CN114557958A - 一种刺激响应型聚两性离子纳米凝胶的制备方法与应用 - Google Patents
一种刺激响应型聚两性离子纳米凝胶的制备方法与应用 Download PDFInfo
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Abstract
本发明公开一种刺激响应型聚两性离子纳米凝胶的制备方法与应用，通过由透明质酸或其衍生物、类似物修饰双键后与带正电药物形成复合物，或者由聚乙烯亚胺修饰双键后与带负电的药物形成复合物，再通过双键与一端带有双键结构的两性离子、两端带有双键结构的响应型交联剂通过原位自由基聚合的方式形成聚两性离子纳米凝胶，得到包覆蛋白多肽类药物或核酸类药物的聚两性离子纳米笼。该纳米凝胶具有优异的抗污性能，能够抵抗非特异性生物大分子的吸附，显著延长血液循环时间，可在肿瘤特定刺激下发生裂解，实现治疗药物的靶向递送。该载体具有生物相容性好、载药率高、肿瘤部位高效富集以及药物可控释放等特点，达到高效治疗肿瘤的目的。
Description
技术领域
本发明涉及药用高分子材料制备方法及用途，特别涉及一种刺激响应型聚两性离子纳米凝胶的制备方法与应用。
背景技术
随着生物技术与多肽合成技术的日臻成熟，越来越多的多肽、核酸、蛋白质等大分子药物被开发并应用于临床。蛋白多肽类药物是指具有治疗或控制疾病的蛋白质或多肽片段，主要包括酶、细胞因子、激素等，可用于治疗癌症、代谢和自身免疫性疾病等。与小分子药物相比，蛋白多肽类药物具有低毒性、靶向性、特异性等优点。然而，该类药物同时还存在易被血液中蛋白酶降解、消除速度快、稳定性差等缺点(Tiwari G，Tiwari R，SriwastawaB，et al.Drug delivery systems：An updated review[J].International journal ofpharmaceutical investigation，2012，2(1)：2.)。药物载体可以保护药物不被降解，并且有效递送到靶部位，提高药物的安全性和有效性，从而增强药物治疗效果。因此，研究开发大分子药物递送系统对于大分子药物临床应用具有重要意义。
纳米药物载体在体内的递送需经历三个阶段：全身血液循环、肿瘤部位的富集渗透和靶部位药物释放。纳米药物最终治疗效果取决于以上每个阶段的效率。研究发现，两性离子材料由于其强烈的水化作用可有效减少蛋白的吸附和网状内皮系统对纳米颗粒的清除。与PEG及其衍生物相比，两性离子材料形成的水化层更加稳定和致密，具有更好的水溶性，同时可以避免体内产生PEG材料因多次注射引起的PEG抗体。此外两性离子材料还具有良好的生物相容性、低免疫原性，这些特性使两性离子材料被广泛用于纳米载体的构建。
在体内循环过程中，过早或过快的药物释放都将使纳米载体药代动学优势丧失或对正常组织毒性增加，而到达靶部位后过迟或过缓的药物释放则会使药物的治疗效果降低。利用肿瘤组织的独特性质，如低pH、各种酶的过表达、较高的还原性以及活性氧水平，研究者们开发了多种刺激响应型的纳米药物，不仅可以保证纳米药物在体内循环中的稳定性，同时还实现了纳米药物在靶部位的可控释放，极大提高了药物的生物利用度。如Wang等人合成了二硫键连接的聚磷酸胆碱(-聚β-己内酯(PCL-s-s-PMPC)聚合物并以此为基础构建了GSH响应型聚合物纳米胶束用于阿霉素的递送。利用两性离子PMPC优异的亲水性，PCL-s-s-PMPC胶束在血液循环中能够有效抵抗蛋白吸附、提高稳定性从而延长血液循环半衰期。此外，该胶束有效提高了阿霉素的载药率和载药效率(22.1％，95.9％)；被细胞摄取后在GSH的作用下快速释放阿霉素，表现出高效的肿瘤杀伤能力。(Wang W，Wang B，Liu S，etal.Bioreducible polymer nanocarrier based on multivalent choline phosphatefor enhanced cellular uptake and intracellular delivery of doxorubicin[J].ACSapplied materials&interfaces，2017，9(19)：15986-15994.)因此，开发一种能够在肿瘤部位高效富集并长时间滞留，且能够在肿瘤部位快速释放出药物的纳米载体对肿瘤治疗具有重大意义。
发明内容
发明目的：本发明目的是提供一种刺激响应型聚两性离子纳米凝胶的制备方法。
本发明另一目的是提供所述刺激响应型聚两性离子纳米凝胶的用途。
技术方案：本发明所述的刺激响应型聚两性离子纳米凝胶的制备方法，首先由透明质酸或其衍生物、类似物或者带有羧基聚合物修饰双键后与带正电药物形成复合物，或者由聚乙烯亚胺或带氨基聚合物修饰双键后与带负电药物形成复合物，或者由两个或多个带相反电荷的药物形成多药纳米结晶，然后通过复合物中双键与一端有双键结构的两性离子、两端有双键结构的肿瘤部位敏感的响应型交联剂通过原位聚合的方式形成聚两性离子纳米凝胶，得到包覆带电药物的聚两性离子纳米笼。
进一步地，所述的透明质酸或其衍生物、类似物或带羧基聚合物的分子量为7～100KDa；聚乙烯亚胺或带有氨基的聚合物的分子量为0.6～70KDa。作为优选所述的透明质酸的分子量为2600Da，聚乙烯亚胺的分子量为10KDa。
进一步地，所述的两性离子为丙烯酰胺羧基甜菜碱(CBAA)、丙烯酰胺磺基甜菜碱(SBAA)或2-甲基丙烯酰氧乙基磷酸胆碱(MPC)，结构如下：
进一步地，所述的肿瘤部位敏感的响应型交联剂包括：
所述的含还原敏感或酸敏感的交联剂选自如下结构所示的化合物：
R1选自H或CH3。
所述的活性氧敏感的交联剂选自如下结构所示的化合物：
其中，R1选自H或CH3。
所述的基质金属蛋白酶(MMP)敏感的交联剂选自：丙烯酸修饰的MMP-9敏感多肽(Acrylic acid)-GPLGLK-(Acrylic acid)或马来酰亚胺修饰的MMP-9敏感多肽Mal-GPLGLK-Mal。
所述的组织蛋白酶-B(cathepsin-B)敏感的交联剂选自：丙烯酸修饰的Cathepsin-B敏感多肽(Acrylic acid)-GFLG-(Acrylic acid)或马来酰亚胺修饰的Cathepsin-B敏感多肽Mal-GFLG-Mal。
所述的药物纳米晶体以3-溴丙酮酸-甲氨蝶呤(3BP-MTX)为例，其制备过程为：由甲氨蝶呤与3-溴丙酮酸按质量比1∶3混合，在冰浴条件下逐滴缓慢滴入至10mL水中，并以1000rpm转速快速搅拌5min。
进一步地，所述带电药物包括带正电小分子药物、蛋白及多肽类药物；带负电药物包括带负电的核酸类药物、小分子药物以及蛋白类药物。所述的多药纳米结晶制备过程为：将两个或多个带相反电荷的小分子药物按比例混合，在零摄氏度冰浴的条件下，用注射器逐滴缓慢滴入水中并快速搅拌。
进一步地，所述两性离子与多肽药物的质量比为1∶(0.05-0.5)，两性离子与药物纳米结晶的质量比为1∶(0.2-0.5)，两性离子与带负电的核酸类药物的质量比为1∶(0.05-0.5)。
作为优选：两性离子与多肽的质量比为1∶0.1，两性离子与药物纳米结晶的质量比为1∶0.4，两性离子与siRNA的质量比为1∶0.1。
进一步地，两性离子与透明质酸及其衍生物、类似物或者带羧基聚合物的质量比为1∶(0.25-2.5)，两性离子与聚乙烯亚胺或其他带氨基聚合物的质量比为1∶(0.4-4)。两性离子与肿瘤部位敏感的响应型交联剂的质量比为1∶(0.10-0.8)。引发剂为核黄素(VB2)或过硫酸铵(APS)；催化剂为N，N，N’，N’-四甲基乙二胺(TEMED)；两性离子与引发剂、催化剂的质量比为1∶(0.01-0.5)∶(0.05-0.5)。引发剂为核黄素(VB2)或过硫酸铵(APS)；催化剂为N，N，N’，N’-四甲基乙二胺(TEMED)；两性离子与引发剂、催化剂的质量比为1∶(0.01-0.5)∶(0.05-0.5)。
作为优选，
两性离子与透明质酸或其衍生物、类似物或者带有羧基的聚合物的质量比为1∶0.5，两性离子与聚乙烯亚胺或带有氨基的聚合物的质量比为1∶0.5。
两性离子与肿瘤部位敏感的响应型交联剂的质量比为1∶0.5。
两性离子与引发剂过硫酸铵的质量比为1∶0.2，两性离子与引发剂核黄素的质量比为1∶0.01，两性离子与催化剂N，N，N’，N’-四甲基乙二胺的质量比为1∶0.15。
所述反应条件为氮气保护下反应3h，转速为300rpm。
有益效果：本发明与现有技术相比，具有如下优势：
1、该纳米凝胶由于两性离子带电基团的强烈水合作用而具有优异的抗蛋白吸附性能，并且引入刺激响应性交联剂增加了纳米凝胶的稳定性，显著延长了体内循环时间。
2、该纳米凝胶可通过静电作用负载带电药物分子，并且可在肿瘤部位特异性快速释放药物，实现治疗药物的高效靶向递送。
3、该纳米载体具有生物相容性好、载药率高、体内循环时间长、肿瘤部位富集多以及肿瘤部位释放快等特点，可达到高效抗肿瘤的目的。
4、本发明制备方法简便，制备过程无毒无害，该纳米载体生物相容性好、载药率高，可以实现在肿瘤部位的高效富集和长时间滞留以及药物快速响应释放，从而提高药物抗肿瘤效果。
5、通过原位聚合法制备了粒径均一的聚两性离子纳米药物载体。
6、该纳米凝胶具有优异的抗污性能，能够抵抗非特异性生物大分子的吸附，显著延长血液循环时间，并且可在肿瘤特定刺激下发生裂解，实现治疗药物的靶向递送。
7、该载体使用一锅法合成，操作简单且制备过程无毒无害，成本较低，可重复性高，避免了复杂的合成和纯化过程，易于放大到工业生产中，具有广阔的市场应用前景。
附图说明
图1实施例中谷胱甘肽敏感交联剂N，N’-双(丙烯酰)胱胺的氢核磁谱图；
图2实施例1和2中MEL/HA复合物和MEL/HA@NG纳米粒子的粒径图；
图3实施例1和2中MEL/HA复合物和MEL/HA@NG纳米粒子的透射电镜图(左：MEL/HA复合物，右：MEL/HA@NG；)；
图4实施例3和4中MDNCs@NG纳米粒子的粒径图；
图5实施例6中纳米凝胶在10mM GSH 1h和10mM GSH 3h条件下的粒径变化；
图6实施例6中纳米凝胶在10mM GSH 1h和10mM GSH 3h条件下的透射电镜图(左：10mM GSH 1h，右：10mM GSH 3h)；
图7实施例7中空白纳米凝胶对小鼠乳腺癌细胞4T1细胞的细胞毒性结果图；
图8实施例7中载药纳米凝胶对小鼠乳腺癌细胞4T1细胞的细胞毒性结果图。
具体实施方式
实施例1：制备MEL/HA@NG(引发剂为核黄素)
步骤一：制备MEL/HA复合物
将1mg蜂毒肽(MEL，0.35μmol)溶于1mL去离子水中，与500μL的透明质酸(HA，10mg/mL，1.92μmol)混合，搅拌1h。在制备前，由于HA的粘性使其难以均匀分散，因此对HA溶液进行超声5min。MEL/HA复合物经去离子水透析(MWCO：7000Da)12h，除去游离的HA与MEL。
步骤二：制备MEL/HA@NG
将上述制备好的MEL/HA复合物2mL(MEL，0.5mg/mL)在持续搅拌的情况下，依次加入2-甲基丙烯酰氧乙基磷酰胆碱(MPC，10mg，34μmol)，N，N’-双(丙烯酰)胱胺(CBA，5mg，19μmol)，引发剂核黄素(VB2，0.2mg，0.5μmol)，催化剂N，N，N’，N’-四甲基乙二胺(TEMED，1.574mg，13.5μmol)。通氮气10min后，使用紫外灯(20mW/cm2)照射30min。采用去离子水透析(MWCO：7000Da)24h，除去游离的单体，得到MEL/HA@NG溶液。
实施例2：制备MEL/HA@NG(引发剂为过硫酸铵)
步骤一：制备MEL/HA复合物
将1mg蜂毒肽(MEL，0.35μmol)溶于1mL去离子水中，与500μL的透明质酸(HA，10mg/mL，1.92μmol)混合，搅拌1h。在制备前，由于HA的粘性使其难以均匀分散，因此对HA溶液进行超声5min。MEL/HA复合物经去离子水透析(MWCO：7000Da)12h，除去游离的HA与MEL。动态光散射仪测得该纳米凝胶的平均粒径为116nm，粒径分布指数为0.19(图2)。
步骤二：制备MEL/HA@NG
将上述制备好的MEL/HA复合物2mL(MEL，0.5mg/mL)在持续搅拌的情况下，依次加入2-甲基丙烯酰氧乙基磷酰胆碱(MPC，10mg，34μmol)，N，N’-双(丙烯酰)胱胺(CBA，5mg，19μmol)，引发剂过硫酸铵(APS，1.545mg，6.9μmol)，催化剂N，N，N’，N’-四甲基乙二胺(TEMED，1.574mg，13.5μmol)。通氮气3min后，室温下继续搅拌3h。采用去离子水透析(MWCO：7000Da)24h，除去游离的单体，得到MEL/HA@NG溶液。动态光散射仪测得该纳米凝胶的平均粒径为160nm，粒径分布指数为0.17(图2)。
图3为MEL/HA复合物和MEL/HA@NG纳米粒子的透射电镜图(左：MEL/HA复合物，右：MEL/HA@NG)，可以看到MEL/HA复合物的粒径均一，粒径在80nm左右，MEL/HA@NG为球形的粒径均一的纳米粒子，粒径在130nm左右。
实施例3：制备聚两性离子包覆的多药纳米晶体MDNCs@NG(引发剂为核黄素)
称取甲氨喋呤(MTX，0.3mg，6.6μmol)与3-溴丙酮酸(3-BP，0.9mg，5.4μmol)超声混合，于冰浴条件下，用注射器将混合液逐滴缓慢滴入至10mL水中，以1000rpm转速快速搅拌5min后，将转速调为300rpm，依次加入丙烯酰胺羧基甜菜碱(CBAA，4.8mg，20.9μmol)、N，N’-双(丙烯酰)胱胺(CBA，2.4mg，9.2μmol)和核黄素(VB2，0.1mg，0.25μmol)。通氮气10min后，使用紫外灯(20mW/cm2)照射30min，经去离子水透析(MWCO：7000Da)24h，除去游离的药物、两性离子与交联剂，得到聚两性离子包覆的多药纳米晶体MDNCs@NG。
实施例4：制备聚两性离子包覆的多药纳米晶体MDNCs@NG(引发剂为过硫酸铵)
称取甲氨喋呤(MTX，0.3mg，6.6μmol)与3-溴丙酮酸(3-BP，0.9mg，5.4μmol)超声混合，于冰浴条件下，用注射器将混合液逐滴缓慢滴入至10mL水中，以1000rpm转速快速搅拌5min后，将转速调为300rpm，依次加入丙烯酰胺羧基甜菜碱(CBAA，4.8mg，20.9μmol)N，N’-双(丙烯酰)胱胺(CBA，2.4mg，9.2μmol)，引发剂过硫酸铵(APS，0.78mg，3.4μmol)，催化剂N，N，N’，N’-四甲基乙二胺(TEMED，0.78mg，6.7μmol)。通氮气3min后，于室温下继续搅拌3h。采用去离子水透析(MWCO：7000Da)24h，除去游离的药物、两性离子与交联剂，得到聚两性离子包覆的多药纳米晶体MDNCs@NG。动态光散射仪测得该纳米凝胶的平均粒径为170nm，粒径分布指数为0.12(图4)。
实施例5：制备PEI/siRNA@NG(以萤光素酶siRNA-siGL3为例)
将1mg siGL3溶于1mL无酶水中，与1mL的聚乙烯亚胺(PEI，5mg/mL，0.5μmol)混合，搅拌1h。在制备前，由于PEI的粘性使其难以均匀分散，因此对PEI溶液进行超声5min。将上述制备好的PEI/siRNA复合物2mL(siGL3，0.5mg/mL)在持续搅拌的条件下，依次加入2-甲基丙烯酰氧乙基磷酰胆碱(MPC，10mg，34μmol)，N，N’-双(丙烯酰)胱胺(CBA，5mg，19μmol)，引发剂核黄素(VB2，0.2mg，0.5μmol)，催化剂N，N，N’，N’-四甲基乙二胺(TEMED，1.574mg，13.5μmol)。通氮气10min后，使用紫外灯(20mW/cm2)照射30min。采用去离子水透析(MWCO：10KDa)24h，除去游离的单体，得到PEI/siRNA@NG溶液。
实施例6：一种刺激响应型聚两性离子纳米凝胶(MEL/HA@NG)对含氨基药物分子的负载，以抑制剂药物GSK2606414为例。
步骤一：制备MEL/HA/GSK复合物
将1mg蜂毒肽(MEL，0.35μmol)溶于1mL去离子水中，与500μL透明质酸(HA，10mg/mL，1.92μmol)水溶液、100μLGSK(1mg/mL，0.22μmol)乙醇溶液混合搅拌1h。在制备前，由于HA的粘性使其难以均匀分散，因此对HA溶液进行超声5min。所得MEL/HA/GSK复合物采用去离子水透析(MWCO：7000Da)12h，除去游离的HA、GSK与MEL。
步骤二：制备MEL/HA/GSK@NG
将上述制备好的MEL/HA/GSK复合物2mL(MEL，0.5mg/mL)在持续搅拌的条件下，依次加入2-甲基丙烯酰氧乙基磷酰胆碱(MPC，10mg，34μmol)、N，N’-双(丙烯酰)胱胺(CBA，5mg，19μmol)、引发剂过硫酸铵(APS，1.545mg，6.9μmol)和催化剂N，N，N’，N’-四甲基乙二胺(TEMED，1.574mg，13.5μmol)；通氮气3min后，于室温下搅拌反应3h。采用去离子水透析(MWCO：7000Da)24h，除去游离的单体，得到MEL/HA/GSK@NG溶液。载药量(DLC)和包封率(DLE)通过以下公式计算得到：
载药量(w.t.％)＝(纳米粒子中药物质量/纳米粒子中聚合物与药物的质量总和)×100％
包封率(％)＝(纳米粒子中药物质量/投入的药物质量)×100％
表1.包裹蜂毒肽(MEL)和GSK2606414的纳米凝胶的表征a
a纳米凝胶最终浓度为1mg/mL。
b平均粒径和粒径分布在25℃、pH 7.4条件下通过动态光散射仪测定。
由表1可知，该纳米凝胶对MEL和GSK的载药量分别为8.6％和5％。
实施例7：刺激响应型聚两性离子纳米凝胶(MEL/HA@NG)在10mM GSH(pH 7.4)条件下粒径的变化
取制备好的的纳米凝胶(1mg/mL，1mL)，加入一定量的GSH溶液调至所需缓冲液环境。将样品置于37℃恒温摇床(200rpm)中。于指定时间点采用动态光散射仪测定其粒径的变化。
图5为纳米凝胶在10mM GSH(pH 7.4)条件下放置不同时间的粒径图。
图6为纳米凝胶在10mM GSH 1h和10mM GSH3h条件下的透射电镜图(左：10mM GSH1h，右：10mM GSH3h)，结果显示MEL/HA@NG在10mM GSH(pH 7.4)条件下孵育1小时粒径显著增大，达到500nm左右，说明纳米凝胶发生了明显的溶胀；到3小时，纳米凝胶结构完全破坏。
实施例8：刺激响应型聚两性离子纳米凝胶(MEL/HA@NP)对4T1细胞毒性测试(MTT)
纳米凝胶(MEL/HA@NP)在4T1细胞中的毒性通过MTT法测定。首先将100μL细胞的1640悬浮液(1640培养基中含10％胎牛血清、100IU/mL青霉素和100μg/mL链霉素)铺于96孔培养板中，并置于37℃，5％二氧化碳条件下培养24h使单层细胞的覆盖率达到70～80％。然后向每孔中加入10μL不同浓度的纳米凝胶(MEL/HA@NP)的PB溶液。待继续培养24h后，向每孔中加入10μL 3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(MTT)的PBS溶液(5mg/mL)，并放入培养箱继续培养4h以使MTT与活细胞作用。随后去除含有MTT的培养液，向每孔中加入150μL DMSO以溶解活细胞与MTT产生的紫色甲瓒结晶，并使用酶标仪(SpectraMax i3x)测定每个孔在570nm处的吸收。细胞相对存活率通过与只有空白细胞的对照孔在570nm处的吸收相比得到。实验数据均是平行三组得到的。
细胞存活率(％)＝(OD570样品/OD570对照)×100％
图7为未包覆药物的空白聚两性离子纳米凝胶对4T1细胞的细胞毒性实验结果图。结果表明：该聚两性离子纳米载体是没有毒性的，有较好的生物相容性。
图8为包覆蜂毒肽的聚两性离子纳米凝胶对4T1细胞的细胞毒性实验结果图。结果表明：游离的蜂毒肽药物对4T1细胞有较高的毒性，半数致死浓度约为6μg/mL，包覆蜂毒肽的聚两性离子纳米凝胶(MEL-NP)对4T1细胞有较高的毒性，半数致死浓度约为8μg/mL，具有较高的抗肿瘤效果。
Claims (10)
1.一种刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：首先由透明质酸或其衍生物、类似物或者带有羧基聚合物修饰双键后与带正电药物形成复合物，或者由聚乙烯亚胺或带氨基聚合物修饰双键后与带负电药物形成复合物，或者由两个或多个带相反电荷的药物形成多药纳米结晶，然后通过复合物中双键与一端有双键结构的两性离子、两端有双键结构的肿瘤部位敏感的响应型交联剂通过原位聚合的方式形成聚两性离子纳米凝胶，得到包覆带电药物的聚两性离子纳米笼。
2.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：所述的透明质酸或其衍生物、类似物或带羧基聚合物的分子量为7～100KDa；聚乙烯亚胺或带有氨基的聚合物的分子量为0.6～70KDa。
3.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：两性离子为丙烯酰胺羧基甜菜碱(CBAA)、丙烯酰胺磺基甜菜碱(SBAA)或2-甲基丙烯酰氧乙基磷酸胆碱(MPC)，结构如下：
4.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：所述的肿瘤部位敏感的响应型交联剂包括：
还原敏感或酸敏感的交联剂，选自如下所示结构的化合物：
其中，R1选自H或CH3；
或活性氧敏感的交联剂，选自如下所示结构的化合物：
其中，R1选自H或CH3；
或基质金属蛋白酶(MMP)敏感的交联剂，选自：
丙烯酸修饰的MMP 9敏感多肽(Acrylic acid)-GPLGLK-(Acrylic acid)或马来酰亚胺修饰的MMP 9敏感多肽Mal-GPLGLK-Mal；
或组织蛋白酶-B(Cathepsin-B)敏感的交联剂，选自：
丙烯酸修饰的Cathepsin-B敏感多肽(Acrylic acid)-GFLG-(Acrylic acid)或马来酰亚胺修饰的Cathepsin-B敏感多肽Mal-GFLG-Mal。
5.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：所述带正电药物包括带正电小分子药物、蛋白及多肽类药物；带负电药物包括带负电的核酸类药物、小分子药物以及蛋白类药物。
6.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：所述的多药纳米结晶制备过程为：将两个或多个带相反电荷的小分子药物按比例混合，在零摄氏度冰浴的条件下，用注射器逐滴缓慢滴入水中并快速搅拌。
7.根据权利要求1或5所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：所述两性离子与多肽药物的质量比为1∶(0.05-0.5)，两性离子与药物纳米结晶的质量比为1∶(0.2-0.5)，两性离子与带负电的核酸类药物的质量比为1∶(0.05-0.5)。
8.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：
两性离子与聚合物的质量比分别为如下：
两性离子与透明质酸及其衍生物、类似物或者带羧基聚合物的质量比为1∶(0.25-2.5)，两性离子与聚乙烯亚胺或其他带氨基聚合物的质量比为1∶(0.4-4)。两性离子与肿瘤部位敏感的响应型交联剂的质量比为1∶(0.10-0.8)。
9.根据权利要求1所述的刺激响应型聚两性离子纳米凝胶的制备方法，其特征在于：引发剂为核黄素(VB2)或过硫酸铵(APS)；催化剂为N，N，N’，N’-四甲基乙二胺(TEMED)；两性离子与引发剂、催化剂的质量比为1∶(0.01-0.5)∶(0.05-0.5)。
10.权利要求1-9任一项方法制备得到的刺激响应型聚两性离子纳米凝胶在制备抗肿瘤药物中的应用。
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