| Skin wounds caused by surgery,trauma,or metabolic diseases have become common disease types.The repair of wounded skin involves a series of sequential,overlapping and precisely programmed phases involving the interaction of several cells,growth factors and cytokines;therefore,the realization of an efficient and high-quality wound healing has become one research challenges in recent years.Gene therapy,which regulates the expression of target genes by introducing exogenous functional genes into target cells via gene delivery system,represents an attractive alternative approach due to the continuously production of growth factors with high biologic activity and in situ supplement of skin wounds.Safe and efficient gene delivery system is critical to the success of gene therapy.Compared to viral vectors,polycationbased vectors——benefitting from their chemical composition and structural diversity,facile functionalization strategies,and low host immunogenicity——have attracted more and more attention.In recent years,polysaccharides and peptides with high biocompatibility have been explored as gene delivery system.Therefore,functional peptides can be designed by tuning amino acids with different characteristics,and further combined with natural polysaccharide via a simple and stable synthesis method,to develop a low-toxic and high-efficient gene delivery system,which is suitable for skin wound therapy and to explore the treatment methods/applications in wound healing.In Chapter two,a cation peptide-grafted dextran conjugate(D-R3H7)was constructed via a simple two-step reaction of transesterification and ThiolMichael,where dextran(Dex)with plentiful amounts of hydroxy serves as a backbone.The combination of highly biocompatible Dex and cation short peptides endowed D-R3H7 with low cytotoxicity in mouse fibroblasts(L929)and rat primary fibroblasts(RFB).Due to the positive arginine(R)and the high proton-buffer-capacity histidine(H),D-R3H7 realized high DNA loading capacity and excellent gene transfection performance.In vitro experiments demonstrated that D-R3H7 achieved the efficient delivery of therapeutic plasmid(ph EGF)encoding human epidermal growth factor and enhanced the expression of exogenous human EGF & endogenous mouse EGF in L929 cells.The therapeutic effect of ph EGF delivered by D-R3H7(D-R3H7/ph EGF)was investigated in detail in a rat full-thickness-skin-defect model.The model of normal rat showed that both of single-high plasmid dose and multiple-low plasmid doses delivery by D-R3H7/ph EGF promoted skin healing,and the former achieved a better effect.The mechanism of this different therapeutic effect was initially revealed: compared with the continuous supplementation of exogenous growth factors,the efficient delivery of exogenous growth factors(single-high plasmid dose in this study)during the initial stage of wound can promote the secretion of rat-EGF and accelerate the wound repair more effectively.In the rabbit ear defect model with less contractures,the effective healing effect of D-R3H7/ph EGF with single-high dose model is further confirmed,which is promising for clinical applications.The commonly-used polymeric-based delivery systems suffer from a critical shortcoming upon dilution(rapidly/non-linear decreased transfection efficiency at low dose),which is advised to their in vivo application and clinical translation.In Chapter 3,an anionic peptide(CH2E5)containing glutamate(E),was designed and grafted onto the Dex backbone to prepare an anionic peptidegrafted dextran conjugate(Dex-H2E5).A ternary gene delivery system(HPD)with Dex-H2E5 as the smart coating was constructed via a classic electrostatic assembly process from the binary cationic polymer-based gene delivery systems.The in vitro results showed that Dex-H2E5 coating decreased the surface and enhanced the cytocompatibility of binary delivery systems.Moreover,HPD achieved the high transfection efficiency under high-serum and low-DNA-dose transfection conditions,as well as high serum stability.In addition,the transfection of ternary complexes formed by other typical polymeric gene vectors can also be improved by the Dex-H2E5 coating.The mechanism of this potent Dex-H2E5 coating was reveled to the favorable high-serum/low-dose stability,cell uptake efficiency,endo/lysosome escape ability,and DNA releasing ability.Based on the high gene transfection performance of HPD system,the therapeutic effect of HPD delivering lower dose of therapeutic plasmid(ph EGF)on rat full-thickness–skin–defect models were investigated in Chapter 4.In vitro experiments showed that HPD effectively delivered ph EGF in L929 cells,upregulated the expression of EGF and promoted cell proliferation.In rat fullthickness skin defect,the application of HPD with single-low plasmid dose realized the effective expression of h EGF in the wounds,and promoted the wound repair,which confirms the high in vivo transfection efficiency of HPD.At the same time,aiming at the problem of less residual cells in severe ulcer or burn wounds,the ex vivo gene therapy of HPD was explored to promote wound healing.In vitro results showed that HPD effectively delivered the reporter plasmid in RFB with dose independence.The allogeneic RFB was pre-delivered ph EGF in vitro by HPD——which was made into cell suspension for wound treatment after genetically modification——and achieved efficient and highquality wound healing.In summary,aimed at the development of low-toxic,high-efficient gene delivery system via stable synthetic methods for skin wound repair,peptidegrafted dextran conjugates were constructed via a simple two-step method of transesterification and Thiol-Michael,where dextran with high biocompatibility and facile modification of hydroxy as a backbone and short cationic peptide was consisting of different structural/functional amino acids.The low cytotoxicity and high transfection efficiency of the delivery system were proved by in vitro experiments.The therapy method,therapeutic effect,and biological safety of gene therapy in skin healing were explored and verified in full-thickness skin defect models of rat and rabbit ear.This work not only provides the promising treatment for the clinical application of gene therapy for skin diseases,but also new approach for the development of efficient gene delivery system. |
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