修敬雅,王晓萍,李丹,乔明曦
XIU Jingya,WANG Xiaoping,LI Dan,QIAO Mingxi

• 1:沈阳药科大学药学院
• 2:沈阳医学院附属中心医院药剂科

摘要(Abstract)：

目的 构建一种M2型巨噬细胞膜包裹的共载化疗药阿霉素及免疫佐剂咪喹莫特的仿生型纳米粒作为肿瘤原位疫苗，通过刺激肿瘤细胞发生免疫原性细胞死亡提供肿瘤相关抗原，激活系统抗肿瘤免疫反应实现肿瘤免疫治疗。方法 通过乳化-溶剂挥发法制备共载阿霉素(doxorubicin, DOX)和咪喹莫特(R837)的聚乳酸-羟基乙酸共聚物纳米粒(doxrubicin-R837-nanoparticles, DRNPs),采用细胞膜提取试剂盒提取M2型巨噬细胞膜，通过共挤出法制备巨噬细胞膜包裹的聚乳酸-羟基乙酸共聚物纳米粒(M2-doxrubicin-R837-nanoparticles, MDRNPs),以两种药物包封率及载药量为考察指标，优化载体的处方工艺，对优化后原位疫苗的粒径、电位、形态及药物释放进行测定，对构建的原位疫苗体外细胞毒性、细胞摄取及诱导免疫原性细胞死亡相关指标进行考察。结果 优化后DRNPs的处方工艺：药载质量比为1∶10,初乳水相聚乙烯醇浓度为2%,分散相PVA浓度为0.5%,初乳分散相体积比为1∶6,优化后DRNPs中DOX和R837包封率分别为84.3%和81.3%,巨噬细胞包裹原位疫苗MDRNPs的粒径为(196.3±5.8)nm,电位为(-15.3±1.2)mV,呈球形或类球形，该制剂在生理条件下可缓慢释放DOX和R837。MDRNPs对4T1肿瘤细胞IC_(50)值为11.18 mg·L~(-1),且可通过巨噬细胞膜作用有效靶向至肿瘤细胞。MDRNPs可显著增加肿瘤细胞钙网蛋白外翻，高迁移速率蛋白1外流及胞内ATP外流进而诱导树突状细胞成熟以激活肿瘤细胞发生免疫原性细胞死亡。结论 MDRNPs可有效靶向至肿瘤细胞并诱导肿瘤细胞发生免疫原性细胞死亡，从而激活抗肿瘤免疫响应。
Objective To fabricate M2 macrophage membrane coated biomimetic nanoparticles as in situ cancer vaccine for doxorubicin and immune adjuvant imiquimod co-delivery.Methods Emulsion/solvent evaporation method was used to prepare DOX/R837 loaded PLGA nanoparticles DRNPs.The M2 macrophage membrane(M2m)was extracted by extraction kit, and macrophage membrane coated PLGA nanoparticle MDRNPs were prepared using co-extrusion method.To optimize the formulation process of the carriers, drug loading ratio, aqueous phase PVA concentration, dispersed phase PVA concentration, colostrum/dispersed phase volume ratio were selected as variables, and the encapsulation efficacy and loading content were selected as indicators.Furthermore, the optimized in situ vaccine was characterized by particle size, surface potential, morphology and drug release behavior.The cell cytotoxicity, cellular uptake and relative ICD markers were also investigated to evaluate the therapeutic potential of MDRNPs.Results The optimized prescription of DRNPs was as follows: drug loading ratio was 1∶10,PVA concentration in colostrum aqueous phase was 2%,PVA concentration in dispersed phase was 0.5%,and colostrum/dispersed phase volume ratio was 1∶6.The encapsulation efficiencies of DOX and R837 were 84.3%and 81.3%,respectively.The particle size and surface potential of MDRNPs were(196.3±5.8)nm and(-15.3±1.2)mV,respectively.MDRNPs was uniform spheroid-like structure, which displayed sustained release behavior in physiological conditions.IC_(50) value of MDRNPs against 4T1 cell line was 11.18 mg·L~(-1).MDRNPs could increase CRT expression, HMGB1 and ATP secretion, which could subsequently induce DCs maturation and active cancer immunogenic cell death process.Conclusion In situ cancer vaccine MDRNPs could effectively target tumor cell and induce immunogenic cell death to activate antitumor immune response.

关键词(KeyWords)： 原位疫苗;巨噬细胞膜;阿霉素;咪喹莫特
in situ vaccine;macrophage membrane;doxorubicin;imiquimod

Abstract:

Keywords:

基金项目(Foundation): 沈阳市科技计划项目(19-112-4-097)

作者(Author): 修敬雅,王晓萍,李丹,乔明曦
XIU Jingya,WANG Xiaoping,LI Dan,QIAO Mingxi

DOI: 10.14066/j.cnki.cn21-1349/r.2022.1174

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