Cấu trúc, tính chất sợi polymer electrospun và ứng dụng y sinh

STRUCTURE AND PROPERTIES OF ELECTROSPUN POLYMER FIBERS AND APPLICATIONS IN BIOMEDICAL ENGINEERING by Cheryl L. Casper A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy with a major in Materials Science and Engineering Fall 2005 Copyright 2005 Cheryl L. Casper All Rights Reserved UMI Number: 3200539 UMI Microform 3200539 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 STRUCTURE AND PROPERTIES OF ELECTROSPUN POLYMER FIBERS AND APPLICATIONS IN BIOMEDICAL ENGINEERING by Cheryl L. Casper Approved: __________________________________________________________ John F.

Chairperson, Department of Materials Science and Engineering Approved: __________________________________________________________ Eric W. Dean of the College of Engineering Approved: __________________________________________________________ Conrado M. Gempesaw II, Ph. Vice Provost for Academic and International Programs I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy.

Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Bruce Chase, Ph. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy.

Signed: __________________________________________________________ Darrin Pochan, Ph. Member of dissertation committee ACKNOWLEDGMENTS I would like to thank my advisor, Dr. John Rabolt, for his help and support during my graduate studies. My thanks to Dr.

Bruce Chase for his guidance and insightful discussions. I would also like to thank the other members of my committee, Dr. Darrin Pochan and Dr. Kristi Kiick, for the time and effort they put into being a part of my committee.

I would like to thank Dr. Kristi Kiick and Dr. Nori Yamaguchi for their insights, which greatly contributed to my research. I would also like to thank Dr.

Kirk Czymmek (DBI), Debbie Powell (DBI), and Gerald Poirier (Department of Physics and Astronomy) for their assistance with FESEM and LSCM. A sincere thanks to Dr. Cindy Farach-Carson, Dr. Weidong (William) Yang, and Anissa Brown in the Department of Biology for their time and expertise in the field of biology.

A special thank you to Dr. Patricia Cotts (Dupont) for GPC analyses and Nancy Tassi (Dupont) for her AFM expertise. I would like to thank NSF (NIRT and DMR) and IGERT for financial support of this work. I would like to thank the members of the Rabolt Research Group and the MSE staff for their support and friendship, which has enriched my graduate experience.

A special thank you to Dr. Lori Stephans and Amy Carpinelli for the introduction into the world of science and their mentoring along the way. I would also like to thank my family and friends for being so supportive throughout all my endeavors. iv Lastly, and most importantly, I am forever grateful to my parents for their unwavering support and encouragement, without them none of this would be possible.

This manuscript is dedicated to my parents, Walter and Christine Casper. v TABLE OF CONTENTS LIST OF TABLES. ix LIST OF FIGURES. xiv Chapter 1 Introduction .1 Introduction to Electrospinning.2 The Electrospinning Process .3 Effects of Experimental Parameters on the Electrospinning Process.1 Effect on Fiber Diameter.2 Factors Affecting Fiber Shape and Surface Morphology.

15 2 Influence of Humidity and Molecular Weight on Fiber Formation and Surface Morphology.1 Influence of Polymer Molecular Weight on Fiber Formation .2 Effect of Humidity on Surface Morphology .1 Varying Humidity Level.2 Effect of Molecular Weight on Pore Formation. 55 3 Functionalizing Electrospun Fibers with Biologically Relevant Macromolecules for Potential Biomaterial Applications .2 Synthesis of PEG-LMWH-Dye and LMWH-Dye Conjugates .1 Toluidine Blue Assays .2 Growth Factor Binding Assays .3 Growth Factor Release Assays.4 Cell Proliferation Assay .1 Electrospinning Dye-Labeled LMWH with PEO .2 Incorporation of PEG-LMWH-dye in PEO Electrospun Fibers .3 Changing the Carrier Polymer.4 Preliminary Cell Studies. 102 4 Attachment of Proteins to Electrospun Collagen and Gelatin Membranes for Use as Tissue Engineering Scaffolds.1 Determining Extent of Crosslinking via Kaiser Test .4 Synthesis and Attachment of Proteins to Electrospun Fibers .1 Heparin-BSA Synthesis and Biotinylation.2 Dot Blot Assay .3 Attachment of Heparin-BSA-Biotin and PlnDI- Biotin to Electrospun Fibers.4 Assessing Bioactivity of Heparin-BSA-Biotin and PlnDI via Growth Factor Binding Assays .3 Results and Discussion .1 Preparation and Crosslinking of Electrospun Collagen and Gelatin Membranes .2 Synthesis and Attachment of Proteins to Electrospun Matrices. 140 5 Conclusions and Future Work.

146 viii LIST OF TABLES Table 1.1 Examples of Polymers Fabricated into Fibers via Electrospinning .1 Electrospinning Various PS Molecular Weights in THF .2 Applying the Mark-Houwink Equation to Various PS Molecular Weights.3 Applying the Mark-Houwink Equation to Various PEO Molecular Weights.4 Pore Diameters of 190,000 g/mol PS/THF Fibers under Varying Humidity Levels.5 Pore Diameters of 560,900 g/mol PS/THF Fibers under Varying T Humidity Levels.6 Micro- and Nanopore Diameters of 31,600 g/mol PS/THF Beads T under Varying Humidity Levels .1 Samples Tested via Kaiser Test. 113 ix LIST OF FIGURES Figure 1.1 Schematic representation of the electrospinning apparatus .1 FESEM micrographs of (a)31,600 g/mol, (b) 44,100 g/mol, (c) 75,700 g/mol, (d) 171,000 g/mol, (e) 560,900 g/mol, and (f) 1,800,000 g/mol PS/THF electrospun fibers.2 FESEM micrograph of 31,600 g/mol PS electrospun from an 80 wt% solution of PS/THF.3 Scanning electron micrographs of 100,000 g/mol PEO/water solutions electrospun at various concentrations (a) 0.4 Scanning electron micrographs of 300,000 g/mol PEO/water solutions electrospun at various concentrations (a) 5 wt% and (b) 10 wt%.5 FESEM micrographs of 190,000 g/mol PS/THF fibers electrospun under varying humidity: (a) <25%, (b) 31-38%, (c) 40-45%, (d) 50-59%, (e) 60-72% .6 Pore diameter distributions of pores found on 190,000 g/mol PS/THF electrospun fibers at varying humidity ranges: (a) 31- 38%, (b) 40-45%, (c) 50-59%, (d) 60-72%.7 FESEM micrograph of 171,000 g/mol PS/THF fiber electrospun in 50% humidity .8 FESEM image of 560,900 g/mol fiber electrospun in (a) 31-38% humidity and (b) 40-45% humidity.9 FESEM image showing the two pore sizes on 31,600 g/mol PS/THF bead, 60-72% humidity .10 AFM Images, topography (left), phase (right) (a) 190,000 g/mol T PS fibers under 50-59% humidity, (b) 190,000 g/mol PS fibers under 60-72% humidity, (c) 560,900 g/mol PS fibers under 50- 59% humidity, (d) 560,900 g/mol PS fibers under 60-72% humidity .1 Scanning electron micrograph of 10 wt% PEO/H2O electrospun B B fibers.2 Scanning electron micrographs of (a) PEO/ LMWH-dye electrospun fibers and (b) beaded section of the fiber .3 Multiphoton microscopy images of electrospun PEO fibers (a) reflection mode and (b) fluorescence (5 µm scale bars).4 Multiphoton microscopy image of electrospun PEO/LMWH-dye fibers (5 µm scale bar).5 Scanning electron micrographs of electrospun PEO/PEG-LMWH- dye (a) fibers and (b) beaded section of the fiber.6 Multiphoton microscopy image of PEG-LMWH-dye/PEO electrospun fibers (5 µm scale bar).7 Scanning electron micrographs of (a) PLGA electrospun fibers, (b) PLGA/LMWH-dye fibers, and (c) PLGA/PEG-LMWH-dye fibers.8 Multiphoton microscopy images of electrospun PLGA fibers (a) transmitted light and (b) fluorescence (10 µm scale bars).9 Multiphoton microscopy images of electrospun (a) LMWH- dye/PLGA fibers (10 µm scale bar), (b) PEG-LMWH-dye/PGLA fibers (10 µm scale bar) and (c) z-stack of PEG-LMWH/PLGA fibers (20 µm scale bar).10 bFGF binding on electrospun fibers.11 Scanning electron microscopy images of PEG-LMWH-dye/PLGA fibers and LMWH-dye/PLGA fibers after incubation in PBS for 6 h, 1 day, 3 days, and 14 days.12 Retention of heparin within electrospun matrices as observed by multiphoton microscopy for the PEG-LMWH-dye/PLGA fibers and LMWH-dye/PLGA fibers after incubation in PBS for 6 h, 1 day, 3 days, and 14 days (10 µm scale bars).13 Retention of PEG-LMWH-dye within depth of the electrospun matrix after 14 days of incubation in PBS as imaged via multiphoton microscopy.14 Release of bFGF from PLGA, LMWH-Dye, and PEG-LMWH- Dye electrospun fibers after 5 h and 24 h incubation in PBS .15 bFGF binding on PLGA, LMWH-Dye, and PEG-LMWH-Dye electrospun fibers after 5 h and 24 h incubation in PBS .16 Digital picture displaying the visual differences observed in the binding of bFGF to PLGA, LMWH-Dye, and PEG-LMWH-Dye electrospun fibers after 5 h and 24 h incubation in PBS.17 Results from the BrdU cell proliferation assay .18 LSCM images of (a) LMWH/PLGA and (b) PEG-LMWH/PLGA fibers (blue) incubated for 8 days with MG63 cells (green) (10 µm scale bars).1 Schematic of the crosslinking apparatus .2 Sulfo-NHS(-LC)-Biotin reaction scheme.3 EDC/NHS reaction scheme .4 FESEM micrographs of electrospun (a) collagen and (b) gelatin.5 Scanning electron micrographs of crosslinked electrospun gelatin fibers before (left hand column) and after (right hand column) immersion in ethanol/water solution.6 LSCM images of gelatin scaffold seeded with MG63 cells.7 Image displaying the dot blot assay results.8 Digital image of heparin-BSA-biotin presence on electrospun mats .9 Detection of heparin-BSA-biotin complex in electrospun collagen and gelatin membranes.10 bFGF binding on collagen/heparin-BSA-biotin and gelatin/heparin-BSA-biotin electrospun membranes .11 Detection of biotin-containing proteins on electrospun collagen and gelatin fibers coupled via EDC/NHS reaction.12 bFGF binding to electrospun collagen and gelatin membranes coated with either heparin-BSA-biotin or PlnDI.13 Digital image of bFGF binding to fibers containing heparin-BSA- biotin or PlnDI. 137 xiii ABSTRACT Increased interest in nanotechnology has revived a fiber processing technique invented back in the 1930’s. Electrospinning produces nanometer to micron size fibers that are not otherwise achievable using conventional fiber spinning methods.

Due to small fiber diameters, high surface area, tailorable surface morphology, and the creation of an interconnected fibrous network, electrospun fibers have found use in a variety of applications. However, a multitude of parameters directly affect the electrospinning process thus requiring a fundamental understanding of how various parameters affect the process and resulting fiber properties. Accordingly, the focus of this dissertation is to provide insight on how solution characteristics and processing parameters directly affect the electrospinning process, and then apply this knowledge to create electrospun membranes for biomedical applications. Solution characteristics (polymer/solvent, molecular weight, concentration, viscosity) were found to directly affect the electrospinning process.

The effect of molecular weight, concentration, and viscosity was examined and it was determined that the intrinsic viscosity ([η]), which is a function of molecular weight, and solution concentration (C) directly affect the fiber formation process. If [η]C< 10, beads are formed instead of continuous fibers. This study illustrated the critical role that chain xiv entanglements and other intermolecular interactions play in the fiber formation process. Another fundamental study was carried out to investigate the effect of relative humidity on the surface morphology of electrospun fibers.

It was found that electrospinning in an atmosphere of 30% or more relative humidity creates porous features on the surface of polystyrene fibers. Pores were approximately 85 nm in diameter and increased to 135 nm with increasing humidity. Not only do pore diameters change with increasing humidity, but pore size distributions, shape, and pore depths are also affected by humidity levels. AFM studies revealed that pore depths increase from 48 nm to 220 nm as the relative humidity during electrospinning increased.

The effect of humidity on surface morphology is mainly attributed to evaporative condensation. The increases in pore diameter as a function of increasing molecular weight is believed to result from reduced polymer diffusion times since the diffusion coefficient is inversely proportional to the square of the polymer molecular weight. These fundamental studies provided insight on how to control the electrospinning process; this knowledge was then utilized to electrospin fibrous membranes for biomedical applications. One aspect of this work focused on incorporating low molecular weight heparin (LMWH) into electrospun fibers.