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博碩士論文 etd-0101116-101829 詳細資訊
Title page for etd-0101116-101829
論文名稱
Title
利用具前瞻性的奈米材料於生物醫學的應用:光熱治療、藥物傳遞及生物標記感測
Taping the potential of nanomaterials for biomedical applications: Photothermal therapy, Drug delivery and biomarker detection.
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
181
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-01-26
繳交日期
Date of Submission
2016-02-01
關鍵字
Keywords
腫瘤、藥物傳遞、碳奈米粒子、金奈米管、石墨烯
Carbon dots, Drug delivery, Tumor, Gold Nanorods, Graphene Oxide
統計
Statistics
本論文已被瀏覽 5720 次,被下載 35
The thesis/dissertation has been browsed 5720 times, has been downloaded 35 times.
中文摘要
本研究論述奈米粒子在生物研究上的應用,針對多樣化的奈米粒子,如氧化石墨烯和金奈米棒在光熱治療上的應用、碳量子點則做為基質和藥物遞送的載體。源自於石墨烯衍生物的氧化石墨烯(GO),因為獨有的生物相容性,且具備了優良的攜帶藥物能力、抗細菌、真菌和高水溶性的特性,幫助了藥物的遞送,因此在藥物傳遞上有著廣泛的應用,而我們透過晶種法來合成GO@GNRs,成功的鍵結氧化石墨烯(GO)與金奈米棒(GNRs)。透過上述的合成物,在室溫(28±2 °C)時,阿黴素(DOX)可依附到合成物上。藉由增強近紅外光誘導藥物釋放和光熱屬性觀察中,發現fGO@GNRsDOX同時是化療和光熱治療的理想選擇。水溶性碳量子點(Cdots)藉由高生物相容性發揮了關鍵作用於治療中可以良好的控制藥物釋放於生理條件下。目前,希望善用前述釋放藥物的能力,利用C-dots來攜帶氯化氫多巴胺的釋放(Dopaminehydrochloride, DA),一個潛在的神經傳遞介質,以其來研究相關藥物來治療神經系統疾病,如阿爾茨海默氏症和帕金森症。本研究使用Nero 2A細胞,作為了解C-dots-DA在生理條件中的影響力。
氧化石墨烯(GO)的光熱療法在抗菌、抗真菌和控制傷口感染的治療研究中使用近紅外雷射YAG(1064nm) 並專注在各種致病性細菌(Pseudomonas aeruginosa, Staphylococcus aureus)和真菌(Saccharomyces cerevisiae, Candida Utilis)。利用蛋白質分析,光密度(OD600),標準微稀釋程式,透射電子顯微鏡和螢光顯微鏡測量細胞毒性。氧化石墨烯(GO)雷射介質表面活化被有效利用於抗真菌和抗菌治療策略,展現了不容置疑的影響和廣泛的適用性。處理具感染力微生物的傷口感染治療是整個治療裡最具挑戰的問題之一。主要是由於病原體的快速突變能力和演化出高耐藥性與抗菌特性。因此,我們提出一種新方法是藉由金奈米棒(AuNRs)搭配YAG雷射(1064nm)產生的光熱,在傷口感染的小鼠上作用,嘗試殺死致病細菌(銅綠假單胞菌,Pseudomonasaeruginosa)。目前的方法可以控制在感染嚴重的皮膚傷口耐抗生素的病原菌的數量。
當紫外線在 220-350nm範圍時,碳量子點(C-dots)表現出較強的吸收,使用MALDI-MS的N2 雷射 (337 nm)來轉換能量藉由MALDI-MS快速檢測分析物。由於這種強大的功能和極小的體積 (2-4 nm),它們被用來提高低分子量生物信號在血清中的強度在MALDIMS分析上。在此研究中,我們利用C-dots的特殊屬性來做為基質給Serotonin Acid(Sr)、Glutamic Acid (GA)和Dopamine Hydrochloride(DA)等在MALDIMS檢測中使用。這些化學物質是基本的生物指標或生物標誌在一些重大疾病上如阿茲海默症等。
Abstract
This thesis presents the exploration of nanoparticles in the use of biological application. Here in this work various nanoparticles are employed like Graphene oxide and Gold nanorods use for photothermal therapy. Carbon dots use as a matrix and drug delivery vehicle.
Graphene oxide (GO) is a close derivative of graphene has unlocked many pivotal steps in drug delivery due to their inherent biocompatibility, excellent drug loading capacity, antibacterial, antifungal and high water solubility. we have conjugated them with gold nanorods (GNRs) using in situ synthesis of GO@GNRs via seed mediated method. To the above conjugate, Doxorubicin (DOX) was attached at ambient temperature (28±2°C). The enhancement in NIR induced drug release and photothermal property was observed which indicates that the fGO@GNRs-DOX method is an ideal choice for chemotherapy and photothermal therapy simultaneously. Delivery of therapeutic moieties using water soluble Carbon dots (C-dots) has been pivotal to control the release of the drugs under physiological condition due to their high biocompatibility. Controlled Dopamine hydrochloride (DA), a potential neurotransmitter using C-dots as carriers is studied in the present work, in order to highlight its potential to deliver drugs related with neurological disorders such as Alzheimer’s and Parkinson’s disease. In order to understand the impact of the C-dots-DA conjugate under physiological conditions, Nero 2A cells were taken under consideration.
Photothermal treatment of graphene oxide (GO) for antibacterial, antifungal and controlling the wound infection treatment using near infrared laser Nd-YAG (1064 nm) were reported. Various pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) and fungal (Saccharomyces cerevisiae and Candida Utilis) were investigated. The Cytotoxicity was measured using the proteomic analysis, optical density (OD600), standard micro dilution procedures, TEM and Epifluorescence microscopy. The laser mediated surface activation of GO was achieved for efficient antifungal and antibacterial therapeutic strategy. GO provided unassailable effects and wide applicability.
Wound infection treatment is one of the most challenging problems to be addressed in infectiously microbiological treatment. This is mainly due to the pathogen’s ability for fast mutation and generating severely antibiotic resistance to antimicrobial treatment. Therefore, we have proposed a novel method by using gold nanorods (Au NRs) to assist the Nd-YAG laser (1064 nm) for photothermal killing pathogenic bacteria (Pseudomonas aeruginosa) for directly healing the wound infection on the (albino) mice. The current approach can be used to control severe skin infections from antibiotic resistant pathogens in wounds.
Carbon dots (C-dots) exhibit strong absorbance in the UV (220-350nm) range, which was exploited to transfer the energy from N2 laser (337 nm) of Matrix-assisted laser desorption/ionization-Mass Spectroscopy (MALDI-MS) to analytes for their rapid detection. Due to this strong feature and extremely small size (2- 4 nm), they were used to enhance the signal intensity of MALDI-MS peaks of low molecular weight biomarkers in serum. In this study, we utilized the extraordinary property of C-dots as a matrix for the detection of Serotonin (Sr), Glutamic Acid (GA) and Dopamine Hydrochloride (DA) by using MALDI-MS.
目次 Table of Contents
Chapter 1 Page


Introduction

Nanotechnology: An emerging revolutionary science 1
Metal Nanoparticles and Light: Surface Plasmon Resonance 2
Synthesis of Metal Nanoparticles 6
Bottom up Approach 6
Top down approach 8
Highlights of thesis 11
UV-Visible spectroscopy 12
Photoluminescence 13
FTIR 14
XRD 15
TEM 16
RAMAN 16
MALDI-MS 17
MALDI-MS used for the detection of biomarker 18
Photothermal Therapy 19
Nd-YAG laser (1064 nm) 20
Drug Delivery 21
Wound healing mechanism 22
Culturing of bacteria 24
Colony Forming Units 24
Acridine Orange 25
References 26
Chapter 2 Page
Graphene Oxide@Gold Nanorods for Controlled Release of Doxorubicin in mice tumor 2.1 Introduction 30
Materials and Methods 32
Characterization 33
Synthesis and purification of Graphene Oxide (GO) 33
Synthesis of Gum arabic functionalized GO (fGO) 34
Synthesis of fGO@GNRs conjugate) 34
Synthesis of fGO@GNRs-DOX conjugate 34
Drug loading capacity 35
Drug Release Studies 35
Near Infrared (NIR) radiation induced drug release 35
Cytotoxicity studies 36
Animal experiments for controlling the tumor 36
Results and Discussion 37
UV-VIS spectral analysis 37
Morphological analysis 41
Interaction and attachment studies 43
Drug Release Kinetics 48
Cytotoxicity and in vitro photothermal therapy 52
Trypan blue test for viability 54
Microtomy of mice vital organs 55
Conclusion 57
References 57
Chapter 3 Page
Controlled delivery of dopamine hydrochloride using surface modified carbon dots for neuro diseases
Introduction 62
Material and Methods 64
Synthesis of Carbon dots 65
Attachment of DA on C-dots (preparation of the nano-conjugate) 65
Drug loading association efficiency 65
In vitro drug release 66
Cytotoxicity study 66
Culturing Neuro 2A cells and interaction with DA 67
Drug release and kinetics of DA 67
Result and discussion 67
Biocompatibility of C-dots-DA conjugates 71
Drug release study and Kinetics 72
Fluorescence microscopy of Neuro 2A cells with C-dots-DA 73
In- vivo Toxicity by Histology 75
Body weight analysis 76
Conclusion 77
References 77









Chapter 4 Page

Near infrared (NIR) laser mediated surface activation of graphene oxide nanoflakes for efficient antibacterial, antifungal and wound healing treatment
Introduction 80
Materials, methods and synthesis of GO 82
Proteomic analysis of bacterial cells 82
Epifluorescence measurements 83
In vivo Assay 83
Infection for mice wound 84
Result and Discussion 84
Characterization of GO 85
In vitro cytotoxicity study of GO 88
MALDI-MS studies 89
Epifluorescence studies 91
Total viable counts studies 93
Nd-YAG laser used in triple wound sites of mice 95
Morphology changes in S. aureus using TEM images 98
Mechanisms 101
Conclusion 102
4.16 References 103










Chapter 5 Page


Highly efficient Gold nanorods assisted Laser phototherapy for rapid treatment on mice wound infected by pathogenic bacteria
Introduction 107
Wound: Classification, Stages, Healing factors 107
Photothermal therapy 110
Material methods 110
Instrumentation 111
Nd-YAG Laser for wound infection treatment 113
Pseudomonas aeruginosa collection from wound site 113
Nd-YAG Laser for wound infection treatment 114
Synthesis of Gold Nanorods (Au-NRs) 114
Optimization of laser for time and Au NR’s 115
Animal experiments for wound healing by laser 116
Differentiate between bacterial proteins and wound proteins 117
Wound size determination 118
Result and Discussion 118
Characterization of the Au NRs 118
Nd-YAG laser used in triple wounds on mice skin 120
MALDI-MS studies of wound condition in mice 124
Histological studies 130

Conclusions 134
References 134







Chapter 6 Page


Exploring the ability of water soluble carbon dots as matrix for detecting neurological disorders using MALDI-TOF MS
Introduction 138
Material methods 141
Synthesis of C-dots 142
Mass spectrometric analysis 142
Characterization 143
Sample preparation for MALDI-MS analysis 143
Quantification of Sr, GA and DA in serum 143
Result and discussion 144
Study of C-dots as a matrix by MALDI-MS 148
Conclusions 158
References 158



Figure Captions

Chapter 1 Page

Fig. 1.1 Quantum dots of different size and their respective florescence. The color purely depends upon the size and quantum confinement 3
Fig. 1.2 Quantum confinements for quantum dots 4
Fig. 1.3 Size dependent color of gold nanoparticles 5
Fig. 1.4 Top down and bottom up approach for nanomaterials 9

1.5 Highlights of each chapter 10
Fig. 1.6 Schematic representation of UV-Visible spectrometric 13
Fig. 1.7 Jablonski diagram. After an electron absorbs a high energy photon the system is excited electronically and vibrationally. The system relaxes vibrationally, and eventually fluoresces at a longer wavelength 14
Fig. 1.8 Schematic representation of a FTIR 15
Fig. 1.9 Energy level states involved in Raman signal 17
Fig 1.20 Schematic representation of MALDI-MS 18
Fig. 1.21 Photothermal therapy employed on cells for apoptosis 20
Fig. 1.22 Photon excitation from ground state to excited state 21
Fig. 1.23 shows the mechanism of light induced wound healing 23
Fig. 1.24 shows the dilution factor for colony forming unit 25






Chapter 2 Page

Fig. 2.1 Schematic representation of the important steps involved in synthesis of the drug delivery vehicle based on graphene oxide and gold nanorods (a) Colors of the solution at various stages of reactions to form final conjugate viz i-GO, ii-GA, iii-fGO, iv-fGO@GNRs and v-fGO@GNR-DOX (b) fGO conjugate (GO-GA) synthesis after reaction of GO with GA
(c) Incorporation of fGO in the GNRs (fGO@GNRs) during zipping mechanism (d) Loading of anti-cancer drug DOX on fGO@GNRs complex 38
Fig. 2.2 UV-Vis spectra of (a) Graphene Oxide, Gum arabic and their complex and (b) Gold nanorods and its conjugation with fGO 40
Fig. 2.3 Electron micrograph showing (a) TEM image of graphene oxide (GO), (b) fGO (c) bare GNRs, (d) FE-SEM image of fGO@GNRs, (e) TEM image of fGO@GNRs displaying magnified view of interactions, (f) enlarged contrasted view of highlighted area of (e) showing clear view of dog-bone shaped GNR on a thin layer of fGO and (g) another TEM image showing dog-bone shaped GNR with other anisotropic nanostructures on a thin sheet of fGO 42

Fig. 2.4 Fourier Transform Infrared (FTIR) spectra of (a) pure gum arabic extract, (b) pure graphene oxide, (c) GO-GA (fGO) complex, (d) bare GNR solution, (e) fGO@GNR complex,
(f) pure doxorubicin (DOX) and (g) fGO@GNR-DOX complex in aqueous solution 46
Fig. 2.5 (A) Cyclic voltammograms of (a) fGO, (b) GNR and (c) fGO@GNR. (B) (a) TGA of fGO@GNR and final complex and (C) Zeta potential values various components involved in formation of final complex where A: fGO, B: Pure GNRs, C: fGO@GNR & D: fGO@GNR- DOX 49
Fig. 2.6 (A) (a) Effect of irradiation time on photothermal temperature of different components in vitro and (b) Infrared image showing increase in temperature of final complex fGO@GNR after 15 min. (B) Temp enhancement IR images of PBS, GO, GNR , GO@GNR by using FLIR camera 50
Fig. 2.7 Percentage Drug Release with respect to time (a) without NIR and (b) with NIR irradiation 52
Fig. 2.8 IC50 values on in vitro cell lines (a) without NIR irradiation and (b) with NIR irradiation where A: GNRs, B: fGO@GNR, C: fGO@GNR-DOX & D: Free DOX 53
Fig. 2.9 Trypan blue treatment of A549 cancer cells by GO@GNR and laser 54
Fig. 2.10 Microtomy of (A) Tumor with different condition, i. Control ii. Dox treatment iii. fGO@GNR-DOX (B) different section of organs treated by Dox and fGO-GNR-Dox 56
Chapter 3 Page
Fig. 3.1 Optical properties of C-dots (a) UV-Vis spectra of C-dots (black), DA (green), C- dots-DA (blue). (b) Showing the fluorescence of C-dots (red), C-dots-cyst Hyd (black), C- dots-DA (green). (c) TEM image of purified C-dots (d) XRD of C-dots 70
Fig. 3.2 (a) RAMAN of C-dots. (b) Zeta potential of C-dots, C-dots with Cystamine Hydrochloride and C-dots with Cystamine Hydrochloride and conjugate with Dopamine Hydrochloride.(c) FTIR spectra of C-dots showing their typical functionalization pattern with Cystamine hydrochloride and Dopamine hydrochloride 71
Fig. 3.3 Biocompatibility of normal cells after injecting different conc of DA and C-dots-DA--
-----------------------------------------------------------------------------------------------------------------72
Fig. 3.4 Percentage release of C-dots-DA and Dopamine with respect to time in PBS at pH- 7.4 73

Fig. 3.5 Epifluorescent microscopic images normal cells treated by PBS, DA, and C-dots-DA-
----------------------------------------------------------------------------------------------------------------74
Fig. 3.6 Tissue section (stained by Haemotoxolin and Eosin) of brain, heart, liver, kidney and spleen were treated by DA and C-dots-DA 75
Fig. 3.7 showing the percentage body weight of mice during the 45 days after injecting DA and C-dots-DA 76
Chapter 4 Page
Fig. 4.1 Characterization of GO using (A) UV-Vis spectra, (B) FTIR spectra, (C) TEM, (D) SEM, inset represent EDX analysis (E) Raman spectroscopy, and (F) 2D Raman peak 86
Fig. 4.2 Proteomic analysis using MALDI-MS spectra of (A) Pseudomonas aeruginosa , (B) Staphylococcus aureus , (C) Saccharomyces cerevisiae and (D) Candida utilis cells before (a) and after incubation with (b) GO (c) Nd-YAG laser alone (d) GO+ Nd-YAG laser 91
Fig. 4.3 Epifluorescence micrographs of Pseudomonas aeruginosa, Staphylococcus aureus, Saccharomyces cerevisiae and Candida utilis cells after treatment with Graphene oxide, Nd- YAG laser and Graphene oxide with Nd-YAG laser. Scale bar equal 10 µm 93
Fig. 4.4 Standard Micro dilution protocol of the antibacterial activities of GO and their photothermal treatment 96
Figure 4.5 TEM images of S. aureus during the treatment with GO and Nd-YAG (1064 nm) laser (a) control, (b) incubation with GO, (c) exposure to laser and (d) exposure to laser combine with GO 101
Chapter 5 Page
Fig. 5.1 Schematic diagram showing the methodology of whole experiment 112
Fig. 5.2 (a) UV-Visible spectra of Au NRs showing both the absorbtion of longitudinal surface Plasmon resonance and transverse Plasmon resonance. (b) TEM images of Au NRs.
(c) Temp enhancement curve of Au NRs upon heating by Nd-YAG laser. (d) Thermal image
was taken using thermal imaging camera (FLIR) upon irradiation of Nd-YAG laser (1064 nm) 119

Fig. 5.3 The optimization of laser employed in the experiment. (a) Setup picture of working Nd-YAG laser on bacterial suspension. (b) The microtiter plate utilized for bacterial suspension for optimized condition. (c) The Nd-YAG laser exposed on the mice wound (d) IR images taken during the irradiation of Nd-YAG laser and Au NRs 121
Fig. 5.4 (a) TEM images of control P. aeruginosa. (b) Treated by only Nd-YAG laser for 240 sec, upto this exposure time, the morphology remain same. (c) Exposure of P. aeruginosa with Au NRs and Nd-YAG laser, morphology changed including disruption in plasma membrane 122
Fig. 5.5 Third day of MALDI-MS analysis on (a) Control cells of P. aeruginosa (b) pure blood sample for reference of blood peak (c) Wound treated with Nd-YAG (1064 nm) laser only (d) wounds treated with Nd-YAG (1064 nm) laser and Au NRs 125
Fig. 5.6 Sixth day of MALDI-MS analysis on (a) Control cells of P. aeruginosa (b) pure blood sample for reference of blood peak (c) Wound treated with Nd-YAG (1064 nm) laser only (d) wounds treated with Nd-YAG (1064 nm) laser and Au NRs 126
Fig. 5.7 Twelfth day- of MALDI-MS analysis on (a) Control cells of P. aeruginosa (b) pure blood sample for reference of blood peak (c) Wound treated with Nd-YAG (1064 nm) laser only (d) wounds treated with Nd-YAG (1064 nm) laser and Au NRs 127
Fig. 5.8 (a) Three fresh wounds namely as W1, W2, and W3 on mice model. (b) Pseudomonas aeruginosa infection on W1, W2 and W3 wounds. (c) After twelve days of the treatment on W1, W2 and W3. (d), (e) and (f) show changed in the treatment and location of wounds on the dorsal surface of animal model by using Au NRs and Nd-YAG laser 129
Fig. 5.9 Photographs of control and treated wound tissue section stained by Haemotoxolin and Eosin taken after 3rd and 12th day exposure by Au NRs and Nd-YAG laser (10X objective, scale bar = 100 µm) 131
Fig. 5.10 The correlation of wound size area by treatment of laser therapy. (a) W1-Control wound (b) W2-Nd-Yag laser treated (c) W3-Treated by Au NR's and Nd-YAG laser 132
Fig. 5.11 Schematic representation of the wound healing process 133
Chapter 6 Page

Fig. 6.1(a) UV-Vis spectra of C-dots also showing photographs under normal light and UV light (b) Showing the fluorescence of C-dots (c) TEM image of purified C-dots 145
Fig. 6.2(a) Raman (b) XRD spectra of purified C-dots displaying their typical morphological features 146
Fig. 6.3 FTIR spectra of C-dots showing their typical functionalization pattern 147


Fig. 6.4 MALDI-MS spectra of Dopamine Hydrochloride with (a) Bare C-dots (b) DHB matrix (c) DHB- Dopamine Hydrochloride and (d) C-dots in dopamine hydrochloride -----------------------------------------------------------------------------------------------------------149


Fig. 6.5 MALDI-MS spectra of Dopamine Hydrochloride with (a) Bare C-dots (b) DHB matrix (c) DHB- Dopamine Hydrochloride in serum (d) C-dots- Dopamine Hydrochloride in serum ----------------------------------------------------------------------------150

Fig. 6.6 Detection of Serotonin by using DHB and C-dots MALDI MS spectra of (a) C-dots (b)DHB matrix (c) DHB- Serotonin and (d) C-dots- Serotonin ----------------------151

Fig. 6.7 Detection of Serotonin by using DHB and C-dots MALDI MS spectra of (a) C-dots (b)DHB matrix (c) DHB- Serotonin in serum (d) C-dots – Serotonin in serum ---152

Fig. 6.8 MALDI-MS spectra of Glutamic acid using (a) pure C-dots (b) DHB matrix (c) DHB- Glutamic acid and (d) C-dots-Glutamic acid ------------------------------------------154

Fig. 6.9 MALDI-MS spectra of Glutamic acid in human serum using (a) pure C-dots (b) DHB matrix (c) DHB- Glutamic acid and (d) C-dots- Glutamic acid in serum ---155

Table 1 Tabulated all peaks in MALDI-MS spectrum of Serotonin, Glutamic acid and Dai n C-dots matrix Vs DHB matrix.

Table 2 Determination of LOD (nM) for Serotonin, Glutamic acid and dopamine hydrochloride by using two matrices.

Chapter 7
Conclusion 156
Publications 159
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