Transcutaneous co-delivery of tumor antigen and resiquimod in solid

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Transcutaneous co-delivery of tumor antigen and resiquimod in solid-in-oil nanodispersions promotes anti-tumor immunity Rie Wakabayashi, Hidetoshi Kono, Shuto Kozaka, Yoshiro Tahara, Noriho Kamiya, and Masahiro Goto ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00260 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Biomaterials Science & Engineering

Transcutaneous co-delivery of tumor antigen and resiquimod in solid-in-oil nanodispersions promotes anti-tumor immunity Rie Wakabayashi,[a,c] Hidetoshi Kono,[a] Shuto Kozaka,[a] Yoshiro Tahara,[a] Noriho Kamiya,[a,b,c] and Masahiro Goto*[a,b,c] AUTHOR INFORMATION Corresponding Author *TEL:

+81

92

802

2806.

FAX:

+81

92

802

2810.

E-mail:

m-

School

of

[email protected].

[a]

Department

of

Applied

Chemistry,

Graduate

Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. [b] Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 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

Page 2 of 44

[c] Center for Advanced Transdermal Drug Delivery System Center, Kyushu

University,

744

Motooka,

Nishi-ku,

Fukuoka

819-0395,

Japan.


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ABSTRACT

Cancer

vaccines

aim

to

prevent

or

inhibit

tumor

growth

by

inducing an immune response to tumor-associated antigens (TAAs) encoded

by

or

present

in

the

vaccine.

Previous

work

has

demonstrated that effective anti-tumor immunity can be induced using

a

co-delivery

system

in

which

nonspecific

immunostimulatory molecules are administered together with TAAs. In

this

study,

we

investigated

the

anti-tumor

effects

of

a

solid-in-oil (S/O) nanodispersion system containing a model TAA, ovalbumin (OVA), and resiquimod (R-848), a small molecular Tolllike receptor 7/8 ligand, which induces an antigen-nonspecific cellular immune response that is crucial for the efficacy of cancer vaccines. R-848 was contained in the outer oil phase of S/O

nanodispersion.

mouse

skin

indicated

after that

Analysis

of

application

R-848

rapidly

OVA

of

an

and

R-848

R-848

permeated

S/O the

permeation

in

nanodispersion skin

and

pre-

activated Langerhans cells, resulting in efficient uptake of OVA and migration of antigen-loaded Langerhans cells to the draining lymph nodes. Transcutaneous immunization of mice with an R-848 S/O nanodispersion inhibited the growth of E.G7-OVA tumors and prolonged

mouse

survival

to

a

greater

extent

than

did

immunization with an S/O nanodispersion containing OVA alone. Consistent with this observation, antigen-specific secretion of


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Page 4 of 44

the Th1 cytokine interferon-γ and cytolytic activity were both high in splenocytes isolated from mice immunized with R-848 S/O. Our

results

thus

demonstrate

that

co-delivery

of

R-848

significantly amplified the anti-tumor immune response induced by antigen-containing S/O nanodispersions, and further suggest that S/O nanodispersions may be effective formulations for codelivery of TAAs and R-848 in transcutaneous cancer vaccines.

KEYWORDS Transcutaneous vaccine, cancer immunotherapy, emulsion, solidin-oil, resiquimod.


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1. INTRODUCTION Cancer

vaccines

are

designed

to

deliver

tumor-associated

antigens (TAAs) that can induce a tumor-specific adaptive immune response,

leading

cancer.1-2

Dendritic

presenting

cells

to

suppression cells

that

(DCs)

potently

and/or are

eradication

professional

activate

naïve

T

of

the

antigencells

and

promote their differentiation into mature T cells with effector and memory properties. In general, peptide antigens presented by major histocompatibility complex (MHC) class II molecules induce the differentiation of naïve CD4+ T cells into T helper (Th) cells, whereas antigen-MHC class I complexes induce CD8+ T cell differentiation into cytotoxic T lymphocytes (CTLs). The antitumor activity of TAA-specific CTLs is considered to be a key player which

in

cancer

TAAs

are

processed

for

immunotherapy. taken

up

presentation

by onto

Therefore,

cross-priming,

antigen-presenting MHC

class

I

cells

molecules

in and

to

T

cells, is crucial for inducing an effective anti-tumor immune response. One powerful strategy to boost the efficacy of cancer vaccine is to co-deliver TAAs with additional immunostimulatory factors, such

as Toll-like

receptor

(TLR) ligands3, that activate the

immune system in an antigen-nonspecific manner. In this regard, vaccines have been developed that combine TAAs with endosomal TLR ligands, such as CpG oligodeoxynucleotides, using carriers


5


such

as

nanoparticles,4-5

polymer

Page 6 of 44

nanocages,6

protein

self-

assembling peptides,7 lipid nanomaterials,8-9 and inorganic nano/micro-particles.10-12 In this strategy, co-administration of the TLR ligand and TAA per se is not the crucial factor; rather, it is their co-delivery in a form capable of being taken up by the same DCs that is crucial for the induction of a strong adaptive response,6,

immune

nonspecific

9

in

activation

of

part the

because immune

it

system

avoids

aberrant

by

ligands,

TLR

which can lead to autoimmune disease.13 Local administration of TLR ligands at the site of TAA injection is another strategy to control induction of the immune response. Candidates for this strategy

include

imidazoquinoline

ligands

of

TLR7/8,

such

as

imiquimod and resiquimod (R-848), which are small hydrophobic molecules and are amenable to topical delivery using, e.g. gel formulations.14 Transcutaneous (t.c.) co-delivery of TAA and a TLR ligand is a promising approach to cancer immunotherapy, particularly because t.c.

vaccination

is

a

safe,

noninvasive,

and

cost-effective

alternative to conventional subcutaneous (s.c.) or intramuscular injections.15-16 However, the barrier properties of the skin are a major

hindrance

in

the

development

of

t.c.

vaccines;

in

particular, the highly hydrophobic nature of the outermost layer of

the

skin,

stratum

corneum

(SC),

inhibits

permeation

of

proteinaceous TAAs to layers containing Langerhans cells (LCs),


6

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the most abundant DCs in the skin.17-18 To overcome this issue, we have previously described the development of nanodispersions of antigens

in

an

oil

nanodispersions.19-20 proteins/peptides promote

vehicle,

S/O

solid-in-oil

nanodispersions

surrounded

efficient

termed

by

permeation

hydrophobic

of

the

are

(S/O)

composed

surfactants

encapsulated

of that

antigens

through the skin and thus facilitate antigen delivery to LCs. Construction

of

S/O

nanodispersions

is

flexible,

and

various

molecules can be co-delivered by co-encapsulation of hydrophilic molecules hydrophobic

within

particles21

the

molecules

to

outer

and/or oil

by

addition

vehicle.22

Thus,

of S/O

nanodispersions represent an excellent candidate formulation to co-deliver combinations of TAAs and TLR ligands. Our previous study showed that an S/O nanodispersion carrying a model antigen, ovalbumin (OVA), could induce an adaptive immune response against OVA-expressing E.G7-OVA cancer cells in mice, thereby

preventing

tumor

growth.23

We

also

showed

that

co-

delivery of TAAs with R-848 enhanced the suppression of melanoma growth compared with S/O nanodispersions containing TAA alone.24 However, the mechanism by which co-delivery with R-848 amplified the potency of TAA-S/O nanodispersions was not investigated in detail. Here, we prepared S/O nanodispersions encapsulating OVA with or without R-848 in the outer oil phase. Using in vitro and in vivo techniques, we investigated the effect of co-delivery of


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Page 8 of 44

OVA and R-848 on the efficiency of t.c. delivery, activation of LCs, CTLs, and Th cells, and suppression of tumor growth in mice. Our results indicate that co-delivery of R-848 not only enhances augments

the

efficiency

the

of

induction

t.c.

of

antigen

an

delivery

antigen-specific

but

also

anti-tumor

cellular immune response.

2. MATERIALS AND METHODS 2-1. Materials OVA,

copper(II)

sulfate

5(6)-carboxyfluorescein were

purchased

Cyclohexane,

from

sodium

solution,

diacetate

disulfate

N-succinimidyl

Sigma-Aldrich dodecyl

G418

(St.

sulfate

salt,

ester

Louis,

(SDS),

and

(CFSE)

MO,

USA).

acetonitrile,

ethanol, 2-mercaptoethanol, sodium pyruvate, phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS), and bovine serum albumin (BSA, fraction V) were purchased from Wako Pure Chemical

Industries

(Osaka,

Japan).

R-848

was

purchased

from

Enzo Life Sciences (Farmingdale, NY, USA), sucrose laurate (L195) was from Mitsubishi-Kagaku Foods (Tokyo, Japan), isopropyl myristate (IPM) was from Tokyo Kasei (Tokyo, Japan), and acetone was

from

Nacalai

Tesque

(Kyoto,

Japan).

4,4-Difluoro-5,7-

dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic FL

C5),

RPMI-1640

medium,

antibiotic-antimycotic

fetal

solution

bovine were

serum

from

acid

(BODIPY

(FBS),

Thermo

and

Fisher


8

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Scientific (Waltham, MA, USA). Hydrochloric acid solution was obtained from Kishida Chemical (Osaka, Japan). All chemicals and solvents were used as received. 2-2. Cell culture The mouse lymphoma cell lines E.G7-OVA and EL4 were obtained from American Type Culture Collection (Manassas, VA, USA). E.G7OVA cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, antibiotic-antimycotic solution, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 400 μg/mL G418. EL4 cells

were

grown

in

RPMI-1640

supplemented

with

10%

heat-

inactivated FBS and antibiotic-antimycotic solution. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. 2-3. Animals Female C57BL/6N mice (5–6 weeks old) were purchased from Kyudo (Saga,

Japan)

and

maintained

under

standard

conditions.

All

animal experiments were carried out with the authorization of the Ethics Committee for Animal Experiments of Kyushu University (approval no. A28-273-0) and in accordance with the Guide for the

Care

and

Use

of

Laboratory

Animals

(Science

Council

of

Japan). 2-4.

Preparation

and

characterization

of

solid-in-oil

(S/O)

nanodispersions OVA in water (0.5 mg/mL) and L-195 in cyclohexane (12.5 mg/mL) were mixed at a 1:2 (v:v) ratio and homogenized using a polytron


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Page 10 of 44

homogenizer (Kinematica AG, Luzern, Swiss) at 26,000 rpm for 2 min. The resultant water-in-oil emulsion was immediately frozen in liquid nitrogen and then freeze dried for 1 day to yield a white solid of OVA–L195 complex. The complex was dispersed in IPM alone to give the OVA-S/O nanodispersion or in 0.2 mg/mL R848

in

IPM

to

generate

the

OVA/R-848-containing

S/O

nanodispersion. The particle size of S/O nannodispersions was analyzed using a Zetasizer Nano ZS (Malvern, Worcestershire, UK.) with an S/O nanodispersion

containing

0.04

mg/mL.

For

analysis

of

S/O

nanoparticle morphology by scanning electron microscopy (SEM), an

OVA-S/O

nanodispersion

(1

mg/mL)

was

prepared

using

cyclohexane instead of IPM and then drop-cast on a STEM grid with an elastic carbon film (Okenshoji, Tokyo, Japan), washed with

cyclohexane,

and

dried

under

ambient

conditions.

The

specimen was then sputter-coated with platinum (MSP-1S, Vacuum Device, Ibaraki, Japan) and imaged using a Helios NanoLab 600i system (FEI, Hillsboro, OR, USA) at an acceleration voltage of 2 kV. 2-5. In vitro analysis of OVA release from S/O nanodispersions OVA release was analyzed using a Franz diffusion cell with a diffusion area of 0.785 cm2. A polycarbonate membrane (0.1 μm pore size, Avanti Polar Lipids, Alabaster, AL, USA) was placed between

the

donor

and

receiver

compartments.

The

receiver


10

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compartment was filled with 5 mL of 1% SDS in PBS and maintained at

37°C

constructed

with

gentle

using

stirring.

Cy3-labeled

OVA

S/O

nanodispersions

(prepared

using

Cy3

were mono-

reactive Dye Pack, GE Healthcare, Chicago, IL, USA), and 250 μL was

placed

in

the

donor

compartment.

At 1,

3,

6,

and 24 h

intervals, 150 μL of the receiver phase was withdrawn and fresh medium was added. The concentrations of released Cy3-OVA were determined

using

a

PerkinElmer

LS55

fluorescence

spectrophotometer with λex = 550 nm and λem = 570 nm. 2-6.

In

vitro

analysis

of

skin

permeation

of

OVA

by

S/O

nanodispersions The skin permeation of OVA by S/O nanodispersions was evaluated essentially

as

reported

previously.23

Briefly,

frozen

Yucatan

Micro Pig (YMP) skin (Charles River Laboratories Japan, Kanagawa, Japan)

was

thawed

at

room

temperature,

subcutaneous

fat

was

removed, cut into ~2  2 cm2, and placed on a Franz diffusion cell with the SC facing the donor phase. An aliquot of 250 μL of PBS solution or S/O nanodispersion of Cy3-labeled OVA was added to the donor chamber, and the receiver chamber was filled with PBS maintained at 32.5°C with gentle stirring. After 24 h, the skin was removed from the cell, washed with ethanol and PBS, minced, and soaked in PBS:acetonitrile:methanol (2:1:1, v:v:v) for 12 h to extract Cy3-OVA. The concentrations of extracted


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Page 12 of 44

Cy3-OVA were determined using a PerkinElmer LS55 with λex = 550 nm and λem = 570 nm. 2-7. In vivo analysis of skin permeation depth of OVA by S/O nanodispersions S/O nanodispersions with or without R-848 were prepared with Cy5-OVA (Cy5 mono-reactive Dye Pack, GE Healthcare) as described above using concentrations of 4 mg/mL Cy5-OVA and

0 or 0.2

mg/mL R-848. Hand-made patches composed of gauze (~0.5  1 cm2, Hakujuji, Tokyo, Japan) and Cathereep FS dressing tape (~1.5  3 cm2,

Nichiban,

Tokyo,

Japan)

were

immersed

in

25

μL

of

PBS

solution or S/O nanodispersions, placed on both ear auricles of C57BL/6N mice, and held in place by surgical tape (Kyowa, Osaka, Japan). After 24 h, the patches were removed, the mice were sacrificed, and the ear auricles were harvested and washed with ethanol

and

PBS.

The

Cutting

Temperature

skin

pieces

(O.C.T.)

were

compound

embedded

(Sakura

in

Finetek

Optimal Japan,

Tokyo, Japan), frozen in liquid nitrogen, and sectioned (20 μm thickness) with a CM1860UV cryostat (Leica Biosystems, Wetzlar, Germany). After fixing in cold acetone for 10 min, the sections were

incubated

in

4’,6-diamidino-2-phenylindole

(DAPI,

2.5

μg/mL) for 15 min at room temperature to label the nuclei. The skin

sections

were

imaged

on

a

BZ-9000

microscope

(Keyence,

Osaka, Japan) with filter sets for DAPI-B (EX 360/40, DM 400, BA


12

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460/50) and Cy5 (EX 620/60, DM 660, BA 700/75). Depth of Cy5-OVA permeation was analyzed using ImageJ software.25 2-8.

Comparison

of

skin

permeation

of

OVA

and

an

R-848

surrogate by S/O nanodispersions To compare the permeation of OVA and R-848, we performed the same mouse skin permeation analysis as described in section 2.7 using

S/O

nanodispersions

composed

of

Cy5-OVA

and

the

fluorescent dye BODIPY FL C5 (BP), which has a similar molecular weight and hydrophobicity to R-848, as a surrogate of R-848. S/O nanodispersions with and without BP were prepared and applied to mouse ear auricles for up to 24 h as described above. The skin sections were processed and imaged on a BZ-9000 microscope with filter sets for GFP-B (EX 470/40, DM 495, BA 535/50) and Cy5 (EX 620/60, DM 660, BA 700/75) for BP and Cy5-OVA, respectively, and the images were analyzed by using ImageJ software. For analysis of the permeation pathway, skin pieces were directly mounted on slides

and

imaged

on an

LSM700

microscope (Carl Zeiss) with

diode lasers (488 nm for BP, 639 nm for Cy5). 2-9. OVA uptake by LCs and migration to lymph nodes Patches were immersed in 25 μL of L-195 in IPM (L-195/IPM), R848 in IPM (R-848/IPM), Cy5-OVA in PBS (OVA/PBS), Cy5-OVA S/O (S/O), or Cy5-OVA/R-848 S/O (R-848 S/O)(4 mg/mL Cy5-OVA, 200 mg/mL L-195, and 0.2 mg/mL R-848) and then placed on both ear auricles

(50

μL/mouse)

of

C57BL/6N

mice

for

24

h.

After


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Page 14 of 44

sacrificing the mice, the ear auricles and submandibular lymph nodes

were

isolated

and

The

sections

were

above.

sections fixed

were

in

cold

prepared

as

acetone

described

for

10

min,

blocked with 2% BSA in PBS for 1 h, and then incubated with a 1:50

dilution

antibody BSA/PBS

of

(Clone at

4°C

phycoerythrin 4C7,

(PE)-labeled

BioLegend,

overnight.

San

After

Diego,

washing

anti-mouse CA,

the

USA)

CD207 in

sections

2%

were

incubated with DAPI (2.5 μg/mL) for 15 min at room temperature and then imaged on an LSM700 microscope (Carl Zeiss) with diode lasers (405 nm for DAPI, 488 nm for PE, 639 nm for Cy5). 2-10. Therapeutic immunization and suppression of tumor growth in vivo E.G7-OVA

cells

(1

×

106

cells/mouse,

100

μL

of

HBSS)

were

injected s.c. into the dorsal skin of C57BL/6N mice. On day 7 after the inoculation, the mice were randomly assigned to six groups to receive (i) no treatment (control), (ii) OVA/PBS patch (OVA aq.), (iii) R-848/IPM patch (R-848/IPM), (iv) OVA/PBS s.c. injection (injection), (v) OVA-S/O patch (S/O), or (vi) OVA/R848-S/O patch (R-848 S/O). Patches were prepared as described in section 2-9 using 4 mg/mL OVA, 200 mg/mL L-195, and 0.2 mg/mL R848 and were placed on both ear auricles (50 μL/mouse, 200 μg OVA/mouse)

for

24

h.

For

s.c.

injection,

100

μL

of

OVA/PBS

solution (2 mg/mL; 200 μg OVA/mouse) was injected at the base of earlobes. The mice were immunized in the same manner on day 14.


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Tumor sizes were measured every 2 days using a Vernier caliper, and the volumes were calculated using the following formula: Volume [mm3] = (major axis [mm]) × (minor axis [mm])2 × 0.5 Animals were sacrificed when the tumor volume exceeded 2500 mm3 or when ulceration was apparent. 2-11. Splenocyte preparation and cytokine secretion C57BL/6N mice were injected s.c. into the dorsal skin with E.G7-OVA cells in HBSS (1 × 106 cells/100 μL/mouse). Five days later, mice were randomly assigned to six groups and immunized as described in section 2-10. The immunization was repeated on day 12. On day 19, the mice were sacrificed and spleens were isolated from the mice, and splenocyte cell suspensions were prepared. Briefly, the spleens were washed in HBSS, treated with Tris-buffered ammonium chloride to lyse red blood cells, and washed. The cells were resuspended in HBSS, filtered through an EASYstrainer

(70

μm

mesh,

Greiner

Bio-one),

washed,

and

resuspended in RPMI-1640/10% FBS. The cells were added (1 × 106 cells/well) to a 12-well microplate (AGC Techno Glass, Shizuoka, Japan) containing 100 μg/mL OVA and incubated at 37°C for 72 h. The plate was then centrifuged and the supernatants were removed for

quantification

enzyme-linked

of

secreted

immunosorbent

interferon-γ

assay

(ELISA)

(IFN-γ) kit

using

an

(Ready-Set-Go!

Mouse IFN-γ kit, eBioscience) according to the manufacturer’s instructions.


15


Page 16 of 44

2-12. Measurement of serum OVA-specific IgG concentrations Blood samples were collected from the tail veins of mice on days −1 and 18 and serum was prepared. OVA-specific IgG levels were

evaluated

by

ELISA,

as

described

previously.21

The

peroxidase-conjugated secondary antibodies used were anti-mouse IgG (H&L) (rabbit polyclonal, 610-4302), anti-mouse IgG1 (rabbit polyclonal, 610-4340), and anti-mouse IgG2a (rabbit polyclonal, 640-4341), all obtained from Rockland (Limerick, PA, USA). The antibody titer was defined as the serum dilution factor at which the

optical

density

was

equal

to

that

of

the

serum

samples

obtained preimmunization on day −1. 2-13. Antigen specificity of suppression of tumor growth in vivo Naïve C57BL/6N mice were randomly assigned to three groups of 4 mice and immunized as follows on days 0 and 7: (i) no treatment (control),

(ii)

OVA/PBS

s.c.

injection

(injection),

or

(iii)

OVA/R-848-S/O patch (R-848 S/O). Concentrations and OVA dose are same

as

2-10

(200

μg

OVA/mouse).

On

day

14,

the

mice

were

injected s.c. into the left or right side of the dorsal skin with

EL4

or

E.G7-OVA

cells,

respectively,

in

HBSS

(1

×

106

cells/100 μL/mouse). Tumor sizes were measured every 2 days and the volumes were calculated as described in section 2-10. 2-14. In vitro CTL assay


16

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To generate cytotoxic effector cells, naïve C57BL/6N mice were vaccinated as described in section 2-13 (200 μg OVA/mouse). On day 14, the mice were sacrificed, and splenocytes were prepared as described in section 2-11, except that red blood cells were lysed

with ammonium-chloride-potassium buffer.

The cells

were

resuspend in RPMI-1640/10% FBS, added at 1 × 107 cells/mL/well in 24-well microplates (MS-9024X, Sumitomo Bakelite), and cultured in the presence of 10 U/mL IL-2 and 50 μg/mL OVA for 72 h. To generate target cells, EL4 or E.G7-OVA cancer cells (2 × 106 cells/mL) were incubated with 25 μM CFSE in HBSS for 15 min at 37°C,

mixed

with

centrifuged. washed

The

twice

an

equal

volume

supernatant

with

was

RPMI-1640/10%

of

RPMI-1640/10%

removed FBS.

and

The

FBS,

and

the

cells

were

cells

were

then

resuspended in the same medium and incubated at 37°C overnight. For the cytotoxicity assay, the effector cells (E) (50 μL/well, 2 × 105 cells/mL) and target cells (T) were resuspended in RPMI1640/10%

FBS

(Thermo

Fisher

and

added

to

Scientific)

96-well at

a

round-bottom

ratio

of

1:100

microplates E:T

(104:106

cells/100 μL/well). The plates were centrifuged at 1500 rpm for 2 min and the cells were incubated at 37°C for 6 h. Aliquots of RPMI-1640/10% FBS were added at 100 μL/well and the plate was centrifuged

at

1500

rpm

for

2

min.

Supernatant

samples

(50

μL/well) were removed and transferred to white 96-well half-area microplates (Greiner) and mixed with 50 μL/well of 0.5% (w/v)


17


Page 18 of 44

Triton X-100 in RPMI-1640 (designated Experimental wells). Wells containing target cells without effector cells were processed in the same manner (Target spontaneous). Target cells lysed with 1% (w/v)

Triton

X-100

served

as

the

control

for

maximum

CFSE

release (Target maximum). The final concentration of Triton X100

in

all

samples

was

0.25%

(w/v).

CFSE

present

in

the

supernatants or lysates was quantified using a Perkin Elmer LS55KG

with

λex

=

495

nm

and

λem

=

515

nm.

Cytotoxicity

was

calculated using the following formula: % Cytotoxicity = (Experimental − Target spontaneous)/(Target maximum − Target spontaneous)  100 2-15. Statistical analysis GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) was used

for

statistical

analysis.

Significance

was

evaluated

by

one-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. P < 0.05 was considered statistically significant. Data are expressed as the mean ± standard error of the mean unless specified.

3. RESULTS AND DISCUSSION 3-1. Preparation and skin permeation of S/O nanodispersions carrying OVA with or without R-848 S/O

nanodispersions

were

prepared

to

contain

a

fixed

concentration of OVA as a TAA in the presence or absence of R-


18

Page 19 of 44 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


848 in the outer IPM phase (Fig. S1). R-848 is a TLR7/8 ligand and

known

to

induce

a

cellular

immune

response.26-27

OVA

was

homogeneously dispersed in both formulations (Fig. 1A). The S/O nanoparticles were spherical in morphology (Fig. 1B) and the dispersions showed mono-modal distributions in the particle size analysis

(Fig.

1C).

The

average

particle

size

was

~100

nm

regardless of the presence of R-848 (Fig. 1C, Table 1). These results

suggest

that

the

S/O

particles

were

successfully

dispersed in the oil phase without marked aggregation.

Figure

1.

Characterization

and

skin

permeation

of

S/O

nanodispersions. (A) Physical appearance of (i) free OVA in IPM (precipitated),

(ii)

S/O

nanodispersion,

and

(iii)

R-848

S/O

nanodispersion. (B) SEM image of OVA-S/O particles. Bar: 500 nm.


19


Page 20 of 44

(C) Particle size distribution in S/O (dotted line) and R-848 S/O (solid line) nanodispersions. (D) Permeation of Cy3-OVA in YMP

skin

solution

following (OVA

24

aq.),

h

incubation

S/O

in

vitro

nanodispersion, **p

nanodispersion. N = 3, mean ± SE,

or

with

OVA/PBS

R-848

S/O

< 0.01. (E) Release of Cy3-

OVA from OVA-S/O (dotted line) and OVA/R-848-S/O (solid line) nanodispersions. N = 3, mean ± SE.

Table 1. Size analysis of S/O nanoparticles with and without R848.

Sample

Average [nm]

Diameter

S/O

98.1 ± 2.6

0.104-0.254

R-848 S/O

99.1 ± 2.3

0.063-0.167

PDI

PDI = polydispersity index. mean ± SE of n = 3.

To evaluate the ability of OVA to permeate the skin, we applied Cy3-OVA to Yucatan Micro Pig (YMP) skin in three forms: as an aqueous solution in PBS, as an S/O nanodispersion, or as an R848 S/O nanodispersion. After the 24 h of treatment, the amount of

Cy3-OVA

application

permeated of

OVA

into as

an

the S/O

skin

was

quantified.

nanodispersion

Notably,

enhanced

the

permeation of Cy3-OVA into the skin by ca. 5-fold compared with


20

Page 21 of 44 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


OVA/PBS (Fig. 1D).

This enhancement is likely due to increased

penetration

layer

of

SC

by

the

hydrophobic

nature

of

S/O

nanodispersions compared with the aqueous solution, highlighting the

importance

of

TAA

encapsulation

in

enhancing

skin

permeation. Since the water content of the skin increases at depths below the SC, the release of encapsulated drug is also crucial in the skin permeation using the S/O technique. We have previously reported that the use of the surfactant L-195, which contains C12 alkyl chains, yields S/O particles that facilitates the release of encapsulated proteins.24,

28

We confirmed that the

release of OVA from S/O nanodispersions was not affected by the addition of R-848 (Fig. 1E), suggesting OVA both in S/O and R848 S/O formulations can be released from S/O nanodispersions to permeate into the deeper region of the skin. Using fluorescence microscopy, encapsulated

we

assessed

Cy5-OVA

the

permeated

depth

to

the

which

mouse

skin

aqueous

or

sections

S/O

(Fig.

S2). This analysis demonstrated that Cy5-OVA delivered by S/O nanodispersions

was

found

at

the

deeper

region

in

the

skin

(throughout the SC layer), whereas most Cy5-OVA applied in PBS remained

at

the

skin

surface

(Fig.

S2).

Collectively,

these

results suggest that S/O nanodispersions enhance the permeation of OVA through the hydrophobic SC layer, enabling release of the encapsulated antigen at greater depth.


21


3-2.

Transcutaneous

delivery

of

OVA

to

Page 22 of 44

skin

LCs

and

their

migration to draining lymph nodes To effectively induce an immune response, t.c. vaccines must deliver

TAAs

to

antigen-presenting

cells

resident

in

the

epidermis, particularly LCs.29 The ability of S/O nanodispersions to deliver OVA to LCs was determined by applying patches soaked in

various

formulations

of

Cy5-OVA

and/or

R-848

to

the

ear

auricles of C57BL6/N mice for 24 h. Skin sections were then prepared and co-stained with anti-CD207-PE to identify langerin+ DCs,

such

as

LCs and langerin+ dermal

DCs. Langerin+ DCs in

intact skin were found to locate throughout the viable epidermis and dermis (Fig. 2A). As expected, we found that application of control

patches

containing

L-195/IPM

alone

had

little

to

no

influence on the location of langerin+ DCs in skin sections, which appeared similar to the samples from untreated mice (Fig. 2B).

In

contrast,

administration

of

R-848/IPM

induced

accumulation of langerin+ DCs just beneath the SC layer (topmost layer of viable epidermis) and almost no langerin+ DCs were found at

the

regions

below

(Fig.

2C).

A

similar

change

in

the

localization of langerin+ DCs was observed in skin treated with the R-848 S/O nanodispersions (Fig. 2F) but not with OVA/PBS (Fig.

2D)

or an

S/O

nanodispersion lacking R-848 (Fig. 2E).

Thus, the TLR7/8 ligand R-848 effectively induced relocation of langerin+ DCs, such as LCs, in the skin.


22

Page 23 of 44 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


Figure 2. Transcutaneous delivery of OVA to LCs in the skin. Light

and

fluorescence

microscopy

images

of

sections

of

ear

auricles from C57BL/6N mice at 24 h after application of patches soaked in solutions or S/O nanodispersions. Skin sections were co-stained with anti-CD207-PE to detect LCs and DAPI to detect nuclei. (A) No patch, (B) L-195/IPM, (C) R-848/IPM, (D) OVA/PBS, (E) S/O nanodispersion, and (F) R-848 S/O nanodispersion. Bars: 100 μm.


23


Page 24 of 44

Activation of LCs has been reported to induce elongation of their dendrites to penetrate tight junctions.30 Although the skin permeation of S/O nanoparticles is slow and requires ca. 24 h to permeate

below

hydrophobic

the

R-848

“pre-activating”

SC,31

it

molecules the

LCs

is

possible

may to

diffuse enhance

that much

the

small

faster,

antigen

and

thereby

uptake

and

presentation. To determine whether the rate of R-848 permeation from S/O nanodispersions differs from that of OVA, we compared their penetration of skin using patches soaked in Cy5-OVA S/O nanodispersions containing the fluorescent dye BODIPY FL C5 (BP), which was used as a surrogate for R-848 because of its similar molecular

weight

(Mw

314.4

and

320.1

for

R-848

and

BP,

respectively) and hydrophobicity (Log P 2.1 for R-848 and 2.5 (pH 5.0) or 1.2 (pH 7.4) for BP32). Since the skin permeation of small molecules shows dependence on their molecular weights and hydrophobicity,33 we assumed R-848 would permeate into the skin in a similar manner with BP.

After application of patches for

6, 12, and 24 h, we evaluated the diffusion of Cy5-OVA and BP in skin sections by fluorescence microscopy (Fig. S3). We found that both Cy5-OVA and BP gradually permeated into the deeper layers of the skin (Fig. S3A), but BP permeated more rapidly and was detectable at a greater depth below the SC layer compared with Cy5-OVA (Fig. S3B). Both Cy5-OVA and BP were observed in intracellular region of corneocytes, which are characterized by


24

Page 25 of 44 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


hexagonal

structure,34

indicating

the

intercellular

permeation

pathway (Fig. S3C). Taken together, these results suggest that the smaller hydrophobic molecule R-848 diffuses rapidly through the skin and pre-activates LCs beneath the SC layer to ensure efficient uptake of protein antigens. The activation of LCs was confirmed by the increased expression of costimulatory factors CD80

and

CD86

in

the

skin

administrated

with

R-848

S/O

nanodispersion (Fig. S4). Following

antigen

uptake,

LCs

migrate

to

local

lymph

nodes

where they present processed antigen to naïve T cells. To assess this,

we

isolated

submandibular

lymph

nodes

from

mice

after

application of patches soaked in the various Cy5-OVA and/or R848 formulations to the ear auricles for 24 h. The lymph node sections were then co-stained with anti-CD207-PE to detect LCs and examined by fluorescence microscopy. Notably, the lymph node sections from mice treated with S/O nanodispersions containing OVA

(Fig.

3E)

or

OVA/R-848

(Fig.

3F)

showed

increased

co-

localization of Cy5-OVA and anti-CD207-PE compared with sections from mice treated with OVA/PBS (Fig. 3D) or control applications (Fig. 3A and B). To assess the migration of LCs holding OVA from the skin, the skin was sensitized with FITC/dibutyl phthalate and sections of the draining lymph node were imaged. After the treatment with S/O nanodispersions, increased number of FITClabeled DCs (i.e., skin-derived DCs) were detected (Fig. S5).


25


Page 26 of 44

Moreover, LCs were more dominant than dermal CD207+ DCs among Cy5-OVA+ cells (Fig. S6). These results suggest that increased number of LCs holding antigen migrated to lymph nodes by t.c. immunization using S/O nanodispersions. We observed a similar increase in the migration of LCs to the lymph nodes in mice treated

with

R-848/IPM

(Fig.

3C),

presumably

because

of

the

local activation of LCs by R-848 as reported previously.35 These results clearly indicate that R-848 plays an important role in activating and promoting LC migration to lymph, and that antigen uptake by LCs is more efficient when OVA is applied in S/O nanodispersions compared with aqueous solutions.


26

Page 27 of 44 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


Figure

3.

Migration

of

LCs

to

lymph

nodes.

Fluorescence

micrographs of submandibular lymph node sections from C57BL/6N mice at 24 h after application of patches to the ear auricles. Sections were co-stained with anti-CD207-PE to detect LCs and DAPI to detect nuclei. Samples: (A) no treatment, (B) L-195/IPM, (C) R-848/IPM, (D) OVA/PBS, (E) S/O nanodispersion, (F) R-848 S/O nanodispersion. Bars: 100 μm.


27


Page 28 of 44

3-3. Suppression of tumor growth in vivo by immunization with S/O nanodispersions Next, we asked whether immunization with S/O and/or R-848 S/O nanodispersions

could

suppress

the

growth

of

OVA-expressing

tumor cells in a mouse model. Mice were injected with E.G7-OVA tumor cells on day 0 and received the following treatments on days 7 and 14: no treatment, OVA/PBS patch, R-848/IPM patch, S/O patch,

R-848

formulations

S/O

patch,

contained

or

200

s.c. μg

injection

of

OVA.

of

We

OVA.

All

observed

OVA

strong

inhibition of tumor growth in mice immunized with OVA via s.c. injection

or

t.c.

nanodispersions, significant

patches

among

inhibition

containing

which

R-848

(Fig.

S/O

S/O

4A).

In

and

R-848

showed

the

contrast,

S/O most t.c.

administration of OVA/PBS or R-848/IPM had little influence on tumor inhibition (Fig. 4A), suggesting that efficient permeation of both OVA and R-848 into the skin is necessary to mount a response

and,

further,

that

t.c.

co-administration

via

S/O

nanodispersions may be an effective delivery method for cancer immunotherapy. Consistent with the inhibition of tumor growth, we observed prolonged survival of mice in the s.c. injection, S/O, and R-848 S/O groups (median survival 28, 27.5, and >29 days, respectively) compared with the untreated control group (20.5 days). Of particular note, the R-848 S/O immunized mice


28

Page 29 of 44 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


showed a remarkable response, with 100% of the mice surviving for >30 days after tumor inoculation (Fig. 4B). To verify activation of T cells in the immunized tumor-bearing mice, we examined secretion of IFN-γ after 72 h stimulation of the

splenocytes

with

OVA

in

vitro.

As

shown

in

Fig.

4C,

splenocytes from mice immunized with R-848 S/O nanodispersion secreted significantly more IFN-γ compared with the control or OVA/PBS-immunized

mice.

This

result

suggests

that

co-

administration of R-848 with OVA in S/O nanodispersions promotes efficient activation of T cells during the anti-tumor immune response. In support of this, we also detected an increase in OVA-specific IgG (Fig. S7A), particularly IgG2a, indicative of a Th1-biased immune response (Fig. S7B, Table S1), which was most significant in mice immunized with the R-848 S/O nanodispersion. These data demonstrate that t.c. delivery of antigen via R-848 S/O nanodispersion induces an efficient and markedly superior cellular anti-tumor immune response compared with delivery via s.c. injection, whereby much more antigen should be delivered into the body than t.c. route (Fig. 4A and B). We

have

previously

encapsulating

antigen

immunization

IgG

with

without

levels.36

that

the

efficiency

nanodispersions serum

shown

CpG,

TLR

S/O

ligand

6–9-fold as

Therefore,

CpG

compared

measured we

nanodispersions

by

performed

co-

enhances

the

with

S/O

antigen-specific a

preliminary


29


comparison

of

the

efficacy

of

CpG

Page 30 of 44

S/O

and

R-848

S/O

nanodispersions using the same protocol as that shown in Figure 4.

Since

CpG

is

a

hydrophilic

oligonucleotide,

we

co-

encapsulated CpG and OVA in S/O nanodispersions. Of note, the R848

S/O

nanodispersion

effectively

than

did

the

inhibited CpG

tumor

S/O

growth

nanodispersion

much (data

more not

shown). This result suggests that small, hydrophobic TLR ligands such as R-848 are more effective than hydrophilic ligands in S/O formulations, presumably because they more rapidly permeate the skin to activate LCs.

Figure 4. Anti-tumor response of mice immunized with OVA by s.c. injection or t.c. patch. immunized

twice

(Control),

OVA/PBS

(arrows patch

(A and B) Mice bearing E.G7-OVA were in (OVA

A)

as

aq.),

follows: R-848/IPM

no

treatment

patch,

OVA/PBS

s.c. injection (Injection), S/O patch, or R-848 S/O patch. Tumor growth and survival were monitored for up to 30 days. (C) Mice were treated as described for A and B and spleens were harvested


30

Page 31 of 44 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


on day 19. Splenocytes were incubated with OVA in vitro for 72 h and IFN-γ secretion was quantified by ELISA. N = 4, mean ± SE, *p < 0.05, **p < 0.01, ***p < 0.001.

3-4. Antigen specificity of the immune response induced by S/O nanodispersions Finally, we investigated the antigen specificity of the antitumor immune response induced by S/O nanodispersions. For this, we inoculated mice with EL4 cells, a tumor cell line that does not express OVA, or E.G7-OVA cells, and then immunized the mice on days 7 and 14 by s.c. injection or by t.c. S/O patches as described unaffected

in by

Figure

4.

Notably,

immunization

(Fig.

growth 5A),

of

EL4

whereas

tumors

E.G7-OVA

was tumor

growth was significantly inhibited by immunization by both s.c. and t.c. routes, although the R-848 S/O nanodispersion was more effective (Fig. 5B). To confirm these results, we performed a cytotoxicity assay using CFSE-labeled EL4 or E.G7-OVA target cells. As expected, CTL

activity

against

E.G7-OVA,

but

not

EL4,

was

markedly

increased by immunization by s.c. injection or t.c. patch (Fig. 5C

and

D).

Moreover,

examination

of

tumor

sections

showed

abundant CD8+ cells at the site of E.G7-OVA tumors, but not EL4 tumors,

consistent

with

the

accumulation

of

antigen-specific


31


effector

cells

in

the

tumor

Page 32 of 44

environment

(Fig.

S8).

Taken

together, these data demonstrate that immunization with an R-848 S/O nanodispersion inhibits tumor growth in an antigen-specific manner,

at

least

in

part

by

promoting

a

cytotoxic

T

cell

response. Administration of TLR7/8 ligands can suppress tumor growth without the need to co-administer TAAs.27 Our data are consistent with

these

findings,

administrated

with

as

R-848

demonstrated

(R-848/IPM)

in

showing

mice the

solely

ability

to

retard tumor growth and prolong survival to a degree similar to that induced by OVA/PBS group (Fig. 4A and B). Splenocytes from the R-848/IPM treated mice also secreted high levels of IFN-γ, suggesting

that

the

TLR

ligand

induces

a

Th1-like

immune

response (Fig. 4C). Thus, the inhibition of tumor growth in mice immunized

with

an

R-848

S/O

nanodispersion

appears

to

be

mediated by an immune response with both antigen-nonspecific and -specific components. However, the nonspecific response has only limited efficacy, and it is clear that the enhanced delivery of OVA by S/O nanodispersions is a crucial element in the antitumor response. The data shown here, combined with our previous study using melanoma-derived CTL epitope peptide as the TAA,24 demonstrate the utility of this co-delivery strategy for cancer vaccines

regardless

of

the

origin

or

the

size

(peptides

or

proteins) of the TAAs.


32

Page 33 of 44 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


Figure 5. Antigen-specific immunization of C57BL/6N mice by S/O nanodispersions.

(A

and

B)

Groups

of

mice

were

untreated

(Control) or immunized by OVA/PBS s.c. injection (Injection) or t.c. R-848 S/O nanodispersion patch (R-848 S/O) on days 0 and 7. On day 14, the mice were injected with

EL4 or E.G7-OVA and

tumor growth was monitored. N = 4, mean ± SE, *p < 0.05. (C and D)

Mice

were

treated

as

described for

A

and

B. On day

14,

spleens were harvested and splenocytes were incubated for 72 h with OVA to prepare effector cells. Cytotoxicity was measured by


33


Page 34 of 44

mixing effector cells with CFSE-labeled EL4 (C) or E.G7-OVA (D) target cells (effector:target cell ratio = 100:1) for 6 h. N = 3, mean ± SE.

4. CONCLUSIONS In conclusion, we evaluated the effect of transcutaneous codelivery

of

TAA

and

R-848

in

S/O

nanodispersions

on

cancer

immunotherapy. R-848 present in the outer oil phase of the S/O formulation

rapidly

permeated

the

skin

to

pre-activate

LCs,

resulting in efficient uptake of OVA and migration of LCs to the lymph

nodes.

Transcutaneous

co-delivery

induced

a

strong

cellular immune response and significantly reduced tumor growth in an antigen-specific manner. Our results clearly demonstrate the

superiority

nanodispersions

of

transcutaneous

compared

with

vaccination

conventional

s.c.

with

S/O

injections,

particularly in the presence of costimulatory additives such as TLR ligands. This safe and effective formulation will be a good candidate for the development of transcutaneous preventive and therapeutic vaccines.

ASSOCIATED CONTENT Supporting Information Available. The following files are available free of charge.


34

Page 35 of 44 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


Chemical structures of chemical components and preparation of S/O nanodispersions; figures showing skin permeation depth, kinetics, and pathway of transcutaneous delivery of OVA using S/O nanodispersions; serum IgG levels in immunized mice; accumulation of T cells in tumors of immunized mice.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Japan Society for the Promotion of Science (JSPS) KAKENHI No. JP16H06369 ACKNOWLEDGMENT This work was supported by the Japan Society for the Promotion of

Science

(JSPS)

KAKENHI

No.

JP16H06369.

The

authors

thank

Professors Y. Katayama, T. Mori, and T. Niidome for facility support for the animal experiments. The authors also thank Mr. Kaku for the assistance with SEM measurement, and the Analysis Center

of Fukuoka Industry-Academia Symphonicity

for facility

support.


35


Page 36 of 44

ABBREVIATIONS TAA, tumor-associated antigen; DC, dendritic cell; APC, antigen presenting cell; Th, T helper; CTL, cytotoxic T lymphocyte; TLR, Toll-like receptor; t.c., transcutaneous; s.c., subcutaneous; SC, stratum corneum; S/O, solid-in-oil; OVA, ovalbumin; IPM, isopropyl myristate; LC, Langerhans cell. REFERENCES 1.

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C.;

Manjili,

M.

H.;

Subjeck,

J.

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Riley, R. S.; June, C. H.; Langer, R.; Mitchell, M. J.,

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Kawai,

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Table of Contents Use Only

Transcutaneous co-delivery of tumor antigen and resiquimod in solid-in-oil nanodispersions promotes anti-tumor immunity Rie Wakabayashi, Hidetoshi Kono, Shuto Kozaka, Yoshiro Tahara, Noriho Kamiya, and Masahiro Goto


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Transcutaneous co-delivery of tumor antigen and resiquimod in solid

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