腫瘤結合性治療廣泛地應用在生醫領域以及大幅度改善癌症治療效率，我們開發了多功能性聚多巴胺修飾之星形奈米粒子當作藥物載體 (NS-D@PPFA)，應用於標靶結合性治療。星形奈米粒子擁有高光熱轉換能力，可作為光熱試劑的角色。除此之外，聚多巴胺以及HS-PEG-FA的表面修飾，改善了星形奈米粒子在藥物載附及標靶性的能力。在細胞實驗中，NS-D@PPFA比起沒有標靶性的藥物載體更容易藉由受器所引導之胞吞作用進入細胞。此外，經由近紅外光雷射照射下，載體誘導的光熱效果及藥物控制釋放達到化學結合光熱治療的效果，此部分在MCF-7 (人類乳癌細胞)、MCF-7/ADR (抗藥性人類乳癌細胞)及HUVEC (人類臍帶內皮細胞)細胞上都有明顯毒殺的作用。相反地，結合性治療在NIH/3T3及HaCaT等正常細胞下則無明顯細胞毒性，顯示標靶性奈米材料在選擇性的優勢。除此之外，藥物載體對於血管增生因子VEGF有良好的親和力 (Kd = 2.68 × 10-10 M)，此數值相較於一般蛋白質高了約兩個級數。因此，藉由載體對於VEGF專一性吸附，抑制血管新生下游端訊號的活化。細胞實驗也證明在有藥物載體的條件下，可以有效抑制VEGF所誘導的血管新生途徑，同時達到抗腫瘤結合抗血管新生之效果。整體來說，此研究開發多功能藥物載體透過多樣結合性方法達到有效治療多重抗藥性腫瘤的效果。

Cancer combination therapy can improve treatment efficacy and have been widely utilized in the biomedical field. In this study, we proposed a facile strategy to develop a multifunctional polydopamine coated gold nanostar as a chemodrug carrier (NS-D@PPFA) for cancer targeting and combination therapy. Gold nanostar (NS) shows high photothermal conversion efficiency due to a tip-enhanced plasmonic effect. The modification of polydopamine and folic acid (HS-PEG-FA) on NS surface also improves the drug loading efficiency and targeting capability. In vitro, NS-D@PPFA can be easily uptake by targeted breast cancer MCF-7 cells relative to non-targeted cells through a receptor-mediated endocytosis pathway. In combination with the photo-thermal effect induced by near infrared (NIR) laser irradiation, controlled payload release can be activated in response to both internal (acid) and external (photothermal) stimuli, leading to an efficient chemo-photothermal action against MCF-7 cells, drug-resistant MCF-7/ADR and Human Umbilical Vein Endothelial Cells (HUVEC). Conversely, cellular damage is less obvious in the cases of normal HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts). In addition, payload-free NS@PPFA also exhibits high binding affinity (Kd = 2.68 × 10-10 M) toward vascular endothelial growth factor (VEGF-A165), which is at least two orders of magnitude stronger than that of high abundant plasma proteins such as human serum albumin. In vitro study further demonstrated that NS@PPFA can effectively inhibit the VEGF-A165-induced proliferation, migration and tube formation of HUVECs, resulting in additional therapeutic benefits in combating tumor via a simultaneous anti-angiogenic action in chemo-photothermal treatment. Overall, our “all-in-one” nanoplatform is highly promising for potential tumor therapy, allowing effective treatment against multidrug resistant cancers.

Abstract I
Chapter 1. Introduction 1
1.1 Nano-technology and biomedical application 1
1.1.1 Introduction of Nanomaterials 1
1.1.2 Nanocarriers 1
1.1.3 Stimuli-responsive nanocarriers 2
1.2 Cancer therapy and its application 5
1.2.1 Introduction of cancer 5
1.2.2 Chemo-photothermal therapy 6
1.2.3 Antiangiogenic therapy 8
1.2.4 Antitumor combined antiangiogenic therapy 10
1.3 Polydopamine and gold nanostar applied in cancer therapy 11
1.3.1 Gold nanostar 11
1.3.2 Polydopamine 12
1.3.3 The application of Au@PDA nanocomposites in drug delivery 14
1.4 Research motivation and purpose 15
Chapter 2. Experimental Method 17
2.1 Material and instrument 17
2.1.1 Experiential agent 17
2.1.2 Instrument 17
2.2 Synthesis and characterization of NS-D@PPFA 18
2.2.1 Synthesis of NS 18
2.2.2 Preparation of NS-D@P 18
2.2.3 Preparation of NS-D@PPFA 19
2.2.4 Characterization of NS-D@PPFA. 19
2.3 Properties of NS-D@PPFA in buffer solution 20
2.3.1 Long-termed stability of NS-D@PPFA 20
2.3.2 Photothermal effect of NS-D@PPFA 20
2.3.3 DOX release of NS-D@PPFA 20
2.4 The interaction of NS-D@PPFA toward MCF-7 cells 21
2.4.1 Cell culture 21
2.4.2 Cellular uptake of NS-D@PPFA 21
2.4.3 Intracellular DOX release of NS-D@PPFA 22
2.4.4 Cytotoxicity of NS-D@PPFA 22
2.4.5 The evaluation of cell recurrent 23
2.4.6 Cell apoptosis assay of NS-D@PPFA 23
2.5 The interaction of NS@PPFA toward HUVEC cells 24
2.5.1 Adsorption of VEGF on NS@PPFA 24
2.5.2 Adsorption of BSA on NS@PPFA 24
2.5.3 Proliferation assay of HUVEC cells 25
2.5.4 Migration assay of HUVEC cells 25
2.5.5 Tube formation assay of HUVEC cells 26
Chapter 3. Result and Discussion 27
3.1 Synthesis and characterization of NS-D@PPFA 27
3.1.1 Synthesis of NS-D@PPFA 27
3.1.2 Characterization of NS-D@PPFA 27
3.2 Properties of NS-D@PPFA in buffer solution 29
3.2.1 Photothermal effect of NS-D@PPFA 29
3.2.2 Drug release of NS-D@PPFA 30
3.3 The interaction of NS-D@PPFA toward breast cancer MCF-7 cells 31
3.3.1 Cellular uptake of NS-D@PPFA 31
3.3.2 Intracellular DOX release 32
3.3.3 Cytotoxicity of NS-D@PPFA 33
3.4 The interaction of NS-D@PPFA toward HUVEC cells 34
3.4.1 Cellular toxicity of NS-D@PPFA toward HUVEC cells 34
3.4.2 Antiangiogenic effect of NS-D@PPFA toward HUVEC cells 35
3.4.3 Binding affinity of NS@PPFA toward VEGF 36
Chapter 4. Conclusion 38
Figure Caption 39
Reference 64

Reference

1. Ferrari, M., Cancer nanotechnology: opportunities and challenges. Nature reviews cancer, 2005. 5(3): p. 161.
2. Koo, O.M., I. Rubinstein, and H. Onyuksel, Role of nanotechnology in targeted drug delivery and imaging: a concise review. Nanomedicine: Nanotechnology, Biology and Medicine, 2005. 1(3): p. 193-212.
3. Car, A., et al., pH-Responsive PDMS-b-PDMAEMA Micelles for Intracellular Anticancer Drug Delivery. Biomacromolecules, 2014. 15(9): p. 3235-3245.
4. Tomlinson, R., et al., Polyacetal− doxorubicin conjugates designed for ph-dependent degradation. Bioconjugate chemistry, 2003. 14(6): p. 1096-1106.
5. Ge, Z. and S. Liu, Functional block copolymer assemblies responsive to tumor and intracellular microenvironments for site-specific drug delivery and enhanced imaging performance. Chemical Society Reviews, 2013. 42(17): p. 7289-7325.
6. Lee, S.H., et al., Current Progress in Reactive Oxygen Species (ROS)‐Responsive Materials for Biomedical Applications. Advanced healthcare materials, 2013. 2(6): p. 908-915.
7. Li, Z.-Y., et al., A redox-responsive drug delivery system based on RGD containing peptide-capped mesoporous silica nanoparticles. Journal of Materials Chemistry B, 2015. 3(1): p. 39-44.
8. Huang, X., et al., Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. Journal of the American Chemical Society, 2006. 128(6): p. 2115-2120.
9. Yuan, H., A.M. Fales, and T. Vo-Dinh, TAT peptide-functionalized gold nanostars: enhanced intracellular delivery and efficient NIR photothermal therapy using ultralow irradiance. Journal of the American Chemical Society, 2012. 134(28): p. 11358-11361.
10. Kawano, T., et al., PNIPAM gel-coated gold nanorods for targeted delivery responding to a near-infrared laser. Bioconjugate chemistry, 2009. 20(2): p. 209-212.
11. Shi, J., et al., A tumor-targeting near-infrared laser-triggered drug delivery system based on GO@ Ag nanoparticles for chemo-photothermal therapy and X-ray imaging. Biomaterials, 2014. 35(22): p. 5847-5861.
12. Cho, H.J., M. Chung, and M.S. Shim, Engineered photo-responsive materials for near-infrared-triggered drug delivery. Journal of Industrial and Engineering Chemistry, 2015. 31: p. 15-25.
13. Peer, D., et al., Nanocarriers as an emerging platform for cancer therapy. Nature nanotechnology, 2007. 2(12): p. 751.
14. Sharma, A. and U.S. Sharma, Liposomes in drug delivery: progress and limitations. International journal of pharmaceutics, 1997. 154(2): p. 123-140.
15. Trédan, O., et al., Drug resistance and the solid tumor microenvironment. Journal of the National Cancer Institute, 2007. 99(19): p. 1441-1454.
16. Zhang, L., et al., A multifunctional platform for tumor angiogenesis-targeted chemo-thermal therapy using polydopamine-coated gold nanorods. ACS nano, 2016. 10(11): p. 10404-10417.
17. Wang, X., et al., Multi-responsive photothermal-chemotherapy with drug-loaded melanin-like nanoparticles for synergetic tumor ablation. Biomaterials, 2016. 81: p. 114-124.
18. Weis, S.M. and D.A. Cheresh, Tumor angiogenesis: molecular pathways and therapeutic targets. Nature medicine, 2011. 17(11): p. 1359.
19. Ellis, L.M. and D.J. Hicklin, VEGF-targeted therapy: mechanisms of anti-tumour activity. Nature reviews cancer, 2008. 8(8): p. 579.
20. Folkman, J., Tumor angiogenesis: therapeutic implications. New england journal of medicine, 1971. 285(21): p. 1182-1186.
21. Yu, D.-H., et al., IF7-Conjugated nanoparticles target annexin 1 of tumor vasculature against P-gp mediated multidrug resistance. Bioconjugate chemistry, 2015. 26(8): p. 1702-1712.
22. Takahashi, H. and M. Shibuya, The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clinical science, 2005. 109(3): p. 227-241.
23. Fukumura, D. and R.K. Jain, Tumor microvasculature and microenvironment: targets for anti-angiogenesis and normalization. Microvascular research, 2007. 74(2-3): p. 72-84.
24. Bartczak, D., et al., Manipulation of in vitro angiogenesis using peptide-coated gold nanoparticles. Acs Nano, 2013. 7(6): p. 5628-5636.
25. Mukherjee, P., et al., Antiangiogenic properties of gold nanoparticles. Clinical Cancer Research, 2005. 11(9): p. 3530-3534.
26. Lai, P.-X., et al., Ultrastrong trapping of VEGF by graphene oxide: anti-angiogenesis application. Biomaterials, 2016. 109: p. 12-22.
27. Day, E.S., et al., Vascular-targeted photothermal therapy of an orthotopic murine glioma model. Nanomedicine, 2012. 7(8): p. 1133-1148.
28. Shi, S., et al., VEGFR targeting leads to significantly enhanced tumor uptake of nanographene oxide in vivo. Biomaterials, 2015. 39: p. 39-46.
29. Lv, L., et al., Enhanced antiglioblastoma efficacy of neovasculature and glioma cells dual targeted nanoparticles. Molecular pharmaceutics, 2016. 13(10): p. 3506-3517.
30. Dahmani, F.Z., et al., Multifunctional Polymeric Nanosystems for Dual‐Targeted Combinatorial Chemo/Antiangiogenesis Therapy of Tumors. Advanced healthcare materials, 2016. 5(12): p. 1447-1461.
31. Yao, J., et al., Neovasculature and circulating tumor cells dual-targeting nanoparticles for the treatment of the highly-invasive breast cancer. Biomaterials, 2017. 113: p. 1-17.
32. Chen, D., et al., Dual targeting luminescent gold nanoclusters for tumor imaging and deep tissue therapy. Biomaterials, 2016. 100: p. 1-16.
33. Li, X., et al., Tumor vascular-targeted co-delivery of anti-angiogenesis and chemotherapeutic agents by mesoporous silica nanoparticle-based drug delivery system for synergetic therapy of tumor. International journal of nanomedicine, 2016. 11: p. 93.
34. Xu, B., et al., Combined tumor-and neovascular-“dual targeting” gene/chemo-therapy suppresses tumor growth and angiogenesis. ACS applied materials & interfaces, 2016. 8(39): p. 25753-25769.
35. Yuan, H., et al., Gold nanostars: surfactant-free synthesis, 3D modelling, and two-photon photoluminescence imaging. Nanotechnology, 2012. 23(7): p. 075102.
36. Xie, J., J.Y. Lee, and D.I. Wang, Seedless, surfactantless, high-yield synthesis of branched gold nanocrystals in HEPES buffer solution. Chemistry of materials, 2007. 19(11): p. 2823-2830.
37. Hao, F., et al., Plasmon resonances of a gold nanostar. Nano letters, 2007. 7(3): p. 729-732.
38. Link, S. and M.A. El-Sayed, Shape and size dependence of radiative, non-radiative and photothermal properties of gold nanocrystals. International reviews in physical chemistry, 2000. 19(3): p. 409-453.
39. Chirico, G., M. Borzenkov, and P. Pallavicini, Gold nanostars: synthesis, properties and biomedical application. 2015: Springer.
40. Hong, S., et al., Non‐covalent self‐assembly and covalent polymerization co‐contribute to polydopamine formation. Advanced Functional Materials, 2012. 22(22): p. 4711-4717.
41. Liu, Y., K. Ai, and L. Lu, Polydopamine and its derivative materials: synthesis and promising applications in energy, environmental, and biomedical fields. Chemical reviews, 2014. 114(9): p. 5057-5115.
42. Miao, Z.-H., et al., Intrinsically Mn2+-chelated polydopamine nanoparticles for simultaneous magnetic resonance imaging and photothermal ablation of cancer cells. ACS applied materials & interfaces, 2015. 7(31): p. 16946-16952.
43. Tao, W., et al., Polydopamine-based surface modification of novel nanoparticle-aptamer bioconjugates for in vivo breast cancer targeting and enhanced therapeutic effects. Theranostics, 2016. 6(4): p. 470.
44. Zhuang, H., et al., Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic Imaging and Chemo-Photothermal Synergistic Therapy. ACS Biomaterials Science & Engineering, 2017. 3(8): p. 1799-1808.
45. Jin, Z. and H. Fan, The modulation of melanin‐like materials: methods, characterization and applications. Polymer International, 2016. 65(11): p. 1258-1266.
46. Mrówczyński, R., Polydopamine-based multifunctional (nano) materials for cancer therapy. ACS applied materials & interfaces, 2017. 10(9): p. 7541-7561.
47. Kumar, A., et al., Oxidative nanopeeling chemistry-based synthesis and photodynamic and photothermal therapeutic applications of plasmonic core-petal nanostructures. Journal of the American Chemical Society, 2014. 136(46): p. 16317-16325.
48. Wang, S., et al., Biologically inspired polydopamine capped gold nanorods for drug delivery and light-mediated cancer therapy. ACS applied materials & interfaces, 2016. 8(37): p. 24368-24384.
49. Yu, Y., et al., Ultrasmall dopamine-coated nanogolds: preparation, characteristics, and CT imaging. Journal of experimental nanoscience, 2016. 11(sup1): p. S1-S11.
50. Zeng, Y., et al., Lipid-AuNPs@ PDA nanohybrid for MRI/CT imaging and photothermal therapy of hepatocellular carcinoma. ACS applied materials & interfaces, 2014. 6(16): p. 14266-14277.
51. Wang, F., et al., Polydopamine‐Functionalized Graphene Oxide Loaded with Gold Nanostars and Doxorubicin for Combined Photothermal and Chemotherapy of Metastatic Breast Cancer. Advanced healthcare materials, 2016. 5(17): p. 2227-2236.
52. Black, K.C., et al., Polydopamine-enabled surface functionalization of gold nanorods for cancer cell-targeted imaging and photothermal therapy. Nanomedicine, 2013. 8(1): p. 17-28.
53. Li, D., et al., Construction of polydopamine-coated gold nanostars for CT imaging and enhanced photothermal therapy of tumors: an innovative theranostic strategy. Journal of Materials Chemistry B, 2016. 4(23): p. 4216-4226.
54. Du, B., et al., Hybrid of gold nanostar and indocyanine green for targeted imaging-guided diagnosis and phototherapy using low-density laser irradiation. Journal of Materials Chemistry B, 2016. 4(35): p. 5842-5849.
55. Li, C., Z. Liu, and P. Yao, Gold nanoparticles coated with a polydopamine layer and dextran brush surface for diagnosis and highly efficient photothermal therapy of tumors. RSC Advances, 2016. 6(39): p. 33083-33091.
56. Tian, Y., et al., Mussel‐Inspired Gold Hollow Superparticles for Photothermal Therapy. Advanced healthcare materials, 2015. 4(7): p. 1009-1014.
57. de Puig, H., et al., Extinction coefficient of gold nanostars. The Journal of Physical Chemistry C, 2015. 119(30): p. 17408-17415.
58. Cheng, W., et al., pH-Sensitive delivery vehicle based on folic acid-conjugated polydopamine-modified mesoporous silica nanoparticles for targeted cancer therapy. ACS applied materials & interfaces, 2017. 9(22): p. 18462-18473.
59. Gref, R., et al., The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres. Advanced drug delivery reviews, 1995. 16(2-3): p. 215-233.
60. Dam, D.H.M., R.C. Lee, and T.W. Odom, Improved in vitro efficacy of gold nanoconstructs by increased loading of G-quadruplex aptamer. Nano letters, 2014. 14(5): p. 2843-2848.
61. Liu, X., et al., Mussel-inspired polydopamine: a biocompatible and ultrastable coating for nanoparticles in vivo. ACS nano, 2013. 7(10): p. 9384-9395.
62. Nam, J., et al., Chemo-photothermal therapy combination elicits anti-tumor immunity against advanced metastatic cancer. Nature communications, 2018. 9(1): p. 1074.
63. Liu, Q., et al., Highly selective uptake and release of charged molecules by pH‐responsive polydopamine microcapsules. Macromolecular bioscience, 2011. 11(9): p. 1227-1234.
64. Maruyama, K., Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Advanced drug delivery reviews, 2011. 63(3): p. 161-169.
65. Poinard, B., et al., Polydopamine Nanoparticles Enhances Drug Release for Combined Photodynamic and Photothermal Therapy. ACS applied materials & interfaces, 2018.
66. Della Vecchia, N.F., et al., Building‐block diversity in polydopamine underpins a multifunctional eumelanin‐type platform tunable through a quinone control point. Advanced Functional Materials, 2013. 23(10): p. 1331-1340.
67. Liu, F., et al., Facile Preparation of Doxorubicin‐Loaded Upconversion@ Polydopamine Nanoplatforms for Simultaneous In Vivo Multimodality Imaging and Chemophotothermal Synergistic Therapy. Advanced healthcare materials, 2015. 4(4): p. 559-568.
68. Dong, Z., et al., Polydopamine nanoparticles as a versatile molecular loading platform to enable imaging-guided cancer combination therapy. Theranostics, 2016. 6(7): p. 1031.
69. Wang, X., et al., A polydopamine nanoparticle-knotted poly (ethylene glycol) hydrogel for on-demand drug delivery and chemo-photothermal therapy. Chemistry of Materials, 2017. 29(3): p. 1370-1376.
70. Cui, J., et al., Immobilization and intracellular delivery of an anticancer drug using mussel-inspired polydopamine capsules. Biomacromolecules, 2012. 13(8): p. 2225-2228.
71. Zhang, R., et al., Smart micelle@ polydopamine core–shell nanoparticles for highly effective chemo–photothermal combination therapy. Nanoscale, 2015. 7(46): p. 19722-19731.
72. Wei, Y., et al., Polydopamine and peptide decorated doxorubicin-loaded mesoporous silica nanoparticles as a targeted drug delivery system for bladder cancer therapy. Drug delivery, 2017. 24(1): p. 681-691.
73. Ochs, C.J., et al., Dopamine-mediated continuous assembly of biodegradable capsules. Chemistry of Materials, 2011. 23(13): p. 3141-3143.
74. Belitsky, J.M., et al., Small molecule modulators of aggregation in synthetic melanin polymerizations. Bioorganic & medicinal chemistry letters, 2012. 22(17): p. 5503-5507.
75. Zhong, X., et al., Polydopamine as a biocompatible multifunctional nanocarrier for combined radioisotope therapy and chemotherapy of cancer. Advanced Functional Materials, 2015. 25(47): p. 7327-7336.
76. Li, Y., et al., Targeted polydopamine nanoparticles enable photoacoustic imaging guided chemo-photothermal synergistic therapy of tumor. Acta biomaterialia, 2017. 47: p. 124-134.
77. Jordans, S., et al., Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions. BMC biochemistry, 2009. 10(1): p. 23.
78. Xing, Y., et al., Mesoporous polydopamine nanoparticles with co-delivery function for overcoming multidrug resistance via synergistic chemo-photothermal therapy. Nanoscale, 2017. 9(25): p. 8781-8790.
79. Kusano, R., et al., α-Amylase and lipase inhibitory activity and structural characterization of acacia bark proanthocyanidins. Journal of natural products, 2010. 74(2): p. 119-128.
80. Jeong, Y., et al., Sprayable Adhesive Nanotherapeutics: Mussel Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy. ACS nano, 2018.
81. Fu, X., et al., IONP-doped nanoparticles for highly effective NIR-controlled drug release and combination tumor therapy. International journal of nanomedicine, 2017. 12: p. 3751.
82. Pan, J. and S.-S. Feng, Targeting and imaging cancer cells by folate-decorated, quantum dots (QDs)-loaded nanoparticles of biodegradable polymers. Biomaterials, 2009. 30(6): p. 1176-1183.
83. Kang, M.J., et al., Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation. International journal of nanomedicine, 2013. 8: p. 1155.
84. Yang, K.K., et al., Folate-modified–chitosan-coated liposomes for tumor-targeted drug delivery. Journal of Materials Science, 2013. 48(4): p. 1717-1728.
85. Hasanpourghadi, M., et al., Mechanisms of the anti-tumor activity of Methyl 2-(-5-fluoro-2-hydroxyphenyl)-1 H-benzo [d] imidazole-5-carboxylate against breast cancer in vitro and in vivo. Oncotarget, 2017. 8(17): p. 28840.
86. Tomankova, K., et al., In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines. International journal of nanomedicine, 2015. 10: p. 949.
87. Dang, Y., et al., Substrate independent coating formation and anti-biofouling performance improvement of mussel inspired polydopamine. Journal of materials chemistry B, 2015. 3(20): p. 4181-4190.
88. Zaccaria, A., et al., Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition. International journal of nanomedicine, 2015. 10: p. 1869.