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研究生:郭婉如
研究生(外文):Wan-Ju Kuo
論文名稱:載有光敏藥物之載體應用於癌症治療之研究
論文名稱(外文):Study on photosensitizer loaded drug vehicles for cancer treatment
指導教授:劉澤英
指導教授(外文):Tse-Ying Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:52
中文關鍵詞:二氧化矽原紫質IX光動力療法
外文關鍵詞:silicaprotoporphyrin IXphotodynamic therapy
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本研究以一種簡單的方法製備出載有光敏藥物 (Protoporphyrin IX, PpIX) 之二氧化矽奈米球,並利用穿透式電子顯微鏡、動態粒徑分析法、表面電位測量、吸收/螢光光譜和雷射掃描共軛焦顯微鏡進行載體性質的分析,有趣的是我們發現當以365 nm或是555 nm進行激發時,載體可以增強PpIX的螢光性質,此外,載體進入細胞後可與粒線體及溶酶體共定位 (co-localize) 。本研究以載有PpIX之二氧化矽奈米球進行光動力療法的治療效益之探討,從細胞存活率的結果顯示,在光動力治療上,因載體改善了PpIX的疏水特性,以及載體表面帶正電這兩項因素都使得載體能大量累積於細胞內,而達到比純藥物更好的治療效果。在In vivo的實驗中,驗證了載有PpIX之二氧化矽奈米球確實能發揮EPR (enhanced permeability and retention) 效應,有效的累積在腫瘤部位,表示本研究製備之載體有潛力應用於腫瘤的治療。
A facile method was developed to synthesize protoporphyrin IX (PpIX) loaded silica nanospheres, and transmission electron microscopy (TEM), dynamic light scattering (DLS) method, zeta potential measurements, absorbance / fluorescence spectrums and laser scanning confocal microscope were used to characterize the vehicles. Interestingly, we found that PpIX loaded silica nanospheres could enhance the fluorescence of PpIX as the excitation wavelength at 365 nm or 555 nm, and moreover, PpIX loaded silica nanospheres would co-localize with mitochondria and lysosomes after cellular internalization. In this study, we investigated the therapeutic effects of PpIX loaded silica nanospheres in photodynamic therapy using cell viability assay. PpIX loaded silica nanospheres could largely accumulate in tumor cells because vehicles improved the hydrophobicity of PpIX and vehicles were positively charged, which led to better therapeutic effects for vehicles than free PpIX in PDT. The enhanced permeability and retention (EPR) effect of PpIX loaded silica nanospheres was confirmed in an in vivo experiment of nude mice, which suggested the prepared vehicles had potential to be used in cancer treatment.
Contents
Acknowledgments...............I
Chinese abstract...............III
Abstract...............VI
Contents...............V
Figure contents...............VII
Table contents...............X
Chapter 1. Introduction...............1
Chapter 2. Literature review...............5
2.1 Photodynamic therapy...............5
2.1.1 Mechanism of PDT...............5
2.1.2 Challenges encountered in PDT...............7
2.2 Sonodynamic therapy...............9
2.2.1 Mechanism of SDT...............10
2.2.2 Challenges encountered in SDT...............10
2.3 Radiosensitization effect...............11
2.4 Gold nanoparticles...............12
2.4.1 Gold nanoparticle as a radiosensitizer...............13
2.4.2 Gold nanoparticle-enhanced PDT or SDT...............14
2.5 Drug vehicles...............14
2.5.1 Tumor microenvironment...............15
2.5.2 Silica-based nano-carriers...............16
Chapter 3. Material and methods...............17
3.1 Materials...............17
3.2 Preparation of APTES conjugated PpIX...............17
3.3 Synthesis of PpIX loaded silica nanospheres...............18
3.4 Deposition of Au NPs onto PpIX loaded silica nanospheres...............18
3.5 Characterization of PpIX loaded Au@SN...............19
3.6 PpIX loading efficiency measurements...............19
3.7 In vitro cytotoxicity assessment...............20
3.8 Cellular uptake in vitro and PpIX accumulation assessment...............20
3.9 Subcellular localization of PpIX loaded Au@SN...............21
3.10 UV induced PDT effects in vitro...............21
3.11 In vitro SDT and X-ray induced PDT effects...............22
3.12 Combination therapy of SDT and X-ray induced PDT in vitro...............23
3.13 In vivo tumor targeting of PpIX loaded Au@SN...............24
3.14 Statistical analysis.....24
Chapter 4. Results and discussion...............25
4.1 Synthesis and characterization of PpIX loaded Au@SN...............25
4.2 Spectroscopic properties of PpIX loaded Au@SN...............31
4.3 Conjugation and loading efficiency of PpIX loaded Au@SN...............34
4.4 In vitro cytotoxicity assessment...............35
4.5 Cellular uptake in vitro and PpIX accumulation assessment...............36
4.6 Subcellular localization of PpIX loaded Au@SN...............38
4.7 UV induced PDT effects in vitro...............39
4.8 In vitro SDT and X-ray induced PDT effects...............41
4.9 In vivo tumor targeting of PpIX loaded Au@SN...............44
Chapter 5. Conclusions...............45
References...............46

Figure contents
Fig. 2-1. Profile of PDT treatment...............5
Fig. 2-2. The mechanism of ROS generation in PDT...............6
Fig. 2-3. Cellular signaling pathways leading to apoptosis in cells after PDT...............6
Fig. 2-4. Possible mechanism of SDT...............10
Fig. 2-5. Physiological characteristics of tumor tissue and vasculatures...............15
Fig. 2-6. Passive targeting of nano-vehicles to tumor cells through EPR effect...............16
Fig. 4-1. The overall synthesis process of PpIX loaded Au@SN...............25
Fig. 4-2. TEM images of silica nanospheres prepared from (A) 1.35 mL and (B) 0.45 mL of ammonia. Size distribution of silica nanospheres prepared from (C) 1.35 mL and (D) 0.45 mL of ammonia...............26
Fig. 4-3. TEM images of (A) PpIX loaded SN and (B) its higher magnification. TEM images of (C) PpIX loaded Au@SN and (D) its higher magnification. The lattice fringe of the gold nanoparticles on the surface of silica was shown in the lower right of (D)...............27
Fig. 4-4. EDX spectra of (A) PpIX loaded SN and (B) PpIX loaded Au@SN...............28
Fig. 4-5. XRD pattern of silica nanosphers and PpIX loaded Au@SN...............28
Fig. 4-6. Zeta potential measurements of silica nanospheres, PpIX loaded SN and PpIX loaded Au@SN in deionized water...............30
Fig. 4-7. Diagram describing the interaction between amine modified silica nanospheres and gold nanoparticles in the water at pH = 7...............30
Fig. 4-8. (A) Absorbance spectra, (B) fluorescence spectra with λex = 410 nm and (C) fluorescence spectra with λex = 555 nm of silica nanospheres, PpIX, PpIX loaded SN and PpIX loaded Au@SN. The solvent was the mixture of absolute ethanol, 5% HCl and deionized water (v/v/v = 1:1:1)...............32
Fig. 4-9. Photographs of (A)(E) silica nanospheres, (B)(F) free PpIX, (C)(G) PpIX loaded SN and (D)(H) PpIX loaded Au@SN in Eppendorf tubes. The lower picture was taken in the dark, and (E)-(H) were under UV light irradiation (λex = 365 nm). (I) Fluorescence spectra with λex = 365 nm of silica nanospheres, PpIX, PpIX loaded SN and PpIX loaded Au@SN. Free PpIX and samples loaded with PpIX were at the same PpIX concentration, and the solvent was the mixture of absolute ethanol, 5% HCl and deionized water (v/v/v = 1:1:1)...............33
Fig. 4-10. FTIR spectra of silica nanospheres, PpIX and PpIX loaded Au@SN...............34
Fig. 4-11. Standard curve of PpIX at λex = 405 nm and λem = 610 nm. The solvent was the mixture of absolute ethanol, 5% HCl and deionized water (v/v/v = 1:1:1)...............35
Fig. 4-12. Dark cytotoxicity of PpIX loaded Au@SN. HeLa cells were incubated with PpIX loaded Au@SN with different PpIX concentrations for 24 h...............35
Fig. 4-13. The fluorescence intensity of PpIX in HeLa cells co-incubated with free PpIX and PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) for 6 h and 24 h...............37
Fig. 4-14. Confocal images of HeLa cells incubated with (A) no drug, (B) 0.5 μg/mL of free PpIX and (C) PpIX loaded Au@SN at the PpIX concentration of 0.5 μg/mL for 6 h. (D) The Z-axis image of HeLa cells...............37
Fig. 4-15. Confocal images of HeLa cells incubated with PpIX loaded Au@SN at the PpIX concentration of 0.5 μg/mL for 6 h and the cells were stained with (A) rhodamine 123 and (B) lysotracker blue...............38
Fig. 4-16. DCF fluorescence intensity of HeLa cells incubated with free PpIX (0.5 and 2 μg/mL), silica nanospheres (49.4 ug/mL) and PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) for 6 h prior to UV light irradiation (40-80 mW/cm2) for 10 seconds...............40
Fig. 4-17. Cell viability of HeLa cells incubated with free PpIX (0.5, 1 and 3 μg/mL) and PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) in the dark for 6 h prior to UV light exposure (40-80 mW/cm2) for 0, 10, 20 and 30 seconds...............40
Fig. 4-18. DCF fluorescence intensity of HeLa cells co-incubated with PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) in the dark for 6 h and then treated with different treatments (XDT: X-ray induced PDT, X-ray radiation with a total dose of 2 Gy; SDT: sonodynamic therapy, ultrasound at 1 MHz, 0.4 W/cm2 and 20% duty ratio for 10 min; SXDT: SDT + X-ray induced PDT)...............42
Fig. 4-19. Cell viability of HeLa cells co-incubated with PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) in the dark for 6 h after different treatments. (XDT: X-ray induced PDT, X-ray radiation with a total dose of 2 Gy; SDT: sonodynamic therapy, ultrasound at 1 MHz, 0.4 W/cm2 and 20% duty ratio for 10 min; SXDT: SDT + X-ray induced PDT)...............43
Fig. 4-20. Cell viability of HeLa cells incubated with free PpIX (0.5 μg/mL) and PpIX loaded Au@SN (at the PpIX concentration of 0.5 μg/mL) in the dark for 6 h with or without SXDT treatment (SXDT: cells were exposed with US at 1 MHz, 0.4 W/cm2 and 20% duty ratio for 10 min, and then X-ray radiation at a total dose of 2 Gy)...............43
Fig. 4-21. Ex vivo IVIS image of tumor and different organs from a nude mouse after intravenous injection with PpIX loaded Au@SN for 18 h (λex = 675 nm; λem = 740 nm)...............44

Table contents
Table 2-1. Examples of nanoparticle-based delivery systems for photosensitizers...............8
Table 2-2. Sonosensitizers used in SDT...............9
Table 2-3. The X-ray dose and photosensitizers used in radiation therapy...............12
Table 2-4. In vitro studies of GNP radiosensitisation with ionising radiation...............13
Table 2-5. In vivo studies of GNP radiosensitisation with ionising radiation...............14
Table 2-6. Compounds used for surface modification of silica...............16

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