CN114712493A - 一种疫苗递送载体及其制备方法和应用 - Google Patents
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Abstract
本发明属于生物医药的技术领域，公开了一种疫苗递送载体及其制备方法和应用。所述疫苗递送载体为免疫佐剂键合聚合物，其结构如下。本发明还公开了疫苗递送载体的制备方法。本发明的疫苗递送载体能将蛋白抗原递送到抗原呈递细胞胞质中。本发明的递送载体提高了抗原胞质递送效率，有效促进了DC细胞抗原交叉提呈。本发明的疫苗递送载体用于制备纳米疫苗。所述纳米疫苗包括疫苗递送载体和蛋白抗原。本发明的疫苗为预防肿瘤和/或抗肿瘤的疫苗。本发明的疫苗通过诱导特异性免疫应答抑制肿瘤生长，其特异性好，治疗效率高。
Description
技术领域
本发明属于生物医药技术领域，涉及疫苗技术领域，具体涉及一种疫苗递送载体及其制备方法和应用。
背景技术
肿瘤免疫治疗通过调节或刺激机体的免疫响应来杀伤肿瘤细胞，相比于化疗、放疗等手段，免疫治疗具有较强的肿瘤特异性，对正常组织的损伤较小。在三种免疫治疗方法中，肿瘤疫苗模拟机体受到病原体侵染的过程来激活免疫应答，同时通过免疫记忆功能产生长期的免疫保护，一直以来都受到广泛关注。
肿瘤疫苗通常由肿瘤抗原、免疫佐剂、递送载体三部分组成，载体保护抗原免受降解和清除，并有助于将其递送至淋巴结(LN)和抗原呈递细胞(APCs)，以主要组织相容性复合体(MHC)I类和II类途径呈递抗原，同时在免疫佐剂提供的共刺激信号作用下激活T、B淋巴细胞，刺激肿瘤特异性免疫应答。
APC细胞有效的抗原交叉提呈对提高疫苗作用效率至关重要，这一方面要求载体材料具备高效的抗原胞质递送能力，另一方面要求抗原与佐剂在同一时间进入同一个免疫细胞。已成熟的方法是通过纳米材料共包载抗原与免疫佐剂，达到二者的时空协同递送，另一种开发趋势是发展具有自佐剂功能的递送载体，载体兼具递送与免疫激活功能，使三元化的疫苗组成精简为二元化，提高疫苗作用效率。但是用于高效递送抗原的材料还非常有限，而且自佐剂功能载体的开发往往需要大规模筛选，其过程伴随着偶然性，设计困难，如何构建高效的精简化肿瘤疫苗仍充满挑战。
发明内容
本发明的目的是为了克服上述精简化疫苗构建的技术问题，提供一种高效的疫苗递送载体及其制备方法。所述疫苗递送载体为肿瘤疫苗递送载体，特别是将蛋白抗原递送到抗原呈递细胞(APCs)胞质中的递送载体。
本发明的另一个目的在于提供上述疫苗递送载体的应用。所述疫苗递送载体用于制备纳米疫苗，特别是纳米肿瘤疫苗。另外，所述递送载体在蛋白胞质递送中的应用。
本发明的目的通过以下技术方案实现：
一种疫苗递送载体为免疫佐剂键合聚合物，其中免疫佐剂为瑞喹莫德(Resiquimod,R848)，它作为一种内体Toll样受体(endosomal toll-like receptor,TLRs)7和8的小分子激动剂，可以有效激活先天免疫。所述聚合物由聚酰胺-胺树枝状聚合物(poly(amidoamine)(PAMAM)dendrimers)与瑞喹莫德的二硫结构衍生物(HSEA-R848)通过麦克尔加成反应得到。
所述HSEA-R848的结构式为：
CH2＝CH-C(O)-O-C2H4-S-S-C2H4-O-C(O)-R848即
所述免疫佐剂键合聚合物的结构式为：
PAMAM-(NH-C2H4-C(O)-O-C2H4-S-S-C2H4-O-C(O)-R848)n即
结构中-NH-来自PAMAM聚合物末端的胺基-NH2，n表示PAMAM表面接枝HSEA-R848分子的个数，n为大于0的整数。
所述n优选为5～30。
PAMAM聚合物为第4代或第5代PAMAM聚合物，优选为第5代。
PAMAM为聚酰胺-胺树枝状分子，其分子量为517-58048道尔顿。
一种疫苗递送载体，即上述佐剂键合树枝状聚合物，将蛋白抗原递送到抗原呈递细胞(APCs)胞质中，具体为树突状细胞(DCs)胞质。
本发明的疫苗递送载体为肿瘤疫苗递送载体，特别是将蛋白抗原递送到抗原呈递细胞(APCs)胞质中的递送载体。所述胞质为树突状细胞(DCs)胞质。
本发明的递送载体是将蛋白抗原递送到抗原提呈细胞胞内；所述蛋白抗原递送到抗原提呈细胞胞内是指蛋白抗原转导进入抗原提呈细胞的细胞质。
所述疫苗递送载体的制备方法，包括以下步骤：
在有机溶剂中，将PAMAM树枝状聚合物与碳碳双键封端的R848二硫衍生物进行反应，后续处理，获得疫苗递送载体。所述PAMAM树枝状聚合物与碳碳双键封端的R848二硫衍生物的摩尔比为以1:20～1:100，所述有机溶剂为二甲亚砜或甲醇，所述反应的温度为40～60℃，反应的时间为24～72h；所述后续处理是指通过在甲醇和水中透析，或者利用水相凝胶柱纯化。
一种纳米疫苗，包括上述免疫佐剂键合聚合物和蛋白抗原，能够在体外和体内有效激活特异性免疫响应。
所述蛋白抗原具体表示既为肿瘤抗原又为蛋白质的物质。
在本发明中蛋白质类型的抗原都可以和免疫佐剂键合聚合物形成疫苗抑制肿瘤。
所述疫苗的制备方法，是在水中将免疫佐剂键合聚合物与蛋白抗原进行复合得到；所述复合是指在搅拌或者涡旋条件下将聚合物与抗原混合。
所述免疫佐剂键合聚合物与蛋白抗原的质量比≥1/8，优选为(0.5～10)：1。
所述免疫佐剂键合聚合物与蛋白抗原相互作用并复合形成颗粒状。
所述蛋白抗原为卵清蛋白(OVA)，肿瘤细胞裂解液，肿瘤组织裂解液，肿瘤细胞膜等。
本发明的纳米疫苗为预防肿瘤和/或抗肿瘤的疫苗。
本发明的疫苗用于制备预防肿瘤和/或抗肿瘤的产品(如：药物)。
本发明的疫苗在肿瘤免疫治疗中具有较好的效果。
本发明的疫苗对B16F10黑色素瘤、4T1乳腺癌等都有效果。
所述递送载体用于将蛋白质货物递送到细胞质。
一种用于将蛋白质货物递送到细胞质的组合物，包括免疫佐剂键合聚合物和蛋白质货物，所述组合物为用于将蛋白质递送到细胞质并保留蛋白质的生物活性的组合物。
所述免疫佐剂键合聚合物与蛋白质货物通过静电和/或疏水作用方式连接。
所述免疫佐剂键合聚合物与蛋白质货物相互作用并复合形成颗粒状。
所述免疫佐剂键合聚合物与蛋白质货物的质量比≥1/8。
所述蛋白质货物为牛血清蛋白(BSA)，核糖核酸酶A(RNase A)，β-半乳糖苷酶(β-Gal)，绿色荧光蛋白(GFP)，细胞色素C(Cyt C)，藻红蛋白(R-PE)等。
本发明针对肿瘤疫苗中蛋白抗原递送效率不足，精简化疫苗构建复杂的问题，以PAMAM树枝状聚合物为骨架，在表面修饰免疫佐剂，R848佐剂分子既能激活DC细胞，其自身的多环结构和衍生物中的二硫结构又能分别提升聚合物在抗原结合和抗原胞质释放上的功能，从而实现高效的抗原胞质递送；这种佐剂键合聚合物同时作为抗原递送载体和免疫佐剂，可以与蛋白抗原结合形成精简化纳米疫苗；本发明通过研究证实这种纳米疫苗显示出高效的抗原递送能力，同时有效激活免疫细胞表面受体，促进交叉提呈，诱导机体产生肿瘤抗原特异性免疫应答，达到抑制和杀伤肿瘤的目的。
与现有技术相比，本发明具有如下优点及有益效果：
1)本发明的免疫佐剂键合PAMAM聚合物提高了抗原胞质递送效率，有效促进了DC细胞抗原交叉提呈，这种聚合物与肿瘤抗原形成纳米疫苗，并通过诱导特异性免疫应答抑制肿瘤生长，其特异性好，治疗效率高；
2)现有的精简化疫苗中，自佐剂递送材料的合成与筛选需要较大的工作量，开发难度较大，开发结果不可控，本发明选取的佐剂分子键合于聚合物表面，使其既发挥免疫刺激功能，又充分利用其化学结构特点，以满足抗原胞质递送对递送材料结构的要求，这种设计思路能够进一步拓展到其他疫苗体系，增加疫苗递送材料构建结果的可预测性，减小精简化疫苗设计与开发的难度。
附图说明
图1为R848二硫衍生物以及R848衍生物键合PAMAM聚合物(PAMAM-R848)的合成路线示意图；
图2为PAMAM-R848的1H NMR图；
图3为PAMAM-R848聚合物与蛋白抗原形成纳米疫苗的示意图以及聚合物与OVA模式抗原形成的纳米疫苗(记为PRO)的粒径表征结果；
图4为纳米疫苗(PRO)在小鼠骨髓来源树突状细胞(BMDCs)中的免疫激活和抗原交叉提呈结果；其中图A为不同比例的纳米疫苗(PRO)促进BMDCs成熟的流式结果，图B为疫苗在BMDCs上的细胞摄取和内涵体逃逸情况，图C为疫苗在BMDCs上的抗原交叉提呈结果；
图5为纳米疫苗(PRO)在体内的免疫激活结果；其中，图A为免疫检测的示意图，其中“Day”表示天数，Day 5表示5天后，Day 7表示7天后；图B为淋巴结DC细胞成熟情况；图C为淋巴结DC细胞的抗原交叉提呈结果；图D为脾脏中抗原特异性T细胞比例的流式图；图E为脾脏中抗原特异性T细胞比例的流式定量结果；
图6为纳米疫苗(PRO)在B16-OVA肿瘤模型中的肿瘤预防效果；其中A为疫苗接种和肿瘤细胞接种的示意图，其中“Day”表示天数，Day-21表示第一次注射疫苗，此时距离接种肿瘤细胞的时间为21天，Day-14表示第二次注射疫苗，此时距离接种肿瘤细胞的时间为14天，Day-7表示最后一次注射疫苗，此时距离接种肿瘤细胞的时间为7天；B为各组的肿瘤生长曲线；C为各组中每只小鼠的肿瘤生长曲线；
图7为PAMAM-R848聚合物对蛋白货物的胞质递送结果；其中，图A为在HeLa细胞中递送BSA-FITC后的胞内平均荧光强度；图B为在HeLa细胞中递送BSA-FITC后的胞内荧光图；图C为β半乳糖苷酶(β-Gal)水解底物产生蓝色物质的示意图；图D为在HeLa细胞中递送β-Gal后的胞内蓝色产物情况。
具体实施方式
下面结合具体实施例对本发明作进一步详细描述，但本发明的实施方式不限于此。
免疫佐剂键合聚合物的结构为
PAMAM-(NH-C2H4-C(O)-O-C2H4-S-S-C2H4-O-C(O)-R848)n即
结构中-NH-来自PAMAM聚合物末端的胺基-NH2，n表示PAMAM表面接枝HSEA-R848分子的个数，n为大于0的整数。所述n优选为5～30，更优选为20。
实施例1：PAMAM-R848聚合物的合成
免疫佐剂键合聚合物是由树枝状PAMAM聚合物表面氨基与R848二硫衍生物的双键通过迈克尔加成反应得到。
本实施例中树枝状PAMAM聚合物为第五代PAMAM聚合物，第五代PAMAM聚合物的分子量为28826g/mol，1mol五代PAMAM中含有128mol的氨基。PAMAM-R848的合成路线如图1所示。
免疫佐剂键合聚合物(即PAMAM-R848)的具体制备步骤如下：将15mg五代PAMAM(G5-PAMAM)与17.1mg的HSEA-R848溶解于1.0mL的无水DMSO中，HSEA-R848与G5-PAMAM的摩尔比为60:1，在40℃油浴锅中反应48小时；产物以超纯水为流动相过PD-10凝胶柱，纯化得到产物的水溶液，通过冻干获得PAMAM-R848聚合物。
PAMAM-R848聚合物的1H NMR表征图谱如图2所示。
实施例2：PAMAM-R848聚合物与抗原蛋白制备纳米疫苗
PAMAM-R848聚合物与蛋白抗原通过静电作用和疏水作用形成纳米疫苗的示意图如图3中A所示。
在验证实验中，配置浓度为0.1-1mg/mL的PAMAM-R848水溶液，然后分别以2:1、1:1、1:2的质量比(聚合物:抗原蛋白)加入OVA模式抗原，涡旋混匀(转速为500-2500rpm都行，混匀即可)并静置孵育10min得到PAMAM-R848/OVA(记为PRO)纳米疫苗。以2:1质量比制备得到的PRO纳米疫苗记为PRO(2:1)，它的动态光散射(DLS)和透射电镜(TEM)表征结果如图3中B所示，结果显示PRO(2:1)颗粒粒径约为100nm，并且形貌较为均匀、致密。
PRO(1:1)颗粒的粒径约为150nm，PRO(1:2)颗粒的粒径约为250nm。
实施例3：纳米疫苗的体外免疫激活和抗原递送效果
R848作为TLR 7/8激动剂能够促进DC细胞成熟，为了在细胞水平鉴定PRO的免疫激活功能，本发明选用通过实施例2的制备方法得到的三种质量比的纳米疫苗：PRO(1:2)、PRO(1:1)、PRO(2:1)。本发明将这三种纳米疫苗与诱导成功的BMDCs共孵育24h，OVA浓度均为10μg/mL，然后用CD11c、CD80和CD86抗体标记BMDCs表面分子，用流式细胞术检测共刺激信号分子表达水平，流式统计结果如图4中A所示。与对照组(未加入疫苗和OVA)和游离OVA组(未加入免疫佐剂)相比，PRO疫苗能够有效促进体外BMDCs成熟，且PRO(2:1)的效果最优，将其用于后续的体外和体内实验。
G5-PAMAM与OVA复合并与R848分子物理混合作为R848+G5/OVA组，其中，游离R848含量与PRO中接枝R848含量相同。将各组疫苗与体外诱导成功的BMDCs共孵育6h，OVA浓度10μg/mL。图4中B为孵育后的激光共聚焦扫描显微镜结果(即疫苗在BMDCs上的细胞摄取和内涵体逃逸情况)，其中蓝色荧光为DAPI染色的细胞核，绿色荧光为标记了FITC的OVA，红色荧光为LysoTracker Red DND-99染色的内涵体和溶酶体。共聚焦图像显示，PRO(2：1)组中BMDCs有效摄取了抗原，且绿色与红色荧光信号重叠较少，大部分OVA位于胞质中，表明PRO疫苗有效促进了抗原的内涵体逃逸，然而R848+G5/OVA组和游离OVA组的胞内绿色荧光较少，且大部分与红色荧光信号重叠，未实现有效的抗原摄取和内涵体逃逸。结果表明，PRO纳米疫苗具备优异的抗原胞质递送能力，且这种功能与聚合物键接了R848密切相关，PAMAM与R848物理混合是无效的，说明正是R848及其衍生物的化学结构赋予了递送材料良好的递送性能。
抗原交叉提呈过程中，外源性抗原发生内涵体逃逸进入到DC胞质后，被进一步加工和呈递给MHC I分子以激活CD8+T细胞，OVA蛋白257-264位短肽含有可识别的抗原表位，可用来检测交叉提呈水平。将各组疫苗与BMDCs共孵育24h，OVA浓度10μg/mL，然后用CD11c和SIINFEKL-H-2Kb抗体孵育标记，图4中C的流式统计结果显示，PRO疫苗显著提高了BMDCs的交叉提呈水平，优于游离OVA组和R848+G5/OVA组。
图4为纳米疫苗(PRO)在小鼠骨髓来源树突状细胞(BMDCs)中的免疫激活和抗原交叉提呈结果；其中图A为不同比例的纳米疫苗(PRO)促进BMDCs成熟的流式结果，图B为疫苗在BMDCs上的细胞摄取和内涵体逃逸情况，图C为疫苗在BMDCs上的抗原交叉提呈结果。
实施例4：PRO疫苗的体内免疫激活效果
图5中A为免疫检测的示意图。将4至6周龄的雌性C57BL/6小鼠随机分为4组：对照组、游离OVA组、游离R848+OVA组和PRO组，尾基皮下注射相同体积、相同OVA剂量(每只50μg)和相同R848剂量(每只14μg)的疫苗。5天后，牺牲小鼠(每组6只)并取出注射部位同侧的腹股沟淋巴结，研磨成单细胞悬液后取1×106细胞用CD45、CD11c、MHC II、CD80、CD86、SIINFEKL-H-2Kb抗体孵育标记，通过流式检测DC表面分子的表达；以同样的方式注射小鼠，7天后牺牲小鼠(每组6只)并取出脾脏，研磨成单细胞悬液后取1×106细胞用CD3、CD8、H-2KbOVA Tetramer抗体孵育标记，通过流式检测抗原特异性T细胞比例。从图5中B和图5中C可以看到，经PRO疫苗免疫的小鼠中，淋巴结DC的成熟和抗原交叉提呈效果均优于其他组，与体外结果一致。同时，如图5中D和图5中E所示，PRO组小鼠脾脏内抗原特异性T细胞的比例发生了明显的升高。由此可见，本发明的PRO纳米疫苗可以有效刺激体内DC的抗原提呈并激活抗原特异性T细胞。
图5中B为淋巴结DC细胞成熟情况；图5中C为淋巴结DC细胞的抗原交叉提呈结果；图5中D为脾脏中抗原特异性T细胞比例的流式图；图5中E为脾脏中抗原特异性T细胞比例的流式定量结果。
实施例5：PRO疫苗预防肿瘤生长
使用特异性表达OVA抗原的B16-OVA作为小鼠肿瘤模型进行肿瘤预防实验，实验分为对照组、游离OVA组、游离R848+OVA组和PRO组。图6中A展示了疫苗接种和肿瘤细胞接种的具体流程，疫苗接种时在尾基皮下注射相同体积、相同OVA剂量(每只50μg)和相同R848剂量(每只14μg)的疫苗，最后一次接种7天后在小鼠背部皮下接种3.5×105个B16-OVA细胞，并观察肿瘤生长情况。图6中B和图6中C分别为各组的肿瘤生长曲线和各组中每只小鼠的肿瘤生长曲线，结果显示PRO纳米疫苗能够有效抑制肿瘤的生长，而OVA和R848+OVA几乎无法抑制肿瘤生长。由此证明，在B16-OVA小鼠肿瘤模型中，PRO作为预防型疫苗可以有效地抑制肿瘤生长。
图6为纳米疫苗(PRO)在B16-OVA肿瘤模型中的肿瘤预防效果；其中A为疫苗接种和肿瘤细胞接种的示意图；B为各组的肿瘤生长曲线；C为各组中每只小鼠的肿瘤生长曲线。
实施例6：PAMAM-R848聚合物用于蛋白胞质递送
为了证明PAMAM-R848聚合物对蛋白货物的胞质递送能力，将实施例1制备的PAMAM-R848聚合物与FITC标记的BSA蛋白以1:2的质量比混合(BSA蛋白替换抗原蛋白，其他制备条件同实施例2)得到PAMAM-R848/BSA复合物；将G5-PAMAM与BSA-FITC复合并与R848分子物理混合作为R848+G5/BSA组，其中，游离R848含量与PAMAM-R848中接枝R848含量相同；将各组复合物与HeLa细胞共孵育4h，BSA浓度10μg/mL，并且设置两种孵育条件：培养基中含有或者不含血清。孵育后用0.2mg/mL的台盼蓝处理细胞以淬灭胞外荧光，然后分别使用流式细胞术和共聚焦显微镜检测胞内平均荧光强度和蛋白的胞内分布，由图7中A所示，PAMAM-R848/BSA组的平均荧光强度远高于游离BSA组和R848+G5/BSA组，与商业化的蛋白递送试剂PULSin相当，值得注意的是，当培养基中含有血清时，PAMAM-R848/BSA能够保持80％的胞内荧光，而其他组的荧光都有较大的减弱，说明PAMAM-R848聚合物与蛋白货物有较大的结合力，一旦形成复合物后就不易被杂质蛋白交换而提前释放。图7中B显示，PAMAM-R848/BSA组的胞内荧光信号较强，且均匀地分布在细胞胞质中，而当培养基中含有血清时，PAMAM-R848/BSA组的胞内荧光信号未发生明显减弱，这与流式结果一致。
为了验证蛋白货物在进入胞质后仍然保持生物活性，选用β-Gal作为模型蛋白，它可以催化无色X-Gal底物生成蓝色沉淀(图7中C)，蓝色产物可以用光学显微镜观察并比较颜色深浅。以本发明中实施例2的制备方法将PAMAM-R848聚合物与β-Gal蛋白以1:2的质量比混合得到PAMAM-R848/β-Gal复合物，将G5-PAMAM与β-Gal复合并与R848分子物理混合作为R848+G5/β-Gal组，其中，游离R848含量与PAMAM-R848中接枝R848含量相同；将各组复合物与HeLa细胞共孵育4h，β-Gal浓度10μg/mL，设置两种孵育条件：培养基中含有或者不含血清。由图7中D所示，PAMAM-R848/β-Gal组在两种条件下，胞质中均含有大量的蓝色催化产物，且远多于其他组，说明递送进入胞质的蛋白仍然保留了生物活性。由以上结果可见，PAMAM-R848聚合物具有优异的蛋白递送能力，可作为递送载体应用于蛋白胞质递送。
图7为PAMAM-R848聚合物对蛋白货物的胞质递送结果；其中，图A为在HeLa细胞中递送BSA-FITC后的胞内平均荧光强度；图B为在HeLa细胞中递送BSA-FITC后的胞内荧光图；图C为β半乳糖苷酶(β-Gal)水解底物产生蓝色物质的示意图；图D为在HeLa细胞中递送β-Gal后的胞内蓝色产物情况。
Claims (10)
1.一种疫苗递送载体，其特征在于：为免疫佐剂键合聚合物，其结构为PAMAM-(NH-C2H4-C(O)-O-C2H4-S-S-C2H4-O-C(O)-R848)n即
结构中-NH-来自PAMAM聚合物末端的胺基-NH2，n表示PAMAM表面接枝HSEA-R848分子的个数，n为大于0的整数。
2.根据权利要求1所述疫苗递送载体，其特征在于：所述n为5～30；
PAMAM聚合物为第4代或第5代PAMAM聚合物；
PAMAM为聚酰胺-胺树枝状分子，其分子量为517-58048道尔顿。
3.根据权利要求1所述疫苗递送载体，其特征在于：
所述疫苗递送载体是将蛋白抗原递送到抗原呈递细胞胞质中的递送载体；
所述疫苗递送载体为肿瘤疫苗递送载体。
4.根据权利要求1～3任一项所述疫苗递送载体的制备方法，其特征在于：包括以下步骤：
在有机溶剂中，将PAMAM树枝状聚合物与碳碳双键封端的R848二硫衍生物进行反应，后续处理，获得疫苗递送载体；
所述碳碳双键封端的R848二硫衍生物即HSEA-R848的结构式为：CH2＝CH-C(O)-O-C2H4-S-S-C2H4-O-C(O)-R848即
5.根据权利要求4所述疫苗递送载体的制备方法，其特征在于：所述PAMAM树枝状聚合物与碳碳双键封端的R848二硫衍生物的摩尔比为1:20～1:100，所述有机溶剂为二甲亚砜或甲醇中一种以上，所述反应的温度为40～60℃，反应的时间为24～72h；
所述后续处理是指通过在甲醇和水中透析，或者利用水相凝胶柱纯化。
6.一种纳米疫苗，其特征在于：包括疫苗递送载体和蛋白抗原；所述疫苗递送载体如权利要求1～3任一项所定义。
7.根据权利要求6所述纳米疫苗，其特征在于：
所述疫苗递送载体与蛋白抗原的质量比≥1/8；
所述蛋白抗原为卵清蛋白，肿瘤细胞裂解液，肿瘤组织裂解液，肿瘤细胞膜中一种以上；
所述纳米疫苗为预防肿瘤和/或抗肿瘤的疫苗。
8.根据权利要求6所述纳米疫苗的制备方法，其特征在于：包括以下步骤：在水中，将疫苗递送载体与蛋白抗原进行复合，得到纳米疫苗；
所述疫苗递送载体与蛋白抗原相互作用并复合形成颗粒状。
9.根据权利要求6或7所述纳米疫苗的应用，其特征在于：所述纳米疫苗用于制备预防肿瘤和/或抗肿瘤的产品。
10.一种用于将蛋白质货物递送到细胞质的组合物，其特征在于：包括免疫佐剂键合聚合物和蛋白质货物，所述组合物为用于将蛋白质递送到细胞质并保留蛋白质的生物活性的组合物；
所述免疫佐剂键合聚合物如权利要求1或2所定义；
所述免疫佐剂键合聚合物与蛋白质货物通过静电和/或疏水作用方式连接；
所述蛋白质货物为牛血清蛋白，核糖核酸酶A，β-半乳糖苷酶，绿色荧光蛋白，细胞色素C，藻红蛋白中一种以上。
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