CN117045787A - 一种自组装型纳米光敏剂及制备方法和应用 - Google Patents

一种自组装型纳米光敏剂及制备方法和应用 Download PDF

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CN117045787A
CN117045787A CN202311258719.0A CN202311258719A CN117045787A CN 117045787 A CN117045787 A CN 117045787A CN 202311258719 A CN202311258719 A CN 202311258719A CN 117045787 A CN117045787 A CN 117045787A
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王奎
黄景彬
张蓉
许汝福
王振强
王凤玲
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Second Affiliated Hospital Army Medical University
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Abstract

本发明公开了一种自组装型纳米光敏剂及制备方法和应用，该纳米光敏剂，其组成为TPP‑Ppa@ATO@CAT@RGD‑PEG‑BSA，主要由线粒体靶向的卟啉TPP‑Ppa、阿托伐醌(ATO)、过氧化氢酶(CAT)和具有细胞主动靶向功能的RGD‑PEG‑BSA自组装而得。该光敏剂能通过双重靶向及双重途径增加氧气的方式增强光动力治疗效果，并通过光动力治疗引发免疫原性死亡效应与Anti‑PD‑L1抗体联用，从而实现乳腺癌逐级增强的光动力‑免疫治疗。

Description

一种自组装型纳米光敏剂及制备方法和应用
技术领域
本发明属于医药技术领域，具体涉及一种自组装型纳米光敏剂及制备方法和应用。
背景技术
癌症仍然是世界范围内发病率和死亡率很高的公共卫生问题，化疗仍然是主流，但其副作用和缺乏持久反应促使了新疗法的发展。近年来，光动力治疗(PDT)与免疫治疗等新方法备受关注，PDT需要光敏剂(PSs)、与光敏剂匹配的光源以及氧气分子来共同作用生成具有细胞毒性的活性氧形态(reactive oxygen species，ROS)，从而杀伤肿瘤细胞。然而，由于肿瘤细胞的大量增殖使得肿瘤细胞快速消耗血液中供应的氧气从而形成一个乏氧的微环境，导致PDT对于肿瘤的治疗效果有限。免疫治疗在肿瘤治疗上展现出了巨大潜力，然而，基于免疫检查点阻断的免疫疗法在临床上仅有10-30％的响应率，极少有实体瘤患者得到完全和持久的缓解。研究发现PDT有利于增强肿瘤的免疫治疗研究，因此，PDT联合免疫治疗对实现肿瘤的高效抑制具有重要意义。
PDT利用光敏剂、氧气和光照产生ROS。然而，传统的光敏剂在水溶液中聚集导致猝灭并降低ROS的产生，而且体内应用代谢迅速，不利于其血液循环与肿瘤富集。因此，制备纳米化的光敏剂具有重要意义。现今发展到第四代纳米型的光敏剂，主要以卟啉或酞菁为构筑基元制备出纳米材料，例如，以卟啉或酞菁单体自组装类型的纳米材料、金属有机框架或共价有机框架类型的纳米材料，从而解决其疏水性，提高其生物利用效率。此外，因为ROS的半衰期通常很短(40ns)而且只能在很短的距离内有效(＜20nm)。对于没有直接暴露在激光照射下的隐藏肿瘤是无法有效控制其转移。研究发现，PDT与免疫治疗的联合治疗对于抑制肿瘤的转移有着显著的作用。因此，为了克服上述PDT与免疫治疗的局限性，本发明人运用分子自组装的方法构建出高效的纳米药物进行生物医学应用。
发明内容
本发明的目的在于提供一种自组装型纳米光敏剂及制备方法，该纳米光敏剂可以用于治疗肿瘤。
本发明是利用TPP-Ppa(具有线粒体靶向功能的卟啉分子)、RGD-PEG-BSA(具有主动靶向以及血液长循环的分子，即RGD肽-聚乙二醇-牛血清白蛋白)、ATO(阿托伐醌，一种抑制线粒体的呼吸减少肿瘤细胞内氧气消耗的分子)、CAT(过氧化氢酶，可催化过氧化氢产生氧气)四组分通过分子间的弱作用力(亲疏水、π-π堆积力等)巧妙设计出多功能一体化的纳米光敏剂，实现对肿瘤增强的光动力治疗效果。
为实现本发明的目的，提供了如下实施方案。
在一实施方案中，本发明提供了一种自组装型纳米光敏剂，其组成为TPP-Ppa@ATO@CAT@RGD-PEG-BSA，主要由TPP-Ppa、阿托伐醌(ATO)、过氧化氢酶(CAT)和RGD-PEG-BSA自组装而得，所述TPP-Ppa的化学结构如下所示：
其中，TPP-Ppa：ATO：CAT：RGD-PEG-BSA投料的质量比为1:0.5:0.5:2。在另一实施方案中，本发明的一种自组装型纳米光敏剂的制备方法，所述纳米光敏剂组成为TPP-Ppa@ATO@CAT@mPEG-BSA，该制备方法包括：
1)将RGDfC与Mal-PEG-NHS在溶液中反应，再加入BSA，搅拌反应；
2)反应结束后，将反应液透析、冻干，得到RGD-PEG-BSA；
3)将ATO与TPP-Ppa分别溶解在有机溶剂中制得ATO溶液和TPP-Ppa溶液，再混合成ATO@TPP-Ppa混合溶液；
4)将RGD-PEG-BSA与CAT分别用水溶解后再混合成水溶液，在超声条件下将ATO@TPP-Ppa溶液滴加到CAT与RGD-PEG-BSA的混合溶液中，制备完成后透析制得纳米光敏剂，
其中，所述TPP-Ppa的化学结构如下所示：
上述本发明的制备方法，步骤1)中，所述溶液为PBS溶液，RGDfC与Mal-PEG-NHS和BSA的质量比为17：147：400。
上述本发明的制备方法，步骤1)中所述的反应，反应时间为12小时。
上述本发明的制备方法，步骤3)中，ATO与TPP-Ppa的质量比为1：2。
上述本发明的制备方法，步骤3)中，所述有机溶剂为二甲基亚砜。
上述本发明的制备方法，步骤3)中，ATO溶液的浓度为10mg/mL，TPP-Ppa溶液的浓度为20mg/mL。
上述本发明的制备方法，步骤3)中，RGD-PEG-BSA、CAT和ATO的质量比为2：0.5：0.5。
另一方面，本发明还提供一种上述本发明的光敏剂和上述本发明的制备方法制备的光敏剂制造抗肿瘤药物中的应用，
上述本发明的应用，进一步包括与Anti-PD-L1单抗联用，或组成复方制剂。
又一方面，本发明提供一种药物组合物，含有上述本发明的光敏剂和上述本发明的制备方法制备的光敏剂，任选的，和药物上可接受的辅料。
术语：“TPP-Ppa@ATO@CAT@RGD-PEG-BSA”中的“@”表示物质的各组分连接。
本发明的光敏剂可引发肿瘤的免疫原性死亡(ICD)效应，刺激DC成熟，进一步与Anti-PD-L1抗体联用，增强乳腺癌的免疫治疗效果，从而实现乳腺癌逐级增强的光动力-免疫治疗。
本发明纳米光敏剂具有如下几点创新：
(1)通过分子间的亲疏水与π-π堆积力等弱相互作用力提高光敏剂的装载效率，解决光敏剂疏水以及易聚集的问题；
(2)RGD-PEG-BSA与TPP-Ppa分子实现纳米光敏剂的逐级靶向，不仅靶向肿瘤细胞，同时靶向线粒体，增强PDT效果；
(3)装载过氧化氢酶，不仅分解肿瘤内的过氧化氢产生氧气，而且通过ATO减少线粒体呼吸链中氧气的消耗，通过增产量减消耗的双重路径增加氧气含量，从而增强PDT效果；
(4)在PDT的作用下实现原发肿瘤的杀伤，实现肿瘤细胞的ICD效应与促进肿瘤相关抗原(TAA)的释放，进一步刺激DC的成熟，从而增强Anti-PD-L1抗体的免疫治疗效果，抑制肿瘤的转移。
附图说明
图1为TPP-Ppa的核磁共振谱图。
图2为纳米光敏剂TPP-Ppa@ATO@CAT@RGD-PEG-BSA的粒径分布图。
图3为纳米光敏剂TPP-Ppa@ATO@CAT@RGD-PEG-BSA的透射电镜图。
图4为有肿瘤细胞靶向和无肿瘤细胞靶向的纳米光敏剂在不同时间点(2及4小时)的内吞效果图。
图5为纳米光敏剂对线粒体靶向的内吞评价测试结果图。
图6为利用CCK-8进行了各组实验对肿瘤细胞的治疗效果图。
图7为利用光动力治疗肿瘤细胞后引起钙网蛋白(CRT)产生的能力评价和产生荧光强度的定量评价效果图。
图8为双侧肿瘤模型各实验组的肿瘤抑制曲线图。
具体实施方式
以下实施例是典型的，用于进一步理解和说明本发明的精神实质，但不以任何方式限制本发明的范围。
实施例1TPP-Ppa的制备
TPP-Ppa的合成路线如下：
将焦脱镁叶绿酸盐a(140mg)、EDC.HCI(70mg)、HOBt(50mg)、三乙胺(0.05mL)、BOC-乙二胺(200mg)混合在二氯甲烷溶液中(25mL)后在0℃条件下搅拌1小时，随后继续常温反应24小时，加水溶液调节pH值为酸性，加入二氯甲烷萃取，饱和NaCl洗三次，水洗2次，硫酸钠干燥，旋蒸浓缩得到物质后过柱分离(3％甲醇+CH2Cl2)，得到化合物3。
将化合物3(60mg)和TFA(5mL)加入到溶剂二氯甲烷(5mL)中在0℃下搅拌5小时，随后将反应溶液旋蒸去除溶剂得到化合物4。将化合物5(61mg，2eq)、PyBOP(72mg，2eq)、DIPEA(18mg)混合在二氯甲烷溶液中(10mL)，搅拌1小时，随后加入化合物4(40mg)继续搅拌24小时，加入二氯甲烷萃取，饱和NaCl洗三次，水洗2次，硫酸钠干燥，旋蒸浓缩得到物质后过柱分离(展开剂：3％CH3OH+CH2Cl2至10％CH3OH+CH2Cl2梯度洗脱)，得到TPP-Ppa。所得产品TPP-Ppa的磁共振谱如图1。
实施例2光敏剂TPP-Ppa@ATO@CAT@RGD-PEG-BSA的制备
1)高分子RGD-PEG-BSA以及mPEG-BSA的制备
将RGDfC(17mg)与Mal-PEG-NHS(147mg)在PBS溶液中反应，随后将BSA(400mg)加入到上述溶液中继续搅拌12小时，反应结束后进行透析冻干，从而得到具有细胞主动靶向功能的RGD-PEG-BSA。
同时将mPEG5000-NHS(150mg)与BSA(400mg)在PBS溶液中搅拌反应12小时，反应结束后进行透析冻干，从而制备出无靶向高分子mPEG-BSA为对照组。
2)纳米型光敏剂的制备
将ATO及TPP-Ppa分别溶解在二甲基亚砜中，配制成ATO溶液(10mg/mL)和TPP-Ppa溶液(20mg/mL)，各取0.05mL等体积混合，制得ATO-@TPP-Ppa混合溶液。
将RGD-PEG-BSA和CAT分别溶解在水中，配制成RGD-PEG-BSA溶液(10mg/mL)和CAT溶液(1mg/mL)，取RGD-PEG-BSA溶液(10mg/mL，0.2mL)与CAT溶液(1mg/mL，0.5mL)混合，定容体积到5mL，制得水溶液。
将ATO-@TPP-Ppa混合溶液在超声条件(5％，5min，超5s停5s)下滴加到上述水溶液中，制得TPP-Ppa@ATO@CAT@RGD-PEG-BSA，随后进行透析制备出纳米光敏剂。
对纳米光敏剂TPP-Ppa@ATO@CAT@RGD-PEG-BSA的粒径、形貌进行表征。采用粒径电位仪及透射电镜分别考察纳米光敏剂的粒径和形貌，结果见图2和图3。结果表明纳米光敏剂具有纳米尺度的粒径和形貌。
实施例3抗肿瘤细胞活性体外评价
实施例2的纳米型光敏剂的体外活性评价
(1)细胞靶向与PDT效果的评价
通过流式细胞术与激光共聚焦成像的内吞实验证明纳米型光敏剂的肿瘤细胞靶向功能；在细胞水平，利用荧光探针标记线粒体，通过共聚焦实验证明纳米光敏剂的线粒体靶向能力。
利用共聚焦技术进行了肿瘤细胞的靶向内吞评价,测试结果见图4，表明了有肿瘤细胞靶向(TPP-Ppa@ATO@CAT@RGD-PEG-BSA，缩写为TAC@RPB)及无肿瘤细胞靶向的纳米光敏剂(TPP-Ppa@ATO@CAT@mPEG-BSA，缩写为TAC@PB)在不同时间点(2及4小时)的内吞效果。
以及进行了线粒体靶向的内吞评价，测试结果见图5，表明了有线粒体靶向(TPP-Ppa@ATO@CAT@RGD-PEG-BSA，缩写为TAC@RPB)及无线粒体靶向纳米光敏剂(Ppa@ATO@CAT@RGD-PEG-BSA，缩写为PAC@RPB)的内吞效果。
以上结果证明纳米光敏剂(TAC@RPB)具有优异的肿瘤细胞靶向及线粒体靶向能力。
(2)纳米光敏剂进行肿瘤细胞的治疗效果评价及线粒体靶向的PDT诱导肿瘤细胞ICD能力的评价
利用CCK-8试剂进行各组实验对肿瘤细胞的治疗效果评价，通过流式细胞术、共聚焦显微镜成像、Western Blot等实验手段验证纳米粒子实施线粒体靶向的光动力治疗后CRT、HMGB1与ATP的变化，从而表征PDT诱导肿瘤细胞ICD的能力；进一步通过抗体检测DC细胞成熟的标志物，从而验证肿瘤细胞光动力治疗后产生肿瘤相关抗原，增强Anti-PD-L1的免疫治疗。
结果：利用CCK-8试剂进行了各组实验对肿瘤细胞的治疗效果评价，评价结果如图6所示，相比于TC@RPB+Laser或者TA@RPB+Laser组(TPP-Ppa@CAT@RGD-PEG-BSA，缩写为TC@RPB；TPP-Ppa@ATO@RGD-PEG-BSA，缩写为TA@RPB)，TAC@RPB+Laser组在乏氧条件下具有优异的协同杀伤肿瘤细胞的能力。
通过共聚焦成像技术评价，利用钙网蛋白(CRT)抗体进行了PDT杀伤肿瘤细胞后产生CRT能力的评价，结果如图7所示。相比于图A中各组(Control、TC@RPB、TA@RPB、TAC@RPB)治疗肿瘤细胞后引起钙网蛋白(CRT)产生的能力，纳米光敏剂加激光照射组(TAC@RPB+Laser)具有最佳的产生肿瘤细胞免疫原性死亡的能力；图B为对图A各组产生荧光强度的定量评价。
以上结果表明纳米光敏剂具有优异的肿瘤细胞杀伤能力及产生肿瘤细胞免疫原性死亡的能力。
实施例4光动力-免疫治疗的协同抗肿瘤研究动物评价
构建4T1双侧的皮下肿瘤模型，即1×106与2×106的4T1细胞分别注射进入Balb/c小白鼠左侧与右侧皮下组织处，将其分别称为远端瘤与原始瘤，从而建立双侧瘤肿瘤模型。当原始瘤体积达到大约100mm3，小鼠分为6组(5只/组)，注射纳米型光敏剂(5mg/kg)，根据体内荧光实验结果，进行右侧原始肿瘤部位的激光照射(660nm,0.5W/cm2,5min)，Anti-PD-L1抗体每间隔2天进行腹腔注射(间隔2天一次，共计3次，抗体用量为750μg/kg)，每隔两天远端瘤与原始瘤的肿瘤尺寸用游标卡尺进行测定，同时称量体重。治疗14天后处理小鼠得到离体肿瘤进行拍照与称重，对肿瘤组织切片进行H&E染色，并且检测肿瘤组织中各类免疫细胞的含量。取出脾脏与淋巴结，检测脾脏与淋巴结中各类免疫细胞的含量。
我们进行了双侧肿瘤模型的抑制评价，对原发肿瘤进行光照，远端肿瘤不光照，监测两端肿瘤体积随时间的变化，结果表明该纳米光敏剂结合抗体实施的光动力-免疫治疗不仅能够抑制原发瘤的生长，而且能够通过免疫治疗有效抑制远端瘤的生长，结果如图8所示。图8中A为各实验组的原发瘤抑制曲线，B为各实验组的远端瘤抑制曲线。结果表明，相比于PBS对照组，PDT加Anti-PD-L1联合治疗组不仅能够抑制原发瘤的生长，同时能够通过免疫治疗的作用实现远端未光照肿瘤的抑制。

Claims (10)

1.一种自组装型纳米光敏剂，其组成为TPP-Ppa@ATO@CAT@RGD-PEG-BSA，主要由TPP-Ppa、阿托伐醌(ATO)、过氧化氢酶(CAT)和RGD-PEG-BSA通过自组装而得，其中，所述TPP-Ppa的化学结构如下所示：
2.如权利要求1所述的纳米光敏剂，TPP-Ppa：ATO：CAT：RGD-PEG-BSA的投料质量比为1:0.5:0.5:2。
3.一种自组装型纳米光敏剂的制备方法，所述纳米光敏剂组成为TPP-Ppa@ATO@CAT@RGD-PEG-BSA，该制备方法包括：
1)将RGDfC与Mal-PEG-NHS在溶液中反应，再加入BSA，搅拌反应；
2)反应结束后，将反应液透析、冻干，得到RGD-PEG-BSA；
3)将ATO与TPP-Ppa分别溶解在有机溶剂中制得ATO溶液和TPP-Ppa溶液，再混合成TPP-Ppa@ATO混合溶液；
4)将RGD-PEG-BSA与CAT分别用水溶解后再混合溶液，在超声条件下将TPP-Ppa@ATO混合溶液滴加到RGD-PEG-BSA与CAT的混合溶液中，制得TPP-Ppa@ATO@CAT@RGD-PEG-BSA经透析得到纳米光敏剂，
其中，所述TPP-Ppa的化学结构如下所示：
4.如权利要求3所述的制备方法，步骤1)中，所述溶液为PBS溶液，RGDfC与Mal-PEG-NHS和BSA的质量比为17：147：400，所述反应的时间为12小时。
5.如权利要求3所述的制备方法，步骤3)中，ATO与TPP-Ppa的质量比为1：2。
6.如权利要求3所述的制备方法，步骤3)中，所述有机溶剂为二甲基亚砜，所述ATO溶液的浓度为10mg/mL，所述TPP-Ppa溶液的浓度为20mg/mL。
7.如权利要求3所述的制备方法，步骤3)中，RGD-PEG-BSA、CAT和ATO的质量比为2：0.5：0.5。
8.权利要求3的光敏剂和权利要求2的制备方法制备的纳米光敏剂在制造抗肿瘤药物中的应用。
9.如权利要求8所述的应用，包括与Anti-PD-L1单抗联用，或组成复方制剂。
10.一种药物组合物，含有权利要求1或2的光敏剂和权利要求3的制备方法制备的光敏剂，任选的，药物上可接受的辅料。
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