阳离子聚合物在医用金属表面的可控构建及其抗菌性能研究

【作者】 刘莉；

【导师】 刘平生；

【作者基本信息】 南京师范大学 ， 高分子化学与物理， 2020， 硕士

【摘要】 金属材料因具有良好的机械强度、优异的力学性能和良好的生物相容性而被广泛用于生物医学领域。然而,由于缺乏有效的抗菌性能,其表面存在容易引起细菌滋生并导致感染的严重问题,从而极大地降低金属医用材料及器械在体内的留滞时间以及诊疗的成功率。因此,表面抗菌性能的提升是植/介入金属医用材料及器械亟待解决的关键问题之一。表面改性是一种提高材料表面性能的有效手段。它能在保持基材基础特性不变的情况下显著提高其表面的性能。近年来,为提高医用金属生物材料的抗菌性能,大量的表面改性抗菌策略被报道。通过硅烷偶联剂偶联和多巴胺聚合将功能聚合物接枝到金属材料表面是提高表面性能的常用方法。然而,硅烷偶联剂与金属形成的Si-O-Ti键的水解稳定性弱;而基于多巴胺聚合涂层的稳定性仍然是需解决的问题。近年来,膦酸（酯）化合物因能够与金属及金属氧化物形成牢固的配位作用受到广泛关注。因此,本论文利用膦酸酯结构与金属材料形成稳定的配位作用,结合阳离子聚合物优异的广谱抗菌性能以及两性离子材料优异的抗生物粘附性能,设计并制备了一系列抗菌聚合物涂层材料,并将其构建到金属材料表面,系统考察了共聚物涂层对金属表面抗菌/防污性能的影响,具体研究内容如下:（1）膦酸酯/季铵盐阳离子嵌段共聚物涂层对金属表面抗菌性能的影响研究。通过可逆加成-断裂链转移聚合（RAFT）和季铵化反应制备了3种含不同长度季铵盐链段的膦酸酯阳离子嵌段共聚物。采用¹H NMR对嵌段共聚物的化学结构进行准确表征;通过静态水接触角（WCA）和XPS测试对膦酸酯/季铵盐阳离子嵌段共聚物修饰的钛合金表面的亲水性能和表面元素组成进行了考察;采用体外抗菌实验（平板计数法、SEM、CLSM观察）系统研究了不同链段长度的嵌段共聚物对钛合金表面抗菌性能的影响;对共聚物涂层的细胞毒性进行了评价。在此基础上,将共聚物涂层构建在不同金属表面,进一步研究共聚物涂层对提高医用金属表面抗菌性能的普适性。¹H NMR实验结果表明精准合成了不同链段长度的膦酸酯/季铵盐阳离子嵌段共聚物pDEMMP-b-pTMAEMA,其中膦酸酯链段长度为15,季铵盐阳离子链段长度分别为8,45和70;WCA和XPS测试结果表明pDEMMP-b-pTMAEMA成功构建到钛合金表面;体外抗菌实验（平板计数法、SEM、CLSM观察）结果一致表明,改性后的钛合金表面具有高效、广谱的抗菌能力:随着时间的延长和季铵盐链段长度的增长,杀菌效果更显著,接触8小时后,季铵盐阳离子聚合物涂层(P₇₀)对其表面初始接种密度为1.5×10⁴ CFU/cm²的S.aureus的杀菌率为96%和初始接种密度为0.5×10⁴ CFU/cm²的E.coli的杀菌率为93%;细胞毒性测试结果表明共聚物涂层具有良好的生物相容性;不同金属表面抗菌性能测试结果表明pDEMMP-b-pTMAEMA涂层对提高多种医用金属表面的抗菌性能具有普适性。（2）膦酸酯/季铵盐阳离子无规共聚物涂层对金属表面抗菌性能的影响研究。通过无规自由基聚合制备了7种不同配比的(n_DEMMP:n_DMAEMA=5:95～95:5)膦酸酯/第三胺无规共聚物（pDEMMP-co-pDMAEMA）,进一步通过季铵化得到膦酸酯/季铵盐阳离子无规共聚物（pDEMMP-co-pTMAEMA）。并通过一步法将pDEMMP-co-pTMAEMA构建到钛合金表面。采用¹H NMR对聚合物的化学结构进行精确表征;采用静态水接触角（WCA）和XPS测试对膦酸酯/季铵盐阳离子无规共聚物修饰过的钛合金表面的亲水性能和表面元素组成进行了考察;采用体外抗菌实验（平板计数法、SEM、CLSM观察）系统研究了不同配比的无规共聚物对钛合金表面抗菌性能的影响;对共聚物涂层的细胞毒性进行了评价;在此基础上,将共聚物涂层构建在不同金属表面,进一步研究共聚物涂层对提高医用金属表面抗菌性能的普适性。¹H NMR实验结果表明精准合成了不同配比的膦酸酯/季铵盐阳离子无规共聚物pDEMMP-co-pTMAEMA;WCA和XPS测试结果表明pDEMMP-co-pTMAEMA成功构建到钛合金表面;体外抗菌实验（平板计数法、SEM、CLSM观察）结果一致表明,改性后的钛合金表面具有高效、广谱的抗菌能力:接触8小时后,摩尔比介于n_DEMMP:n_TMAEMA=5:95～90:10的pDEMMP-co-pTMAEMA能够充分协同地发挥膦酸酯结构的“锚定”作用以及季铵盐结构的杀菌功能,对其表面初始接种密度为1.5×10⁴ CFU/cm²的金黄色葡萄球菌的杀菌率≥87%和初始接种密度为0.5×10⁴ CFU/cm²的大肠杆菌的杀菌率≥93%;细胞毒性测试结果表明共聚物涂层具有良好的生物相容性;不同金属表面抗菌性能测试结果表明pDEMMP-co-pTMAEMA涂层对提高多种医用金属表面的抗菌性能具有普适性。（3）膦酸酯/两性离子/季铵盐阳离子嵌段共聚物涂层对金属表面抗菌和防污性能的影响。通过RAFT聚合和季铵化反应制备了5种不同链段长度的膦酸酯/两性离子/季铵盐阳离子嵌段共聚物;采用¹H NMR对聚合物的化学结构进行精确表征;采用静态水接触角（WCA）和XPS测试对共聚物修饰过的钛合金表面的亲水性能和表面元素组成进行了考察;利用抗菌/防污等实验（CFU、SEM、CLSM观察）系统表征了嵌段共聚物涂层对钛合金表面抗菌、防污性能的影响;对聚合物涂层的细胞毒性进行了评价;在此基础上,将共聚物涂层构建在不同金属表面,进一步研究共聚物涂层对提高医用金属表面抗菌/防污性能的普适性。¹H NMR结果表明精准合成了不同链段长度的pDEMMP-b-p（SBMA-co-TMAEMA）;WCA和XPS测试结果表明pDEMMP-b-p（SBMA-co-TMAEMA）成功构建到钛合金表面;体外抗菌/防污实验（CFU、SEM、CLSM观察）一致表明:在两性离子聚合物和季铵盐聚合物的协同（杀菌-防污双重功能）作用下,五种聚合物涂层改性后的钛合金表面均具有优良抑制细菌在基材表面的粘附及生物被膜的形成;同时,体外细胞毒性测试结果表明共聚物涂层具有良好的生物相容性;不同金属表面抗菌/防污性能测试结果表明pDEMMP-b-p（SBMA-co-TMAEMA）涂层对提高多种医用金属表面的抗菌/防污性能具有普适性。更多还原

【Abstract】 Due to good mechanical properties and biocompatibility,metallic substrates have been widely used as biomedical implants.However,the lack of antibacterial ability make it prone to trigger bacterial infection,which may largely shorten the indwelling time of metallic medical devices in body and compromise the success rate of treatment.Therefore,improving the antibacterial property is one of essential requirements for biomedical metals.Surface modification is a promising strategy to improve the surface property of the substrate without compromising the inherent bulk properties.In recent years,in order to improve the antibacterial performance of medical metal,numerous surface modification strategies have been reported.Silane coupling and dopamine polymerization are two common methods to“graft”functional polymers on metallic substrates to improve the surface properties.However,the stability of Si-O-Ti bond associated with silane coupling agent is weak,while the stability is still a remaining issue of the dopamine based surface coating.In recent years,phosphorus-containing compounds have attracted great attention for their strong coordination with metals and metal oxides.Therefore,take advantage of the ability of phosphonate group to react with metals and metal oxides,combined with the good antibacterial properties of cationic polymer and the excellent antifouling properties of the zwitterionic materials,a series of functional antibacterial&antifouling polymers were prepared.The polymers were constructed on the surfaces of metal substrates,and the effects of the functional coating on the antifouling/antibacterial properties of metallic substrates were systematically investigated.The detailed research contents are as follows:（1）Effect of phosphonate/quaternary ammonium cationic block copolymers coating on antibacterial properties of the surface metallic substrates.Three phosphonate/quaternary ammonium cationic block copolymers with different quaternary ammonium salt segments were prepared by RAFT polymerization and quaternization reaction.The chemical structures of the polymers were characterized by ¹H NMR.The surface wettability and elemental composition of TC4 surfaces were characterized by WCA and XPS.The effect of block copolymer with different chain lengths on the surface antibacterial properties of TC4 substrates were systematically investigated（plate counting,SEM and CLSM）.The toxicity of copolymer coating to cells was tested in vitro.The generality of the block copolymer to improve the antibacterial performance of different metal substrates were also investigated.¹H NMR results showed the precise synthesis of phosphonate/quaternary ammonium cationic block copolymers with different chain lengths（pDEMMP-b-pTMAEMA）.WCA and XPS experiments results showed pDEMMP-b-pTMAEMA were successfully constructed on the surface of TC4 substrates.In vitro antibacterial tests showed the broad and high-efficiency antibacterial ability of modified TC4 substrates:the longer of contact time and the cationic segment length,the better bactericidal property endowed to TC4 substrates.After 8 hours of contact with bacteria,the polymer coatings(TC4-P₇₀)were able to kill 96%to the S.aureus（initial inoculation density≤1.5×10⁴ CFU/cm²）and 93%to the E.coli（initial inoculation density≤0.5×10⁴ CFU/cm²）.In addition,pDEMMP-b-pTMAEMA coating could be a general antibacterial coating for biomedical metals.（2）Effect of phosphonate/quaternary ammonium cationic random copolymers coating on the antibacterial properties of metallic substrate.Seven random copolymers pDEMMP-co-pDMAEMA(n _DEMMP:n _DMAEMA=5:95～95:5)were prepared by random radical polymerization and quaternization reaction.The chemical structures of the polymer were characterized by ¹H NMR.The surface wettability and elemental composition of TC4 surfaces were characterized by WCA and XPS.The effect of random copolymers with different proportions coating on the surface antibacterial properties of TC4 substrates were investigated（plate counting,SEM and CLSM）.The toxicity of copolymer coating to cells was tested in vitro.The generality of the random copolymer to improve the antibacterial performance of different metal substrates were also investigated.¹H NMR results showed the precise synthesis of phosphonate/quaternaryammoniumcationicrandomcopolymers（pDEMMP-co-pTMAEMA）with varied compositions.WCA and XPS experiments results showed pDEMMP-co-pTMAEMA were successfully constructed on the surface of TC4 substrates.In vitro antibacterial tests consistently indicated that the broad and high-efficiency antibacterial ability of the coating on TC4 substrates:after8-hour contacting with bacteria,the copolymers(n _DEMMP:n _TMAEMA=5:95～90:10)can efficiently kill the bacterial attached on TC4 substrates（87%for S.aureus and93%for E.coli）without significant compromising the cytocompatibility of TC4substrates.In addition,pDEMMP-co-pTMAEMA coating could be a general antibacterial coating for biomedical metals.（3）Effect of phosphonate/zwitterionic/quaternary ammonium cationic block copolymers coating on the antibacterial/antifouling properties of metallic substrates.Five phosphonate/zwitterion/quaternary ammonium salt cationic block copolymers were prepared by RAFT polymerization and quaternization reaction.The chemical structures of the polymers were characterized by ¹H NMR.The surface wettability and elemental composition of TC4 surfaces were characterized by WCA and XPS.The effect of block copolymer coating on the surface antibacterial and antifouling properties of TC4 substrates were systematically investigated（plate counting,SEM and CLSM）.The toxicity of copolymer coating to cells was tested in vitro.The generality of the block copolymer to improve the antibacterial/antifouling performance of different metal substrates were also investigated.¹H NMR results showed the precise synthesis of pDEMMP-b-p（SBMA-co-TMAEMA）.WCA and XPS experiments results showed pDEMMP-b-p（SBMA-co-TMAEMA）were successfully constructed on the surface of TC4 substrates.In vitro antibacterial and antibacterial tests indicated the all five polymer modified TC4 substrates can efficiently inhibit the adhesion of bacterial and consequent biofilm formation.Furthermore,the polymer coatings can significantly improve the antibacterial property without significantly compromising the cytocompatibility of the substrates.In addition,pDEMMP-b-p（SBMA-co-TMAEMA）coating could be a general antibacterial/antifouling coating for biomedical metals.更多还原