Dissolvable hydrogel-based wound dressings for in vivo applications

Controlled hydrogel dissolution allows for: 1) atraumatic material removal after it served its function, 2) site-specific delivery of encapsulated therapeutics (e.g., proteins, small molecules), and 3) a tailored administration of an agent with high efficiency. Dissolution of covalently crosslinked hydrogels has been accomplished by incorporating cleavable moieties that undergo ester hydrolysis or enzymatic degradation. Recently, thiol-disulfide exchange, retro Michal-type reactions, retro Diels-Alder reactions, and thiol-thioester exchange chemistries have gained attention, as they provide a responsive synthetic handle for engineering hydrogel dissolution rates. We synthesized, characterized and tested in vivo two on-demand dissolvable dendritic thioester hydrogel dressings for second-degree burn care and hemorrhage control. The hydrogels are composed of lysine-based dendrons and PEG-based crosslinkers, which were prepared in high yields. In context of hemorrhage, there is an unmet clinical need for an on-demand dissolvable sealant for non-compressible hemorrhage or areas of body not amenable to treatment with a torniquet. In a model of in vivo hemorrhage control of intra-abdominal wounds, our hydrogel reduced blood loss by 33% in severe hepatic hemorrhage and by 22% in aortic injury, as compared to untreated controls. There is an unmet clinical need for a second-degree burn dressing that can be removed atraumatically and serve as a barrier to bacterial infection. When our hydrogel was used as a dressing, local and systemic bacterial proliferation after wound contamination was significantly lower than in the untreated group. The total bacterial burden of the burn wound in the positive controls was significantly higher than in the hydrogel group and the negative controls (1.39x10E8 ± 8.30x10E7 CFU/g v. 4.04x10E3 ± 3.99x10E3 CFU/g v. 6.88x10E2 ± 6.38x10E2 respectively; P = 0.009). Also, the total systemic bacterial burden in the positive controls was significantly higher than the hydrogel group and the negative controls (9x10E2 ± 7.76x10E7 CFU/g v. 5x10E1 ± 0 CFU/g v. 5x10E1 ± 0 CFU/g, respectively; P = 0.031). A unique feature of both hydrogel systems is their capability to be dissolved on-demand via thiol-thioester exchange reaction with a biocompatible solution following its initial application – thus the wound area can be re-exposed to allow for definitive surgical care

Biocompatibility and Physiological Thiolytic Degradability of Radically Made Thioester-Functional Copolymers: Opportunities for Drug Release

Being nondegradable, vinyl polymers have limited biomedical applicability. Unfortunately, backbone esters incorporated through conventional radical ring-opening methods do not undergo appreciable abiotic hydrolysis under physiologically relevant conditions. Here, PEG acrylate and di(ethylene glycol) acrylamide-based copolymers containing backbone thioesters were prepared through the radical ring-opening copolymerization of the thionolactone dibenzo[c,e]oxepin-5(7H)-thione. The thioesters degraded fully in the presence of 10 mM cysteine at pH 7.4, with the mechanism presumed to involve an irreversible S–N switch. Degradations with N-acetylcysteine and glutathione were reversible through the thiol–thioester exchange polycondensation of R–SC(═O)–polymer–SH fragments with full degradation relying on an increased thiolate/thioester ratio. Treatment with 10 mM glutathione at pH 7.2 (mimicking intracellular conditions) triggered an insoluble–soluble switch of a temperature-responsive copolymer at 37 °C and the release of encapsulated Nile Red (as a drug model) from core-degradable diblock copolymer micelles. Copolymers and their cysteinolytic degradation products were found to be noncytotoxic, making thioester backbone-functional polymers promising for drug delivery applications

Application of Dynamic Combinatorial Chemistry to Identify New Compounds that Bind G-Quadruplex DNA & Probing the Role of the Cation-π Interaction Between the HP1 Chromodomain and Methylated Lysine Using Unnatural Amino Acids

This dissertation discusses two different projects. The first project involves the development of cyclic-peptide acridine conjugates in the effort to identify molecules that can selectively bind to and stabilize G-quadruplex DNA. The second project seeks to probe the role of the cation-π interaction in the binding of the HP1 chromodomain to trimethylated lysine 9 of histone 3 (H3). In recent years, interest in the G-quadruplex DNA structure has increased enormously due to the unique physical properties of this secondary DNA structure as well as the presence of guanine-rich sequences in biologically functional regions of many genomes. Given the propensity of G-quadruplex structures to control many biological functions, it has become desirable to identify small molecules that can bind to and stabilize G-quadruplexes. This work aims to develop quadruplex ligands that exhibit selectively over not only duplex DNA but also over various quadruplex sequences. It was believed that cyclic peptides could deliver selectivity for the quadruplex structure over duplex DNA while also providing the added advantages of mimicking native protein structure, displaying enhanced metabolic stability and possessing structural preorganization. Using a strategy that has been developed in our lab, we propose screening libraries of cyclic peptides generated in situ using thiol-thioester exchange for dynamic combinatorial chemistry (DCC). These libraries can efficiently be screen against different quadruplex sequences as well as duplex DNA in order to determine the selectivity of each species. In the second project, we sought to characterize the noncovalent interactions responsible for the recognition of trimethylated lysine 9 of histone 3 by the aromatic pocket of the HP1 chromodomain. Lysine can exist in three distinct methylation states under the control of highly specific methyl transferases or demethylases. These methylation states serve to turn on specific protein-protein interactions with partners that specifically recognize the methylated side chain. Recognition and affinity is mainly derived from cation-π interactions between the positively charged cationic side chain and the electron rich π surfaces of nearby aromatic rings. This interaction can be quantified by incorporation of fluorinated derivatives of the aromatic amino acids responsible for the binding of H3K9Me3 to the HP1 chromodomain. The cation-π interaction between the aromatic pocket of the HP1 chromodomain and H3K9Me3 can be revealed by incorporation of a series of fluorinated amino acid analogues. A linear correlation between binding affinity and the calculated magnitude of the cation-pi interaction of those groups indicates a cation-π interaction. Because many reader proteins for methylated lysine have an aromatic cage in their binding pockets, findings from the investigation of the HP1 chromodomain will provide broad insight into this class of proteins.Doctor of Philosoph

In situ dissolvable hydrogels for biomedical applications

Hydrogels are hydrophilic, three-dimensional polymeric networks prepared through chemical or physical conjugation. Hydrogels are recognized for their tunable properties, specifically through changes in the backbone of the polymers, such as 1) modifying the number of hydrophobic chain lengths, 2) adding or removing cleavable linkages, 3) varying reactive-end groups, 4) increasing or decreasing the weight percent of the hydrogel, and 5) combining two or more hydrogel networks into one, namely creating an interpenetrating network. We synthesized and characterized on- and off-demand, dissolvable hydrogels for use as burn wound dressings, polypectomy bandages, and vascular occlusion devices, and within interpenetrating networks. The hydrogels are composed of PEG-based crosslinkers, and PEI-based hyperbranched macromers which were prepared in high yields. In context of burn wound dressings, there is an unmet need for an adherent dressing with ease of removal, such as a dissolvable hydrogel dressing. In a model of in vivo porcine burn wounds, our hydrogel shows superior burn healing relative to traditional dressings such as sterile gauze pad and non-adherent foam dressings. When our hydrogel was removed, no newly formed tissue adhered to the dressing, and immunohistochemical stains exhibit improved inflammation and necrosis. When our hydrogel was used as an in vivo polypectomy sealant, we observed ease of application and adhesion to the colon, despite peristalsis. In in vitro studies, we observe no migration of bacteria through the hydrogel. As a vascular occlusion device, our hydrogels withstand an ex vivo burst pressure of up to 440mmHg on average, over 3x that of arterial pressure. Furthermore, we prepared an interpenetrating network from two hydrogel formulations both using SN2 chemistry with tunable mechanical properties. The hydrogel formulations highlighted in this work vary in gelation, mechanical properties, swelling, dissolution, and adhesion based on the structure of the polymer and reactive groups. These hydrogels represent a future direction in wound dressings and sealants as they prevent bacterial migration into an open wound, adhere to tissue, provide a moist wound environment, demonstrate structure-function relations allowing for tunable mechanical properties, and are biocompatible.2022-03-10T00:00:00

In Situ Designer Lipid Production: Integration of Novel Characteristics and Behaviors into Synthetic Cell Membranes

This thesis investigated the coupling of lysolipids and functionalized tails for in situ formation of synthetic liposomes designed to enable specific characteristics or behaviors in applications ranging from drug delivery to the advancement of artificial cell development. Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) mediated lipid coupling was improved via incorporation of a photoinitiation system. Here, photo-CuAAC enabled spatiotemporal control over liposome assembly, an over 400-fold increase in formation density, and control over the maximal cross-sectional area of the liposomes formed. Thiol-Michael mediated lipid coupling was enabled using thiol-functionalized lysolipids and acrylate tails where phospholipids were produced over 48 hours with approximate 90% conversion. Coupling was achieved using the thiol-Michael addition reaction for designer lipid synthesis by forming lipids bearing terminal alkyne functionalities in the presence of a visible light-sensitive photoinitiator over 48 hours to reach approximately 90% conversion followed by irradiation to homopolymerize the lipid tails. Dynamic lipid bilayers were formed using thiol-thioester exchange for in situ liposome formation between thiol-functionalized lysolipids and phenyl thioester-functionalized aliphatic tails. Two tails, a C7 phenyl thioester and a C11 phenyl thioester, reacted with thiol lysolipid to greater than 90% conversion over 48 hours and 12 hours, respectively. These phospholipid products produced liposomes with differences in self-assembly behavior and enhanced permeability was found in the C11 thioester-containing phospholipid system. Following phospholipid formation with the C11 tail, addition of C7 phenyl thioester enabled exchange to convert between 30 and 40% of C11 thioester-containing phospholipids into C7 thioester-containing phospholipids over 96 hours. Finally, photo-cleavable lipids synthesized using CuAAC-mediated coupling were mixed with natural lipids to enable photo-induced pinocytosis behavior in liposomes formed either via lipid film hydration or the pull-down technique. The lipid film hydration method liposomes displayed consistent pinocytosis in liposomes with pearled structures or aspect ratios greater than 2, indicating that they possessed a critical volume-to-surface area ratio. Morphological transitions and 31x greater liposome formation using an asymmetric formation technique with photocleavable lipid in the outer leaflet indicate a dependence upon asymmetric lipid distribution, decreasing the outer leaflet to inner leaflet ratio during irradiation, to cause engulfment. Pinocytosis in spherical, unilamellar systems using osmotic pressure followed by irradiation lead to an average of 44% of the imaged population undergoing pinocytosis, making this approach potentially applicable to protocell and artificial cell systems

Expanding the Toolbox for Probing Dynamic Behavior in Covalent Adaptable Networks

Covalent Adaptable Networks are an important and growing class of polymer materials that enable reconfiguration of what would otherwise be a static three-dimensional network through the incorporation of dynamic bonds. Such adaptability enables the design of stimuli responsive materials and crosslinked networks that can processed and reprocessed in ways that are typically attributed to linear and branched polymers. Two areas were explored in this thesis with respect to covalent adaptable networks. The first was the development and characterization of the thiol-ene-disulfide polymerizations of linear disulfides, which combines the thiol-ene and disulfidation reactions in one network-forming system to make tunable dynamic materials. The second was implementation of dielectric analysis to characterize the dynamic behavior of various dynamic networks. this was done to understand the impact of thiol substitution on dynamic thioester reactions and explore the utility of DEA as a tool for understanding the differences in dynamic for different mechanisms of dynamic bonding, A combination of FTIR, DMA, NMR, rheology, and stress relaxation were implemented to develop and study these materials and techniques. It was found that thiol-ene-disulfide photopolymerizations were a viable approach for making dual-cure and dynamic networks. A thorough kinetics analysis showed that the thiol-ene reaction is about 30 times faster than the disulfide-ene reaction, producing a dual-cure system with spatial and temporal control. Inherent to this approach, radical-disulfide exchange slowed the thiol-ene step, and the specific thiol/disulfide pair significantly impacted the rate of both the thiol-ene and disulfide-ene stages of the polymerization, depending on the relative stability of the thiyl radical formed by the thiol and disulfide. Exchange also enabled shrinkage stress reduction during the polymerization and induced stress relaxation that could be controlled by network structure and choice of disulfide core. Although thiol substitution has been shown to have little impact on thiol-ene the thiol-ene reaction, it was found to have a significant impact on dynamic reactions involving thioesters. In networks only capable of thioester exchange, stress relaxation was significantly slower for the secondary thiol/thioester networks than for the primary analogues. Dielectric analysis indicated that the slowed rate was due to a combination of steric hinderance, which slows nucleophilic attack onto the thioester, and the decrease in polarity associated with the large number of additional methyl groups, which is known to create a less conducive environment for thioester exchange in general. For thiol-anhydride-ene networks, which are capable reversible addition and reversible exchange, substitution biased dynamic bonding toward the reversible thiol-anhydride addition at a given temperature. Finally, analysis of dielectric and stress relaxation spectra showed that DEA can detect differences between reversible addition and reversible exchange mechanisms. Time-temperature-superposition of dielectric spectra for Diels-Alder (addition), thioester (exchange), and thiol-anhydride (both) networks showed that the spectra were superimposable if the equilibrium number of crosslinks was not significantly impacted by the temperature.</p

Covalent Adaptable Networks to Create Dynamic, Tunable, and Actuatable Materials from Molecular Self Assembly

The work in this thesis focuses on molecular-level self-assembly to achieve autonomous shape change in polymer materials. The breadth of this work ranges from macroscopic to microscopic shape programming of polymers. These autonomous, shape switching materials rely on post-polymerization modifications to the polymer network facilitated by dynamic covalent chemistry (DCC) incorporated into crosslinked polymers to form covalent adaptable networks (CANs). The DCC significantly utilized in this work is light activated addition-fragmentation chain-transfer (AFT), which provides spatial and temporal control of bond rearrangement. Furthermore, the self-assembly capabilities afforded by liquid crystals (LCs) tethered to a polymer network create the structure through which these micro and macroscopic shape changes are made possible, enabling molecular assembly to generate 3D structures. By disrupting LC alignment tethered to the polymer network in a liquid crystal elastomer (LCE), reversible shape switching is achieved by heating past the LC phase transition temperature. These shape switching structures were programmed by simple mechanical deformations under illumination and deployed via a thermal stimulus to reversibly actuate the material. Investigating the complex LC alignment also allows for the achievement of visible color changes linked to structure, that are reversibly modified. AFT-LCEs on the order of millimeters in length, were designed and programmed into a variety of complex shapes post polymerization. These programmed shapes were also erased by 3 irradiating the materials above their phase transition temperature to erase any alignment that existed prior. Furthermore, incorporation of a chiral LC monomer induced a handedness of alignment throughout the thickness of the film, which resulted in a Bragg reflection on visible wavelengths of light. The cholesteric LCE (CLCE) exhibited a red reflective character and upon uniaxial deformation to modify the underlying structure, resulted in a blue shift of the material. Light exposures performed programmed the strain and color change in the material. Color was erased by irradiating the CLCE above the phase transition temperature, resulting in a transparent, clear film. CANs were then translated to micro-networks. AFT-LCEMPs were generated via a thiol- Michael dispersion polymerization, yielding particles with a diameter of 7&nbsp;&plusmn; 2 &mu;m . These particles doped with photoinitiator were compressed and irradiated with light to program them into a variety of geometries including prolate and oblate, by tuning the temperature with which programming was done. Switchable shapes were investigated, and these programmed shapes were also erased by exposure of the particles to light above their phase transition temperature, which erased any programming or alignment that existed prior. Amorphous CAN microparticles with a diameter of 4.0&nbsp;&plusmn; 0.4 &mu;m were generated by a thiol- Michael dispersion polymerization. These particles were designed with DCC to enable interparticle bond exchange to generate new permanent structures. The DCC utilized here was the thiol-thioester exchange, which is base catalyzed. The ultra-violet visible spectroscopy (UV-vis) of the particle coalescence process indicated that change to the transmission cease to occur at 3 days. The integrity of the films generated from microparticle coalescence was evaluated by performing tensile tests, finding that films made directly from monomer and films made from 4particles had similar mechanical properties after 7 days. The modulus values were measured to be 5 &plusmn; 1 MPa and 6&nbsp;&plusmn; 1 MPa for the monomer-films and particle-films respectively.Ultimately in this work, investigation of the stress relaxation mechanism for network reorganization enables the programming of new, permanent, and temporarily reversible structures post-polymerization, while simultaneously utilizing molecular self-assembly to generate 3D structures. Demonstrations of programming and re-programming were reported, showcasing the adaptability achieved by polymer networks designed as CANs.</p

Analysis and Control of Degradation in Covalent Adaptable Networks

This thesis explores polymer degradation within covalent adaptable networks (CANs), focusing on degradation and properties controlled by the thiol-thioester exchange, the thiol-thioaminal exchange, and the thioaminal scission reactions. By comparing mass loss profiles and mechanical property changes to theoretical models developed herein, this work explores the degree to which polymer network structure and reaction kinetics tune the degradation process. The first part of this thesis focuses on the base-catalyzed thiol-thioester exchange reaction and how this reaction was used to degrade thioester-containing CANs. Statistical models quantitatively depicted the degradation process where these models incorporated the thiol-thioester exchange reaction kinetics, polymer structure, and mass gained from the exchanging thiol. A single reaction rate constant (k) fit experimental mass loss profiles to model predictions, and only varied from 0.0024 &ndash; 0.0051 M-1min-1 throughout all degradation conditions studied including changing crosslinking density, reactant concentration, oligomer lengths, and oligomer distributions. Using these parameters, degradation within thioesters networks could be tuned to occur on timescales from 2.5 h to near infinity, shift the degradation mechanism from surface to bulk, enable &gt; 90 % selective recovery of fillers, and mediate controlled mass release. The second part of this thesis focuses on how thioaminal groups enable degradation within CANs. First, the thiol-thioaminal exchange reaction was explored as a new, reversible-exchange CAN chemistry. The exchange reaction was monitored by a small molecule system, showing the choice of thiol impacted four kinetic parameters (kf, kr, Keq, t1/2,eq). Meanwhile, thioaminal-containing networks were found to stress relax (10 s at 95&nbsp;&deg;C), degrade rapidly when exposed to neat excess thiol (from 4 &ndash; 380 min), and exhibit temperature-independent crosslink density. Second, the thioaminal scission reaction was used as a means to create constructing-then-destructing CANs. Exposing thioaminal small molecules to photoradicals probed polymerization and scission reactions and found the exposure resulted in thioamide formation. Increasing thiol substitution (1&deg;&nbsp;&ndash; 3&deg;) resulted in greater extent of scission (5 &ndash; 39 %), with scission occurring semi-orthogonally to the thiol-ene polymerization reaction. Constructing-then-destructing CANs that depended on total light dose exposure were created, switching between construction and destruction at a light dose of 2 J/cm2 under the selected conditions.</p

Electrospun nanofiber meshes: applications in oil absorption, cell patterning, and biosensing

Nanofabrication techniques produce materials with enhanced physicochemical properties through a combination of nanoscale roughness and the use of chemically diverse polymers which enable advanced applications in separation science (air/water purification), tissue engineering, and biosensing. Since the late 1990’s, electrospinning has been extensively studied and utilized to produce nano- to microfiber meshes with 3D porosity on the gram scale. By combining a high surface area to volume ratio and tunable surface chemistry, electrospinning is a facile platform for generating non-woven polymeric fibers for many biomedical and industrial applications. This thesis describes three applications of electrospun nano- and microfiber meshes spun from both commercially available and novel polymer systems for: 1) oil and water separation after an accidental oil spill; 2) ultraviolet light controlled protein and cell patterning throughout 3-dimensional nanofiber meshes; and 3) novel diagnostic platform by combining electrospun nanofiber meshes with solid state nanopores for enhanced single molecule nucleic acid and protein detection. Each application embodies the philosophy that electrospun materials have the potential to solve a wide variety of problems by simply tuning the physicochemical properties and mesh morphologies towards the design requirements for a specific problem. For example, to solve the problem of recovering crude oil after an oil spill while generating a minimal waste burden, a hydrophobic and biodegradable microfiber mesh was designed to repeatedly separate oil and water and naturally biodegrade after use. In order to solve the problem of spatiotemporal placement of cells within a 3-dimensional tissue engineering construct, an ultraviolet light activated mesh was designed to transition from hydrophobic (water impermeable) to hydrophilic (water permeable) upon exposure to ultraviolet light facilitating protein and cell patterning. Finally to address two problems with single molecule solid state nanopore biosensors, namely rapid nucleic acid translocation rates and limited protein identification capabilities, a new biosensor platform was developed based on two novel polymeric systems which were synthesized and electrospun into high surface area nanofiber mesh coatings.2018-02-17T00:00:00

PNA-protein conjugates for nano-scale modeling of protein aggregates

Abstract Programmable assembly of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this research, the unique characteristics of PNA were applied to create controllable protein assemblies directed by precise PNA-DNA hybridization. The signatures of assembly were studied via FRET, providing a powerful tool which should be effective in live system imaging. Two model systems were developed in this study. In the first model system, site-selective conjugation of monomeric teal fluorescent protein (mTFP) to PNA was achieved by covalent linkage of mTFP to PNA via expressed protein ligation. The mTFP-PNA conjugates were efficiently aligned on a DNA beacon, to create a hetero-FRET system. The FRET indicated by decrease of fluorescence intensity and lifetime of the donor and an increase of donor anisotropy. The assembly of similar multiple mTFP-PNA constructs on DNA scaffolds provided dimeric and oligomeric forms which were studied by SEC-HPLC and SDS-PAGE. A decrease of anisotropy was exhibited due to homo-FRET following induced formation of dimers and oligomers. In the second model system, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers and oligomers