Study On The Preparation And Properties Of Polyphosphazene Based Hydrogels For Tissue Repair

Tissue repair hydrogels(TRHs)show a good application prospect in the field of biomedicine due to their controlled structure,drug loading and biocompatibility.Currently,TRHs have been widely reported,but they usually have a single function,which is difficult to meet differential needs of dynamic repair in different stages of trauma(hemostasis,inflammation,proliferation and remodeling).The preparation of hemostatic,antibacterial,anti-inflammatory and healing promoting TRHs is an important way to solve the above problems.In this thesis,a series of TRHs with antibacterial,anti-inflammatory and tissue adhesion properties were prepared by covalent cross-linking or dynamic cross-linking based on polyphosphazene with good biocompatibility,controllable structure and biodegradation,and their properties and applications in tissue repair were studied,and the possible mechanism was discussed.(1)Linear polydichlorophosphazene(PDCP)was prepared by thermal ringopening polymerization of HCCP.Using PDCP,N,N-dimethylethylenediamine(DMAEA)and 3-bromopropene as raw materials,the macromolecular crosslinking agent poly(dimethylaminoethyl-allyl ammonium bromide)phosphazene(AFP)was prepared by two-step nucleophilic substitution reaction.The structure of AFP was characterized by 1H NMR and 31P NMR.Using N-vinylpyrrolidone(NVP)as monomer,AFP as crosslinking agent,2-hydroxy-4’-(2-hydroxy-ethoxy)-2-methylphenylacetone(Irgacure 2959)as photoinitiator,quaternary ammonium group functionalized AFPNVP hydrogels were prepared by free radical polymerization.AFP-NVP hydrogels were impregnated with standard iodine solution to prepare AFP-NVP-I hydrogels.The swelling behavior,composition,mechanical properties,bacteriostatic properties,biocompatibility and promoting healing of infected wounds were investigated,respectively.The results showed that the AFP has been successfully synthesized and iodine has been successfully complexed in the hydrogels.The swelling behavior,mechanical properties and contact antibacterial properties of the obtained hydrogels were all affected by the content of AFP and NVP in the hydrogels.The bacteriostasis rate of AFP3-NVP20 against E.coli reached 98.6±1.6%by the quaternary ammonium group on the surface of the uniodized hydrogel.After iodine complexation,AFPiNVP20-Ⅰ hydrogel achieved almost 100%bacteriostasis after contacting with E.coli for 18 h through the synergistic action of quaternary ammonium group and iodine on the surface.In the rat wound model infected with S.aureus,AFP1-NVP20 and AFP1-NVP20I hydrogels could inhibit 92.7 ± 1.8%and 93.7± 0.6%of S.aureus.Compared with the wound closure rate of Tegaderm film group(90.1± 2.7%)on the 12th day,the wound closure rate of AFP1-NVP20 and AFP1-NVP20-I hydrogels groups were as high as 97.7 ± 1.1%and 98.5 ± 0.5%.The results indicated that the prepared hydrogels had a good application prospect as an anti-infection wound dressing.(2)Using PDCP,DMAEA and 3-bromomethyl phenylboric acid(BPBA)as raw materials,poly[(N,N-dimethyl ethylenediamine)-g-(N,N,N,N-dimethylamine ethyl methyl benzene boric acid ammonium bromide)]phosphazene(PPBA)was obtained by nucleophilic substitution reaction.The structure of PPBA was characterized by 1H NMR and 31P NMR.Using PPBA and polyvinyl alcohol(PVA)as raw materials,PPBA-PVA hydrogels with dynamic borate ester bond as crosslinking bond was prepared by simple mixing.The composition,morphology,self-healing properties,tissue adhesion properties,antibacterial properties,biocompatibility,hemostatic properties and wound healing promoting properties of PPBA-PVA were investigated.The results showed that PPBA-PVA hydrogel had been synthesized successfully.The adhesion strength of PPBA-PVA hydrogel to tissues was up to 47.1 ± 2.5 kPa,and the 2 h contact inhibition efficiency of PPBA-PVA hydrogels to E.coli was more than 95%,indicating that the hydrogels obtain good tissue adhesion and inherent antibacterial properties.The prepared PPBA20-PVA samples can also achieve good adhesion strength in extreme underwater environment and exhibit long-term stable adhesion.In addition,PPBA-PVA was able to reduce 87.5%blood loss in rat hepatic hemorrhage models.Compared with the wound closure rate of Tegaderm film group on the 9th day(75.8 ± 2.3%),the wound closure rate of PPBA30-PVA sample was up to 92.9±3.5%.The prepared hydrogel material has good hemostatic and promoting healing properties.(3)Taking PPBA and tannic acid(TA)as raw materials,the drug cojugates PPBATA with reactive oxygen species(ROS)responsive borate bond as bridging linker were prepared by simple mixing the raw materials.The formation of PPBA-TA was characterized by fluorescence spectrophotometer.Multistage ROS response PPBA-TAPVA drug conjugated hydrogel was prepared by simple mixing the PPBA-TA conjugates and PVA solutions.FTIR,SEM,ROS scavenging test,ROS responsive degradation test,contact inhibition test,cell migration test,CCK-8,wound healing promotion test in diabetic rats,q-PCR and ELISA were used to characterize the composition,morphology,ROS scavenging performance,ROS response degradation performance and antibacterial efficiency of PPBA-TA-PVA,respectively.Cell migration promotion,biological toxicity and anti-inflammatory activity in vivo were studied.The results showed that the scavenging rate of 1,1-diphenyl-2trinitrophenylhydrazide(DPPH)was up to 98.5± 1.2%at 20 h,and the scavenging rate of OH radical was up to 97.0 ± 0.6%at 10 h,which indicating hydrogels obtain good free radical scavenging performance.ROS degradation tests showed that the degradation rate of PPBA-TA-PVA was accelerated with the increase of TA concentration.In the wound healing experiment of diabetic rats,compared with the wound closure rate of Tegaderm film group on the sixth day(44.0±4.4%),the wound closure rate of PPBA-TA-PVA drug-coupled hydrogel group reached 84.5± 6.0%,significantly accelerating the healing speed of diabetic wounds.PPBA-TA-PVA can inhibit the expression of inflammatory factors(IL-6 and IL-1β)in early wounds,and promote the rapid proliferation of diabetic wounds.The prepared hydrogel material has good performance in promoting wound healing of chronic diabetes mellitus.(4)Using PPBA and ethyl bromoacetate(EBA)as raw materials,Poly(N,Ndimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl ammonium bromide(ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl acetate ethyl ammonium bromide)]phosphazene(PZBA)was prepared by quaternization reaction.The structure of PZBA was characterized by 1H NMR and 31P NMR.By reacting PZBA with TA,epigallocatechin(EGC)and epigallocatechin gallate(EGCG),three drug conjugates were obtained by using reactive oxygen species(ROS)responsive borate bonds as conjugated chemical bonds.The drug conjugations were characterized by fluorescence spectrophotometer.Three biodegradable drug conjugated hydrogels PZBA-TA-GG,PZBA-EGC-GG and PZBA-EGCG-GG were prepared by reacting the above three drug conjugated compounds with biodegradable guar gum(GG),respectively.The micromorphology,injectable performance,self-healing performance,ROS clearance performance,ROS clearance performance,biotoxicity,adhesion prevention performance after Achilles tendon operation and anti-inflammatory activity of drugcoupled hydrogels were investigated by SEM,rheology test,ROS clearance test,CCK8 test and q-PCR.The results showed that the drug-coupled hydrogels had good injectable and self-healing properties,and had a good application prospect in the field of minimally invasive surgery.In addition,the DPPH clearance rates of PZBA-TA-GG,PZBA-EGC-GG and PZBA-EGCG-GG hydrogels were 21.3 ± 2.1%,87.2±4.5%and 82.5± 1.4%for 2 h.The 4 h scavenging rates of OH free radical were 33.6±1.4%,73.1± 4.1%and 90.5± 2.2%,respectively,which indicated that the obtained drugcoupled hydrogels had good free radical scavenging performance.In the test of antiadhesion of Achilles tendon surgery,PZBA-TA-GG,PZBA-EGC-GG and PZBAEGCG-GG drug conjugate hydrogels can effectively prevent postoperative adhesion in Achilles tendon bruising and Achilles tendon rupture models,and PZBA-EGCG-GG drug conjugate hydrogels can reduce the grade of adhesion of Achilles tendon from grade 5 to grade 1.In the early stage of injury,the expression of inflammatory factors(TNF-1α,HIF-1α,IL-1β and IL-6)is inhibited,which has good anti-inflammatory activity.In the late stage of injury,the expression of cytokines related to collagen regeneration(TGF-β,COL-1 and COL-3)was promoted,and the prepared hydrogel material had good performance in preventing postoperative adhesion of Achilles tendon.