(220e) Skin Permeable Peptide Amphiphiles As an Anti-Aging Agent Conference: AIChE Annual MeetingYear: 2015Proceeding: 2015 AIChE Annual MeetingGroup: Chemical Engineers in MedicineSession: Nanotechnology in Medicine & Drug Delivery Time: Monday, November 9, 2015 - 4:27pm-4:45pm Authors: Mi, G., Northeastern University Webster, T. J., Northeastern University Skin Permeable Peptide Amphiphiles as an Anti-aging Agent Gujie Mi1, Thomas J Webster1,2 1Department of Chemical Engineering, Northeastern University, Boston, USA. 2Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia. Introduction: One of the most predominant symptoms of skin aging is wrinkle formation, which resulted from both intrinsic aging and environmental damage, such as chronic exposure to UV radiation. Current anti-aging and anti-wrinkle materials often induce a toxic response, which results in inflammation, to increase tissue growth under the skin. Although effective, a much safer and more effective way to alleviate the effect of aging and thereby winkle formation need to be developed. Several biomimetic peptide sequences1 (KTTKS, GQPR, GHK etc.) have been identified as structural mimics of collagen type I to be effective in stimulating synthesis of key constituents of extracellular matrix by fibroblasts, however, they typically suffer from poor skin penetration due to the existence of stratum corneum. Incorporation of cell penetrating peptides (CPPs) has been an emerging strategy to transport a variety of cargo across cellular membrane in a dose dependent manner.1 In this study, peptide amphiphiles that consist of both CPPs and biomimetic sequences was designed, synthesized and characterized. The abilities of these peptide amphiphiles to penetrate the skin, to scavenge free radicals, and to promote fibroblast functions (specifically, increasing adhesion, proliferation, collagen synthesis, and decreasing elastase and collagenase synthesis) were determined. Materials and Methods: Material characterization. Self-assembled amphiphiles were characterized by zeta potential to determine their charge, by dynamic light scattering to examine their size, and by TEM to observe their morphology. Cell viability assay. To determine cell viability, human dermal fibroblasts were seeded at a density of 10,000 cells/cm2 in 96-well plate and subsequently co-cultured with peptide amphiphiles at various concentrations (12/20/40/80/160 µM) for 24 hours. MTS assays were used to determine cell density after incubation. Briefly, 20 µl of MTS dye solution was added per 100 µl of solution, and the absorbance readings were taken at 490 nm after 4 hours of incubation. Cell density was then determined with correlation to a standard curve. Cell adhesion and proliferation. To determine cellular adhesion/proliferation, keratinocytes and human dermal fibroblasts were seeded at a density of 10,000 cells/cm2 and cultured respectively in keratinocyte medium supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Hyclone), and DMEM supplemented with 10% FBS and 1% P/S. Both types of cells were incubated under standard cell culture conditions (37°C, humidified, 5% CO2/95% air) for a certain period of time (4h for adhesion; 1,3, and 5 days for proliferation). The MTS assays were used to determine cell density after incubation. Skin penetration/permeation. Skin penetration and permeation was determined using the Static Franz diffusion cell (FD-C) with porcine skin as the membrane between donor and acceptor compartment due to their resemblance to human skin. Peptide amphiphiles were applied in the donor compartment, and PBS was used as the receptor solution. The donor compartment was sealed with aluminum foil and the system was maintained at 37°C in water bath. The skin samples were fixed and sectioned to examine penetration using confocal microscopy. Measurement of total collagens. Sircol soluble collagen assay (Biocolor, UK) was used to quantify total soluble collagens after 7, 14 and 21 days of culturing. All experiments were conducted in triplicate and repeated at least three different times. Results and Discussion: While peptide amphiphiles showed moderate toxicity at a higher concentration, with LC50 values at around 76.66 µM, they provided improved skin penetration across the stratum corneum when compared to using short peptides alone (Figure 1). Also, it was found that these peptide amphiphiles have the ability to promote cell proliferation as well as total collagen synthesis in the long term. Figure 1: Density of human dermal fibroblasts after 1 day of culturing when co-cultured with different concentrations of APNP. Cell was seeded at 10,000 cells/cm2. Values are mean ± SD, n=6. *p<0.005, **p<0.0001 when compared to control group. Conclusions: Through the above experiments, various peptide amphiphiles were identified that can be added to current skin cream formulations to promote penetration of active ingredients, to increase fibroblast growth, as well as to enhance collagen synthesis. References:  Zhao, Xiubo. et al. Molecular self-assembly and applications of desiner peptide amphiphiles. Chem. Soc. Rev., 2010, 39, 3480-3498.  Heitz, F. el al. Twenty Years of Cell- Penetrating Peptides: From Molecular Mechanisms to Therapeutics. Br. J. Pharmacol. 2009, 157, 195–206.