Cell experiment:

The cytotoxicity of SPACE peptide is assessed using the MTT Cell Proliferation Assay. HEKa cells are seeded in 96-well microplates at a density of 5000 cells/well. Cultures are allowed to grow until they reaches ~80% confluency. Cells are then incubated with 150 μL of 10, 5, 2.5, or 1.25 mg/mL SPACE peptide in media. Media only is used as a negative control, and media without cells is used to subtract background. Cytotoxicity is assessed for 1, 4, and 12 h incubation periods[2].

Animal experiment:

Mice: Female BALB/c mice (knockdown of GAPDH protein) are anesthetized using isoflurane inhalation (2-3%), the back skin of animals is shaved, and a cylinder with an exposed skin area of 1.8 cm2 is attached to the back of mice. 200 μL of the SPACE peptide is topically applied in the attached cylinder, and is manually spread over the entire exposure area. Applied test solutions are allowed to incubate with the exposed skin for 6 hours while keeping animals under minimal anesthesia. After 6 hours, the cylinder is carefully removed, and the entire exposure area is covered with sterile gauze and a breathable bandage. After 72 hrs, animals are sacrificed and skin biopsies (5 mm diameter) are collected from treated area of the animal’s skin. Total protein concentration in the skin biopsy tissue is determined[3].

SPACE peptide is a skin penetrating peptide which facilitates the delivery of molecules through the skin.

SPACE peptide, when conjugates to cargoes such as small molecules and proteins, is able to facilitate their penetration across the stratum corneum into epidermis and dermis. The peptide also exhibits increased penetration into various cells including keratinocytes, fibroblasts, and endothelial cells, likely through a macropinocytosis pathway[1]. SPACE enhances cyclosporine A, penetration into the skin significantly. It does not alter the skin lipid barrier. It interacts with skin proteins and induces changes in skin protein secondary structures (α-helices, β-sheet, random coils and turns). SPACE enhances cyclosporine A skin penetration, via a transcellular pathway, enhancing its partitioning into keratin-rich corneocytes through concurrent binding of SPACE with keratin and cyclosporine A. Interaction between SPACE and keratin best correlates with measured cyclosporine A skin transport[2]. SPACE-peptide in combination with a DOTAP-based ethosomal carrier system can enhance skin delivery of siRNA[3]. The SPACE-ethosomal system enhances hyaluronic acid penetration into porcine skin in vitro by 7.8+/-1.1-fold compared to PBS[4].

The efficacy of DOTAP-SES in delivering GAPDH-siRNA into skin is confirmed in BALB/C mice. Topical application of DOTAP-SES on mice skin results in 63.2%±7.7% of GAPDH knockdown, which is significantly higher than that from GAPDH-siRNA PBS (p<0.05) [3].

[1]. Hsu T, et al. Delivery of siRNA and other macromolecules into skin and cells using a peptide enhancer. Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):15816-21. [2]. Kumar S, et al. Peptides as skin penetration enhancers: mechanisms of action. J Control Release. 2015 Feb 10;199:168-78. [3]. Chen M, et al. Topical delivery of siRNA into skin using SPACE-peptide carriers. J Control Release. 2014 Apr 10;179:33-41. [4]. Chen M, et al. Topical delivery of hyaluronic acid into skin using SPACE-peptide carriers. J Control Release. 2014 Jan 10;173:67-74.