Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Diseas...

PhanLeMinhTu TV pdf 107th Doctoral Dissertation Thesis Advisor Tae Jung Park Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Disease August 2020 The Gradu[.]

107th Doctoral Dissertation

Thesis Advisor: Tae Jung Park

Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Disease

August 2020

The Graduate School Chung-Ang University

Department of Chemistry Major in Biochemistry

Le Minh Tu Phan

Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Disease

Presented to the Faculties of the Chung-Ang University in Partial Fulfillment of the Requirement of the

Degree of Doctor of Philosophy

August 2020

The Graduate School Chung-Ang University

Department of Chemistry Major in Biochemistry

Le Minh Tu Phan

Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Disease

by

Le Minh Tu Phan Department of Chemistry Chung-Ang University

Date:

Approved:

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Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Department of Chemistry

in the Graduate School of Chung-Ang University

2020

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ABSTRACT

Advanced Nanomedicine in Sensitive Diagnostic and Therapeutic Fields of Cancer and Infectious Disease

Le Minh Tu Phan Major in Biochemistry Department of Chemistry The Graduate School, Chung-Ang University

Cancer and infectious disease continue to be one of the most difficult global healthcare problems, becoming one of the biggest health challenges facing humanity It is essential to develop platforms that can address diagnostic methods, screening strategies, interventions for prevention or treatment of disease, or strategies to improve the healthcare system in precision medicine Nanomedicine is a new science that allowed investigations of nanomaterials and applied nanotechnology in monitoring, diagnosing, preventing, repairing or curing diseases and damaged tissues in biological systems Herein, nanomaterial based potential diagnostic and therapeutic tools for infectious disease (tuberculosis), carcinogenic heavy metal (hexavalent chromium) and cancer (prostate cancer) were investigated and applied For early and accurate diagnosis of tuberculosis, a facile dot-blot assay for sensitive detection of Mycobacterium tuberculosis antigens (CFP-10, Ag85B) via the formation of copper nanoshell on the AuNPs surfaces was

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investigated The present method was successfully applied to specific visual detection of CFP-10 and Ag85B antigens in clinical samples, which offers that

it can be a promising potential tool for on-site tuberculosis diagnostics For prevention of cancer by heavy metal detoxification, one-step synthetic approach for fabrication of Silicon quantum dots by using a silicon source and l-ascorbic acid as a reducing agent was developed The as-fabricated Si

and biocompatibility for sensing of Cr(VI) in water For therapeutic field of cancer, novel one-pot synthetic approach for the fabrication of polydopamine-folate carbon dots as theranostic nanocarriers for the image-guided photothermal therapy targeting of prostate cancer cells was explored The as-fabricated carbon dots acted as a dual probe in bio-identification and thermal therapeutic products, suggesting the utilization of as-synthesized carbon dots can be used

as promising candidates in biorecognition and thermal treatment applications These new strategies for nanomedicine design exploit unique nano bio interactions to overcome the limitations of conventional medicines, leading it

to be an alternative effective approach that is being exploited globally

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Table of Contents

ABSTRACT i

List of Figures vii

List of Tables xv

List of Abbreviation xvi

Chapter 1 Introduction 1

1.1 Carcinogenic heavy metal 1

1.1.1 Overview of carcinogenic heavy metal 1

1.1.2 Diagnostic methods of carcinogenic heavy metal 4

1.1.3 Perspectives of sensing carcinogenic heavy metal 11

1.2 Tuberculosis 12

1.2.1 Overview of tuberculosis 12

1.2.2 Diagnostic methods for Tuberculosis 14

1.2.3 Perspectives of Tuberculosis diagnosis using nanomaterials 33

Chapter 2 Research objectives 37

Chapter 3 Gold-copper nanoshell dot-blot immunoassay for naked-eye sensitive detection of Tuberculosis specific CFP-10 antigen 40

3.1 Introduction 40

3.2 Materials and methods 45

3.2.1 Materials 45

3.2.2 Synthesis of gold nanoparticles 46

3.2.3 Expression and purification of CFP-10 antigen and GBP-CFP10G2 fusion antibody 46

3.2.4 Conjugation of GBP-CFP10G2 with AuNPs 47

3.2.5 Dot-blot assay using gold nanoparticles 48

3.2.6 Copper nanoshell enhancement 48

3.2.7 Silver nanoshell enhancement 49

3.2.8 Characterization 49

3.3 Results and discussion 51

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3.3.1 Characterization and confirmation of nanocomposites 51

3.3.2 Optimization of protein expression condition 57

3.3.3 Optimization condition for copper enhancement 59

3.3.4 Comparison of enhancement capacity of copper with silver and sensitivity analysis 63

3.3.5 Specificity of CFP-10 antigen in spiked urine sample 71

3.3.6 Real urine sample test from TB patients 73

3.4 Chapter conclusion 74

Chapter 4 Reliable naked-eye detection of Mycobacterium Tuberculosis 85B antigen using gold and copper nanoshell enhanced immunoblotting techniques 75

4.1 Introduction 75

4.2 Materials and methods 78

4.2.1 Reagents 78

4.2.2 Fabrication of GBP-50B14 fusion antibody 78

4.2.3 Conjugation of GBP-50B14 to AuNPs 79

4.2.4 Immunoblot for detection of 85B antigen 80

4.2.5 Characterizaion 81

4.3 Results and discussion 82

4.3.1 Verification of gold and copper nanoshell generations 82

4.3.2 Detection of Ag85B using immunoblotting technique with gold and copper enhancement 87

4.3.3 Specificity of nanoshell enhanced immunoblotting technique 93

4.3.4 Detection of tuberculosis Ag85B from clinical urine specimens 95 4.3.5 Clinical performance of copper enhanced immunoblot for determination of active tuberculosis 97

4.4 Chapter conclusion 102

Chapter 5 Synthesis of fluorescent silicon quantum dots for ultra-rapid and selective sensing of Cr(VI) ion 103

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5.1 Introduction 103

5.2 Materials and methods 107

5.2.1 Reagents 107

5.2.2 Synthesis of silicon quantum dots 107

5.2.3 Instrumentation 108

5.2.4 Fluorescent probe for the detection of Cr(VI) 109

5.3 Results and discussion 109

5.3.1 Optimization of reaction temperature and time 109

5.3.2 Characterization of Si QDs 111

5.3.3 Effect of Si QDs concentration, ionic strength and pH on the fluorescence spectra of Si QDs 117

5.3.4 Detection of Cr(VI) using Si QDs as a fluorescent probe 122

5.3.5 Sensing mechanism for detection of Cr(VI) 126

5.3.6 Selectivity of the probe 129

5.3.7 Analytical application of Si QDs for the detection of Cr(VI) in water samples 131

5.4 Chapter conclusion 133

Chapter 6 One-spot synthesis of carbon dots with intrinsic folate receptor for synergistic imaging- guided photothermal therapy of prostate cancer cells 134

6.1 Introduction 134

6.2 Materials and methods 139

6.2.1 Materials 139

6.2.2 Synthesis of polydopamine-folic acid carbon dots 140

6.2.3 Characterization 140

6.2.4 Photothermal performance 141

6.2.5 Cell culture 142

6.2.6 Cell viability 142

6.2.7 Flow cytometry assay 143

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6.2.8 Target tumor cell imaging using PFCDs 144

6.2.9 In vitro photothermal therapy 144

6.3 Results and discussion 146

6.3.1 Characterization of PFCDs 146

6.3.2 Photophysical properties and photothermal performance of PFCDs 152

6.3.3 Targeting prostate cancer cell imaging using PFCDs 159

6.3.4 In vitro photothermal therapy 163

6.4 Chapter conclusion 167

Chapter 7 Conclusions 168

Publications 173

References 177

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List of Figures

Figure 1.1 The relationship between latent tuberculosis infection and active tuberculosis, and current diagnostic methods used to diagnose tuberculosis in both stages [1]

Figure 1.2 a) Preparation process of the ECL-sensing platform for measuring IFN- -2 [2], b) Schematic representation of the electrochemical detection strategy from DNA extraction to readout A positive DPV signal is generated when there are sufficient AuNPs immobilized on the working electrode surface [3]

Figure 3.1 Schematic illustration for the naked-eye detection of CFP-10 by silver and copper nanoshell formations on the gold nanoparticle catalytic surface via dot-blot immunoassay platform

Figure 3.2 Characterization of AuNPs a) TEM image of AuNPs, b) Size distribution of AuNPs using DLS, c) Zeta potential of AuNPs

Figure 3.3 a) SEM image of copper nanostructure after 10 min growth incubation time (inset: different strategic orientation of copper nanoshell formation on AuNPs and magnified SEM image of single copper nanostructure) b) X-ray diffraction patterns of AuNPs@GBP-CFP10G2 and AuNPs@Cu core-shell structure

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Figure 3.4 a) Normalized absorption spectra of bare AuNPs, GBP-CFP10G2 directional conjugated AuNPs, gold-copper core-shell nanostructure b) FTIR spectra of AuNPs@GBP-CFP10G2 and AuNPs@Cu core-shell structure Figure 3.5 Optimization of protein expression by SDS-PAGE and western blot analyses

Figure 3.6 Screening of the reducing capacity for copper amplification process using different reduction agents Intensity analysis of copper enhanced blot by a) sodium borohydride, b) sodium citrate, c) ascorbic acid, d) sodium ascorbate

Figure 3.7 Optimization of copper enhancing solution condition a) Different concentrations of sodium ascorbate (SA) to induce copper nucleation process, b) Incubation time after immersing into copper enhancing solution

Figure 3.8 Standard calibration curve for quantitative detection of CFP-10 based on direct dot-blot immunoassay using a) only AuNPs, b) silver enhancement and c) copper enhancement Inset: the linear section of standard curve for detection of CFP-10 The upper photographs indicate the naked-eye detection of dot-blot immunoassay to detect CFP-10 using these enhancing methods d) Comparison of three amplification process including only AuNPS, silver and copper nanoshell enhancement

Figure 3.9 a) Quantitative detection of CFP-10 antigen by ChemiDoc imaging system based on direct dot-blot immunoassay using copper enhancement, b) Construction of calibration curve between signal intensity and CFP-10 antigen

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concentration using ChemiDoc imaging system and cell phone camera image

by ImageJ software

Figure 3.10 Verification of successful dot-blot immunoassay on nitrocellulose membrane corresponding to AuNPs aggregation on membrane and copper nanoshell enhancement SEM images of a) commercial nitrocellulose membrane, b) negative sample without CFP-10, c) positive sample with AuNPs and after immersion into silver d) or copper e) enhancing solution

Figure 3.11 Specificity of copper-enhanced gold nanoparticle-based dot blot immunoassay for detection of M tuberculosis specific antigen CFP-10 M tuberculosis antigen 85B (Ag85B), alpha-fetoprotein (AFP), prostate-specific antigen (PSA), glucose and urea were used to test the specificity of the dot blot immunoassay

Figure 3.12 Quantification of TB antigen, CFP-10, concentrations from the real patient specimens with ImageJ software using a cellular phone camera Figure 4.1 Schematic presentation for naked-eye detection of Ag85B using direct immunoblotting techniques with copper and gold nanoshell enhancement

Figure 4.2 Confirmation of GBP-50B14 antibody conjugated AuNPs Absorption spectra (A) and FTIR spectra (B) of AuNPs and AuNPs@GBP-50B14

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Fig 4.3 Absorption spectra of AuNPs, copper and gold nanoshell after particle enlargement

Figure 4.4 Particle size characterization A) TEM image of AuNPs, inset: size distribution by dynamic light scattering SEM image of copper nanoshell (B) and gold nanoshell (C) after enlargement, inset: size distribution by SEM Figure 4.5 Immunoblotting performance for quantitative detection of Ag85B (A) Photographs of immunoblot using AuNPs with gold and copper intensification Standard calibration curve of quantitative detection of Ag85B using AuNPs immunoblotting technique (B) with copper nanoshell enhancement (C) and gold nanoshell enhancement (D) The error bars represent the standard deviations of three independent measurements

Fig 4.6 Corroboration of successful nanoshell formation on AuNPs inside the nitrocellulose paper SEM images of (A) nitrocellulose paper, (B) nitrocellulose paper after immunoblot with AuNPs, (B) copper nanoshell and (C) gold nanoshell enhancement

Fig 4.7 Specificity of copper enhanced AuNPs immunoblot for detection of tuberculosis Ag85B, compared to others biomarker: tuberculosis CFP10 antigen, human serum albumin (HSA), bovine serum albumin (BSA), phosphate-buffered saline (PBS)

Figure 4.8 Quantitative measurement of tuberculosis Ag85B from clinical urine specimens

Figure 5.1 Effect of reaction temperature and time on the size of Si QDs

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Figure 5.2 a) HR-TEM image (inset: diameter distribution of Si QDs measured by dynamic light scattering), b) FT-IR spectrum of Si QDs

Figure 5.3 EDX spectrum of Si QDs

Figure 5.4 XPS spectrum of a) Si QDs High resolution XPS spectra of b) C 1s, c) N 1s, d) O 1s, and e) Si 2p

Figure 5.5 a) UV visible absorption, emission spectrum of Si QDs when

and under

different excitation wavelengths

Figure 5.6 Calibration curve for fluorescence emission spectra of Si QDs and quinine sulfate with various absorbance below 0.1

Figure 5.7 Fluorescent emission peak of Si QDs at a) 10-fold dilution, b) 50-fold dilution and c) 100-50-fold dilution when excitation wavelength range of 350

- 400 nm

Figure 5.8 a) Emission spectra of Si QDs at different dilutions (pristine, 10- and 100-fold), b) Effect of NaCl concentration (100

the emission intensity of Si QDs

Figure 5.9 Effect of reaction time on the fluorescence emission spectra of Si QDs with and without Cr(VI)

Figure 5.10 a) Fluorescence emission spectra of Si QDs in the presence of Cr(VI) at various concentrations (1.25

calibration graph was plotted between the concentration of Cr(VI) and