Construction Of Liposomes Transdermal Drug Delivery System And Study On Its Anti-aging Effects Loaded With Resveratrol And Carnosine

Skin aging is a complex process with many factors and mechanisms,which characterized by skin damage such as dryness,laxity,roughness and wrinkles.The main obstacle of delaying skin aging by drugs is the cuticle.Therefore,the core problem of transdermal drug delivery is how to select drugs and get most of the drugs pass through the skin barrier.Currently,the efficacy of common single-mechanism administration remains limited.Researches have demonstrated that oxidative stress and glycosylation damage are the two main inducements of aging,while resveratrol(RES)and carnosine(CAR)show powerful antioxidant and anti-glycosylation effects,respectively.From these two perspectives,it is not yet clear that the two drugs can play a unique synergistic effect.Thus,it is particularly important to clarify the therapeutic effect of the combination of the two drugs.Liposomes have the ability to co-load insoluble and water-soluble components.Meanwhile,they show great advantages over other transdermal forms owing to there small particle size and similar structure to cell membranes.Nevertheless,traditional liposomes still exist the disadvantages of easy leakage and poor stability.Wrapping chitosan(CS)around the liposomes can not only solve the above problems,but also increase the penetration depth of skin and the degree of closeness to the skin.After modifying with catechol group,water solubility and adhesion with skin are increased.Therefore,the rational design of catechol chitosan coated liposomes is of great significance for the co-delivery of two drugs and the treatment of skin aging.In this study,catechol chitosan coated resveratrol and carnosine co-loaded liposomes were prepared.The synergistic effect of the two drugs and the good efficacy of the drug-loaded liposomes were demonstrated in vivo and in vitro.The study contains three parts:(1)HPLC methods for the content determination of RES and CAR were established and the oil-water distribution coefficients of the two drugs were measured.By comparing the DPPH free radical scavenging ability,ABTS free radical scavenging ability,AGEs inhibition ability and iron reduction ability of RES and CAR when they were used alone or combined with different mass ratios,the optimal mass ratio of the two drugs was 2:1.Catechol modified chitosan(Cat-CS)was synthesized and characterized by infrared,UV-Vis and X ray diffraction spectra.Cat-CS coated co-loaded liposomes(RES+CAR@Cat-CS-LP)was prepared,subsequently.The optimum prescription and preparation parameters of liposomes were determined by single factor investigations and star design-response surface method.The spherical morphology of the liposomes was observed under transmission electron microscopy and the particle size,Zeta potential,polydispersity index(PDI),encapsulation efficiency(EE),loading capacity(LC),stability and drug release properties of each liposomes were further evaluated.RES+CAR@Cat-CS-LP exhibited the particle size of 177.82±2.51 nm,the Zeta potential of 17.58±0.49 m V and the PDI of 0.268±0.014.The EE and LC of RES were 82.39±1.54%and 6.04±0.13%,respectively.The EE and LC of CAR were 74.18±0.82%and 2.71±0.24%,respectively.The drug release was significantly slower than that of free drugs,which could be simulated by Weibull model.The release mechanism was the combination of diffusion and dissolution with the cumulative release amount over 75%in 36 h.The above results proved that the prepared liposomes had good basic properties and drug release properties.(2)Human skin fibroblasts(HSF)and human immortalized keratinocytes(Ha Ca T)were used to simulate the dermis and epidermis to evaluate the anti-aging ability of RES,CAR,RES+CAR,RES+CAR@LP,RES+CAR@CS-LP and RES+CAR@Cat-CS-LP.The best irradiation dose was chosen on the basis of cell survival rate and microscopic morphology.The dose of UVA irradiation on HSF cells was selected as 6 J/cm~2.At this time,the cell survival rate was 55.2%with the morphology became expanded,round and the cytoplasm got dark.When UVB irradiated Ha Ca T cells at the dose of 40 m J/cm~2,the cell survival rate was 54.5%accompanied by the decreased density and the rounded shape.The optimal drug concentration was determined according to the survival rate of HSF and Ha Ca T cells,which were 20μg/m L and 30μg/m L,respectively.ROS levels were observed by inverted fluorescence microscope.The activity or content of SOD,CAT,GSH-Px,MDA,HYP and AGEs were measured by the kits.The results showed that drug administration could significantly reduce the ROS level,increase the activity or contents of SOD,CAT,GSH-Px,HYP and decrease the contents of MDA and AGEs.Moreover,the synergistic effect of the two drugs was greater than that of drug administration alone and the effect was better after giving and improving the dosage form.(3)Guinea pigs were used as animal models to evaluate the anti-aging abilities of RES,CAR,RES+CAR,RES+CAR@LP,RES+CAR@CS-LP and RES+CAR@Cat-CS-LP.Skin appearance,texture,moisture,elasticity,barrier damage index,darkness index,firmness index,thickness and subcutaneous blood vessels were recorded.Skin tissue sections were stained with HE,Masson and Sirius scarlet,respectively.The levels of HYP,MDA,CAT,GSH-Px,SOD,AGEs and MMP-1 in skin tissues were measured.The penetration depth of the carrier was observed by laser confocal scanning.The experimental results showed that the administration groups could improve the skin condition and texture arrangement,reduce wrinkles,reduce subcutaneous vascular dilation,reduce thickness,increase moisture and elasticity to varying degrees.Through the three stained skin tissue sections,it could be observed that the deep skin morphology and the tightness of collagen fibers were improved.The percentage of type I collagen was reduced and type III collagen was increased.The activity or contents of SOD,CAT,GSH-Px,HYP was increased and the contents of MDA,AGEs,MMP-1 was decreased.In addition,the improvement degree of the above indexes emerged RES+CAR group>RES group and CAR group,which proved that the greater synergistic effect of two drugs than that of single drug administration.RES+CAR@Cat-CS-LP group>RES+CAR@CS-LP group>RES+CAR@LP group>RES+CAR group,which certified that the drug displayed better effect after encapsulating in liposomes.The anti-aging effect was also gradually enhanced after CS and Cat-CS coating.Laser confocal scanning results exhibited that skin penetration depth from high to low was Cat-CS-LP≈CS-LP>LP>free drug.In summary,combined administration of RES and CAR can play a synergistic effect.Cat-CS-LP can carry drugs deep into the skin layer to enhance the anti-aging effect.Therefore,RES+CAR@Cat-CS-LP is expected to become a potential anti-aging drug and provide new ideas for delaying aging.