MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING 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: Inorganic Chemistry Code: 8440113 MASTER THESIS SUPERVISOR: Assoc. 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 1 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 do 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 2 Tran Minh Chien – Master Thesis TABLE OF CONTENT DECLARATION OF INTERESTS. 2 TABLE OF CONTENT. 3 LIST OF FIGURES. 6 LIST OF TABLES.
8 LIST OF ABBREVIATION. 12 CHAPTER 1: LITERATURE REVIEW. 15 CHAPTER 2: MATERIALS AND METHODS. PREPARATION AND CHARACTERIZATION OF PCL-AG SUSPENSIONS.
Preparation of PCL-Ag suspensions. Characterization of PCL-Ag suspensions. FABRICATION AND CHARACTERIZATION OF PCL-AG MEMBRANES. Morphological observation of electrospun membranes.
Mechanical properties of electrospun membranes. In vitro Ag release kinetic. 22 3 Tran Minh Chien – Master Thesis 2. PREPARATION OF PCL-AG-COS MEMBRANE.
Preparation of PCL-Ag/POX membrane. Preparation of COS/PVP solution. COS/PVP coating on PCL-Ag/POX membrane. CHARACTERIZATION OF PCL-AG-COS MEMBRANE.
Fourier-transform infrared spectroscopy (FTIR) analysis. In vitro Ag release kinetic. 26 CHAPTER 3: RESULTS AND DISCUSSION. CHARACTERIZATION OF PCL-AG SUSPENSIONS.
CHARACTERIZATION OF ELECTROSPUN MEMBRANES. Morphology of electrospun membranes. Mechanical properties of PCL-Ag membranes. In vitro Ag release kinetic.
CHARACTERIZATION OF PCL-AG-COS MEMBRANE. 41 4 Tran Minh Chien – Master Thesis 3. Water absorbability and moisture vapor transmission rate. In vitro silver release kinetic.
51 CHAPTER 4: CONCLUSION AND IMPLICATIONS. 64 5 Tran Minh Chien – Master Thesis LIST OF FIGURES Figure 2. Graphical illustration of the trilayer PCL-Ag-COS membrane fabrication process. (a) Photographs of PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm irradiated at gamma dose of 7.
Images were taken at 7-day intervals for 28 days. Comparison of UV-Vis spectra of PCL-Ag suspensions at day 0 (b) and day 28 (c). 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).
X-ray diffraction (XRD) patterns of SNPs incorporated PCL membranes 32 Figure 3. 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). Tensile strength - strain curves of PCL-Ag compared with raw PCL (n = 3). 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, *: p<0. 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. Aliquots were taken after 1, 3, 6, 12, and 24 hours, and quantified by ICP-MS technique (data = mean ± SD, n=3).
(a) Photographs of the inhibition zones of raw PCL, PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm against P. aureus strains and (b) inhibition zone diameters (Scale bar: 10 mm, data = mean ± SD, n=4, *: p<0. 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, *: p<0. 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 6 Tran Minh Chien – Master Thesis Figure 3. FT-IR spectra of PCL-Ag 500 ppm, PCL-Ag/POX and PCL-Ag-COS membranes. (a) Images of water droplets on PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS over time. Tensile strength-strain curves of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS (n = 3).
Water absorbability of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS membranes (data = mean ± SD, n = 5, ns: p> 0. 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. Aliquots were taken after 1, 3, 6, 12, and 24 hours, and quantified by ICP-MS technique (data = mean ± SD, n=3). Image of (a) the Zones of inhibition formed by the raw PCL, PCL-Ag 500 ppm, and PCL-Ag-COS membranes against P.
aureus and (B) the measured zone diameters (Scale bar: 10 mm, data = mean ± SD, n = 4, ns: p > 0. 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. 51 7 Tran Minh Chien – Master Thesis LIST OF TABLES Table 3. Viscosity of PCL, PCL-Ag suspensions before and after gamma exposure.
The deviations between samples were lower than the machine’s error range. Silver content of PCL-Ag 250 ppm, PCL-Ag 500 ppm, and PCL-Ag 1000 ppm membrane. Tensile properties of electrospun PCL membranes incorporated with different concentration of SNPs (data = mean ± SD, n = 3). Moisture vapor transmission rate of PCL-Ag membranes (data = mean ± SD, n=5).
Average fiber diameter and pore size of PCL-Ag 500 ppm and PCL-Ag/POX membranes (data = mean ± SD, n=30). Tensile properties of PCL-Ag 500 ppm, PCL-Ag/POX, and PCL-Ag-COS membranes (data = mean ± SD, n = 3). 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 8 Tran Minh Chien – Master Thesis EDS: Energy-dispersive X-ray spectroscopy XRD: X-ray diffraction TEM: Transmission electron microscopy FT-IR: Fourier-transform infrared spectroscopy ICP-MS: Inductively coupled plasma-mass spectrometry MVTR: Moisture vapor transmission rate 9 Tran Minh Chien – Master Thesis Abstract ABSTRACT In the first stage, I proposed a straightforward electrospun polycaprolactone (PCL) loaded with silver nanoparticles (SNPs) membrane fabrication process, in which SNPs were directly synthesized from silver nitrate (AgNO3) in PCL-acetone mixture by gamma irradiation. The insolubility of AgNO3 in PCL solution was solved using an auxiliary dimethyl sulfoxide solvent. As a physical approach, gamma rays readily converted silver ions into SNPs without the addition of harmful reduction agents, which reduced the cytotoxicity of the synthesized material.
By avoiding some processes such as purification, solvent removal, or redispersion of SNPs, this method was more timesaving compared to other related studies. SNPs formation was confirmed by both UV-Visible spectrum (UV-Vis) and X-ray diffraction analysis. Scanning electron microscopy (SEM) revealed that the addition of SNPs significantly reduced the fiber diameter of PCL-Ag membranes compared to that of raw PCL. Uniform spherical- shaped SNPs incorporated in PCL fibers were observed under transmission electron microscopy (TEM).
The tensile test proved that the electrospun PCL-Ag membranes exhibited good mechanical characteristics. Moisture easily penetrated the porous microstructure of PCL-Ag, facilitating wound humidity regulation. Inductively coupled plasma-mass spectroscopy (ICP-MS) was employed to study the release profiles of SNPs at different time intervals. Overall, the PCL-Ag 500 ppm sample exerted excellent antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus strains and good in vitro biocompatibility.
Hence, the PCL-Ag 500 ppm is chosen as the basement layer to build up a complete antibacterial wound dressing. In the next stage, I introduced a simple approach to fabricate a trilayer membrane based on the optimized PCL-Ag 500 ppm. Oligomer chitosan (COS), synthesized from chitosan via microwave-assisted oxidative degradation, and Poly (N-vinyl pyrrolidone) (PVP) were prepared and coated onto the PCL-Ag 500 ppm membrane. Due to the weak link between COS/PVP and PCL-Ag 500 ppm layers, the system had a high tendency to be separated.
To overcome the challenge, Poloxamer 407 was added to the system in form of a connective intermediate layer. The morphology of the final product (PCL-Ag-COS) was observed under Scanning Electron Microscopy (SEM) to ensure COS/PVP solution evenly distributing across the membrane. Fourier-transform infrared spectroscopy (FTIR) was employed to identify the presence of functional groups. PCL-Ag-COS possessed many valuable properties such as asymmetric wettability, 10 Tran Minh Chien – Master Thesis Abstract hygroscopicity, and moisture permeability.
These characteristics allowed the membrane to both prevent polluted liquid from entering the wound as well as kept the moisture and body fluids at an appropriate level. By introducing PCL-POX and COS/PVP, the Ag release rate of PCL-Ag-COS was slower than that of PCL-Ag 500 ppm for 24 hours. PCL-Ag-COS had good antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus strains. The results of the biocompatibility assay showed that extract solution from PCL-Ag-COS at 100% concentration kills most of the L929 cells.
However, an in vivo experiment is required to decide whether the PCL-Ag-COS membrane is suitable for wound dressing application or not. Keywords: Electrospun membrane; polycaprolactone (PCL); silver nanoparticles (SNPs); gamma irradiation; oligomer chitosan (COS); Poly (N-vinyl pyrrolidone) (PVP); antibacterial wound dressing.