«by PARIMAL V. BAPAT RENATO P. CAMATA, COMMITTEE CHAIR SUSAN L. BELLIS S. AARON CATLEDGE DERRICK R. DEAN YOGESH K. VOHRA A DISSERTATION Submitted to ...»
GAS PHASE LASER SYNTHESIS AND PROCESSING OF CALCIUM PHOSPHATE
NANOPARTICLES FOR BIOMEDICAL APPLICATIONS
PARIMAL V. BAPAT
RENATO P. CAMATA, COMMITTEE CHAIR
SUSAN L. BELLIS
S. AARON CATLEDGE
DERRICK R. DEAN
YOGESH K. VOHRA
A DISSERTATIONSubmitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy
GAS PHASE LASER SYNTHESIS AND PROCESSING OF CALCIUM PHOSPHATE
NANOPARTICLES FOR BIOMEDICAL APPLICATIONS
PARIMAL V. BAPAT
ABSTRACTBiochemical processes make pervasive use of calcium and phosphate ions.
Calcium phosphate salts that are naturally nontoxic and bioactive have been used for several medical applications in form of coatings and micropowders. Nanoparticle-based calcium phosphates have been shown to be internalized by living cells and be effective in DNA transfection, drug delivery, and transport of fluorophores for imaging of intracellular processes. They are also expected to interact strongly with cell adhesive proteins and are therefore promising elements in approaches to mimic the complex environment of the extra cellular matrix of bone. Harnessing this biomedical potential requires the ability to control the numerous characteristics of nanophase calcium phosphates that affect biological response, including nanoparticle chemical composition, crystal phase, crystallinity, crystallographic orientation of exposed faces, size, shape, surface area, number concentration, and degree of aggregation. This dissertation focuses on the use of laser-induced gas-phase synthesis for creation of calcium phosphate nanoparticles, and corresponding nanoparticle-based substrates that could offer new opportunities for guiding biological responses through well-controlled biochemical and topological cues.
ii Gas-phase synthesis of nanoparticles has several characteristics that could enhance control over particle morphology, crystallinity, and surface area, compared to liquid-phase techniques. Synthesis from gas-phase precursors can be carried out at high temperatures and in high-purity inert or reactive gas backgrounds, enabling good control of chemistry, crystal structure, and purity. Moreover, the particle mean free path and number concentration can be controlled independently. This allows regulation of inter- particle collision rates, which can be adjusted to limitaggregation. High-temperature synthesis of well-separated particles is therefore possible. In this work high power lasers are employed to vaporize microcrystalline calcium phosphate materials to generate an aerosol of nanoparticles which is further processed and deposited using principles of aerosol mechanics.
Particles and resulting particle-based systems are analyzed by transmission electron microscopy, atomic force microscopy, X-ray diffraction, and optical absorption.
Obtained substrates are functionalized with cell adhesive peptides. Findings show that laser-induced gas-phase synthesis provides attractive new dimensions in the controlled fabrication of calcium phosphate nanoparticles, including manipulation not only of size and chemical composition, but also crystal phase make-up, fractal structure, and nanotopography of derived substrates.
I would like to thank my advisor Dr. Renato P. Camata for everything he has done for me as a graduate mentor. I am grateful for his guidance, support, encouragement and patience he has given me throughout my years of graduate study. I am privileged to have developed as a researcher under his leadership. I must thank Dr. Yogesh K. Vohra for his constant support as my research committee member as well as being our graduate program director. I particularly enjoyed his lectures on solid state physics. I would also like to extend my gratitude towards my research committee members Dr. Aaron S.
Catledge, Dr. Susan L. Bellis, and Dr. Derrick R. Dean for their valuable time and suggestions throughout my research.
I would like to thank Dr. David Shealy, the department chair, and rest of the physics department faculty and staff members. I must thank Jerrie McCurry, Amanda Dickinson and Mark Case for their valuable support. My special thanks to Mr. Jerry Sewell of the physics department machine shop for building my experimental equipment.
I also enjoyed my relationship with Dr. Vinoy Thomas who always helped and guided me sometime as a researcher, sometime as a friend.
I would like to thank my lab group members Dr. Jonathan Williams and Dr.
Marco Bottino for training me in x-ray diffraction and atomic force microscopy. I would like to thank Finn Perkins for helping me with UV-Vis spectroscopy. I would also like to thank REU student Becky Kraft who helped me in designing and implement my
thank Bonnie Culpepper for helping me with peptide studies.
I enjoyed the time that I have spent with my colleagues: Dr. Gopi Samudrala, Walter Uhoya, Pramesh Singh, Parul Tyagi, Anitha Armugam, Sonal Singh, Leigh Booth, Sunil Karna and Hadiyah Green. I want to thank all my friends, roommates and particularly my girlfriend, Ketaki More who have always supported me during my graduate studies.
I would like to thank the Alabama Commission on Higher Education for the award of a fellowship from the Graduate Research Scholarship Program. This work was supported by the Brazilian Synchrotron Light Laboratory (LNLS)/Brazilian Biosciences National Laboratory (LNBio) under proposal D12A-XRD1-9943. Authors are thankful to Prof. Daniel Zanetti de Florio, Eduardo Souza Santos, and Pedro Ivo Braun Ferreira for assistance during experiments at LNLS, to Dr. Saulius Drukteinis for assembling the system to control the partial pressure of water, and to Justin T. Marbutt for processing of the AFM data. Instrumentation used in this research was supported by the National Science Foundation under grant number DMR-0116098.
Finally, my deepest gratitude goes to my parents for their unending love, support, encouragement and sacrifices made for my studying and living abroad.
LIST OF TABLES
LIST OF FIGURES
1.1. Background and Motivation
1.2. Statement of Dissertation Objectives
1.3. Dissertation Outline
2. CALCIUM PHOSPHATE BIOCERAMICS AND NANOPARTICLES................. 7
2.2. Fundamental Characteristics of Calcium Phosphate Bioceramics
2.3. Calcium Phosphate Bioceramics in the Form of Nanoparticles
2.4. Interaction of Calcium Phosphate Nanoparticles with Biological Systems..... 17
2.5. Research Needs and Opportunities in Calcium Phosphate Bioceramic Nanoparticles
3. GAS PHASE LASER SYNTHESIS AND CHARACTERIZATION OF CALCIUMPHOSPHATE NANOPARTICLES
3.2. Generation of Nanoparticles in the Gas Phase by Laser Ablation
3.2.1. Basic Physical Phenomena
3.2.2. The Aerosol State
3.2.3. Dynamics of Calcium Phosphate Nanoparticle Aerosols
3.3. Processing and Deposition of Nanoparticles from the Aerosol State............... 33 3.3.1. Temperature Control
vii 3.3.2. Synthesis in Reactive Environments
3.3.3. Charging of Aerosol Nanoparticles
3.3.4. Electrical Mobility Classification
3.3.5. Deposition of Aerosol Nanoparticles on Substrates
3.4. Laser Aerosol Synthesis of Calcium Phosphate Nanoparticles
3.5. Characterization of Calcium Phosphate Nanoparticles
3.5.1. Transmission Electron Microscopy
3.5.2. Atomic Force Microscopy
3.5.3. X-ray Diffraction
3.5.4. Optical Absorption
4. FRACTAL-LIKE NANOPARTICLES FORMED BY LASER ABLATION OF CALCIUM PHOSPHATE
4.2. Synthesis Configuration
4.3. General Nanoparticle Characteristics
4.4. Effect of Laser Energy Density
4.5. Effect of Total Pressure
4.6. Summary of Results
5. AGGREGATION-FREE SIZE-CONTROLLED HYDROXYAPATITE NANOPARTICLES
5.2. Synthesis Configuration
5.3. Nanoparticle Morphology and Size Control
5.4. Nanoparticle-based Substrates with Controlled Nanotopography
5.5. Effect of Temperature and Partial Pressure of Water on Nanoparticle Crystal Structure
5.6. Peptide Adsorption on HA Nanoparticle-based Substrates
5.7. Optical Absorption Measurements
5.8. Summary of Results
6. NANOPARTICLE-BASED BIPHASIC CALCIUM PHOSPHATE SUBSTRATES
6.2. Post-deposition Processing and Crystal Phase Make-up Analysis
6.3. Summary of Results
7. SUMMARY AND CONCLUSIONS
8. FUTURE STUDIES
The solubility product constant, K, and the Ca/P molar ratio of calcium phosphate I.
II. Distortions in unit cell parameters of TTCP structure as a function of effect of synthesis temperature.
1 Crystal structure of hydroxyapatite. Projection along the (001) direction of the hexagonal structure. In this projection the c-axis of the crystal is normal to the surface of the page.
2 Crystal structure of tetracalcium phosphate. Projection along the (001) direction of the monoclinic structure. In this projection the a-axis of the structure is normal to the page.
3 Illustration of cellular uptake of genetic material enabled by plasmid DNA conjugation with calcium phosphate nanoparticles..
4 Schematic representation of the main processes involved in laser-induced gasphase calcium phosphate nanoparticle synthesis and the preparation of substrates comprising the corresponding size-selected nanoparticles on bioinert surfaces... 29 5 Diagram of the ablation chamber used for calcium phosphate nanoparticle generation, featuring a rotating target holder in a tube furnace. The laser beam has access to the chamber through a UV window. A continuous flow of Ar/H2O is maintained through the chamber.
6 Schematic diagram of the apparatus used for creating a controlled Ar/H2O mixture. MFC, MFC 1, MFC 2 are the mass flow controllers used for monitoring and controlling the Ar gas flow through the chamber.
8 Diagram of laser aerosol process showing laser ablation followed by neutralization of the aerosol by a Kr-85 diffusion charger and subsequent flow into the differential mobility analyzer.
9 Schematic diagram of differential mobility analyzer featuring coaxial capacitor in which charged nanoparticles from a polydisperse aerosol migrate across a particle-free laminar sheath gas flow due to an applied electric field................... 44 10 Schematic diagram of apparatus used for gas-phase synthesis of calcium phosphate nanoparticles featuring temperature and humidity-controlled ablation chamber, differential mobility analyzer (DMA), electrostatic precipitator, and aerosol electrometer. MFC, MFM, V, and VP refer to mass flow controller, mass flow meter, manual valve, and vacuum pump, respectively.
11 Photograph of the apparatus gas-phase laser synthesis shown schematically in Fig.
10 and implemented at UAB in the Center for Nanoscale Materials and Biointegration.
12 Transmission electron microscopy (TEM) image of 10 nm calcium phosphate nanoparticles deposited on a TEM grid
13 Atomic force microscopy scan of well separated 30-nm calcium phosphate nanoparticles deposited on a silicon substrate. Nominal areal number concentration based on deposition time and gas-phase concentration is 2 × 108 cm-2.
14 Height profiles of select nanoparticles (denoted by Green, Blue and orange lines) obtained from a 5 µm × 5 µm scan area on sample size-selected for 30 nm........55 xii 15 Transmission electron microscopy images of nanoparticles generated by laser ablation of hydroxyapatite at room temperature in an inert ambient. The images underscore the variety of nanoparticle morphologies that can be generated using the laser/aerosol method.
16 Transmission electron microscopy images showing effects of laser energy density and total pressure on nanoparticle shape and morphology.
(a) TEM image of nanoparticles synthesized with laser energy density of 5 J/cm2, 17 p H 2O =160 mbar, and T = 800°C with DMA set at a voltage of –324 V. Inset shows particle size distribution from image. Solid line fitted to the data points represents two log-normal distributions. (b) Close-ups of select nanoparticles.
Size distributions of samples deposited with DMA voltage of (c) –231 V and (d) – 430 V. In parts (c) and (d), the solid lines fitted to the data points represent superposition of two log-normal distributions (dotted lines). (e) TEM image of polydisperse nanoparticles and conrresponding size distribution (log-normal fit)
AFM scans of samples produced with laser energy density of 5 J/cm2, p H 2O =160 18 mbar, and T = 800°C with DMA at a voltage of –324 V. Samples deposited for (a) 5 min., (b) 30 min., and (c) 90 min