CN110711250B - 一种双靶向多模协同治疗纳米载药体系的构建方法 - Google Patents
一种双靶向多模协同治疗纳米载药体系的构建方法 Download PDFInfo
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Abstract
本发明属于人源血清蛋白负载无机纳米药物体系制备技术领域，尤其为一种双靶向多模协同治疗纳米载药体系的构建方法，其包括以下步骤：S1：ICG‑Pt配位物的制备：将ICG与顺铂和去离子水按照一定比列混合后，室温下避光搅拌，去离子水条件下，用分子量为1KD的透析袋对反应液进行透析，得到ICG‑Pt配合物溶液；S2：HSA‑ICG‑DDP纳米粒子的制备：在室温条件下，将S1中所述的制备的配合物溶液，加入到GSH与HSA的溶液中，37℃下搅拌，后向此反应液中加入醇溶液使HSA‑ICG‑DDP纳米粒子沉淀析出。本发明设计制备的抗肿瘤HSA‑ICG‑Pt纳米载药微球不仅具有显著的抗肿瘤效果，而且对正常细胞的毒副作用比较小，同时能够实现实时监测肿瘤成像的效果，且制备方法简单可靠。
Description
技术领域
本发明涉及人源血清蛋白负载无机纳米药物体系制备技术领域，尤其涉及一种双靶向多模协同治疗纳米载药体系的构建方法。
背景技术
口腔癌症是危害人类十大恶性肿瘤之一。全球每年新发口腔癌263900例，死亡128000病例，其中口腔鳞状细胞癌(Oral squamous cell carcinoma,OSCC)最为常见，约占80％～90％。目前，以手术为主的，辅以化疗或放疗等多种手段的多学科综合治疗是公认的OSCC患者最佳治疗策略，但近30年内，OSCC患者5年生存率仍然在60％左右徘徊。临床实践中发现，一旦手术中肿瘤切除不彻底，术后复发将大大增加，而化疗，放疗等治疗手段，由于异质性肿瘤内含有的癌细胞亚群对单一治疗的抵抗效应，使得单一模态治疗方法并不能完全有效消除肿瘤，最终导致治疗失败。因此探索新型治疗方法，借助新型技术整合多种抗肿瘤手段，尽可能克服肿瘤对于单一治疗耐受性，减少肿瘤复发成为临床医生及科研人员研究关注的核心问题。
对于晚期OSCC，复发性OSCC以及无法耐受手术的患者，化疗是治疗或延长患者生命的主要手段之一。肿瘤细胞及微环境对化疗药物耐药性及化疗药物缺乏靶向性而产生的全身毒性，是化疗的两大难题。目前多使用几种化疗药物协同来增强抗肿瘤效果，但在临床应用中，多种药物联用对正常细胞毒性并没有降低，甚至出现增加可能，使得部分OSCC患者死于化疗而非疾病本身。光化学治疗将光敏剂和化疗药物制备入单一的纳米结构，实现了光动力治疗(PhotodynamicTherapy，PDT)和化疗协同抗肿瘤效应。基于纳米载体的光化学治疗较单一化疗具有明显优越性，因此设计一种具有同一纳米载体下的光化学协同治疗纳米载药体系或许是解决单一化疗的两大难题的一条有效途径，具有广阔的应用前景。
HSA以及ICG均是已经被FDA批准可用于静脉注射的药物，构建的纳米粒子同样具有生物安全性。HSA-ICG-DDP纳米粒子结合了光学治疗及化疗的优势，能够靶向性的在肿瘤基质内蓄积及被肿瘤细胞摄取，提高ICG及DDP在肿瘤组织内的含量，与游离的ICG及DDP相比，治疗效果将优于单独的光学治疗或化疗，且能够克服游离ICG的光学不稳定性，降低DDP的全身毒副反应，为口腔癌患者提供了另一创伤小、副作用低的治疗手段，因此我们提出了一种双靶向多模协同治疗纳米载药体系的构建方法用于解决上述问题。
发明内容
本发明的是为克服现有技术不足而提供的一种光动力促进ICG-Pt配位键断裂的HSA纳米载药体系的制备方法，本发明制备的抗肿瘤HSA-ICG-Pt纳米载药微球不仅具有显著的抗肿瘤效果，而且对正常细胞的毒副作用比较小，同时能够实现实时监测肿瘤成像的效果；本发明的制备方法简单可靠，所有使用材料均是FDA批准的，能够保证本发明产品的质量。
为了实现上述目的，本发明采用了如下技术方案：
一种双靶向多模协同治疗纳米载药体系的构建方法，包括以下步骤：
S1：ICG-Pt配位物的制备：将ICG与顺铂和去离子水按照一定比列混合后，室温下避光搅拌，去离子水条件下，用分子量为1KD的透析袋对反应液进行透析，得到ICG-Pt配合物溶液；
S2：HSA-ICG-DDP纳米粒子的制备：在室温条件下，将S1中所述的制备的配合物溶液，加入到GSH与HSA的溶液中，37℃下搅拌，后向此反应液中加入醇溶液使HSA-ICG-DDP纳米粒子沉淀析出，悬浮液在室温下继续剧烈搅拌后，反应液用分子量为1kD的透析膜在去离子水中透析，除去醇溶液以及游离的ICG以及GSH，得到的透析溶液即为HSA-ICG-DDP的纳米粒子。
优选的，所述S1中，所述的ICG与顺铂和去离子水的质量比为1：(0.1-1.0):(50.0-250.0)。
优选的，所述S1中，所述的ICG与顺铂和去离子水的搅拌时间为3h-24h。
优选的，所述S2中，所使用的GSH浓度为50nM，37℃下搅拌时间为2h-12h。
优选的，所述S2中，所使用的醇溶液为甲醇、乙醇中的一种，所选用的载体为HSA。
本发明中所述的一种双靶向多模协同治疗纳米载药体系的构建方法的有益效果为：
(1)将临床可应用的HSA作为纳米载体，并将同样已应用于临床的ICG和DDP进行配位连接，实现光控断裂ICG-DDP配位化学键，最大程度减少纳米载体及化疗药物的毒性；
(2)HSA纳米载体可以和SPARC结合，具有定点靶向作用，增加了ICG和DDP在肿瘤内蓄积；
(3)HSA-ICG-DDP纳米粒子结合了光学治疗及化疗的优势，利用ICG光动力治疗产生ROS，改变口腔癌微环境低氧状态及克服可能存在的DDP耐药性问题；为进一步说明本发明与现有技术对比具有良好的效果，以HSA-ICG-DDP的纳米粒子，对生物学实验进行了验证；
本发明设计制备的抗肿瘤HSA-ICG-Pt纳米载药微球不仅具有显著的抗肿瘤效果，而且对正常细胞的毒副作用比较小，同时能够实现实时监测肿瘤成像的效果，且制备方法简单可靠。
附图说明
图1为纳米载药体系的制备方法流程示意图；
图2为纳米粒子透射电镜图与动态光散射图；
图3为光控促进配位键断裂的体外释放图；
图4为HSA-ICG-DDP的纳米粒子在口腔癌细胞的体内成像图；
图5为HSA-ICG-DDP的纳米粒子在口腔癌细胞的体内抑瘤效果评价图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例一：
一种双靶向多模协同治疗纳米载药体系的构建方法，包括以下步骤：
S1：ICG-Pt配位物的制备：室温下20mg的DDP与20mg的ICG首先溶解于1mL的去离子水中，避光反应24h，用分子量为1KD的透析袋对反应液进行透析，除去游离的ICG以及顺铂，即可得到ICG-Pt配合物溶液。
S2：在室温条件下，将S1中所述的制备的配合物溶液，加入到50nM的GSH与80mg的HSA的溶液中，37℃下搅拌1h后，后向此反应液中加入2mL的乙醇溶液使HSA-ICG-DDP纳米粒子沉淀析出，悬浮液在室温下继续剧烈搅拌30min后，反应液用分子量为1kD的透析膜在去离子水中透析24h，除去醇溶液以及游离的ICG以及GSH，得到的透析溶液即为HSA-ICG-DDP的纳米粒子。
实施例二：
一种双靶向多模协同治疗纳米载药体系的构建方法，包括以下步骤：
S1：ICG-Pt配位物的制备：室温下10mg的DDP与100mg的ICG首先溶解于5mL的去离子水中，避光反应24h，用分子量为1KD的透析袋对反应液进行透析，除去游离的ICG以及顺铂，即可得到ICG-Pt配合物溶液。
S2：在室温条件下，将S1中所述的制备的配合物溶液，加入到100nM的GSH与80mg的HSA的溶液中，37℃下搅拌1h后，后向此反应液中加入5mL的甲醇溶液使HSA-ICG-DDP纳米粒子沉淀析出，悬浮液在室温下继续剧烈搅拌30min后，反应液用分子量为1kD的透析膜在去离子水中透析24h，除去醇溶液以及游离的ICG以及GSH，得到的透析溶液即为HSA-ICG-DDP的纳米粒子。
实施例三：
一种双靶向多模协同治疗纳米载药体系的构建方法，包括以下步骤：
S1：CG-Pt配位物的制备：室温下10mg的DDP与100mg的ICG首先溶解于5mL的去离子水中，避光反应10h，用分子量为1KD的透析袋对反应液进行透析，除去游离的ICG以及顺铂，即可得到ICG-Pt配合物溶液。
S2：在室温条件下，将S1中所述的制备的配合物溶液，加入到100nM的GSH与80mg的HSA的溶液中，37℃下搅拌10h后，后向此反应液中加入5mL的乙醇溶液使HSA-ICG-DDP纳米粒子沉淀析出，悬浮液在室温下继续剧烈搅拌30min后，反应液用分子量为1kD的透析膜在去离子水中透析10h，除去醇溶液以及游离的ICG以及GSH，得到的透析溶液即为HSA-ICG-DDP的纳米粒子。
本发明经过反复实验验证，取得了满意的应用效果，现以HSA-ICG-DDP、HSA-ICG、DDP、ICG为例，其应用效果图示如下：
如图1所示是本发明提出的制备一种光动力促进ICG-Pt配位键断裂的HSA纳米载药体系的制备方法流程示意图：首先将ICG与顺铂制备成配合物溶液，然后加入到GSH还原后的HAS溶液中进行自组装，形成纳米粒子。
如图2所示是本发明的HSA-ICG-DDP的纳米粒子透射电镜图与动态光散射图：图2中可见，所制备的HSA-ICG-DDP纳米粒子(NPs)为较为规则的圆形，粒径比较均匀，尺寸约为20-30nm，与动态光散射测试的结果相一致。
如图3所示是本发明光控促进配位键断裂的体外释放图：图3可见在近红外光808nm激发照射条件下，体外释放溶液中只有游离的Pt,而ICG并未游离出来，显示ICG-Pt之间的配位键发生了断裂，顺铂表现出缓慢释放的特点，这样有利于纳米药物能够在肿瘤部位集聚，从而达到定点释放，起到杀伤肿瘤的作用。
如图4所示是本发明的HSA-ICG-DDP的纳米粒子在口腔癌细胞的体内成像图：将4-6周雄性裸鼠(重量约20g)背部皮下注射1×106的HSC3细胞，构建荷瘤裸鼠皮下瘤模型，当肿瘤生长到0.5×0.5×0.5cm3时，在两组小鼠中分别尾静脉注射5mg/kg的游离ICG，以及含有同等含量ICG的HSA-ICG-DDP纳米粒子，在注射后的不同时间点(1,3,6,12,24,48h)，通过小动物活体成像仪检测小鼠体内不同组织的荧光变化，并利用近红外光谱仪采集不同试验组中的ICG荧光强度及光谱数据，图4结果显示HSA-ICG-DDP纳米粒子较比单纯的ICG，能够较长时间的在体内循环，为后续药物能够长时间滞留在肿瘤部位提供了保证。
如图5所示是本发明的HSA-ICG-DDP的纳米粒子在口腔癌细胞的体内抑瘤效果评价图：将4-6周雄性裸鼠(重量约20g)背部皮下注射1×106的HSC3细胞，构建荷瘤裸鼠皮下瘤模型。设置不同治疗组分组：HSA-ICG-DDP/NIR，ICG/NIR，DDP，PBS，每组3只。当肿瘤生长至14天时，分别在第0、2、4天经裸鼠尾静脉注射HSA-ICG-DDP纳米粒子，以及与HSA-ICG-DDP纳米粒子中同等含量的游离ICG，DDP，通过激发光808nm(3min，2W/cm2)照射相应组别裸鼠，定期记录荷瘤裸鼠体重以及肿瘤大小变化情况，3周后安乐处死，分离出移植瘤，比较各个治疗组的治疗效果。各组药物如图5所示，其中ICG的剂量为5mg/kg,Pt的剂量为0.5mg/kg。HSA-ICG-DDP/NIR，ICG/NIR，DDP中的ICG与Pt剂量等同裸药，经过两周注射后，图5中可见体内实验结果显示HSA-ICG-DDP纳米粒子的光照组产生了显著优于单纯裸药以及非光照对照组的抑瘤效果。
本发明所提出的一种用于靶向SPARC高表达恶性肿瘤的光能控制配位键断裂的双靶向多模协同治疗纳米载药体系的构建方法，其具体步骤为：利用谷胱甘肽(Glutathione,GSH)对二硫键具有还原的能力将HAS(人源血清蛋白)在GSH作用下还原成含二硫键的HSA；通过ICG上磺酸基团与顺铂[Pt(NH3)2Cl2]的配位能力形成ICG-DDP配合物,最后将还原型HSA与ICG-DDP在水溶液中进行自组装，形成HAS-ICG-DDP的纳米粒子；所制备的HSA-ICG-DDP纳米粒子平均尺寸为20-30nm,在无近红外光的激发下，此纳米粒子几乎无生物毒性；在808nm激光照射下能够有效的促进配位键的断裂，实现光能控制下肿瘤部位定点释放和杀伤；HSA-ICG-DDP纳米粒子结合了光动力治疗，光热治疗及化疗协同治疗的优势，能够通过被动靶向先行渗透入肿瘤机制内并蓄积，然后主动靶向SPARC高表达的肿瘤基质，肿瘤细胞和肿瘤相关成纤维细胞并被主动摄取，提高了ICG及DDP在肿瘤组织内的含量，与游离的ICG及DDP相比，治疗效果明显优于单一光学治疗或化疗，且能够克服游离ICG的光学不稳定性，降低DDP的全身毒副反应，为口腔癌患者提供了另一创伤小、副作用低的治疗手段。
本实施例中，本发明的作用原理是：第一，ICG结构中存在两个磺酸根离子，配位能力较弱，磺酸根的配位能力主要是利用磺酸基的三个氧原子可以从不同方向上与金属离子配位，利用这种性能，通过ICG与Pt(NH3)2Cl2进行配位，制备ICG-Pt的配合物对近红外光具有良好的影响性；第二，利用具有生物相容性的人血清白蛋白(HumanSerumAlbumin,HSA)在水溶液中能够自发的与ICG-Pt发生自组装形成纳米粒子；第三，所制备的HSA-ICG-DDP纳米体系能够具有靶向口腔癌细胞、CAF(肿瘤相关成纤维细胞)和肿瘤组织间隙的作用，增加了ICG和DDP在肿瘤内蓄积，同时该纳米药物具有单药物或者单药所不能比拟的低毒副作用，进一步减少化疗药物全身毒性；第四，HSA-ICG-DDP纳米粒子结合了光学治疗及化疗的优势，利用ICG光动力治疗产生活性氧(ROS)，改变口腔癌微环境低氧状态及克服可能存在的DDP耐药性问题。
本实施例中，将临床可应用的HSA作为纳米载体，并将同样已应用于临床的ICG和DDP进行配位连接，实现光控断裂ICG-DDP配位化学键，最大程度减少纳米载体及化疗药物的毒性；HSA纳米载体可以和SPARC结合，具有定点靶向作用，增加了ICG和DDP在肿瘤内蓄积；HSA-ICG-DDP纳米粒子结合了光学治疗及化疗的优势，利用ICG光动力治疗产生ROS，改变口腔癌微环境低氧状态及克服可能存在的DDP耐药性问题；为进一步说明本发明与现有技术对比具有良好的效果，以HSA-ICG-DDP的纳米粒子，对生物学实验进行了验证。
Claims (5)
1.一种双靶向多模协同治疗纳米载药体系的构建方法，其特征在于，包括以下步骤：
S1：ICG-Pt配位物的制备：将ICG与顺铂和去离子水按照一定质量比混合后，室温下避光搅拌，去离子水条件下，用分子量为1KD的透析袋对反应液进行透析，得到ICG-Pt配合物溶液；
S2：HSA-ICG-DDP纳米粒子的制备：在室温条件下，将S1中所述的制备的配合物溶液，加入到GSH与HSA的溶液中，37℃下搅拌，后向此反应液中加入醇溶液使HSA-ICG-DDP纳米粒子沉淀析出，悬浮液在室温下继续剧烈搅拌后，反应液用分子量为1kD的透析膜在去离子水中透析，除去醇溶液以及游离的ICG以及GSH，得到的透析溶液即为HSA-ICG-DDP的纳米粒子。
2.根据权利要求1所述的一种双靶向多模协同治疗纳米载药体系的构建方法，其特征在于，所述S1中，所述的ICG与顺铂和去离子水的质量比为1：(0.1-1.0):(50.0-250.0)。
3.根据权利要求1所述的一种双靶向多模协同治疗纳米载药体系的构建方法，其特征在于，所述S1中，所述的ICG与顺铂和去离子水的搅拌时间为3h-24h。
4.根据权利要求1所述的一种双靶向多模协同治疗纳米载药体系的构建方法，其特征在于，所述S2中，所使用的GSH浓度为50nM，37℃下搅拌时间为2h-12h。
5.根据权利要求1所述的一种双靶向多模协同治疗纳米载药体系的构建方法，其特征在于，所述S2中，所使用的醇溶液为甲醇、乙醇中的一种。
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