Nghiên cứu chế tạo và đánh giá băng gạc kháng khuẩn ba lớp định hướng ứng dụng trong điều trị vết thương

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY TRẦN MINH CHIẾN Trần Minh Chiến INORGANIC CHEMISTRY INVESTIGATION OF TRILAYER MEMBRANE ORIENTATED FOR ANTIBACTERIAL WOUND DRESSING: FABRICATION, CHARACTERIZATION, AND EVALUATION MASTER THESIS Inorganic Chemistry 2021 Ho Chi Minh city - 2021 MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY Tran Minh Chien INVESTIGATION OF TRILAYER MEMBRANE ORIENTATED FOR ANTIBACTERIAL WOUND DRESSING: FABRICATION, CHARACTERIZATION, AND EVALUATION Major: Code: Inorganic Chemistry 8440113 MASTER THESIS SUPERVISOR: Assoc Prof Nguyen Thi Hiep Ho Chi Minh City – 2021 Tran Minh Chien – Master Thesis DECLARATION OF INTERESTS I hereby declare that this thesis represents my own work which has been done after registration for the Master degree at Graduate University of Science and Technology, and has not been previously included in a thesis or dissertation submitted to this or any other institution for a degree, diploma or other qualifications All the results are correct and impartial, if wrong, I take full responsibility Ho Chi Minh city, September 10th, 2021 Trần Minh Chiến Tran Minh Chien – Master Thesis ACKNOWLEDGMENTS This thesis has received numerous guidance and assistance from my supervisors and colleagues at Tissue Engineering & Regenerative Medicine laboratory (TERM) It would not have been possible without these individuals who contributed generously to the completion of this thesis First of all, I am especially thankful to my supervisor, Prof Nguyen Thi Hiep for providing me this interesting topic, for the opportunity to work independently, and for valuable discussions She had always trusted and encouraged me during this thesis Her guidance helped me in all the time of research and writing of this thesis I would also thank her for teaching me how to work efficiently, how to solve problems, and how to research independently Secondly, I want to express my gratitude to Prof Nguyen Phuong Tung, who offered me the research position at Nanomaterials and Petroleum additives Lab, Institute of Applied Materials Science Even though the time I worked with her was short, she gave me many precious lessons and experiences If it was not for her encouragement, I could never engage further in science I would like to give my sincere thanks to all the lecturers from Graduate University of Science and Technology for their invaluable guidance throughout my studies They provided me with many in-depth insights into the field of Chemistry that aided my research Next, I would like to thank all the help from my seniors, especially Hieu Minh, Khanh Vinh, and Thao Nhi who welcomed and helped me during my time at TERM I am also grateful to the Department of Biomedical Engineering at the International University for the facility support Last but not least, I would like to express my deepest gratitude to my family They supported and gave me all the best things during my thesis work Their mental and physical supports helped me overcome many difficulties It is my honor to have all of you supporting me Without any of you, I may not make a successful thesis like this Once again, thank you very much Sincerely Yours, Tran Minh Chien Tran Minh Chien – Master Thesis TABLE OF CONTENT DECLARATION OF INTERESTS ACKNOWLEDGMENTS TABLE OF CONTENT LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATION ABSTRACT 10 INTRODUCTION 12 CHAPTER 1: LITERATURE REVIEW 15 CHAPTER 2: MATERIALS AND METHODS 19 2.1 MATERIALS 19 2.2 PREPARATION AND CHARACTERIZATION OF PCL-AG SUSPENSIONS 19 2.2.1 Preparation of PCL-Ag suspensions 19 2.2.2 Characterization of PCL-Ag suspensions 19 2.3 FABRICATION AND CHARACTERIZATION OF PCL-AG MEMBRANES 20 2.3.1 Electrospinning 20 2.3.2 Morphological observation of electrospun membranes 20 2.3.3 Nanoparticles analysis 20 2.3.4 Mechanical properties of electrospun membranes 21 2.3.5 Wettability 21 2.3.6 Moisture permeability 21 2.3.7 In vitro Ag release kinetic 21 2.3.8 Antibacterial activity 21 2.3.9 Cytotoxicity assay 22 Tran Minh Chien – Master Thesis 2.4 PREPARATION OF PCL-AG-COS MEMBRANE 22 2.4.1 Preparation of PCL-Ag/POX membrane 22 2.4.2 Preparation of COS/PVP solution 22 2.4.3 COS/PVP coating on PCL-Ag/POX membrane 23 2.5 CHARACTERIZATION OF PCL-AG-COS MEMBRANE 23 2.5.1 Morphological study 23 2.5.2 Fourier-transform infrared spectroscopy (FTIR) analysis 23 2.5.3 Asymmetric wettability 23 2.5.4 Mechanical properties 24 2.5.5 Water absorbability 24 2.5.6 Moisture permeability 24 2.5.7 In vitro Ag release kinetic 24 2.5.8 Antibacterial activity 25 2.5.9 Cytotoxicity assay 25 2.6 STATISTICAL ANALYSIS 26 CHAPTER 3: RESULTS AND DISCUSSION 27 3.1 CHARACTERIZATION OF PCL-AG SUSPENSIONS 27 3.2 CHARACTERIZATION OF ELECTROSPUN MEMBRANES 29 3.2.1 Morphology of electrospun membranes 29 3.2.2 Nanoparticles analysis 31 3.2.3 Mechanical properties of PCL-Ag membranes 33 3.2.4 Wettability 35 3.2.5 Moisture permeability 36 3.2.6 In vitro Ag release kinetic 37 3.2.7 Antibacterial activity 38 3.2.8 Cytotoxicity assay 39 3.3 CHARACTERIZATION OF PCL-AG-COS MEMBRANE 41 Tran Minh Chien – Master Thesis 3.3.1 Morphological study 41 3.3.2 FTIR study 42 3.3.3 Asymmetric wettability 43 3.3.4 Mechanical properties 44 3.3.5 Water absorbability and moisture vapor transmission rate 46 3.3.6 In vitro silver release kinetic 48 3.3.7 Antibacterial activity 49 3.3.8 Cytotoxicity assay 51 CHAPTER 4: CONCLUSION AND IMPLICATIONS 53 REFERENCES 54 APPENDIX 64 Tran Minh Chien – Master Thesis LIST OF FIGURES Figure 2.1 Graphical illustration of the trilayer PCL-Ag-COS membrane fabrication process 26 Figure 3.1 (a) Photographs of PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm irradiated at gamma dose of 7.5, 15, 25 kGy, respectively Images were taken at 7-day intervals for 28 days Comparison of UV-Vis spectra of PCL-Ag suspensions at day (b) and day 28 (c) 27 Figure 3.2 SEM micrographs of electrospun raw PCL (a1), PCL-Ag 250 ppm (a2), PCL-Ag 500 ppm (a3) and PCL-Ag 1000 ppm (a4) (Scale bar: 50 µm) Histogram of fiber diameter and pore size distribution of raw PCL (b1, c1), PCL-Ag 250 ppm (b2, c2), PCL-Ag 500 ppm (b3, c3) and PCL-Ag 1000 ppm (b4, c4) (n=30) 30 Figure 3.3 X-ray diffraction (XRD) patterns of SNPs incorporated PCL membranes 32 Figure 3.4 Transmission electron microscopy (TEM) micrographs of PCL-Ag 250 ppm (a1), PCL-Ag 500 ppm (b1), PCL-Ag 1000 ppm (c1), and their size distribution histograms (a2, b2, c2) (Scale bars: 200 nm- a1, 500 nm-b1, c1, n=30) 33 Figure 3.5 Tensile strength - strain curves of PCL-Ag compared with raw PCL (n = 3) 34 Figure 3.6 Contact angles of raw PCL, PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm membranes The photographs above each column illustrate the water droplets on the membrane surface (data = mean ± SD, n=5, *: p0.05) 35 Figure 3.7 Quantification of the in vitro release of silver from the PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm in PBS solution (pH=5.5) Aliquots were taken after 1, 3, 6, 12, and 24 hours, and quantified by ICP-MS technique (data = mean ± SD, n=3) 37 Figure 3.8 (a) Photographs of the inhibition zones of raw PCL, PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm against P aeruginosa and S aureus strains and (b) inhibition zone diameters (Scale bar: 10 mm, data = mean ± SD, n=4, *: p0.05) 38 Figure 3.9 Cytotoxicity test of PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm on L929 murine fibroblast cell (data = mean ± SD, n=3, *: p0.05) 40 Figure 3.10 SEM micrographs of electrospun PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS membranes from (a) top-down view and (b) cross-section view (Scale bar: 10 µm) 41 Tran Minh Chien – Master Thesis Figure 3.11 FT-IR spectra of PCL-Ag 500 ppm, PCL-Ag/POX and PCL-Ag-COS membranes 42 Figure 3.12 (a) Images of water droplets on PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS over time (b) Dynamic contact angle of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS (n=3) 44 Figure 3.13 Tensile strength-strain curves of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS (n = 3) 45 Figure 3.14 Water absorbability of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS membranes (data = mean ± SD, n = 5, ns: p> 0.05, *: p< 0.05) 47 Figure 3.15 Quantification of the in vitro release of silver from the PCL-Ag 500 ppm, PCL-Ag/POX and PCL-Ag-COS in PBS solution (pH=5.5) Aliquots were taken after 1, 3, 6, 12, and 24 hours, and quantified by ICP-MS technique (data = mean ± SD, n=3) 49 Figure 3.16 Image of (a) the Zones of inhibition formed by the raw PCL, PCL-Ag 500 ppm, and PCL-Ag-COS membranes against P aeruginosa and S aureus and (B) the measured zone diameters (Scale bar: 10 mm, data = mean ± SD, n = 4, ns: p > 0.05, *: p < 0.05) 50 Figure 3.17 Viability (%) of fibroblasts after 24h of incubation in different concentrations of extracted solution of PCL-Ag 500 ppm and PCL-Ag-COS membranes (data = mean ± SD, n = 3, ns: p> 0.05, *: p< 0.05) 51 Tran Minh Chien – Master Thesis LIST OF TABLES Table 3.1 Viscosity of PCL, PCL-Ag suspensions before and after gamma exposure The deviations between samples were lower than the machine’s error range 29 Table 3.2 Silver content of PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm membrane 31 Table 3.3 Tensile properties of electrospun PCL membranes incorporated with different concentration of SNPs (data = mean ± SD, n = 3) 34 Table 3.4 Moisture vapor transmission rate of PCL-Ag membranes (data = mean ± SD, n=5) 36 Table 3.5 Average fiber diameter and pore size of PCL-Ag 500 ppm and PCL-Ag/POX membranes (data = mean ± SD, n=30) 41 Table 3.6 Tensile properties of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS membranes (data = mean ± SD, n = 3) 46 Table 3.7 Moisture vapor transmission rate of PCL-Ag 500 ppm, PCL-Ag/POX and PCL-Ag-COS membranes (data = mean ± SD, n=5) 46 LIST OF ABBREVIATION PCL: Polycaprolactone SNPs: Silver nanoparticles COS: Oligomer Chitosan PVP: Poly (N-vinyl pyrrolidone) POX: Poloxamer 407 DMSO: Dimethyl sulfoxide S aureus: Staphylococcus aureus P aeruginosa: Pseudomonas aeruginosa DMEM: Dulbecco`s Modified Eagle Media PBS: Phosphate-buffered saline UV-Vis: Ultraviolet- Visible spectroscopy SEM: Scanning electron microscopy Tran Minh Chien – Master Thesis Chapter 3: Results and Discussion Gram-positive and Gram-negative bacteria Several researchers hypothesized that the thicker cell wall of Gram-positive bacteria due to more peptidoglycan composition leads to less susceptibility than Gram-negative bacteria because it can prevent SNPs from anchoring and penetrating the cell wall[95] Likewise, COS was reported to be more effective on Gram-negative bacteria thanks to the more positive charge (NH3+), which promotes higher adsorption through cell walls [96, 97] The inhibition zone formed by PCL-Ag-COS was larger compared to that of PCL-Ag 500 ppm This outcome was more likely due to COS significantly enhancing the antibacterial effect 3.3.8 Cytotoxicity assay Figure 3.17 Viability (%) of fibroblasts after 24h of incubation in different concentrations of extracted solution of PCL-Ag 500 ppm and PCL-Ag-COS membranes (data = mean ± SD, n = 3, ns: p> 0.05, *: p< 0.05) The resazurin reduction assay was carried out to measure cell viability after being cultured in the extract solution for 24 hours, thereby evaluating the cytotoxicity of the materials According to the results shown in Figure 3.17, cell viability was 102% after being cultured in the 50% extract solution from PCL-Ag-COS sample However, being 51 Tran Minh Chien – Master Thesis Chapter 3: Results and Discussion exposed to 100% concentration reduced survival rate of cell population down to 10%, which was significantly lower compared to that of PCL-Ag 500 ppm (76%) The wound dressing must not cause cytotoxicity or damage to the exposed tissue However, the PCL-Ag-COS membrane seemed to be strongly harmful to L929 cells, which killed mostly 90% of the cell population Aside from SNPs effect, this could also be the results of several factors of COS in the culture extract such as concentration, molecular weight, positive charge density, or deacetylation degree [98, 99] In specific, the high molecular weight polymer with extended conformation and positive charge groups could readily bind to the cell membrane and obstruct their metabolism and respiration Despite the extract from the PCL-Ag-COS membrane annihilated most of the cells, we cannot state that PCL-Ag-COS is inappropriate for wound dressing application Since there are vast differences between in vitro and in vivo experiments For example, in the resazurin assay, the cells were entirely exposed in extract solution containing all the COS and SNPs that were released in 24 hours However, in the in vitro tests, COS and SNPs were slowly diffused into the wound bed for a long period, hence, causing less stress for the damaged tissue Therefore, an in vivo experiment is required to accurately evaluate the potential of PCL-Ag-COS in wound treatment 52 Tran Minh Chien – Master Thesis Chapter 4: Conclusion and Implications CHAPTER 4: CONCLUSION AND IMPLICATIONS In the first stage, I succeeded in directly synthesizing of SNPs in PCL solution using gamma irradiation for the electrospinning process of PCL-Ag membranes The physicochemical properties of the electrospun membranes were characterized and their in vitro properties were assessed in this study The SNPs produced by gamma irradiation were stable in PCL solution for 28 days, anticipating the potency to synthesize nanoparticles in a non-aqueous solvent Images captured by SEM implied that the rise in silver components slightly increased the fiber diameter XRD spectra confirmed the existence of the SNP crystalline phase, while TEM micrographs demonstrated the uniform distribution of SNPs across the electrospun fibers Further, the electrospun PCL-Ag membrane possessed surface hydrophobic properties and excellent tensile tolerance The PCL-Ag 500 ppm and 1000 ppm samples expressed excellent antimicrobial effects against both S aureus and P aeruginosa strains However, only PCL-Ag 500 ppm membranes showed good biocompatibility and could be promising for wound dressing applications In the second stage, the study has reported the effect of coating the COS/PVP onto PCL-Ag 500 ppm membrane for wound treatment In conclusion, the PCL-Ag-COS membrane was prepared successfully by coating COS/PVP solution onto the PCL-Ag 500 ppm membrane with the help of the PCL-POX layer Morphology observation illustrated an evenly coated surface of the membrane The supplementation of COS/PVP imparted the membrane with asymmetric wettability, water absorbability, while on the other hand, reduced mechanical strength and water vapor transmission rate The gradual release of SNPs after 24 hours was also confirmed Despite having an excellent bacterial inhibitory, the PCL-Ag-COS membrane showed a limitation in the viability of cells, which might be 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Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T., In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, Biomaterials, 2003, 24 (7), 1121-1131 63 Tran Minh Chien – Master Thesis Appendix APPENDIX Figure 1A: EDS mapping of C, O, and Ag elements in meshes (a) PCL-Ag 250 ppm, (b) PCL-Ag 500 ppm and, (c) PCL-Ag 1000 ppm (d) Ag element ratio analysis Scale bar: µm Table 1A: Concentration of residual acetone in raw PCL and PCL-Ag membrane Sample Acetone concentration (ppm) PCL-Ag 250 ppm 1647.5±50.5 PCL-Ag 500 ppm 511.9±20.5 PCL-Ag 1000 ppm 144.5±7.5 Raw PCL (12%PCL-AC) 76.7±4.2 64 Tran Minh Chien – Master Thesis Appendix Figure 2A: COS release profile from the PCL-Ag-COS membrane in PBS solution (pH=5.5) Aliquots were taken after 5, 10, 15, 20, 40, 60 and 180 minutes, and quantified by Florescence absorbance technique at the wavelength of excitation at 420nm and that of emission at 460nm using microplate reader (Varioskan LUX, ThermoScientific) (data = mean ± SD, n=3) 65 ... are attractive because of their high effectiveness against bacterial growth with a broad bactericidal spectrum [4] Even though the antibacterial mechanisms of SNPs have yet to be fully elucidated... quantified by ICP-MS technique (data = mean ± SD, n=3) 3.3.7 Antibacterial activity The antibacterial activities of the PCL-Ag-COS against two bacterial strains P aeruginosa and S aureus were investigated... measured zone diameters (Scale bar: 10 mm, data = mean ± SD, n = 4, ns: p > 0.05, *: p < 0.05) 50 Figure 3.17 Viability (%) of fibroblasts after 24h of incubation in different concentrations