Preparation,antibacterial Properties Of Antibacterial Polymer And Their Roles In Skin Repairs Of Diabetic Animal’s Ulcers

OBJECTIVE: Guanidine-based polymers were synthesized through chemical reactions,which were further modified based on its functional group reactivity to reduce its cytotoxicity.Finally,we explored the antibacterial properties of various materials,and applied them to the skin of ulcers in diabetic animals to test their effect on promoting skin repair.METHODS: A series of guanidine-containing compounds were synthesized by guanidine hydrochloride and amine compound as raw materials at 160 ℃ for five hours,including polytrimethylene guanidine(PTMG 13),polytetramethylene guanidine(PTMG 14),polyhexamethylene guanidine(PHMG),polyheptamethylene guanidine(PHMG 17),polyoctamethylene guanidine(POMG 18).PHMG was chosen as reactant for further synthesizing two modified products: one was based on hyaluronic acid,which used the characteristics of its carboxyl group to synthesize product(PHMGH)with guanidyl cations on PHMG;The other one is to use chitosan as a raw material,which will crosslink with PHMG in the presence of crosslinking agents glutaraldehyde,resulting into the hydrogel product(PHMGC).Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa(E.coli,MRSA USA 300,PAO 1,PGO2330),which are commonly found in skin infections,were selected as research strains.The antibacterial effect was explored by measuring the minimum inhibitory concentration and the zoon of the inhibition.3T3 fibroblasts and primary animal bone marrow mesenchymal stem cells were selected for cytotoxicity measurement to investigate the biocompatiblility of the materials.ICR male mice were used as experimental animals,streptozotocin(STZ)was used to establish type I diabetes,a full-thickness skin injury model was created without bacteria and a full-thickness skin injury model was infected with bacteria for comparison.After applying PHMG,PHMGH,PHMGC and PBS(control),the wounds gradually healed.In order to explore the effect of bioremediation,we measured and counted the changes in wound diameter,the number of white blood cells in the blood,and performed histological morphological imaging lasting for 28 days.RESULTS: A variety of guanidine-based polymers PTMG 13,PTMG 14,PHMG,PHMG17,and POMG 18 were successfully synthesized.The antibacterial performance test found that with the increase of substrate methylene,the antibacterial ability of the products gradually increased.Polyhexamethylene guanidine(PHMG)synthesized from guanidine hydrochloride and1,6-hexanediamine was selected to be further modified with chitosan and hyaluronic acid.Through antibacterial tests,we found that among the four bacteria tested(E.coli,MRSA USA 300,PAO 1,PGO 2330),both the guanidine-based polymers and modified products showed excellent antibacterial properties,superior to the common chitosan season.The ammonium salt antibacterial agents even approach or exceed the antibacterial effect of the antibiotic drug erythromycin.Cytotoxicity experiments were used to test the effect of samples’ biocompatibility.3T3 fibroblasts and primary mesenchymal stem cells were selected as research objects.The effect of various concentrations of PHMG on the viability of these two cells were tested.The antibacterial ability of PHMG has a certain toxic effect on the two cells.The cytotoxicity experiments for PHMGC and PHMGH showed that the two modified products can significantly reduce the cytotoxicity of the materials.In the experiments on diabetic mice,we found that antibacterial materials(PHMG,PHMGC,PHMGH)have significant in vivo antibacterial effects.In the case of both the uninfected wounds with bacteria and infected with S.aureus.The treatment group by antibacterial materials showed that the animal’s wounds are clean and free of flow pus,no serious secretions,and less redness.After skin injury,it is necessary to go through an inflammatory phase,a tissue hyperplasia phase,and a remodeling phase.Among them,the inflammatory phase is a key stage that determines whether the skin can enter the next few phases.Especially for people with diabetes,their immune system is low and their antibacterial and anti-inflammatory capabilities are reduced.It is much more necessary to use external antibacterial agents to help the body fight against microbial pollution.Therefore,the excellent antibacterial properties of the guanidine-based polymer materials have a positive effect on the healing of the damaged skin of diabetic patients.However,PHMG exhibits a high bactericidal function and also has a killing effect on tissue cells.This has also been found in cell compatibility experiments in vitro that the materials inhibit growth of fibroblasts and primary bone marrow mesenchymal stem cells.However,the modified PHMGH and PHMGC are relatively less toxic to the above two cells.The effect of PHMGC and PHMGH on wound size in animal experiments is small,especially in the middle and late stages of skin repair,which is faster than that of the blank group,reflecting the advantages of antibacterial materials.CONCLUSION: Polyhexamethylene guanidine(PHMG)and the products(PHMGC,PHMGH)obtained by chemical modification of chitosan and hyaluronic acid,are effective in all four bacteria(E.coli,MRSAUSA300,PAO 1,PGO 2330).In the damaged skin repair experiments of diabetic mice,the bacterial infection and uninfected model wounds significantly reduced the degree of wound inflammation in the uncoated blank group of animals,and also facilitated the final wound repair,which played an important role in promoting skin regeneration.The effect of the antibacterial and repair properties can be used as a new type of wound dressing for the treatment of diabetic ulcer skin.