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: _ [ ] _ [ ] _ [ ] _ [ ] _ [ ] 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 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 address diagnostic methods, to develop screening platforms strategies, that can 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 i 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 QDs show several advantages such as rapidity, selectivity and biocompatibility for sensing of Cr(VI) in water For therapeutic field of cancer, novel one-pot synthetic approach for the fabrication of polydopaminefolate 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 ii Table of Contents ABSTRACT i List of Figures vii List of Tables xv List of Abbreviation xvi Chapter Introduction 1.1 Carcinogenic heavy metal 1.1.1 Overview of carcinogenic heavy metal 1.1.2 Diagnostic methods of carcinogenic heavy metal 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 Research objectives 37 Chapter 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 iii 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 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 Synthesis of fluorescent silicon quantum dots for ultra-rapid and selective sensing of Cr(VI) ion 103 iv 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 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 v 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 Conclusions 168 Publications 173 References 177 224 vi 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 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 vii 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 nakedeye 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 viii 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@GBP50B14 ix 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 x 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 UV- at 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) 50fold dilution and c) 100-fold dilution when excitation wavelength range of 350 - 400 nm Figure 5.8 a) Emission spectra of Si QDs at different dilutions (pristine, 10and 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 (F0xi F)/F0 for the detection of Cr(VI) Figure 5.11 Schematic illustration for the detection of Cr(VI) using Si QDs as a fluorescent probe Figure 5.12 a) HR-TEM image of Si QDs with Cr(VI) (inset: DLS of Si QDs with Cr(VI)), b) XPS spectrum of Si QDs before and after adding Cr(VI) Figure 5.13 Selectivity of Si QDs towards Cr(VI), the concentrations of Figure 5.14 Figure 6.1 Schematic illustration for synthesis of PFCDs and synergistic imaging-guided photothermal therapy of prostate cancer cells Figure 6.2 Spectroscopic characterization of PFCDs a) TEM image of PFCDs, (inset: particle size distribution of PFCDs from TEM image) b) FTIR spectra of dopamine, polydopamine, folic acid and PFCDs Figure 6.3 XPS spectrum of PFQDs and high-resolution XPS spectra of C 1s, N 1s, and O 1s Figure 6.4 a) X-ray diffraction (XRD) pattern of PFCDs, b) Thermogravimetric analysis (TGA) of polydopamine and PFCDs Figure 6.5 Photophysical characterization of PFCDs a) UV-visible absorption spectrum, fluorescence excitation and emission spectra of PFCDs Inset: Photographic image of PFCDs under day light and UV-light irradiation xii at 365 nm b) Fluorescence emission spectra of PFCDs after excited with different excitation wavelengths at 400-440 nm Figure 6.6 Fluorescence stability of PFCDs in different pH a) Fluorescence emission curve of PFCDs at pH 4-10 after excited at 420 nm wavelength b) Normalized fluorescence intensity of PFCDs at different pH Figure 6.7 Fluorescence stability of PFCDs after storing for 90 days Figure 6.8 Photothermal performance of PFCDs a) Infrared thermal image of PFCDs aqueous solution at different concentrations (0 500 µg/mL) under 808 nm NIR laser irradiation (1.5 W/cm2) with increasing irradiation time from to 20 b) Temperature increment of PFCDs, deionized water or PBS buffer at various concentrations after 808 nm NIR laser irradiation time at 20 c) Normalized heating curve of a series of concentrations of PFCDs exposed to 808 nm laser irradiation for 20 Figure 6.9 Photothermal performance of PFCDs at different pH a) Infrared thermal image of PFCDs aqueous solution under 808 nm laser irradiation (1.5 W cm-2) with increasing irradiation time from to 20 b) Temperature increment of PFCDs at various pH after 808 nm irradiation time for 20 c) Normalized heating curve of PFCDs in different pH after exposed to 808 nm laser irradiation for 20 Figure 4.10 Relative cell viability of A549, RWPE-1 and LNCaP after incubation with various concentrations of PFCDs for a) hours, b) 12 hours, c) 24 hours xiii Figure 6.11 Intracellular fluorescence images of A549, RWPE-1 and LNCaP cell lines incubated with PFCDs for 2.5 h Bright field images, fluorescence images, merge images were assigned to A549 lung cancer cells (without PSMA), RWPE-1 normal prostate epithelial cells (with low PSMA expression) and LNCaP prostate cancer cells (with over PSMA expression), respectively Figure 6.12 Flowcytometric analysis of A549, RWPE-1 and LNCaP cells after treatment with PFCDs Figure 6.13 Effect of photothermal therapy (under 808 nm laser exposure with power density of 1.5 W/cm2) on cell viability a) Cell viability of LNCaP cells after incubation with different concentrations of PFCDs and treatment with various exposure time of laser irradiation b) Flow cytometry analysis of LNCaP cells apoptosis induced by PFCDs-mediated PTT xiv List of Tables Table 3.1 Evaluation of AuNPs@Cu nanoshell enhancement blot-dot assay with reported various analytical strategies for the detection of tuberculosis CFP-10 antigen Table 3.2 AuNPs@Cu nanoshell dot-blot immunoassay for the detection of CFP10 antigen in spiked urine samples Table 4.1 Limit of detection (LOD) and limit of quantitation (LOQ) by immunoblotting technique Table 4.2 Comparison of different sensing techniques for detection of tuberculosis Ag85B Table 4.3 Determination of tuberculosis Ag85B in spiked urine samples using nanoshell enhancing immunoblot Table 4.4 Clinical sample characteristics of tuberculosis Ag85B using immunoblot technique Table 4.5 Clinical results of Ag85B and CFP10 antigens detection from tuberculosis patients Table 4.6 Diagnostic accuracy of clinical examination to confirm active tuberculosis using copper enhanced immunoblotting technique Table 5.1 Comparison of the present method with the reported methods for the detection of Cr(VI) Table 5.2 Determination of Cr(VI) in DI water and tap water xv List of Abbreviation AA = L-ascorbic acid AuNPs = Gold nanoparticles AuNRs = Gold nanorods CDs = Carbon dots CFP-10 = Culture filtrate protein 10 CRX = Chest X-ray DST = Drug susceptibility testing ESAT-6 = Early secreted antigenic target 6-kDA protein FA = Folic acid GBP = Gold-binding polypeptide GO = Graphene oxide HDA = Helicase-dependent amplification HIV = Human immunodeficiency virus IGRA = Interferon- IL = Interleukin INH = Isoniazid LAM = Lipoarabinomannan LPA = Line probe assay LTBI = Latent tuberculosis infection MB = Magnetic bead MDRTB = Multidrug-resistant TB MGIT = Mycobacteria growth indicator tube MTB = Mycobacterium tuberculosis NAA = Nucleic acid amplification PCs = Prostate cancer cells POCT = Point-Of-Care-Testing QDs = Quantum dots xvi RIF = Rifampicin SA = Sodium ascorbate TB = Tuberculosis TBST = Tris-buffered saline with 0.1% Tween 20 TST = Tuberculin skin test WHO = World Health Organization ZN = Ziehl-Nelseen xvii