A Dual Deformable Liposomal Ointment Functionalized With Retinoic Acid And Epidermal Growth Factor For Enhanced Burn Wound Healing Therapy

Burns is a very common trauma in the world which requires for tedious treatment.Existing burn treatment includes debridement,topical drug application and wound dressing application,bringing conspicuous pain and heavy economic burden for patients.Currently,developing appropriate topical preparations is still crucial for clinical treatment of burns.Epidermal growth factor(EGF)has been widely employed in the burn therapy,but its poor stability and unsatisfactory effect on treating deep burns or massive uclers limit its clinical application.Retinoic acid is a multifunctional drug which exerts positive effect on connective tissue generation and chronic wound healing.There are researches showed that combined application of EGF and TRA will generate a synergistic effect on wound healing for TRA upregulating the expression level of EGFR and HB-EGF.In this study,we prepared TRA deformable liposomes(TRA DLs)or EGF cationic deformable liposomes(EGF CDLs).An ointment containing both liposomes was then produced with the aim of producing synergistic effect of drugs,promoting therapeutic efficacy,reducing side effects,accelerating wound healing and improving the restorative quality.Lecithin was used as the membrane material,and Tween 80 was selected as edge activator to prepared DLs with various contents of surfactant by thin-film dispersion method.The results of physicochemical properties showed that all TRA liposomal formulations featured particle sizes of sub-100 nm,drug entrapment efficiency over 99%,loading content of 3%,and sustained drug release behavior.In comparison to free drug,TRA DLs exhibited superior skin permeation and significantly increased drug deposition in excised rat skin,being favorable for dermal drug delivery.The average particle size of 20%Tween 80 liposomal formulation was about 48.10 nm and could release TRA for more than 48 hours.This specific formulation could increase skin deposition by 2.92 fold with respect to free drug,and retain the excellent permeation when corporated into an ointment base.DOTAP was used as the membrane material,and Tween 80 was selected as edge activator to prepared blank vesicles by thin-film dispersion method,and EGF was loaded into cationic liposomes by electrostatic incubation.The results of physicochemical properties showed that EGF CDLs characterized by a particle size of 16.00 nm,drug entrapment efficiency of 63.73%,and could sustained drug release for 48h.Compared to free EGF,EGF CDLs increased skin deposition by 18.82 fold and retained the excellent permeation when corporated into an ointment base.HaCaT were used as the cell model to investigate cytotoxicity of blank liposomes as well as the promoting effect of two deformable liposomes on cell proliferation and migration.The results demonstrated that the blank liposomes were almost non-toxic to HaCaT cells and featured with excellent biocompatibility.Exclusive application of TRA DLs exhibited a mild effect on promoting cell proliferation while the effect of EGF CDLs was much stronger.Furthermore,combined application exerted a synergistic effect and prominently promoted cell proliferation.No obvious changes were observed on cell migration when TRA DLs were employed,but EGF CDLs significantly improved migration.In addition,similar synergistic effect on promoting cell migration was noticed in occasion of dual usage of two DLs.The ointment containing two deformable liposomes was prepared.Results of in vitro drug release,skin permeation and retention study revealed that the ointment could release TRA for more than 72 hours and exhibited favorable skin permeation ability for its high steady-state flux.Rat model of deep partial-thickness burn wound was established to evaluate therapeutic effects of dual deformable liposomal ointment on burns.The results showed that the dual deformable liposomal ointment stimulated wound closure,promoted formation of skin appendages and increased the production of collagen,so as to improve the healing quality.The expression of PCNA and Keratin 6 were elevated remarkably as showed in results of immumohistochemical staining.Finally,the TRA significantly up-regulated the expression of EGFR and HB-EGF for enhancing the therapeutic effect of EGF on wound sites.In summary,the dual liposomal ointment could effectively encapsulate TRA and EGF,and achieve sustained release of drugs.The dual liposomal ointment brought synergistic effect of drugs into full play,significantly promoted cell proliferation and migration,so as to accelerate wound healing and improve the healing quality.It is expected to be applied as a safe and efficient treatment of burns.