CN116531499A - 多功能一体化聚肽治疗性纳米疫苗及其制备方法和应用 - Google Patents
多功能一体化聚肽治疗性纳米疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了多功能一体化聚肽治疗性纳米疫苗及其制备方法和应用，该疫苗为由聚半胱氨酸‑接枝‑(2‑甲基丙烯酰氧乙基磷酸胆碱)‑接枝‑硫亚硝基、三价铁离子和没食子酸共混配位反应而成的聚肽纳米疫苗溶液，其中聚半胱氨酸‑接枝‑(2‑甲基丙烯酰氧乙基磷酸胆碱)‑接枝‑硫亚硝基的结构如式(1)所示，制备方法简单，应用于制备治疗三阴性乳腺癌的治疗性疫苗药物，通过铁死亡‑一氧化氮‑光热‑免疫治疗一体化抗肿瘤模式有效抑制肿瘤复发和肿瘤转移，为聚肽纳米疫苗重塑免疫抑制微环境开发多效合一的治疗手段提供巨大潜力，特别是在三阴性乳腺癌治疗领域具有广阔的应用前景。
Description
技术领域
本发明属于生物医药领域，具体涉及多功能一体化聚肽治疗性纳米疫苗及其制备方法和应用。
背景技术
三阴性乳腺癌是一类转移性高、易于复发、临床难治愈的致命性疾病，仍是临床癌症治疗的挑战性难题。基于免疫检查点抑制剂的免疫治疗在实体瘤治疗中取得一些进展，但临床响应率通常低于20％。因此，基于肿瘤细胞免疫死亡(ICD)效应的原理，开发治疗性纳米疫苗，触发免疫响应和免疫记忆效应，具有重要的科学意义和临床应用价值。另一方面，尽管国际上已有几种聚肽纳米药物进入癌症治疗的临床Ⅰ-Ⅲ期，但由于治疗效果偏低，阻碍进一步进入临床应用。因此，基于聚肽材料优异的生物相容性、降解性以及良好的生物安全性，开发聚肽治疗性纳米疫苗具有重要的临床应用前景，必将为三阴性乳腺癌等实体瘤的协同高效治疗提供新的思路和策略。
文献调研发现，Chunzheng Yang等人在题目<Heterostructural NanoadjuvantCuSe/CoSe2 for Potentiating Ferroptosis and Photoimmunotherapy throughIntratumoral Blocked Lactate Efflux>(设计一种纳米佐剂整合铁死亡、光热疗法和光动力疗法多种治疗方式于一体，实现乳腺癌细胞的代谢调控，铁死亡和免疫疗法之间的有效级联)的论文(Chunzheng Yang,Man Wang,Mengyu Chang,Meng Yuan,Wenying Zhang,Jia Tan,Binbin Ding,Ping’an Ma,*and Jun Lin,*J.Am.Chem.Soc 2023,145,7205-7217)报道了一种异质结构纳米疫苗，不仅可以原位引发乳腺癌细胞发生铁死亡，还具有光热和光催化性能，引发免疫原性细胞死亡；同时依靠负载的阻断剂来阻断肿瘤细胞乳酸外排增强铁死亡的杀伤效果并逆转免疫抑制微环境。但是，上述报道的纳米疫苗不仅使用昂贵的免疫阻断剂，制备过程复杂、纳米材料尺寸和形貌难于控制，而且在体内的肿瘤治疗效果不佳，既不能完全清除肿瘤，又存在肿瘤转移和复发风险。
发明内容
为解决现有的纳米疫苗制备成本高、治疗方式单一、临床治疗效果差以及逆转肿瘤免疫抑制微环境能力不足的问题，本发明的主要目的是提供多功能一体化聚肽治疗性纳米疫苗(PCSFG)，同时实现铁死亡-一氧化氮-光热-免疫治疗一体化抗肿瘤模式，即不仅实现温和光热疗、铁死亡治疗、一氧化氮气体治疗的一体化功能，还能显著放大乳腺癌细胞的免疫原性细胞死亡效应(ICD)，启动强烈的免疫响应和免疫记忆效应，有效抑制肿瘤复发和肿瘤转移，为聚肽纳米疫苗重塑免疫抑制微环境开发多效合一的治疗手段提供巨大潜力。
本发明的另一目的是提供上述多功能一体化聚肽治疗性纳米疫苗的制备方法，利用聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基和三价铁离子、没食子酸共混配位形成稳定均匀的聚肽纳米疫苗溶液，制备方法简单。
本发明的再一目的是提供上述多功能一体化聚肽治疗性纳米疫苗在制备治疗实体瘤的治疗性疫苗药物中的应用。
为实现上述目的，本发明可以通过以下技术方案实现：
本发明提供多功能一体化聚肽治疗性纳米疫苗，为由聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基、三价铁离子和没食子酸共混配位反应而成的聚肽纳米疫苗溶液，其中：
所述聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的结构如式(1)所示：
作为优选，所述聚肽纳米疫苗溶液中纳米粒子的数均粒径为45-50nm。
本发明还提供所述多功能一体化聚肽治疗性纳米疫苗的制备方法，包括以下步骤：
(1)将聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的水溶液和三氯化铁水溶液室温共混，充分搅拌进行反应；
(2)向上述溶液中加入没食子酸溶液，充分搅拌进行共混配位反应，透析，获得均一的聚肽纳米疫苗溶液。
作为优选，步骤(1)中，搅拌置于旋涡振荡器上进行，搅拌温度为25-50℃，优选37℃；反应时间为4-12h，优选8h。
作为优选，步骤(1)中，聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的水溶液和三氯化铁水溶液的质量比为0.5-3：1，优选为1:1。
作为优选，步骤(2)中，搅拌温度为25-50℃，优选37℃；反应时间为5-60min，优选30min。
作为优选，步骤(2)中，三氯化铁和没食子酸的质量比为1-6:1，优选4:1，稳定均匀的纳米疫苗中铁离子主要具有不饱和的单双共混配位模式。
作为优选，步骤(2)中，透析采用的透析袋截留分子量为1000-5000道尔顿，透析液为pH7.4、10mM的PBS溶液，透析3-6次，透析总时间为6-24h；优选透析4次，透析总时间24h。
本发明还提供所述多功能一体化聚肽治疗性纳米疫苗在制备治疗实体瘤的治疗性疫苗药物中的应用。
作为优选，通过所述多功能一体化聚肽治疗性纳米疫苗实现铁死亡-一氧化氮-光热-免疫治疗多功能一体化抑制实体瘤转移和复发。
作为优选，所述实体瘤为三阴性乳腺癌。
与现有技术相比，本发明具有如下有益效果：
(1)和临床中使用的纳米疫苗相比，本发明的多功能一体化聚肽治疗性纳米疫苗不需要使用昂贵的抗原蛋白或抗原肽，制备过程简单，易于储存和使用，大大降低成本。
(2)和临床使用的放疗、化疗、光动力治疗相比，本发明仅通过聚肽纳米材料设计，赋予其兼具温和光热疗、铁死亡治疗、一氧化氮气体治疗的功能，并能显著地实现ICD效应的放大作用。
(3)本发明仅通过一次静脉给药和一次近红外光照，就能实现转移性乳腺癌的完全无痕消融，启动强烈的免疫响应和免疫记忆效应，实现铁死亡-一氧化氮-光热-免疫治疗一体化的协同高效抗肿瘤效应，从而达到根除肿瘤、抑制肿瘤转移和复发的功效，为发展具有优异的治疗效果和良好的生物安全性治疗性纳米疫苗提供新策略。
(4)相比于化疗、放疗、纳米药物需要多次给药，本发明的疫苗仅需要一次或少次用药，临床上病人的依从性好，易于临床转化，在三阴性乳腺癌和其它类型的乳腺癌治疗领域具有广阔的应用前景。
附图说明
图1为实施例1中多功能一体化聚肽治疗性纳米疫苗的动态光散射图谱。
图2为实施例2中多功能一体化聚肽治疗性纳米疫苗对4T1肿瘤生长抑制作用的结果。
图3为实施例3中多功能一体化聚肽治疗性纳米疫苗对再挑战4T1肿瘤生长抑制作用的结果。
图4为实施例4中多功能一体化聚肽治疗性纳米疫苗对4T1肿瘤肺器官转移抑制作用的结果。
图5为实施例1中多功能一体化聚肽治疗性纳米疫苗的制备工艺流程图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
实施例1
本实施例按图5所示的多功能一体化聚肽治疗性纳米疫苗制备工艺，制备多功能一体化聚肽治疗性纳米疫苗，具体步骤如下：
步骤一：取5mL的聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基水溶液(0.8mg/mL)于25mL玻璃瓶中，缓慢加入100μL的三氯化铁溶液(40mg/mL)，在37℃下搅拌8h。
步骤二：向上述溶液中加入50μL的没食子酸溶液(20mg/mL)，置于旋涡振荡器上充分震荡30min。反应结束后，用10mM，1000mLPBS(pH＝7.4)溶液透析24h(4×1L，透析袋MW＝1000)，得到稳定均匀的聚肽治疗性纳米疫苗溶液。
本实施例制得的聚肽治疗性纳米疫苗的动态光散射图谱如图1所示，其数均粒径为45±4nm，PDI为0.28±0.08。
实施例2
实施例1制得的聚肽治疗性纳米疫苗体内对三阴性乳腺癌生长的影响实验。
将4T1荷瘤小鼠随机分为四组：PBS、PBS+NIR、聚肽治疗性纳米疫苗(2.75mg/mL)、聚肽治疗性纳米疫苗+NIR(2.75mg/mL)。第0天通过尾静脉注射一次纳米粒子，并于注射6小时后对PBS+NIR和聚肽治疗性纳米疫苗+NIR进行光照(808nm,1W/cm2，10min)，之后每两天对小鼠进行称重并对肿瘤体积进行测量。21天治疗结束后，将各治疗组小鼠的肿瘤摘取并绘制肿瘤体积生长曲线(图2)，从绘制出来的肿瘤体积曲线可以发现：聚肽治疗性纳米疫苗治疗组中单一的铁死亡疗法肿瘤抑制率为40％，然而，聚肽治疗性纳米疫苗+NIR组的小鼠肿瘤在第3天完全消融(TIR＝100％)，且无疤痕留下，并且在接下来的治疗期间内完全无复发无转移。
以上结果表明，实施例1制得的聚肽治疗性纳米疫苗通过一次静脉给药和一次低剂量的近红外光照(808nm,10min，1W/cm2)，便能实现4T1实体瘤的完全无痕消融，表现出对三阴性乳腺癌优异的抑制作用，在动物水平上证明铁死亡-一氧化氮-光热-免疫治疗的一体化抗肿瘤效果。
实施例3
实施例1制得的聚肽治疗性纳米疫苗体内对再攻击三阴性乳腺癌生长影响的实验。
将4T1荷瘤小鼠随机分为四组：PBS、PBS+NIR、聚肽治疗性纳米疫苗(2.75mg/mL)、聚肽治疗性纳米疫苗+NIR(2.75mg/mL)。第0天通过尾静脉注射一次纳米粒子，并于注射6小时后对PBS+NIR和聚肽治疗性纳米疫苗+NIR进行光照(808nm,1W/cm2，10min)，之后第35天对聚肽治疗性纳米疫苗+NIR组无瘤小鼠和小鼠左侧接种4T1细胞(2×105个细胞)。每两天记录一次肿瘤体积，实验终点定义为死亡或肿瘤体积大于1000mm3。分别绘制聚肽治疗性纳米疫苗+NIR组和/>组小鼠肿瘤体积生长曲线(图3)，从绘制出来的肿瘤体积曲线可以发现：与/>组小鼠肿瘤的快速生长相比，聚肽治疗性纳米疫苗+NIR组小鼠100％排斥4T1肿瘤生长至第55天。
以上结果表明，使用实施例1制得的聚肽治疗性纳米疫苗，通过一次静脉给药和一次低剂量的近红外光照(808nm,10min，1W/cm2)，可以产生强大的免疫记忆效应来防止肿瘤复发。
实施例4
实施例1制得的聚肽治疗性纳米疫苗体内对肿瘤肺器官转移影响的实验。
将4T1荷瘤小鼠随机分为四组：PBS、PBS+NIR、聚肽治疗性纳米疫苗(2.75mg/mL)、聚肽治疗性纳米疫苗+NIR(2.75mg/mL)。第0天通过尾静脉注射一次纳米粒子，并于注射6小时后对PBS+NIR和聚肽治疗性纳米疫苗+NIR进行光照(808nm，1W/cm2，10min)，第1天给各组小鼠尾静脉注射4T1细胞(5×105个细胞)，建立肺转移模型。14天后摘取出各治疗组荷瘤小鼠的肺组织，统计肺结节个数。从各组肺结节个数(图4)可以看出，PBS和PBS+NIR两组小鼠肺转移明显，聚肽治疗性纳米疫苗组肺器官转移有所抑制，对于聚肽治疗性纳米疫苗+NIR，肺器官转移明显抑制，抑制率高达90％。
以上结果表明，使用实施例1制得的聚肽治疗性纳米疫苗，通过一次静脉给药和一次低剂量的近红外光照(808nm，10min，1W/cm2)，可以阻止肿瘤肺器官转移，在三阴性乳腺癌的治疗中具有临床应用前景。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.多功能一体化聚肽治疗性纳米疫苗，其特征在于，为由聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基、三价铁离子和没食子酸共混配位反应而成的聚肽纳米疫苗溶液，其中：
所述聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的结构如式(1)所示：
2.根据权利要求1所述的多功能一体化聚肽治疗性纳米疫苗，其特征在于，所述聚肽纳米疫苗溶液中纳米粒子的数均粒径为45-50nm。
3.权利要求1或2所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，包括以下步骤：
(1)将聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的水溶液和三氯化铁水溶液室温共混，充分搅拌进行反应；
(2)向上述溶液中加入没食子酸溶液，充分搅拌进行共混配位反应，透析，获得均一的聚肽纳米疫苗溶液。
4.根据权利要求3所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，步骤(1)中，搅拌置于旋涡振荡器上进行，搅拌温度为25-50℃，反应时间为4-12h，聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的水溶液和三氯化铁水溶液的质量比为0.5-3：1。
5.根据权利要求4所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，步骤(1)中，搅拌温度为37℃，反应时间为8h，聚半胱氨酸-接枝-(2-甲基丙烯酰氧乙基磷酸胆碱)-接枝-硫亚硝基的水溶液和三氯化铁水溶液的质量比为1:1。
6.根据权利要求3所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，步骤(2)中，搅拌温度为25-50℃，反应时间为5-60min，三氯化铁和没食子酸的质量比为1-6:1。
7.根据权利要求6所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，步骤(2)中，搅拌温度为37℃，反应时间为30min，三氯化铁和没食子酸的质量比为4:1。
8.根据权利要求3所述多功能一体化聚肽治疗性纳米疫苗的制备方法，其特征在于，步骤(2)中，透析采用的透析袋截留分子量为1000-5000道尔顿，透析液为pH7.4、10mM的PBS溶液，透析3-6次，透析总时间为6-24h。
9.权利要求1或2所述多功能一体化聚肽治疗性纳米疫苗在制备治疗三阴性乳腺癌的治疗性疫苗药物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述治疗性疫苗药物通过所述多功能一体化聚肽治疗性纳米疫苗实现铁死亡-一氧化氮-光热-免疫治疗多功能一体化抑制三阴性乳腺癌转移和复发。
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