(220e) Skin Permeable Peptide Amphiphiles As an Anti-Aging Agent

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:
[1] Zhao,
Xiubo. et al. Molecular self-assembly and applications
of desiner peptide amphiphiles. Chem. Soc. Rev., 2010, 39,
3480-3498. [2] Heitz, F. el al. Twenty Years of Cell- Penetrating
Peptides: From Molecular Mechanisms to Therapeutics. Br. J. Pharmacol. 2009, 157, 195–206.