CN112574343B - 一种缺氧响应的阳离子聚合物及其制备方法和应用 - Google Patents
一种缺氧响应的阳离子聚合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种缺氧响应的阳离子聚合物及其制备方法和应用，所述阳离子聚合物的结构如式(I)所示，通过4‑硝基苄溴或4‑硝基苄氯与聚合物PDEA季胺化得到。本发明提供的阳离子聚合物带大量正电荷，能够很好地包裹DNA,并且进入肿瘤细胞后能够在肿瘤细胞内的缺氧条件发生电荷改变，快速释放出DNA进行转染，具有高效、低毒的特点，具有良好的应用前景。
Description
技术领域
本发明涉及高分子和生物技术领域，具体涉及一种缺氧响应去电荷的阳离子聚合物、制备方法及其作为基因输送载体的应用。
背景技术
基因治疗是指将外源正常基因导入靶细胞，以纠正或补偿缺陷和异常基因引起的疾病，以达到治疗目的。对于肿瘤，特别是肝癌这种缺少靶向药物的肿瘤，基因治疗被认为是最具有前景的治疗方法之一。目前常用的输送载体可以分为病毒载体和非病毒载体。
非病毒载体包括阳离子的脂质体、聚合物、树枝状大分子等，与病毒载体相比具有良好的安全性，低免疫原性，生物相容性和大量生产成本低等优点。但是较之病毒载体，较低的转染效率一直是困扰其应用的瓶颈，在体外具有良好效果的非病毒载体，往往在体内无法取得良好的转染效果。
在非病毒载体中，阳离子聚合物常用来中和DNA的负电并将其压缩成纳米颗粒从而保护DNA不被降解，并帮助其进入细胞。然而通过正/负静电相互作用形成的阳离子聚合物/核酸药物复合物纳米颗粒是热力学稳定的，该纳米复合物进入细胞后很难解离释放出核酸药物，导致核酸药物难以发挥药效。
公开号为CN105153339A的中国发明专利申请公开了一种氧化响应去正电荷的阳离子聚合物，包括如下片段：
该硼酸(酯)苄基季胺化阳离子聚合物在细胞内ROS作用下季铵盐会变为三级胺,随后通过自催化酯键水解生成带负电的羧酸基,促使与DNA的核酸药物解离而使其有效转染。
肿瘤组织活性氧(ROS)水平高(A Biocompatible Oxidation-Triggered CarrierPolymer with Potential in Therapeutics[J].Journal of the American ChemicalSociety,2011,133(4):756-758)，而由于肿瘤组织血管分布的特点，实体肿瘤往往由于肿瘤的过度生长导致内部高度缺氧，存在乏氧区域，实体肿瘤的平均氧分压为15mmHg,而正常组织的平均氧分压为35mmHg。因此利用缺氧环境来改变进入肿瘤组织的纳米复合物电荷从而释放核酸，能很好地利用肿瘤微环境特点，达到肿瘤组织特异性释放的目的。
因此，需要设计能够响应细胞内微环境而能够快速释放出核酸药物的载体，提高核酸药物的药效。
发明内容
本发明提供了一种缺氧响应的阳离子聚合物及其制备方法和应用，该阳离子聚合物进入肿瘤细胞后能够在肿瘤细胞内的缺氧条件发生电荷改变，快速释放出DNA进行转染。
本发明的技术方案为：
一种缺氧响应的阳离子聚合物，所述阳离子聚合物的结构如式(I)所示；其中，n代表聚合度，取值范围为5-500；阴离子为溴离子或氯离子；
式(I)所示阳离子聚合物在细胞内低氧条件下，硝基会还原成氨基，随后发生1,4-消除反应，生成的三级胺聚合物会发生自催化水解生成聚丙烯酸，实现电荷由正到负的转变，从而快速释放出负载的基因。
所述的阳离子聚合物的制备方法，包括如下步骤：丙烯酸N,N-二乙基氨基乙酯在引发剂的作用下进行聚合反应得到聚合物PDEA；聚合物PDEA与式(II)化合物季胺化得到式(I)所示的阳离子聚合物；其中，式(I)中，n代表聚合度，取值范围为5-500，阴离子为溴离子或氯离子；X为Br或Cl；
所述聚合物PDEA与式(II)化合物的投料质量比为1:1～2。
所述的引发剂为2-溴异丁酸乙酯、偶氮二异丁腈或过硫酸铵。
本发明还提供了所述的阳离子聚合物在输送DNA类核酸短链中的应用。
本发明提供了一种纳米复合物，所述纳米复合物包括本发明所述的阳离子聚合物与质粒DNA。
所述的质粒DNA为pEGFP质粒DNA或luciferase质粒DNA。
所述的纳米复合物的制备方法，包括如下步骤：将式(I)所示的阳离子聚合物溶于缓冲液中得到载体溶液，将质粒DNA溶于缓冲液中得到DNA溶液，将载体溶液加入DNA溶液中，振荡后静置得到含有所述的纳米复合物的溶液。
所述的阳离子聚合物与质粒DNA之间的N/P摩尔比为2～32；优选为16～32。
本发明还提供了所述的阳离子聚合物与质粒DNA的纳米复合物在标记肿瘤细胞中的应用。
相对于现有技术，本发明具有以下有益的技术效果：
1、本发明提供的缺氧响应的阳离子聚合物结构简单、制备方便。
2、不同于一般的季胺化载体，本发明提供的阳离子聚合物能够在肿瘤细胞内的缺氧条件发生电荷改变，促使DNA与聚合物解离，快速释放出DNA进行转染。
3、本发明提供的阳离子聚合物与质粒DNA的纳米复合物具有较高转染活性，低氧响应，能靶向肿瘤微环境进行转染，实现肿瘤细胞标记。
附图说明
图1为测试例1.1中N-PDEA/DNA纳米复合物的凝胶阻滞实验电泳图，其中NC代表单纯DNA质粒。
图2为测试例1.2中N-PDEA/DNA纳米复合物的粒径分布和Zeta电位图。
图3为测试例1.3中N-PDEA/DNA纳米复合物氮磷比为16条件下的透射电子显微镜图。
图4为测试例2中PEI、聚合物N-PDEA、N-PDEA/DNA纳米复合物的细胞毒性结果图。
图5为本发明实施例3.1中不同N/P比的N-PDEA/DNA纳米复合物的细胞转染效果图，其中图A为不同细胞的流式检测荧光分布图，图B为绿色荧光蛋白阳性细胞百分比与N/P之间的关系，图C为细胞绿色荧光平均强度与N/P之间的关系。
图6为本发明实施例3.2中N/P比16的N-PDEA/DNA纳米复合物与对照组的细胞转染效果图，其中图A为不同细胞的流式检测荧光分布图，图B为不同载体之间绿色荧光蛋白阳性细胞百分比的比较。
图7为本发明实施例3.3中N/P比16的N-PDEA/DNA纳米复合物的的细胞转染效果图，其中NC代表单纯DNA质粒。
图8为本发明测试例4中N-PDEA/DNA纳米复合物在缺氧条件下的细胞转染图，其中图A为不同细胞的流式检测荧光分布图，图B为N-PDEA/DNA纳米复合物在常氧、缺氧条件下绿色荧光蛋白阳性细胞百分比的比较，图C为N-PDEA/DNA纳米复合物在常氧、缺氧条件下细胞绿色荧光平均强度的比较。
图9为本发明测试例5中N-PDEA/DNA纳米复合物的瘤内注射体内转染图。
具体实施方式
实施例1、N-PDEA的合成
称取减压蒸馏纯化后的丙烯酸N,N-二乙基氨基乙酯单体(5.0g,29.2mmol)和偶氮二异丁腈(AIBN,0.02g,0.12mmol)，置于安瓿瓶中，氩气鼓泡30分钟除氧后密封，置于65℃油浴进行聚合，反应24小时，终止聚合后，用冰正己烷沉淀三次，烘干，得到数均分子量为20kDa(n＝120)的聚合物PDEA3.8g，产率为76％。
称取1.0g聚合物PDEA和4-硝基苄溴(1.9g，8.83mmol)，溶于N,N-二甲基甲酰胺中，室温下搅拌过夜。将反应液倒入过量四氢呋喃中，过滤收集沉淀并用截留分子量为3.5kDa的透析袋置于超纯水中透析，冻干得到1.6g聚合物N-PDEA，阴离子为溴离子，产率为70％。
1H-NMR,D2O：δ＝7.6-8.2(4H,ArH),δ＝4.2-4.7(4H,ArHCH2N(CH2CH3)2CH2CH2OOCCH-),δ＝2.9-3.8(6H,ArHCH2N(CH2CH3)2CH2CH2OOCCH-),δ＝2.3-2.6(1H,-CH2CH-),δ＝1.6-2.2(-CH2CH-),δ＝0.8-1.5(6H,ArHCH2N(CH2CH3)2CH2CH2OOCCH-)。
实施例2、N-PDEA/DNA纳米复合物的制备
称取一定量的聚合物N-PDEA溶于HEPES缓冲溶液(pH值为7.4,10mM),浓度为2mg/mL(记为N-PDEA溶液)，将pEGFP质粒DNA(吉凯基因)用HEPES缓冲溶液(pH值为7.4,10mM)稀释成40μg/mL的溶液(记为DNA溶液),按照设定的一系列氮磷比(N/P摩尔比为0.5、1、2、4、8、16、32)将N-PDEA溶液稀释成相应的浓度，按体积比1:1迅速加入到DNA溶液中,涡旋振荡10秒，室温静置30分钟，得到一系列不同氮磷比的纳米复合物溶液。
测试例1、对实施例2制得的纳米复合物溶液的理化性质表征：
1.1、凝胶阻滞实验
取制备好的一系列不同氮磷比(分别选取N/P摩尔比为0.5、1、2、4、8的样品)的纳米复合物溶液20μL,同时取10μL纯pEGFP质粒DNA作对照，将6个样品分别上样于1％琼脂糖(含有0.5mg/mL溴化乙锭)制备的凝胶孔中，在1x TAE缓冲液中120mV,电泳30min。电泳结束后，置于紫外凝胶成像系统拍摄，得到电泳图如图1所示。
从图1中可以看出，N-PDEA可以很好地结合DNA,在N/P大于1的时候即可以很好地阻滞DNA的迁移。
1.2、粒径分布和Zeta电位测定
取制备好的一系列不同氮磷比(分别选取N/P摩尔比为2、4、8、16、32的样品)的纳米复合物溶液利用动态光散射仪在25℃条件下对其尺寸，Zeta电位以及聚集性能进行测定。所得结果是经过DTS软件计算得出。每组实验重复3次并取平均值。结果如图2所示，粒径可以被压缩到50nm,Zeta电位分布在20～30mV之间。
1.3、N-PDEA/DNA的透射电子显微镜观察
取制备好的N/P摩尔比为16的纳米复合物，滴一滴到400目铜网上，用滤纸吸除多余液体，室温自然晾干，用透射电子显微镜来观察样品的形态，记录透射电镜图。结果如图3所示，通过静电自组装形成的纳米复合物，呈现出类球形的纳米颗粒，形态规则、均匀，粒径在50nm左右，跟动态光散射仪所测的结果基本一致。
测试例2、N-PDEA及其纳米复合物的细胞毒性实验
采用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(CCK-8)法来检测聚合物在7721细胞的体外细胞毒性。
将细胞培养于96孔板中，细胞密度为5000个/孔，每孔中加入100μL培养基(10％(v/v)胎牛血清)，细胞置于5％CO2浓度和95％湿度的37℃恒温培养箱中培养24h。
24h后，每个孔中加入100μL不同浓度N-PDEA、纳米复合物(实施例2制得的N-PDEA/DNA纳米复合物(吉凯基因)，氮磷摩尔比为16)培养基溶液(使聚合物N-PDEA终浓度分别为5mg/ml、10mg/ml、20mg/ml、40mg/ml、80mg/ml)，空白组加入100μL的培养基溶液，聚醚酰亚胺(PEI，25KDa)作为对照组，细胞继续培养48h。
培养48h时后，弃尽每个孔中的培养基，加入100μL的含10％CCK-8试剂的培养基，细胞继续培养1h。最终用酶标仪测试样品在450nm处的吸光度。
细胞存活率(百分比)是以实验组的吸光度值去除以空白组的吸光度值来表示。每一组数据均为同组试样三个孔的平均值。
结果如图4所示，N-PDEA对7721细胞的毒性随着浓度的增大而增大，在相同浓度下细胞存活率比PEI(25KDa)高，且聚合物N-PDEA与DNA组成纳米复合物后可减少聚合物对细胞的毒性。
测试例3、N-PDEA/DNA纳米复合物的细胞绿色荧光蛋白基因转染实验
3.1确定转染效率最佳的氮磷比
将7721细胞培养于6孔板中，细胞密度为300000个/孔，每孔1.75mL培养基，并将细胞置于5％CO2浓度和95％湿度的37℃恒温培养箱中培养24h。之后将每孔中培养基替换为1.75mL无血清培养基，并加入制备好的一系列氮磷比的纳米复合物溶液(实施例2制得的N-PDEA/DNA纳米复合物，DNA为pEGFP质粒(吉凯基因)，氮磷摩尔比分别为2、4、8、16、32)250μL(含pEGFP质粒DNA 5μg)，培养4h。然后弃尽板中含有纳米复合物的培养基，替换为新鲜的培养基继续培养至48h。
培养完成后，用0.25％胰酶/0.03％EDTA溶液将细胞消化收集到1.5mL离心管中，PBS溶液清洗两次，最后将细胞悬浮于500μL PBS溶液中，移入流式检测管中进行流式细胞仪检测。绿色荧光蛋白的激发波长为488nm,发射波长为510nm。结果如图5A～5C所示，N/P为16或者32的N-PDEA/DNA纳米复合物的细胞转染效率最佳，该基因输送载体确实能有效地将DNA释放出来促进转染。
3.2转染效率验证
将7721细胞培养于6孔板中，细胞密度为300000个/孔，每孔1.75mL培养基，并将细胞置于5％CO2浓度和95％湿度的37℃恒温培养箱中培养24h。之后将每孔中培养基替换为1.75mL无血清培养基，并加入制备好的纳米复合物溶液(实施例2制得的N-PDEA/DNA的纳米复合物，DNA为pEGFP质粒(吉凯基因)，氮磷摩尔比为16)250μL(含pEGFP质粒DNA 5μg)，培养4h。然后弃尽板中含有纳米复合物的培养基，替换为新鲜的培养基继续培养至48h。
培养完成后，用0.25％胰酶/0.03％EDTA溶液将细胞消化收集到1.5mL离心管中，PBS溶液清洗两次，最后将细胞悬浮于500μL PBS溶液中，移入流式检测管中进行流式细胞仪检测。绿色荧光蛋白的激发波长为488nm,发射波长为510nm。实验中，阳性对照为PEI/DNA纳米复合物及商业化转染试剂lipofectamine3000(Thermo Fisher Scientific)，DNA为pEGFP质粒(吉凯基因)，阴性对照为不经任何处理的细胞。每一组数据均为同组试样三个孔的平均值。
结果如图6A～6B所示，N/P为16的N-PDEA/DNA纳米复合物的转染值为99.99％，高于阳性对照组PEI/DNA(17.14％)及Lipo3000组(49.01％)。
3.3激光共聚焦观察绿色荧光蛋白表达
将7721细胞按照300000/孔的细胞浓度培养于半径15mm玻璃底培养皿中，每孔1.75mL培养基，并将细胞置于5％CO2浓度和95％湿度的37℃恒温培养箱中培养24h。之后将每孔中培养基替换为1.75mL培养基(血清含量为0％)，并加入制备好的纳米复合物溶液(N-PDEA的纳米复合物，氮磷摩尔比分别为16)250μL(含pEGFP质粒DNA 5μg)，培养4h。然后弃尽板中含有纳米复合物的培养基，替换为新鲜培养基继续培养至48h。
培养完成后，置于激光共聚焦显微镜下观察。绿色荧光蛋白的激发波长为488nm,发射波长为510nm,所有照片均在20倍物镜下拍摄，始终使用同一光强。
结果如图7所示，PEI/DNA纳米复合物只转染了少数细胞，lipofectamine3000转染稍多的细胞，只是这些细胞的亮度很高，而N-PDEA/DNA纳米复合物转染的细胞更均一化，这对基因治疗方面有很大意义。对于治疗基因来说，大部分细胞都能转染产生治疗蛋白远远好于只有一少部分细胞表达大量蛋白，因为这些蛋白往往只需要很少量就可以引起细胞的凋亡，达到治疗效果。
测试例4、N-PDEA/DNA纳米复合物在缺氧条件作用下的细胞转染实验
将7721细胞培养于6孔板中，细胞密度为300000个/孔，每孔1.75mL培养基，并将细胞置于5％CO2浓度和95％湿度的37℃恒温培养箱中培养24h。之后将每孔中培养基替换为1.75mL无血清培养基，并加入制备好的纳米复合物溶液(N-PDEA/DNA纳米复合物，氮磷摩尔比为16)250μL(含pEGFP质粒DNA 5μg)；缺氧组在1％O2浓度的缺氧培养箱中培养4h；对照组在常氧培养箱中培养4h。
然后弃尽板中含有纳米复合物的培养基，替换为新鲜培养基在常氧培养箱继续培养至48h。
培养完成后，用0.25％胰酶/0.03％EDTA溶液将细胞消化收集到1.5mL离心管中，PBS溶液清洗两次，最后将细胞悬浮于500μL PBS溶液中，移入流式检测管中进行流式细胞仪检测。绿色荧光蛋白的激发波长为488nm,发射波长为510nm。
结果如图8A～8C所示，缺氧组的细胞转染效率高于常氧组，证明细胞在缺氧条件下，纳米复合物中的DNA更容易释放。
测试例5、N-PDEA/DNA纳米复合物的瘤内注射体内转染实验。
6周的BALB/c雌性裸鼠腋下接种5x 106个7721肿瘤细胞，待肿瘤长至约200mm3后将其随机分为两组，每组六只，分别瘤内注射N-PDEA/DNA和PEI/DNA纳米复合物溶液60μL(均含luciferase质粒DNA 15μg，吉凯基因，制备方法参照实施例2，不同之处在于将pEGFP质粒DNA替换为luciferase质粒DNA，N/P为16),48小时后处死裸鼠，剥离肿瘤，加入4倍体积的Ix细胞裂解液，剪碎匀浆，13500g离心10分钟，取上清，加入荧光素酶底物，用多功能酶标仪测定化学发光强度。蛋白浓度用BCA蛋白检测试剂盒测定。化学发光强度利用蛋白浓度归一化，单位是每毫克蛋白发光强度(RLU/mg protein)。结果如图9所示，N/P为16的N-PDEA/DNA纳米复合物的体内转染比PEI/DNA纳米复合物高23倍，计算公式为N-PDEA/DNA纳米复合物组的平均每毫克蛋白发光强度除以PEI/DNA纳米复合物组的平均每毫克蛋白发光强度。证明N-PDEA/DNA纳米复合物一旦到达肿瘤部位就能进行有效转染。
Claims (8)
1.一种缺氧响应的阳离子聚合物，其特征在于，所述阳离子聚合物的结构如式(I)所示；其中，n代表聚合度，取值范围为5-500；阴离子为溴离子或氯离子；
2.根据权利要求1所述的阳离子聚合物的制备方法，其特征在于，包括如下步骤：丙烯酸N,N-二乙基氨基乙酯在引发剂的作用下进行聚合反应得到聚合物PDEA；聚合物PDEA与式(II)化合物季胺化得到式(I)所示阳离子聚合物；其中，式(I)中，n代表聚合度，取值范围为5-500，阴离子为溴离子或氯离子；X为Br或Cl；
3.根据权利要求2所述的阳离子聚合物的制备方法，其特征在于，所述聚合物PDEA与式(II)化合物的投料质量比为1:1～2。
4.根据权利要求1所述的阳离子聚合物在输送DNA类核酸药物中的应用。
5.一种纳米复合物，其特征在于，包括权利要求1所述的阳离子聚合物与质粒DNA，所述的质粒DNA为pEGFP质粒DNA或luciferase质粒DNA。
6.根据权利要求5所述的纳米复合物的制备方法，其特征在于，包括如下步骤：将式(I)所示的阳离子聚合物溶于缓冲液中得到载体溶液，将质粒DNA溶于缓冲液中得到DNA溶液，将载体溶液加入DNA溶液中，振荡后静置得到含有所述纳米复合物的溶液。
7.根据权利要求6所述的纳米复合物的制备方法，其特征在于，所述的阳离子聚合物与质粒DNA之间的N/P摩尔比为2～32。
8.根据权利要求5所述的纳米复合物在标记肿瘤细胞中的应用。
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