具程序催化與抗原捕獲之粘附性奈米凝膠應用於增強免疫治療

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研究生：	巴努倪羅莎 Bhanu Nirosha Yalamandala
論文名稱：	具程序催化與抗原捕獲之粘附性奈米凝膠應用於增強免疫治療 Programmed Catalytic Therapy and Antigen Capture-Mediated Cancer Immunotherapies by In Situ-Forming Adhesive Nanoreservoirs
指導教授：	胡尚秀 Hu, Shang-Hsiu
口試委員:	張建文姜文軒黃玠誠陳冠宇
學位類別：	博士 Doctor
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	115
中文關鍵詞：	藥物傳遞、金屬有機骨架、奈米凝膠、抗原捕獲、免疫反應、T細胞浸潤、肺轉移腫瘤
外文關鍵詞：	Drug delivery, MOF, nanogel, antigen capture, immune response, T cell infiltration, lung metastasis
相關次數：	點閱：3 下載：0
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免疫療法是一種很有發展性的癌症治療療法，其利用自身免疫系統的活化來對抗癌細胞。免疫療法能使T細胞去識別癌細胞，並抑制腫瘤及誘導產生抗原相關的免疫反應和長久的免疫記憶。然而，癌細胞的免疫豁免和低免疫原性會減少腫瘤中淋巴細胞的存在，尤其是侵入性轉移型腫瘤。為了解決這個問題，一個策略是使用免疫原性補充劑來提高抗原免疫原性。本研究的主軸分為兩部分: (1) 雙金屬有機框架 (MOF) 的奈米劑型在肺轉移模型中的程序化催化療法，藉由ROS的產生和促進T細胞侵入腫瘤來達到治療效果；(2)藉由MOF奈米劑型原位形成的催化療法和奈米黏合劑之抗原捕獲能力，將腫瘤抗原攜帶至淋巴結的樹突狀細胞以達到癌症免疫療法。
在第一部分中，我們討論了雙金屬有機框架奈米劑型對肺轉移腫瘤的程序化催化免疫治療。雙MOF 與氯喹(CQ)在癌細胞中產生類芬頓反應，通過識別腫瘤相關抗原，可產生ROS並毒殺肺轉移腫瘤。在這項研究中，提出了一種功能性核-殼金屬-有機骨架奈米立方體（雙 MOF）兼作催化劑和 T 細胞侵入誘導劑，可對 ROS 進行編程並抑制自噬作用。雙MOF 利用普魯士藍 (PB) 包覆含鐵 (Fe2+) 金屬有機骨架（MOF, MIL88）作為癌細胞中的程序化過氧化物誘導物，持續的促進ROS生成。在氯喹的協助下，通過抑制溶酶體自噬作用，破壞癌細胞自我防禦代謝機制，進一步提高細胞毒性。此材料設計的目的是抑制腫瘤自噬和產生ROS，最終提高T細胞聚集並用於肺轉移腫瘤的免疫治療。邊緣化和內化介導的癌細胞攝取改善了體內轉移性腫瘤雙 MOF 的積累。雙 MOF結合自噬作用的抑制及ROS的催化作用在轉移型肺腫瘤中引起約3倍的T細胞聚集。肺轉移模型中顯示，雙MOF藉由 T細胞聚集和ROS生成的協同作用，成功的抑制了肺部90%以上的腫瘤灶。
在第二部分中，我們提出了一種原位形成的粘附催化納米凝膠(CN)，攜帶MnO2作為磁熱誘導的抗原庫，用於增強免疫治療效果。T淋巴細胞聚集和侵入腫瘤區能抑制最具破壞性的轉移型腫瘤。然而，癌細胞的免疫豁免和低免疫原性通常會減少腫瘤中淋巴細胞的存在，尤其是對於侵入性的轉移型腫瘤。我們在此研究中提出了一種含有二氧化錳(MnO2) 和兒茶酚功能化磁性金屬有機骨架(MOF)的粘合催化奈米凝膠 (CN)，用於抗原捕獲和遞送。靜脈注射的CN通過邊緣目標和磁熱效應驅動的原位形成凝膠在腫瘤處累積。在腫瘤部位，CN通過消耗穀胱甘肽(GSH)和類芬頓反應釋放 Mn2+ 進行氧化還原反應，即化學動力學療法 (CDT)。伴隨磁熱治療和化學動力學療法促進腫瘤釋放腫瘤相關抗原，包括新抗原和損傷相關分子模式（DAMPs）。接著，帶有兒茶酚基團的凝膠能充當抗原庫，將腫瘤相關抗原遞送至樹突狀細胞，實現持續的免疫刺激。原位形成的肺轉移催化奈米凝膠作為磁熱誘導的抗原庫在 60 天內有效抑制了腫瘤並提高了存活率。

Immunotherapy is a promising cancer suppressive therapy that uses the activation of the immune system to fight off cancer cells. It enables T cells to recognize cancer cells, potentially suppressing tumors as well as inducing antigen-related immune responses and long-lasting immune memory. However, the immune privilege and low vaccine immunogenicity usually reduce the presence of lymphocytes in tumors, especially for invading metastatic clusters. To address this issue, one simple strategy is to use immunogenic supplements to improve antigen immunogenicity. The topic of this research is divided into two parts. (1) Programmed Catalytic Therapy-Mediated ROS Generation and T-Cell Infiltration in Lung Metastasis by a Dual Metal-Organic Framework (MOF) Nanoagent (2) Programmed Catalytic Therapy and Antigen Capture-Mediated Dendritic Cells Harnessing Cancer Immunotherapies by In Situ-Forming Adhesive Nanoreservoirs.
In the first part, we addressed, programmed catalytic immune therapy in lung metastasis by the dual MOF nanoagent. Fenton-like reaction in cancer cells by dual MOF with chloroquine allows the potential for ROS generation & catalytic immune therapy of lung metastasis via the recognition of tumor-associated antigens. In this study, a functional core-shell metal-organic framework nanocube (dual MOF) doubling as a catalytic agent and T cell infiltration inducer that programs ROS and inhibits autophagy is reported. The dual MOF integrated a Prussian blue (PB)-coated iron (Fe2+)-containing metal-organic framework (MOF, MIL88) as a programmed peroxide mimic in the cancer cells, facilitating the sustained ROS generation. With the assistance of Chloroquine (CQ), the inhibition of autophagy through lysosomal deacidification breaks off the self-defense mechanism and further improves the cytotoxicity. The purpose of this material design was to inhibit autophagy and ROS efficacy of the tumor, and eventually improve T cell recruitment for immune therapy of lung metastasis. The margination and internalization-mediated cancer cell uptake improve the accumulation of dual MOF of metastatic tumors in vivo. The effective catalytic dual MOF integrated dysfunctional autophagy at the metastasis elicits the ~3-fold recruitment of T lymphocytes. Such synergy of T cell recruitment and ROS generation transported by dual MOF during the metastases successfully suppresses more than 90% of tumor foci in the lung.
In the second part, we proposed an in situ forming adhesive catalytic nanogel (CN) loaded with MnO2 as a magnetothermal-induced antigen reservoir for enhanced immunotherapy. T lymphocyte recruitment and infiltration promises to suppress the most devastating metastatic tumors for immunotherapy. However, the immune privilege and low vaccine immunogenicity usually reduces the presence of lymphocytes in tumors, especially for invading metastatic clusters. Here, an adhesive catalytic nanogel (CN) containing manganese dioxide (MnO2) and catechol-functionalized magnetic metal organic framework (MOF) for the antigens capture and delivery is reported. The intravenously injected CN accumulates at tumor via the marginated target and in situ forming gel actuated by a magnetothermal effects. At tumor site, CN releases Mn2+ for redox reactions by depleting glutathione (GSH) and Fenton-like activity, i.e., chemodynamic therapy (CDT). Accompanying with hyperthermia and CDT promote the tumor to release the tumor-associated antigens including neoantigens and damage-associated molecular patterns (DAMPs). Then, the gels with catechol groups act as antigen reservoirs and deliver the autologous tumor-associated antigens to dendritic cells, achieving sustained immune stimulation. The in situ-forming catalytic nanogel at lung metastasis as a magnetothermal-induced antigen reservoir effectively inhibited the tumor in 60 days and increased the survival rate.

中文摘要    ii
致谢    vii
Acknowledgment    viii
Table of Contents    ix
List of Figures    xii
List of Tables    xv
Chapter 1 Introduction    1
Chapter 2 Literature Review and Theory    4
1 Introduction of Lung Metastasis    4
1.1 Role of immunotherapy in lung metastasis    5
1.2 Metal-Organic Frameworks (MOFs)    6
1.3 Autophagy Protein Expression (Regulation of Autophagososmes)    12
1.4 Margination    15
1.5 Nanoparticle Shape Effect    15
2 Introduction of Injectable Materials- Hydrogel    17
2.1 Antigen Capture and Immune Stimulation    20
2.2 Glutathione (GSH) Depletion    20
2.3 Alternating magnetic field (AMF)    22
2.4 Chemodynamic Therapy (CDT)    23
2.5 Dendritic cell (DC)    24
Chapter 3 Experimental Section    25
1 Materials    25
1.1 Materials used in synthesis and MOF nanoparticles (PB and NH2MIL88B):    25
1.2 Materials used in Fenton reaction experiment:    25
1.3 Materials used for in-vitro studies:    26
1.4 Materials used in in vivo studies    26
2 Apparatus and Instruments    27
3 Synthesis of Dual MOFs (MlL88@PB)    30
4 Synthesis of CN (Catalytic Nanoreservoir)    31
5 Material Characterization    31
5.1 Scanning Electron Microscopy (SEM)    31
5.2 Transmission Electron Microscopy (TEM)    32
5.3 Fourier Transform Infrared Spectroscopy (FTIR) for nanoparticles    32
5.4 Powder X-ray Diffraction Spectroscopy (XRD)    32
5.5 Dynamic Light Scattering (DLS)    32
5.6 X-ray Photoelectron Spectroscopy (XPS)    32
5.7 Fenton reaction    33
6 In Vitro    34
6.1 Cell Culture    34
6.2 Cell viability assay    34
6.3 Cellular uptake    35
6.4 Autophagy expression (LC3B)    35
6.5 Antigen release    36
7 In Vivo    36
7.1 In Vivo Imaging System (IVIS)    36
7.2 Tissue Section Immunostaining    37
7.3 Flow Cytometry Analysis    38
7.4 Animal Survival Test    39
7.5 Statistical Analysis    39
Chapter 4    40
1 Abstract    40
2 Introduction    41
3. Method    44
3.1. Materials    44
3.2 Synthesis of PB MOF    45
3.3 Synthesis of NH2-MIL88    45
3.4 Synthesis of MIL88@PB (Dual MOFs)    45
3.5 Characterizations    46
3.6 In Vitro Drug Release    46
3.7 In Vitro Cellular Toxicity    47
3.8 In Vitro Cellular Uptake    47
3.9 In Vitro LC3B Autophagy Protein Expression (Regulation of Autophagosomes)    48
3.10 In Vitro Catalytic Performance of Dual MOFs    48
3.11 Tissue Section Immunostaining    49
3.12 In Vivo Flow Cytometry Analysis    50
4. Results and Discussion    51
4.1 Characterization of PB, MIL88, and Dual MOF Nanoparticles    51
4.2 In Vitro Drug Release    56
4.3 In Vitro Cellular Uptake and Cytotoxicity of PB, MIL88, and Dual MOF    56
4.4 In Vitro Cellular Uptake and LC3B Protein expression (Regulation of Autophagosomes)    58
4.5 CQ Inhibition of LC3B Protein Expression    60
4.6 In Vivo Study of Mice Bearing Lung Metastasis Treated with Dual MOF    61
5 Conclusions    66
Chapter 5    67
1 Abstract    67
2 Introduction    67
3 Results and Discussion    70
3.1 Synthesis and Characterization of Catalytic Nanoreservoirs    70
3.2 Physicochemical Characterization of MOF, cMOF, cMOF@PND and CN    72
3.3 Cytotoxicity and Cell Uptake of MOF, cMOF, cMOF@PND and CN    78
3.4 Chemodynamic Effect of CN    80
3.5 In Vivo Lung accumulation of MOF, cMOF, cMOF@PND and CN    84
3.6 In Vivo mice bearing Lung Metastasis treated by MOF, cMOF, cMOF@PND and CN    86
3.7 T cell recruitment in lung metastasis    88
3.8 Antigen capture by adhesive nanoreservoir and immune activation    90
4 Conclusion    96
Chapter 6    97
References:    99
Curriculum Vitae    110

                                

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