可靶、可视、可控的血小板载药系统的设计及其抗淋巴瘤效应的研究

More attention has been focused on the targetd therapy of lymphoma.CD22 antigen was found to be highly expressed on the surface of lymphoma cell in many studies. Compared with nano-biotechnology, platelets which are used as carrier of anti-lymphoma drug (DOX) as delivery of drug to the targeted site an essential blood component having a natural affinity for cancer cells, can achieve higher loading efficiency, more efficient biocompatibility and better targeted. Our initial studies have proved that DOX was nicely combined with platelets to construct platelets-DOX for drug delivery system with the loading efficiency of 50.08±2.36%, which was pH sensitive. In this study, a new type active targeted delivery system was presented to target lymphoma cells: (1) we will construct CD22-platelet-DOX which is a new drug delivery system with active targeted ability and pH sensitive, by optimizing the reactive conditions of CD22Ab, DOX and platelets; (2) The targeted anti-tumor ability against types of lymphomas cells and animals will be observed. Biocompatibility, pharmacokinetic and distribution will also be studied at the same time. CD22Ab-Platelet-DOX will be an effective approach to the clinic targeted therapy of lymphoma.

恶性淋巴瘤分子靶向治疗是目前研究的热点。B细胞系恶性淋巴瘤细胞膜表面高表达CD22抗原，与CD20不同，CD22抗原与抗体结合后能迅速内化。我们前期研究结果显示以血小板作为药物载体的载药量远高于一般纳米载体。本研究拟在前期基础上构建一种新型血小板主动靶向载药体系：1. 在血小板表面修饰CD22抗体(Fab’)片段，即能实现主动靶向性，又可避免抗体Fc段介导的吞噬细胞的吞噬作用，使血循环中的载药血小板寿命延长至正常生理9 天左右；2.通过不断优化CD22(Fab’)片段、DOX、血小板的装载条件，制备出可靶、可视、可控的载药体系CD22(Fab’)-血小板-DOX；3. 观察其对淋巴瘤Raji细胞及动物模型的靶向治疗作用，同时对其药代动力学、生物相容性及体内分布机制进行深入的研究，为临床淋巴瘤靶向治疗提供有力依据。

目前，淋巴瘤主要治疗手段是化疗，然而化疗药物在杀伤肿瘤细胞的同时，往往易对正常组织和器官造成不可逆的损害。CD22抗体可作为一种具有相对特异性的靶向配体，与药物载体交联后将抗淋巴瘤药物特异性的转运至淋巴瘤细胞发挥药效。本研究拟将血小板（plt）表面修饰CD22单抗，通过包载阿霉素（DOX），构建新型靶向生物载药体系（DOX-plt-CD22），探讨其抗淋巴瘤疗效及安全性。将DOX、CD22单抗及血小板以不同体积、反应条件进行一系列生化反应，以有效的靶向效应为前提，摸索达到最大载药量的反应条件。通过共聚焦显微镜、考马斯亮蓝染色等手段考察CD22单抗的修饰效率，考察DOX是否包载在血小板内，并且对血小板的形态及功能有无明显影响。采用激光共聚焦考察载药体系的体外靶向作用，CCK-8法测定其对淋巴瘤Raji细胞的生长抑制作用，流式细胞术、DAPI染色等评估细胞凋亡情况；构建Burkitt淋巴瘤荷瘤小鼠模型，采用活体成像技术检测DOX-plt-CD22的体内靶向功能，评估肿瘤组织生长抑制和凋亡情况，并评估主要脏器损伤情况；进一步用Western Blot和免疫荧光检测凋亡相关蛋白表达水平，阐明其作用的分子机理。实验结果证实CD22单抗成功修饰在血小板表面；DOX可被包载于血小板内，且载药率较高，同时载药后的血小板形态与单纯的血小板形态相似；在肿瘤的酸性环境中，药物释放明显多于在中性环境的正常组织中的释放；在CD22-plt-DOX处理组，Raji细胞中DOX的蓄积最为明显，并且细胞凋亡也最为显著，表明CD22修饰后的新型血小板载药体系可以增加阿霉素的抗肿瘤效应；动物实验也表明CD22-plt-DOX可靶向肿瘤组织，使药物在肿瘤组织富集，从而显著抑制肿瘤生长及促进其凋亡，并减轻DOX的心脏毒性。Western blot和免疫荧光结果一致表明该载药体系的抗肿瘤活性与Bcl-2/Caspase-9/Caspase-3凋亡通路相关。综上所述，本项目构建的CD22-plt-DOX新型载药体系为淋巴瘤治疗提供了一种新的思路和方法，具有重要的学术意义和应用价值。