CN109200294A - 类病毒基因—药物共传递纳米载体及其制备方法和应用 - Google Patents
类病毒基因—药物共传递纳米载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开类病毒基因—药物共传递纳米载体及其制备方法和应用，采用聚阳离子聚乙烯亚胺与DNA复合形成纳米复合物，通过静电作用力将DOX纳米粒子修饰在PEI/DNA复合物的表面，构建一种具有病毒粗糙表面的纳米载体。在治疗癌症时，单一疗法存在许多不可避免的缺陷，该纳米载体可以同时装载化疗药物和治疗基因，最终实现药物和基因的联合治疗。本发明可用于抗肿瘤制剂的制备，且其具有生物毒性低、转染效率高、治疗能力强和操作简便等优点。
Description
技术领域
本发明涉及一种新型的具有肿瘤选择性多功能纳米制剂的制备方法，属于生物医药领域。
背景技术
癌症，也称恶性肿瘤，如今己经成为人类身体健康和生存寿命的重大威胁。目前,临床上常用的癌症治疗手段主要包括有外科手术、放射治疗、化学治疗和基因治疗。然而传统的化疗药物毒副作用大，肿瘤靶向性差，还容易产生多药耐药性。随着分子生物学的迅猛发展和纳米技术的日渐成熟，基因治疗已然成为根治癌症的强有力手段。目前基因治疗面临的一个亟待解决的问题仍是如何使治疗基因高效的输送进入宿主细胞，为此，需要一个合适的载体作为协助。聚阳离子载体一直是最具前景的非病毒基因载体之一，但是他们的转染效率还有待提高。DNA与载体的复合物进入细胞的能力是影响基因转染效率的主要因素之一，所以人们高度期望能够有一种策略可以促进DNA纳米复合物的细胞摄取能力。近年来，随着人们对病毒结构与功能关系的深化理解，为我们进行人工载体的设计提供了有用的信息，使得通过提高跨膜转运能力来实现高水平转染成为了可能。研究表明，许多病毒，如流感病毒，单纯疱疹病毒(HSV)和人类免疫缺陷病毒(HIV)等均含有一个被尖锋糖蛋白覆盖的粗糙表面，而这种尖锋糖蛋白组成的纳米级粗糙病毒表面对细胞膜是“友好的”，有利于细胞内吞。为此构建一个类病毒表面的纳米基因载体将有可能实现高转染，最终提高癌症的治疗效率。
近年来随着人们对病毒结构与功能关系的深入了解，人们意识到病毒的粗糙表面有利于其进入细胞，目前已有许多类病毒纳米载体相继被设计。例如，Yu等模拟病毒的粗糙表面，将小粒径硅球修饰在大硅球表面以增加其粗糙程度，制备了一个类病毒表面的纳米载药系统。研究表明具有粗糙表面的纳米硅球更容易进入细胞。冯俊课题组将金纳米粒子通过静电作用复合在负载有DNA的纳米复合物表面，形成了一种类病毒表面的有机无机杂化纳米载药系统，提高了聚阳离子基因载体进入细胞的能力，同时实现了癌症的基因治疗和光热治疗的协同治疗。纵观所有载体的设计多是通过硬质材料来实现类病毒表面纳米载体的设计，很少有人利用软质材料来进行类病毒表面纳米载体的设计。
阿霉素(DOX)作为一种常用的临床癌症化疗药物，由于其疏水性强，毒副作用大以及载药量低等缺陷，极大的限制了其在临床上的应用。近年来许多研究表明，自由的DOX可以自组装成纳米粒子，粒径约10nm左右，且带负电。例如有人利用DOX自组装形成一种无载体的纳米载体。实验表明该纳米载体有90.47％的载药量，而且具有良好的肿瘤治疗效果。同时，已有研究和临床数据表明单一疗法(化疗和基因治疗)不可避免的存在固有的不足，多种治疗手段的联合使用因此近年成为肿瘤治疗手段的重要发展方向。将多种不同治疗机制的治疗因子，装载在同一纳米载体中运载到疾病部位，实现定点同时给药，可以互补机制缺陷或者实现治疗增效。但药物与基因的装载和传递机制截然不同，构建高效低毒且具有良好生物相容性的共传递体系成为目前有关协同治疗研究的突出挑战之一。
发明内容
本发明的目的在于克服现有技术的不足，要解决的技术问题是提供一种具有类病毒粗糙表面的、可实现基因/药物共传递、高效低毒无载体纳米制剂及其制备方法，将DOX自组装纳米载体通过静电作用力引入到DNA与聚乙烯亚胺(PEI)复合物的表面，一方面改变了DNA/PEI复合物的光滑表面，以促进其进入细胞的能力，提高其转染效率；另一方面DOX的介入，使得该纳米载体实现了基因与药物的同时装载，有望实现肿瘤的基因治疗与化学治疗的协同治疗，最终实现癌症的高效治疗。
类病毒基因—药物共传递纳米载体，将药物自组装为药物纳米粒子，通过静电作用力将基因与阳离子聚合物复合形成纳米复合物，再利用静电作用力将药物纳米粒子复合在纳米复合物表面，形成同时装载有基因和药物的具有类病毒粗糙表面的纳米载体。
在上述技术方案中，药物为阿霉素。
在上述技术方案中，基因为鱼精DNA，或者p53基因。
在上述技术方案中，阳离子聚合物为PEI，Mw＝1800。
在上述技术方案中，类病毒基因—药物共传递纳米载体的大小为140—150nm，整体呈现负电势。
在进行制备时，按照下述步骤进行：
步骤1，将药物自组装为药物纳米粒子
在步骤1中，药物为阿霉素，将盐酸阿霉素进行脱盐处理，再通过再沉淀法得到自组装阿霉素纳米粒子：采用微型注射器在避光条件下，向超纯水中边搅拌边缓慢滴加脱盐阿霉素，搅拌5h，搅拌速度为1000rpm/5min，递交速度为每分钟1—5ml，温度为室温20—25摄氏度。
步骤2，通过静电作用力将基因与阳离子聚合物复合形成纳米复合物
在步骤2中，基因为鱼精DNA，或者p53基因；阳离子聚合物为PEI，Mw＝1800；采用浓度为1mg/mL PEI水溶液和浓度为200ng/μL的DNA水溶液进行复合，两者复合时间为10—40min，优选30—40min，温度为室温20—25摄氏度。
在步骤2中，基因和阳离子聚合物体系中的氮磷比为20—30，即N/P比是指载体材料所含N的摩尔数与DNA所含P的摩尔数之比。
步骤3，利用静电作用力将药物纳米粒子复合在纳米复合物表面
阿霉素纳米粒子(DOX NPs)加入含有纳米复合物PEI/DNA的水溶液中，通过静电力作用，以使阿霉素纳米粒子与PEI/DNA进行复合，复合时间为10—15min，温度为室温20—25摄氏度。
在步骤3中，先分别测定DOX与PEI/DNA的电势，再根据两者电势进行不同比例的匹配和混合，得到电中性粒子。
本发明的类病毒基因—药物共传递纳米载体在制备基因治疗和/或化学治疗药物中的应用。
本发明所制备的具有类病毒表面的基因药物共传递载体选用的是低分子量聚乙烯亚胺，毒性低，生物相容性好。DOX NPs纳米粒子通过静电力作用复合在PEI/DNA的表面增加了PEI/DNA纳米复合物的表面粗糙度，使得载体更易进行细胞内吞，提高转染效率。同时，本发明所制备的共传递体系可以实现基因与药物的共传递，在癌症治疗领域实现基因治疗与化疗的协同治疗。本发明制备方法简单，毒性低转染效率高，并且可提高药物和基因的利用率，进一步提高对肿瘤细胞的杀伤效果。
附图说明
图1是本发明中使用的PEI1800的化学结构式。
图2是本发明制备的类病毒粗糙表面基因药物共传递纳米载体PEI/DNA@DOX的DLS图像(粒径分布图)。
图3是本发明制备的类病毒粗糙表面基因药物共传递纳米载体PEI/DNA@DOX的TEM图像，其中A为DOX NPs、B为PEI/DNA、C为PEI/DNA@DOX。
图4是本发明制备的类病毒粗糙表面基因药物共传递纳米载体PEI/DNA@DOX的HeLa细胞毒性图像，分别为PEI/DNA、DOX NPs和PEI/DNA@DOX的细胞毒性示意图。
图5是本发明实施例中PEI/DNA在HeLa细胞中培养4小时后的激光共聚焦图像，其中绿色为YoYo-1标记的DNA，蓝色为细胞核。
图6是本发明实施例中类病毒粗糙表面基因药物共传递纳米载体PEI/DNA@DOX的在HeLa细胞中培养4小时后的激光共聚焦图像，其中绿色为YoYo-1标记的DNA，蓝色为细胞核，红色为DOX。
具体实施方式
下面结合具体实施例进一步说明本发明的技术方案。
阿霉素纳米粒子的制备(DOX NPs)
将10mg的DOX·HCl加入到9mL二甲基亚砜(DMSO)中，再向溶液中加入1mL三乙胺(TEA)，避光条件下搅拌12h，制备1mg/mL的DOX脱盐溶液。
取200μL的1mg/mL的DOX脱盐溶液，在1000rpm/5min的剧烈搅拌下，用微型注射器缓慢滴加到10mL水中，避光条件下搅拌12h，使用之前用220nm滤头对其进行过滤。具体制备详见参考文献Wang C,Cheng L,Liu Z.Drug delivery with upconversionnanoparticles for multi-functional targeted cancer cell imaging and therapy.[J].Biomaterials,2011,32(4):1110-20。
PEI/DNA纳米复合物的制备
配制分子量为1800的PEI水溶液(1mg/mL)，浓度为200ng/μL的DNA水溶液(鱼精DNA，购买自：天津市凯通试剂化学有限公司，规格：5g)。然后按照PEI与DNA的氮磷比(N/P)为20的纳米复合物。即：将2.6μgPEI加入到5.0μg的DNA溶液中，加入92.4mL超纯水，将复合物涡旋30s并置于37℃的环境中孵育30min。之后用含有超纯水将复合物稀释至1mL。
PEI/DNA@DOX纳米载体的制备
向1mL的DNA/PEI纳米复合物溶液中加入80μL的DOX NPs溶液，涡旋30s，然后将该复合物置于37℃恒温箱中孵育15min，使其通过静电力复合。
PEI/DNA@DOX纳米载体的粒径和电势分析
使用动态光散射粒度仪(DLS)测试纳米粒子的粒径和电势。DOX NPs的DOX的粒径为10nm，PDI为0.384，电势为-34.1mV。PEI/DNA的粒径为140nm左右，电势为+22.0mV。不同浓度DOX与PEI/DNA复合物复合后的粒径和电势，以使最终PEI/DNA@DOX纳米载体为电中性粒子，即先分别测定DOX与PEI/DNA的电势，再根据两者电势进行不同比例的匹配和混合，得到电中性粒子。随着加入DOX NPs溶液体积的不断增加，PEI/DNA@DOX的粒径有一个增大的趋势，电势不断的降低。粒径的增大和电势的降低说明DOX NPs成功的复合到了PEI/DNA上，增加了PEI/DNA的体积且中和了其正电性。当DOX NPs加入体积为80μL时，纳米粒子的电势为-5.58mV，接近电中性，生物相容性最好。同时其平均粒径为152.4nm，也符合纳米粒子的尺寸要求。考虑到电势与粒径对纳米粒子进入细胞的影响，以及DOX的载药量，选择DOX体积为80μL的复合物作为样品模型，并做进一步的研究。
PEI/DNA@DOX纳米载体对HeLa细胞毒性的研究
PEI/DNA在复合DOX NP前后的细胞毒性通过MTT法检测，同时加上阿霉素纳米粒子，共计三组样品PEI/DNA、DOX NPs和PEI/DNA@DOX。HeLa细胞以6.0×103个细胞每孔的密度接种在96孔板中在加入复合物溶液之前，在100μL含有10％FBS的DMEM培养基中培养24小时。采用在DLS实验部分所描述的方法将聚合物与1μg的鱼精DNA在N/P比为20的不同的条件下复合。并加入不同浓度的DOX(10μL、30μL、50μL、70μL、80μL)，在37℃恒温箱中复合15min后，将其与细胞共培养48小时后，用200μL新鲜的含有10％FBS的DMEM培养基替代原来的培养基，并在每孔中加入20μL MTT(5mg/mL in PBS buffer)溶液，在37℃培养箱中继续培养4小时，弃去培养基，每孔中加入150μL DMSO，在酶标仪(Bio-Red,Model 550,USA)上检测该溶液在570nm处的吸光强度。细胞的相对存活率按如下公式计算：Cell viability(％)＝(OD570(smple)/OD570(control))×100％，其中OD570(sample)和OD570(Control)分别代表无样品处理和有样品处理后的溶液吸光值。
由图中可以看出，PEI/DNA复合物中的细胞存活率高达98％以上，证明PEI/DNA复合物基本没有毒性。而由于DOX纳米粒子的引入，细胞存活率明显下降，且随着DOX纳米粒子浓度的增加细胞毒性明显增大。证明PEI/DNA@DOX的毒性是由DOX NPs引起的。DOXNPs的体积为80μL时其毒性达到最大，故进一步确定DOX NPs加入量为80μL。PEI/DNA@DOX的细胞毒性大于裸DOX NP，从另一个角度证明DOX NPs的引入改变了纳米粒子的表面粗糙程度，引起了细胞内吞行为的变化，最终增强了其对肿瘤细胞的杀伤能力。
PEI/DNA@DOX纳米载体对HeLa细胞和COS7细胞的内吞行为的研究
采用激光共聚焦(CLSM)研究纳米粒子在HeLa细胞和COS7细胞内的内吞行为。此实验中选择PEI与DNA的最佳转染比20来研究。首先将细胞以1×105的浓度接种在三个单独的小培养皿中，37℃条件下在含有10％FBS的DMEM培养基培养24小时。1μg的DNA在与聚合物复合之前，与2.5μL的浓度为1×10-5YoYo-1在37℃下作用15min以进行荧光标记，然后将其按照PEI/DNA复合物制备实验以及DLS测试实验中的方法将其与PEI以及DOX NPs进行复合，并将其在37℃条件下孵育30min。随后用含有10％FBS的DMEM培养基将DOX NP、PEI/DNA、PEI/DNA@DOX复合物稀释至1mL并立即加入到接种有细胞的单个培养皿中。培养4小时后，弃去培养基并用500mL PBS轻轻润洗几次，然后在激光共聚焦显微镜(Nikon C1-si TE2000,Japan)下观察红光、绿光和蓝光的分布情况，绿光的激发波长为488nm,发射波长是515-530nm；红光的激发波长为561nm；蓝光的激发波长为405nm。细胞图像通过EZ-C1 3.70软件记录。为了避免发生荧光猝灭，实验全程进行避光处理。
如图5和6所示，绿色代表YoYo-1标记的鱼精DNA，红色代表DOX的荧光，蓝色代表33342标记的细胞核。图像清晰显示绿色荧光分散HeLa细胞的细胞质中，不难发现图6绿色荧光较图5中的绿色荧光也有一定的增强，说明表面修饰的软质材料DOX NPs引入到PEI/DNA纳米复合物的表面增加了其表面粗糙程度，对PEI/DNA@DOX的内吞行为起到了一定的促进作用。同时复合纳米粒子的红色荧光强度也高于纯DOX NPs的荧光强度，与图4结果相吻合，也进一步证明类病毒纳米基因/药物共载体系的设计可以有效提高进入细胞的能力，最终提高其抗癌能力。图6红色荧光与绿色荧光的同时增强，也进一步说明了该纳米载体可同时将DNA和DOX运载进入细胞，也从另一个侧面进一步表明DOX NPs很好的修饰在了PEI/DNA复合物表面，该纳米载体具有同时实现基因治疗和化学治疗的潜力。
根据本发明内容记载进行工艺参数的调整，均可实现本发明载体的制备且表现出基本一致的性能，如选择用作治疗的DNA：p53基因，购买自：北京赛业生物科技有限公司，规格：20μg，制备具有治疗功能的载体。以上对本发明做了示例性的描述，应该说明的是，在不脱离本发明的核心的情况下，任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。
Claims (8)
1.类病毒基因—药物共传递纳米载体，其特征在于，将药物自组装为药物纳米粒子，通过静电作用力将基因与阳离子聚合物复合形成纳米复合物，再利用静电作用力将药物纳米粒子复合在纳米复合物表面，形成同时装载有基因和药物的具有类病毒粗糙表面的纳米载体。
2.根据权利要求1所述的类病毒基因—药物共传递纳米载体，其特征在于，药物为阿霉素；基因为鱼精DNA，或者p53基因；阳离子聚合物为PEI，Mw＝1800。
3.根据权利要求1所述的类病毒基因—药物共传递纳米载体，其特征在于，类病毒基因—药物共传递纳米载体的大小为140—150nm，整体呈现负电势。
4.类病毒基因—药物共传递纳米载体的制备方法，其特征在于，按照下述步骤进行：
步骤1，将药物自组装为药物纳米粒子；
步骤2，通过静电作用力将基因与阳离子聚合物复合形成纳米复合物，基因和阳离子聚合物体系中的氮磷比为20—30；
步骤3，利用静电作用力将药物纳米粒子复合在纳米复合物表面，先分别测定药物纳米粒子与纳米复合物的电势，再根据两者电势进行不同比例的匹配和混合，得到电中性粒子。
5.根据权利要求4所述的类病毒基因—药物共传递纳米载体的制备方法，其特征在于，在步骤1中，药物为阿霉素，将盐酸阿霉素进行脱盐处理，再通过再沉淀法得到自组装阿霉素纳米粒子：采用微型注射器在避光条件下，向超纯水中边搅拌边缓慢滴加脱盐阿霉素，搅拌5h，搅拌速度为1000rpm/5min，递交速度为每分钟1—5ml，温度为室温20—25摄氏度。
6.根据权利要求4所述的类病毒基因—药物共传递纳米载体的制备方法，其特征在于，在步骤2中，基因为鱼精DNA，或者p53基因；阳离子聚合物为PEI，Mw＝1800；采用浓度为1mg/mL PEI水溶液和浓度为200ng/μL的DNA水溶液进行复合，两者复合时间为10—40min，优选30—40min，温度为室温20—25摄氏度。
7.根据权利要求4所述的类病毒基因—药物共传递纳米载体的制备方法，其特征在于，在步骤3中，阿霉素纳米粒子加入含有纳米复合物PEI/DNA的水溶液中，通过静电力作用，以使阿霉素纳米粒子与PEI/DNA进行复合，复合时间为10—15min，温度为室温20—25摄氏度。
8.如权利要求1所述的类病毒基因—药物共传递纳米载体在制备基因治疗和/或化学治疗药物中的应用。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111920947A (zh) * | 2020-08-17 | 2020-11-13 | 福州大学 | 一种花菁染料介导的核酸/抗癌药物复合物的制备方法 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012009448A2 (en) * | 2010-07-13 | 2012-01-19 | The Regents Of The University Of California | Cationic polymer coated mesoporous silica nanoparticles and uses thereof |
| CN102851304A (zh) * | 2011-06-30 | 2013-01-02 | 复旦大学 | 基于静电包衣的三元非病毒核酸递释系统及其制备方法 |
| CN103305549A (zh) * | 2013-06-19 | 2013-09-18 | 四川大学 | 具有多重氧化还原刺激响应的纳米粒子基因载体系统及其制备方法和应用 |
| CN104524597A (zh) * | 2014-11-27 | 2015-04-22 | 郑州大学 | 基于dna生物材料的蒽环类抗肿瘤药物纳米复合物构建方法及其应用 |
| CN105561334A (zh) * | 2014-10-11 | 2016-05-11 | 中国人民解放军第二军医大学 | 一种基于温度靶向的纳米凝胶基因递送复合物及其制备方法与应用 |
-
2017
- 2017-07-06 CN CN201710547692.5A patent/CN109200294A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012009448A2 (en) * | 2010-07-13 | 2012-01-19 | The Regents Of The University Of California | Cationic polymer coated mesoporous silica nanoparticles and uses thereof |
| CN102851304A (zh) * | 2011-06-30 | 2013-01-02 | 复旦大学 | 基于静电包衣的三元非病毒核酸递释系统及其制备方法 |
| CN103305549A (zh) * | 2013-06-19 | 2013-09-18 | 四川大学 | 具有多重氧化还原刺激响应的纳米粒子基因载体系统及其制备方法和应用 |
| CN105561334A (zh) * | 2014-10-11 | 2016-05-11 | 中国人民解放军第二军医大学 | 一种基于温度靶向的纳米凝胶基因递送复合物及其制备方法与应用 |
| CN104524597A (zh) * | 2014-11-27 | 2015-04-22 | 郑州大学 | 基于dna生物材料的蒽环类抗肿瘤药物纳米复合物构建方法及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| CAITONG YU等: "Smart doxorubicin nanoparticles with high drug payload for enhanced chemotherapy against drug resistance and cancer diagnosis", 《NANOSCALE》 * |
| HUI-ZHEN JIA等: "Virus-Surface-Mimicking Surface Clustering of AuNPs onto DNA-Entrapped Polymeric Nanoparticle for Enhanced Cellular Internalization and Nanocluster-Induced NIR Photothermal Therapy", 《ADVANCED SCIENCE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111920947A (zh) * | 2020-08-17 | 2020-11-13 | 福州大学 | 一种花菁染料介导的核酸/抗癌药物复合物的制备方法 |
| CN111920947B (zh) * | 2020-08-17 | 2022-09-13 | 福州大学 | 一种花菁染料介导的核酸/抗癌药物复合物的制备方法 |
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Application publication date: 20190115 |