利用奈米材料調控硼中子捕獲治療及光動力於治療腫瘤上之應用

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研究生：	那力士 Naresh, Kuthala
論文名稱：	利用奈米材料調控硼中子捕獲治療及光動力於治療腫瘤上之應用 Nanomaterial-Mediated Boron Neutron Capture Therapy and Photodynamic Therapy for the Destruction of Tumors
指導教授：	黃國柱 Hwang, Kuo-Chu
口試委員:	趙瑞益 Chao, Jui-I 袁俊傑 Yuan, Chiun-Jye 江啟勳 Chiang, Chi-Shiun 宋信文 Sung, Hsing-Wen
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	184
中文關鍵詞：	硼中子俘獲療法、光動力療法
外文關鍵詞：	Boron neutron capture therapy, Photodynamic therapy
相關次數：	點閱：3 下載：0
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摘要

近幾年來，藉由奈米材料進行光動力治療(photodynamic therapies)及光熱治療(photodynamic therapies)達到診斷及治療癌症的方法，已經成為熱門的研究主題且具備根除癌症的潛力，使該領域備受重視。光照治療(phototherapy)使用的探針能夠有效地吸收光能，進而產生活性氧物質(reactive oxygen species)及熱能，達到殺死惡性癌細胞的效果。在許多文獻中，作者利用紫外光及可見光達到破壞癌細胞的效果，由於紫外光和可見光在人體的穿透深度較淺，限制了其在臨床上的應用。具有吸收第一生物窗及第二生物窗波長的光照治療試劑的相關研究較少，此外也沒有研究探討關於第三生物窗內的光動力治療，為了達到診療人體較深處的癌細胞，發展此類奈米材料的研究平台愈發重要。在眾多的材料中，以奈米金結構在近紅外光區對癌細胞的治療最廣為討論。然而，光在人體內的穿透深度依然有限，因此對於一些埋藏在體內較深處的癌細胞，如: 膠質母細胞瘤，胰腺癌和肺腺癌等無法使用常見的光照治療。為了治療此類癌症，因而建立了硼中子捕獲治療的方法，而利用奈米材料進行硼中子捕獲治療的相關研究依然有限，因此本論文將對此作深入探討。

第一章中，利用在硼10豐富的奈米粒子表面上修飾精甘天冬氨酸肽(RGD peptide)且其上以FITC標定，故將此材料命名為10BSGRF NPs，此奈米粒子能通過血腦屏障並標靶性地作用於多形性膠質母細胞瘤(Glioblastoma Multiforme)上，此材料能順利進入癌細胞內，平均每克的細胞內含有50.5微克的硼10。利用10BSGRF NPs不僅能提高核磁共振成像的對比度，並成功地抑制小腦腫瘤生長，將小鼠的半生期從22天(對照組)延長至39天。

第二章中，藉由微波合成硼10豐富的磷酸硼奈米粒子(10BPO4 NPs)，其大小約為50奈米，並利用此材料對頭部和頸部的癌症進行標靶性治療。此材料具有良好的藥物傳遞性，平均每克細胞中含有63微克的硼10，且其對頭部頸部的癌細胞破壞性也優於臨床上使用的硼中子捕獲治療藥物(BPA-F)。為了提升細胞對磷酸硼奈米粒子的包吞作用，在奈米粒子表面上修飾anti-EGFR抗體，在體外測試中，在修飾anti-EGFR抗體後的磷酸硼奈米粒子條件下，頭頸部癌細胞致死率高達72 %，遠高於在BPA-F條件下的致死率(30%)。

第三章中，我們合成了獨一無二的金奈米花生(AuNPN)，奈米花生的殼層間具有間隙，並在間隙內填滿了氯化鈉，由於氯化鈉具有表面電漿增強特性，因此利用此材料進行癌細胞的治療。實驗中分別合成間隙為2奈米及6.5奈米的金奈米花生，兩者皆具有表面電漿共振增強的效果且吸收峰紅移，但在間隙6.5奈米的材料中更為明顯，且此材料也有較佳的光動力治療及光熱治療效果。透過氯化鈉和奈米花生的交互作用，改變表面電漿共振的特性，使材料在近紅外光的照射下能夠有效抑制生物體內癌細胞的生長。

第四章中，我們發現六硼化鑭奈米立方體(LaB6 CNPs)能夠在第三生物窗中的光照射下產生氫氧自由基，並能在缺氧的環境下進行光動力治療。在體內實驗中，六硼化鑭奈米立方體同時具有光動力治療及光熱治療的能力，可以有效地摧毀具有多重抗藥性的肺癌細胞。六硼化鑭奈米立方體能夠有效地吸收近紅外光並產生單重態氧。此外，這是第一篇探討利用奈米材料對多重抗藥性的肺癌細胞(NCI-H23)進行光動力治療及光熱治療的相關研究
和CT成像。

In the recent years nanotechnology based nanomaterials become pathfinder to diagnose and eradicate complicated cancers through phototherapies like photodynamic (PDT) and photo thermal (PTT) therapies. The phototherapy probe with efficient absorption of light triggers the generation of reactive oxygen species (ROS) and heat to eradicate the malignant tumors. In literature numerous therapeutic models demolish tumors by UV and visible light which will limits their application in clinical view due to their poor penetration depth than the NIR (near infrared) light. The photo therapy agents explored in both the biological window I (650-950 nm), window II (1000-1350 nm) are very rare and no literature explored the PDT in the biological window III (1550-1870 nm), and researchers need to develop such kind of nanoplatforms to treat the deep tissue buried tumors with minimum invasiveness. Among all, the gold nanostructures like AuNRs, Au-nanoshells, Au-nanocages and nanohexapods attracted much interest to treat cancer cells and malignant tumors in NIR biological window I due to the low toxicity levels. Even though, the deep buried tumor like glioblastoma, pancreatic cancer, and lung adenocarcinoma can’t be treated with conventional photodynamic/photo thermal therapy where the light can’t be penetrated such a deep buried cancer tumor cells. To treat such a deep buried tumor alternative therapy model like boron neutron capture therapy with the nanotechnology based platform to be established. To date there are limited amount of reports published on nanotechnology based boron neutron capture therapy.
In the chapter one, we have demonstrated that an unprecedented 10B-enriched (96% 10B enrichment) boron nanoparticle nanomedicine (10BSGRF NPs) surface-modified with a FITC-labeled RGD-K peptide can pass through the brain blood barrier, selectively target at Glioblastoma multiforme brain tumor sites and deliver high therapeutic dosage (50.5 μg 10B/g cells) of boron atoms to tumor cells with a good tumor-to-blood boron ratio of 2.8. The major challenge in treating GBMs using BNCT is to achieve selective imaging, targeting and sufficient accumulation of boron-containing drug at the tumor site so that effective destruction of tumor cells can be achieved without harming the normal brain cells. To tackle this challenge, The 10BSGRF NPs not only can enhance the contrast of MR imaging to help diagnose the location/size/progress of brain tumor, but also effectively suppress murine brain tumors via magnetic resonance (MR) imaging-guided boron neutron capture therapy (BNCT), prolonging the half-life of mice from 22 days (untreated group) to 39 days. This work sheds light on a new way to treat patients with complicated and ‘difficult-to-treat’ brain tumors via MR imaging-guided BNCT.
In the chapter two, we have synthesized 10B rich boron phosphate nanoparticles (10BPO4 NPs) with microwave arcing method with particle size of ~ 50 nm within the single step and this 10B-enriched boron phosphate (96% 10B enrichment) employed as nanomedicine for head and neck cancer tumor targeting ability, which can enables to deliver huge amount (~63 μg/g cells) of 10B accumulation than the clinical BNCT molecular (BPA-F) drugs and exerts higher degree of destruction to the head and neck cancer tumors than the currently using clinical molecular BNCT drug, BPA-F. As the 10B-enriched boron phosphate NPs were modified with anti-EGFR antibody to enable the targeting ability through the EGFR mediated uptake at cell membrane. In vitro cell death (72 %) for 10BPO4-anti-EGFR NPs treated head and neck cancer cells is ~ 2.4 folds higher than (30%) for BPA-F. The mice xenograft BNCT model, 10BPO4-anti-EGFR NPs exerts the higher degree of destruction to the head and neck tumors. The median survival of the BNCT treated mice with 10BPO4-anti-EGFR NPs extends to 75 days is far better than the mice treated with BPA-F (33 days), blank + NR mice (25), and blank mice (23 days).
In the Chapter three, we have explored a unique peanut type gold nano structure called AuNPN with the gaps 2.0 and 6.5 nm by filled with NaCl into the gaps in order to enhance the plasmonic field enhancement and it was further utilized for tumor therapeutic models. As fabricated AuNPN 2.0, AuNPN 6.5 exhibits drastic plasmonic field enhancement by the introduction of NaCl to induce the plasmonic effect in the far near IR region, which pronounced more in the AuNPN 6.5. Interestingly, we observe that, AuNPN 6.5(NaCl) exhibits better therapeutic results by the plasmonic field enhanced photodynamic and photothermal therapy. This plasmonic effect with NaCl AuNPN highlights the in vivo study upon NIR laser irradiation to inhibit the tumor recurrence.
In the chapter four, we have performed that LaB6 CNPs can generate ROS species hydroxyl radical in biological window III to address the unresolved penetration depth issue and hypoxia environment tumors with photodynamic therapy. In the in vivo, LaB6 CNPs can exert combination of photodynamic (PDT) and photothermal (PTT) therapeutic treatment for curing intrinsic multi-drug resistant (MDR) lung tumors completely. Lanthanum Hexaboride (LaB6) cubic nanoparticles can absorb near infra-red (NIR) light (400~1700 nm) very efficiently and sensitize formation of singlet oxygen as well as heat in biological windows II and III. To the best of our knowledge, this is the first literature example of the nanomaterial-mediated photodynamic and photothermal therapy for treating intrinsic multi-drug resistant lung cancers (NCI-H23) with bimodal MR and CT imaging.

Chapter 1: Engineering Novel Targeted Boron-10-Enriched Theranostic Nanomedicine to Combat against Murine Brain Tumors via MR Imaging-Guided Boron Neutron Capture Therapy

1 Background and Introduction……………………………………………..…1

1.1.Boron neutron capture therapy using molecular drugs/nanomaterials.............1
2 Experimental Section………………………..………………………………..7

2.1 Synthesis of 10BSGRF NPs...............................................................................7
2.2 Synthesis of BPA-Fructose complex solution..................................................8
2.3 Characterization of nanoparticles ....................................................................9
2.4 Estimation of RGD-K-FITC peptide on the surface of 10BSGRF NPs using Bradford assay...........................................................................................................9
2.5 Cell culture………………………………………………………………….10
2.6 Cell viability assay………………………………………..……………..…10
2.7 Intracellular uptake of 10BSGRF NPs using confocal microscopy…...….….11
2.8 In vitro Calcein-AM and PI staining………………………………………..12
2.9 In vitro targeting of 10BSGRF NPs……………………………..……….…..13
2.10 In vitro detection of apoptotic and necrotic cells………………..………...13
2.11 In vitro cell cycle analysis…………………………………..……………..13
2.12 In vivo intracranial injection of ALTS1C1 cells into the brain.….......……14
2.13 T1 weighted MR imaging..……………………………...…………………14
2.14 In vivo biodistribution 10BSGRF NPs and BPA-F by Inductively coupled mass spectrometry (ICP-MS)…………………………………..............................15
2.15 In vivo boron neutron capture therapy….……………….………………...16
2.16 Histology and Caspase-3 staining………………….………………………16
3 Results and Discussion………………………………………………………17

3.1 Synthesis and characterization of the 10BSGRF NPs.....................................17
3.2 In vitro cellular uptake experiments and boron neutron capture therapy using the 10BSGRF NPs…………………………………………………………………23
3.3 In vivo bio-distribution kinetics and MR-imaging-guided boron neutron capture therapy of 10BSGRF NPs.……………………..………………………….29
4 Conclusions.......................................................................................................43
5 References.........................................................................................................46

Chapter 2: Single step synthesis of 10B rich Boron phosphate nanoparticles for effective Boron neutron capture therapy of recurrent head and neck cancer

1 Background and Introduction………………………………………………53

2 Experimental Section………………………………………………………..57
2.1 Synthesis of 10BPO4 NPs.........................…………….……………………..57
2.2 Surface modification of 10BPO4 NPs……….....………...…………………..58
2.3 Synthesis of BPA-Fructose complex solution……………….……….……..58
2.4 Characterization of  10BPO4 NPs…………………………………………....59
2.5 Cell culture……………………………….…………...………………….....59
2.6 Cytotoxicity evaluation of  10BPO4-EGFR NPs.…………...…………….....60
2.7 Calcein-AM and PI staining………...………………...…………………….60
2.8 Invivo BNCT xenograft model with head and neck cancer cell line ….……61
2.9 In vivo biodistribution for 10BPO4 NPs …………………….………………61
2.10 In vivo boron neutron capture therapy.……………………..……………..61
2.11 Histology and Caspase-3 staining …………………..…………………......62

3 Results and Discussion………………………………………………………63

3.1 Synthesis and Characterization of 10BPO4 NPs........................……………..63
3.2 In vitro boron neutron capture therapy with 10BPO4 NPs in head and neck cancer cells.....................……………...…………………………………………..70
3.3 In vivo bio-distribution of 10BPO4 NPs in head and neck cancer implanted mice model.....................……………...…………………………………………..72
3.4 In vivo boron neutron capture therapy model of 10BPO4-anti-EGFR NPs.…74

4 Conclusions.......................................................................................................80

5 References…………………………………………………………………….83

Chapter 3: Plasmonic Field Enhanced NIR Light Absorption, Surface Enhanced Raman Scattering, Dual Modal Photodynamic and Photothermal Therapies in Biological Windows I and II using Gold Nanopeanuts

1 Background and Introduction........................................................................87

2 Experimental Section..………………………………………………………91

2.1 Synthesis of AuNRs ….……………………………………………………..91
2.2  Synthesis of Au-Ag core shell NRs...………………………………………92
2.3 Synthesis of Au NPNs.………………………...…….…………….………..92
2.4 Synthesis of Au NPNs tuned LSPR in the broad NIR region………………93
2.5 Surface modification of AuNPNs……………………...................................93
2.6 Characterization of AuNPNs ……………………………………………….93
2.7 Singlet oxygen phosphorescence measurements …………………………...94
2.8 Calculation of molar extinction coefficient of AuNPNs………………..…..94
2.9 Cell culture methods…………………………….……………………….....94
2.10 Cytotoxicity assays………………………………………………………..95
2.11 Cellular uptake evaluation-CLSM …………..…...…………………….....95
2.12 ROS-in vitro detection ……………..……………………………………...96
2.13 Heat-shock protein in vitro analysis ……….………….………………..…96
2.14 In vivo PDT/PTT …………………………………...…………………..…96
2.15 Histology analysis …….……………………………...……………………97
2.16 In vivo thermal imaging …………………………..………………………97
2.17 Singlet O2 phosphorescence Quantum yield measurements…….…………97
3 Results and Discussion....................................................................................99

3.1 Characteristic investigations of AuNPNs 2.0 and 6.5 nm ……….…………99
3.2 XRD and SERS enhancement….………………………...…………….….105
3.3 In vitro PDT/PTT cellular experiments ..…………………...……….…….110
3.4 Au NPNs- enhanced plasmonic field photodynamic therapy………..…….116
4 Conclusions………………………………………………………………….120

5 References…………………………………………………………………...121

Chapter 4: Lanthanum Hexaboride Nano Platform for Photodynamic Therapy in Hypoxia Mimicking and Intrinsic Drug Resistant Tumors by Generating Oxygen Independent Hydroxyl Radical in Biological Window III with dual MR and CT Imaging
1 Background and Introduction…………….……………………………….126

2 Experimental Section…..…………………………………………………..130

2.1 Preparation of anti-EGFR conjugated LaB6 cubic nanoplates.……………130
2.2 Singlet oxygen phosphorescence measurements..........................................130
2.3 Calculation of molar extinction coefficient of LaB6 CNPs.……………….131
2.4 Cell culture, Cytotoxicity assays ……………….…………………………131
2.5 ROS detection.…………………..…………………...…………………….132
2.6 Heat-shock protein expression analysis …………………………………...132
2.7 In vivo PDT/PTT .…………………………………...…………..………...133
2.8 Histology analysis……………………………………...…………………..133
2.9 In vivo thermal imaging ..……………………………..…………………..134
2.10 T2-MR imaging …………………………………….….…………………134
2.11 CT-imaging ………………………………………….…….……………..134
2.12 EPR measurements …………………………………...………………….135
2.13 Hypoxia in vitro study in biological window III.….…….…………...…..135
2.14 ROS-ID hypoxia detection.…………………..……………………….….136
2.15 Penetration depth in vitro study……..….………………………………..137
2.16 Evidences for anti-h-EGFR targeting on anti-EGFR-LaB6 CNPs ..……..137
2.17 Flow cytometer analysis ..………………………………………………..137
2.18 Confocal imaging ………………………………………………………..138
2.19 DPBF quenching experiments …………………..…….…………………138
2.20 SOSG experiments …………………………….………………….……..139
2.21 Singlet O2 phosphorescence quantum yield ………….………………….139
2.22 Photothermal conversion efficiency ……………....…….…………...…..140
3 Results and Discussion…..…………………………………………………142

3.1 Characterization and Optical Properties of LaB6 CNPs.…………...………145
3.2 Singlet O2 generation in II biological window.…………………………....146
3.3 Hydroxyl radical generation in III biological window in hypoxia and……….. normoxia ……………………………………………………..………………….149
3.4 Surface modification to target NCI-H23 cells ……………………...……..152
3.5 Imaging modality of LaB6 CNPs- MRI and CT imaging………………….154
3.6 In vitro photodynamic and photothermal therapy ……………….…….….160
3.7 Cell viability assay in normoxia ……………………………………….….160
3.8 Cell viability assay in hypoxia ………………………………...……….….164
3.9 Cell viability assay mimicking deeper tissue penetration ………….….….167
3.10 In vitro evaluation of ROS and HSP-70 ……………..………….…….…168
3.11 In vivo photodynamic and photothermal therapy ……………….…….…169
4 Conclusions………………………………………………………………….176

5 References…………………………………………………………………...177

                                

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