CN111658785B - 一种基因载体及其制备方法和应用 - Google Patents
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Abstract
本发明属于纳米生物材料领域，公开了一种基因载体，包括PEG‑PEI共聚物和其所包裹的SPIO，PEG：PEI：SPIO的质量比为(20‑25)：1：3，所述基因载体的粒径为420‑450nm。所述基因载体与基因结合能力强，毒性小、转染效率高，并且可实现磁共振成像，可应用于制备靶向性基因探针或基因治疗药物。
Description
技术领域
本发明属于纳米生物材料领域，特别涉及一种基因载体及其制备方法和应用。
背景技术
基因治疗被认为是一种有前景的方法，不仅用于治疗具有遗传缺陷的疾病，而且可用于治疗和预防诸如神经退行性疾病，癌症，心血管疾病和类风湿性关节炎等后天性疾病。然而，核酸以及其他聚阴离子物质会被核酸酶迅速降解，并且当在递送时呈现细胞的摄取力低下，靶向性不强，这就导致了用于传送基因的载体系统的研发至关重要。
迄今使用的基因载体系统包括病毒系统和非病毒系统，病毒系统常见的几种病毒载体包括逆转录酶病毒(retroviruses)/慢病毒(lentiviruses，LV)，腺病毒(adenovirus，Ad)和腺相关病毒(adeno-associated virus，AAV)，非病毒系统则包括传统的脂质体和聚合物。与非病毒载体相比，病毒载体具有较高的转染效率，如AAV载体是神经系统中最常用的基因治疗载体，转染效率高，同时在动物和人类实验中都证实了其部分的安全性，但其转染能力有限，并且由于体积小，只能包装少量基因，且在部分组织中可能会产生严重的免疫反应。
与病毒载体相比，采用传统脂质非病毒载体的基因疗法，它们虽然安全、大部分不引起免疫反应以及可携带大分子的基因，但是其导入外源性基因的效率低下、不能长时间表达目的基因，多用于基础研究。而聚合物非病毒载体相比其他传统非病毒载体和病毒载体，它安全性更强，且有良好的灵活性和重复给药的可能性，可以大规模设计具有明确结构和化学性质的载体；尤其是其中可生物降解的聚合物，无需用外科手术来移除递送系统，因此越来越受到人们的关注。
在现有研究中，可生物降解的聚合物非病毒基因载体仍存在下列问题的一种或多种，例如无法造影成像、稳定性和活性较差、转染效率低、细胞毒性过大等。
因此，希望提供一种与基因结合能力强，可造影成像，毒性小且转染效率高的基因载体。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种基因载体，其与基因结合能力强，毒性小、转染效率高，并且可实现磁共振成像。
本发明还公开了一种基因载体的制备方法。
一种基因载体，包括PEG-PEI共聚物和其所包裹的SPIO，PEG：PEI：SPIO的质量比为(20-25)：1：3，所述基因载体的粒径为420-450nm。
其中PEI(聚乙烯亚胺)是一种合成的阳离子聚合物，被认为是具有最高阳离子电荷潜力的有效基因载体，PEI的支链由25％，50％和25％的伯胺，仲胺和叔胺组成，不仅可以在体外也可以在体内对基因进行凝聚和传递。其伯胺通过与DNA磷酸基团的相互作用参与PEI-DNA复合物的形成，仲胺和叔胺由于均可被质子化使得在生理条件下PEI具有良好的缓冲作用。但由于PEI是阳离子聚合物，细胞内正电荷容易引起细胞聚集死亡，一些在体实验证明也可以表明PEI-DNA聚合物的细胞毒性可能是细胞死亡和基因表达减弱的原因，因此单一的PEI-DNA聚合物不能适用于实际的需求。
其中PEG为聚乙二醇，具有较低的免疫原性和抗原性，将其作为共聚物的组成成分，可一定程度上提高溶解性和降低细胞毒性。
SPIO(超顺磁性氧化铁粒子)是一系列由磁性氧化铁组成的晶状物质，该化合物通式为Fe3+ 2O3M2+O，其中最常研究的为Fe2+组成的Fe3O4，铁磁性微粒的粒径一般在0.1-10μm，当铁的氧化物远小于铁磁性微粒时，呈现出超顺磁性，Fe3O4晶体呈现超顺磁性的临界直径为50nm。超顺磁性氧化铁纳米粒子以其较高的生物相容性和优良的磁学性质广泛应用于生物医学领域，如靶向药物载体、核磁共振成像造影剂、磁性细胞分离以及DNA的纯化等。
上述基因载体的制备方法，包括以下步骤：
(1)配置EDC的DMSO溶液，并加入PEI和DSPE-PEG-COOH，搅拌后透析，冻干，得到DSPE-PEG-PEI；
(2)将SPIO的DMSO溶液、DSPE-PEG-Mal以及步骤(1)制得的DSPE-PEG-PEI混合，超声，透析，冻干，制得基因载体。
其中EDC为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，DMSO为二甲基亚砜，DSPE为二硬脂酰磷脂酰乙醇胺，Mal为马来酰亚胺。
优选的，步骤(1)中PEI使用MES缓冲液进行溶解。MES缓冲液的中文名称2-(N-吗啡啉)乙磺酸，主要作为生物缓冲剂。
优选的，步骤(1)中DSPE-PEG-COOH使用DMSO进行溶解，并与EDC混合。
优选的，步骤(1)中搅拌的方式为磁力搅拌，搅拌的时间为20-30h。
优选的，步骤(1)中使用蒸馏水进行透析，透析袋的截留分子量为3500。
优选的，步骤(2)超声的频率为15-25HZ，超声的时间为1-2h。
优选的，步骤(2)中使用蒸馏水进行透析，透析袋的截留分子量为2000。
所述基因载体在制备靶向性基因探针中的应用。本发明所述基因载体为可视化纳米复合物，包含有磁性特质的SPIO，可通过磁共振成像对基因载体进行示踪，因此可根据需要装载目的基因，制得靶向性的基因探针。
所述基因载体在制备基因治疗药物中的应用。
一种基因治疗药物，包括转录因子、靶向分子和所述基因载体。
优选的，所述转录因子为TFEB，所述靶向分子为VCAM-1。所述基因治疗药物中通过添加有转录因子TFEB以及靶向分子VCAM-1，可实现对血管源性异常物质堆积性疾病，包括脑血管及颈动脉粥样硬化等疾病的探查与治疗。
转录因子TFEB(transcription factor EB)是调控自噬过程最重要的转录因子，TFEB属于bHLH-LZ类转录因子中MiTF亚家族成员，与其家族其他成员相比，转录因子TFEB更加广泛的存在于各种组织细胞中，在自噬、溶酶体的生物发生、应激、癌症、免疫等各方面发挥重要作用。TFEB可通过与溶酶体及自噬相关基因启动子区CLEAR元件结合，调控溶酶体与自噬相关基因的表达，参与调节自噬体与溶酶体的形成及融合、底物的降解等自噬过程多个阶段，在各种细胞和动物实验中证实了TFEB为溶酶体的主控基因，通过参与自噬-溶酶体途径的调控，能全面调控细胞内清除再循环利用和能量代谢，在细胞自噬、溶酶体功能调节、脂质代谢等方面起重要作用。转录因子TFEB在自噬溶酶体障碍疾病以及脂质代谢等疾病的治疗中有着不可或缺的作用。VCAM-1(血管细胞粘附分子-1)可作为靶向分子，对疾病的探查起到靶向性的作用。
所述基因治疗药物的制备方法，包括以下步骤：
(1)将上述基因载体用PBS缓冲液分散，加入VCAM-1，搅拌，透析，冻干，得到SPIO-PEI-PEG-VCAM；
(2)将步骤(1)制得的SPIO-PEI-PEG-VCAM与TFEB混合进行反应，制得基因治疗药物SPIO-PEI-PEG-VCAM/TFEB。
优选的，步骤(1)中基因载体与VCAM-1的质量比为(10-12)：1。
优选的，步骤(1)中使用蒸馏水进行透析，透析袋的截留分子量为3500。
优选的，步骤(2)中SPIO-PEI-PEG-VCAM与TFEB的质量比为(3-10)：1。其中TFEB需用DEPC水(纯水，不含RNA酶、DNA酶和蛋白酶)溶解，再与SPIO-PEI-PEG-VCAM进行混合，并加入pH缓冲液。
相对于现有技术，本发明的有益效果如下：
(1)本发明所述基因载体的细胞毒性小，具备良好的安全性；
(2)本发明所述基因载体的转染效率高，与基因的结合能力好；
(3)本发明所述基因载体为可视化纳米复合物，包含有磁性特质的SPIO，可通过磁共振成像对基因载体进行示踪；
(4)本发明所述基因治疗药物SPIO-PEI-PEG-VCAM/TFEB可实现对血管源性异常物质堆积性疾病，包括脑血管及颈动脉粥样硬化等疾病的靶向性治疗和探查。
附图说明
图1表示细胞对SPIO和实施例1制得的基因载体的摄取情况；
图2表示SPIO-PEI-PEG-VCAM对TFEB的结合能力。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例1
一种基因载体，包括PEG-PEI共聚物和其所包裹的SPIO，其中PEG：PEI：SPIO的质量比约为23：1：3，基因载体的粒径为430nm。其制备方法包括以下步骤：
SPIO可通过市面购买或自行制备的方式获取，其自行制备的方法为：将2mmol Fe(acac)3、1mmol Mn(acac)2与10mmol的1,2-十六烷二醇装入反应容器中混合，再分别加入3mmol油酸和3mmol油胺，并加入10mL二苄醚作溶剂，在氩气保护下磁力搅拌，加热至200℃保温2h，然后加热至300℃回流反应0.5h。冷却至室温后，在反应产物中加入40mL乙醇后离心(8000r/min，10min)得到棕黑色的沉淀物，除去滤液，向沉淀中加入0.05mL油酸和0.05mL油酰胺后，用正己烷将沉淀物溶解分散。离心(8000r/min，10min)后除去未分散的杂质，再次在无水乙醇中沉淀离心(8000r/min，10min)，真空干燥箱干燥，备用。
制备DSPE-PEG-PEI：称取1g的PEI溶解于50mL的MES缓冲溶液中，配制成浓度为20mg/mL的PEI溶液，备用；分别称取EDC 100mg，DSPE-PEG-COOH 50mg，溶解在5mL的DMSO中，室温下搅拌15-30min；然后按质量比PEI：DSPE-PEG-COOH＝12.5：1，向DMSO溶液中加入PEI溶液，室温下磁力搅拌24小时，然后蒸馏水透析48小时(截留分子量：3500)，-80℃冷冻干燥，即得DSPE-PEG-PEI，置于干燥器存放备用；
制备基因载体SPIO-PEI-PEG：称取5mg SPIO溶于四氢呋喃(2-3mL)中，然后加入20mg以上步骤所制备的DSPE-PEG-PEI，并与20mg DSPE-PEG-Mal混合均匀，边超声边滴入40mL的纯水中，超声1h后，产物移入透析袋(截留分子量：2000)中，透析三天，每天换纯水两次，将透析液冷冻干燥后，得到固体产物，即为基因载体SPIO-PEI-PEG。
实施例2
一种基因治疗药物，包括转录因子TFEB、VCAM-1和实施例1所制得的基因载体。其制备方法包括以下步骤：
(1)称取20mg的基因载体SPIO-PEI-PEG，通过超声分散在PBS缓冲溶液中，然后按质量比SPIO-PEI-PEG:VCAM-1＝10:1，加入VCAM-1，4℃下磁力搅拌24小时，产物移入透析袋(截留分子量：3500)中，透析三天，每天换纯水两次，将透析液冷冻干燥后，收集固体产物于4℃下保存备用，记作SPIO-PEI-PEG-VCAM；
(2)在无菌的条件下，将10nmol的TFEB用100μL的DEPC水溶解制得浓度为0.1nmol/μL的TFEB，与SPIO-PEI-PEG-VCAM混悬液混合，然后用Hepes缓冲溶液补足至1mL，混合后至于37℃培养箱静置30min，即得基因治疗药物SPIO-PEI-PEG-VCAM/TFEB。
产品效果测试
粒径测试
将实施例1所制备的基因载体SPIO-PEI-PEG溶于1mL去离子水中，分散均匀，用Nano ZS90 Malvern仪器进行DLS粒径测试，测定SPIO-PEI-PEG水溶液的粒径及其分布，激光波长为658nm，角度为90°，多分散系数为1.33，测试温度为25℃。扫描电镜的结果显示，SPIO-PEI-PEG呈规则圆球形结构，通过对其进行粒径分析可知，实施例1制得的SPIO-PEI-PEG平均粒径为430.47nm。
SPIO和基因载体SPIO-PEI-PEG的振动样品磁强分析
将SPIO和基因载体SPIO-PEI-PEG彻底干燥，精确称重，然后用VSM测定。测定条件：300K，N2环境，外加磁场范围从20000KOe到-20000KOe。
SPIO和基因载体SPIO-PEG-PEI的磁滞曲线都通过原点，证明剩磁为零，均具有超顺磁性。SPIO纳米粒子饱和磁化强度为56emu/g，证实单分散超顺磁性纳米粒子具有较高的饱和磁化强度。而包载有SPIO的基因载体SPIO-PEI-PEG，其饱和磁化强度降低为10emu/g，证明SPIO纳米粒子被PEG-PEI聚合物有效包覆，使得饱和磁化强度发生降低。
基因载体SPIO-PEI-PEG的毒性试验
将成纤维细胞L929以5000个/孔的密度接种于96孔板中，然后将其置于二氧化碳培养箱中培养贴壁过夜。随后，吸出原有的培养基，换上新鲜的含有不同浓度的SPIO和SPIO-PEI-PEG的完全培养基，所选的样品的浓度范围为10-160μg/mL，每个浓度设置5个平行。在培养箱中培养24h后，用PBS将细胞洗涤一次后并向各孔中加入100μL的新鲜培养基(含有10％的CCK-8试剂，CCK-8试剂的主要成分是2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐)。置于培养箱中孵育一段时间，最后使用酶标仪检测并记录在450nm波长处的吸光度，通过以下公式计算细胞存活率：细胞存活率(％)＝(实验组吸光度-空白组吸光度)/(阴性对照组吸光度-空白组吸光度)×100％。
通过分析细胞毒性试验表明，SPIO对小鼠成纤维细胞L929存在一定的毒性，且细胞存活率与药物成浓度依赖关系。而SPIO-PEI-PEG在不同浓度下均未表现出细胞毒性，说明该基因载体不会对正常细胞产生杀伤效果，具有良好的安全性。
L929细胞对SPIO和基因载体SPIO-PEI-PEG的摄取情况
通过普鲁士蓝染色观察L929细胞细胞对SPIO和实施例1中制得的基因载体SPIO-PEI-PEG的摄取情况，具体测试方法为：细胞爬片：4％多聚甲醛固定15min，PBS漂洗3次，5min/次。加破膜液破膜10-15min，PBS漂洗3次，5min/次。染色：取等量2％亚铁氢化钾和2％盐酸等比例混合，切片入染液中染色10-20min；流水冲洗2min，蒸馏水洗。复染：0.1％核固红复染核5-10min，蒸馏水洗数分钟。脱水封片：孔板内依次加入95％酒精5min-95％酒精5min-无水乙醇5min-无水乙醇5min，将爬片从孔板内挑出来，晾干后将有细胞的一面朝下用中性树胶封固在载玻片上。通过正置光学显微镜进行镜检，图像采集分析；染色后，细胞核呈红色，铁离子呈蓝色。
试验结果如图1所示，图1A表示在放大倍数为200倍时，SPIO被L929细胞的摄取情况；图1B表示在放大倍数为400倍时，SPIO被L929细胞的摄取情况；图1C表示在放大倍数为200倍时，基因载体SPIO-PEI-PEG被L929细胞的摄取情况；图1D表示在放大倍数为400倍时，基因载体SPIO-PEI-PEG被L929细胞的摄取情况。
从图1中可以看出，在不同倍数下，基因载体SPIO-PEI-PEG组中细胞内部的蓝色部分均明显多于SPIO组，证实基因载体SPIO-PEI-PEG被L929细胞摄取量明显多于SPIO，具有良好的转染效率。
SPIO-PEI-PEG-VCAM对TFEB的结合能力测试：
对实施例2中所制得的SPIO-PEI-PEG-VCAM/TFEB复合物中SPIO-PEI-PEG-VCAM与TFEB的质量比(N/P比)进行调整，并通过琼脂凝胶电泳的方法测试SPIO-PEI-PEG-VCAM对TFEB的结合能力。
如图2所示，对照组为TFEB的电泳条带，不同质量比的SPIO-PEI-PEG-VCAM/TFEB复合物表现出不同的DNA迁移情况；在N/P比小于3的SPIO-PEI-PEG-VCAM/TFEB复合物中，可以明显观察到白色亮条带从孔槽中迁出，这说明在质量比小于3的SPIO-PEI-PEG-VCAM不能与TFEB形成稳定的复合物；而质量比≥3时，TFEB已难以在电场作用的条件下从复合物中迁出，说明在该质量比及以上，SPIO-PEI-PEG-VCAM能够很好地包裹基因，与TFEB所形成的复合物稳定性良好，表明该基因载体与所携带基因具有良好的结合能力。
Claims (1)
1.一种基因治疗药物SPIO-PEI-PEG-VCAM/TFEB，其特征在于，包括基因载体、TFEB和VCAM-1；
所述基因载体由PEG-PEI共聚物和其所包裹的SPIO组成，PEG：PEI：SPIO的质量比为（20-25）：1：3，所述基因载体的粒径为420-450nm；
所述基因治疗药物SPIO-PEI-PEG-VCAM/TFEB的制备方法，包括以下步骤：
（1）称取1g的PEI溶解于50mL的MES缓冲溶液中，配制成浓度为20mg/mL的PEI溶液，备用；分别称取EDC 100mg，DSPE-PEG-COOH 50mg，溶解在5mL的DMSO中，室温下搅拌15-30min；然后按质量比PEI：DSPE-PEG-COOH＝12.5：1，向DMSO溶液中加入PEI溶液，室温下磁力搅拌24小时，然后蒸馏水透析48小时，所述透析的截留分子量为3500Da，-80℃冷冻干燥，即得DSPE-PEG-PEI，置于干燥器存放备用；
（2）称取5mg SPIO溶于2-3mL的四氢呋喃中，然后加入20mg步骤（1）所制得的DSPE-PEG-PEI，并与20mg DSPE-PEG-Mal混合均匀，边超声边滴入40mL的纯水中，超声1h后，产物移入截留分子量为2000Da的透析袋中，透析三天，每天换纯水两次，将透析液冷冻干燥后，得到固体产物，即为基因载体SPIO-PEI-PEG，所述超声的频率为15-25HZ；
（3）称取20mg步骤（2）所制得的基因载体SPIO-PEI-PEG，通过超声分散在PBS缓冲溶液中，然后按质量比SPIO-PEI-PEG:VCAM-1＝10:1，加入VCAM-1，4℃下磁力搅拌24小时，产物移入截留分子量为3500Da的透析袋中，透析三天，每天换纯水两次，将透析液冷冻干燥后，收集固体产物于4℃下保存备用，记作SPIO-PEI-PEG-VCAM；
（4）在无菌的条件下，将10nmol的TFEB用100μL的DEPC水溶解制得浓度为0.1nmol/μL的TFEB，再加入步骤（3）所制得的SPIO-PEI-PEG-VCAM，所述SPIO-PEI-PEG-VCAM与TFEB的质量比为(3-10)：1，然后用Hepes缓冲溶液补足至1mL，混合后置于37℃培养箱静置30min，即得基因治疗药物SPIO-PEI-PEG-VCAM/TFEB。
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