CN107137762A - 一种医用钛或钛合金表面抗菌涂层的构建方法 - Google Patents

一种医用钛或钛合金表面抗菌涂层的构建方法 Download PDF

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CN107137762A
CN107137762A CN201710331528.0A CN201710331528A CN107137762A CN 107137762 A CN107137762 A CN 107137762A CN 201710331528 A CN201710331528 A CN 201710331528A CN 107137762 A CN107137762 A CN 107137762A
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antimicrobial coating
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方立明
赵亮
陈海婷
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South China University of Technology SCUT
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Abstract

本发明公开了一种医用钛或钛合金表面抗菌涂层的构建方法，包括以下步骤：（1）将医用钛或钛合金样品表面用砂纸打磨抛光；（2）将抛光后的样品表面用piranha溶液处理；（3）以无水甲苯溶解带有氨基的硅烷偶联剂，得到硅烷偶联剂溶液，将样品在此溶液中反应，然后固化；（4）将含有Mal‑PEGn‑NHS的DMF溶液滴至步骤（3）的样品表面进行反应；（5）将含有抗菌多肽的点击反应溶液滴至步骤（4）的样品表面进行反应，再超声清洗，得到抗菌涂层。本发明提高了抗菌多肽的利用率，使抗菌效果可以长时间的维持。本发明显著提高了金属钛及钛合金的抗菌性能，且具有优异的生物相容性，可广泛用于临床医用抗菌材料。

Description

一种医用钛或钛合金表面抗菌涂层的构建方法
技术领域
本发明属于生物医用材料技术领域，具体涉及一种医用钛或钛合金表面抗菌涂层的构建方法。
背景技术
医用钛及钛合金由于其优异的生物学性能、综合力学性能及耐腐性，被广泛应用于外科植入手术，常作为如牙种植体、人工关节和骨创伤产品等人体硬组织替代物和修复物的首选材料。但随着植入材料的广泛应用，植入物相关感染已经成为临床上一个非常棘手的问题。研究表明细菌在生物材料表面的黏附、繁殖并形成细菌生物膜是引发植入物相关感染的主要原因之一。因此，对钛及钛合金植入材料进行表面改性，从而赋予其优异抗菌性能是目前生物医用材料领域的一个研究热点。随着抗生素滥用导致了严重的细菌耐药性问题，传统抗生素已不能满足要求，因此具有高效、广谱抗菌且不易产生耐药性的抗菌多肽成为抗感染植入材料研究的热点趋势之一。抗菌多肽广泛存在于生物体内，一般有10～50个氨基酸序列组成，带正电荷并呈疏水性，一般认为抗菌多肽杀菌机理主要是抗菌多肽的正电荷与细菌的细胞膜的负电荷发生作用,致使细菌细胞膜破碎,导致细菌胞内物溢出而使细菌死亡。由于其杀菌机理是直接破坏细菌细胞膜，导致不易产生耐药性而受到广泛的关注。而由于物理吸附抗菌多肽涂层，会有突释、药物利用率低、毒性大等缺点，化学接枝可以很好地克服这些问题。但直接在植入物材料表面接枝抗菌多肽，会导致抗菌多肽不能完全达到和游离的抗菌多肽的构象，从而影响其抗菌性能，因此通过PEG作为链接剂可以很好地解决这一问题。
发明内容
为了克服现有技术的上述缺点与不足，本发明的目的在于提供一种医用钛及钛合金表面抗菌涂层的构建方法。该方法通过对样品表面的piranha溶液处理，硅烷化，PEG的接枝和抗菌多肽的接枝，实现了抗菌多肽成功的接枝在金属钛及钛合金表面，使抗菌多肽在化学接枝的情况下仍能保持原有的抗菌构象，能够显著提高钛及钛合金的抗菌性能，并使抗菌效果长时间维持，提高抗菌多肽的利用率，并使其保持良好的生物相容性。
本发明的目的通过以下技术方案实现。
一种医用钛或钛合金表面抗菌涂层的构建方法，包括以下步骤：
(1)将医用钛或钛合金样品用砂纸打磨抛光进行表面处理；
(2)将抛光后的样品表面用piranha溶液(70％H2SO4+30％H2O2)处理；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂，得到硅烷偶联剂溶液，将步骤(2)所得样品浸泡于硅烷偶联剂溶液中反应，然后固化；
(4)将含有Mal-PEGn-NHS(马来酰亚胺-聚乙二醇-羟基琥珀酰亚胺)的DMF溶液滴至步骤(3)所得样品表面进行反应；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)所得样品表面进行反应，再超声清洗，得到抗菌涂层；所述含有抗菌多肽的点击反应溶液的浓度为0.5～3μM，每平方厘米样品表面滴加1～4μL。
优选的，步骤(1)所述打磨抛光至样品表面Ra小于40nm。
优选的，步骤(2)所述piranha溶液处理的时间为5min～30min。
优选的，步骤(3)所述硅烷偶联剂溶液的体积浓度为2％～5％。
优选的，步骤(3)所述反应的条件为：时间1-3h，温度60-90℃，反应中含氧量低于2ppm。
优选的，步骤(4)所述含有Mal-PEGn-NHS的DMF溶液的浓度为0.5～3μM，每平方厘米样品表面滴加1～4μL。
优选的，步骤(4)所述反应是在室温下反应6-12h。
优选的，步骤(4)所述Mal-PEGn-NHS中PEG的分子量为100～10000。
优选的，步骤(5)所述反应是在室温下反应12-24h。
优选的，步骤(5)所述的抗菌多肽为C-PEG2-PEG2-GRRRRSVQWCA；其中C-PEG2-PEG2-GRRRRSVQWCA的结构式如下：
本发明与现有技术相比具有以下优点：
(1)本发明的抗菌涂层的构建方法得到的医用钛及钛合金表面具有优异的抗菌性能及生物相容性。
(2)本发明使用点击化学反应接枝抗菌多肽，反应条件温和，效率高。
(3)本发明使用化学法接枝抗菌多肽，提高了抗菌多肽的利用率并提高了有效抗菌时间。
(4)本发明使用PEG作为链接剂接枝抗菌多肽，可以使抗菌多肽保持游离的构象从而达到更好的抗菌效果。
附图说明
图1为本发明所用的抗菌多肽的分子式结构图。
图2a为本发明医用钛对金黄色葡萄球菌的扫描电镜图。
图2b为本发明实施例1所得涂层对金黄色葡萄球菌的扫描电镜图。
图2c为本发明实施例2所得涂层对金黄色葡萄球菌的扫描电镜图。
图2d为本发明实施例3所得涂层对金黄色葡萄球菌的扫描电镜图。
图2e为本发明实施例4所得涂层对金黄色葡萄球菌的扫描电镜图。
图3为本发明实施例1～4所得涂层对金黄色葡萄球菌的抗菌效果图。
具体实施方式
下面结合具体施例，对本发明作进一步的详细说明，但本发明的实施方式不限于此。
以下所用的抗菌多肽的分子式结构图如图1所示。
实施例1
(1)将待处理的医用钛样品进行表面处理，即用砂纸打磨抛光至Ra小于40nm；
(2)将抛光后的样品表面放入piranha溶液中处理20min；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂KH-550，得到硅烷偶联剂溶液(体积浓度为2％)，将样品浸泡于硅烷偶联剂溶液中反应2h，反应温度为70℃，含氧量小于2ppm，然后在常温下固化2h；
(4)将含有Mal-PEGn-NHS的DMF溶液滴至步骤(3)的样品表面，在常温下反应6h；PEG分子量为250，Mal-PEGn-NHS的DMF溶液浓度为1μM，每平方厘米表面滴加1μL；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)的样品表面，在常温下反应12h，超声清洗，在医用钛表面得到抗菌涂层；含有抗菌多肽的点击反应溶液的浓度为1μM，每平方厘米表面滴加1μL。
在医用钛表面培养金黄色葡萄球菌(菌液浓度为106CFU/ml)2h后的扫描电镜图如图2a所示。图2b为在该抗菌涂层表面培养金黄色葡萄球菌(菌液浓度为106CFU/ml)2h后的扫描电镜图。和图2a相比，表面细菌数量有所减少，表明该抗菌图层具有一定的抗菌性能。
实施例2
(1)将待处理的医用钛样品进行表面处理，即用砂纸打磨抛光至Ra小于40nm；
(2)将抛光后的样品表面放入piranha溶液中处理20min；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂KH-550，得到硅烷偶联剂溶液(体积浓度为2％)，将样品浸泡于硅烷偶联剂溶液中反应2h，反应温度为70℃，含氧量小于2ppm，然后在常温下固化2h；
(4)将含有Mal-PEGn-NHS的DMF溶液滴至步骤(3)的样品表面，在常温下反应6h；PEG分子量为1000，Mal-PEGn-NHS的DMF溶液的浓度为1μM，每平方厘米表面滴加1μL；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)的样品表面，在常温下反应12h，超声清洗，在医用钛表面得到抗菌涂层；含有抗菌多肽的点击反应溶液的浓度为1μM，每平方厘米表面滴加1.5μL。
如图2c所示为在该抗菌涂层表面培养金黄色葡萄球菌(菌液浓度为106CFU/ml)2h后的扫描电镜图。和图2a相比，表面细菌数量有所减少，表明该抗菌图层具有一定的抗菌性能。
实施例3
(1)将待处理的医用钛样品进行表面处理，即用砂纸打磨抛光至Ra小于40nm；
(2)将抛光后的样品表面放入piranha溶液中处理20min；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂KH-550，得到硅烷偶联剂溶液(体积浓度为5％)，将样品浸泡于硅烷偶联剂溶液中反应2h，反应温度为70℃，含氧量小于2ppm，然后在常温下固化2h；
(4)将含有Mal-PEGn-NHS的DMF溶液滴至步骤(3)的样品表面，在常温下反应6h；PEG分子量为3400，Mal-PEGn-NHS的DMF溶液浓度为1μM，每平方厘米表面滴加1μL；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)的样品表面，在常温下反应12h，超声清洗，在医用钛表面得到抗菌涂层；含有抗菌多肽的点击反应溶液的浓度为1μM，每平方厘米表面滴加1.5μL。
如图2d所示为在该抗菌涂层表面培养金黄色葡萄球菌(菌液浓度为106CFU/ml)2h后的扫描电镜图。和图2a相比，表面细菌数量明显减少，表明该抗菌图层具有优异的抗菌性能。
实施例4
(1)将待处理的医用钛样品进行表面处理，即用砂纸打磨抛光至Ra小于40nm；
(2)将抛光后的样品表面放入piranha溶液中处理20min；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂KH-550，得到硅烷偶联剂溶液(体积浓度为5％)，将样品浸泡于硅烷偶联剂溶液中反应2h，反应温度为70℃，含氧量小于2ppm，然后在常温下固化2h；
(4)将含有Mal-PEGn-NHS的DMF溶液滴至步骤(3)的样品表面，在常温下反应6h；PEG分子量为10000，Mal-PEGn-NHS的DMF溶液浓度为1μM，每平方厘米表面滴加1μL；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)的样品表面，在常温下反应12h，超声清洗，在医用钛表面得到抗菌涂层；含有抗菌多肽的点击反应溶液的浓度为1μM，每平方厘米表面滴加1.5μL。
如图2d所示为在该抗菌涂层表面培养金黄色葡萄球菌(菌液浓度为106CFU/ml)2h后的扫描电镜图。和图2a相比，表面细菌数量明显减少，表明该抗菌图层具有优异的抗菌性能。
由图3可知，与医用钛相比，本发明实施例1-4所制得的涂层对金黄色葡萄球菌具有更好的抗菌效果。

Claims (10)

1.一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，包括以下步骤：
(1)将医用钛或钛合金样品用砂纸打磨抛光进行表面处理；
(2)将抛光后的样品表面用piranha溶液处理；
(3)以无水甲苯为溶剂溶解带有氨基的硅烷偶联剂，得到硅烷偶联剂溶液，将步骤(2)所得样品浸泡于硅烷偶联剂溶液中反应，然后固化；
(4)将含有Mal-PEGn-NHS的DMF溶液滴至步骤(3)所得样品表面进行反应；
(5)将含有抗菌多肽的点击反应溶液滴至步骤(4)所得样品表面进行反应，再超声清洗，在医用钛或钛合金表面得到抗菌涂层；所述含有抗菌多肽的点击反应溶液的浓度为0.5～3μM，每平方厘米样品表面滴加1～4μL。
2.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(1)所述打磨抛光至样品表面Ra小于40nm。
3.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(2)所述piranha溶液处理的时间为5min～30min。
4.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(3)所述硅烷偶联剂溶液的体积浓度为2％～5％。
5.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(3)所述反应的条件为：时间1-3h，温度60-90℃，反应中含氧量低于2ppm。
6.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(4)所述含有Mal-PEGn-NHS的DMF溶液的浓度为0.5～3μM，每平方厘米样品表面滴加1～4μL。
7.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(4)所述反应是在室温下反应6-12h。
8.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(4)所述Mal-PEGn-NHS中PEG的分子量为100～10000。
9.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(5)所述反应是在室温下反应12-24h。
10.根据权利要求1所述的一种医用钛或钛合金表面抗菌涂层的构建方法，其特征在于，步骤(5)所述的抗菌多肽为C-PEG2-PEG2-GRRRRSVQWCA；其中C-PEG2-PEG2-GRRRRSVQWCA的结构式如下：
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CN110585482A (zh) * 2019-08-02 2019-12-20 大连理工大学 一种具有抗菌性能的杂萘联苯聚芳醚腈及其表面改性方法
CN111035806A (zh) * 2018-10-12 2020-04-21 上海市静安区闸北中心医院 一种抗感染生物材料及其制备方法
CN114748692A (zh) * 2022-03-11 2022-07-15 华南理工大学 一种基于介孔二氧化硅的表面功能化钛基植入体及其制备方法与应用

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Publication number Priority date Publication date Assignee Title
CN108124904A (zh) * 2018-01-05 2018-06-08 天津喜诺生物医药有限公司 一种鲎素肽-载银复合抗菌剂及制备方法
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CN110585482A (zh) * 2019-08-02 2019-12-20 大连理工大学 一种具有抗菌性能的杂萘联苯聚芳醚腈及其表面改性方法
CN110585482B (zh) * 2019-08-02 2022-03-01 大连理工大学 一种具有抗菌性能的杂萘联苯聚芳醚腈及其表面改性方法
CN114748692A (zh) * 2022-03-11 2022-07-15 华南理工大学 一种基于介孔二氧化硅的表面功能化钛基植入体及其制备方法与应用

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