Skin dendritic cells (DCs) such as Langerhans cells and dermal dendritic cells have a pivotal role in inducing antigen-specific immunity; therefore, transcutaneous cancer vaccines are a promising strategy to prophylactically prevent the onset of a variety of diseases, including cancers. The largest obstacle to delivering antigen to these skin DC subsets is the barrier function of the stratum corneum. Although reverse micellar carriers are commonly used to enhance skin permeability to hydrophilic drugs, the transcutaneous delivery of antigen, proteins, or peptides has not been achieved to date because of the large molecular weight of drugs. To achieve effective antigen delivery to skin DCs, we developed a novel strategy using a surfactant as a skin permeation enhancer in a reverse micellar carrier. In this study, glyceryl monooleate (MO) was chosen as a skin permeation enhancer, and the MO-based reverse micellar carrier enabled the successful delivery of antigen to Langerhans cells and dermal dendritic cells. Moreover, transcutaneous vaccination with the MO-based reverse micellar carrier significantly inhibited tumor growth, indicating that it is a promising vaccine platform against tumors.

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 coloading 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-2, as a peptide antigen against melanoma. Transcutaneous vaccination of the S/O nanodispersion coloaded 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.

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 antitumor immunity can be induced using a codelivery system in which nonspecific immunostimulatory molecules are administered together with TAAs. In this study, we investigated the antitumor effects of a solid-in-oil (S/O) nanodispersion system containing a model TAA, ovalbumin (OVA), and resiquimod (R-848), a small molecular Toll-like 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. Analysis of OVA and R-848 permeation in mouse skin after application of an R-848 S/O nanodispersion indicated that R-848 rapidly permeated the skin and preactivated 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 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 codelivery of R-848 significantly amplified the antitumor 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.

The skin loses its moisture with advancing age, causing cosmetic issues such as wrinkles. In addition, the loss of moisture leads to hypersensitivity to external stimuli such as UV light. Transcutaneous supplementation with hyaluronic acid (HA) is an effective and safe method of recovering the moisturizing function and elasticity of the skin. However, the transcutaneous delivery of HA remains challenging owing to the barrier function of the stratum corneum (SC) layer. To penetrate the SC barrier, we used a reverse micelle formulation that does not require high energy consumption processes for preparation. We aimed to enhance the skin permeability of HA by incorporating glyceryl monooleate—a skin permeation enhancer—into the formulation. A fluorescently-labeled HA-loaded reverse micelle formulation showed significantly enhanced permeation across Yucatan micro pig skin. Fourier transform infra-red spectroscopy of the surface of the skin treated with the reverse micelle formulation showed blue shifts of the CH2 symmetric/asymmetric stretching peaks, indicating a reduction in the barrier function of the SC. Further study revealed that HA was released from the reverse micelles at the hydrophobic/hydrophilic interface between the SC and the living epidermis. The results demonstrated that our reverse micellar system is an easy-to-prepare formulation for the effective transcutaneous delivery of HA.

Lipidic cubic phase (LCP) is a self-assembled system composed of lipids with interpenetrated aqueous channels, and its potential in drug delivery systems has been investigated. Although LCP was shown to improve transcutaneous penetration of hydrophilic molecules of up to 1203 Da so far, the transcutaneous delivery of larger molecules such as proteins has not been achieved. This is likely because proteins are usually larger than the aqueous channels of LCP (∼37.2 Å in diameter), which limits the molecular diffusion in LCP. In this report, we overcome this issue by adding N-octyl-β-d-glucopyranoside to glyceryl monooleate–water-based LCP to give swollen LCP (SLCP), which has larger aqueous channel diameters (∼65.6 Å). First, we systemically evaluated the effect of swelling on drug diffusion in LCP/SLCP. The release kinetics of various peptides and proteins whose sizes ranged from 9.14 to 55.28 Å in diameter were evaluated, and the diffusion coefficients (D) were calculated by the Fickian diffusion model. As expected, all peptides and proteins diffused faster in SLCP than in LCP. A more detailed analysis revealed a negative linear relationship between log D and the ratio of the radius of gyration of the proteins to the aqueous channel radius, indicating that swelling of a cubic nanostructure is an effective strategy to enhance D. Next, the skin penetration of proteins encapsulated in LCP and SLCP was evaluated. The skin penetration of ovalbumin (42.9 kDa), for example, was enhanced by SLCP but not by LCP, and a positive correlation between D and the amount of skin penetration was found. Collectively, this study provides an effective measure for designing LCP systems that enhance transcutaneous penetration of biomacromolecules.