Enhanced Cancer Vaccination by In Situ Nanomicelle-Generating

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Enhanced Cancer Vaccination by In Situ Nanomicelle-Generating Dissolving Microneedles Nak Won Kim, Sun-Young Kim, Jung Eun Lee, Yue Yin, Jong Han Lee, Su Yeon Lim, E Seul Kim, Huu Thuy Trang Duong, Hong Kee Kim, Sohyun Kim, Jung-Eun Kim, Doo Sung Lee, Jaeyun Kim, Min Sang Lee, Yong Taik Lim, and Ji Hoon Jeong ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04146 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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Enhanced

Cancer

Vaccination

by

In

Situ

Nanomicelle-Generating Dissolving Microneedles Nak Won Kim†,‡, Sun-Young Kimǁ,‡, Jung Eun Lee†, Yue Yin†, Jong Han Lee†, Su Yeon Lim†, E Seul Kim†, Huu Thuy Trang Duong#, Hong Kee Kim§, Sohyun Kimǁ, Jung-Eun Kimǁ, Doo Sung Lee#, Jaeyoon Kim#, Min Sang Lee†,*, Yong Taik Limǁ,* and Ji Hoon Jeong†,*

†

ǁ

School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea

SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon

16419, Republic of Korea

#

Theranostic Macromolecules Research Center, School of Chemical Engineering,

Sungkyunkwan University, Suwon 16419, Republic of Korea

§

‡

*

Raphas R&D Center / Raphas Co., Ltd., Seoul 07793, Republic of Korea

The authors contributed equally to this work

Correspondence

should

be

addressed

to

M.S.L.

([email protected]),

Y.T.L.

([email protected]) or J.H.J. ([email protected]).

ABSTRACT: Efficient delivery of tumor antigens and immunostimulatory adjuvants into lymph nodes is crucial for the maturation and activation of antigen-presenting cells (APCs), 1 ACS Paragon Plus Environment

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which subsequently induce adaptive antitumor immunity. Dissolving microneedle (MN) has been considered as an attractive method for transcutaneous immunization due to its superior ability to deliver vaccines through the stratum corneum in a minimally invasive manner. However, since dissolving MNs are mostly prepared using water-soluble sugars or polymers for their rapid dissolution in intradermal fluid after administration, they are often difficult to formulate with poorly water-soluble vaccine components. Here, we develop amphiphilic triblock copolymer-based dissolving MNs in situ that generate nanomicelles (NMCs) upon their dissolution after cutaneous application, which facilitate the efficient encapsulation of poorly water-soluble toll-like receptor 7/8 agonist (R848) and the delivery of hydrophilic antigens. The sizes of NMCs range from 30 to 40 nm, which is suitable for the efficient delivery of R848 and antigens to lymph nodes and promotion of cellular uptake by APCs, minimizing systemic exposure of the R848. Application of MNs containing tumor model antigen (OVA) and R848 to the skin of EG7-OVA tumor-bearing mice induced a significant level of antigenspecific humoral and cellular immunity, resulting in significant anti-tumor activity.

KEYWORDS: cancer vaccine, dissolving microneedle, nanomicelle, toll-like receptor agonist, lymphatic delivery

Vaccines have been considered one of the first-line treatments for the control of cancer as well as infectious disease. Antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages and B cells, are known to play a central role in inducing efficient antitumor or antiviral immune responses after vaccination. For maturation and activation of APCs, the efficient delivery of antigens and immunomodulators called adjuvants is critical. 2 ACS Paragon Plus Environment

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Microneedles (MNs) have emerged as an attractive way to overcome the skin barrier for cutaneous drug delivery in a minimally invasive manner.1,2 MNs represent an array of needles of several hundred microns in length, which can achieve efficient transdermal delivery of biomacromolecules, such as protein or DNA, across the stratum corneum.3-9 MNs can avoid unnecessary pain since the length of the MN is long enough to pierce skin but does not stimulate dermal nerves and may not require professional training for administration.10-12 MNs are especially useful for the delivery of vaccines since transdermal vaccine delivery has advantages over intramuscular or subcutaneous administrations due to a relative abundance of resident Langerhans cells and APCs, including DCs and macrophages, in the epidermis and dermis of skin.13-17 Among various types of MNs, dissolving MNs have attracted much attention due to their ease of preparation and improved safety.18,19 Unlike solid and hollow MNs that pose the risk of breakage in the skin, dissolving MNs do not leave the debris of needles underneath the skin, minimizing the risk of inflammation caused by foreign materials.20-21 Dissolving MNs also enable efficient cutaneous drug delivery via the rapid dissolution of the matrix upon contact with moisture in the intradermal region, which allows recovery of the damaged skin layer before removal of the patch to prevent potential infection.22 Since dissolving MNs should undergo rapid dissolution in intradermal fluid, their matrices mostly consist of water-soluble sugars and polymers. The use of water-soluble materials as a matrix, however, often limits the formulation of active ingredients with limited aqueous solubility. To address this problem, nanoparticles such as liposomes containing a hydrophobic drug were incorporated into the MN matrix.23,24 However, the incorporation of nanoparticles may compromise the mechanical integrity of MNs during long-term storage due to an unstable interface between the matrix and the nanoparticle surface.

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Resiquimod (R848), an immunomodulating imidazoquinoline derivative, is a specific ligand for human toll-like receptor 7 and 8 (TLR7/8) or murine TLR7 expressed on immune cells such as DCs, macrophages, natural killer cells and B-lymphocytes.25-27 The TLR7/8 agonist can stimulate immune cells to produce Th1-type cytokines such as interleukin-12, interferon-γ and tumor necrosis factor-α and to enhance antigen-specific humoral and Th1type cellular immune responses when used as an adjuvant, leading to strong anti-viral and anti-tumor effects.28,29 Despite the potent immune stimulation activity of R848, the use of it as vaccine adjuvants was limited due to its poor solubility in an aqueous medium and systemic cytotoxicity.28-31 Poorly water-soluble imidazoquinolines have been incorporated in micro- or nano-size particles to deliver the adjuvant to immune cells.30,31 In this study, we employed an amphiphilic triblock copolymer, Pluronic F127, as a main matrix material for dissolving MNs. The polymer is approved for clinical use and can be readily dissolved in both organic and aqueous solvents, which facilitates the preparation of MNs containing hydrophilic antigen and the hydrophobic adjuvant TLR7/8 agonist (R848) in the same matrix. The dissolution of MNs after intradermal administration led to the formation of nanomicelles (NMCs), which further facilitated the dissolution of R848 in the intradermal fluid. In addition, the sizes of the NMCs ranging from 30 to 40 nm were in the hydrodynamic diameter range known to be optimal for efficient transition to and retention in lymph nodes, allowing efficient migration to lymph nodes to stimulate resident immune cells.32-34 When formulated with the combination of ovalbumin (OVA) and R848 as a model therapeutic cancer vaccine, the MNs showed enhanced antigen-specific humoral and cellular immune responses and significant anti-tumor activity in E.G7-OVA tumor-xenograft mice (Scheme 1).


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RESULTS AND DISCUSSION Improved lymphatic delivery of hydrophobic TLR7/8 agonist To achieve improved lymphatic delivery of the hydrophobic TLR7/8 agonist for enhanced transdermal immune stimulation, we designed a dissolving MN that can generate NMCs upon injection into the skin. The NMCs with a size of ca. 30 nm can efficiently migrate to the vicinal lymph nodes, stimulating resident professional APCs such as DCs and macrophages. NMC-generating dissolving MNs were prepared based on an amphiphilic triblock copolymer of

poly(ethyleneoxide)-b-poly(propyleneoxide)-b-poly(ethyleneoxide)

(Pluronic

F127)

blended with poly(ethylene glycol) (PEG, Mw 6000). PEG was employed to improve the micelle-forming behavior of F127 in intradermal fluid since water molecules tend to decrease their tendency to form hydrogen bonds with F127 in the presence of PEG, facilitating the formation of micelles.35 In addition, blending of PEG with F127 was also found to improve the mechanical properties of dissolving MN after molding. The fabrication of MN using a film rehydration and micromolding method was shown schematically in Figure S1. To load hydrophobic adjuvant in MN matrix, R848 dissolved in ethanol was mixed with F127 dissolved in dichloromethane. A thin film was formed by evaporating the solvents under reduced pressure. The final mixture solution for the MNs was prepared by rehydrating the film in an aqueous solution containing PEG 6000 and antigen (OVA). The mixing stoichiometry of F127 to PEG was 7:3. The resulting mixture was then applied to a polydimethylsiloxane (PDMS) mold and dried at a room temperature under a vacuum to form the dissolving MNs. The fabrication process was designed to minimize undesirable denaturation of the antigen by the organic solvent and heat. The morphology of the MNs was observed under a scanning electron microscope (SEM) and stereo-microscope (Figure 1a, 1b). The MN array included 49 pyramid-shape needles in a 1 × 1 cm patch. The length of the 5 ACS Paragon Plus Environment

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MNs was 350 µm, which is enough to penetrate the mouse stratum corneum (~5 µm in thickness) and efficiently deliver the adjuvant and antigen into the intradermal space. The mechanical strength of the MNs was evaluated by applying axial forces using a texture analyzer. The MN height was gradually reduced as the applied force was increased (Figure S2). The tip of the MN was compressed to eventually collapse rather than fracture in response to the axially applied forces. This phenomenon may have been due to the pyramid shape of the MN. The addition of PEG to the MN matrix led to significant improvement of the mechanical strength. The MNs with a blend ratio (F127/PEG) of 7:3 showed the lowest reduction in MN height and the efficient creation of a micro-conduit when applied to mouse skin with a gentle thumb pressure (Figure 1c). The skin penetration efficiency of MNs is generally considered to increase with increased forces applied per MN.36,37 The gentle force applied by thumb was enough for the MN to penetrate the topmost layers of the skin (Figure S3), leading to dissolution of the needles within 30 min (Figure 1d). When MNs containing a hydrophobic fluorescent probe, 1,1'-dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine, 4chlorobenzenesulfonate salt (DiD), were applied to mouse back skin, the fluorescence signal disappeared slowly over time (Figure 1e), suggesting the dissolution and migration of the hydrophobic DiD entrapped in the core of the NMCs. The DiD-loaded MNs were readily dissolved in aqueous solution via the formation of NMCs with a diameter of 33 ± 8.73 nm (Figure 1f). The DiD-loaded NMCs (DiD@NMCs) could be readily taken up by cells (HCT116) and localized in the cytosolic space, while the fluorescent probe dissolved in DMSO and mixed with the cell culture medium only stained the cell membrane, suggesting an enhanced cellular uptake of NMCs via endocytosis (Figure 2a). Increased cytosolic accumulation of the fluorescence over time also evidenced the endocytosis-mediated cellular delivery of NMCs (Figure 2b).


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NMCs generated from MNs can medicate the efficient delivery of a hydrophobic TLR7/8 agonist To evaluate the capability of R848-loaded NMCs (R848@NMC) to activate and stimulate RAW264.7 cells, the cells were stimulated with R848 and R848@NMC. Treatment with the immune stimulants induced the secretion of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (Figure 2c and d). TNF-α and IL-6 are considered to be important cytokines for antitumor activity.38,39 To assess the maturation effect, we observed the expression of co-stimulatory molecules of RAW264.7 cells using flow cytometry. The expression of CD40, CD86 and CD80 were significantly up-regulated in macrophages (Figure 2e-g). It should be noted that R848@NMC was able to elicit a much stronger immune stimulation effect for macrophage maturation and cytokine secretion than a soluble form of R848. In addition, the cellular uptake of fluorescently labeled OVA by RAW264.7 cells was significantly increased by R848@NMC (Figure 2h and i). These results demonstrated that the NMCs could efficiently deliver not only R848 but the antigen to immune cells, leading to enhanced maturation of immune cells, and secretion of proinflammatory cytokines.

NMCs generated from MNs were able to migrate to lymph nodes Based on the in vitro results, we hypothesized that the NMCs generated from the MNs could mediate efficient delivery of a hydrophobic TLR7/8 agonist, R848, to lymph nodes, where R848 induces immune response-stimulating immune cells via activation and secretion of cytokines that lead to the activation of CD8 cytotoxic T lymphocytes. Therefore, OVA and R848 were used as an antigen and an immunomodulatory agent, respectively, and loaded in 7 ACS Paragon Plus Environment

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the F127/PEG MNs. The OVA maintained its structural integrity after entrapment in the MN matrix (Figure S4). Figure 3a shows that the fluorescently labelled OVA was evenly distributed over the matrix of the MN array and almost completely released after injection into mouse skin. The OVA and R848 were released from the MN over 60 min when the MN was placed in PBS at 37 °C (Figure 3b and c). Upon the injection, the MN would be dissolved to generate NMCs in the intradermal space. To further investigate the in-situ generation of NMCs from the MN, the intradermal situation after MN injection was simulated using a Transwell culture plate in which Matrigel was layered on the porous membrane of the Transwell insert and RAW264.7 cells were cultured on the bottom of the lower chamber of the plate as illustrated in Figure S5a. The generation and cellular delivery of NMCs were observed by fluorescence resonance energy transfer (FRET) technique. The MN containing hydrophobic FRET pairs, DiO (FRET donor) and DiI (FRET acceptor), was placed on the surface of Matrigel to allow the dissolution of MN and the generation of NMC containing both DiO and DiI in its hydrophobic core (DiO/DiI@NMC). The DiO/DiI@NMC migrated through the gel matrix and supporting membrane with a pore size of 8 µm and diffused into the lower chamber where the cells are located. After 6 h, the FRET fluorescence (λex = 488 nm and λem = 565 nm) was observed in the cytoplasm of the cells in the bottom chamber, suggesting that the DiO/DiI@NMC could migrate through the Matrigel, an artificial extracellular matrix (ECM), to be transferred to the cells without losing its structural integrity (Figure S5b). The distribution of DiD@NMCs in areas of epidermis and dermis after injection further confirmed the efficient migration of the NMCs through the skin layers (Figure 3d). To observe whether the NMCs generated from MNs were able to migrate to lymph nodes, DiD was incorporated in the MN matrix as a hydrophobic fluorescent probe for in vivo tracking of DiD@NMCs to the lymph node. We analyzed dissected lymph nodes in which DiD@NMCs were taken up by macrophages and DCs 24 h after transdermal 8 ACS Paragon Plus Environment

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application of the DiD-containing MNs. Strong fluorescence signals were observed in the dissected lymph nodes, suggesting that the DiD@NMCs generated from the dissolution of the MNs could efficiently migrate to the lymph nodes (Figure S6). We further investigated the localization of DiD@NMCs to DCs (DEC205+, upper panel), subcapsular macrophages (CD169+, middle panel), medullary macrophages (F4/80+, middle panel), and Langerhans cells (CD207+, lower panel) in the lymph nodes (Figure 3e). The DiD@NMCs generated from MNs after transdermal administration were able to co-localize with the APCs in lymph nodes. These results suggested that the transdermal application of MNs resulted in the generation of NMCs that could efficiently deliver a hydrophobic model adjuvant to APCs in the lymph node through lymphatic vessels due to the optimized size of NMCs for lymphatic transition.

Anti-tumor immune responses can be elicited by MN vaccination The ability of R848 loaded in MNs to induce immunity was evaluated in vivo by monitoring the immunogenicity of OVA as a model antigen. The average doses of OVA and R848 were 90.65 µg and 44.15 µg, respectively. The MN injection into mouse skin was performed manually by gentle pressure applied by thumb. The MN can deliver more than 95% of the dose into the skin in 60 min. The addition of R848 to the MNs significantly increased the production of antigen-specific antibodies (IgG) (Figure 4a). The secretion of IFN-γ by CD8+ T cells was also significantly elevated in OVA/R848 MNs after in vitro restimulation (Figure 4b). However, the OVA MN groups did not show increased IFN-γ secretion. The transdermal delivery of OVA and R848 using the MNs (OVA/R848 MNs) resulted in the generation of significantly higher levels of antigen-specific antibodies and cytotoxic T cells than subcutaneous injection of OVA and R848 using a hypodermic syringe 9 ACS Paragon Plus Environment

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(OVA/R848 SC) (Figure 4c). Furthermore, the OVA/R848 MNs demonstrated no significant induction of an inflammatory cytokine, interleukin-6 (IL-6), in serum after the transdermal application (Figure 4d). In contrast, 6 h after subcutaneous injection (OVA/R848 SC), the serum concentration of IL-6 was more than 10 times higher than that with OVA/R848 MNs (Figure 4d). The reduced production of systemic inflammatory cytokines may have been due to the efficient NMC-mediated delivery of R848 to lymph nodes and promotion of cellular uptake by APCs, minimizing systemic exposure of the TLR 7/8 ligand. This result is in agreement with previous observations of the reduction of systemic inflammatory responses of the TLR 7/8 ligand using nanoparticles as a carrier.40 These results suggest that transdermal administration using MNs may facilitate the efficient delivery of poorly water-soluble adjuvants to lymph nodes and the in situ generated NMCs containing the TLR 7/8 ligand can significantly enhance a specific cytotoxic cell-mediated immune response to facilitate the clearance of cancer cells expressing a target antigen. The induction of immune responses against tumor-specific antigen has been considered one of the most promising strategies for therapeutic and prophylactic vaccination for cancer. The therapeutic anti-tumor effect of OVA/R848 MNs was assessed in a mouse tumor xenograft model. The mouse tumor model was generated by injecting E.G7-OVA mouse lymphoma cells constitutively synthesizing and secreting OVA into the flank of C57BL/6 mice. When the tumor size reached approximately 100 mm3, formulations containing OVA antigen either with or without R848 were administered through transdermal routes using the MN patch. For therapeutic vaccination, six injections were given to the tumor-bearing mice following the schedule shown in Figure 5a. The administration of OVA/R848 resulted in a significant retardation of tumor growth (Figure 5b). The body weight of the mice was not significantly influenced by the administration of the vaccine formulations during the experimental period (Figure S7). Massive areas of necrosis were observed in the tumor tissues treated with OVA/R848 MNs 10 ACS Paragon Plus Environment

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(Figure 6a). However, there were no detectable signs of toxicity or pathological changes in response to OVA/R848 MNs in major organs, including heart, liver, spleen, kidneys, and lungs (Figure 6b). Prophylactic cancer vaccines may have advantages over conventional therapeutic approaches since there are a relatively small number of malignant cells as a target of immunity in the early phase of cancer.41,42 Therefore, as an alternative approach, we assessed the utility of OVA/R848 MNs as a prophylactic cancer vaccine. To evaluate the prophylactic anti-cancer effect, mice were immunized three times with formulations containing OVA antigen either with or without R848 using the MNs. Seven days after the last immunization, E.G7-OVA cells were administered into the flack of the immunized mice, and the growth of the E.G7-OVA tumors was monitored for 26 days (Figure 7a). The OVA/R848 MN groups showed an effective tumor prevention effect (Figure 7b). All the control mice were dead at 56 days after tumor inoculation, whereas all the mice treated with OVA/R848 MN survived (Figure 7c).

CONCLUSIONS In this study, the NMC-generating MNs based on the poloxamer was developed and used as a transdermal delivery vehicle for hydrophobic adjuvant as well as hydrophilic antigen. It is noteworthy that a hydrophobic TLR7/8 agonist, R848, loaded in the MNs could be efficiently transited to the lymph nodes in the form of NMCs with a diameter ranging from 30 to 40 nm, stimulating immune cells including DCs, macrophages and Langerhans cells. The efficient migration of NMCs to the lymph node may also be able to reduce the risk of inflammatory cytokine-mediated systemic toxicity of the TLR7/8 agonist. Since the administration of OVA antigen and R848 efficiently induces both humoral and cellular immune responses against the antigen, OVA/R848 MNs could be utilized as both therapeutic and prophylactic vaccines for 11 ACS Paragon Plus Environment

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cancer. Considering the advantages of MNs over the conventional injection using a hypodermic needle, such as improved patient compliance, the NMC-generating dissolving MNs could be used as a potential clinical platform for transdermal vaccination for therapeutic and prophylactic purposes.

EXPERIMENTAL SECTION Materials: The microneedle (MN) mold was obtained from Miti systems (Daejeon, Korea). Pluronic F127, polyethylene glycol (PEG, Mw 6000), formalin solution, neutral buffered, 10%, trypan blue solution (0.4%), Dulbecco’s Modified Eagle’s Medium (DMEM), PBS, and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). DAPI (4',6diamidino-2-phenylindole), DiD and FITC-OVA were obtained from Molecular Probes (Eugene, OR). Resiquimod was purchased from Sigma-Aldrich (St. Louis, MO). Ovalbumin was purchased from InvivoGen (San Diego, CA). Disposable plastic tissue embedding molds were obtained from Polysciences (Warrington, PA 18976). Optimal cutting temperature (OCT) compounds were purchased from Sakura Finetek (Torrance, CA 90501). Micro-slide glasses were obtained from Matsunami (Osaka). High-profile microtome blades were purchased from Leica Biosystems Nussloch GmbH (Nussloch). Mounting medium for fluorescence with DAPI was from Vector Laboratories (Burlingame, CA 94010). All other chemicals and solvents were of analytical grade and were used without further purification. Fabrication of the Pluronic-based dissolving MN patches containing hydrophobic molecules and antigens: MN was fabricated by combining a film rehydration and micromolding method (Figure S1). Pluronic F127 (530 mg) was dissolved in 4 ml anhydrous dichloromethane (CH2Cl2). Either DiD (2.0 mg) or R848 (7.0 mg) were dissolved in 1 ml 12 ACS Paragon Plus Environment

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ethanol (CH3CH2OH) and mixed with the Pluronic F127 solution to homogeneity. Solvents were then removed from the mixture in a round-bottomed flask using a rotary evaporator. After obtaining a clear film, the remaining solvent was completely evaporated with nitrogen. An aqueous solution (3 ml) containing OVA (4.7 mg/ml) and PEG (75.7 mg/ml) was prepared to obtain the Pluronic F127/PEG blend ratio of 7:3 (w/w). Sonication was applied to generate a homogenous resuspension of the film. The resulting solution was filtered through 0.8 µm filter to remove unnecessary materials. The solution (0.15 ml) was placed on a 1 cm × 1 cm reusable polydimethylsiloxane (PDMS) mold at room temperature. This mold was then placed under a vacuum for 3 min at 35 ℃ and left for 2 hours to remove air from the mixture and to improve filling of all the microholes with the mixture. After drying and cooling completely at room temperature, a 2 cm × 2 cm adhesive tape was attached on the base plate for reinforcement. A sheet of hydrophobic molecules and antigen-loaded MNs was obtained by peeling off the mold. Morphology of MN: The morphologies and dimensions of the fabricated MN arrays were observed by SEM (JSM-7600F, JEOL, Tokyo, Japan) and a fluorescence stereo microscope (Carl Zeiss Microscopy GmbH, Gottingen, Germany). The images of the MN patches before and after insertion into mouse skin were determined using the same devices mentioned above. Characterization of micelles released from MN: The morphology and diameter of hydrophobic molecule-loaded micelles released from degradable MNs was investigated. DiDloaded MN array (1 cm×1 cm) was immersed in 1 ml PBS (pH 7.4) and incubated for dissolution at an ambient temperature for 30 min. A TEM device (JEM-3010, ZEOL, Tokyo, Japan) was used for structural analysis of the micelles. The solution (10 µl) were dropped onto copper grid and allowed to stand for 10 min. A 1% diluted aqueous solution of uranyl acetate (5 µl) was added to the grid for 10-15 seconds and dried. TEM images were observed 13 ACS Paragon Plus Environment

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at an accelerating voltage of 100 kV. A size and distribution analysis of hydrophobic molecule encapsulated micelles was measured by DLS (Malvern Nano ZS, Malvern, UK). Stability of OVA antigen during the MN fabrication: For the evaluation of OVA stability during fabrication of MN, freshly prepared MN array (1 cm × 1 cm) was dissolved in 1 ml PBS. For extrinsic fluorescence spectra, 200 µl of the solution containing the released OVA (1.5 µM) was mixed with 20 µl of ANS (40 µM) at 25ºC in PBS. An OVA solution dissolved in PBS (1.5 µM) was used as a control. Fluorescence spectroscopy of the protein was performed using an Infinite® 200 PRO multimode microplate reader (TECAN, Mannedorf, Switzerland) at 25˚C. The experiments were carried out at 370 nm, and emission spectra were recorded between 400-600 nm with an emission slit width of 2 nm each. Release of OVA and R848 from MN: To observe the in vitro release profiles of OVA and R848 from MN, MN array (1 cm × 1 cm) was immersed in in 1 ml PBS and incubated at 37 ˚C. Samples were collected at pre-determined time intervals (0, 1, 2, 3, 4, 5, 10, 20, 30, 60 and 90 min), and the medium was replaced with the same volume of fresh release medium. The amount of released R848 was determined by monitoring a peak absorbance for R848 at 327 nm using UV-Vis microplate reader (TECAN Infinite® M500, Männedorf, Switzerland). The release of OVA was monitored by the bicinchoninic acid (BCA) assay using a microplate reader at 590 nm (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland). Measurement of the mechanical strength of MN: To observe the effect of PEG blend on the mechanical strength of MN, MNs having different PEG blending ratios (Pluronic F127/PEG = F127 only, 9:1, 8:2 and 7:3, w/w) were prepared. Each MN array (1 cm × 1 cm) was carefully fixed to the moveable probe (length 5 cm, cross-sectional area 1.5 cm2) of the TA-XT2 Texture Analyzer (Stable Micro Systems, Surrey, UK) using a double-sided adhesive tape. An axial compression load was then applied. The test station pressed the MN 14 ACS Paragon Plus Environment

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against an aluminum block at a speed of 0.5 mm/s for 30 seconds with different forces (0.05 N, 0.15 N, 0.3 N and 0.5 N/needle). MNs were visualized after the compression test using a stereoscopic microscope and the changes in the height of the needles were expressed as the percent reduction in height of the MNs (means ± S.D., n= 3). Mice and cells: Female C57BL/6 (7 weeks old) mice were obtained from Jungang Lab Animal Inc. (Seoul, Korea). All the mice were maintained in a temperature, humidity and light-controlled room (22-25 °C; 55-60% humidity; 12-h light/dark cycle), and all experimental protocols were approved by the SKKU School of Pharmacy Institutional Animal Care and Use Committee. Human colorectal cancer cells (HCT116) and murine macrophage cells (RAW 264.7) were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultivated in DMEM supplemented with 10% fetal bovine serum (FBS) containing penicillin/streptomycin and maintained at 37 ˚C in a humidified 5% CO2 incubator. Cutaneous permeation test: The skin penetration capability of degradable MNs was tested by inserting MNs into a mouse back skin. A mouse was sacrificed, and a piece of back skin was shaved with depilatory cream, cut off using a scalpel and cleaned with distilled water. The fresh cut skin was affixed to a polystyrene plate. MN patch was manually inserted into the skin by applying gentle thumb pressure and left in place for 30 min and then peeled off. After MN application, the surface of the skin was exposed to a trypan blue solution for 10 min to stain and identify the sites of stratum corneum perforation. After wiping out the residual dye solution with dry paper, the stained site was further wiped out with wet tissue to remove unnecessary stain. The injection spots on the skin were observed using a stereomicroscope.


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In vivo delivery of hydrophobic molecules by MN: The cutaneous delivery and intradermal clearance of hydrophobic molecules after MN injection was visualized using an in vivo optical imaging system (Optix MX3 system, Advance Research Technologies, Montreal, QC). MN containing DiD as a hydrophobic model molecule was manually injected into the back skin of C57BL/6 mouse, and the mouse was imaged at predetermined time intervals (30 min, 60 min and 120 min). Fluorescence intensities were analyzed and quantified using OptiView® software. The in vivo trafficking of DiD@NMC generated from MN was observed histologically assessed by microscopy. Sixty minutes after the injection of MN containing DiD into C57BL/6 mouse, the mouse back skin was taken and frozen by embedding the skin in OCT compound (Tissue-Tek®), followed by storage at -80 °C until cryo-sectioning. The frozen tissue block was transferred to a cryotome cryostat (-20 °C), and 25-µm sections were obtained. The sections were placed on glass slides and mounted in a mounting solution (Fisher Scientific, Waltham, MA). The tissue sections were investigated using a confocal microscope (Zeiss LSM 510, Carl Zeiss MicroImaging GmbH, Germany). Cellular uptake of micelles released from MN: The cellular uptake of DiD@NMC released from MNs was visualized by confocal fluorescence microscopy. HCT116 cell lines were cultured and used for in vitro cellular uptake studies of micelles. After the culture reached 70% confluency, the cells were trypsinized, seeded in a 6-well microplate at a density of 5.0 × 105 cells in DMEM containing 10% FBS and cultured for 24 h. After replacing the culture medium with a serum-free medium, the cells were treated with either DiD (final concentration = 5 µM) or DiD@NMC released from MN. After the incubation for 4 h, the cells were washed three times with PBS and treated with DAPI for 5 min to stain the nucleus. The cells then washed tree times with PBS and fixed in 10% formalin solution for 24 h at 4 °C. The cells were observed by confocal microscopy. 16 ACS Paragon Plus Environment

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FRET analysis: To investigate the in-situ generation of NMCs from the MN, MatrigelTM (BD Biosciences, San Diego, CA) was layered on the porous membrane of the TranswellTM insert (membrane pore size = 8 µm, Corning Inc., Corning, NY) and RAW264.7 cells were cultured on the bottom of the lower chamber of the plate (Figure S5a). The generation and cellular delivery of NMCs were observed by fluorescence resonance energy transfer (FRET) technique. MN fabricated to contain hydrophobic FRET pairs, DiO (FRET donor, 5 µM) and DiI (FRET acceptor, 5 µM) was placed on the surface of the Matrigel in the Transwell insert. After incubation for 6 h, the cells were washed with PBS, stained with DAPI and fixed in 10% formaldehyde. FRET fluorescence signal was observed using a confocal microscope at an excitation wavelength (λex) of 488 nm and emission wavelength (λem) of 565 nm. Effect of vaccine adjuvant on antigen internalization: Cellular uptake of antigens was examined in RAW 264.7 cells that were activated with the immune response modifier, R848, as an adjuvant. Cells were randomly separated into four groups: mock (PBS alone), no adjuvant, free adjuvant (R848), or adjuvant released from MNs (R848@NMC). The macrophages were plated in 24-well plates in 500 µl (1.0 × 105 cells) per well for FACS and fluorescence intensity analysis and seeded onto confocal dishes at 2 ml (5.0 × 105 cells) for confocal fluorescence microscopy. The cells were pretreated with R848 molecules at a final concentration of 1 µg or 2 µg for 24 h at 37 °C with 5% CO2. After the cell medium was replaced with serum-free medium, the activated cells were further incubated with 10 µg FITC-OVA protein for 4 hr at 37 °C with 5% CO2. For FACS analysis, FITC-OVAcontaining macrophages were isolated from plates and diluted in PBS to determine the appropriate number of cells for detection. To obtain quantitative data, the lysates were collected from FITC-OVA-treated macrophages, and the fluorescence intensity was


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determined using a fluorescence spectrophotometer. Confocal microscopy was utilized to observe dye-labeled proteins that were taken up by macrophages as well as cell structure. Cytokine and maturation assay: RAW264.7 cells were cultured in 6-well plates at a density of 4.5 × 105 cells per well in the culture medium and allowed to adhere overnight. R848 and R848 loaded in R848@NMC were added to the wells in 1 ml volume. Then, 24 h post-treatment, the culture supernatants were harvested and analyzed for TNF-α and IL-6 secretion using cytokine-specific OptEIATM ELISA kits (BD Biosciences) according to the manufacturer’s instructions. Cytokine concentrations were quantified using a VersaMax apparatus at OD450 according to the manufacturer’s recommendations. The effect of R848 and micelle-R848 on the expression of RAW264.7 cells maturation markers in vitro was measured by flow cytometer (AcurriTM, BD Biosciences, San Diego, CA) after 24 h of treatment. RAW264.7 cells were stained with PE-anti-CD40 (Clone: 3/23), PE-anti-CD80 (Clone: 16-10A1), and PE-anti-CD86 (Clone: GL1) (BD Pharmingen). Control samples were not treated with antibody. A minimum of 10,000 events were collected (n = 6). Anti-OVA IgG production: ELISA was used to evaluate total anti-OVA antibody levels. Blood samples were obtained 7 days after the third immunization of mice with OVA or OVA and R848 via subcutaneous and transdermal MN injection methods. Vaccination was performed once a week. To confirm the differences in antibody production over time, serum samples were collected after 1, 2 and 4 weeks from each final injection. Microtiter plates (96well, Dynatech, Alexandria, VA) were coated with 100 µl of OVA (10 µg/mL) in PBS at 4 ˚C overnight. The wells were then washed two times with PBST (PBS containing 0.01% Tween 20) and incubated with 100 µl of blocking solution (5% skimmed milk solution in PBST) at 37˚C for 1 h. After washing two times with PBST, the plates were incubated with 100 µl of serially diluted mouse serum at 37 ˚C for 2 h. After three washes with PBST, the plate was 18 ACS Paragon Plus Environment

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incubated with 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (diluted 1:10,000 in ELISA buffer, Covance, Emeryville, CA) for 1 h at 37°C. The plate was then washed four times with PBST, and 100 µl of a 3,3’,5,5’-tetramethylbenzidine (TMB) liquid substrate was added to each well for 30 min at room temperature. Finally, 100 µl of 1 M sulfuric acid (H2SO4) solution was used to stop the sample development. Absorbance was monitored at 450 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland). Systemic cytokine detection: Blood sample were isolated from immunization of mice with OVA and R848 via subcutaeonus and transdermal MN injection methods. The serum samples were obtained from the blood by centrifugation at 14,000 rpm for 15 min. IL-6 were analyzed using cytokine-speicific OptEIATM ELISA kits (BD Biosciences) according to the manufacturer’s instructions. Cytokine concentrations were quantified using a VersaMax apparatus at OD450 according to the manufacturer’s recommendations (n = 5). Immunohistofluorescence analysis of lymph nodes: To confirm the localization of NMCs in the lymph nodes, the inguinal lymph nodes were dissected 24 h after transdermal injection of MN containing DiD as a fluorescent probe (DiD@NMCs) and embedded in Tissue-Tek OCT compound (SAKURA, Tokyo, Japan) followed by snap-frozen in liquid nitrogen. Cryosections (5 µm) were prepared using a Leica cryostat CM1850 (Leica Microsystems, Wetzlar, Germany) and transferred to poly-L-lysine coated glass slides. The sections were fixed with ice-cold acetone for 5 min, dried, and frozen at −20°C until use. The slides were washed in PBS and blocked with PBS containing 5% fetal bovine serum (FBS) for 1 h at room temperature. After additional washing with PBS, the slides were stained with rat anti-mouse CD205 (DEC-205, Clone: NLDC-145, Biolegend, San Diego, CA), CD169 (Siglee-1, Clone: 3D6.112, Biolegend, San Diego, CA), F4/80, (Clone: BM8, eBioscience, 19 ACS Paragon Plus Environment

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San Diego, CA) and CD207 (Langerin, Clone: eBioRMUL.2, Biolegend) for overnight at 4°C to label the DCs and macrophages, respectively. The slides were then stained with FITCconjugated anti-rat IgG secondary antibodies (Jackson Laboratory, Bar Harbor, ME) 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 mounting medium (Dako, CA). Tissue sections (n = 3) were visualized with fluorescence microscope (Olympus IX71, Olympus Optical, Tokyo, Japan) and DeltaVision PD (GE Life Sciences, Marlborough, WA) with the filter set (DAPI: λex=360 nm, λem=455 nm; FITC: λex=490 nm, λem=525 nm; Cy-5: λex=645 nm, λem=705 nm; Omega Optical, Brattleboro, VT). Intracellular Staining: C57BL/6 mice were immunized three times per week with OVA/R848 MN. At 7 d post last immunization, splenocytes were collected and restimulated with 10 µg/ml OVA peptide (SIINFEKL, Invivogen, San Diego, CA) in medium containing GolgiPlug (BD Biosciences) for 12 h in round bottom 96-well plates (Nunc, Roskilde, Denmark) at a density of 5 × 105 cells per well (200 µl). Cells were washed with PBS and stained with PE-anti-CD8α (Clone: 53-6.7, BD Pharmingen) for 30 min at 4°C. After washing

with

PBS,

cells

were

permeabilized

using

Cytofix/Cytoperm

Plus

Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer’s instructions for 20 min at 4°C. Next, cells were washed twice with washing buffer (BD Biosciences) and stained with APC-anti-IFN-γ (Clone: XMG1.2, BD Pharmingen) for 30 min at 4°C. After washing (BD Biosciences), the cells were analyzed using an AcurriTM flow cytometer (BD Biosciences, San Diego, CA). A minimum of 100,000 events were collected (n = 6). Antitumor immunity of OVA and R848: The amount of OVA an R848 in the needle tips of MN was calculated based on the volume fraction of the pyramid-shaped needle structure. The MN array was consisted of 49 needle tips (0.38×0.38×0.35 mm, L×W×H, total volume 20 ACS Paragon Plus Environment

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of needle tips= 0.83 mm3 (= 0.017 mm3 × 49)) and a square needle base (10×10×0.05 µm, L×W×H, volume of needle base = 5 mm3). The average amounts of OVA and R848 in each needle tip were 1.85 ± 0.03 µg and 0.90 ± 0.02 µg, respectively (n = 20 of 1 × 1 patches). The MN patch was manually administered into the back skin of mouse under anesthesia by applying gentle thumb pressure and left in place for 60 min and then peeled off. The amounts of OVA and R848 delivered into the skin by MN injection were determined using a mass balance by measuring the amount of the remaining agents in the needle base. Tumor-bearing mice were divided into three experimental groups (n=5): mock-treated group (PBS), OVA MN group and OVA/R848 MN group. For the therapeutic antitumor vaccination, E.G7-OVA cells at a density of 1×106 were subcutaneously injected into the right flank of C57BL/6 mice (day 0). Once the tumor volume had reached approximately 100 mm3, the treatments were initiated. OVA MN or OVA/R848 MN were manually administered into the left back skin of the tumor-bearing mice on days 7, 9, 11, 13, 15 and 17. The average doses of OVA and R848 were 90.65 µg and 44.15 µg, respectively. Tumor size was determined using a Vernier caliper, and the volume of the tumor (V) was calculated with the following formula: V=0.5×W2×L, where W and L are the minor and major axis of the tumor, respectively. Solid tumor volumes and body weights were measured every three day. The tumors were dissected on day 26 for observation of tumor shapes, sizes and weights. For the prophylactic tumor vaccination, the left flank of C57BL/6 mice was vaccinated 3 times (on days 0, 14 and 21) with the indicated formulations. At one week after the last immunization (day 28), E.G7-OVA cells were subcutaneously injected (1×106 cells) into the right dorsal flank of C57BL/6 mice. When the tumor mass became palpable (approximately 3-5 mm in length), the tumor volume was monitored as mentioned above. A survival curve


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(Kaplan-Meier) was generated using the GraphPad Prism program. All treated groups consisted of five mice. Statistical analysis: To analyze significant differences between two groups, an unpaired Student’s t-test was used. All values are expressed as the means ± standard deviations (SD).

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Schematic diagram of the fabrication of microneedles using a film rehydration and micromolding method; Mechanical properties of NMC-generating dissolving MNs in the presence of different blend ratios of Pluronic and PEG 6000; Histology (H&E) staining showing the microneedle penetration; Structural integrity of OVA released from MNs; In vitro simulation showing the generation and cellular uptake of NMCs; In vivo trafficking of DiD@NMCs to lymph nodes; Changes in the body weight of tumor-bearing mice during therapeutic vaccine treatment. Financial interest statements. The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] 22 ACS Paragon Plus Environment

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*E-mail: [email protected] Author Contributions ‡

Nak Won Kim and Sun-Young Kim contributed equally to this work.

ACKNOWLEDGMENTS This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (Grant number: 2016R1A2B4015056, 2010-0027955, 2017M3A9F5032628,

2018M3A9B5021319,

2017R1A2A1A17069277,

2017R1A5A1014560). Industrial Strategic Technology Development Program (10077704) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea). Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (HI18C1174) funded by the Ministry of Health & Welfare.

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Scheme 1. Schematic illustration of the efficient delivery of hydrophobic immunomodulators and tumor antigens through in situ generated nanomicelles (NMCs) from dissolving microneedles (MNs) for enhanced cancer immunotherapy.


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Figure 1. Characterization of NMC-generating dissolving MNs. (a) Scanning electron microscope (SEM). Scale bars = 1 mm, 100 µm (25×, 150×) and (b) fluorescence stereo microscope images of DiD-loaded MNs. Scale bars = 500 µm. (c) Penetration of microneedles into mouse back skin. The area of penetration was stained with trypan blue solution. Scale bars = 1 mm (d) SEM images of microneedle before and after application to mouse skin. Scale bars = 1 mm (e) In vivo distribution of DiD-loaded NMCs from MNs after injection. The MN array of a 1×1 cm patch was applied on the back skin of mice. The timedependent disappearance of fluorescence was monitored using an in situ optical imaging device. (f) Size distribution and TEM images of NMCs generated by the dissolution of MNs in PBS.


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Figure 2. (a) Representative confocal microscope images of HCT-116 cells incubated with 5 µM DiD (red) for cell membrane staining and with 5 µM DiD-loaded NMCs (red) generated from MNs. Cell nuclei (blue) were stained with DAPI. Scale bars = 5 µm. (b) The timedependent cellular uptake of DiD-loaded NMC generated from MNs. The fluorescence intensity in HCT-116 cells (λex = 640 nm; λem = 700 nm) was monitored at different time intervals. Data represent the mean ± SD of triplicate experiments. (c, d) Secretion of TNF-α (c) and IL-6 (d) by RAW264.7 cells after incubation for 24 h with varying concentration of R848. The concentration of cytokines in the culture medium was determined using ELISA. (e-g) Effect of R848 and R848@NMC on R848-induced up-regulation of maturation markers of RAW264.7 cells. The expression of the markers CD40 (e), CD86 (f), and CD80 (g) were assessed by the mean fluorescence intensity (MFI) using flow cytometry. (h, i) Effect of R848 and R848@NMCs on cellular uptake of OVA-FITC by RAW264.7 cells. RAW 264.7 cells were pre-incubated with PBS (no adjuvant), R848 and R848@NMCs released from MNs for 24 h, followed by addition of OVA-FITC (green) and incubation of the cells for 4 h prior to the indicated analysis. (h) Cellular uptake of OVA-FITC with RAW264.7 cells after stimulation of R848 and R848@NMC and observation by confocal microscopy. Cell nuclei (blue) were dyed with DAPI. Scale bars = 5 µm. (i) Dose-dependent effect of R848 and R848@NMCs on cellular uptake of OVA-FITC by RAW264.7 cells, as determined by flow cytometry. Data represent the mean ± SD of triplicate experiments. (*P

Enhanced Cancer Vaccination by In Situ Nanomicelle-Generating

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