNP-ICrhodamine B (5 µg/mL) in an ibidi µ-slide 8-well microscopy chamber (ibidi, Martinsried, Germany) at a density of 2 × 104 cells per well at 37°C for 3 h. After washing with PBS, the cells were fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature and stained with 2 µg/mL solution of Hoechst 33342 (trihydrochloride, trihydrate; Thermo Scientific) in 6

ACCEPTED MANUSCRIPT PBS for 10 min. Florescence images were obtained using DeltaVisionTM PD (GE Life Sciences, Marlborough, MA, USA). BMDCs or BMMΦs were seeded on 6-well plates at a density of 2 × 106 cells per well. SVNP-OVA-FITC (5 µg/mL) or SVNP-IC-rhodamine B (5 µg/mL) was added to each well and incubated for 3 h. After washing with PBS, the cells were analyzed using an AcurriTM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). To

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detect the intracellular localization of SVNP-IC-rhodamine B, cells were stained with LysoTracker Blue (Invitrogen) for 30 min. Fluorescence images were obtained as described above.

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2.7. In vitro cytokine assay

BMDCs and BMMΦs were cultured in 6-well plates at a density of 2 × 106 cells/well with γ-

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PGA-CH-NH2 NPs (2.5–5 µg/mL), poly (I:C) (5–10 µg/mL), or SVNP-IC (5–10 µg/mL). After 24 h, the culture supernatants were collected; TNF-α and IL-6 levels were analyzed using cytokine-specific OptEIA™ ELISA kits (BD Biosciences), and the IFN-β level was analyzed using a VeriKine Mouse Interferon Beta ELISA Kit (PBL Assay Science, Piscataway, NJ, USA) according to the manufacturer’s instructions. Cytokine concentrations

recommendations.

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were quantified using a VersaMax apparatus at OD450 according to the manufacturer’s

2.8. Quantitative PCR for cytokines

Total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany), and 1 µg of

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the total RNA was used for the first-strand cDNA synthesis using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA), according to the manufacturer's

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instructions. Quantitative PCR was performed using the StepOnePlusTM Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). Quantitative PCR amplification was conducted in a reaction volume of 20 µL, containing 10 µL of SYBR Green PCR Master Mix (Applied Biosystems), 7 µL distilled water, 5 pmol each of forward and reverse oligonucleotide primers, and 1 µL of cDNA template. The following primers were specific to a conserved region: mouse TNF-α 5′-TCCCAGGTTCTCTTCAAGGGA-3′ (forward)

and

5′-GGTGAGGAGCACGTAGTCGG-3′

ACAACCACGGCCTTCCCTACTT-3′ CACGATTTCCCAGAGAACATGTG-3′

(reverse);

(forward) (reverse);

mouse

IL-6

and mouse

IFN-α4

5′ 5′5′-

GCTTTCCTCATGATCCTAGTAA-3′ (forward) and 5′-AGGAGGTTCCTGCATCACA-3′ 7

ACCEPTED MANUSCRIPT (reverse); and mouse IFN-β 5′-TTCAAGTGGAGAGCAGTTGAG-3′, (forward) and 5′CATCAACTATAAGCAGCTCCA-3′ (reverse) (Bioneer, Daejeon, Korea). GAPDH expression levels served as a reference to normalize target mRNA levels. The samples were run in triplicate, and melting curve analysis was performed to confirm the amplification

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specificity of the PCR products.

2.9. In vivo fluorescence imaging

For in vivo imaging, C57BL/6 mice were anesthetized with 300 µL of 2.5% avertin solution (2,2,2-tribromoethanol-tert-amyl alcohol, Sigma-Aldrich), and the imaging areas were treated

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with a depilatory cream. Thereafter, SVNP-OVA-IRDye800 or OVA-IRDye800 solutions (25 µg in 50 µL of water) were intradermally injected into the forepaw pad. SVNP-OVA-

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IRDye800 or OVA-IRDye800 were tracked using a custom-made whole body optical imaging system at various experimental time points. NIR images (0.02 s exposure) of the axillary lymph nodes were acquired using a 785 nm, 500 mW diode laser as an excitation light source and 835/45 nm band-pass emission filter. All images were processed using Simple PCI software (Compix, Inc., Cranberry Township, PA, USA). Fluorescence signals of the lymph nodes were quantitatively analyzed using image analysis software (Image J,

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Wayne Rasband, National Institutes of Health, USA) (n = 3).

2.10. In situ histofluorescence

The in situ distribution of SVNPs was analyzed after dissection of the axillary LN, 30 min

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post administration of 50 µg of SVNP-OVA-FITC and SVNP-IC-rhodamine B, followed by embedding in Tissue-Tek OCT compound (SAKURA, Tokyo, Japan) and freezing in liquid

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nitrogen. Cryosections (5 µm) were prepared using a Leica cryostat CM1850 (Leica Microsystems, Wetzlar, Germany) and transferred to glass slides. The sections were fixed with cold acetone for 5 min, dried, and frozen at −20°C until use. The slides were washed in phosphate-buffered saline (PBS) and blocked with PBS containing 1% bovine serum albumin for 1 h at room temperature. After additional washing, the slides were stained with rat antimouse F4/80 (Serotec, Oxford, UK), CD169 (Siglec-1, Serotec), and CD205 (DEC-205, Serotec) overnight at 4°C to label the macrophages and DCs, respectively. The slides were then stained with TRITC-conjugated anti-rat IgG secondary antibodies (BD Biosciences) for 1 h at room temperature. The slides were washed twice with PBS and then treated with 2 µg/mL Hoechst 33342 in PBS for 10 min. After the final wash, the slides were mounted in 50% 8

ACCEPTED MANUSCRIPT glycerol (in PBS) and examined using a fluorescence microscope (Olympus IX71, Olympus Optical, Tokyo, Japan) and DeltaVision PD (n = 3).

2.11. Assessment of innate immune response C57BL/6 mice (6–8 weeks old) were injected subcutaneously at the tail base with poly (I:C)

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(50 µL/mice, 50 µg of poly (I:C)) or SVNP-IC diluted in PBS (50 µL/mice, 50 µg of poly (I:C) and 25 µg of γ-PGA-CH-NH2 NPs). Twenty-four hours after injection, inguinal lymph nodes were removed and processed as follows. Briefly, organ-fragments were dissociated with 1.0 mg/mL collagenase A (Sigma), 40 µg/mL DNase I (Roche, Indianapolis, IN, USA),

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and 10% FBS in RPMI 1640 medium at 37°C for 30 min while vigorously shaking at 250 rpm. The digested mixture was passed through a nylon mesh to remove undigested tissue and

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single-cell suspensions were subsequently pooled in RPMI 1640 medium supplemented with 10% FBS. To analyze the cytokine expression levels, the total RNA was extracted from the isolated lymph node cells and quantitative RT-PCR for TNF-α, IL-6 and IFN-β was performed as described above (n = 3). To further analyze the maturation of NK cells, 1 × 106 cells diluted in RPMI-1640 were stained with PE-labeled anti-NK1.1 and APC-labeled antiCD69 (for NK cell activation) antibodies (BD Biosciences). After washing with PBS, the

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cells were analyzed using an AcurriTM flow cytometer (n = 5).

2.12. Assessment of adaptive immune response To investigate the effects of SVNP-OVA and SVNP-IC on the induction of adaptive immune

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response, C57BL/6 mice were immunized twice, with a 1-week interval between both immunizations, at the tail base with SVNP-IC (50 µL/mice, 50 µg of poly (I:C)) and SVNP-

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OVA (50 µL/mice, 25 µg of OVA) in sequence with a 30-min interval between injections. Seven days after the last injection, splenocytes were collected and re-stimulated with 10 µg/mL OVA peptide (SIINFEKL, Invivogen, San Diego, CA, USA) in medium containing GolgiPlug (BD Biosciences) for 12 h in round-bottom 96-well plates (Nunc, Roskilde, Denmark) at a density of 5 × 105 per well (200 µL). Cells were washed with PBS and stained with PE-anti-CD8α (BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min at 4°C. After washing with PBS, the cells were permeabilized for 20 min at 4°C using the Cytofix/CytopermTM Plus Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer’s instructions. Next, the cells were washed twice with washing buffer (BD Biosciences) and stained with APC-anti-IFN-γ (BD Biosciences) for 30 min at 4°C. After 9

ACCEPTED MANUSCRIPT washing buffer, the cells were analyzed using an AcurriTM flow cytometer (n = 3).

2.13. In vivo tumor challenge To study the prophylactic effect, C57BL/6 mice were immunized twice, with a 1-week interval between both immunizations, at the tail base with SVNP-IC and SVNP-OVA. One

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week after the second immunization, 1 × 106 EG7–OVA cells were injected into the right back of C57BL/6 mice. To assess the therapeutic efficacy, 1 × 106 EG7–OVA cells were inoculated into the right flank of C57BL/6 mice. On days 1, 3, and 7 after inoculation, mice were injected at the tail base with SVNP-IC and SVNP-OVA. Tumor growth was monitored

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by measuring the major and minor axes of tumors by using an electronic digital caliper. Tumor volume was calculated using the following formula: tumor volume (mm3) = major

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axis × minor axis2 × 0.5. The results are expressed as the mean ± SEM of six mice per group. On day 21 or 23, mice were euthanized and the tumors were dissected and photographed (n = 6).

2.14. Statistical analysis

Differences between groups were determined by the analysis of variance (ANOVA), and

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means were compared using Student’s t-test. P values of <0.05, <0.01, and <0.001 were regarded as significant. Animal survival rates were compared with the log-rank test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

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3. Results and discussion

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3.1. Characterization of synthetic vaccine nanoparticle (SVNPs) γ-PGA-based SVNPs that can deliver antigens and immunostimulatory adjuvants to the lymph nodes were developed. To synthesize self-assembled nanoparticles, amphiphilic γPGA was synthesized by conjugating cholesterol-NH2 to a γ-PGA backbone (Supplementary Fig. S1, S2). When γ-PGA-CH was dispersed in aqueous solution, NPs with size of 121.0 ± 11.8 nm, with a zeta potential of -32.6 ± 1.06 mV (Supplementary Fig. S3, Table 1), were generated. To facilitate the loading of tumor antigen (OVA) and double-strand RNA virus (poly (I:C)), the carboxyl groups of γ-PGA-CH were partially aminated using ethylene diamine. The amination of the carboxylic group of γ-PGA-CH induced the reduction in size 10

ACCEPTED MANUSCRIPT (23.4 ± 1.7 nm) and the inversion in net charge (34.7±3.7 mV) in γ-PGA-CH-NH2 NPs (Fig. 2A, B, Table 1). In fact, γ-PGA has abundant carboxylate groups and is classified as a superabsorbent polymer that can absorb and retain extremely large amounts of water relative to their own mass through hydrogen bonding with water molecules [23]. However, the

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amination of carboxylated surfaces inhibits the hydrogen bonding between γ-PGA-CH-NH2 NPs and water molecules, resulting in the shrinkage of the NPs. The experimental result suggested that the physical properties of γ-PGA-CH-NH2 NPs were optimal for loading anionic tumor antigens and double strand RNA as well as for rapid entry into lymphatic vessels and migration into lymph nodes, as reported previously [13, 14]. Although OVA

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protein was studied in this study as model antigen, various antigen that have different charges and sizes should be systematically investigated to expand the current SVNPs platforms into

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various vaccine development. To determine the optimal conditions to prepare a stable complex of antigen and adjuvants, γ-PGA-CH-NH2 NPs were mixed with OVA and Poly (I:C) at various mass ratios; this was followed by the measurement of the size and zeta potential (Fig. 2C, D). The γ-PGA-CH-NH2/OVA (SVNP-OVA) complex showed aggregation at ratios of 1:1 and 1:2. In this study, the selected complex (indicated by an

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asterisk) had an optimal ratio of 1:0.5 and a small size of 37.74 ± 9.5 nm. In the case of the γPGA-CH-NH2/Poly (I:C) (SVNP-IC) complex, a 1:2 mixing ratio was selected to generate stable and small-sized NPs (59.0 ± 5.6 nm) (Supplementary Fig. S4, Table 1).

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3.2. Intracellular uptake of SVNP-IC and SVNP-OVA The internalization of SVNP-IC and SVNP-OVA in bone marrow-derived DCs (BMDCs)

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was investigated using fluorescence microscopy. For these experiments, SVNPs were formulated with rhodamine B-labeled poly (I:C) and fluorescein (FITC)-labeled OVA. The resulting SVNP-OVA (SVNP-OVA-FITC) and SVNP-IC (SVNP-IC-rhodamine B) were incubated with BMDCs for 3 h. After cellular uptake, the fluorescence signals from SVNPIC-rhodamine B and SVNP-OVA-FITC revealed that SVNP-ICs were taken up by the same cells that took up SVNP-OVA. Most of the SVNPs were localized in the same region when the cells were simultaneously treated with both SVNP-IC and SVNP-OVA (Fig. 3A). To confirm the intracellular localization of SVNP-IC, APCs were stained with LysoTracker Blue. The fluorescence microscopy images (Supplementary Fig. S5A) suggested that SVNP-IC was co-localized with LysoTracker Blue). To further evaluate the uptake-efficiency, BMDCs and 11

ACCEPTED MANUSCRIPT bone marrow-derived macrophages (BMMΦs) were incubated with SVNP-IC and SVNPOVA for 3 h, and the fluorescence intensities of the cells were analyzed using flow cytometry. As shown in Fig. 3B,C and Supplementary Fig. S5B,C, a significant increase in fluorescence intensity was observed for the groups treated with SVNP-IC and SVNP-OVA compared to those treated with soluble poly (I:C) and OVA. The results showed that both

3.3. In vitro immunostimulatory activity of SVNP-IC

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SVNP-IC and SVNP-OVA could efficiently deliver poly (I:C) and OVA protein into APCs.

The immunostimulatory effect of SVNP-IC on immune cells was investigated after treating

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APCs with SVNP-IC. After 24 h of treatment, the culture supernatants were harvested and the expression levels of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α

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and interleukin (IL)-6, were measured; these molecules have important roles in the immune systems [24-27]. As shown in Fig. 4A and B, SVNP-IC significantly induced the activation of APCs in a dose-dependent manner. The secretion of TNF-α and IL-6 from BMDCs treated with SVNP-IC was much higher than those from cells treated with soluble poly (I:C) or undecorated NPs. Similarly, SVNP-IC significantly enhanced the production of cytokines in BMMΦs (Fig. 4A,B and Supplementary Fig. S6). Type-I IFN plays a critical role in initiating

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immune responses by stimulating natural killer (NK) cells, Th1 responses, and DC maturation for CD8+ T cells.[28-30] Type I IFNs comprise IFN-α proteins, IFN-β, and other less-investigated IFNs. The success of conventional chemotherapeutics, targeted anticancer agents, and immunological adjuvants is highly dependent on type I IFN signaling [31, 32].

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Thus, we also assessed the expression profile of type I IFN-related genes in BMDCs. We found that SVNP-IC induced a significant increase in IFN-α4 and IFN-β expression, while

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the expression of type I IFN-related genes was negligible in the soluble poly (I:C) and undecorated NP groups (Fig. 4C). Enzyme-linked immunosorbent assay (ELISA) results confirmed that BMDCs treated with SVNP-IC exhibited a more significant increase in IFN-β production than soluble poly (I:C) (Fig. 4D). These results suggest that SVNP-IC can trigger both pro-inflammatory and type I IFN responses.

3.4. In vivo trafficking of SVNP-OVA and SVNP-IC to the lymph nodes Carriers that can deliver antigens and immunostimulatory molecules to lymph nodes constitute an important technology platform that can improve the therapeutic efficacy of cancer immunotherapy. As the SVNPs had an optimal size for transit through lymphatic 12

ACCEPTED MANUSCRIPT vessels to the lymph nodes [8, 9, 13], we investigated whether SVNPs could be used to target lymph nodes. For the experiments, IRDye800 dye-labeled SVNP-OVA (SVNP-OVA-IR800) (upper panel) or IRDye800-labeled OVA (IR800-OVA) (lower panel) were subcutaneously injected into the right paw of mice and the migration of SVNPs was imaged in vivo using a near-infrared (NIR) optical imaging system for 7 days (168 h). A brighter NIR fluorescence

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signal was detected in the axillary lymph nodes of SVNP-OVA-IR800-injected mice compared to that in OVA-IR800-injected mice, suggesting the effective migration and delivery of SVNP-OVA-IR800 into the immune cells in lymph nodes (Fig. 5A). Furthermore, the NIR signal of SVNP-OVA-IR800 in axillary lymph nodes was maintained until 7 days

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post-injection, while the signal disappeared in the case of OVA-IR800-treated mice (Fig. 5A). The experimental results also suggested that the retention of vaccine could be increased

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to several days by the use of SVNPs. This is expected to maximize the adaptive immune response.

The distribution of SVNPs in the lymph nodes of mice that had been injected with SVNP-OVA-FITC was examined. At 24 h post-injection of SVNP-OVA-FITC, strong FITC signals were observed in dissected lymph nodes, indicating the potency of SVNP-OVA to target the lymph node region (Fig. 5B). In contrast, weak FITC signals were detected in the

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lymph nodes of mice injected with FITC-OVA in the presence of γ-PGA-CH NPs (without amine groups) (Supplementary Fig. S7). The experimental results suggest that the positive amine moiety of γ-PGA-CH-NH2 NPs had an important role in delivering negatively charged

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OVA. The localization characteristic of SVNP-OVA-FITC within the lymph nodes was investigated using anti-DC or macrophage antibodies. In general, DCs and macrophages were localized and dispersed throughout the subcapsular and medullary space of the lymph nodes.

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As shown in Fig. 5B, SVNP-OVA-FITCs were significantly co-localized with subcapsular macrophages (CD169+ cells, upper panel), medullary macrophages (F4/80+ cells, middle panel), and DCs (CD205+ cells, lower panel). A flow cytometry profile representing the percentage of FITC-associated cells confirmed that similar percentages of DCs and macrophages were associated with SVNP-OVA-FITC (Fig. 5C). It should be emphasized that fluorescence signals from SVNP-IC and SVNP-OVA were co-localized in the lymph node when both SVNP-IC and SVNP-OVA were simultaneously injected (Fig. 5D). These results suggested that the SVNPs could enter lymphatic drainage because of their optimally engineered size. In fact, many researchers have previously attempted to encapsulate antigens and adjuvants into one-pot NPs. This has been driven by the fact that the effective activation 13

ACCEPTED MANUSCRIPT of immune cells can be obtained when antigens and adjuvants are delivered into immune cells simultaneously [33-36]. However, it is difficult to independently control the loading amount of antigens and adjuvants in one-pot particles. Although multiple compounds can be simultaneously encapsulated into one NP, the reproducibility and commercialization of NPbased drugs remain limited. Because the chemical properties, such as molecular size and

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hydrophilicity of antigens and adjuvants, vary, different encapsulating materials and protocols should be adopted. In this study, two types of vaccine NPs (SVNP-OVA and SVNP-IC) were developed to control the loaded amount of antigens and adjuvants independently. A possible drawback in the loading of antigens and adjuvants in separate

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delivery carrier is the fact that the probability of simultaneous and colocalized delivery of them into the same site of cells may be not high. However, immunohistological assays and

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fluorescence microscopy image analysis revealed that the two SVNPs were successfully colocalized in the same position of the immune cells. The cocktail approach may be extended to tailor personalized or polarized immune response according to the target disease.

3.5. In vivo immunostimulatory activity of SVNP-IC

SVNPs enabled the delivery of both antigen and adjuvants to APCs in lymph nodes.

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Therefore, we examined the in vivo local immunostimulatory effect after immunization with SVNP-IC. To investigate the targeted effects in the lymph nodes, the inguinal lymph nodes draining the subcutaneous injection site were harvested and the activation level of immune cells was analyzed [37, 38]. The mRNA levels of TNF-α (Fig. 6A), IL-6 (Fig. 6B), and IFN-β

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(Fig.6C) in the lymph nodes were high in the mice treated with SVNP-IC. The activation markers (CD86) were highly expressed in CD11c+ lymph node cells as confirmed by flow

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cytometry analysis (Fig. 6D). Comparison of the expression levels of CD69 in NK cells (NK1.1+ cells) (based on flow cytometric data) showed that SVNP-IC were more effective than soluble poly (I:C) in promoting NK cell expansion (Fig. 6E) and activation (Fig. 6F) in lymph nodes. The experimental results suggested that the immunostimulatory effect of poly (I:C) was improved owing to the enhanced delivery-capacity of SVNPs. In fact, type I IFNs are known to be key factors in initiating immune responses by stimulating NK cells [28-30]. Furthermore, poly (I:C), oncolytic viruses, TLR7, and TLR8 agonists induce the secretion of type I IFNs by cancer cells and/or myeloid cells [19-20]. Thus, these results indicate that enhanced therapeutic outcomes in cancer immunotherapy approaches can be obtained by 14

ACCEPTED MANUSCRIPT taking advantage of a sophisticated defense system that originally evolved to clear virusinfected cells.

3.6. Anti-cancer adaptive immunotherapy with lymph node-targeting SVNPs We investigated whether the adaptive immune responses could be achieved in vivo by

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simultaneous administration of both SVNP-OVA and SVNP-IC (Fig. 7A). CD8+ T cells have important roles in cancer immunotherapy [39], thus, we assessed the antigen (OVA)-specific CD8+ T cell response by intracellular staining and flow cytometric analysis after restimulation with 5 µg/mL OVA peptide (SIINFEKL), a major histocompatibility complex

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class I cytotoxic T cell epitope. The capability of CD8+ T cells to produce IFN-γ is important for inducing a specific cytotoxic response in order to clear infectious pathogens and cancer

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cells. Higher secretion of IFN-γ was observed in mice simultaneously treated with SVNPOVA and SVNP-IC than in any other group (Fig. 7B). These results suggested that the lymph node-targeting SVNPs induced a strong antigen-specific cellular immune response. The antitumor immunity of lymph node-targeting SVNPs was also evaluated by tumor challenge experiments following the simultaneous administration of SVNP-OVA and SVNP-IC to EG7-OVA (OVA epitopes expressing EL4 tumor) tumor-bearing mice. In a

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preventive model, we simultaneously injected SVNP-OVA and SVNP-IC at the tail base, twice each week. Seven days after the final injection, EG7-OVA tumor cells were inoculated and tumor growth was evaluated. As shown in Fig. 7C, D, tumor growth was dramatically inhibited in mice injected with both SVNP-OVA and SVNP-IC. In contrast, the tumor cells

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proliferated in mice injected with SVNP-OVA alone and both SVNP-OVA and poly (I:C). These observations suggested that the targeted and simultaneous delivery of antigen

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and immunostimulatory components are crucial to induce enhanced antitumor immunity. To conduct the in vivo tumor challenge in a therapeutic model, we subcutaneously injected both SVNP-OVA and SVNP-IC at the tail base at 1, 4, and 7 days after EG7-OVA tumor inoculation. As shown in Fig. 8A, simultaneous injection of SVNP-OVA and SVNP-IC significantly inhibited the tumor growth compared to the injection of SVNP-OVA alone and both SVNP-OVA and poly (I:C); notably, this inhibition was maintained for the entire observation period of 18 days. Moreover, the simultaneous injection of SVNP-OVA and SVNP-IC prolonged the 50% survival of mice until day 35, which was higher than that of other mice groups (Fig. 8B). These experimental results suggest that SVNPs induced targeted 15

ACCEPTED MANUSCRIPT delivery of both antigens and adjuvant simultaneously and efficiently to the lymph nodes and enhanced the cytotoxic T cell response and cancer immunotherapeutic effects.

4. Conclusion In this study, we successfully engineered the size and surface character of SVNPs for the

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efficient loading and subsequent delivery of model antigen and immunostimulatory adjuvants into lymph nodes. The positively charged SVNPs with tunable size showed excellent performance in the loading and delivery of negatively charged tumor antigen and immunostimulatory materials. After rapid migration into the lymph nodes, SVNP-OVA and

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SVNP-IC were efficiently taken up by DCs and macrophages, which secreted higher levels of pro-inflammatory cytokines (TNF-α, IL-6) and type I IFN (IFN-α, IFN-β). The immunization

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of SVNP-OVA and SVNP-IC also induced NK cell activation (as a part of innate immunity) and CD8+ T cell response (in adaptive immunity), which are essential for effective cancer immunotherapy. In response to therapeutic tumor challenges, SVNP-OVA and SVNP-IC induced higher antitumor immunity and inhibited tumor growth. The enhanced antitumor immunity was obtained by tuning the size and surface character of SVNPs for the effective loading and delivery of vaccine components. These SVNPs may be combined with various

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tumor antigens (recombinant proteins, peptides, genes) and immunostimulatory compounds (other TLRs, nucleotide oligomerization domain-like receptors, STING agonist, antibodies) to induce enhanced antitumor as well as anti-viral immunity[4-7, 40, 41].

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Author contribution

Notes

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S.-Y. Kim, Y.-W. Noh and T. H. Kang contributed equally to this work.

The authors declare no conflicts of interest.

Acknowledgements This work was supported by grants from the National Research Foundation of Korea (NRF) funded

by

the

Korean

government

(MSIP)

(no.

2014R1A2A1A10049960,

2015R1A2A1A15051980, no. 2016R1A5A2012284, no. 2013M3A9B6075888).

Appendix A. Supplementary data 16

no.

ACCEPTED MANUSCRIPT Supplementary data related to this article can be found at ELSEVIER Publications website.

Reference

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[1] Irvine DJ, Swartz MA, Szeto GL. Engineering synthetic vaccines using cues from natural immunity. Nat Mater. 2013;12:978-90. [2] Conde J, Bao C, Tan Y, Cui D, Edelman ER, Azevedo HS, et al. Dual Targeted Immunotherapy via In Vivo Delivery of Biohybrid RNAi ‐ Peptide Nanoparticles to Tumor‐Associated Macrophages and Cancer Cells. Adv Funct Mater. 2015;25:4183-94. [3] Moyano DF, Liu Y, Peer D, Rotello VM. Modulation of Immune Response Using Engineered Nanoparticle Surfaces. Small. 2015;12:76-82. [4] Dobrovolskaia MA, McNeil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol. 2007;2:469-78. [5] Hubbell JA, Thomas SN, Swartz MA. Materials engineering for immunomodulation. Nature. 2009;462:449-60. [6] Noh YW, Hong JH, Shim SM, Park HS, Bae HH, Ryu EK, et al. Polymer Nanomicelles for Efficient Mucus Delivery and Antigen‐Specific High Mucosal Immunity. Angew Chem. 2013;125:7838-43. [7] Naahidi S, Jafari M, Edalat F, Raymond K, Khademhosseini A, Chen P. Biocompatibility of engineered nanoparticles for drug delivery. J Controlled Release. 2013;166:182-94. [8] Zhou Q, Zhang Y, Du J, Li Y, Zhou Y, Fu Q, et al. Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses. ACS nano. 2016;10:2678-92. [9] Wu X, He C, Wu Y, Chen X, Cheng J. Nanogel‐Incorporated Physical and Chemical Hybrid Gels for Highly Effective Chemo–Protein Combination Therapy. Adv Funct Mater. 2015;25:6744-55. [10] Smith DM, Simon JK, Baker Jr JR. Applications of nanotechnology for immunology. Nat Rev Immunol. 2013;13:592-605. [11] Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol. 2005;5:617-28. [12] Bousso P. T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nat Rev Immunol. 2008;8:675-84. [13] Reddy ST, van der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O'Neil CP, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol. 2007;25:1159-64. [14] Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA. In vivo targeting of dendritic cells in lymph nodes with poly (propylene sulfide) nanoparticles. J Controlled Release. 2006;112:26-34. [15] Noh Y-W, Kong S-H, Choi D-Y, Park HS, Yang H-K, Lee H-J, et al. Near-infrared emitting polymer nanogels for efficient sentinel lymph node mapping. ACS nano. 2012;6:7820-31. [16] Liu H, Moynihan KD, Zheng Y, Szeto GL, Li AV, Huang B, et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature. 2014;507:519-22. [17] De Koker S, Cui J, Vanparijs N, Albertazzi L, Grooten J, Caruso F, et al. Engineering Polymer Hydrogel Nanoparticles for Lymph Node‐Targeted Delivery. Angew Chem Int Ed. 2016;55:1334-9. 17

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[18] Jewell CM, López SCB, Irvine DJ. In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles. Proc Natl Acad Sci. 2011;108:15745-50. [19] Ammi R, De Waele J, Willemen Y, Van Brussel I, Schrijvers DM, Lion E, et al. Poly (I: C) as cancer vaccine adjuvant: knocking on the door of medical breakthroughs. Pharmacol Ther. 2015;146:120-31. [20] DeMuth PC, Garcia‐Beltran WF, Ai‐Ling ML, Hammond PT, Irvine DJ. Composite dissolving microneedles for coordinated control of antigen and adjuvant delivery kinetics in transcutaneous vaccination. Adv Funct Mater. 2013;23:161-72. [21] Hafner AM, Corthésy B, Merkle HP. Particulate formulations for the delivery of poly (I: C) as vaccine adjuvant. Adv Drug Del Rev. 2013;65:1386-99. [22] Noh Y-W, Jang Y-S, Ahn K-J, Lim YT, Chung BH. Simultaneous in vivo tracking of dendritic cells and priming of an antigen-specific immune response. Biomaterials. 2011;32:6254-63. [23] Sung MH, Park C, Kim CJ, Poo H, Soda K, Ashiuchi M. Natural and edible biopolymer poly‐γ‐glutamic acid: synthesis, production, and applications. Chem Rec. 2005;5:352-66. [24] Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev. 2002;13:155-68. [25] Matsumoto M, Tatematsu M, Nishikawa F, Azuma M, Ishii N, Morii-Sakai A, et al. Defined TLR3-specific adjuvant that induces NK and CTL activation without significant cytokine production in vivo. Nat Commun. 2015;6;2680. [26] Conforti R, Ma Y, Morel Y, Paturel C, Terme M, Viaud S, et al. Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands. Cancer Res. 2010;70:490-500. [27] Seya T, Shime H, Takeda Y, Tatematsu M, Takashima K, Matsumoto M. Adjuvant for vaccine immunotherapy of cancer–focusing on Toll‐like receptor 2 and 3 agonists for safely enhancing antitumor immunity. Cancer Sci. 2015;106:1659-68. [28] Tel J, Sittig SP, Blom RA, Cruz LJ, Schreibelt G, Figdor CG, et al. Targeting uptake receptors on human plasmacytoid dendritic cells triggers antigen cross-presentation and robust type I IFN secretion. J Immunol. 2013;191:5005-12. [29] Kadowaki N, Antonenko S, Liu Y-J. Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c− type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. J Immunol. 2001;166:2291-5. [30] Le Bon A, Etchart N, Rossmann C, Ashton M, Hou S, Gewert D, et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat Immunol. 2003;4:1009-15. [31] Kranz LM, Diken M, Haas H, Kreiter S, Loquai C, Reuter KC, et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature. 2016;534:396-401. [32] Miyashita A, Fukushima S, Nakahara S, Kubo Y, Tokuzumi A, Yamashita J, et al. Immunotherapy against Metastatic Melanoma with Human iPS Cell–Derived Myeloid Cell Lines Producing Type I Interferons. Cancer Immunol Res. 2015;4:248-58. [33] Kreutz M, Tacken PJ, Figdor CG. Targeting dendritic cells—why bother? Blood. 2013;121:2836-44. [34] Luo Z, Wang C, Yi H, Li P, Pan H, Liu L, et al. Nanovaccine loaded with poly I: C and STAT3 siRNA robustly elicits anti-tumor immune responses through modulating tumorassociated dendritic cells in vivo. Biomaterials. 2015;38:50-60.

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TE D

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[35] Rosalia RA, Cruz LJ, van Duikeren S, Tromp AT, Silva AL, Jiskoot W, et al. CD40targeted dendritic cell delivery of PLGA-nanoparticle vaccines induce potent anti-tumor responses. Biomaterials. 2015;40:88-97. [36] Tincer G, Yerlikaya S, Yagci FC, Kahraman T, Atanur OM, Erbatur O, et al. Immunostimulatory activity of polysaccharide–poly (I: C) nanoparticles. Biomaterials. 2011;32:4275-82. [37] Ebihara T, Azuma M, Oshiumi H, Kasamatsu J, Iwabuchi K, Matsumoto K, et al. Identification of a polyI: C-inducible membrane protein that participates in dendritic cell– mediated natural killer cell activation. J Exp Med. 2010;207:2675-87. [38] Akazawa T, Ebihara T, Okuno M, Okuda Y, Shingai M, Tsujimura K, et al. Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells. Proc Natl Acad Sci. 2007;104:252-7. [39] Klebanoff CA, Gattinoni L, Restifo NP. CD8+ T‐cell memory in tumor immunology and immunotherapy. Immunol Rev. 2006;211:214-24. [40] Lee IH, Kwon HK, An S, Kim D, Kim S, Yu MK, et al. Imageable Antigen‐Presenting Gold Nanoparticle Vaccines for Effective Cancer Immunotherapy In Vivo. Angew Chem Int Ed. 2012;51:8800-5. [41] Liu H, Kwong B, Irvine DJ. Membrane anchored immunostimulatory oligonucleotides for in vivo cell modification and localized immunotherapy. Angew Chem Int Ed. 2011;123:7190-3.

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Fig. 1. Schematic illustration of synthetic vaccine nanoparticle (SVNP)-based cancer

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immunotherapy. A) Chemical synthesis of poly(γ-glutamic acid) (γ-PGA)-based SVNPs. γPGA was conjugated with the cholesterol-NH2, and it formed γ-PGA-CH NPs (~120 nm in diameter) in aqueous solutions by self-assembly. The partial amination of the carboxylic group of the γ-PGA-CH NPs induced a reduction in NP size (to ~20 nm in diameter) owing to the increased hydrophobicity at the γ-PGA-CH-NH2 NP–water interface and the resulting shrinkage of NPs. By loading the model tumor antigen (OVA) and toll-like receptor 3 (TLR3) agonist (poly (I:C)) onto γ-PGA-CH-NH2 NPs, SVNP-OVA and SVNP-IC, respectively, were generated. B) SVNPs carrying OVA and poly (I:C) into lymph nodes efficiently modulated both innate and adaptive immune responses for effective cancer immunotherapy. 20

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Fig. 2. Characterization of synthetic vaccine nanoparticles (SVNPs). A) Particle diameter and

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zeta potential were analyzed by dynamic light scattering. B) Representative image of SVNPs from transmission electron microscopy. Transmission electron microscopy images of γ-PGA-

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CH-NH2 NPs were obtained by negative staining with uranyl acetate. C, D) Optimizing the formulation of γ-PGA-CH-NH2 NPs with poly (I:C) and OVA. Effects of the mass ratio of C) γ-PGA-CH-NH2 NPs to OVA and of D) γ-PGA-CH-NH2 NPs to poly (I:C) on the particle size (blue) and zeta potential (red) of complexes. The complexes showed aggregation at γPGA-CH-NH2 NPs:OVA mass ratios of 1:1 and 1:2 (zeta potential, not shown) (asterisk: ratio of formulation optimization).

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Fig. 3. Cellular uptake of synthetic vaccine nanoparticles (SVNPs). A) Representative fluorescence microscopy images of SVNP-OVA and SVNP-IC revealed co-localization of

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OVA (labeled with FITC for green fluorescence) and poly (I:C) (labeled with rhodamine B for red fluorescence) after cellular uptake. Bone marrow-derived dendritic cells (BMDCs) were incubated with SVNP-OVA and SVNP-IC for 3 h. Nuclei were stained with Hoechst 33342 (DAPI, blue); scale bars = 15 µm. B, C) Cellular uptake efficiency was analyzed by

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flow cytometry analysis in BMDCs (left) and bone marrow-derived macrophages (BMMΦ; right). BMDCs were incubated with SVNP-OVA-FITC (5 µg/mL) (B) or SVNP-IC-

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rhodamine B (5 µg/mL) (C) for 3 h. (MFI = mean fluorescence intensity). Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

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Fig. 4. In vitro activation effects of SVNP-IC. A, B) Bone marrow-derived dendritic cells (BMDCs; left) and bone marrow-derived macrophages (BMMΦs; right) were incubated with the indicated materials at a concentration of poly (I:C) )(5–10 µg/mL) for 24 h. Concentrations of TNF-α (A) and IL-6 (B) cytokines in the culture medium were assessed

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using ELISA kits. (C) In vitro type I interferon (IFN-α4 and IFN-β) gene expression levels were analyzed by real-time PCR. (D) Concentration of IFN-β in the culture medium after 24

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h incubation with SVNP-IC (10 µg/mL poly (I:C)) was measured by ELISA. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

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Fig. 5. In vivo trafficking of SVNP-OVA and SVNP-IC to lymph nodes. A) An in vivo NIR fluorescence image of IRDye800-labeled SVNP-OVA (upper) and IRDye800-labeled OVA

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(lower) at 1, 24, 48, 168 h after administration into the footpad of mice. (triangle: footpad, circle: axillary lymph node). MFI, Mean fluorescence intensity. B) Immunohistofluorescence

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analysis of dissected lymph nodes from a mouse injected with SVNP-OVA-FITC. Slides were stained with anti-CD169 (macrophage, upper panel), anti-F4/80 (macrophage, middle panel), or anti-CD205 (dendritic cell, lower panel) antibodies. DIC, differential interference contrast; scale bars represent 90 µm. C) Uptake of SVNP-OVA-FITC by immune cells in the draining lymph node of mice at 24 h after injection. D) Histological analysis of lymph nodes at 24 h after simultaneous injection of SVNP-OVA-FITC and SVNP-IC-rhodamine B. Data are presented as mean ± SD (*P < 0.05).

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Fig. 6. In vivo innate immune responses as a result of SVNP-IC administration. A, B) TNF-α, IL-6 and C) IFN-β cytokine mRNA expression. mRNA levels in the lymph node were measured by RT-PCR. D) Dendritic cell activation in the lymph nodes. E, F) NK cell proliferation and activation in the lymph nodes were analyzed by flow cytometry (MFI =

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< 0.001).

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mean fluorescence intensity). Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P

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Fig. 7. In vivo adaptive immune response as a result of SVNP-OVA and SVNP-IC

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administration. (A) Immunization schedule. C57BL/6 mice were simultaneously immunized with SVNP-OVA and SVNP-IC twice, with a 1-week interval between immunizations. (B) Analysis of OVA-specific adaptive immune response. At 7 days after the last injection, splenocytes were collected and restimulated in vitro for 12 h with 5 µg/mL OVA peptide (SIINFEKL). Staining intensity of CD8+ T cells was detected by intracellular staining for INF-γ. Presented data refer to numbers of CD8+ INF-γ+-producing cells. C, D) C57BL/6 mice were simultaneously immunized with both SVNP-OVA and SVNP-IC twice, with a 1-week interval between immunizations. At 7 days after the last injection, EG7-OVA tumor cells were inoculated for the tumor challenge (C). Survival rate of EG7-OVA tumor-bearing mice

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< 0.05, **P < 0.01).

Fig. 8. In vivo analysis of therapeutic effect after the tumor challenge. Analysis of therapeutic

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effect in EG7-OVA tumor-bearing mice after three injections of both SVNP-OVA and SVNP-IC (A). Photographs of the dissected tumor tissue from each group are shown in the graph. Injection time points are highlighted by black arrows. Survival rate of EG7-OVA

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tumor-bearing mice was determined by Kaplan-Meier survival analysis (B). Data are presented as mean ± S.D. (*P < 0.05).

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Table 1. Characterization of nanoparticles employed in this study. Data are presented as

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mean ± SD.

Determined by a) DLS, b) H-NMR, c) Elemental analysis (γ-PGA-CH : C (44.27±1.21), H

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(10.02±0.50), N (5.46±0.04), γ-PGA-CH-NH2 (NP): C (44.2±1.13), H (19.02±1.73), N

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(7.37±0.49)).

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Supplementary data

Fig. S1. 1H NMR spectra of cholesterol-NH2. 1H NMR (700 MHz. chloroform-d): a) cholestetyl chloroformate; b) cholesterol-NH2. The conjugation of cholesterol-NH2 was demonstrated by a new peak of conjugated ethlenediamine at 2.9-3.0 ppm (peak, e) and cholesterol at 5.4 ppm (peak, a).

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Fig. S2. 1H NMR spectra of γ-PGA-CH. 1H NMR (700 MHz, Dimethyl sulfoxide-d): a) cholesterol-NH2; b) γ-PGA; c) γ-PGA-CH. The conjugation of cholesterol-NH2 to γ-PGA was demonstrated by a new peak indicating a cholesterol group at 5.4 ppm (peak, e) and a proton peak in γ-PGA at 2.3 ppm (peak, b).

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Fig. S3. Characterization of γ-PGA-CH nanoparticles. A) Particle diameter was analyzed using dynamic light scattering and transmission electron microscopy. The transmission electron microscopic images of γ-PGA-CH nanoparticles were obtained following negative

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staining with uranyl acetate.

Fig. S4. The number distribution of SVNP-IC (γ-PGA-CH-NH2 : Poly (I:C) ratio = 1:2) and SVNP-OVA (γ-PGA-CH-NH2 : OVA ratio = 1:0.5) as shown on the dynamic light scattering plots.

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Fig. S5. Cellular uptake of synthetic vaccine nanoparticles (SVNPs). A) Representative fluorescence microscopy images of LysoTracker Blue and poly (I:C) (labeled with rhodamine B for red fluorescence) after cellular uptake of SVNP-IC. Bone marrow-derived dendritic

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cells (BMDCs) were incubated with SVNP-IC for 3 h. scale bars = 5 µm. B, C) Cellular uptake efficiency was analyzed by flow cytometry analysis in BMDCs (left) and bone marrow-derived macrophages (BMMΦ; right). BMDC and BMMΦ were incubated with 5 µg/mL SVNP-OVA (B) or 5 µg/mL SVNP-IC (C) for 3 h. (MFI = mean fluorescence

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intensity). Data are presented as mean ± SD

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Fig. S6. Comparision of cytokine levels induced by control & NP (as negative control), LPS (100 ng/ml as positive control). BMDCs (left) and BMMΦ (right) were incubated with the

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indicated materials at a concentration of poly (I:C) (10 µg/mL) for 24 h. The concentrations

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of TNF-α and IL-6 cytokines in the culture medium were assessed using ELISA kits.

Fig. S7. Histological analysis of the lymph node at 24 h after intra-lymph node administration of OVA-FITC (left), γ-PGA-CH with OVA-FITC (middle), or SVNP-OVA-FITC (right). Scale bars represent 90 µm. 33