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Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study as its polyether backbone provides it with many unique properties that are still not fully understood, and are not seen with other similar polymers. PEG has been shown to exhibit amphiphilic character, due to its high conformational freedom, and the ability to hydrogen-bond 2-3 water molecules for each ethylene oxide subunit, creating a very structured water shell and large hydrodynamic radius. Together, the properties formed the hypothesis for the possibility for PEG to control drug release and its environment, expanding its potential in biomedical applications. This hypothesis was investigated with PEG in three states – free PEG, conjugated and blended. Free PEG was determined to inhibit melanoma cell viability by activating apoptosis via PEG effects on the osmolality of the cell medium (Chapter 3). Novel silicone hydrogels incorporating methacrylated PEG as the sole hydrophilic component showed advantageous properties for biomedical applications across a range of formulations (such as low contact angle and protein deposition), as well as altering the release of highly hydrophilic antibiotics from the materials, presumably via PEG-drug hydrogen bonding (Chapter 4). Novel siloxane-PEG blended materials were shown to have the ability to influence drug release of hydrophilic, hydrophobic and drug salts through the structure of PEG (Chapter 5). Overall, the work within this thesis expanded understanding of the abilities and limitations of PEG based on its distinct structure, and expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive. / Thesis / Doctor of Philosophy (PhD) / Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study due to its unique exhibition of amphiphilic character and the ability to hydrogen-bond multiple water molecules, that together suggest the possibility for PEG to control drug release and its environment. Through strategic experimental designs, greater understanding of the abilities and limitations of PEG was established and shown to be the result of the distinct structure of PEG. Specifically, two novel drug delivery systems were developed with demonstrated understanding of the structure-function relationship between polymers and drugs, and the activity of PEG as a melanoma cell viability inhibitor was discovered and found correlated to the PEG structure. Overall the work within this thesis expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive.

Elastin like peptides (ELPs)—derived from the protein elastin—are widely used as thermoresponsive components in biomaterials due to their LCST (lower critical solution temperature) behavior at a characteristic transition temperature (Tt). While linear ELPs have been well investigated, few reports focused on branched ELPs. Using lysine (Lys, with an additional side-chain amine) as branching units, ELP dendrimers were synthesized by solid-phase peptide synthesis (SPPS) with up to 155 amino acid residues. A secondary structure change with decreasing ratio of random coil and increasing ratio of β-turn upon heating, which is typical of linear ELPs, was confirmed by circular dichroism spectroscopy for all peptides. Conformational change did not show evident dependence on topology, while a higher Tt was observed for dendritic peptides than for their linear control peptides with the same number of GLPGL repeats. Variable-temperature small-angle X-ray scattering (SAXS) measurements showed a size increase and fractal dimension upon heating, even below the Tt. These results were further confirmed by cryogenic transmission electron microscopy (cryo-TEM), and micro differential scanning calorimetry (micro-DSC), revealing the presence of aggregates below the Tt. These results indicated the presence of a pre-coacervation step in the LCST phase transition of the ELP dendrimers. We further prepared hydrogels by crosslinking hyaluronic acid (HA) with ELP dendrimers. We invesigated their physical properties with scanning electron microscopy (SEM), swelling tests, SAXS, and model drug loading/release experiments. Most of the HA_denELP hydrogels retained transparent upon gelation, but after lyophilization and reswelling remained opaque for days. This reswelling process was carefully investigated with time-course SAXS studies, and was attributed to forming pre-coacervates in the gelation step, which slowly reswelled during rehydration. We then prepared hydrogels with H2S-releasing aroylthiooxime (SATO) groups and showed human neutrophil elastase-responsive H2S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. Furthermore, we prepared a series of wedge-shaped triblock polyethylene glycol (PEG)-ELP dendrimer-C16 (palmitic acid) conjugate amphiphiles with adjustable Tts. Various techniques were used to investigate their hierarchical structures. The triblock PEG-peptide-C16 conjugate amphiphiles were thermoresponsive and showed a morphology change from small micelles to large aggregates. However, the hydrophilic shell and strong tendency for micelle formation limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. The secondary structure was twisted from conventional β-sheet, and the thermoresponsive trend observed in typical ELP systems was not observed, either. Variable temperature NMR showed evidence for coherent dehydration of the PEG and ELP segments, probably due to the relatively short blocks. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release in hyperthermia. / Doctor of Philosophy / Elastin like peptides (ELPs) are similar to the protein elastin in terms of amino acid sequence. They are used widely as thermoresponsive (change in properties at different temperatures) components in biomaterials due to their abnormally lower solubility at higher temperatures. While linear ELPs have been thoroughly investigated, few investigations in ELP dendrimers have been studied. Dendrimers are molecules that branch in a controlled way to achieve sphere-like structures with rich surface functionalities. We synthesized the ELP dendrimers by using lysine amino acids as branching units. A protein secondary structure change, typical of ELPs, was observed for all peptide dendrimers. Secondary structure transitions showed no dependence on the molecular branching/linear structures, but a higher transition temperature (T_t) was observed for dendritic peptides than for their linear control peptides with the same number of amino acids. Various techniques confirmed the existence of aggregates below the T_ts, which was never reported before. We further fabricated hydrogels that mimic the native extracellular matrix, by connecting hyaluronic acid (HA) with ELP dendrimers. Interestingly, most of the hydrogels studied retained transparent upon gelation, but after freeze-drying and addition of water remained opaque for days. This phenomenon was attributed to forming of small aggregates in the gelation step, which resulted in slow reswelling. We then prepared hydrogels with H₂S-releasing groups, which showed human neutrophil elastase-responsive H₂S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. We then prepared a series of wedge-shaped triblock poly (ethylene glycol) (PEG)- ELP dendrimer-alkyl chain molecules. The triblock molecules were thermoresponsive and showed a change from small spheres to large aggregates. However, the hydrophilic shell limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. We found evidence of coherent assembly of the PEG and ELP parts, probably due to the relatively short polymer chains. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release for cancer treatment.

The inability to accurately reproduce the complexities of the in vivo tumor microenvironment with reductionist-based two-dimensional in vitro cell culture models has been a notable deterrent in identifying therapeutic agents that reliably translate to in vivo animal and human clinical trials. In an effort to address this, a growing number of three-dimensional (3D) in vitro tumor models capable of mimicking specific tumorigenic processes have emerged within the last decade. This concept stems from the understanding that cells cultured within 3D in vitro matrices have the ability to acquire phenotypes representative of the in vivo microenvironment. The objective of this project was to apply a tissue engineering approach towards developing a 3D in vitro tumor angiogenesis model. Initially, different scaffolds were investigated for supporting 3D tumor growth, including bacterial cellulose, electrospun polycaprolactone/collagen I, and highly porous electrospun poly(L-lactic acid). However, cancer cells cultured on these scaffolds demonstrated poor adhesion, sufficient adhesion with poor infiltration, and increased but still inadequate infiltration, respectively. Collagen I hydrogels were chosen as an appropriate scaffold for facilitating 3D in vitro tumor growth for two reasons -- cell-mediated degradation and immediate 3D cell growth. It was hypothesized that cancer cells cultured within collagen I hydrogels could be encouraged to recapitulate key characteristics of in vivo tumor progression. MDA-MB-231 human breast cancer cells were shown to experience hypoxia and undergo necrosis in response to limitations in oxygen diffusion and competition for nutrients. Upregulation of hypoxia-inducible factor-1" resulted in a significant increase in vascular endothelial growth factor gene expression. To capitalize on this endogenous angiogenic potential, microvascular endothelial cells were cultured on the surface of the designated "bioengineered tumors." It was hypothesized that paracrine signaling between tumor and endothelial cells co-cultured within this system would be sufficient for inducing an angiogenic response in the absence of exogenous pro-angiogenic growth factors. Endothelial cells in the co-culture group were shown to invasively sprout into the underlying collagen matrix, forming a capillary-like tubule network. This project culminated with the establishment of an improved in vitro tumor model that can be used as a tool for accurate evaluation and refinement of cancer therapies. / Ph. D.

Yes / In this work we report a facile method to prepare a fluorescence active self-healable hydrogel via incorporation of fluorescence responsive ionic block copolymers (BCPs). Ionic block copolymers were prepared via a combined effect of ring opening polymerization (ROP) of ε-caprolactone and xanthate mediated reversible addition–fragmentation chain transfer (RAFT) polymerization. Here polycaprolactone (PCL) was modified with xanthate to prepare a PCL based macro-RAFT agent and then it was utilized to prepare block copolymers with cationic poly(2-(methacryloyloxy)ethyltrimethyl ammonium chloride) (PCL-b-PMTAC) and anionic poly(sodium 4-vinylbenzenesulfonate) (PCL-b-PSS). During the block formation, the cationic segments were randomly copolymerized with a trace amount of fluorescein O-acrylate (FA) (acceptor) whereas the anionic segments were randomly copolymerized with a trace amount of 9-anthryl methylmethacrylate (AMMA) (donor) to make both the segments fluorescent. The block copolymers form micelles in a DMF : water mixture (1 : 4 volume ratio). The ionic interaction of two BCPs was monitored via Förster resonance energy transfer (FRET) and zeta potential measurements. The oppositely charged BCPs were incorporated into a polyacrylamide (PAAm) based hydrogel that demonstrated self-healing behavior and is also highly fluorescent. / IIT Kharagpur and MRC (MR/N501888/2)

Yes / Functional crosslinked hydrogels were prepared from 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA). The acid monomer was incorporated both via copolymerization and chain extension of a branching, reversible addition–fragmentation chain-transfer agent incorporated into the crosslinked polymer gel. The hydrogels were intolerant to high levels of acidic copolymerization as the acrylic acid weakened the ethylene glycol dimethacrylate (EGDMA) crosslinked network. Hydrogels made from HEMA, EGDMA and a branching RAFT agent provide the network with loose-chain end functionality that can be retained for subsequent chain extension. Traditional methods of surface functionalization have the downside of potentially creating a high volume of homopolymerization in the solution. Branching RAFT comonomers act as versatile anchor sites by which additional polymerization chain extension reactions can be carried out. Acrylic acid grafted onto HEMA–EGDMA hydrogels showed higher mechanical strength than the equivalent statistical copolymer networks and was shown to have functionality as an electrostatic binder of cationic flocculants.

Yes / A set of water-swollen core−shell particles was synthesized by emulsion polymerization of a 1,3-dioxolane functional monomer in water. After removal of the 1,3- dioxolane group, the particles’ shells were shown to swell in aqueous media. Upon hydrolysis, the particles increased in size from around 70 to 100−130 nm. A bicinchoninic acid assay and ζ-potential measurements were used to investigate the adsorption of lysozyme, albumin, or fibrinogen. Each of the core−shell particles adsorbed significantly less protein than the noncoated core (polystyrene) particles. Differences were observed as both the amount of difunctional, cross-linking monomer and the amount of shell monomer in the feed were changed. The core−shell particles were shown to be resistant to protein adsorption, and the degree to which the three proteins adsorbed was dependent on the formulation of the shell. / EPSRC and MRC