熱治療(photothermal therapy, PTT)在癌症治療具有空間與時間精準控制的優勢。此外，光熱效應也可應用於光觸發藥物釋放，因此光熱治療目前在全球臨床醫療與基礎研究上廣為探討與應用。然而，單一光熱治療癌症效果有限，需面臨腫瘤復發的風險；此外，光熱治療藥物的傳遞也受限無法深入腫瘤內部而降低其治療癌症效果。為了克服以上缺點，本論文針對光熱治療提出三種新型藥物傳遞系統，分別探討(1)光熱治療合併化療、(2)合併光動力治療(photodynamic therapy, PDT)與(3)結合生物膜偽裝策略，對於惡性腫瘤之治療效果。
為了增進單一光熱治療於癌症治療之效果，本論文第一項研究為開發一種結合光熱治療與化療之光觸發藥物傳遞系統。將金奈米棒(gold nanorod, AuNR)光熱試劑和阿黴素抗癌藥物(doxorubicin, Dox)透過單層乳化法共同包覆於聚乳酸甘醇酸(poly(lactic-co-glycolic acid), PLGA)核心。為了增進載體穩定度，粒子表面透過逐層修飾法修飾生物可降解的高分子(bioreducible polyethylenimine, SPEI)與具腫瘤標靶性的人類血清白蛋白(human serum albumin, HSA)，以形成HAS/AuNR/Dox-PLGA (HADP)奈米複合物。在給予808 nm雷射照射後，HADP奈米複合物所產生之光熱效應，可加速PLGA載體內之Dox釋放。在體外CT26大腸癌細胞實驗證實，HADP不僅展現低毒性，還能夠透過表面HSA修飾而增進癌細胞對其之攝取。在CT26動物腫瘤模型證實，與free Dox組別相比較，HADP在給予雷射照射後，展現優異的抑制腫瘤生長效果。
腫瘤高內壓與緻密細胞間質環境降低奈米藥物載體對於腫瘤之深入性，因此限制癌症治療效果。著眼於此問題，本論文第二項研究設計透過具腫瘤趨向性與滲透性之幹細胞作為DDS並結合PTT/PDT治療，以提升奈米藥物對於腫瘤之深入能力。為此，我們提出以脂肪幹細胞(adipose-derived stem cells, ADSC)作為金奈米棒(AuNR)-聚乙二醇(polyethylene glycol, PEG)-聚乙烯亞胺(polyethylenimine, PEI) (APP)/Chlorin e6 (Ce6)攜載的新型傳遞系統。APP/Ce6展現低藥物洩漏性、低細胞毒性、良好細胞攝取效率與具細胞內氧化還原反應性釋放的優勢。裝載APP/Ce6的ADSC仍保持良好的腫瘤趨向性，並能夠穿透入三維腫瘤球內部。在給予近紅外808 nm光照可觸發光熱效應，進一步促進Ce6從幹細胞向周圍釋放，增進其治療癌細胞效果。裝載APP/Ce6的ADSC透過預期的光熱和光動力作用可發揮有效的癌細胞殺傷效果。在小鼠CT26腫瘤模型中證實，裝載APP/Ce6的ADSC比APP/Ce6具有更好的抑制腫瘤生長效果。此外，由組織切片結果觀察APP/Ce6的ADSC於注射後並未對體內正常器官造成明顯之損害。
藥物載體表面性質對其腫瘤累積與治療效果扮演著重要角色。因此，本論文第三項研究目標探討製備具長循環能力與腫瘤標靶性之生物膜偽裝奈米光熱藥物，以期將來可應用於癌症治療。本研究中透過簡單的超音波分散作用將氟碳化之超順磁氧化奈米鐵粒子與幹細胞膜透過自組裝作用，形成幹細胞膜包覆之光熱/磁性奈米液滴，其具有適合癌細胞攝取之粒徑(288 nm)與表面電位(-30 mV)。此生物膜奈米液滴核心包含超順磁氧化鐵奈米粒子與IR780光熱藥劑。所製備之粒子具有良好分散性、高細胞相容性與可重複性光熱效應等特點。可透過磁導引提升載體於腫瘤累積量，續以光熱作用有效殺傷癌細胞。由細胞攝取實驗結果證實，生物膜奈米液滴不僅能夠降低巨噬細胞(RAW264.7)對其之攝取，且能為小鼠大腸癌細胞(CT26)所攝取。

Photothermal therapy (PTT) treat cancers in a spatial- and temporal-accurate manner. In addition, photothermal effects can be applied to achieve photo-responsive drug release. The potentials of PTT on clinical and basic research are actively explored. However, single PTT treatment is usually not sufficient to eliminate tumors completely. Besides, the poor tumor penetration of photothermal drugs limited their therapeutic effects. To address these challenges, three drug delivery strategies for cancer therapy were proposed in this dissertation, including: (1) combination of PTT with chemotherapy, (2) cell-based delivery of combined PTT and photodynamic therapy (PDT), and (3) a novel biomembrane camouflage nanocomplexes system.
In order to improve the therapeutic effect of cancer in single PTT, the first part of this dissertation is to develop a phototriggerable drug delivery system (DDS) for combined PTT and chemotherapy. The gold nanorod (AuNR) as a photothermal agent and doxorubicin (Dox) were encapsulated into the core of poly(lactic-co-glycolic acid (PLGA) through a single emulsion process. To improve the colloidal stability, the surface of nanoparticles was modified with bioreducible polyethylenimine (SPEI) and tumor-targeting human serum albumin (HSA) via a layer-by-layer process to form HSA/AuNR/Dox-PLGA (HADP) nanocomplexes. After 808 nm laser irradiation, the photothermal effect accelerated Dox release from the HADP nanocomplexes. In vitro study confirmed that HADP exhibited not only low cytotoxicity but also efficient cellular uptake via the interactions between HSA and the recep¬tor on cancer cells. In vivo study demonstrated that the combined PTT and chemotherapy of HADP exhibited excellent tumor growth suppression on a mouse colon cancer model.
The high interstitial pressure and dense extracellular matrix in tumors obstruct the delivery and penetration of DDS, thereby limiting the therapeutic effects. In the second part of this dissertation, the tumor-tropic and penetrable stem cells were utilized as DDS for combined PTT/PDT on cancer treatment. To this end, the adipose-derived stem cells (ADSC) loaded with AuNR-polyethylene glycol (PEG)-polyethylenimine (PEI) (APP) /Chlorin e6 (Ce6) was proposed. The APP/Ce6 nanocomplexes exhibited low drug leakage, low cytotoxicity, good cellular uptake, and redox-responsive drug release. The APP/Ce6- loaded ADSCs maintained respectable tumor tropism and tumor penetration into the interior of the three-dimensional tumor spheroids. After 808 nm laser irradiation, the generated photothermal effect promoted Ce6 release from ADSC to the surroundings, thereby enhancing the anti-cancer effect. The APP/Ce6-loaded ADSCs exerted an effective cancer-killing effect through the expected photothermal and photodynamic effects in vitro. The in vivo study confirmed that APP/Ce6-loaded ADSCs displayed the superior tumor growth suppression than APP/Ce6 nanocomplexes without any noticeable detrimental effects on normal tissues/organs.
The surface modification of DDS plays a vital role in tumor accumulation and therapeutic effects. In the third part of this dissertation, a biomembrane camouflage strategy was attempted to formulate long-circulating and tumor targeting DDS for PTT on cancer treatment. By a simple ultrasonication procedure, the IR780-loaded-fluorocarbonized superparamagnetic iron oxide nanoparticles (SPION) and stem cell membranes were self-assembled to form stem cell membrane-camouflaged IR780-SPION nanodroplets. The size and surface potential of the nanodroplets were 288 nm and -30 mV, respectively. The nanodroplets displayed excellent colloidal stability, good cell compatibility, and repeatable photothermal effects. In vitro results confirmed that the nanodroplets could by efficiently taken by colorectal cancer cells (CT26) but not macrophages (RAW264.7).

中文摘要 i
Abstract iii
致謝 v
Table of contents vi
List of Figure x
List of tables xiii
1. Introduction 1
1.1. Background 1
1.2. Motivation and Specific Aim 4
2. Literature reviews 12
2.1. Introduction 12
2.2. Drug delivery system for photothermal therapy (PTT) 18
2.1.1. Phospholipid based nanoparticles for PTT 21
2.1.2. Polymeric nanoparticles for PTT 27
2.1.3. Inorganic nanoparticles 33
2.1.4. Cell membrane camouflaged nanoparticles 37
2.3. Drug delivery system for photodynamic therapy (PDT) 42
2.3.1. Phospholipid based nanoparticles for PDT 44
2.3.2. Polymeric nanoparticles for PDT 48
2.3.3. Inorganic nanoparticles for PDT 52
2.3.4. Cell membrane camouflaged nanoparticles for PDT 56
2.4. Drug delivery system for combination of PTT/PDT 59
2.4.1. Single laser irradiation 59
2.4.2. Double laser irradiation 66
2.5. Cell based delivery system for phototherapy 72
2.5.1. Macrophage 75
2.5.2. Stem cell 76
2.6. Conclusion 80
3. Gold Nanorods-Encapsulated Biodegradable Polymeric Matrix for Combined Photothermal and Chemo Cancer Therapy 81
3.1. Abstract 82
3.2. Introduction 83
3.3. Materials and methods 85
3.3.1. Materials 85
3.3.2. AuNRs synthesis and surface modification 86
3.3.3. Preparation of AuNR/DOX/PLGA (ADP) nanoparticles 86
3.3.4. HSA modification and characterization 87
3.3.5. Characterizations and calculation of drug loading content 87
3.3.6. Photothermal effects of HADP 88
3.3.7. Photothermal effect on triggered DOX release 88
3.3.8. Cell culture 89
3.3.9. Cellular uptake of HADP nanoparticles by cancer cells 89
3.3.10. In vitro cytotoxicity assays 89
3.3.11. In vivo photothermal effect on tumor 90
3.3.12. In vivo therapeutic efficacy of HADP with laser treatment 90
3.3.13. Statistical analysis 91
3.4. Results and Discussion 92
3.4.1. Preparation and characterizations of hydrophobic AuNRs 92
3.4.2. Preparation and characterizations of ADP nanoparticles 92
3.4.3. Preparation and characterizations of HADP nanoparticles 93
3.4.4. Photothermal effect of HADP 94
3.4.5. Photothermal effect on triggered Dox release 94
3.4.6. In vitro cellular uptake 95
3.4.7. In vitro anti-cancer effects 95
3.4.8. Therapeutic effects of HADP on animal cancer model 97
3.5. Conclusion 98
4. Stem Cell-Based Delivery of Gold/Chlorin e6 Nanocomplexes for Combined Photothermal and Photodynamic Therapy 111
4.1. Abstract 112
4.2. Introduction 113
4.3. Materials and methods 116
4.3.1. Materials 116
4.3.2. Synthesis of Polymer PEG-SH and PEI-SH 116
4.3.3. Gold nanorods synthesis and characterization 117
4.3.4. Preparation of PEG-PEI-AuNR /Ce6 Nanocomplex 118
4.3.5. Characterizations of photosensitizer Ce6 loaded on APP/Ce6 119
4.3.6. Investigation of the photothermal effect of APP/Ce6 nanocomplex 120
4.3.7. ROS generation 120
4.3.8. Cellular uptake of APP/Ce6 nanocomplexes 120
4.3.9. In vitro photothermal/photodynamic effects 121
4.3.10. Co-culture of CT26 cancer cells with APP/Ce6 loaded ADSC Radiation dosimetry estimation 121
4.3.11. In Vitro migration ability of APP/Ce6 loaded ADSCs 122
4.3.12. Study the effect of 3D cultured APP/Ce6 loaded ADSC on cell size and cell viability 122
4.3.13. In vivo photothermal therapeutic efficacy of APP/Ce6-loaded stem cells 122
4.3.14. Tumor growth inhibition 123
4.3.15. In vivo compatibility of APP/Ce6-loaded ADSC 124
4.3.16. Statistical analysis 124
4.4. Results and Discussion 125
4.4.1. Synthesis and characterization of APP/Ce6 nanocomplexes 125
4.4.2. Cellular uptake of APP/Ce6 nanocomplexes and it impacts on tumor tropism 127
4.4.3. In vitro cancer cell-killing effects by APP-Ce6/ADSCs 128
4.4.4. Therapeutic effects of APP/Ce6-loaded ADSCs on the animal cancer model Radiation dosimetry 129
4.5. Discussion 131
5. Biomimetic Stem Cell Membrane-coated Phase Change Droplets for Cancer Photothermal Therapy 142
5.1 Abstract 143
5.2 Introduction 144
5.3 Materials and Methods 146
5.3.1. Materials 146
5.3.2. Synthesis of SPIONs 146
5.3.3. Synthesis of SPION-TEOS/IR780 147
5.3.4. Synthesis of SPION-TEOS/IR780-F 147
5.3.5. Synthesis of stem cell membrane camouflaged SPION-TEOS/IR780-F nanodroplets (STM/SI-F NDs) 148
5.3.6. Photothermal effect of STM/SI NDs 149
5.3.7. Protein retention by SDS-PAGE 149
5.3.8. Cellular uptake and cytotoxicity of STM/SI NDs 149
5.3.9. Statistical analysis 150
5.4. Results & Discussion 151
5.4.2. Synthesis and Characterization of Stem cell membrane/SPION/ IR780-F nanodroplets 153
5.4.3. In vitro study for Stem cell membrane/SPION/IR780-F nanodroplets 155
5.5. Conclusion 156
6. Conclusion 164
6.1. Gold Nanorods-Encapsulated Biodegradable Polymeric Matrix for Combined Photothermal and Chemo Cancer Therapy 164
6.2. Stem Cell-Based Delivery of Gold/Chlorin e6 Nanocomplexes for Combined Photothermal and Photodynamic Therapy 164
6.3. Biomimetic Stem Cell Membrane (STM) Camouflaged Phase Change Nanodroplets for Photothermal Therapy 165
7. Future work and Perspective 166
7.1. Gold Nanorods-Encapsulated Biodegradable Polymeric Matrix for Combined Photothermal and Chemo Cancer Therapy 166
7.2. Stem Cell-Based Delivery of Gold/Chlorin e6 Nanocomplexes for Combined Photothermal and Photodynamic Therapy 166
7.3. Biomimetic Stem Cell Membrane (STM) Camouflaged Phase Change Nanodroplets for Photothermal Therapy 167
8. References 168

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