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以作者查詢圖書館館藏、以作者查詢臺灣博碩士論文系統、以作者查詢全國書目
作者(中文):邱奕霖
作者(外文):Chiu, Yi-Lin
論文名稱(中文):合成聚硼酸酯聚合物微胞:一種潛在的硼藥物透過結合BNCT和PD-1/PD-L1檢查點阻斷免疫療法以增強癌症治療效果
論文名稱(外文):Synthesis of Polyboronate Ester Polymer Micelles: A Potential Boron Drug for Enhancing Cancer Treatment through the Combination of BNCT and PD-1/PD-L1 Checkpoint Blockade Immunotherapy
指導教授(中文):龔佩雲
指導教授(外文):Keng, Pei-Yuin
口試委員(中文):王子威
吳思翰
口試委員(外文):Wang, Tzu-Wei
Wu, Si-Han
學位類別:碩士
校院名稱:國立清華大學
系所名稱:材料科學工程學系
學號:110031632
出版年(民國):112
畢業學年度:111
語文別:中文
論文頁數:109
中文關鍵詞:硼中子捕獲治療、免疫療法、癌症治療、檢查點阻斷、藥物投遞、奈米藥物
外文關鍵詞:Boron neutron capture therapy、Immunotherapy、Cancer treatment、Checkpoint blockade、Drug delivery、Nanomedicine
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近年來，針對癌症的研究進展迅速，有許多研究集中在癌症的發病機制、癌細胞的生長及轉移方式、周圍環境和癌症細胞的交互作用，以及如何防治癌症等問題。許多研究還集中於癌症治療，開發新型藥物和治療方法，包括基因療法、細胞療法、硼中子捕獲治療、免疫療法等，以提高癌症治療的效果和生存率。其中提到的硼中子捕獲治療（BNCT）是一種放射治療模式，依靠將非放射性硼-10（10B）同位素特定運送到腫瘤部位。經過熱中子照射，硼原子將進行核分裂反應，釋放出高能α粒子，可以局部特異性地摧毀癌細胞。然而，目前的硼藥物在腫瘤中顯示出較低的腫瘤累積和分布不均，導致BNCT後癌症復發。為了克服這個挑戰，在本篇碩士論文中開發了富硼聚硼酸酯聚合物微胞，用於結合BNCT和免疫檢查點阻斷 (PD-1/PD-L1 blockade) 治療的聯合治療，這已被證明可以有效逆轉T細胞抑制並增強體內抗腫瘤免疫力。通過自組裝的方法製備富硼的高分子微胞，聚乙二醇-b-[聚(3-乙烯基苯甲醛)-r-聚(4-乙烯基苯硼酸酯)] (mPEG-b-(PVB-r-PVBE) ，在這個方法中，PVBE重複單元同時作為中子捕獲劑，又用作微胞中疏水段，在選擇性溶劑作用下誘導自組裝的微胞結構。此外，苯甲醛重複單元被安裝作為席夫鹼鍵結的連接點與抗小鼠PD-L1單克隆抗體(anti-mouse PD-L1 mAbs)共價結合。在這種聯合治療中，附著在微胞結構上的抗PD-L1會在進入微酸腫瘤微環境（TME）時因對酸敏感的席夫鹼鍵結斷鍵而釋放出來，然後結合到腫瘤表面的PD-L1軸上進行免疫檢查點阻斷。進行BNCT治療後，大部分原發腫瘤被破壞而釋放出腫瘤特異性抗原，隨後在宿主中啟動可持續的免疫反應循環。根據我們的結果，mPEG-b-(PVB-r-PVBE) 微胞與 BPA 相比，顯示出更長的5.4天的腫瘤生長延遲（TGD）。此外，mPEG-b-(PVB-r-PVBE) 微胞和 anti-PD-L1@mPEG-b-(PVB-r-PVBE) 微胞在 BNCT 和免疫療法聯合治療中表現出更長的 6.6 天和 5.2 天的 TGD。這些結果表明在聯合治療中，mPEG-b-(PVB-r-PVBE) 微胞和 anti-PD-L1@mPEG-b-(PVB-r-PVBE) 微胞對於抑制腫瘤具有優越的效果，相比單獨進行的BNCT或BPA聯合治療效果更好。這表明這些新型富硼聚酯兩親性嵌段共聚物微胞具有潛在的應用前景，可用作硼載體和免疫療法藥物，以增強癌症治療效果並潛在地加強免疫療法的效果。
Over the past few decades, research on cancer has progressed rapidly, with many studies focusing on the mechanisms of cancer development, the growth and metastasis of cancer cells, and the interaction between surrounding environment and cancer cells, and how to prevent the cancer. Many studies also concentrate on cancer treatment, developing new drugs and treatment methods, including gene therapy, cell therapy, boron neutron capture therapy, immunotherapy, and others, to improve the effectiveness and survival rates of cancer treatment. One of the mentioned therapies, boron neutron capture therapy (BNCT) is a radio-therapeutic technique that depends on the specific delivery of non-radioactive boron-10 (10B) isotope to the tumor site. Upon thermal irradiation of neutrons, the B atoms will undergo nuclear fission reactions yielding highly energetic alpha particles that can specifically destroy the cancer cells locally. However, the current boron drugs exhibit low tumor accumulation and poor distribution throughout a solid tumor which resulted in cancer recurrent after BNCT.
To overcome this challenge, our group is developing boron-rich polyboronate ester polymer micelles for combinatorial therapy of BNCT and checkpoint PD-1/PD-L1 (programmed death-1/programmed death-1 ligand) blockade immunotherapy, which has been shown to reverse the T-cell suppression effectively and enhance endogenous antitumor immunity. Specifically, the boron-rich polymer micelles were prepared via the self-assembly of poly(ethylene glycol)-b-[(poly(3-vinylbenzaldehyde)-r-poly (4-vinylphenylboronate ester)) (mPEG-b-(PVB-r-PVBE)). In this approach, the PBA repeating units served as both the neutron capture and the hydrophobic segment in inducing the self-assembly of the micellar structure in selective solvent. Moreover, the benzaldehyde moieties are installed to conjugate anti-mouse PD-L1 monoclonal antibodies (mAbs) via the Schiff’s base bond linker. In this co-therapy, anti-PD-L1 conjugated on the micellar structure will be released and subsequently bind to the PD-L1 axis on the surface of the tumor cells upon entering the tumor microenvironment (TME), as the acid labile Schiff’s base bond is cleaved under slightly acidic pH value. Upon BNCT monotherapy treatment, most of the primary tumor is destroyed and thus releasing tumor specific antigens and subsequently initiates a sustainable cycle of immune response in the host. In our results, mPEG-b-(PVB-r-PVBE) micelle demonstrated more tumor suppression with 5.4 days of tumor growth delay (TGD) compared to BPA with 3.3 days of TGD. Furthermore, both mPEG-b-(PVB-r-PVBE) micelle and anti-PD-L1@mPEG-b-(PVB-r-PVBE) micelle exhibited further tumor suppression with 6.6 days and 5.2 days of TGD in the BNCT and immunotherapy combined therapy. These results indicate that mPEG-b-(PVB-r-PVBE) micelle and anti-PD-L1@mPEG-b-(PVB-r-PVBE) micelle have superior tumor inhibition effects in the combined therapy compared to either BNCT monotherapy or BPA combined therapy. This suggests the potential application of these novel polyboronate ester amphiphilic block copolymer micelles as boron carriers and immunotherapy drug to enhance cancer treatment efficacy and potentiate immunotherapy.
Abstract------------------------------------------------------I
摘要----------------------------------------------------------III
Acknowledgment------------------------------------------------VI
Table of Content----------------------------------------------VII
Figures, Schemes and Table------------------------------------X
Chapter 1 Introduction----------------------------------------1
Chapter 2 Literature Review-----------------------------------4
2.1 Radiotherapy and Boron Neutron Capture Therapy------------4
2.1.1 Radiotherapy--------------------------------------------4
2.1.2 Boron Neutron Capture Therapy---------------------------5
2.2 Immunotherapy---------------------------------------------9
2.3 Nanocarrier for immunotherapy-----------------------------17
2.4 Boron agent of BNCT and future development----------------19
2.4 Combination therapy of boron neutron capture therapy and immunotherapy-------------------------------------------------24
Chapter 3 Materials, Methods, and Instruments-----------------28
3.1 Materials and instruments---------------------------------28
3.2 Preparation of anti-PD-L1 conjugated poly(ethylene glycol)-b-[(poly(3-vinylbenzaldehyde)-r-poly(4-vinylphenylboronate ester)) anti-PD-L1@mPEG-b-(PVB-r-PVBE) polymer micelle---------------------31
3.2.1 Preparation of poly (ethylene glycol) methyl ether 2-bromoisobutyrate mPEG-Br macroinitiator-----------------------31
3.2.2 Protection of 4-vinylphenylboronic acid-----------------32
3.2.3 Synthesis of poly(ethylene glycol)-b-[(poly(3-vinylbenzaldehyde)-r-poly(4-vinylphenylboronate ester)) mPEG-b-(PVB-r-PVBE) amphiphilic block copolymer-----------------------------32
3.2.4 Synthesis of anti-PD-L1 conjugated mPEG-b-(PVB-r-PVBE) amphiphilic block copolymer-----------------------------------34
3.2.5 Preparation of anti-PD-L1@mPEG-b-(PVB-r-PVBE) polymer micelle-----------------------------------------------------------------35
3.2.6 Quantification of anti-PD-L1 in anti-PD-L1@mPEG-b-(PVB-r-PVBE) polymer micelle-----------------------------------------------35
3.3 In vitro assay--------------------------------------------37
3.3.1 In vitro cytotoxicity assay-----------------------------37
3.3.2 In vitro cellular boron uptake--------------------------38
3.3.3 In vitro Boron Neutron Capture Therapy treatment--------39
3.4 In vivo assay---------------------------------------------40
3.4.1 In vivo tumor models------------------------------------40
3.4.2 In vivo biodistribution of boron content----------------41
3.4.3 In vivo boron neutron capture therapy combine with immunotherapy treatment-----------------------------------------------------43
Chapter 4 Results and Discussion------------------------------47
4.1 Characterization of anti-PD-L1@mPEG-b-(PVB-r-PVBE) polymer micelle-------------------------------------------------------47
4.1.1 Characterization of mPEG-b-(PVB-r-PVBE) amphiphilic block copolymer using nuclear magnetic resonance (NMR) spectrum-----49
4.1.2 Characterization of mPEG-b-(PVB-r-PVBE) amphiphilic block copolymer using Fourier-transform infrared spectroscopy (FT-IR)--52
4.1.3 Gel permeation chromatography (GPC) of mPEG-b-(PVB-r-PVBE) amphiphilic block copolymer-----------------------------------54
4.1.4 Size distribution and TEM morphology--------------------55
4.1.5 Enzyme-linked immunosorbent assay (ELISA)---------------57
4.2 In vitro assay against B16-F10 melanoma cells-------------58
4.2.1 Cytotoxic studies of polymeric micelles-----------------58
4.2.2 Boron uptake of B16-F10 melanoma cells------------------60
4.2.3 Cell viability after BNCT treatment---------------------64
4.3 In vivo assay---------------------------------------------66
4.3.1 Biodistribution of boron content in B16-F10 melanoma tumor-bearing mice--------------------------------------------------66
4.3.2 Synergistic therapy of BNCT and immunotherapy-----------68
Chapter 5 Conclusion------------------------------------------79
Chapter 6 Prospective-----------------------------------------81
References----------------------------------------------------83
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