Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 113-116).

This thesis presents a novel ZnO-hydrogel based fluorescent colloidal semiconductor nanomaterial system for potential bio-medical applications such as bio-imaging, cancer detection and therapy. The preparation of ZnO nanoparticles and their surface modification to make a biocompatible material with enhanced optical properties is discussed. High quality ZnO nanoparticles with UV band edge emission are prepared using gas evaporation method. Semiconductor materials including ZnO are insoluble in water. Since biological applications require water soluble nanomaterials, ZnO nanoparticles are first dispersed in water by ball milling method, and their aqueous stability and fluorescence properties are enhanced by incorporating them in bio-compatible poly N-isopropylacrylamide (PNIPAM) based hydrogel polymer matrix. The optical properties of ZnO-hydrogel colloidal dispersion versus ZnO-Water dispersion were analyzed. The optical characterization using photoluminescence spectroscopy indicates approximately 10 times enhancement of fluorescence in ZnO-hydrogel colloidal system compared to ZnO-water system. Ultrafast time resolved measurement demonstrates dominant exciton recombination process in ZnO-hydrogel system compared to ZnO-water system, confirming the surface modification of ZnO nanoparticles by hydrogel polymer matrix. The surface modification of ZnO nanoparticles by hydrogel induce more scattering centers per unit area of cross-section, and hence increase the luminescence from the ZnO-gel samples due to multiple path excitations. Furthermore, surface modification of ZnO by hydrogel increases the radiative efficiency of this hybrid colloidal material system thereby contributing to enhanced emission.

Although medical devices and biomaterial implants are used clinically in a variety of applications, the process of implanting them damages local tissue and initiates a localized non-specific inflammatory response that is detrimental to device performance. Extensive research efforts have focused on developing material surface treatments and systems to deliver anti-inflammatory agents to abrogate such biomaterial-mediated inflammation, yet long-term use of these traditional materials in vivo is limited due to continued inflammation and fibrous encapsulation. This work aims to address these limitations by developing a versatile implant coating with non-fouling properties using a system based on hydrogel microparticles (i.e. microgels). The overall objective of this project was to evaluate host responses to these microgel coatings. Microgel particles were synthesized from poly(N-isopropyl acrylamide) cross-linked with poly(ethylene glycol)-diacrylate and were successfully deposited onto polymeric substrates using a simple and reproducible spin coating technique. We determined that microgel-coated samples adsorbed significantly lower levels of human fibrinogen than controls. Further characterization using an in vitro culture system demonstrated that microgel coatings significantly reduced the adhesion and spreading of murine macrophages and primary human blood-derived monocytes compared to controls. Materials were then evaluated for early cellular responses following implantation in the intraperitoneal cavity of mice to model acute inflammation. Analyses of explanted biomaterials using immunofluorescence staining techniques revealed that microgel-coated samples significantly reduced the density of surface-adherent cells. Additional analysis using flow cytometry revealed that microgel-coated samples exhibited significantly lower levels of pro-inflammatory cytokines in adherent leukocytes compared to controls, indicating that these coatings modulate cellular pro-inflammatory activities. Finally, we implanted samples subcutaneously in rats to determine the efficacy of microgel coatings at longer time points using an established model of chronic inflammation. Explants were processed histologically and stained for various markers. Importantly, staining demonstrated that the microgel coatings significantly reduced fibrous capsule thickness, the capsules appeared less compact and structurally ordered than controls, and also contained significantly fewer cells. Collectively, these results demonstrate that microgel particles can be applied as polymeric coatings to modulate inflammation and achieve more desirable host responses in vivo, with the potential to extend implant lifetime.

Chronic wounds pose a major threat to healthcare systems, and can be caused by a variety of factors, from diabetes to battlefield injuries. Traditional wound care does not account for a patient’s specific circumstances, and is only effective in up to 50% of cases. As such, there is a growing need for smarter wound healing technologies that can be used in a wide array of settings, from low resource hospitals to at home, and that provide customized treatments that can be administered without trained professionals. In this dissertation, we detail the development of technologies for customized treatments to accelerate wound healing. In Aim 1, we used a water-activated, electronics-free dressing to accelerate wound healing in a diabetic mouse model. Electrical stimulation has previously been used to improve wound healing; however, common dressings often require that the wearer be physically connected to large benchtop electronics, are expensive to produce, and/or contain toxic elements. We demonstrated that this device, developed by our collaborators, accelerated time to wound closure by approximately 30%, on par with other, more complex devices, and further improved angiogenesis, collagen intensity, and reduced inflammation when compared with the controls. Furthermore, we demonstrated that this device is biocompatible and does not affect mouse behavior; the device does not heat up when activated, and did not impact the distance the mice traveled during a ten-minute measurement window. We are exploring additional use cases for this technology to further accelerate wound healing, including through iontophoresis, the use of electric currents to transmit drugs through the skin. In Aim 2, we developed ultrasound-responsive, perfluorocarbon-based nanoparticles for spatiotemporal control of payload release. Focused ultrasound can be used to selectively and noninvasively trigger perfluorocarbon vaporization, releasing the payload from the triggered nanoparticles without disrupting nearby nanoparticles. Furthermore, these systems can be designed to remain stable when not in use, and to then release their payloads at safe acoustic pressures. We developed PLGA-coated, perfluorocarbon-based nanoparticles of various sizes, geometries, and with payloads. We used B-mode imaging and acoustic signal analysis to determine the acoustic thresholds of these nanoparticles, and then incorporated the nanoparticles into gels, from which we measured their payload release upon exposure to focused ultrasound. Initial in vivo testing of these nanoparticles showed that they remained stable until application of focused ultrasound. Such a technology has the potential to customize healing treatments, releasing specific payloads when and where they are most needed. In Aim 3, we integrated components of a closed-loop wound healing system in vitro and in vivo. This system comprises an ultrasound bandage to provide both sensing of the wound and treatment to the wound, sensors to analyze the wound state, drug delivery depots to selectively release payload into the wound, and a machine learning algorithm to guide treatment based on sensor values. We developed ultrasound-responsive microcapsules to selectively release drugs, and tested these in vitro and in a diabetic mouse wound model. We tested the ultrasound-responsiveness of alginate-acrylamide hydrogels in vitro and in vivo. We additionally tested versions of the ultrasound bandage and lactate sensors in vivo, and tested various combinations of these technologies. We tested the release of growth factor from the hydrogels using focused ultrasound while collecting ultrasound images for analysis, and demonstrated that a commercial-grade ultrasound probe can differentiate between wound healing states, which suggests that this technology will be translatable beyond the lab. Future work could demonstrate a truly closed-loop system, and could move beyond the diabetic mouse model, to one more similar to healing in humans. In this dissertation, we demonstrated technologies that can, individually or together, be used to improve wound healing in a variety of settings. Overall, this work advances the field of wound healing and demonstrates a suite of tools that can be used to provide customized treatments based on a patient’s needs, towards a vision for closed-loop wound healing systems.

Heart failure is the leading cause of death worldwide. In 2013, the American Heart Association estimated that one American will die of cardiovascular disease every 39 seconds. While heart transplantation is the most viable treatment option, the limited availability of donor hearts has necessitated the search for treatment alternatives such as the use of adult stem cells for cardiac repair and regeneration. Following myocardial infarction (MI), the inflammatory cardiac microenvironment, limited survival of stem/progenitor cells, myocardial scarring and fibrosis affect cardiac regeneration. This dissertation examines adult stem cell based approaches for cardiac regeneration by studying the effect of i) H₂O₂- mediated oxidative stress on mesenchymal stem cells, ii) Notch1 activation in cardiac progenitor cells using a self-assembling peptide hydrogel containing the Notch1 ligand mimic RJ in vitro and functional consequences in a rat model of MI. Through these approaches, the central hypothesis that modulation of stem cell response using cues such as oxidative stress and activation of Notch1 signaling can improve functional outcome following myocardial infarction has been studied.

Biodegradable hydrogels have become very promising materials for a number of biomedical applications, including tissue engineering and drug delivery. For optimal tissue engineering design, the mechanical properties of hydrogels should match those of native tissues as closely as possible because these properties are known to affect the behavior and function of cells seeded in the hydrogels. At the same time, high water-contents, large mesh sizes and well-tuned degradation rates are favorable for the controlled release of growth factors and for adequate transport of nutrients through the hydrogel during tissue regeneration. With these factors in mind, the goal of this research was to develop and investigate the behavior of injectable, biodegradable hydrogels with enhanced stiffness properties that persist even at high degrees of swelling. In order to do this, degradable functionalities were incorporated into photo-crosslinkable poly(ethylene glycol) and poly(acrylic acid) hydrogels, and these two components were used to make a series of double-network hydrogels. Synthesis of the precursor macromers, photopolymerization of the hydrogels, and structural parameters of the hydrogels were analyzed. The composition and the molecular weight between crosslinks (Mc) of the hydrogel components were varied, and the degradation, swelling, thermal and mechanical properties of the hydrogels were characterized over various time scales. These properties were compared to corresponding properties of the component single-network hydrogels.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been widely used in many biomedical applications. Ultraviolet (200-400 nm) or visible (400-800 nm) light can interact with light-sensitive compounds called photoinitiators to form radical species that trigger photopolylmerization. Since UV light has potential to cause cell damage, visible light-mediated photopolymerization has attracted much attention. The conventional method to fabricate hydrogels under visible light exposure requires usage of co-initiator triethanolamine (TEA) at high concentration (∼200 mM), which reduces cell viability. Therefore, the first objective of this thesis was to develop a new method to form poly(ethylene glycol)-diacrylate (PEGDA) hydrogel without using TEA. Specifically, thiol-containing molecules (e.g. dithiothreitol or cysteine-containing peptides) were used to replace TEA as both co-initiator and crosslinker. Co-monomer 1-vinyl-2-pyrrolidinone (NVP) was used to accelerate gelation kinetics. The gelation rate could be tuned by changing the concentration of eosinY or NVP. Variation of thiol concentration affected degradation rate of hydrogels. Many bioactive motifs have been immobilized into hydrogels to enhance cell attachment and adhesion in previous studies. In this thesis, pendant peptide RGDS was incorporated via two methods with high incorporation efficiency. The stiffness of hydrogels decreased when incorporating RGDS. The second objective of this thesis was to fabricate hydrogels using poly(ethylene glycol)-tetra-acrylate (PEG4A) macromer instead of PEGDA via the same step-and-chain-growth mixed mode mechanism. Formation of hydrogels using PEGDA in this thesis required high concentration of macromer (∼10 wt.%). Since PEG4A had two more functional acrylate groups than PEGDA, hydrogels could be fabricated using lower concentration of PEG4A (∼4 wt.%). The effects of NVP concentration and thiol content on hydrogel properties were similar to those on PEGDA hydrogels. In addition, the functionality and chemistry of thiol could also affect hydrogel properties.