CN110420335A - 一种基于多孔碳酸钙的纳米免疫制剂的制备及应用 - Google Patents
一种基于多孔碳酸钙的纳米免疫制剂的制备及应用 Download PDFInfo
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Abstract
本发明属于药物制剂领域,具体涉及一种基于多孔碳酸钙的纳米免疫制剂,以多孔CaCO3纳米颗粒为载体,并在其孔道内部装载免疫阻断抑制剂IDOi,经脂质体层包覆,脂质体层表面携带免疫调节核酸CpG ODNs。本发明合成的纳米免疫制剂生物相容性好、安全性高,针对小鼠乳腺癌具有良好的治疗及防止癌症复发的效果,制备过程简单易实现,应用于抗肿瘤药物制备领域具有良好的应用前景。
Description
技术领域
本发明属于药物制剂领域,具体涉及一种纳米免疫制剂,以多孔CaCO3纳米颗粒为载体,并在其孔道内部装载免疫阻断抑制剂IDOi,表面携带免疫调节核酸CpG ODNs,其合成方法及其在强化乳腺癌疗效并防止其复发领域的应用。
背景技术
癌症是严重威胁人类健康的恶性肿瘤,原发性肿瘤治疗后的复发是大多数癌症病人死亡的最主要原因。现阶段肿瘤的临床常规的治疗方法主要是手术、放疗和化疗,但是利用这些治疗方法治疗后机体仍会残留癌细胞(即微小病灶),而这些微小病灶是癌症复发的最主要原因。因此,发展低毒性的、高效的治疗方法来治疗癌症,同时消灭微小病灶并防止癌症的复发是亟待解决的问题。
肿瘤的免疫治疗作为新一代的肿瘤治疗策略迅速发展并在实际临床应用中表现出巨大潜力。通过调整或刺激机体自身的免疫***可以***,并且利用机体自身免疫***长时间的记忆效应,可以有效预防肿瘤的复发。近年来,已经发展了多种肿瘤疫苗通过激活病人的自身免疫来抑制肿瘤。研究表明现在常用的免疫疫苗法存在以下问题:首先,免疫疫苗引起的免疫反应效果有限,一般需要加入免疫佐剂来加强免疫反应,但是免疫佐剂(如氢氧化铝)的毒性较大。其次,当肿瘤发展到恶性的时候,大部分的肿瘤相关抗原通常都被清除了,这就是所谓的肿瘤免疫逃脱机制。并且肿瘤组织处的免疫抑制微环境通常很难被打破,这很容易导致肿瘤免疫治疗的失败。因此,发展一种安全有效的方法引发机体自身强烈的免疫响应,打破肿瘤的免疫抑制微环境,提高癌症免疫治疗的效率,并利用其记忆效应进一步防止癌症的复发是非常必要的,也是具有挑战性的研究工作。
发明内容
为克服临床常规癌症治疗方法的缺陷,本发明目的在于提供一种基于多孔碳酸钙的纳米免疫制剂,当纳米免疫制剂在血液中循环至肿瘤处时,CaCO3纳米材料会在弱酸性环境中特异性地溶解,在肿瘤部位释放出CpG ODNs、IDOi和Ca2+,三者原位协同作用,增强机体的免疫响应,确保肿瘤组织免疫反应的持续发生并保持记忆效应。这种新型的碳酸钙纳米制剂应用于生物体治疗中,具有强化癌症的免疫治疗的效率,并防止其复发的效果。
本发明的第一方面,提供一种基于多孔碳酸钙的纳米免疫制剂,该纳米制剂的组成为多孔碳酸钙纳米颗粒、免疫阻断抑制剂IDOi、脂质体层及免疫调节核酸CpG ODNs。
优选的,上述免疫制剂中,多孔碳酸钙纳米颗粒作为药物载体,颗粒的孔道内部装载免疫阻断抑制剂IDOi,装载免疫制剂的多孔碳酸钙纳米颗粒被脂质体层所包覆,脂质体层的表面具有免疫调节核酸CpG ODNs修饰。
优选的,上述脂质体层是由DPPC、胆固醇和DSPE-PEG组成。
本发明的第二方面,提供一种基于多孔碳酸钙的纳米免疫制剂的制备方法,步骤如下:
(1)通过气体扩散反应合成多孔碳酸钙纳米颗粒,将制得的多孔碳酸钙纳米颗粒置于无水乙醇中装载IDOi,得到CaCO3@IDOi;
(2)采用脂质体层修饰步骤(1)中制得的CaCO3@IDOi得到CaCO3@IDOi@PEG@PEI;将得到的CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌,既得该纳米制剂。
优选的,上述制备方法中的步骤如下:
(1)将CaCl2·H2O和NH4HCO3同时置于真空干燥室中,将整个***保持在真空环境中,最后通过离心分离制得的碳酸钙纳米颗粒;将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中搅拌过夜,装载IDOi,得到CaCO3@IDOi;
(2)将步骤(1)中制得的CaCO3@IDOi乙醇溶液与DOPA混合,然后将DPPC,胆固醇和DSPE-PEG加入上述溶液中,将溶液中的有机溶剂旋干,将制得的纳米颗粒溶于水溶液中,加入PEI后搅拌以制备CaCO3@IDOi@PEG@PEI;将得到的CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌,既得该纳米制剂。
进一步优选的,步骤(1)中将CaCl2·H2O和NH4HCO3同时置于真空干燥室中,设置温度为25-30℃,将整个***保持在真空环境中20-30h后,通过10000-15000rpm离心分离制得碳酸钙纳米颗粒。
进一步优选的,步骤(1)中将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中装载IDOi,得到CaCO3@IDOi具体步骤为将碳酸钙与IDOi的乙醇溶液搅拌至少24小时。
进一步优选的,步骤(1)中CaCl2·H2O:NH4HCO3质量比为(0.5-2):(30-50),CaCO3和IDOi的质量比为(35-45):(0.5-2.5);更为优选的,CaCl2·H2O:NH4HCO3质量比为1:40,CaCO3和IDOi的质量比为40:1。
进一步优选的,步骤(2)中CaCO3@IDOi:DOPA:DPPC:胆固醇:DSPE-PEG:PEI:CpGODN的质量比为(35-45):(12-18):(12-18):(6-10):(30-35):(0.2-1);更进一步的,为(38-42):(14-16):(14-16):(7-9):(31-33):(0.3-0.5);具体的,为(40-41):16:16:8:32:0.48。
本发明的第三方面,提供上述基于多孔碳酸钙的纳米免疫制剂在制备防治肿瘤药物领域的应用。
优选的,上述防治肿瘤药物为防治乳腺癌药物。
进一步优选的,防治肿瘤药物为强化乳腺癌疗效及防止肿瘤复发领域的药物。
本发明有益效果
1.本发明的多孔碳酸钙纳米免疫制剂具有良好的治疗效果,毒性低、免疫激活效率高,能够消除传统手术治疗方法所残存的微小病灶,提高乳腺癌的免疫治疗效率,有效防止乳腺癌的复发,能够用于生物体治疗。
2.本发明中制剂的合成方法简单,不引入有毒试剂,制备的纳米制剂不存在有毒试剂残留问题,安全性高。制备过程中无需高温加热等复杂操作,适于规模化生产。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。
图1小鼠肿瘤体积变化和体重变化曲线图;
图2实施例2中防止肿瘤复发实验小鼠活体成像图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例与对比例详细说明本申请的技术方案。
实施例1基于多孔碳酸钙纳米制剂的制备
(1)通过气体扩散反应合成多孔CaCO3纳米颗粒:将220毫克CaCl2·H2O和8克NH4HCO3同时置于真空干燥室中,控制温度为25℃,将整个***保持在真空环境中24小时后,获得的CaCO3纳米颗粒通过在12000rpm离心分离。然后将在无水乙醇中CaCO3的孔道中装载IDOi,得到CaCO3@IDOi(缩写为CaI)。
(2)用脂质体修饰上述纳米颗粒。首先,将20毫克CaI的乙醇溶液和2毫克DOPA混合。然后将DPPC,胆固醇和DSPE-PEG以4:4:2M比例加入上述溶液中,用旋转蒸发仪将溶剂旋干,将得到的纳米颗粒溶解在水溶液中以备进一步使用。然后,将获得的纳米颗粒与200毫克PEI一起搅拌以制备CaCO3@IDOi@PEG@PEI(缩写为CaIP)。
(3)将上述CaIP与12OD CpG ODN在水溶液中搅拌,得到最终产物,缩写为CaIPC。
对比例1
作为对照,本发明合成了一种只以脂质体为载体的纳米免疫制剂(LIPC),将IDOi装载入脂质体中,DPPC,胆固醇和DSPE-PEG以4:4:2M比例混合加入有机溶液中搅拌后旋干,后修饰12OD CpG ODN,将制备的纳米免疫制剂在8000rpm下离心10分钟并分散于溶解在生理盐水,配制成LIPC储备液储存待用。
实施例2小鼠造模实验
2.1体内试验
选取雌性Balb/c小鼠作为研究对象,通过皮下注射小鼠乳腺癌细胞制作荷瘤小鼠模型,待肿瘤生长至50mm3开始给药。给药组分别为CaIPC(实验组)、LIPC、生理盐水。通过静脉方式对荷瘤小鼠给药,实验周期中,隔天测量肿瘤体积及称重。实验结果表明,实施例1中CaIPC处理的小鼠肿瘤生长速度最慢,表明本发明可有效抑制小鼠模型中的肿瘤生长。相反,与生理盐水组相比,LIPC组显示出并不是很优异的肿瘤抑制效果,该结果表明,只有本发明处理的组表现出最优的肿瘤治疗效果。体重是评估材料对身体的全身毒性的重要参数,所有组的小鼠体重随着时间延长没有明显变化,这意味着利用本发明进行治疗没有对小鼠产生明显的副作用,适用于活体水平的肿瘤治疗(图1)。
2.2防止肿瘤复发实验
为了研究本发明在实际的手术治疗后防止肿瘤复发的效果,本发明建立了肿瘤术后复发模型。首先,将小鼠乳腺癌细胞接种到雌性Balb/c小鼠腋下,对小鼠肿瘤进行手术切除并留下残留肿瘤病灶。手术后静脉注射和瘤旁注射本发明材料进行术后进一步治疗,观察小鼠的肿瘤复发情况。实验结果表明,生理盐水组的小鼠肿瘤出现了复发。而CaIPC实验组静脉或瘤旁注射均没有肿瘤复发的情况发生,表明该发明有明显地抑制肿瘤复发的能力(图2)。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (10)
1.一种基于多孔碳酸钙的纳米免疫制剂,其特征在于,所述纳米免疫制剂的组成为多孔碳酸钙纳米颗粒、免疫阻断抑制剂IDOi、脂质体层及免疫调节核酸CpG ODNs修饰。
2.如权利要求1所述的纳米免疫制剂,其特征在于,所述免疫制剂以多孔碳酸钙纳米颗粒为载体,所述多孔碳酸钙颗粒的孔道内部装载免疫阻断抑制剂IDOi,被脂质体层包覆后,脂质体层表面修饰免疫调节核酸CpG ODNs。
3.如权利要求1所述的纳米免疫制剂,其特征在于,所述脂质体层由DPPC、胆固醇和DSPE-PEG组成。
4.一种基于多孔碳酸钙的纳米免疫制剂的制备方法,其特征在于,包括如下步骤:
(1)通过气体扩散反应合成多孔碳酸钙纳米颗粒,将制得的多孔碳酸钙纳米颗粒置于无水乙醇中装载IDOi,得到CaCO3@IDOi;
(2)采用脂质体层修饰步骤(1)中制得的CaCO3@IDOi制备CaCO3@IDOi@PEG@PEI,将所述CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌,既得该纳米制剂。
5.如权利要求4所述的制备方法,其特征在于,步骤如下:
(1)将CaCl2·H2O和NH4HCO3同时置于真空干燥室中,将整个***保持在真空环境中一段时间后,通过离心分离制得的碳酸钙纳米颗粒;将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中装载IDOi,得到CaCO3@IDOi;
(2)将步骤(1)中制得的CaCO3@IDOi乙醇溶液与DOPA混合,然后将DPPC,胆固醇和DSPE-PEG加入上述溶液中,将溶液中的有机溶剂旋干,将制得的纳米颗粒溶于水溶液中,加入PEI后搅拌以制备CaCO3@IDOi@PEG@PEI;将所述CaCO3@IDOi@PEG@PEI与CpG ODN在水溶液中搅拌,即得该纳米制剂。
6.如权利要求5所述的制备方法,其特征在于,步骤(1)的具体步骤如下:将CaCl2·H2O和NH4HCO3同时置于真空干燥室中,设置温度为25℃,将整个***保持在真空环境中24h后,通过10000-15000rpm离心分离制得碳酸钙纳米颗粒。
7.如权利要求5所述的制备方法,其特征在于,步骤(1)中所述将分离得到的碳酸钙纳米颗粒置于无水乙醇溶液中装载IDOi,得到CaCO3@IDOi具体步骤为:将碳酸钙与IDOi的乙醇溶液搅拌至少24小时。
8.如权利要求5所述的制备方法,其特征在于,步骤(1)中所述CaCl2·H2O:NH4HCO3质量比为(0.5-2):(3-5),CaCO3和IDOi的质量比例为(35-45):(0.5-2.5);优选的,所述CaCl2·H2O:NH4HCO3质量比例为1:4,CaCO3和IDOi的质量比例为40:1。
9.如权利要求5所述的制备方法,其特征在于,步骤(2)中CaCO3@IDOi:DOPA:DPPC:胆固醇:DSPE-PEG:PEI的质量比为(35-45):(12-18):(12-18):(6-10):(30-35):(0.2-1);优选的,(38-42):(14-16):(14:16):(7-9):(31-33):(0.3-0.5)。
10.权利要求1-3所述基于多孔碳酸钙的纳米免疫制剂在制备防治肿瘤药物领域的应用;优选的,所述防治肿瘤药物为防治乳腺癌药物;进一步优选的,为强化乳腺癌疗效及防止肿瘤复发领域的药物。
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