CN114680136B - 一种Cu2O/CuO@Ag-四环素的制备方法 - Google Patents
一种Cu2O/CuO@Ag-四环素的制备方法 Download PDFInfo
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Abstract
本发明公开了一种Cu2O/CuO@Ag‑四环素的制备方法，将纳米Ag负载在其表面合成Cu2O/CuO@Ag复合材料，通过配位吸附把四环素结合在Cu2O/CuO@Ag表面合成Cu2O/CuO@Ag‑四环素复合材料，研究抑菌活性和应用潜力。与现有技术相比，本发明以有机抗生素特有的大分子结构中含有的氨基和羟基官能团为配体，用纳米Ag和Cu可与氨基和羟基发生配位的原理制备高效且不产生耐药的抑菌剂Cu2O/CuO@Ag‑四环素，其协同作用可对耐药菌产生一定的抑菌活性，具有推广应用的价值。
Description
技术领域
本发明涉及无机协同有机复合抑菌材料，尤其涉及一种Cu2O/CuO@Ag-四环素的制备方法。
背景技术
有机抗菌剂抗生素是生物在新陈代谢过程中产生抗病原体的代谢产物或人工合成的类似物，根据分子结构的不同，可把抗生素分为四环素类、磺胺类、大环内酯类、喹诺酮类和氯霉素类等。而四环素类由于对革兰氏阳性菌、阴性菌、细胞内支原体、衣原体和立克次氏体、病毒等都具有强的抑制活性，且价格低、作用范围广、副作用小、靶向性强等优点被广泛用于农业、水产业、养殖畜牧业等领域。四环素作为重要四环素类抑菌剂其抑菌机制为：其作为多羟基化合物由于分子量小于600D可通过离子通道蛋白结合Mg2+进入离子蛋白，通过自由扩散进入细胞内部与核糖体作用，特异性的结合核糖体70S中的30S亚基上16sRNA的A部位(氨酰基部位)，使氨酰-tRNA的反密码子不能在A部位和mRNA结合，可有效阻断肽链的延长而具有强的杀菌性。但随着四环素的泛滥使用诱导细菌质粒中抗性基因表达而产生核糖体保护蛋白、外排泵、酶失活等耐药体系，致使四环素的药用价值越来越低。目前解决方案之一是结合具有不产生耐药性的无机抑菌剂来提高四环素的药用价值。
无机市售抑菌剂如波尔多液、绿得宝、双效灵、脉舒宝等常见药剂的主要成分为Cu2+和Ag+，Cu2+和Ag+作为生活一次性用药具有杀菌效率高，对人体副作用小，安全可靠等优点被广泛应用于农林畜牧业的病菌防治和生活用药等领域。而Cu2+的抑菌机制是： Cu2+通过铜离子通道进入微生物内部后游离于细胞质中，改变细菌内外的渗透压平衡，破坏细菌细胞器。但Cu2+也作为微生物微量元素，细菌体内也含有微量的用以固定和运输Cu+的伴侣蛋白，正常环境下输送Cu+的伴侣蛋白较少，但大量Cu2+可诱导细菌产生大量的伴侣蛋白而具备拮抗性，且部分植物病原微生物还会在高浓度Cu2+作用下，促进细胞质中抗性基因表达，病菌进化出外排泵蛋白和胞外膜，抑制了Cu2+的毒性而产生耐药，导致Cu2+的抑菌性能越来越低。Ag+因d电子轨道和Cu+相似，也可以通过铜离子通道进入细菌内部，和离子通道蛋白中的硫铁簇结合改变蛋白质空间结构，破坏新陈代谢所需的液质环境等可对细菌造成不可逆损伤，但单独使用Ag+抑菌持久性较差，抑菌活性较低，且循环利用率低等缺点限制了其发展。理论上四环素具有的多羟基和氨基可结合具有抑菌性的金属粒子而具备协同杀菌，但对此类目前研究的较少，目前市售四环素类抗生素主要结合其他有机抗生素协同抑菌用于病菌防治，但容易产生耐药性，而对Cu2O/CuO@Ag-四环素复合的未曾报道，现有的四环素抑菌剂，抑菌活性低，复合其他有机抗生素协同用药在临床领域，其结果易产生耐药菌。
发明内容
本发明的目的就在于为了解决上述问题而提供一种Cu2O/CuO@Ag-四环素的制备方法。将纳米Ag负载在其表面合成Cu2O/CuO@Ag复合材料，通过配位吸附把四环素结合在Cu2O/CuO@Ag表面合成Cu2O/CuO@Ag-四环素复合材料，研究抑菌活性和应用潜力。
本发明通过以下技术方案来实现上述目的：
本发明包括以下步骤：
S1：Cu2O/CuO的制备：硝酸铜和聚乙烯吡咯烷酮加入到超纯水中形成透明溶液，随后水合肼加到上述透明溶液中超声分散均匀，反应得到Cu2O，反应结束后，产物用超纯水和乙醇洗涤数次，然后真空干燥得到Cu2O/CuO；
S2：Cu2O/CuO@Ag的制备：取Cu2O/CuO溶于超纯水中超声，加入银氨溶液反应得到Cu2O/CuO@Ag，产物用超纯水和乙醇洗涤数次，然后在真空干燥；
S3：Cu2O/CuO@Ag-四环素的制备：取Cu2O/CuO@Ag加入到锡箔纸包裹的盐酸四环素中黑暗环境下超声，并陈化后用得到Cu2O/CuO@Ag-四环素，用超纯水洗涤，烘干，用锡箔纸包裹备用。
本发明的有益效果在于：
本发明是一种Cu2O/CuO@Ag-四环素的制备方法，与现有技术相比，本发明以有机抗生素特有的大分子结构中含有的氨基和羟基官能团为配体，用纳米Ag和Cu可与氨基和羟基发生配位的原理制备高效且不产生耐药的抑菌剂Cu2O/CuO@Ag-四环素，其协同作用可对耐药菌产生一定的抑菌活性，具有推广应用的价值。
附图说明
图1是Cu2O/CuO@Ag-四环素透射电镜分析结果；
图2是Cu2O/CuO@Ag-四环素X射线衍射分析结果；
图3是Cu2O/CuO@Ag-四环素光电子能谱分析结果；
图4是Cu2O/CuO@Ag-四环素磁饱红外吸收光谱结果；
图5是材料在浓度在2μg/mL，时对大肠杆菌，金黄色葡萄球菌，耐四环素药菌沙门氏菌抑菌结果；
图6是材料在浓度在5μg/mL时对大肠杆菌，金黄色葡萄球菌，耐四环素药菌沙门氏菌抑菌结果；
图7是材料在浓度在10μg/mL时对大肠杆菌，金黄色葡萄球菌，耐四环素药菌沙门氏菌抑菌结果；
图8是材料Cu2O/CuO@Ag-四环素浓度为20μg/mL对耐药菌沙门氏菌，在0/2/5/10/20/40min时菌落计数抑菌结果。
具体实施方式
下面结合附图对本发明作进一步说明：
本发明包括以下步骤：
S1：Cu2O/CuO的制备：4 g的硝酸铜和2 g聚乙烯吡咯烷酮加入到100 mL超纯水中形成透明溶液，随后80 µL 水合肼加到上述溶液中超声分散均匀，反应15 min得到Cu2O，反应结束后，产物用超纯水和乙醇洗涤数次，然后在80℃真空干燥箱中干燥32 h得到Cu2O/CuO。
S2：Cu2O/CuO@Ag的制备：取Cu2O/CuO 100mg 溶于90mL超纯水中超声30min，加入10mL 5 mg/mL的银氨溶液反应30 min 得到Cu2O/CuO@Ag，产物用超纯水和乙醇洗涤数次，然后在60℃真空干燥箱中干燥8h。
S3：Cu2O/CuO@Ag-四环素的制备：取Cu2O/CuO@Ag 40 mg加入到锡箔纸包裹的50 mL1mg/mL的盐酸四环素中黑暗环境下超声30 min，并陈化4 h后用得到Cu2O/CuO@Ag-四环素，用超纯水洗涤4次，50℃烘干，用锡箔纸包裹备用。
抑菌实验：
材料的抑菌活性用耐四环素沙门氏菌等来监测，耐药沙门氏菌为37℃下活化的指数期细菌，所用的LB培养基超纯水、生理盐水、磷酸缓冲液等生物材料均在高压灭菌锅(121℃, 20min)下灭菌，均在37℃的生化培养箱中培养。
滤纸片扩散实验：
用无菌的纳米材料分散在灭过菌的超纯水中，制备成浓度为2 μg/mL，5 μg/mL，10μg/mL， 20 μg/mL的梯度的溶液。将隔夜活化好的细菌用无菌生理盐水稀释成5×107 CFU(colony-forming units)/mL，取100 µL均匀涂在灭过菌的固体LB培养基上，取8 µL沾有不同材料的抑菌液放置在接过菌的LB培养基上培养12 h，平行做6组观察结果。得到最佳抑菌浓度后，以市售波尔多液抑菌活性为参照，对比复合材料的抑菌活性。
菌落计数法实验：
将纳米材料加入到5×105 CFU/mL菌悬液中，最终质量浓度为20 g/mL，分别混合0min、2 min、5 min、10 min、20min、40min、，磁分离取上层液10 µL均匀涂在灭过菌的固体LB培养基上培养12 h，平行做6组观察结果[18]，抑菌效率(n)为
式中：n代表抑菌效率，B 0 为参照中菌落个数，B是含有不同材料的抑菌结果菌落数。
细菌生长曲线监测法实验：
未检测材料对细菌生长阶段的具体影响，我们用微量热分析检测细菌在适应期，对数期。稳定期和衰退期的释放热量强度的变化来分析材料的抑菌活性，细菌释放热量越多，说明细菌生长活性越强。为此将材料和接过菌的液体LB培养基混合配成5 mL溶液，最终细菌浓度为5×107 CFU，材料浓度为300 μg/mL，37℃下监测细菌的生长和热量释放强度。
细菌PI染色法实验
为了测试细菌细胞细胞膜破坏的完整程度，使用碘化丙啶(PI)作为DNA染色剂。正常的细菌，由于细胞膜完整，不允许PI渗透而没有颜色，另外，如果出现红色，代表细菌受损或者死亡，细胞膜被破坏，PI染色剂进入细菌内部和DNA作用而显示红色荧光。为此，将上述混合液加入50 μg/mL的碘化丙啶(PI) 50 μL黑暗环境下混合15 min，在13000 r/min的离心机下，用磷酸缓冲液洗涤3次，在荧光倒置显微镜下观察细菌的损伤情况。
哺乳细胞的毒理性实验:
10%的标准胎牛血清的HDMEM，且培养基每天更换，细胞在二氧化碳浓度为5%、湿度为95%的37℃环境中培养。具体实验如下，把正常浓度为2000个/孔的乳头细胞加入到96孔板中隔夜粘附培养，除去培养基并加入100 μL含有不同浓度的材料的培养基，培养3天后，加入25 μL的MTT溶液(5 mg/mL in PBS) 培养2 h，移出上清液并加入100 μL的DMSO溶解Formazan结晶，将培养基密封，隔夜培养，用分光光度计监测吸收峰评估材料对细胞的毒理性w。计算公式为：
w = OD(实验组)/OD(对照组)*100%
图1-3为材料的透射电镜结果，证明Cu2O/CuO@Ag-四环素为单分散性的球形材料，图4磁饱和强度结果显示材料具有较强的磁性，UV-vis显示Cu2O/CuO@Ag-和四环素复合构成Cu2O/CuO@Ag-四环素。图5-7时间抑菌结果证明，材料在20min 内抑菌率为100%，图8抑菌结果显示材料在20 μg/mL时对耐四环素沙门氏菌具有强的抑菌活性，80μg/mL时的抑菌活性是同浓度四环素的2.5倍。
以上显示和描述了本发明的基本原理和主要特征及本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (4)
1.一种Cu2O/CuO@Ag-四环素的制备方法，其特征在于，包括以下步骤：
S1：Cu2O/CuO的制备：硝酸铜和聚乙烯吡咯烷酮加入到超纯水中形成透明溶液，随后水合肼加到上述透明溶液中超声分散均匀，反应得到Cu2O，反应结束后，产物用超纯水和乙醇洗涤数次，然后真空干燥得到Cu2O/CuO；
S2：Cu2O/CuO@Ag的制备：取Cu2O/CuO溶于超纯水中超声，加入银氨溶液反应得到Cu2O/CuO@Ag，产物用超纯水和乙醇洗涤数次，然后在真空干燥；
S3：Cu2O/CuO@Ag-四环素的制备：取Cu2O/CuO@Ag加入到锡箔纸包裹的盐酸四环素中黑暗环境下超声，并陈化后得到Cu2O/CuO@Ag-四环素，用超纯水洗涤，烘干，用锡箔纸包裹备用。
2.根据权利要求1所述的Cu2O/CuO@Ag-四环素的制备方法，其特征在于：所述步骤S1中硝酸铜4g，聚乙烯吡咯烷酮2g，超纯水100mL，水合肼80μL，反应时间15min，真空干燥温度80℃，干燥时间32h。
3.根据权利要求1所述的Cu2O/CuO@Ag-四环素的制备方法，其特征在于：所述步骤S2中Cu2O/CuO取100mg，超纯水90mL，超声时间30min，银氨溶液为10mL 5mg/mL，反应时间30min，真空干燥温度60℃，干燥时间8h。
4.根据权利要求1所述的Cu2O/CuO@Ag-四环素的制备方法，其特征在于：所述步骤S3中Cu2O/CuO@Ag取40mg，盐酸四环素为50mL 1mg/mL，超声时间30min，陈化时间4h，超纯水洗涤4次，烘干温度50℃。
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