CN114870034B - 一种具备高效抗感染能力的基因转染纳米材料及其制备 - Google Patents

一种具备高效抗感染能力的基因转染纳米材料及其制备 Download PDF

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CN114870034B
CN114870034B CN202210145204.9A CN202210145204A CN114870034B CN 114870034 B CN114870034 B CN 114870034B CN 202210145204 A CN202210145204 A CN 202210145204A CN 114870034 B CN114870034 B CN 114870034B
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岳冰
王敏琪
曲新华
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Renji Hospital Shanghai Jiaotong University School of Medicine
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Abstract

本发明涉及一种具备高效抗感染能力的基因转染纳米材料及其制备，纳米材料为负载质粒DNA的纳米颗粒，纳米颗粒的外层修饰有聚多巴胺涂层，并通过聚多巴胺涂层接枝抗菌肽。与现有技术相比，本发明构建的基因转染纳米材料具备优异的抗感染作用，可快速杀伤常见细菌；入胞能力较好，可有效杀伤进入细胞内部的胞内菌，更深入地清除隐蔽的难清除细菌；能够使目的基因转染效率明显提高，目的基因表达显著增加；在皮下感染小鼠模型治疗中，表现出更好的抗感染能力；可解决新兴治疗策略基因转染在感染相关疾病治疗中应用过程所面临的巨大挑战，有望为非抗生素的抗感染策略开辟新的方向，具有重大的社会、经济价值。

Description

一种具备高效抗感染能力的基因转染纳米材料及其制备
技术领域
本发明属于基因转染技术领域，涉及一种具备高效抗感染能力的基因转染纳米材料及其制备。
背景技术
骨科植入物在临床上使用频率高，而植入物相关感染作为最常见且后果最严重的并发症之一，是临床上面临的一大难题。目前感染性疾病的治疗仍以抗生素为主，然而近年来，耐药菌的流行极大地削弱了抗生素的疗效；而另一方面，关于疗效更佳的新型抗生素研发遇到瓶颈。因此，非抗生素抗感染策略成为新的研究热点。
作为一种新兴技术，基因转染已得到广泛研究，在抗感染领域，具备抗感染能力的常见目的基因包括β-防御素2、β-防御素3、抗菌肽LL37等，成功转染后均表现出较好的抗感染作用。其中，常用的转染载体包括：1.病毒转染：腺病毒、慢病毒；2.非病毒转染：基因转染纳米平台。非病毒转染载体由于出色的转染效率、较高的生物安全性等优势，表现出优异前景。
基因转染治疗是否发挥疗效，取决于被转染的细胞能否顺利表达目标基因，而在感染环境中，细菌将从多方面影响细胞及周围组织的正常功能，使基因转染体系面临严峻挑战。首先，细菌可分泌多种毒性因子(如内毒素)，直接杀伤周围细胞。细菌还可进入细胞内部形成胞内菌，诱导凋亡程序导致细胞死亡；也可与细胞长期共存，并存在杀死细胞并再次释放到胞外的可能性。因此，考虑到感染相关疾病的特殊性，即细菌影响细胞的正常功能甚至杀死细胞，存在进一步阻碍基因转染发挥治疗作用的可能性，上述现有基因转染技术均不能很好地适用于感染相关疾病的治疗。
发明内容
本发明的目的是针对现有基因转染方法在感染相关疾病治疗中面临的困境，提供一种具备高效抗感染能力的基因转染纳米材料及其制备。本发明中的纳米材料可作为非病毒纳米转染平台，同时实现快速杀菌及高效转染目的基因，改善基因转染方法在感染相关疾病中的应用前景。
本发明的目的可以通过以下技术方案来实现：
一种具备高效抗感染能力的基因转染纳米材料，该纳米材料为负载质粒DNA的纳米颗粒，所述的纳米颗粒的外层修饰有聚多巴胺涂层，并通过聚多巴胺涂层接枝抗菌肽。
进一步地，所述的质粒DNA为抗菌肽基因。
进一步地，所述的抗菌肽包括防御素、LL37或KR12中的一种。
进一步地，所述的纳米颗粒包括介孔二氧化硅或金属有机框架MOFs中的一种。
进一步地，在该纳米材料合成体系中，质粒DNA的含量为8-12μg/ml，聚多巴胺的含量为140-160mg/ml，抗菌肽的含量为8-12mg/ml。优选地，质粒DNA的含量为10μg/ml，聚多巴胺的含量为150mg/ml，抗菌肽的含量为10mg/ml。
一种具备高效抗感染能力的基因转染纳米材料的制备方法，该方法包括以下步骤：
1)将质粒DNA负载在纳米颗粒上，得到负载质粒DNA的纳米颗粒；
2)在步骤1)中得到的负载质粒DNA的纳米颗粒外包裹PDA并接枝抗菌肽；
3)经过后处理，得到所述的纳米材料。
进一步地，步骤1)具体为：步骤1)具体为：将Zn(NO3)与十六烷基三甲基溴化铵溶解在甲醇中得到溶液A，将LL37和2-甲基咪唑溶解在甲醇中得到溶液B，之后将溶液A与溶液B混合。
进一步地，步骤2)具体为：将多巴胺、负载质粒DNA的纳米颗粒、乙醇、三乙醇胺与LL37混合，之后室温下进行搅拌。
进一步地，搅拌时间为3-5小时，优选为4h。
进一步地，步骤3)中，后处理包括洗涤及浸泡。
例如，制备过程为：将2g Zn(NO3)·6H2O和0.15g十六烷基三甲基溴化铵(CTAB)溶解在10ml甲醇溶液中作为溶液A；将100μl LL37质粒(1000ng/ml)和0.34g 2-甲基咪唑溶解在10ml甲醇中形成溶液B，将溶液A与溶液B混合2小时。将0.15g多巴胺、10ml上述混合溶液、10ml乙醇、1.5ml三乙醇胺和10mg/ml的LL37混合，室温下搅拌4小时。之后用无水乙醇洗涤三次，再于20mL无水乙醇中浸泡三天。
目前，以介孔纳米颗粒负载质粒DNA为核心的非病毒基因转染体系，已被证明具备较高效的基因转染作用。本发明在此基础上进一步开发，目的是改善基因转染纳米平台的基因转染效率及抗感染能力。具体而言，本发明在纳米颗粒外层修饰聚多巴胺(PDA)涂层并接枝具备优异抗菌能力的抗菌肽。一方面，接枝的抗菌肽使此纳米材料具备了直接杀菌作用，包括直接清除微环境内的浮游细菌及已进入细胞内部的胞内菌，保证被转染目的基因的细胞不受到细菌的杀伤；另一方面，接枝的抗菌肽可进一步提高此基因转染体系的转染效率，包括增加纳米颗粒入胞、增加溶酶体逃逸等机制。
与现有技术相比，本发明具有以下特点：
1)本发明构建的基因转染纳米材料具备优异的抗感染作用，可快速杀伤MRSA(耐甲氧西林金黄色葡萄球菌)、E.coli(大肠杆菌)等常见细菌，例如，接枝LL37后对MRSA的杀菌率高达99.9％以上。
2)本发明构建的基因转染纳米材料入胞能力较好，可有效杀伤进入细胞内部的胞内菌，更深入地清除隐蔽的难清除细菌。
3)本发明构建的基因转染纳米材料能够使目的基因转染效率明显提高，目的基因表达显著增加。
4)本发明构建的基因转染纳米材料在皮下感染小鼠模型治疗中，表现出更好的抗感染能力。
5)本发明构建的基因转染纳米材料可解决新兴治疗策略基因转染在感染相关疾病治疗中应用过程所面临的巨大挑战，有望为非抗生素的抗感染策略开辟新的方向，具有重大的社会、经济价值。
附图说明
图1为实施例中生物安全性的结果图；
图2为实施例中材料入胞的共聚焦显微镜结果图；
图3为实施例中目的基因在MC3T3-E1细胞中转染表达的结果图；
图4为实施例中治疗皮下感染小鼠模型的结果图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
本发明提供了一种具备高效抗感染能力的基因转染纳米材料，该纳米材料为负载质粒DNA的纳米颗粒，纳米颗粒的外层修饰有聚多巴胺涂层，并通过聚多巴胺涂层接枝抗菌肽。
其中，质粒DNA为抗菌肽基因。抗菌肽包括防御素、LL37或KR12中的一种。纳米颗粒包括介孔二氧化硅或金属有机框架中的一种。该纳米材料中，质粒DNA的含量为8-12μg/ml，聚多巴胺的含量为140-160mg/ml，抗菌肽的含量为8-12mg/ml。
本发明同时提供了上述基因转染纳米材料的制备方法，该方法包括以下步骤：
1)将质粒DNA负载在纳米颗粒上，得到负载质粒DNA的纳米颗粒；
2)在步骤1)中得到的负载质粒DNA的纳米颗粒外包裹PDA并接枝抗菌肽；
3)经过后处理，得到纳米材料。
步骤1)具体为：步骤1)具体为：将Zn(NO3)与十六烷基三甲基溴化铵溶解在甲醇中得到溶液A，将LL37和2-甲基咪唑溶解在甲醇中得到溶液B，之后将溶液A与溶液B混合。
步骤2)具体为：将多巴胺、负载质粒DNA的纳米颗粒、乙醇、三乙醇胺与LL37混合，之后室温下进行搅拌，搅拌时间为3-5小时。
步骤3)中，后处理包括洗涤及浸泡。
实施例：
本实施例提供了一种负载LL37质粒、并在外层制备PDA涂层并接枝LL37抗菌肽的ZIF8颗粒。研究发现，LL37@PDA-修饰后的基因转染纳米平台，在具备优异基因转染效果的同时，表现出良好的抗感染能力，可克服在感染相关疾病发生时，局部细胞所面临的细菌挑战，使新兴的基因治疗方式在感染相关疾病治疗中应用成为可能。
具体研究过程如下：
1、基因转染纳米材料的制备
将2g硝酸锌、0.34g二甲基咪唑、100μg LL37质粒在10ml乙醇中混合，通过一步法制备负载LL37质粒的ZIF8纳米颗粒，并进一步通过PDA涂层在纳米颗粒表面接枝LL37抗菌肽，最后将纳米颗粒离心收集，PBS清洗三遍后待用。
2、基因转染纳米材料的生物安全性检测
将MC3T3-E1细胞以每孔10000个的数量种于96孔板中，贴壁过夜后加入一定浓度上述制备的纳米材料，进一步孵育24小时后，利用CCK8检测各孔细胞活性情况。
图1为生物安全性结果图，图1显示了制得的基因转染纳米颗粒在1024ug/ml浓度下仍不具有明显的细胞毒性。
3、基因转染纳米材料的吞噬及目的基因转染检测
在上述制备的纳米材料表面吸附PI染料(红色，激发波长为535nm，发射波长为615nm)，并与MC3T3-T1细胞孵育3h后，4％多聚甲醛固定，FITC(绿色，激发波长为488nm，发射波长为519nm)染细胞骨架，DAPI(蓝色，激发波长为358nm，发射波长为461nm)对细胞核进行染色。利用激光共聚焦显微镜观察材料的入胞情况。
设计的LL37质粒携带共同表达的GFP基因，即LL37质粒成功转染并被细胞表达后，细胞除了表达目的基因，还会自发表达GFP绿色荧光。利用纳米材料与MC3T3-T1细胞孵育36h后，4％多聚甲醛固定，DAPI对细胞核进行染色，并利用激光共聚焦显微镜对目的基因的表达情况进一步观察。
图2为材料入胞的共聚焦显微镜结果图，图2显示了基因转染纳米颗粒可大量入胞。
图3为目的基因在MC3T3-E1细胞中转染表达的结果图，图3显示了基因转染纳米颗粒入胞后进一步完成目的基因的转染，大量表达的GFP提示目的基因LL37也被大量表达。
4、基因转染纳米材料在小鼠皮下感染模型中的抗感染检测
将上述制备的基因转染纳米材料颗粒利用水凝胶负载于PCL/HA 3D打印支架中，并植入小鼠背部皮肤的皮下，同时加入50ul浓度为108CFUs/ml的MRSA构建皮下植入物相关感染模型。1周后观察对照组及实验组局部感染进展情况。
图4为治疗小鼠皮下植入物相关感染模型的结果图，图4显示了基因转染纳米颗粒在体内表现出显著的抗感染作用。
经过以上一系列实验，验证了本发明的基因转染纳米材料具有较好的抗感染能力，入胞能力优异，可高效转染目的基因，有望对现有的基因转染方法实现进一步改进，使其在感染相关性疾病的治疗应用中有更优的适用性。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (6)

1.一种具备抗感染能力的基因转染纳米材料，其特征在于，该纳米材料为负载质粒的纳米颗粒，所述的纳米颗粒的外层修饰有聚多巴胺涂层，并通过聚多巴胺涂层接枝抗菌肽LL37，对MRSA的杀菌率达99.9%；
所述的质粒为LL37质粒；
所述的纳米颗粒为ZIF8纳米颗粒；
在该纳米材料合成体系中，质粒的含量为8-12μg/ml，聚多巴胺的含量为140-160mg/ml，抗菌肽的含量为8-12mg/ml。
2.一种如权利要求1所述的具备抗感染能力的基因转染纳米材料的制备方法，其特征在于，该方法包括以下步骤：
1）将质粒负载在纳米颗粒上，得到负载质粒的纳米颗粒；
2）在步骤1）中得到的负载质粒的纳米颗粒外包裹PDA并接枝抗菌肽；
3）经过后处理，得到所述的纳米材料。
3.根据权利要求2所述的一种具备抗感染能力的基因转染纳米材料的制备方法，其特征在于，步骤1）具体为：将Zn(NO3)与十六烷基三甲基溴化铵溶解在甲醇中得到溶液A，将LL37质粒和2-甲基咪唑溶解在甲醇中得到溶液B，之后将溶液A与溶液B混合。
4.根据权利要求2所述的一种具备抗感染能力的基因转染纳米材料的制备方法，其特征在于，步骤2）具体为：将聚多巴胺、负载质粒的纳米颗粒、乙醇、三乙醇胺与抗菌肽LL37混合，之后室温下进行搅拌。
5.根据权利要求4所述的一种具备抗感染能力的基因转染纳米材料的制备方法，搅拌时间为3-5小时。
6.根据权利要求2所述的一种具备抗感染能力的基因转染纳米材料的制备方法，其特征在于，步骤3）中，后处理包括洗涤及浸泡。
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