«GENERAL METHOD FOR PRODUCING BIODEGRADABLE NANOPARTICLES AND NANOFIBERS BASED ON NANOPOROUS MEMBRANES By PENG GUO A DISSERTATION PRESENTED TO THE ...»
GENERAL METHOD FOR PRODUCING BIODEGRADABLE NANOPARTICLES AND
NANOFIBERS BASED ON NANOPOROUS MEMBRANES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA© 2011 Peng Guo To my family, for their continued support and love
ACKNOWLEDGEMENTSThe work presented in this dissertation was conducted between August 2006 and May 2011 in graduate school at the University of Florida. I would like to acknowledge every individual who gives me help, advice, and encouragement.
I would like to sincerely thank my advisor, Prof. Charles Martin, who supports and guides me in both my research and life. Prof. Martin is always helpful and prepared to give me guidance. Besides research, I also matured as an individual in many areas; in relationships, such as trusting coworkers, self-confidence, and desire of exploration. My future career and life have and will continue to significantly benefit from working with Prof.
I am very grateful to have had the opportunity to work in the Martin lab, where I met and collaborated with many fantastic students and postdoctoral fellows. Dr. Pu Jin and Dr.
Jillian Perry helped me begin my thesis research. Both taught me many experiments and how to use instruments during my early graduate years. Dr. Jiahai (Jay) Wang also worked with me in the first three years and provided many valuable suggestions. Dr.
Hitomi Mukaibo, Dr. Lloyd Horne, Dr. Lindsay Sexton, Dr. Kaan Kececi, Dr. Dooho Park, Dr. Fan Xu, Gregory Bishop, Funda Mira, William Hardy and Li Zhao are group members who worked with me and provided valuable support and suggestions during my research.
I also would like to sincerely thank Prof. Richard Zare at Stanford University. During my visiting study in the Zare lab between April 2009 and May 2011, Prof. Zare took me in as one of his own graduate students, generously helping and training me to pursue my research. I also thank all the Zare lab group members for their support in my visiting research at Stanford University.
I also acknowledge our collaborators in Stanford Univeristy: Prof. Gerald Fuller and Dr. Michael Maas in Dept. of Chemical Engineering; Prof. Fan Yu and Dr. Michael Keeney in Dept. of Bioengineering; Prof. Ramin Beygui and Dr. Evgenios Neofytou in Medical Center Line: Cardiothoracic Surgery; Prof. A.C. Matin and Matthew Sylvester in Dept. of Microbiology and Immunology; and Prof. Christopher Contag and Dr. Tobi Schmidt in Dept. of Pediatrics. Without these great minds I could not accomplish close to as much.
My family has provided the most important support during my research. I am very fortunate to have such a loving and supportive family. I want to thank my lovely wife Jing Huang. During the long and sometimes challenging graduate study, Jing has always stood beside me, providing encouragement and support, through both the highs and lows.
Her delicate care and tenderness sustained me through every step of my research. I owe much to her. I want to thank my mother Shuying Wang for taking care of me, her kindness and support never wavered.
LIST OF TABLES
LIST OF FIGURES
CHAPTER 1 INTRODUCTION AND BACKGROUND
Current Progress in Biodegradable Nanostructures
Biodegradable Nanoparticles in Drug Delivery
Biodegradable Nanofibers in Tissue Engineering
Methods for Producing Biodegradable Nanoparticles and Nanofibers
Nanoparticle Formation Method
Nanofiber Formation Method
2 FORMULATING HYDROPHOBIC DRUG NANOPARTICLES
Formation of Hydrophobic Drug Nanoparticles
Analysis of Nanoparticles by Electron Microscope
Dynamic Light Scattering (DLS) Measurement
X-Ray Diffraction (XRD) Analysis
Dissolution Rate Measurement
Results and Discussion
3 GENERAL METHOD FOR PRODUCING POLYMERIC NANOPARTICLESUSING NANOPOROUS MEMBRANES
Formation of Ultrafine Chitosan Nanoparticles
Characterization of Chitosan Nanoparticles
Encapsulation of Rhodamine 6G in Chitosan Nanoparticles
Results and Discussion
4 BIODEGRADABLE POLYMERIC NANOPARTICLES AS DRUG DELIVERY VEHICLE
Synthesis and Characterization of PLGA-PEG Diblock Copolymer.................. 70 Formation of Drug Encapsulated Nanoparticles
Characterization of Drug Encapsulated Nanoparticles
Sustained Release Study of Drug Encapsulated Nanoparticles
Fluorescent Microscopy Imaging
In Vitro Cytotoxicity Study by Clonogenic Assay
In Vivo Cytotoxicity Study by Bioluminescence Imaging
Results and Discussion
Chitosan/luciferin Nanoparticles (CS/Luc NPs)
5 FORMATION OF BIODEGRADABLE NANOFIBERS BY NANOPOROUS MEMBRANE
Formation of Collagen Nanofibers
Characterization of Collagen Nanofibers
Isolation and Culture of Cardiac Stem Cells (CSCs)
Microscopy Fluorescent Imaging
Bioluminescence Imaging (BLI)
Results and Discussion
6 ORGANIC/INORGANIC HYBRID NANOFIBERS FOR TISSUE ENGINEERING... 99
Formation of Hybrid Nanofibers
Analysis of Nanofibers by Electron Microscope
Stem Cell Preparation
In Vitro Cytotoxicity Study by Cell Titer Assay
Fluorescent Microscopy Imaging
Results and Discussion
LIST OF REFERENCES
2-1 Summary of the DLS analysis of hydrophobic nanoparticles.
3-1 Statistical size and encapsulation efficiency data for rhodamine 6G loaded chitosan nanoparticles
4-1 DLS data for PLGA-PEG/MCHB and CS/Luc nanoparticles
1-1 Advantages of biodegradable nanoparticles for drug delivery.
(a) Scheme of nanofiber network of natural ECM.118 (b) A typical SEM image 1-2 of neural interconnect and ECM.119 Nerves and nerve bundles (yellow), ECM (red), and ganglion cells (blue).
1-3 Illustration of fabricating biodegradable nanoparticles through nano-emulsion method.120 (Reprinted with permission from Ref ; Copyright 2008 Elsevier.)
1-4 Illustration of fabricating biodegradable nanoparticles through ionic gelation method.121 (Reprinted with permission from Ref ; Copyright 2004 Elsevier.)
1-5 Illustration of fabricating biodegradable nanofibers through electrospinning method.122 (Reprinted with permission from Ref ; Copyright 2008 Brill.).... 39 2-1 Experimental set-up for the hydrophobic drug nanoparticle preparation using nanoporous membrane. M1 Pressure meter. M2 Flow meter
2-2 Chemical structures of three hydrophobic compounds: silybin, beta-carotene, and butylated hydroxytoluene
2-3 Photograph of a typical experimental setup.
2-4 SEM images of nanoporous membranes: anodized aluminum oxide (AAO) membrane with (a) 20 nm inlet and (b) 200 nm outlet.
2-5 Photograph of 40 mg silymarin nanoparticles obtained within 20 min by using AAO nanoporous membrane. A penny serves as a size marker.
2-6 SEM images of SM, BC, and BHT drug nanoparticles. a, d, and g are SM, BC, and BHT NPs via N-I method, respectively; b, e, and h are SM, BC, and BHT NPs via SEDS, respectively; c, f, and i are untreated SM, BC, and BHT, respectively. The scale bar is 500 nm in all the figures.
2-7 Hydrodynamic diameters of (a) SM, (b) BC, and (c) BHT drug nanoparticles determined by DLS
2-8 Effect of flow rate on diameter of the SM NPs obtained.
2-9 XRD pattern of silymarin nanoparticles and untreated silymarin powder............ 53 2-10 Dissolution profiles for silymarin nanoparticles and untreated silymarin powder in PBS (pH 7.4) at 37°C.
3-1 Method for producing chitosan nanoparticles by flow though a nanoporous membrane.
3-2 SEM images of nanoporous membranes: (a) track-etched polycarbonate (PCTE) membrane with 10 nm pores; and anodized aluminum oxide (AAO) membrane with (a) 20 nm inlet and (c) 200 nm outlet.
3-3 Typical TEM images of chitosan nanoparticles (CSNPs) prepared by using (a) the PCTE membrane; and (b) the AAO membrane. In these TEM images, the black area represents the nanoparticle, and the grey area represents the background
3-4 Comparison of size distributions of chitosan nanoparticles (CSNPs) prepared by using different nanoporous membranes determined by dynamic light scattering: (a) size of CSNPs obtained by PCTE membrane; and (b) size of CSNPs obtained by AAO membrane
3-5 Effect of solution flow rate on the diameter of the chitosan nanoparticle obtained
3-6 Effect of the viscosity of the chitosan feed solution on the diameter of the nanoparticles obtained.
3-7 Typical TEM images of chitosan-rhodamine 6G nanoparticles prepared by using (a) the PCTE membrane and (b) the AAO membrane. In these TEM images, the black area represents the nanoparticle, and the grey area represents the background.
3-8 Comparison of size distributions of chitosan-rhodamine 6G nanoparticles prepared by using different nanoporous membranes determined by dynamic light scattering: (a) PCTE membrane; and (b) AAO membrane
4-1 NMR characterization of (a) PLGA, (b) PLGA-PEG diblock copolymer.............. 80 4-2 Typical SEM image of PLGA-PEG/MCHB nanoparticles
4-3 Hydrodynamic diameter of PLGA-PEG/MCHB NPs determined by DLS............ 81 4-4 In vitro sustained release profile of PLGA-PEG/MCHB NPs
4-5 In vitro cytotoxicity study of PLGA-PEG/MCHB NPs.
4-6 Fluorescent image of PC-3 cell incubated with (a) PLGA-PEG/MCHB NPs, and (b) PLGA-PEG/CNOB NPs
4-7 Typical SEM image of CS/Luc nanoparticles.
4-8 Hydrodynamic diameter of CS/Luc NPs determined by DLS.
4-9 In vitro sustained release profile of CS/Luc NPs
4-10 In vivo biotoxicity study of CS/Luc NPs by bioluminescence imaging................. 84 5-1 Method for producing collagen nanofibers by flowing though a nanoporous membrane.
5-2 Typical SEM images of collagen nanofibers prepared by using the PCTE membrane at (a) high magnification and (b) low magnification
5-3 Typical TEM images of (a) a bundle of collagen nanofibers (b) a single collagen nanofiber. Inset is the related selected area electron diffraction pattern (SAED pattern).
5-4 Photograph of collagen nanofibrous scaffold prepared by N-I method in 2 h. A penny is used as a size marker.
5-5 SEM images of (a) collagen nanofibers prepared by N-I method (b) collagen film prepared without nanoporous membrane.
5-6 Effect of nanopore size on the diameter of the collagen nanofibers................... 97 5-7 Rheology study of collagen nanofibrous scaffold
5-8 (a) Fluorescent microscope imaging of CSCs in (A) and (C) blank control (low and high magnification); (B) and (D) collagen nanofibrous scaffold (low and high magnification). In fluorescent microscope images, bright area represents CSCs, and black area represents background. (b) bioluminescence image of CSCs proliferation on blank control and collagen nanofibrous scaffold.............. 98 6-1 Experimental setup and proposed model for the formation of mineralized collagen fibers.
6-2 (a, b) Unmineralized collagen fibers, (c, d) Mineralized collagen fibers (1 mM CaCl2), (e, f) Mineralized collagen fibers (2.5 mM CaCl2) and (g,
h) Mineralized collagen fibers (5 mM CaCl2). Inset images in (b, d, f, h) are SAED patterns
6-3 (a) Bundle of PAA/CaCO3 nanofibers (b) TEM micrograph and SAED pattern of a PAA/CaCO3 (c) Flattened PILP droplets, (d) PAA/Calcium Phosphate nanofibers
6-4 Proliferation of hADSC's on nanofibers. * indicate statistical difference between groups at the same timepoint
6-5 Alkaline phosphatase production from hADSC's cultured on nanofibers. * indicates statistical difference at the same timepoint
6-6 Fluorescent images of hADSC’s cultured on nanofibers. The green indicates actin filaments while blue indicates cell nuclei.
Chair: Charles R. Martin Major: Chemistry Biocompatible and biodegradable nanostructured materials have attracted more and more attention since they offer numerous exciting possibilities in medical sciences, such as drug delivery and tissue engineering. The increasing need for novel drug delivery systems with enhanced specificity/activity and reduced side toxicity has led to the development of nano-sized drug vehicles, which provide the advantage of delivering small molecular drugs, as well as macromolecules, via both targeted delivery and controlled release. For tissue engineering, considerable effort has been made to develop three dimensional artificial nanofibrous scaffolds, which closely resemble the natural protein nanofiber network in the extracellular matrix (ECM).