Preparation Of Deerskin Collagen Polypeptide Liposome Hydrogel And Its Protective Effect On Photoaging In Mice

Purpose: Long-term ultraviolet radiation will cause photoaging of the skin,resulting in the fracture and synthesis reduction of the collagen in the body,which is characterized by dry and rough skin and finally wrinkles.Collagen peptides can synthesize collagen and play an anti-aging role after being absorbed and utilized by the skin.In order to improve the skin permeability and stability of deerskin collagen polypeptide（DCP）,deerskin collagen polypeptide liposome-hydrogel（DCP-LIP-Gel）was prepared to improve the protective effect of skin photoaging.Methods: Collagen（DC）was extracted by acid extraction method,and the content and yield of DC in raw materials were determined by BCA.The extracted DC was characterized by ultraviolet spectrophotometry,infrared spectrophotometry,SDSPAGE and amino acid composition analysis.DCP were extracted by alkaline protease enzymatic hydrolysis,and the molecular weight of DCP was determined by SDS-PAGE method.The in vitro antioxidant activities of DC and DCP were determined by DPPH and ABTS free radical scavenging experiments.The proliferation activity of DCP on L929 mouse fibroblasts was determined by CCK8 cell proliferation test,and the migration activity of DCP on L929 mouse fibroblasts was determined by cell scratch test.Deerskin collagen polypeptide liposomes（DCP-LIP）were prepared by thin film hydration method.The DCP-LIP ware characterized by transmission electron microscope,Malvern particle size analyzer and dialysis method,and the in vitro release and stability of DCP-LIP were studied by characterization methods.DCP-LIP-Gel was prepared by physical mixing method and characterized by appearance observation and scanning electron microscope.The skin permeability of DCP-LIP-Gel was studied by Franz diffusion cell,and the in vitro anti-photoaging effect of DCP-LIP-Gel on L929 mouse fibroblasts was studied.The photoaging model was established by UVB lamp,and the protective effect of DCP-LIP-Gel on skin photoaging was studied in mice.The stability of DCP-LIP-Gel was studied,including the determination of the content of DCP on different time by BCA protein assay,the appearance stability,heat resistance,cold resistance and centrifugal stability.Finally,the safety of DCP-LIP-Gel was studied,including acute transcutaneous toxicity test,multiple skin irritation test and skin allergic reaction test.Results: The yield of DC extracted by acid extraction was 12.62 ±1.34%.The ultraviolet absorption peak of DC measured by ultraviolet scanning was at 234 nm,which was inaccordance with the characteristics of type Ⅰ collagen.The infrared absorption of DC showed that there were amide band A,amide band B,band I,band Ⅱand band Ⅲ.SDS-PAGE gel electrophoresis analysis showed that the molecular weight of DC was about 300 KD.Amino acid composition analysis showed that DC was rich in amino acids of glycine,proline,hydroxyproline and so on.The molecular weight of DCP was less than 14.4 KD determined by SDS-PAGE electrophoresis.DCP exhibited a significant antioxidant activity determined by ABTS free radical scavenging test.The result of CCK8 cell proliferation test and scratch test showed that DCP had the activity of promoting cell proliferation and migration.The entrapment efficiency of DCP-LIP prepared by thin film method was 88.25±3.48%.The DCP-LIP presented uniformly spherical shape observed under the transmission electron microscope.The particle size of DCP-LIP was about 100 nm.The PDI of DCP-LIP was less than 0.3,and Zeta potential showed negative charge.The stability results showed that the DCP-LIP had good stability at 4 ℃.The results observed by scanning electron microscope showed that the DCP-LIPGel presented a three-dimensional porous network structure with large voids,and the DCP-LIPs were uniformly distributed in the hydrogel matrix.The in vitro release results showed that the cumulative release rates of DCP-LIP and DCP-LIP-Gel within24 hours are 35.80% and 28.03%,respectively.The cumulative penetration rates of DCP in DCP-LIP and DCP-LIP-Gel were 24.32% and 21.97%,respectively.The results of anti-photoaging in vitro showed that DCP-LIP could significantly inhibit the production of intracellular ROS.The results of skin photoaging in mice showed that DCP-LIP-Gel had protective effect on photoaging.The results of stability test showed that DCP-LIP-Gel had good physical,and chemical stability,good heat resistance,good cold resistance and centrifugation stability.The safety test results showed that the DCPLIP-Gel had no acute transdermal toxicity,no skin irritation and no skin allergic reaction in rats and guinea pig.Conclusion: DC and DCP were successfully extracted and hydrolyzed with a high yield.DCP showed a high antioxidant activity and cell proliferation activity;DCP-LIP was prepared with high entrapment efficiency and drug loading.The DCP-LIP-Gel was proved strong protective effects on photoaging in mice photoaging model and a good skin safety profile in animals.