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. 2020 Jan 7;10(1):101.
doi: 10.3390/biom10010101.

Cell Penetrating Peptide as a High Safety Anti-Inflammation Ingredient for Cosmetic Applications

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Free PMC article

Cell Penetrating Peptide as a High Safety Anti-Inflammation Ingredient for Cosmetic Applications

Tse-Kai Fu et al. Biomolecules. .
Free PMC article

Abstract

Cosmeceutical peptides have become an important topic in recent decades in both academic and industrial fields. Many natural or synthetic peptides with different biological functions including anti-ageing, anti-oxidation, anti-infection and anti-pigmentation have been developed and commercialized. Current cosmeceutical peptides have already satisfied most market demand, remaining: "cargos carrying skin penetrating peptide with high safety" still an un-met need. To this aim, a cell-penetrating peptide, CPPAIF, which efficiently transported cargos into epithelial cells was exanimated. CPPAIF was evaluated with cell model and 3D skin model following OECD guidelines without using animal models. As a highly stable peptide, CPPAIF neither irritated nor sensitized skin, also did not disrupt skin barrier. In addition, such high safety peptide had anti-inflammation activity without allergic effect. Moreover, cargo carrying activity of CPPAIF was assayed using HaCaT cell model and rapid CPPAIF penetration was observed within 30 min. Finally, CPPAIF possessed transepidermal activity in water in oil formulation without disruption of skin barrier. All evidences indicated that CPPAIF was an ideal choice for skin penetrating and its anti-inflammatory activity could improve skin condition, which made CPPAIF suitable and attractive for novel cosmeceutical product development.

Keywords: anti-inflammation; cell-penetrating peptides; cosmeceutical peptides.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Stability of CPPAIF stored as dry powder at different temperatures. Each sample was dissolved in milliQ H2O, and filtered through 0.2 μm filter, then freeze dried into powder form. The samples (100 µL, 1 mg/mL) were separately incubated at 4 °C and 25 °C for 1, 3, and 7 days. The residual amount of CPPAIF ingredient was quantified by HPLC equipped with C18 column.
Figure 2
Figure 2
Stability of CPPAIF stored in water at different temperature. Stability of CPPAIF in water at various temperature (−20 °C, 4 °C, 30 °C, 50 °C) for different time duration. Each sample (100 µL, 1 mg/mL) was filtered through 0.2 μm filter, and residual amount of CPPAIF ingredient was quantified by HPLC equipped with C18 column.
Figure 3
Figure 3
In vitro skin irritation test of CPPAIF in SkinEthicTM RHE model. 3D reconstructed human epidermis tissues were incubated with growth medium for 2 h followed by treatment with NC, PC, and 1 mM CPPAIF for 42 min. Then the tissues were washed and further incubated in growth medium for 42 h. Afterwards, the growth medium was substituted with maintenance medium containing MTT agent for an additional 3 h incubation. Next, the insert (with tissue) was washed with PBS and air dried. Formazan in tissues was extracted with isopropanol and measured by OD at 570 nm. PBS was applied as negative control (NC) and set as 100% (mock), 5% SDS was applied as positive control (PC). *** p < 0.001 versus the NC.
Figure 4
Figure 4
In vitro skin barrier function test of CPPAIF in SkinEthicTM RHE model. 3Dreconstructed human epidermis tissues were exposed with NC, PC, and 1 mM CPPAIF for 1 h. The CPPAIF was washed with PBS from the surface followed by application of detergent solution (1% Triton X-100) onto surface of the tissues for another 2 h. The tissues were washed with PBS and cell viability was measured by AlamarBlue cell viability assay. PBS was applied as negative control (NC) set as 100% (mock) and 5% SDS was applied as positive control (PC). *** p < 0.001 versus the NC.
Figure 5
Figure 5
Anti-inflammation activity of CPPAIF. Raw264.7 macrophage cells were seeded in 96-well plates (5 × 105 cells/mL) and allowed to attach overnight. After attachment, the cells were incubated with various concentrations of CPPAIF for 1 h followed by stimulation with 1 μg/mL of LPS. The control group was not treated with LPS and its cytokine secretion was set as 100% (mock). The amounts of TNFα (A) and IL-6 (B) in the medium were analyzed by ELISA. Cell viability was measured by AlamarBlue cell viability assay. *** p < 0.001 versus the control group.
Figure 6
Figure 6
Sensitization test of CPPAIF. The sensitization of CPPAIF was evaluated by mast cell degranulation assay. RBL-2H3 mast cells (5 × 105) were treated with various concentrations (0, 0.1, 1, 2, 5, 10 μM) of CPPAIF. The positive control (PC) was treated with 1 mM A23187 (calcimycin) and the amount of its granule release was set as 100% (mock).
Figure 7
Figure 7
Internalization of TMR-CPPAIF. HaCaT cells (1 × 103) were seeded on collagen I coated cover slide and incubated at 37 °C for 16 h. The cells were then incubatd with 20 μM of TMR-CPPAIF at 37 °C for 30 min. After washing and fixing, the cells were mounted and monitored by confocal microscopy. Red: TMR; Blue: nuclear staining with DAPI. (Magnification: 63×).
Figure 8
Figure 8
Transepidermal degree of formulated CPPAIF in SkinEthicTM RHE model. All 3D reconstructed human epidermis tissues were incubated with growth medium for 2 h, then 0.1 mL formulated CPPAIF (0.1 mM) emulsions were added to the top of the tissues and incubated for 1 h. After treatment the liquid beneath the 3D skin model was collected for HPLC quantification. Transepidermal degree was calculated by measuring the amount of CPPAIF in the liquid beneath the 3D skin model using HPLC. ** p < 0.01 versus the W/O formulation group.
Figure 9
Figure 9
In vitro skin barrier function test of formulated CPPAIF in SkinEthicTM RHE model. 3D reconstructed human epidermis tissues were exposed with formulated CPPAIF (0.1 mM CPPAIF) for 1 h. Then CPPAIF was washed by PBS from the surface followed by application of detergent solution (1% Triton X-100) onto surface of the tissues for another 2 h. The tissues were washed and cell viability was measured by AlamarBlue cell viability assay. H2O was applied as negative control (NC) in which cell viability was set as 100% (mock). * p < 0.05 and *** p < 0.001 versus the NC.

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