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基于纳微双尺度构建安全高效的抗肿瘤疫苗
习晓博
Thesis Advisor马光辉
2018-07
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
Keyword自愈微囊,微环境调控,自组装纳米颗粒,淋巴结靶向,肿瘤疫苗
Abstract

在癌症治疗领域,免疫疗法是一种极具潜力的治疗策略,其中肿瘤疫苗是一种极具吸引力的免疫疗法,主要利用疫苗激发机体的免疫系统来攻击和杀伤肿瘤。然而目前多数肿瘤疫苗在临床应用中均没有获得令人满意的治疗效果,其原因是缺乏对局部微环境或者递送路径的调控,未能使疫苗体系发挥最大的潜力。本论文针对上述问题,以提高抗原利用率、激活安全高效的免疫应答为目标,在微米和纳米两种尺度分别构建了一种局部滞留的微囊体系和一种靶向淋巴结的自组装纳米颗粒体系,结合体内外实验对两种体系在肿瘤治疗方面的效果进行了系统性的评价。论文具体开展的研究内容如下:1. 构建局部滞留的多孔自愈合聚乳酸微囊,并实现对不同分子温和有效地装载。首先调控并优化复乳脱溶剂法的众多因素,最终制备出表面孔径~2 μm、粒径30-60 μm、内部贯穿的多孔微球。随后基于升温愈合的方式制得愈合微囊,同时通过扩散进入微球中的抗原被装载在其中。不仅水溶性的抗原,如蛋白质和多肽,而且油溶性的佐剂,如单磷酸酯A(MPLA),均可以被高效地装载在微囊当中。同时聚乳酸-聚乙二醇(PELA)和聚乳酸-聚羟基乙酸(PLGA)等多种材料均可用于该平台制备出不同物理特性的微球,从而拓展微球在不同领域的应用。2. 基于多孔微球与愈合微囊两种体系不同的抗原释放行为,比较二者对抗原利用率的影响。虽然在体内多孔微球与愈合微囊均会形成抗原滞留位点持续地释放抗原,但是微囊在局部释放抗原时间更久,可以实现一个月的有效释放。伴随着材料的降解,抗原递呈细胞(APC)被大量持续的募集到材料周围,从而不断吞噬释放的抗原,抗原利用率提高至10倍,相比单纯抗原组提高至200倍。在局部募集的APC中,由于DC的比例最高,因此抗原利用率相比单纯抗原组提高至800倍。3. 评价微囊降解时产生的酸性微环境对细胞募集、抗原内吞、交叉递呈和细胞因子分泌的影响,考察其对细胞免疫应答的作用。在微囊降解的过程中,局部注射部位的pH一直维持在6.5左右。相比中性微环境,局部酸性微环境会分泌更多的趋化因子募集更多的APC,且DC和巨噬细胞对抗原的内吞量分别提高至3.5倍和1.9倍。同时二者主要组织相容性复合物(MHC)I的表达比MHC-II高40倍,说明抗原的交叉递呈被显著地提升,而且局部分泌的细胞因子偏向于Th1型,最终大量被激活的APC归巢至淋巴结开展后续的免疫应答。4. 在多种动物模型中,系统地证实了自愈合微囊用于肿瘤疫苗时的抗肿瘤效果及安全性。首先在免疫激活的早期,93.8%的CD8+ T细胞分化增殖,分泌了最高水平的颗粒酶B,杀伤了80%的靶细胞E.G7。在多种肿瘤模型中,基于微囊疫苗激发的持续的T细胞高水平增殖和杀伤表现,疫苗在装载低剂量抗原并且一次免疫的情况下显著地抑制了肿瘤的生长和转移。当同时装载低剂量的佐剂MPLA时,免疫效应得到进一步增强。通过检测细胞因子风暴相关的细胞因子分泌、体温及组织损伤等指标,证实了微囊疫苗在体内良好的生物安全性。5. 基于抗原与佐剂的自组装策略,构建了可高效靶向淋巴结,并快速激活免疫应答的纳米疫苗。在电荷相互作用下,抗原OVA与佐剂CpG形成了高装载率的自组装纳米颗粒。小粒径(~80 nm)、较窄的尺寸分布、负电性及优良的稳定性促使纳米颗粒高效地实现淋巴结靶向。相比单纯抗原组,颗粒组中抗原在淋巴结内的累积数量提高了10倍。同时颗粒被淋巴结内APC大量吞噬并促使CD8+ DC成熟,进而60%的CD8+T细胞发生增殖,体外杀伤实验中超过70%的靶细胞被杀伤。因此基于其激发的有效和快速的细胞免疫应答,自组装NPs用于治疗性疫苗极具潜力。;Due to the great potential of treating cancer, immunotherapy has attracted much attention. Especially therapeutic cancer vaccinations, which depended on arousing host immune response to attack and kill tumor cells by vaccines, were emerging as one of the attractive modalities for cancer treatment. However, owing to the lack of modulating microenvironment in situ or delivery paths, most of vaccines were unable to be fully utilized to maximize the potential, which led to unsatisfying performances in clinical trials. Herein, based on delivery paths, two different size systems were constructed for improving vaccine utilization and arousing safe and efficient immune responses. One was micro-sized microcapsules for retention in situ, and the other one was nano-sized self-assembled nanoparticles for targeting lymph nodes. Moreover the performance of both systems on treating cancer were evaluated systematically in vitro and vivo.In detail, this thesis mainly includes the following issues:1. The versatile vaccine platform, which was constructed by self-healing porous polylactic acid (PLA) microcapsules for retention in situ, could load different cargos effectively and mildly. Initially, based on optimizing multiply factors of the double emulsion and solvent extraction method, the resulting gigaporous microspheres (30-60 μm) exhibited numerous open pores (diameter ≈ 2 μm) on their surface with an interconnected porous network inside. Then through a mild sealing process, healed microcapsules were prepared during the superficial pores closed. Meanwhile, antigen molecules, which diffused into microspheres, were encapsulated. Importantly, both hydrophilic and hydrophobic molecules could be loaded for constructing a versatile loading platform. Different polymers, like poly (monomethoxypolyethylene glycol-co-D,L-lactide) and poly (lactic-co-glycolic acid), could be applied to prepare porous microspheres with different physicochemical characteristics to further extend the applications of microspheres.2. Based on two different antigen release behaviors of microspheres and microcapsules, antigen utilizations of both carriers were evaluated. Although both microspheres and microcapsules formed antigen depots at the injection site to release antigen sustainably, microcapsules achieved a more sustained antigen release within one month. Accompanying carrier degradation, more antigen presentation cells (APCs) were recruited to form a cell habitat around microcapsules for capturing fled antigen. Therefore, cellular internalization was consistently increased due to the spatiotemporal confluence of sustained antigen release and cellular recruitment. In this case, the relative antigen utilization was calculated to be 10-fold and 200-fold higher than that of the microspheres and free antigen groups, respectively. Especially, during the highest percentage of APC, antigen utilization of dendritic cells (DCs) was amplified to 800-fold.3. The influence of acidic microenvironment on immune response, which was created by microcapsule degradation, was evaluated via cell recruitment, antigen uptake, cross-presentation and cytokines secretion. During the process of microcapsule degradation, the pH value at vaccination site was maintained at ~6.5. APCs were recruited more efficiently in the acidic microenvironment (AM) group than neutral microenvironment (NM) group due to more chemokine secretions. Meanwhile, the internalization of antigen in the AM group were 3.5-fold higher for DCs and 1.9-fold higher for macrophages. The expression of major histocompatibility complex (MHC)-I was 40-fold higher than that of MHC-II in the AM group, indicating the significant promotion of antigen cross-presentation. The superiority of the AM was also reflected in the preference of Th1 type cytokines. Finally a flow of activated APCs homed to the lymph nodes for facilitating subsequent cellular response.4. Based on multiply tumor-bearing models, the therapeutic effects and safety of these self-healed microcapsules for potent cancer vaccinations were systematically verified. At the early stage of immune response, the most robust proliferation was observed in self-healed microcapsules with 93.8% of OVA specific CD8+ T cell division, the highest percentage of granzyme B-secreting CD8+ T cells and 80% of target cells (E.G7) lysis. Owing to the sustained proliferation and lysis behavior of T cells, microcapsule vaccine exhibited an outstanding inhibition on tumor growth and metastasis with a low dose antigen and a single immunization. A further step was achieved during a low dose of MPLA was embedded in the matrix of microcapsules. Based on the monitor of cytokine-storm related cytokines, body temperature and organ damages, the vaccine exhibited excellent biosafety in vivo.5. Based on a self-assembly strategy of antigen and adjuvant, nano-vaccines were constructed for efficiently targeting lymph nodes to quickly arouse immune response. The assembled nanoparticles, which were composed of antigen (OVA) and adjuvant (CpG) with high loading efficiency, were formed due to the electronic interaction. The small size (~80 nm), narrow distribution, negative charge and good stability endowed these nano-vaccines with superior capacity for lymph node targeting. Correspondingly, the accumulation at lymph node could be improved to 10 fold. Subsequently, due to the sufficient APCs internalization and maturation in lymph nodes, ~60% of T cells were stimulated to proliferate in vivo, and above 70% of target cells were specifically killed in a vitro specific lysis test. Base on the effective and quick cellular immune response, the assembled nanoparticles exhibited the great potential as therapeutic vaccines. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26890
Collection中国科学院过程工程研究所
研究所(批量导入)
Recommended Citation
GB/T 7714
习晓博. 基于纳微双尺度构建安全高效的抗肿瘤疫苗[D]. 中国科学院研究生院,2018.
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