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研究生:鄭穎琳
研究生(外文):Cheang Weng Lam
論文名稱:Photothermal effects of irradiated upconversion nanoparticles on the fat droplets in 3T3 cells
論文名稱(外文):Photothermal effects of irradiated upconversion nanoparticles on the fat droplets in 3T3 cells
指導教授:薛特
指導教授(外文):Surojit Chattopadhyay
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電研究所
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:2017
畢業學年度:106
語文別:英文
論文頁數:113
中文關鍵詞:上轉換奈米粒子
外文關鍵詞:fat dropletsupconversion nanoparticles
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Table of contents
Acknowledgements……i
Abstract (Chinese)……iii
Abstract……iv
Motivation……v
List of figures……vii
Table of contents……xvi
Chapter 1: Nanostructured materials in Photothermal and Photodynamic therapy……1
1.1 Hyperthermia……1
1.1.1 Photothermal therapy basics……2
1.1.2 Photodynamic therapy basics……4
1.2 Magnetic hyperthermia……6
1.3 Metal nanoparticles in PDT/PTT……7
1.4 Graphene nanoparticle in PDT/PTT……17
1.5 Semiconductor quantum dots in PDT/PTT……19
Chapter 2: Introduction to upconversion nanoparticles and its application in PDT/PTT……22
2.1 Fluorescence……22
2.2 Fluorescent materials nanoparticles……24
2.2.1 Organic dye……25
2.2.2 Organic dye doped nanoparticles……26
2.2.3 Quantum dots……28
2.2.4 Core-shell QDs……30
2.2.5 Graphene quantum dots……31
2.3 Multiphoton absorption……33
2.3.1 Upconversion nanoparticles (UCNPs)……34
2.4 Synthesis of UCNPs……36
2.5 Biocompatibility of UCNPs……36
2.5.1 Hydrophilic processing……37
2.5.2 Surface functionalization……37
2.6 Biomedical application……38
2.6.1 Cell imaging……38
2.6.2 Tumor targeting……40
2.6.3 Photodynamic therapy (PDT)……42
2.6.4 Photothermal therapy (PTT)……43
Chapter 3: Experimental Details……46
3.1 Synthesis of NaYF4: Yb3+, Er3+ nanoparticles……46
3.1.1 Materials……46
3.1.2 Synthesis of NaYF4: Yb, Er core nanoparticles……47
3.2 Synthesis of the SiO2 shell on the NaYF4: Yb3+, Er3+ nanoparticles……47
3.3 Transmission and scanning electron microscopy (TEM, SEM)……48
3.3.1 Sample preparation for cell TEM……49
3.4 Photothermal effect of UCNPs in butter……50
3.5 3T3 cell culture……51
3.6 Incubation of 3T3 cells with UCNPs……52
3.7 MTT and Elisa Reader……52
3.8 Oil Red O assay and Elisa Reader……53
3.9 Microscopy of Oil Red O and Hematoxylin stained 3T3 cells……54
3.10 Time lapse video of 3T3 cells with and without the internalized UCNPs under 980 nm laser irradiation……55
3.11 Time lapse video of 3T3 cells with and without the internalized UCNPs under external heating……57
Chapter 4: Results and discussion I……59
4.1 Morphology of upconversion nanoparticles (core and core shell)……59
4.2 Photoluminescence emission (core and core shell)……61
4.3 Butter experiment……63
4.4 Conclusion……66
Chapter 5: Results and discussion II……67
5.1 3T3 cell proliferation, and differentiation……67
5.2 Cytotoxicity of UCNPs towards 3T3 cells with/without IR irradiation……69
5.3 The bioTEM of 3T3 cells incubated with UCNPs (core shell)……74
5.4 Inverted Microscopy Imaging of the 3T3 cells with and without nuclear staining
in presence and absence of UCNP, and 980 nm irradiation……75
5.5 ELISA-ORO assay of the 3T3 cells in presence and absence of UCNP, and 980 nm irradiation……88
5.6 Videography of the 980 nm irradiated 3T3 cells without and with the UCNPs……91
5.7 Conclusion……97
Chapter 6: Summary and future direction……99
References……102


List of figure
Figure 1.1 Overview of the triggered release of nucleic acids inside cells. Enz-TGR: Enzyme-triggered gene release, L-TGR: Light-triggered gene release, US-TGR: Ultrasound-triggered gene release, and M-TGR: Magnetic-triggered gene release. Reprinted from ref [6]....2
Figure 1.2 Schematic diagram of the variety of effects caused by the different thermal treatments as classified by the corresponding operating temperature. Reprinted from ref [7]....3
Figure 1.3 Mechanism of PDT cytototoxicity: PS administration (step I) following the photophysical reactions represented by modified Jablonski diagram (step II) (vibrational levels omitted). Reprinted from ref [12]....5
Figure 1.4 Schematic illustration of the therapeutic strategy using magnetic nanoparticles, and an external magnetic field. Reprinted from ref [19]....6
Figure 1.5 Schematic illustration of surface plasmons in irradiated metal nanoparticles. E, H, and K denote the direction of electric field, magnetic field, and propagation vector, respectively. +sign denotes positive ion cores, and e- denotes electrons....8
Figure 1.6 Normalized, to maximum, extinction coefficients of three aqueous solutions of spherical gold nanoparticles with diameters of 22, 48, and 99 nm. Reprinted from ref [7]....9
Figure 1.7 Modes of photosensitizer binding by gold nanoparticle transporters. Reprinted from ref [33]....11
Figure 1.8 Schematic diagram of the GNR-AlPcS4 complex for NIR fluorescence imaging, and tumor phototherapy. Reprinted from ref [36]....13
Figure 1.9 (a) Infrared thermal images of tumor-bearing mice with PBS, and Pd-HS-PEG injection under 0.4 W/cm2 808 nm laser irradiation. (b) Relatively tumor volume of different groups. (c) Photographs of the Pd-HS-PEG nanoparticles, and PBS treated mice taken before, and 9 days after laser irradiation. Reprinted from ref [41]....15
Figure 1.10 Schematic Illustration of the Preparation Procedure, and Function Mechanism of ICG-Ag@PANI Theranostic Nanocomposites for Photoacoustic/Fluorescence Imaging-Guided Photothermal, and Photodynamic Therapy. Reprinted from ref [44]....17
Figure 1.11 In vivo PA imaging of ICG-PDA-rGO. (a) Schematic illustration of i.t. injected of ICG-PDA-rGO. (b) In vivo PA imaging of tumor treated with PBS, GO, PDA-rGO, and ICG-PDA-rGO. (C) Statistics of mean PA intensity of the samples measured from in vivo PA imaging. Error bars were taken from three parallel experiments. (*) p<0.05, (**) p<0. 01. Reprinted from ref [47]....18
Figure 1.12 (a) Bright-field image, and (b) red-fluorescence image after subcutaneous injection of GQDs in different areas. The excitation wavelength was 502–540 nm, and fluorescence collected in 695–775 nm. (c) Photographs of mice after various treatments on the 1st, 9th, 17th, and 25th day. (PDT: GQDs + light irradiation; C1: GQDs only; C2: light irradiation only.) (d) Time-dependent tumor growth P<0.05 for each group. Reprinted from ref [49]....21
Figure 2.1 The Jablonski diagram of luminescence where S0,1,2 represents singlet ground states, and excited states, and T1 is for triplet states. Reprinted from ref [50]....24
Figure 2.2 Confocal images of PI stained HCT-116 cells. Reprinted from ref [60]....26
Figure 2.3 The tumor therapy, and bioimaging in vivo of the drug-loaded HPSN-Salphdc-FA nanosystem. Reprinted from ref [62]....28
Figure 2.4 Top: Sixteen emission colors from small (blue) to large (red) CdSe QDs excited by a near-ultraviolet l size of QD can be from ~1 nm to ~10 nm (depends on several parameters, see text for details). Bottom: Photoluminescence spectra of some of the CdSe QD. Reprinted from ref [63]....29
Figure 2.5 In vivo NIR fluorescence imaging of U87MG tumor-bearing mice (arrows) injected with (a) ZCIS/ZnS QDs, and (b) ZCIS/ZnS-cRGD QDs. Reprinted from ref [67]....31
Figure 2.6 (a) General mechanism of cleavage of oxidized graphene during deoxidation. (b) PL peaks of GQDs in different solvents. (c, d) Amino-functionalized GQDs with different reaction temperature (from the left, 150, 120, 70, 90, and 60 °C in 2X conc. ammonia solution). Reprinted from ref [68]....32
Figure 2.7 Mutiphoton absorption. S0, and Si represent ground, and excited states; S1 depicts the final emission state, and the ω‘s depict the frequency of the photons emitted/absorbed. Reprinted from ref [69]....34
Figure 2.8 The schematic of Ln3+ upconversion nanoparticles, showing the three important components: (i) host (NaYF4), (ii) absorber or sensitizer (Yb3+), and (iii) emitter or activator (Er3+). Reprinted from ref [71]....35
Figure 2.9 Confocal fluorescence imaging of MCF-7 cells using silica/NaYF4:Yb,Er nanospheres. a) Bright-field (left), confocal fluorescence (middle), and superimposed (right) images of MCF-7 cells incubated with the nanospheres for 24 h. b) Confocal fluorescence images of MCF-7 cells with the nanospheres, excited by a 980 nm laser with different power intensities. Reprinted from ref [77]....39
Figure 2.10 a) Illustration of the targeted imaging of cancer cells with Lipo-UCNPs-FA. b, c) Transmission, and luminescence microscopy images of HeLa cells treated with b) Lipo-UCNPs-FA, and c) Lipo- UCNPs without folate ligand. For UCNP images, lex = 980 nm, and emission was collected in the range l = 510–560 nm. Scale bar = 5 mm. UCL = upconversion luminescence. Reprinted from ref [85]....40
Figure 2.11 In vivo upconversion luminescence imaging of subcutaneous S-180 tumor (right foreleg) borne by mice after intravenous injection of (a) ZwitLipo-UCNPs or (b) NegaLipo-UCNPs in 8 h. Both images were acquired under the same conditions. Reprinted from ref [86]....41
Figure 2.12 Illustration of multifunctional nano-bioprobes based on rattle-structured organosilica-shelled UCNPs. Reprinted from ref [87]....43
Figure 2.13 Schematic of csUCNP@C for accurate PTT at facile temperature. The csUCNP@C exhibit both UCL emission, and photothermal effect. With temperature-sensitive UCL emission, csUCNP@C was used to monitor the change in microscopic temperature of the photoabsorber (carbon shell) under 730-nm irradiation. The eigen temperature of csUCNP@C was much higher than the apparent temperature observed macroscopically, indicating that csUCNP@C acted as a nano-hotspot at the microscopic level. By utilizing the high eigen temperature during photothermal process, accurate PTT, which prevent the damage to normal tissues can be realized. Reprinted from ref [88]....45
Figure 3.1 (a) Optical photograph of the experimental setup by studying UCNP absorption added butter melting. (b) A schematic of the experimental setup....51
Figure 3.2 (a) Optical photograph of the experimental setup to studying the 3T3 cells with the UCNPs @ SiO2 irradiated with 980 nm light. A schematic of the experimental setup of ORO microscopy....55
Figure 3.3 (a) Optical photograph of the experimental setup to videograph the 3T3 cells with the UCNPs @ SiO2 irradiated with 980 nm light. (b) A schematic of the experimental setup....56
Figure 3.4 Optical photograph of the experimental setup to videograph the 3T3 cells with the UCNPs @ SiO2 under external heating. (a) Inverted microscope, (b) Temperature controller, and (c) the Incubator. (d) The temperature profile used during the videography of the 3T3 cells under external heating....57
Figure 4.1 TEM images of (a) core, and (c) core shell UCNPs. Optical photograph showing the green emission from the when excited by using 980 nm light for (b) core, and (d) core-shell UCNPs....60
Figure 4.2 Fluorescence spectra of (a) core, and (b) core shell UCNPs measured from 0.5 to 3.0 W....62
Figure 4.3 Variation of temperature with time using 980 nm laser exposure at different powers on (a) pure butter, (b), and (c) butter mixed with 15μL of core and core shell nanoparticles, respectively. The different powers used are mentioned in each plot....65
Figure 5.1 Light microscopy (using an inverted microscope) images of 3T3 cells cultured in different conditions. (a) Day 0-day 2, (b) day 3-day 5, (c) day 6-day 8, and (d) day 9. Yellow encircled area indicates initial fibroblast during the culture; the red circles indicate fat droplets that grow in size and density as differentiation progresses....68
Figure 5.2 Histograms of 3T3 cell viability, by MTT assay, co-cultured for 24 h with UCNPs @ SiO2, core-shell nanoparticles at different concentration from 0 to 400 ppm. The cell viability was >80 % for all UCNP concentration from 25-200 ppm. N=6....69
Figure 5.3 Histograms of cell viability of 3T3 cells under (a) only laser (980 nm), and (b) laser + UCNPs@SiO2 with powers of 0.5, 0.75, and 1.0 W for 5 mins. A control of the 3T3 cells without the UCNPS, and 980 nm laser is shown as control (C) in (a). A control of the 3T3 cells with the UCNPS, but without the 980 nm laser is shown as control (C) in (b). N=6....73
Figure 5.4 TEM images of core-shell nanoparticles in the 3T3 cells at different magnification: (a) 12 X, (b, c) 30 X, (d) 15 X, (e, g) 30 X, (f) 50 X, (h) 120 X, and (i) 300 X. The features indicated by RED are the golgi bodies, by GREEN are the mitochondria, and by PURPLE are the cluster of core-shell UCNP@SiO2 nanoparticles....75
Figure 5.5 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in pristine 3T3 cells, collected over four locations near the center of the well. Inset in (b) shows a schematic of the experimental procedure....77
Figure 5.6 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 3T3 cells incubated with 50 ppm of UCNP@SiO2, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure....79
Figure 5.7 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 0.5 W, 980 nm laser irradiated pristine 3T3 cells, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the
experimental procedure. Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (0.5 W)....80
Figure 5.8 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 0.75 W, 980 nm laser irradiated pristine 3T3 cells, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (0.75 W)....81
Figure 5.9 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 1.0 W, 980 nm laser irradiated pristine 3T3 cells, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (1.0 W)....82
Figure 5.10 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 0.5 W, 980 nm laser irradiated 3T3 cells, incubated with 50 ppm of UCNP@SiO2, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure. Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (0.5 W)....84
Figure 5.11 Inverted microscopy images of (a-d) ORO stained fat droplets without nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 0.75 W, 980 nm laser irradiated 3T3 cells, incubated with 50 ppm of UCNP@SiO2, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure. Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (0.75 W)....85
Figure 5.12 Inverted microscopy images of (a-d) ORO stained fat droplets without
nuclear stain, and (e-h) ORO stained fat droplets with hematoxylin stained nucleus (purple) in 1.0 W, 980 nm laser irradiated 3T3 cells, incubated with 50 ppm of UCNP@SiO2, collected over four locations near the center of the wells. Inset in (b) shows a schematic of the experimental procedure. Five spots (1 minute/spot), near the center of the well, were irradiated with 980 nm laser (1.0 W)....87
Figure 5.13 Histograms of relative density of oil red emission, measured by ELISA in 3T3 cells irradiated by laser with different powers having (a) 0 UCNPs, and (b) 50 ppm UCNP. The laser powers used were 0.5, 0.75, and 1.0 W for a total of 5 mins. The term (C) indicates control group (a) for cells (w/o UCNPs, w/o laser), and (b) for cells (w/ UCNPs, w/o laser). N=9....89
Figure 5.14 Optical photograph of the 980 nm laser spot irradiated near the center of an empty well in a 24 well culture plate. It shows that the whole well could not be irradiated with the laser. The inverted microscopic images shown in Figs. 5.5-5.12 were taken from the area irradiated with the laser....91
Figure 5.15 Still shots collected from inverted microscope videography of 980 nm laser (1 W) irradiated pristine (without UCNPs) 3T3 cells at different time points (from t=0, to t=180 minutes)....92
Figure 5.16 Still shots collected from inverted microscope videography of 980 nm laser (1 W) irradiated 3T3 cells with the UCNPs at different time points (from t=0, to t=180 minutes). The circled areas show either death of the cells or rapid movement/agitation of the fat droplets within the cells, or blebbing of the cell wall....94
Figure 5.17 Still shots collected from inverted microscope videography of the pristine 3T3 cells, without the UCNPs, at different time points (from t=0, to t=1440 minutes) from 37 to 45°C. Selected circled areas show either shrinking of the cell surface area (Yellow), or rapid movement/agitation of the fat droplets within the cells (Red)....96
Figure 5.18 Still shots collected from inverted microscope videography of the 3T3 cells, with the UCNPs, at different time points (from t=0, to t=1440 minutes) from 37 to 45°C. Selected circled areas show either shrinking of the cell surface area (Yellow), or rapid movement/agitation of the fat droplets within the cells (Red)....97
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