CN115300459A - 纳米酶复合水凝胶滴眼液的制备方法 - Google Patents

纳米酶复合水凝胶滴眼液的制备方法 Download PDF

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CN115300459A
CN115300459A CN202210964063.3A CN202210964063A CN115300459A CN 115300459 A CN115300459 A CN 115300459A CN 202210964063 A CN202210964063 A CN 202210964063A CN 115300459 A CN115300459 A CN 115300459A
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周庆军
史伟云
宋方英
王红卫
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Shandong First Medical University Affiliated Eye Research Institute Shandong Provincial Eye Research Institute Shandong First Medical University Affiliated Qingdao Eye Hospital
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Abstract

本发明属于滴眼液药物领域,针对引起角膜炎的病原体对常见眼部抗生素的耐药性呈现增加趋势,亟需新型抗菌药物的问题,本发明提供一种纳米酶复合水凝胶滴眼液的制备方法,先利用六水氯化钴与氨水的配位反应以及硝酸银与单宁酸的氧化还原反应合成一种单宁酸配合的银钴复合纳米粒子;将银钴复合纳米粒子和丙烯酸酯修饰明胶混合并超声溶解,然后通过紫外光照射制备出纳米酶复合水凝胶滴眼液。本发明制备方法操作简便且制备时间短;所得滴眼液对多种细菌和真菌显示了广谱杀菌的效果,并对细菌性和耐药菌角膜感染具有良好的预防治疗效果。

Description

纳米酶复合水凝胶滴眼液的制备方法
技术领域
本发明属于滴眼液药物领域,具体涉及纳米酶复合水凝胶滴眼液的制备方法。
背景技术
细菌性角膜炎是由于细菌侵袭角膜引发的感染,发病迅速,严重者会引发角膜溃疡甚至穿孔,是发展中国家主要致盲性眼病之一。最常见的诱发因素包括:使用隐形眼镜,特别是夜间或长期佩戴隐形眼镜以及镜片消毒不充分;外伤;眼部手术特别是角膜手术;慢性眼表疾病;全身性疾病如糖尿病和/或外用皮质类固醇的长期使用。在常见细菌性病原体中,引起角膜炎最常见的病原体包括金黄色葡萄球菌、铜绿假单胞菌、肺炎链球菌和沙雷氏菌,其中以铜绿假单胞菌性角膜炎感染发病最为严重并且难以治疗。临床上对铜绿假单胞菌性角膜炎的治疗以病灶清除和局部抗生素的频繁用药为主,以尽可能高效快速的控制住角膜溃疡发展。但是,抗生素的使用亦存在一定程度的风险和问题。一项对外用抗生素治疗细菌性角膜炎的临床实验回顾分析显示,与氟喹诺酮类药物相比,氨基糖苷类头孢菌素类药物引起眼部不适或化学结膜炎等轻微不良事件的相对风险有所增加。对眼表分离铜绿假单胞菌耐药性回顾分析表明,铜绿假单胞菌对环丙沙星(9%)、庆大霉素(22%)和头孢他啶(13%)等常见眼部抗生素的平均耐药率虽然相对较低,但对于其耐药性有逐年升高的趋势。所以,对于研发针对铜绿假单胞菌性角膜炎治疗的新型抗菌药物仍是紧迫且必需的。
发明内容
针对引起角膜炎的病原体对常见眼部抗生素的耐药性呈现增加趋势,亟需新型抗菌药物的上述问题,本发明提供一种纳米酶复合水凝胶滴眼液的制备方法,先利用六水氯化钴与氨水的配位反应以及硝酸银与单宁酸的氧化还原反应合成一种单宁酸配合的银钴复合纳米粒子(TCN);以水为媒介,将TCN和丙烯酸酯修饰明胶混合并超声至均匀,然后通过紫外光照射一步制备出纳米酶复合水凝胶材料(TCNH);进而与过氧化氢溶液混合均匀即得TCNH滴眼液。所得滴眼液对多种细菌及真菌显示了广谱杀菌的效果,并对细菌性和耐药菌角膜感染具有良好的预防治疗效果。
本发明提供的纳米酶复合水凝胶滴眼液的制备方法包括以下步骤:
纳米酶复合水凝胶滴眼液的制备方法,包括以下步骤:
(1)向氯化钴水溶液中滴加少量氨水,反应一段时间,再滴加氯化钴水溶液继续反应;
(2)将醋酸银加入到氨水溶液中并完全溶解,将该溶液缓慢滴加到步骤(1)中,搅拌一段时间后滴加单宁酸溶液,继续搅拌,析出沉淀;离心、清洗、干燥沉淀并于350-450℃下煅烧1-4h,即得单宁酸配合的银钴复合纳米粒子;
(3)将制得的单宁酸配合的银钴复合纳米粒子加入到5wt%-7.5wt%的丙烯酸酯修饰明胶的水溶液中,并加入安息香二甲醚乙醇溶液作为引发剂,超声至均匀,然后将上述混合物置于紫外光下照射,形成凝胶状溶液,再加入过氧化氢溶液并在室温下超声至均匀,即得纳米酶复合水凝胶滴眼液。
进一步的,所述氯化钴水溶液的浓度为20-30mmol/L。
进一步的,所述氨水为含氨20~28%的水溶液。
进一步的,所述步骤(1)向氯化钴水溶液中滴加氨水,逐步升温至55-65℃,并继续搅拌12-18min;再滴加氯化钴水溶液继续反应1-4h。
进一步的,所述步骤(2)缓慢滴加银氨溶液,搅拌15min,缓慢滴加单宁酸溶液,室温搅拌16h。
进一步的,所述步骤(2)依次用水和乙醇清洗,60℃干燥4h,然后置于400℃下煅烧3h。
进一步的,所述丙烯酸酯修饰明胶为甲基丙烯酸酯化明胶。
进一步的,所述步骤(3)混合物中,丙烯酸酯修饰明胶的质量浓度为2.0%-6.0%。
进一步的,所述步骤(3)紫外光下照射时间为12-18min。
进一步的,所述步骤(3)混合物中,丙烯酸酯修饰明胶与单宁酸配合的银钴复合纳米粒子的质量比为8-12:1。
本发明的有益效果:制备方法操作简便且制备时间短;所得滴眼液对多种细菌和真菌显示了广谱杀菌的效果,并对细菌性和耐药菌角膜感染具有良好的预防治疗效果。
附图说明
图1为实施例制备的TCN纳米酶的扫描电镜照片;纳米酶为球状堆积形貌。
图2为实施例制备的TCNH滴眼液的扫描电镜照片;滴眼液形貌为典型的水凝胶数十微米大孔的形貌。
图3为实施例制备的TCNH滴眼液的光学照片;纳米酶均匀分散在滴眼液中。
图4为实施例制备的TCNH滴眼液的体外抑菌实验-平板实验。
图5为实施例制备的TCNH滴眼液的体外抑菌实验-对照组和H2O2组被染成绿色的活细菌数数量。
图6为实施例制备的TCNH滴眼液的体外抑菌实验-扫描电镜下对照组和H2O2组细菌形态。
图7为实施例制备的TCNH滴眼液的体内抑菌实验(铜绿假单胞菌)-对照组和H2O2组角膜明显感染情况。
图8为实施例制备的TCNH滴眼液的体内抑菌实验(铜绿假单胞菌耐药菌)。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
实施例
(1)向离心管中加入238.0mg六水氯化钴;
(2)向步骤(1)的离心管中加入50.0mL水并超声至完全溶解;
(3)向步骤(2)的离心管中逐滴加入1.0mL 25%氨水溶液,逐步升温至60℃,并继续搅拌15min;
(4)向步骤(3)的离心管中加入20.0mL25 mmol/L六水氯化钴溶液,继续反应3h;
(5)250.3mg醋酸银加入到10mL 25%氨水溶液中,超声至完全溶解;
(6)将步骤(5)中溶液缓慢滴加到步骤(4)的溶液中,并继续搅拌15min;
(7)2.34g单宁酸加入到40.0mL水中,超声至完全溶解;
(8)将步骤(7)中溶液缓慢滴加到步骤(6)的溶液中,室温搅拌16h;
(9)将步骤(8)中溶液离心,并用水和乙醇清洗,60℃干燥4h,然后置于400℃下煅烧3h,即得TCN;
(10)向离心管中加入7.5wt%150mg丙烯酸酯修饰的明胶;
(11)向步骤(10)的离心管中加入3.0mL水并超声至完全溶解;
(12)向离心管中加入15.0mg TCN、及2μL的浓度10wt%安息香二甲醚乙醇溶液,并超声至均匀;
(13)将步骤(12)的离心管置于紫外光下照射15min,安息香二甲醚作为光引发剂,丙烯酸酯修饰的明胶浓度较低,因此发生轻微交联,形成凝胶状水溶液,TCN均匀分散在凝胶水溶液中;
(14)向步骤(13)的离心管中加入过氧化氢溶液并在室温下超声至均匀即得TCNH滴眼液。
抑菌实验
将所得实施例制备的TCNH滴眼液应用于体外大肠杆菌、铜绿假单胞菌、耐药性铜绿假单胞菌和白色念珠菌评价以及体内铜绿假单胞菌和耐药铜绿假单胞菌角膜感染的预防治疗实验。
图4-图6为TCNH滴眼液的体外抑菌实验结果。铜绿假单胞菌、耐药铜绿假单胞菌、大肠杆菌和白色念珠菌的抑菌效果:如图4所示,对照组和H2O2组细菌长满了平板,TCNH和TCNH+H2O2组平板无细菌菌落生成。如图5所示,对照组和H2O2组被染成绿色的活细菌数目较多,被染成红色的死细菌很少,TCNH和TCNH+H2O2组细菌个数显著减少,视野里只有零星几个。如图6所示,扫描电镜下对照组和H2O2组细菌形态规则完好,TCNH和TCNH+H2O2组细菌形貌变得不规则,有明显皱缩甚至凹陷。
TCNH滴眼液的体内抑菌实验(铜绿假单胞菌),结果如图7所示,对照组和H2O2组角膜明显感染,TCNH组有轻微感染,TCNH+H2O2组无感染,角膜中细菌数量统计表明TCNH组细菌数量级显著降低。
TCNH滴眼液的体内抑菌实验(铜绿假单胞菌耐药菌),结果如图8所示,对照组、庆大霉素组(Gen)和妥布霉素组(Tob)均有明显感染,TCNH+H2O2组无感染,角膜中细菌数量统计表明,TCNH组耐药细菌数量级显著降低。
以上所述的实施例仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。

Claims (10)

1.纳米酶复合水凝胶滴眼液的制备方法,其特征在于,包括以下步骤:
(1)向氯化钴水溶液中滴加少量氨水,反应一段时间,再滴加氯化钴水溶液继续反应;
(2)将醋酸银加入到氨水溶液中并完全溶解,将该溶液缓慢滴加到步骤(1)中,搅拌一段时间后滴加单宁酸溶液,继续搅拌,析出沉淀;离心、清洗、干燥沉淀并于350-450℃下煅烧1-4h,即得单宁酸配合的银钴复合纳米粒子;
(3)将制得的单宁酸配合的银钴复合纳米粒子加入到5wt%-7.5wt%的丙烯酸酯修饰明胶的水溶液中,并加入安息香二甲醚乙醇溶液作为引发剂,超声至均匀,然后将上述混合物置于紫外光下照射,形成凝胶状水溶液,再加入过氧化氢溶液并在室温下超声至均匀,即得纳米酶复合水凝胶滴眼液。
2.根据权利要求1所述的制备方法,其特征在于,所述氯化钴水溶液的浓度为20-30mmol/L。
3.根据权利要求1所述的制备方法,其特征在于,所述氨水为含氨20~28%的水溶液。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)向氯化钴水溶液中滴加氨水,逐步升温至55-65℃,并继续搅拌12-18min;再滴加氯化钴水溶液继续反应1-4h。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)缓慢滴加银氨溶液,搅拌15min,缓慢滴加单宁酸溶液,室温搅拌16h。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤(2)依次用水和乙醇清洗,60℃干燥4h,然后置于400℃下煅烧3h。
7.根据权利要求1所述的制备方法,其特征在于,所述丙烯酸酯修饰明胶为甲基丙烯酸酯化明胶。
8.根据权利要求1所述的制备方法,其特征在于,所述步骤(3)混合物中,丙烯酸酯修饰明胶的质量浓度为2.0%-6.0%。
9.根据权利要求1所述的制备方法,其特征在于,所述步骤(3)紫外光下照射时间为12-18min。
10.根据权利要求1或2所述的制备方法,其特征在于:所述步骤(3)混合物中,丙烯酸酯修饰明胶与单宁酸配合的银钴复合纳米粒子的质量比为(8-12):1。
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