载负IL-12的pH/温度双重响应磁性纳米凝胶结合磁感应热疗用于胶质瘤治疗的研究

Gliomas are the most common primary brain tumors in adults. The patients have poor life qualities and high tumor recurrence rates even after the combination treatments of surgical resection, radiotherapy, and adjuvant chemotherapy. Accordingly, novel treatment modalities are urgently required. One of these is immunotherapy, which is the hot spot in the current research of glioma. However, several considerable challenges should be overcome to improve the effect of the immunotherapy of glioma, including: the therapeutic agents usually unstable in the body, cannot cross the blood brain barrier, lack or targeting capabilities to glioma, and cannot response to the special immune escape and immunosuppression of glioma. Based on the previous studies on stimuli-responsive nanogels and targeting diagnosis of glioma, the purpose of this project is to develop IL-12 loaded pH/temperature responsive magnetic nanogels, combing with magnetic hyperthermia for the immunotherapy of glioma. The pH/temperature responsive nanogels could provide the passive targeting capability and improve stability and circulation time of therapeutic agents. Lactoferrin could offer the active targeting ability to glioma and enhance the permeability of the blood–brain barrier. Besides, the magnetic hyperthermia under external alternating magnetic field could address the special immune microenvironment of glioma and also enhance the permeability of the blood–brain barrier. Thus, this novel multimodal therapy of glioma could address the challenges and finally improve the treatment effect.

胶质瘤是颅内常见、严重危害人类健康、致残和死亡率高的疾病，常规治疗的效果不佳。免疫治疗是胶质瘤治疗的有效新方法，但存在一些问题：治疗物质体内稳定性低、无法穿越血脑屏障、缺乏对胶质瘤的靶向性、无法针对性地应对胶质瘤的特殊免疫逃逸和免疫抑制行为。本项目拟构建偶联乳铁蛋白且载负IL-12的pH/温度双重响应性磁性纳米凝胶，结合磁感应热疗，将纳米凝胶的优势（长循环、增加稳定性和被动靶向）、乳铁蛋白的优势（主动靶向性和促进穿越血脑屏障）以及磁感应热疗的优势（调节免疫功能应对特殊的胶质瘤免疫环境和有助于穿越血脑屏障）相结合，针对性地解决胶质瘤免疫治疗中的问题，实现对胶质瘤的综合治疗，增强治疗效果，并初步阐明疗效增强作用的机制。

胶质瘤是颅内常见、严重危害人类健康、致残和死亡率高的疾病，常规治疗效果不佳。免疫治疗通过增强机体对肿瘤的免疫反应，协同机体免疫系统杀伤肿瘤，是胶质瘤治疗的有效新方法。本研究构建了偶联乳铁蛋白且载负IL-12的pH/温度双重响应性磁性纳米凝胶（IL-12@Lf-MPNA纳米凝胶），平均粒径92.4 ± 4.4 nm，流体动力学直径102.5 ± 6.2 nm，PDI为0.186。对基本理化性质和磁学性能进行评价，VPTT为34.3度（pH 6.8）和40.4度（pH 7.4），刺激响应性符合预期，外加磁场作用下可以达到热疗温度（42-46度）。药物载负和释放研究表明Cy5.5-IL-12@Lf-MPNA纳米凝胶具有良好药物载负能力，通过调控温度和pH可调节药物释放行为，37度pH 7.4下累计释放率34.2%，37度pH 6.8下累计释放率61.3%。体外靶向研究表明IL-12@Lf-MPNA纳米凝胶具有主动靶向性和被动靶向性，通过交变磁场，可进一步增强肿瘤细胞对药物和材料摄取。C6细胞（LRP1 表达阳性）对IL-12@Lf-MPNA纳米凝胶摄取量显著高于对照组。在体内对其靶向和综合治疗能力进行评价，显示可以显著增加血脑屏障透过性，促进更多药物到达肿瘤部位（对照组425.7 pg/mg 组织，治疗组683.4 pg/mg 组织）。治疗效果上，与对照组相比，磁热效应对Treg增加明显，而IL-12以及和IL-12和磁热效应联合可显著增加Th和Treg，还可显著增加CTL数量。以肿瘤大小（进展）以及荷瘤大鼠生存期作为评价指标，显示IL-12@Lf-MPNA纳米凝胶结合磁热效应可以显著延缓胶质瘤进展，延长生存期。第15天对照组肿瘤进展率为327.3%，而治疗组为127.4%。对照组中位生存期为22天，治疗组为31天。安全性评价研究表明，未载药Lf-MPNA纳米凝胶对肿瘤细胞和正常细胞存活率无影响，体内实验表明IL-12@Lf-MPNA纳米凝胶对其他器官的淋巴细胞无显著影响，仅对脑部产生显著影响，不会产生全身免疫反应。综上所述，本研究构建IL-12@Lf-MPNA 纳米凝胶，结合磁感应热疗，将该纳米凝胶环境响应性（长循环和被动靶向）、Lf主动靶向性和穿越BBB能力以及磁感应热疗的优势结合起来，有助于穿越屏障并增强免疫治疗效果，解决了胶质瘤免疫治疗中存在的问题，能够有效增强胶