Study On Nanoliposomes Co-delivering Multiple Bioactive Peptides For Skin Anti-aging

Skin aging is a biological process caused by intrinsic and extrinsic factors.Oxidative stress contributes to the loss of collagen and the extracellular matrix,as well as damage to skin cells,thus playing a major role in skin aging.Moreover,excessive stimulation of muscle fibers leads to the formation of wrinkles in the face,which are regarded as a conspicuous sign of skin aging.Bioactive peptides can regulate many biological processes with relevance to skin aging and these peptides are generally classified as antioxidative peptides,signal peptides which can stimulate matrix protein production,or neurotransmitter inhibitor peptides based on their mechanisms.Carnosine is a naturally-sourced dipeptide that can neutralize ROS,and it is a powerful antioxidant.Palmitoyl tripeptide-5（Pal-KVK）,one of the signal peptides,can up-regulate transforming growth factor beta and stimulate matrix protein production.Acetyl hexapeptide-3（Argireline）,can inhibit the release of neurotransmitter including catecholamine（CA）,relaxing muscle fibers and reducing wrinkles.However,bioactive peptides do not readily penetrate the stratum corneum barrier,reaching the targeted skin tissues or cells,and are susceptible to degeneration and inactivation.Moreover,multiple bioactive peptides with different mechanisms of action must be delivered simultaneously to achieve effective skin anti-aging treatment.In the present study,a nanoliposome system for topical codelivery of carnosine,palmitoyl tripeptide-5 and acetyl hexapeptide-3（CPA-NLPs）was developed for comprehensive and enhanced anti-aging effect,and for improved stability.The characterization,drug release profiles,skin penetration capability and cellular uptake of CPA-NLPs were studied.The in vitro anti-oxidation and skin anti-aging efficacy of the CPA-NLPs were investigated,and a clinical trial was also conducted to study the skin anti-aging efficacy of the CPA-NLPs on human skin.The main research works are as follows:（1）Nanoliposome for topical codelivery of carnosine,Pal-KVK and Argireline was developed and characterized.The mean partlcle size of CPA-NLPs was 62.4±1.2 nm,PDI was 0.273±0.010,and the zeta potential was-32.7±2.0 m V.The loading capacity of carnosine,pal-KVK and Argireline were 7.60%,1.06%and 0.74%,respectively.The encapsulation efficacy of carnosine,pal-KVK and Argireline in the CPA-NLPs were69.8%,92.3%and 72.6%,respectively.The results of stability test demonstrated that encapsulation of bioactive peptides within CPA-NLPs improved their thermal stability.The bioactive peptides were slow and sustained released from the CPA-NLPs.In vivo rat skin penetration study indicated that CPA-NLPs could significantly enhance the penetration of bioactive peptides and deliver the cargoes into the deeper layers of skin.（2）The results of cytotoxicity study showed that CPA-NLPs were safe and no cytotoxicity in HaCaT and HSF cells.The cellular uptake of FITC-loaded nanoliposomes（FITC-NLPs）by HSF cells was also investigated.Cellular uptake of FITC-NLPs by HSF cells was significantly increasd in comparison with free FITC,indicating that the prepared nanoliposomes effectively enhance the delivery efficiency of loaded ingredients into HSF cells,which could enhance the anti-aging efficacy of the bioactive peptides.（3）In vitro skin anti-aging efficacy of CPA-NLPs was studied.The CPA-NLPs protected HSF cells from H₂O₂-induced oxidative stress damage,decreased intracellular ROS level and increased their expression levels of Col I and HA.In addition,treatment with CPA-NLPs inhibited CA release in PC12 cells.And these anti-aging effects were enhanced compared with free bioactive peptides.（4）Clinical study of the anti-aging effect of CPA-NLPs was performed.The clinical study indicated that application of CPA-NLPs in face led to a remarkable reduction of the volume,area and area ratio of wrinkles,and an improvement of skin elasticity.Results in this thesis demonstrated that encapsulation of bioactive peptides within CPA-NLPs improved their stability,skin penetration ability and cellular uptake.The combination of bioactive peptides exhibited synergistic effect through multiple pathways which involved anti-oxidation,stimulation of the collagen production and inhibition of neurotransmitter release.These results would provide theoretical and experimental basis for the clinical application of the nanoliposomes codelivering multiple anti-aging bioactive peptides.