Solid-in-Oil Peptide Nanocarriers for Transcutaneous Cancer Vaccine

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Solid-in-Oil Peptide Nanocarriers for Transcutaneous Cancer Vaccine Delivery against Melanoma Rie Wakabayashi, Masato Sakuragi, Shuto Kozaka, Yoshiro Tahara, Noriho Kamiya, and Masahiro Goto Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00894 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Molecular Pharmaceutics

Solid-in-Oil Peptide Nanocarriers for Transcutaneous Cancer Vaccine Delivery against Melanoma Rie Wakabayashi†‡¶, Masato Sakuragi†¶, Shuto Kozaka†, Yoshiro Tahara†, Noriho Kamiya†‡§, and Masahiro Goto*†‡§ †

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University,

Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan ‡

Advanced Transdermal Drug Delivery System Center, Kyushu University, Motooka 744,

Nishi-ku, Fukuoka 819-0395, Japan §

Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395,

Japan *Corresponding Author Address: 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Tel: +81 92-802-2806, Fax: +81 92-802-2810. E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT.

Cancer vaccines represent a prophylactic or therapeutic method of suppressing cancer by activating the adaptive immune system. The immune response is initiated by the delivery of tumor antigens to antigen presenting cells (APCs). The use of peptides, as vaccine antigens, is advantageous especially in the availability and productivity of pure and defined antigens. However, their limited immunogenicity remains a major drawback and therefore the utilization of nanocarriers as a means of delivering antigens to target cells and/or the addition of immune stimulants have been investigated as an efficient peptide-based cancer vaccine. We have developed a solid-in-oil (S/O) nanodispersion as a transcutaneous nanocarrier for hydrophilic molecules. This system has attractive features as a peptide nanocarrier for cancer vaccines, including transcutaneous targeting of professional APCs in the skin, high encapsulation efficacy of hydrophilic molecules, and capacity for co-loading with a variety of immune stimulants such as adjuvants. We therefore sought to utilize the developed S/O nanodispersion for the delivery of the tyrosine-related protein 2 peptide, TRP-2180-188, as a peptide antigen against melanoma. Transcutaneous vaccination of the S/O nanodispersion co-loaded with adjuvant R-848 was associated with a significant inhibition of melanoma growth and suppression of lung metastasis in tumor-bearing mice. Our findings indicate the potential of S/O nanodispersions as an endogenous peptide carrier for cancer vaccines.

KEYWORDS. Cancer vaccine; transcutaneous immunization; solid-in-oil; melanoma; TRP-2 peptide; emulsion.


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INTRODUCTION Cancer vaccines represent the prophylactic or therapeutic suppression of cancer by delivering tumor antigens to antigen presenting cells (APCs) to activate the host immune system. Compared with other methodologies for cancer therapy, cancer vaccines employing the adaptive immune system is believed to be associated with prolonged effectiveness, low risk of adverse effects, and suppression of metastasis and relapse. The use of peptides as vaccine antigens, as compared with other forms of antigens such as whole proteins, offers several advantages, including the availability of fully synthetic, pure epitope peptides without deleterious sequences, the potential for large-scale production, and easy modification of the peptides.1-3 However, peptide-based vaccines are limited by their immunization efficacy and therefore require the use of a delivery system and/or additional immuno-stimulators, such as adjuvants. To date, various nanocarriers for antigen delivery have been developed, including polymer-based nanoparticles and nanogels,46

lipid-based nanoparticles,7-9 and emulsions.10 The use of delivery carriers can prolong the

retention of antigens at the delivery site, thus enabling a longer exposure to APCs. The vaccine administration route also requires consideration. Dendritic cells (DCs) are known as professional APCs because of their capacity for efficient antigen presentation and activation of T cells.11 Given that several types of DCs, including Langerhans cells (LCs) and dermal DCs, are found in the skin, the transcutaneous administration of vaccines represents a promising method of delivery.12-13 The major obstacle to transcutaneous vaccine delivery, however, is the barrier function of the skin. The outermost layer of the skin, the stratum corneum (SC), functions as a hydrophobic barrier and disturbs the permeation of large hydrophilic molecules.14-15 To overcome this issue, we developed a solid-in-oil (S/O) nanodispersion16-18 comprising an oil dispersion of a surfactant-drug complex, formed by coating hydrophilic drugs with hydrophobic


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surfactants. S/O nanodispersions have previously been prepared with high encapsulation efficacy using small molecular drugs as well as proteins and oligonucleotides.18-19 In addition, hydrophobic molecules can be added to the outer oil phase.20 These features of S/O nanodispersions are advantageous for vaccine formulations because a variety of adjuvants can be co-loaded with the hydrophilic antigen. As a platform for a cancer vaccine, we previously developed an S/O nanodispersion loaded with a model cancer antigen, ovalbumin (OVA).21 Mice vaccinated with OVA-S/O were challenged with E.G7-OVA thymoma cells expressing OVA as a surrogate tumor antigen, and significant inhibition of tumor growth in the vaccinated mice was observed, indicating that S/O nanocarrier is a promising transcutaneous antigen carrier for cancer vaccines. However, OVA is a highly immunogenic foreign antigen, while tumor-associated antigens are typically selfantigens. Induction of immune system against those self-antigens remains challenging because of self-tolerance mechanisms and therefore less immunogenic nature of self-antigens. In light of the current situation, in this study, we aimed to develop a nanocarrier to deliver endogenous cancer antigens transcutaneously. We applied our S/O system to an antigen against melanoma, the tyrosine-related protein 2 peptide, TRP-2180-188. TRP-2180-188 is a differentiation antigen and an MHC class I-binding motif within the TRP-2 protein, thus enabling its use as a cancer vaccine.22 We determined the optimal S/O formulation for efficient TRP-2 peptide delivery into the epidermal and dermal layers of the skin. We also evaluated resiquimod R-848 as an immune adjuvant. R-848 is an agonist of Toll-like receptor (TLR) 7/8, which induces cellular immunity.23-24 Since R-848 is a small, hydrophobic molecule, it was added to the outer oil phase of the S/O nanodispersion and co-delivered with the TRP-2 peptide to enhance the vaccine efficacy.


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EXPERIMENTAL SECTION Materials. H-Leu-Trt (2-Cl) -Resin, Fmoc-Trp(Boc)-OH, Fmoc-Val, Fmoc-Phe-OH, FmocAsp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)OH, Fmoc-β-Ala-OH, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium, 1hydroxybenzotriazole monohydrate, dichloromethane, N,N-dimethylformamide (DMF), diisopropylethylamine, 20 % (v/v) piperidine/DMF, and trifluoroacetic acid were obtained from Watanabe Chemical Industries (Hiroshima, Japan). Triisopropyl silane, acetonitrile, ethanol, and sodium dodecyl sulfate (SDS) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Methanol, diethyl ether, and cyclohexane were obtained from Kishida Chemical Co. (Osaka, Japan). Isopropyl myristate (IPM) was purchased from Wako Pure Chemical Industries (Kyoto, Japan) and Sucrose laurate (L-195) and sucrose erucate (ER-290) were kindly provided by Mitsubishi-Kagaku Foods (Tokyo, Japan). Cell culture. The mouse melanoma B16 F10 cell line was purchased from the RIKEN cell bank (Tsukuba, Japan). Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), antibiotic-antimycotic (Thermo Fisher Scientific), and 0.05 mM 2-mercaptoethanol (Wako) at 37°C under 5% CO2 atmosphere. Animals. Female C57BL/6N mice (5–6 weeks old) were obtained from Kyudo, Co. (Saga, Japan) and they were maintained under standardized conditions. All animal experiments were carried out with the authorization of the Ethics Committee for Animal Experiments of Kyushu University (approval no. A26-267-2, A28-273-0) and in accordance with the Guide for the Care and Use of Laboratory Animals (Science Council of Japan).


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Preparation and physical properties of S/O nanodispersion carrying melanoma antigen peptide. Melanoma antigen peptides with and without FITC labeling, FITC-β-AlaKKKGSVYDFFVWL (FITC-K-TRP-2) and KKKGSVYDFFVWL (K-TRP-2), respectively, were synthesized by a standard N-α-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis method. The obtained peptides were analyzed by HPLC (Inertsil ODS-3 column, GL science) and MALDI TOF MS (Autoflex-III, Bruker). K-TRP-2 or FITC-K-TRP-2 aqueous solution (0.5 mg/mL) and L-195 or ER-290 cyclohexane solution (12.5 mg/mL) were added in a vial (aqueous:cyclohexane phase = 1:2, v/v) and homogenized using a Polytron homogenizer PT2500E (Kinematica AG, Lucerne, Switzerland) at 26,000 rpm for 2 min. The resultant waterin-oil (W/O) emulsions were frozen rapidly in liquid nitrogen, followed by lyophilization overnight to yield a peptide-surfactant waxy complex. Addition of IPM into the complex successfully yielded S/O nanodispersions. Particle size was measured by a dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, Worcestershire, UK.). S/O nanodispersions in IPM ([K-TRP-2] = 0.01 mg/mL) were used for the measurement. The refractive index values for IPM, L-195 S/O, and ER-290 S/O are 1.43, 1.46, and 1.44, respectively, which were experimentally determined using a refractometer RA-500 (KEM, Kyoto, Japan). Morphological analysis was performed by scanning electron microscopy (SEM) using a Helios NanoLab 600i system (FEI, Hillsboro, OR, USA) and transmission electron microscopy (TEM) using a JEM-2010 (JEOL, Tokyo, Japan). Briefly, S/O particles dispersed in cyclohexane (1 mg/mL) were drop-cast onto a STEM grid with an elastic carbon film (Okenshoji Co., Tokyo, Japan), washed twice with cyclohexane, and dried. For SEM, the grid was sputter-coated with platinum and imaged at an acceleration voltage of 2 kV. For


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TEM, the specimen was stained with 2% uranyl acetate, dried in vacuo, and imaged at an accelerating voltage of 120 kV. Peptide encapsulation efficiency in S/O nanodispersions. The encapsulation efficiency of antigen peptides in S/O nanodispersions was evaluated as previously reported.25 Briefly, 0.5 mL of ice-cold water was added to freeze-dried peptide-surfactant complex containing 1 mg peptide. The mixture was shaken for 10 min and cooled on ice for 5 min. After repeating these steps three times, the mixture was centrifuged (4°C, 18,000 g, 10 min) and the peptide concentration in the supernatant solution was analyzed by HPLC (Inertsil ODS-3 column, 30%–50% acetonitrile in water containing 0.1% TFA). The encapsulation efficiency was calculated using the following equation: Encapsulation efficiency [%] = ([Antigen]feed − [Antigen]supernatant) / ([Antigen]feed × 100)

Peptide release kinetics from S/O nanodispersions. Peptide release from S/O nanodispersions was examined using a Franz diffusion cell with a diffusion area of 0.785 cm2. Polycarbonate membrane (Avanti Polar Lipids, Inc., Alabaster, AL, USA, 0.1 µm pore size) was placed between the receiver and donor compartments. The receiver phase was filled with 5 mL of PBS containing 1% SDS. S/O nanodispersions carrying 1 mg/mL FITC-K-TRP-2 (0.2 mL) were added to the donor phase, which was maintained at 37°C with a gentle stirring. Samples were collected from the receiver phase at 3, 6, 12, 24, and 48 h and FITC-K-TRP-2 release was evaluated by photoluminescence measurement using a Perkin Elmer LS55 spectrometer (λex = 488 nm). In vitro skin permeation study. The back skin of female C57BL/6N mice was used for an in vitro skin permeation study. Hair was removed using a shaver a day before the skin collection


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and the skin segments were collected and stored at –80°C until use. For permeation studies, skin segments were thawed at room temperature and placed on a Franz diffusion cell, with PBS in the receiver phase. The donor compartment was filled with 0.2 mL of S/O nanodispersion or PBS solution containing 1 mg/mL FITC-K-TRP-2. The skin permeation study was carried out at 32.5°C for 4 h. For the quantification of FITC-K-TRP-2 in the skin, skin segments were washed several times with ethanol and PBS and cut into pieces. The skin pieces were immersed in 500 µL of extraction buffer (PBS:acetonitrile:methanol = 2:1:1, v/v/v) and the extraction was performed at room temperature with vigorous shaking. After 24 h, the supernatant was filtered through a 0.2 µm syringe filter. The concentration of FITC-K-TRP-2 in the extraction buffer was calculated by photoluminescence (Perkin Elmer LS55, λex = 488 nm, λem = 522 nm). For fluorescent imaging, skin segments were washed with ethanol and PBS after the permeation study and immobilized in 4% paraformaldehyde for 6 h. The skin segments were then embedded in O.C.T. compound (Sakura Finetek Japan, Tokyo) and frozen in liquid nitrogen. Sections of 20 µm thickness were parepared with a Leica CM1860UV cryostat (Leica Biosystems, Wetzlar, Germany) and imaged on a Keyence BZ-9000 fluorescence microscope (Keyence Co., Osaka, Japan) with a GFP filter set (EX 470/40, DM 495, BA 525/50). In vitro skin permeation route. Ear auricles of C57BL/6N mice were harvested and stored at −80°C until use. The ear auricles were thawed at room temperature, and 10 µL of S/O nanodispersion encapsulating 1 mg/mL FITC-K-TRP-2 was mounted on the skin. After 1 h, the ear auricles were cut into pieces and mounted on a glass slide using Entellan® (Merck,


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Darmstadt, Germany). The skin permeation route was imaged using a Zeiss LSM700 microscope (Carl Zeiss, Oberkochen, Germany) with a diode laser (488 nm). Vaccination and evaluation of antitumor efficacy. C57BL/6N mice were vaccinated using a home-made patch composed of tissue paper (ca. 0.5 cm × 1.0 cm) and medical adhesive tape. The tissue paper was impregnated with 25 µL of sample: S/O nanodispersion containing K-TRP2 with or without R-848 and R-848 in IPM without K-TRP-2. The mice were transcutaneously vaccinated with 0.2 mg antigen/mouse by placing the patches on both ear auricles for 24 h. As a control, K-TRP-2 PBS solution (2 mg/mL, 100 µL) was subcutaneously injected at the base of the earlobe. Mice were immunized twice with an interval of 1 week. Seven days after the second immunization, B16 F10 cells (1.0 × 106 cells/mouse, 100 µL in HBSS) were subcutaneously inoculated into the backs of the mice. Tumor size was measured every second day, starting on day 5 after inoculation. Tumor volume was calculated according to the following equation: Tumor volume [mm3] = (Major axis [mm]) × (Minor axis [mm])2 × 0.5 Metastasis inhibition efficacy. C57BL/6N mice were immunized as described above. Seven days after the second immunization, B16 F10 cells (2.0 × 105 cells/mouse, 200 µL in HBSS) were inoculated intravenously. After 20 days, lungs were harvested and immobilized in 4% paraformaldehyde for 6 h, and the number of tumor nodules was manually counted. Statistical analysis. Statistical analysis was performed using GraphPad Prism version 6 software (GraphPad Software, Inc., La Jolla, CA). Statistical significance was evaluated by one-


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way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons.

RESULTS AND DISCUSSION Preparation of S/O nanodispersion carrying melanoma antigen peptide K-TRP-2. In this study, we used the K-TRP-2 peptide, melanoma MHC class I-binding peptide TRP-2180-188 modified with three lysine residues (KKKGSVYDFFVWL: underlined text indicates the TRP2180-188 sequence), as an encapsulating peptide (Figure 1a, Supplemental information, Figure S1), and the sucrose esters, sucrose laurate (L-195) and sucrose erucate (ER-290), as surfactants (Figure 1a). The S/O nanodispersion was prepared by the emulsification of K-TRP-2 aqueous solution and surfactant cyclohexane solution to form a W/O emulsion, the simultaneous removal of water and cyclohexane by freeze-drying, and the dispersion of the K-TRP-2-surfactant complex in an oil phase (Figure 1b). IPM was employed as an oil vehicle to enhance skin permeation. Sucrose fatty acid esters and IPM are widely used in cosmetics and pharmaceuticals products and therefore S/O nanodispersions are safe formulations for transcutaneous vaccine application. Moreover, this emulsion-based preparation scheme, without a specific interaction between encapsulating drugs and surfactants, is advantageous in that it enables a highly efficient encapsulation of variety of hydrophilic drugs. Nanosized particle formation was confirmed for both S/O dispersions by size distribution analysis using DLS and morphological investigation using SEM and TEM (Figure 2, Supplemental information, Figure S2). The formation of spherical objects was clearly seen in SEM images. The average particle diameters were 68.6 nm and 61.8 nm for L-195 S/O and ER-290 S/O, respectively (Table 1). The slightly smaller size of


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particles produced using ER-290 may be explained by the smaller size of the W/O emulsion droplets, prepared using surfactants with longer alkyl length (C12 for L-195 and C22 for ER290).26

Figure 1. (a) Chemical structures of K-TRP-2, surfactants, and immune adjuvant R-848 used in this study and (b) preparation of S/O nanodispersion carrying melanoma antigen peptide K-TRP2.


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Figure 2. (a) Physical appearance of K-TRP-2 in isopropyl myristate. Left, free; middle, L-195 S/O; right, ER-290 S/O. (b, c) SEM image of L-195 S/O (b) and ER-290 S/O (c) (bars: 200 nm). (d) Dynamic light scattering profiles of L-195 S/O and ER-290 S/O.

Peptide encapsulation efficiency was examined by extracting free peptide from the peptidesurfactant complex in water. An almost quantitative encapsulation was observed for both L-195 and ER-290 S/O, and the efficiency was much higher than that observed for polymer nanoparticle-based carriers of TRP-2 peptide (Table 1).27-28 Additionally, L-195 S/O nanodispersion incubated at 40°C in an oil phase showed >90% encapsulation of K-TRP-2 peptide after 2 weeks (Supplemental information, Figure S3). S/O system encapsulation efficiency has previously been shown to be quantitative for both small molecular drugs and


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proteins.25, 29 In the S/O system, the drug-surfactant complex is prepared by emulsification followed by freeze-drying. Because hydrophilic drug in the aqueous solution is enforced to be surrounded by the surfactants, the encapsulation of hydrophilic drugs shows quantitative.

Table 1. Size and encapsulation efficiency of S/O nanodispersions. n = 3, mean ± SE.

Z-average [nm]

PDI

Encapsulation efficiency [%]

L-195 S/O

68.6±10.1

0.14–0.37

99.5±0.1

ER-290 S/O

61.8±4.5

0.11–0.24

>99.9

In vitro skin permeation of FITC-K-TRP-2 peptide by S/O nanodispersion. Next, in vitro skin permeation was evaluated using a FITC-labeled peptide, FITC-K-TRP-2. An S/O nanodispersion or PBS solution of FITC-K-TRP-2 peptide was applied to segments of back skin from C57BL/6N mice, set in a Flanz diffusion cell. As shown in Figure 3a, FITC-K-TRP-2 peptide was localized mostly to the skin surface when PBS solution was applied, and only a slight increase in fluorescence intensity in the SC was observed with ER-290 S/O. In contrast, with L-195 S/O, intense signal was detected in the SC and the underlying epidermis and dermis layers. Image analysis was conducted by plotting the fluorescence intensities against the distance from the skin surface (Supplemental information, Figure S4). L-195 clearly enhanced the permeation of FITC-K-TRP-2 peptide into the deeper region of the skin. The total amount of peptide permeated to the skin was determined by extracting the peptide from the whole skin. L-


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195 S/O showed the highest permeation, with a more than 2-fold and almost 4-fold increase from ER-290 S/O and PBS solution, respectively (Figure 3b).

a

b Fluorescent image

Merge ***

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FITC-K-TRP-2 concentration in the whole skin [µg/cm2]

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PBS

ER-290 S/O

L-195 S/O

**

6 5 4 3 2 1 0

PBS

ER-290 S/O L-195 S/O

Figure 3. (a) Skin sectioning fluorescence microscopy images of FITC-K-TRP-2 permeated into mice skin and (b) cumulative amount of FITC-K-TRP-2 in the whole skin. Bars: 100 µm. n = 3, mean ± SE. **p < 0.01, *** p < 0.001.

To investigate the skin permeation mechanism, we examined the release of FITC-K-TRP-2 from S/O nanodispersions under hydrophilic conditions. Figure 4 shows the cumulative release of FITC-K-TRP-2 peptide from S/O nanodispersions to an aqueous PBS solution. Sustained release was observed for both the L-195 and ER-290 S/O nanodispersions. However, the release rate was notably higher with L-195 S/O than with ER-290 S/O, releasing approximately 72% and 26% for L-195 S/O and ER-290 S/O, respectively, at 48 h. These results clearly indicate that L-


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195 S/O nanodispersion is stable in the oil phase but becomes unstable on contact with water, enabling the release of the encapsulated peptide. The water content of the skin increases as the depth increases.30-31 Because of their hydrophobic nature, S/O nanodispersions are largely distributed in the SC region of the skin (Figure 3a). Our previous study showed that S/O nanoparticles and hydrophobic surfactants remained in the SC region, the hydrophobic barrier of the skin, and that only released drug could permeate into the layers below the SC.16, 32 In other words, peptide release is crucial for skin permeation in the S/O system. The gradual release of encapsulated peptides allows their permeation into the deeper regions of the skin, where the SC functions as a reservoir to enable their prolonged absorption.

100

Cumulative release / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


***

80

***

60

** 40

20

* ****

0 0

10

20

30

40

50

Time / h

Figure 4. FITC-K-TRP-2 release from S/O nanodispersion to aqueous solution. Solid line with filled circle: L-195 S/O; dashed line with open circle: ER-290 S/O. n = 3, mean ± SE. *p < 0.05, **

p < 0.01, *** p < 0.001.


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The FITC-K-TRP-2 peptide skin permeation route was further investigated using the ear skin of mice. L-195 S/O carrying FITC-K-TRP-2 peptide was applied to the skin for 1 h and fluorescence imaging was performed. Green fluorescence from FITC-K-TRP-2 was observed as hexagonal shapes, indicating intercellular regions of corneocytes (Supplemental information, Figure S5). The oil vehicle, IPM, is a skin penetration enhancer with a high affinity for intercellular lipids in the SC region, such as ceramides, free fatty acids, and cholesterols.33 Therefore, the increased fluidity of intercellular lipids following treatment with IPM allowed the intracellular permeation of S/O nanoparticles.

In vivo inhibition of melanoma growth by transcutaneous immunization. Prior to in vivo vaccination, the addition of adjuvant as an immune-stimulator was examined. TLR ligands are among the most effective adjuvants used in cancer vaccines, with resiquimod R-848, a TLR7/8 agonist, reported to induce a Th1 type immunoresponse.24, 34-36 In addition, the hydrophobic nature and small molecular size of R-848 facilitate transcutaneous administration.20, 37-38 In the present study, R-848 was added to the outer oil phase of the S/O formulation. The addition of R848 had a negligible influence on the particle size distribution of the S/O nanodispersion (Supplementary Figure S6). Neither skin permeation nor the release of encapsulated FITC-KTRP-2 were significantly different between the S/O nanodispersions with and without R-848 (Supplementary Figure S7), indicating that small hydrophobic adjuvants such as R-848 can be added to the continuous phase of S/O nanodispersions without a marked influence on their physicochemical properties.


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C57BL/6N mice were vaccinated twice by transcutaneous administration of S/O nanodispersions with and without R-848 at an interval of 1 week. The transcutaneous administration of R-848 without K-TRP-2 peptide and the subcutaneous injection of K-TRP-2 PBS solution were used as controls. Immunized mice were inoculated with murine melanoma B16 F10 cells 7 days after the second vaccination. In all immunized groups, tumor growth was inhibited compared with the no treatment group (Figure 5a). Inhibition efficiency in the S/O group was comparable to the injection and R-848 without peptide groups. Distribution of immune cells in harvested tumors were observed using fluorescence microscope. In the S/O group, the infiltration of cytotoxic T lymphocytes was observed, suggesting the induction of T cell response by transcutaneous administration of S/O nanodispersions carrying K-TRP-2 peptide (Supplementary Figure S8). A significant decrease in tumor growth rate was observed in mice vaccinated with S/O containing R-848. Importantly, three of the five mice in the group rejected the tumor implantation until day 31, while in the other mice, tumor started to grow on day 15 at the latest (Supplementary Figure S9). As a result, these three mice survived more than 30 days after tumor cell inoculation (Figure 5b).

a

b 1600

No treat

1400

Injection

1200

R-848

No treat R-848 L-195 S/O (+R-848)

L-195 S/O 1000

L-195 S/O (+R-848)

800

**

600 400 200

Survival rate [%]

Tumor volume [mm3]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Injection L-195 S/O

100 80 60 40 20 0

0 0

5

10

15

0

Days

5

10

15

20

25

30

Days


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Figure 5. Preventive anti-cancer immunity in C57BL/6N mice vaccinated with S/O nanodispersions loaded with K-TRP-2 peptide. Mice were vaccinated on days −14 and −7, and B16 F10 cancer cells were inoculated on day 0. Tumor growth (a) and survival rate (b) of tumorbearing mice. n = 5, mean ± SE, **p < 0.01.

Skin permeation of K-TRP-2 peptide using patches was evaluated by extracting the peptide from mouse ear skin 24 h after administration. An average of 19 µg of peptide was detected in the skin following the administration of S/O formulation (Supplementary Figure S10). Given that a dose of 200 µg was applied, approximately 10% of the peptide was delivered to the skin. Although the amount of antigen peptide internalized following the topical application of S/O is markedly lower than by injection, whereby 100% of peptide is theoretically delivered to the body, the suppression of tumor growth was comparable between the two delivery routes (Figure 5). We assume these results to arise from the sustained action of peptides on LCs and dermal DCs in the epidermal and dermal layers of the skin.39 In essence, the subcutaneous injection of free antigen peptide allows rapid diffusion of the peptide, while the slow release of peptides from S/O nanoparticles in the SC enables prolonged contact between the peptides and the APCs resident under the SC layer. To confirm the uptake of antigen peptide by APCs, sectioning images of the skin and lymph node after administration of S/O nanodispersions were obtained after staining with PE-conjugated anti-mouse CD207 (Supplementary Figures S11 and S12). Merged fluorescence of PE and Cy5 was observed both in the skin and lymph node, suggesting antigen peptides were internalized in LCs and the LCs migrated to lymph node.


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Inhibition of melanoma metastasis in vivo. B16 F10 melanoma cells are highly metastatic and therefore a good candidate for studying the inhibition of metastasis. Following intravenous transplantation, B16 F10 cells form metastases in lung. C57BL/6N mice were vaccinated as described above and B16 F10 cells were subsequently transplanted intravenously. Twenty days after transplantation, lungs were harvested and metastasis was evaluated by the number of nodules observed by visual inspection. Figure 6a shows black nodules in the lungs of mice, corresponding to B16 F10 metastatic tumors. The average number of nodules per lung was approximately 30 for unvaccinated mice. The number of nodules observed in mice vaccinated with injection, L-195 S/O, or L-195 S/O + R-848 was significantly decreased, with an average of 8.5, 6.8, and 3.0 nodules, respectively (Figure 6b). These results clearly indicate that the transcutaneous vaccination of K-TRP-2 using an S/O formulation system induces generalized immunity, which can inhibit metastasis.


19


a 1

2

3

4

5

b ** 40

*

Nodules/Lung

35 30 25 20 15 10 5

-8 48 ) (+

R

S/ O S/ O

L19 5

-8 48 R

n

In je ct io

o

tr ea tm

en t

0

L-

19 5

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 6. Inhibition of metastasis by vaccination with K-TRP-2 peptide. Mice were vaccinated twice at an interval of 1 week, B16 F10 cancer cells were intravenously transplanted 7 days after the second vaccination, and lungs were harvested 20 days after the transplantation. (a) Physical appearance of harvested lungs (1, no treat; 2, injection; 3, R-848; 4, L-195 S/O; 5, L-195 S/O + R-848). (b) Number of nodules formed in lungs. n = 4, mean ± SE, *p < 0.05, **p < 0.01.

CONCLUSIONS We have developed a new S/O formulation loaded with melanoma antigen peptide, K-TRP-2, as a transcutaneous cancer vaccine. S/O encapsulated K-TRP-2 peptide with high efficiency and


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enhanced the skin permeability of the peptide. Vaccinated mice were challenged with mouse melanoma B16 F10 cells, and significant inhibition of tumor growth was observed in mice vaccinated with S/O co-loaded with R-848 as a vaccine adjuvant. Moreover, marked suppression of lung metastasis was achieved. This study indicates the potential of our S/O nanodispersion system as an endogenous antigen peptide carrier for a transcutaneous cancer vaccine. Further investigation of the immunization mechanism will provide a deeper understanding of this novel system.

ACKNOWLEDGMENT The authors thank Professors Y. Katayama, T. Mori, and T. Nidome for their support for the animal experiments, Mr. Kaku for assistance with SEM measurement, and the Analysis Center of Fukuoka Industry-Academia Symphonicity for facility support. Funding Sources Authors R. W., N. K, and M. G. received funding from Grant-in-Aid for Scientific Research (S) 16H06369 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Supporting Information. The following files are freely available: Characterization of K-TRP-2 peptide, morphological analysis of S/O by TEM, stability of S/O nanodispersions, skin permeation profile and route of FITC-K-TRP-2 peptide, characterization of L-195 S/O co-loading R-848, distribution of CD8+ cells in tumors, tumor growth profiles of


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individual mice, quantity of K-TRP-2 peptide delivered into the skin by using patches, transcutaneous delivery of K-TRP-2 peptide to Langerhans cells (file type, PDF). AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval.¶These authors contributed equally. REFERENCES 1.

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TABLE OF CONTENTS/ABSTRACT GRAPHIC

A solid-in-oil (S/O) nanodispersion co-loaded with antigen peptide against melanoma and an immune adjuvant was used for transcutaneous vaccine, leading to a significant suppression of tumor growth in the mice.


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Solid-in-Oil Peptide Nanocarriers for Transcutaneous Cancer Vaccine

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