CN113975459A - 一种纳米酶水凝胶片的制备方法及在创可贴中的应用 - Google Patents
一种纳米酶水凝胶片的制备方法及在创可贴中的应用 Download PDFInfo
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- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
Abstract
本发明公开了一种纳米酶水凝胶片的制备方法，本发明通过制备单宁酸碳点与单宁酸混合作为纳米银还原剂及稳定剂，制备具有纳米酶活性的纳米银，通过Cu,Fe‑N‑C纳米酶的强拟过氧化酶活性，纳米银与Cu,Fe‑N‑C纳米酶表现出很高的催化活性，诱导水凝胶自组装，制备凝胶片；纳米酶保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性；酚羟基还可使纳米酶在水凝胶网络中均匀分布，从而改善其机械性能。纳米酶通过拟氧化酶活性催化反应产生的活性氧和CDs及纳米银的杀菌活性的协同作用赋予水凝胶片抗菌活性；本发明水凝胶片可以有能加速组织再生，防止感染，能应用于创可贴的制备中。
Description
技术领域
本发明涉及纳米材料抗菌技术领域，具体为一种新型纳米酶水凝胶片的制备方法及纳米酶水凝胶片在制备创可贴中的应用。
背景技术
水凝胶是一种具有三维交联网络的生物医用材料，既可以作为机体保护屏障防止细菌感染，又能为组织再生提供合适的微环境。对机体组织和植入物具有双面黏性的水凝胶具有巨大的临床应用潜力。然而，目前水凝胶在医药方面的应用，其研究主要集中在医用敷料上，要获得兼具双面黏性和生物活性的多功能水凝胶仍然面临巨大的挑战，使水凝胶具有高黏附性、好的机械强度及拉伸强度，好的生物相容性，满足“创可贴”的相关要求，研究还较少。崔春燕等研究了“一种高强度速黏纳米杂化水凝胶创可贴”，该研究将N-丙烯酰-2-氨基乙酸(ACG)水溶液与纳米生物活性玻璃(BG)混合，紫外光引发自由基聚合即可快速制备PACG-BG纳米复合水凝胶。通过模仿贻贝、藤壶以及沙堡蠕虫等海洋生物分泌的黏附胶制备的一系列新颖的能够在潮湿环境下黏附多种基质的水凝胶已成为研究的热点。虽然制得的水凝胶具有了一定的黏附性，但成膜性及韧性及抗菌性能有待进一步提高。
纳米酶通过自身具有的类天然酶活性，分别以催化产生强氧化性活性氧自由基破坏细菌生物膜成分、产生次卤酸干扰细菌生存重要的群体感应系统和降解细菌生物膜中外源性DNA 的途径，而达到抗菌的目的。相比于天然酶和传统抗菌剂，纳米酶具有稳定性好、成本低、易于功能化等优点。
发明内容
本发明提供了一种新型纳米酶水凝胶片创可贴的制备方法，该方法利用了纳米酶的催化性、抗菌性，制备新型抗菌凝胶片。
本发明利用碳点具有的抗菌性能、还原性能，单宁酸（TA）的邻苯二酚结构，通过制备单宁酸碳点与单宁酸混合作为纳米银制备的还原剂及稳定剂，制备具有纳米酶活性的纳米银（TA-CDs/AgNPs），通过铜、铁双原子纳米酶（Cu,Fe-N-C）的强拟过氧化酶活性，TA-CDs/AgNPs与Cu,Fe-N-C纳米酶表现出很高的催化活性，诱导水凝胶自组装，制备水凝胶片；纳米酶保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性，类似于贻贝的黏附性；酚羟基还可使纳米酶在水凝胶网络中均匀分布，从而改善其机械性能。纳米酶通过拟氧化酶活性催化反应产生的活性氧和CDs及TA-CDs/AgNPs的本身杀菌活性的协同作用赋予水凝胶抗菌活性。基于这些优点，超小TA-CDs/AgNPs+Cu,Fe-N-C纳米酶催化的水凝胶可以有效地用作粘合剂、抗菌剂，并作为新型“创可贴”来加速组织再生，同时防止感染。
本发明纳米酶水凝胶片的制备方法如下：
（1）单宁酸碳点的合成：称取1.0-2.0g单宁酸溶于20mL超纯水中，超声混合均匀，将溶液转移至聚四氟乙烯内衬水热反应釜中，于180-200℃恒温加热8-10h，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液用0.22μm滤膜除去大颗粒杂质，再经高速离心，上清液真空干燥，得到单宁酸碳点（TA-CDs）；
所述高速离心是在10000 r/min下处理15 min；真空干燥条件为40-60℃干燥24-48h；
（2）银纳米合成（TA-CDs/AgNPs）：9-11mg的单宁酸碳点与9-11mg单宁酸溶于20mL超纯水中，油浴加热至100℃，加入AgNO3，AgNO3在混合液中的浓度为19-22mg/mL，避光搅拌60min，冷却至室温，高速离心，上清液冷冻干燥，得到银纳米（TA-CDs/AgNPs）；
所述高速离心是在10000 r/min下处理15 min；
（3）Cu,Fe-N-C纳米酶制备包括以下步骤：
将2-甲基咪唑酯溶解于甲醇中形成均相A溶液，将Zn(NO3)2.6H2O溶于甲醇中形成B溶液，将A溶液与B溶液混合搅拌24h，离心，固体用甲醇洗洗2-3次，真空干燥，得到ZIF-8，其中2-甲基咪唑酯与Zn(NO3)2.6H2O的质量比为1.5-2.1:1；
所述离心是在4000-6000r/min下处理10-15min；
（4）水凝胶片的合成
将1质量份Cu,Fe-N-C纳米酶与1-3质量份银纳米混合，分散于去离子水中，超声混匀制成质量体积浓度0.02mg/mL的混合纳米酶；将丙烯酸2.7mL、0.01mg/mL的N，N′-亚甲基双丙烯酰胺0.3mL、0.04mg/mL的过硫酸钾1mL、去离子水5mL加到1mL混合纳米酶中，37℃搅拌5-10min，倒入模具，即得到水凝胶片。
所述的离心条件为离心时间为10-15min，离心速率为4000-6000r/min。
所述的真空干燥条件为40-60℃干燥24-48h。
本发明另一目的是将上述方法制得的纳米酶水凝胶片应用在制备用于伤口愈合的创可贴中。
本发明的优点在于：
1、本发明制备的TA-CDs/AgNPs+Cu,Fe-N-C纳米酶在水凝胶片中起到双重作用，一是诱导水凝胶自组装，同时纳米酶保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性，类似于贻贝的黏附性；二是纳米酶的拟过氧化酶活性催化反应产生的活性氧和CDs及TA-AgNPs的本身杀菌活性的协同作用赋予水凝胶抗菌活性；
2、本发明制备的TA-CDs/AgNPs和Cu,Fe-N-C纳米酶催化的水凝胶片可以有效地用作粘合剂、抗菌剂，并作为新型“创可贴”来加速组织再生，同时防止感染，对伤口无刺激、安全、无毒副作用，这主要取决于纳米酶、碳点及银纳米的抗菌、止血、安全、促凝血等功能，且材料本身具有良好生物相容性、低毒性及纳米酶的耐药性。
附图说明
图1为Cu,Fe-N-C纳米酶氧化TMB的紫外可见吸收光谱图；
图2为纳米酶+H2O2+纳米银的活性氧（ROS）产生监控结果示意图；
图3为纳米酶凝胶片粘附性能实验结果示意图；
图4为纳米酶凝胶片细胞毒性实验结果；
图5为Cu,Fe-N-C纳米酶的抗氧化实验结果；
图6为纳米酶凝胶片对大鼠伤口愈合实验结果。
具体实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明，但本发明的保护范围并不仅限于此。
实施例1：本实施例纳米酶水凝胶片的制备及性能
（1）称取1.0g单宁酸溶于20mL超纯水中，超声10min使其混合均匀，将溶液转移至聚四氟乙烯内衬水热反应釜中，于180℃恒温加热10h，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液过0.22μm滤膜以除去大颗粒杂质，再经10000r/min高速离心15min，上清液真空干燥，得到单宁酸碳点（TA-CDs）；
（2）将10mg的单宁酸碳点与10mg单宁酸溶于20mL超纯水中，油浴加热至100℃,按20mg/mL的比例加入AgNO3，避光搅拌60min，冷却至室温，10000r/min高速离心15min，上清液冷冻干燥，得到银纳米（TA-CDs/AgNPs）；
（3）Cu,Fe-N-C纳米酶的制备
（4）水凝胶片的合成
将1质量份的Cu,Fe-N-C纳米酶与2质量份的TA-CDs/AgNPs银纳米混合，分散于去离子水中，超声混合20min，制成质量体积浓度为0.02mg/mL的混合纳米酶；将2.7mL的丙烯酸、0.01mg/mL的N，N′-亚甲基双丙烯酰胺0.3mL、0.04mg/mL的过硫酸钾1mL、5mL的去离子水加到1mL混合纳米酶中，总体积10mL，37℃搅拌10min，倒入模具，即得到水凝胶片；
（5）采用TMB显色反应测定Cu,Fe-N-C纳米酶的过氧化酶活性
将100µg/mL Cu,Fe-N-C纳米酶100μL、100mmol/L的TMB50µL、50µmol/L H2O250µL，加入到pH 7.4磷酸盐缓冲溶液2mL中，充分混匀，室温孵育10min后，离心分离，取上层清液用紫外-可见分光光度计在655nm处测量吸光度，每个样品测量3次，取平均值，结果如图1；从图1中可以看出，在中性条件下，Cu,Fe-N-C纳米酶表现出相当高的过氧化酶活性；
（6）活性氧（ROS）产生量的监控：以抗坏血酸（AA）为探针进行监测，AA在266nm有吸收，但被ROS氧化生成脱氢抗坏血酸后，吸收峰消失；在磷酸缓冲盐溶液( PBS )中共孵育1h后，纳米酶+H2O2+纳米银对抗坏血酸在266nm处的吸光度有很大的降低，且降低程度结果见图2，纳米酶+H2O2+纳米银＞纳米酶+纳米银＞ H2O2。
（7）纳米酶水凝胶片的性能测试
参考文献（韩璐，仿贻贝多功能水凝胶及其生物医学应用的研究，西南交通大学博士论文，2017）中方法对水凝胶片的强度和延展性进行了测量，水凝胶片表现出好的强度和延展性( 348 MPa % )，未加纳米酶的水凝胶片的拉伸强度为86 MPa %；纳米酶结合水凝胶片良好的力学性能主要归因于两个因素。首先，纳米酶均匀分布在聚合物网络中，作为增强纳米填料对水凝胶片进行增强；第二，聚丙烯酸网络，在承受大变形时有效耗散能量。
参照文献（Bioactive Materials 6 (2021) 3962–3975方法，崔春燕等“一种高强度速黏纳米杂化水凝胶“创可贴”，高分子学报，2019，50（6）：613-622）中方法测试纳米酶凝胶片对不同材料表面（钛、聚四氟乙烯、玻璃、猪皮）的粘附性能，结果显示纳米酶凝胶片表现出自粘性，可长时间反复粘附于各种物体表面，试验结果表明水凝胶片对玻璃、钛、聚四氟乙烯（PTFE）和猪皮的粘附强度分别为37、48、58和29kPa (图3 )；
以下菌种分别从北纳创联生物科技有限公司、云南大学微生物研究所及昆明理工大学生命科学与技术学院获得；
实验方法：以金黄色葡萄球菌(S.aureus，ATCC25923)和枯草杆菌(B.subtilis，ATCC6051)为代表革兰氏阳性菌株，大肠杆菌(E.coli，ATCC25922)和铜绿假单胞菌(P.aeruginosa，ATCC27853)为代表革兰氏阴性菌株。此外，耐甲氧西林金黄色葡萄球菌(MRSA) (ATCC BAA-1720) 和多重耐药伤寒沙门菌(MRST)被用作抗生素耐药菌株的代表。对于每个菌株，将3-5个单菌落接种到新鲜的胰蛋白胨大豆肉汤(TSB)中，37℃培养16-18h至稳定期。取40μL菌液用新鲜TSB稀释100倍，37℃培养至对数中期(OD600=0.5-0.7)。细菌细胞收获后，用无菌PBS离心洗涤1次，用无菌PBS调至1.5×106CFU/mL；之后涂覆在固化的琼脂培养基上，将纳米酶凝胶片（20mm ×20mm）+H2O2 (浓度50mmol/L)置于培养基上中，以不加纳米酶水凝胶片的作为对照，在37℃孵育 24h，记录细菌生长情况，观察抑菌圈大小；纳米酶水凝胶片的抑菌圈大小见表1；
表1水凝胶片抑菌圈
结果表明，纳米酶水凝胶片表现出优异的抗菌性能，产生ROS的纳米酶可以规避细菌的耐药机制，因为ROS同时破坏了对细胞功能至关重要的多种小球物质(如核酸、蛋白质、脂类等)，而不是像抗生素那样靶向特定的细胞内代谢途径。
用1，1 -二苯基-2 -丙烯酰肼( DPPH )对其清除自由基能力进行了评价，在室温、黑暗环境中将Cu,Fe-N-C纳米酶和银纳米、Cu,Fe-N-C纳米酶、银纳米分别置于DPPH溶液(3mL )中处理30min，在517nm处测定吸光度，DPPH自由基清除活性测定：清除活性= ( A空白-A样品) / A空白× 100 %；结果如图5，随着Cu,Fe-N-C纳米酶浓度的增加，表现出更高的DPPH清除率。
实施例2：纳米酶凝胶片的制备及用于伤口敷料
（1）称取2.0g 单宁酸溶于20mL超纯水中，超声10min使其混合均匀，将溶液转移至聚四氟乙烯内衬水热反应釜中，于200℃恒温加热8h，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液过0.22μm滤膜以除去大颗粒杂质，再经10000r/min高速离心15min，上清液真空干燥，得到单宁酸碳点（TA-CDs）；
（2）11mg的单宁酸碳点与11mg单宁酸溶于20mL超纯水中，油浴加热至100℃，加入AgNO3，AgNO3在混合液中的浓度为21mg/mL，避光搅拌60min，冷却至室温，高速离心，上清液冷冻干燥，得到银纳米；
（3）Cu,Fe-N-C纳米酶的制备
将220mg ZIF-8加到40mL甲醇中，超声处理40min制得C溶液；将35mg CuCl2·2H2O与20mg FeCl3·6H2O加到10mL甲醇中制得D溶液，超声处理15min；将C溶液与D溶液混合，室温下搅拌12h，离心，固体用甲醇洗洗2次，50℃真空干燥35h，得到纯的CuCl2-FeCl3@ZIF-8；
将步骤
合成的CuCl2-FeCl3@ZIF-8分散于50mL含12mg KOH的甲醇中，超声处理30-40min，室温下搅拌12h，离心，用甲醇洗洗2次，50℃真空干燥35h，得到黄色粉末Cu(OH)2-Fe(OH)3@ZIF-8；
将步骤
合成的黄色粉末Cu(OH)2-Fe(OH)3@ZIF-8置于管式炉中，在800℃、N2保护下煅烧1h制得Cu,Fe-N-C；将Cu,Fe-N-C加到0.5mol/L的H2SO4溶液中，在80℃下孵育12h，离心，水洗涤3次，50℃真空干燥35h，即得Cu,Fe-N-C纳米酶；
（4）水凝胶片的合成：同实施例1；
（5）大鼠创面愈合试验
选用雄性昆明 ( SD )大鼠12只，体重180-220 g，大鼠用戊巴比妥(30mg/kg)麻醉后，将大鼠背侧区域脱毛，75%酒精擦拭消毒皮肤，以直径1cm的皮肤活检器在鼠背部正中距离耳后正中线4cm处形成一个1cm×1cm的圆形伤口，切除范围深达筋膜。用纳米酶水凝胶片、0.9%生理盐水（阴性对照）及白药创可贴（阳性对照）处理，每种材料做四个平行试验，每天换一次药，观察不同时间伤口愈合情况。
实验结果表明，伤口愈合为：纳米酶水凝胶片＞白药创可贴＞0.9%生理盐水，植入15天后，纳米酶水凝胶片组创面缺损得到有效愈合且几乎闭合，这可以解释为纳米酶水凝胶上的酚羟基具有较高的组织亲和力，以皮肤创面闭合率评价创面愈合率，图6结果显示，纳米酶水凝胶在初始愈合期创面闭合率明显高于阴性对照组。
以上结果表明，本发明制备的纳米酶抗菌水凝胶片具有好的机械性能、粘附性能，好的生物相容性、抗菌性能及伤口愈合性能，这主要取决于纳米酶保留了丰富的酚羟基，维持了酚醌的氧化还原动态平衡，为水凝胶提供了长期性和可重复性的黏附性，同时纳米酶的抗菌性能及清除自由基的性能，为伤口愈合提供了保障，该纳米酶抗菌水凝胶片在作为创可贴使用时是安全、有效的。
Claims (4)
1.一种纳米酶水凝胶片的制备方法，其特征在于，步骤如下：
（1）单宁酸碳点的合成：称取1.0-2.0g单宁酸溶于20mL超纯水中，超声混合均匀，将溶液转移至聚四氟乙烯内衬水热反应釜中，于180-200℃恒温加热8-10h，反应完成后自然冷却至室温，得棕色溶液；将棕色溶液用0.22μm滤膜除去大颗粒杂质，再经高速离心，上清液真空干燥，得到单宁酸碳点；
（2）银纳米合成：9-11mg的单宁酸碳点与9-11mg单宁酸溶于20mL超纯水中，油浴加热至100℃，加入AgNO3，AgNO3在混合液中的浓度为19-22mg/mL，避光搅拌60min，冷却至室温，高速离心，上清液冷冻干燥，得到银纳米；
（3）Cu,Fe-N-C纳米酶的制备
将2-甲基咪唑酯溶解于甲醇中形成均相A溶液，将Zn(NO3)2.6H2O溶于甲醇中形成B溶液，将A溶液与B溶液混合搅拌24h，离心，固体用甲醇洗洗2-3次，真空干燥，得到ZIF-8，其中2-甲基咪唑酯与Zn(NO3)2.6H2O的质量比为1.5-2.5:1；
将200-250mg ZIF-8加到40mL甲醇中，超声处理30-40min制得C溶液；将30-40mgCuCl2·2H2O与15-25mg FeCl3·6H2O加到10mL甲醇中制得D溶液，超声处理10-20min；将C溶液与D溶液混合，室温下搅拌12h，离心，固体用甲醇洗洗2-3次，真空干燥，得到纯的CuCl2-FeCl3@ZIF-8；
将步骤
合成的CuCl2-FeCl3@ZIF-8分散于50mL含10-15mg KOH的甲醇中，超声处理30-40min，室温下搅拌12h，离心，用甲醇洗洗2-3次，真空干燥，得到黄色粉末Cu(OH)2-Fe(OH)3@ZIF-8；
将步骤
合成的黄色粉末Cu(OH)2-Fe(OH)3@ZIF-8置于管式炉中，在750-800℃、N2保护下煅烧1-2h制得Cu,Fe-N-C；将Cu,Fe-N-C加到0.5mol/L的H2SO4溶液中，在80℃下孵育12h，离心，水洗涤3次，真空干燥，即得Cu,Fe-N-C纳米酶；
（4）水凝胶片的合成
将1质量份Cu,Fe-N-C纳米酶与1-3质量份银纳米混合，分散于去离子水中，超声混匀制成质量体积浓度0.02mg/mL的混合纳米酶；将丙烯酸2.7mL、0.01mg/mL的N，N′-亚甲基双丙烯酰胺0.3mL、0.04mg/mL的过硫酸钾1mL、去离子水5mL加到1mL混合纳米酶中，37℃搅拌5-10min，倒入模具，即得到水凝胶片。
2.根据权利要求1所述的纳米酶水凝胶片的制备方法，其特征在于：步骤（1）、（2）中高速离心是在10000 r/min下处理15 min。
3.根据权利要求1所述的纳米酶水凝胶片的制备方法，其特征在于：步骤（3）中离心是在4000-6000r/min下处理10-15min。
4.权利要求1-3中任一项所述的纳米酶水凝胶片的制备方法制得的纳米酶抗菌水凝胶片在制备用于伤口愈合的创可贴中的应用。
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| CN115501335A (zh) * | 2022-08-30 | 2022-12-23 | 昆明理工大学 | 光声增强多重酶活性纳米酶水凝胶的制备方法和应用 |
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