Browsing by Subject "Drug delivery"

Showing increased application in biological and medical fields, acoustofluidics is a combined technology between acoustics and microfluidics. The core function of acoustofluidics is a label-free and contact-free manipulation of particles in the fluid, which can be applied as active separation, active mixing, and active concentration. Since in therapeutic and diagnostic applications, contamination in the samples can significantly interference analysis results and treatment outcome, proper per-screening of the sample can significantly decrease the target detection threshold and avoiding interferences come from noise and misreading. The acoustofluidic technology derive a particle manipulation based on physical properties of the particles and fluids, specifically, the size of the particle, densities for the particles and fluid, and the viscosity of the fluid, which generate a screening system that can separate particles with different sizes and densities. By utilizing this property, acoustofluidics has been applied on separating multiple biological particles and objects including circulating cancer cells, red blood cells, and multiple populations of vesicles. These reagent-free and contact-free separations have been demonstrated biocompatible for cells and vesicles and can conserve the cell viabilities and vesicle cargoes including DNA, miRNA, and proteins. However, current achievements on acoustofluidic manipulation focus on general analysis of the separated components, which are not disease specific biomarkers, and the body fluid using for separation are limited to blood and artificial isotonic solutions including phosphate-buffered saline. Although these works demonstrated acoustofluidic technology is eligible for separating bio-particles that have diagnosis and therapeutic functions, lack of real cases related applications and diseases specific investigations still make the technology’s application abilities being restricted to possibilities but not promised functions. To deeply investigate and demonstrate the acoustofluidic technology’s potential on diagnostic application, the technology was evaluated by using samples related with multiple specific diseases. Since the acoustofluidic technology has been demonstrated eligible for isolating exosomes, which are 50-200 nm vesicles secreted from cells, pathology related exosomes were selected for diagnostic application investigation. Exosomes’ vesicle structures make them ideal candidate for diagnosis, since vesicles formed by lipid bilayer membrane contain both proteins or nucleic acids as cargoes inside and transmembrane or membrane proteins and polysaccharides on the surface. Furthermore, the forming and secreting pathologies of exosomes are highly dependent on endocytosis and exocytosis pathologies, which are influenced by cellular metabolism. Exosomes’ cargoes have been found specifically correlated with secreting cells populations, indicates depending on types of cells, like tumor cells or stem cells, the secreted exosomes will contain different molecules that can be used as biomarkers for reversed identifying secreting cells. Except high values on biological and medical research and applications, exosomes’ small size makes the vesicles difficult for isolation and increase the cost on both equipment and time aspects. Since acoustofluidics provides an active approach for separating nanometer sized particles and the isolation is a continuous procedure, the simple and rapid exosome isolation the acoustofluidics can provide makes the technology high valuable. Considering these improvements, the acoustofluidics can provide on exosome related fields, demonstrating acoustofluidic devices separated exosomes containing disease biomarkers and could be used for diagnostic applications become a necessary step for validating the technology’s ability. In this dissertation, the first attempt for validating acoustofluidic exosome separation’s diagnostic potential was made for isolating salivary exosomes aimed at human papillomavirus (HPV) induced oropharyngeal cancer diagnosis. Different with previous research that worked on blood exosome separation, a unique property of this study is achieving exosome separation from saliva, which is a more unstable system on components and physical properties than blood. By isolating salivary exosome using the acoustofluidic technology and processing down-stream digital droplet polymerase chain reaction (PCR) analysis, HPV-16 virus, which has been found can induce oropharyngeal cancer, was found majorly distributed in isolated exosome fractions. Since saliva has complex components that cause inaccuracy analysis result, the application of acoustofluidic technology can increase the diagnostic sensitive and enable saliva based liquid biopsy for early screening of oropharyngeal cancer. In the next work, we further demonstrate the acoustofluidic technology’s advantage on rapid isolation of exosomes benefits the time sensitive diagnosis. The acoustofluidic devices were applied for isolating exosomes from mice models that were induced to traumatic brain injury (TBI), which can develop to chronic diseases or deteriorate in short term. Since these outcomes induced by improper or untimely treatments, fast screening of TBI becomes critical for achieving ideal therapeutic outcomes. By collecting plasma from mice and deriving exosome isolation through the acoustofluidics devices, isolated exosome samples with less contamination were found compared with original plasma. Protein analysis further indicates isolated exosomes keeps several exosome specific and neuron damage specific proteins, indicates the acoustofluidic technology is biocompatible and low harmful for exosome structures and components. High isolation purity achieved by the acoustofluidic technology also benefits downstream analysis by decreasing detection noise. In flow cytometer analysis, the acoustofluidic devices isolated exosomes demonstrated TBI disease biomarker increasing in 24 h after the mice were induced to TBI, while the plasma sample cannot demonstrate this tendency. The success of revealing early stage TBI biomarker changes indicates the acoustofluidic technology not only can benefit diagnosis, but also eligible for achieving diagnosis in a very early stage of the pathology. Since the acoustofluidic technology had demonstrated a promising performance on biocompatibility and rapid separation, other time-sensitive samples, including live virus was applied for evaluating the device’s performance. To achieve better control and eliminate irrelevant variable, we use cultured reverse transcription virus that is used for mammal cells transfection as target for isolation. The acoustofluidic technology showed reliable isolation of the murine leukemia virus and majority of the virus particles were separated out from the original sample. Virus viability was further validated robust based on the transfection experiments that using acoustofluidic separated virus and original virus samples demonstrated similar level transfection rates. This work indicates except vesicles like exosomes, the acoustofluidic technology is also eligible for isolating virus and keeping its viability, which significantly expands the application of the technology. Next, to expend the acoustofluidic technology’s functions, we utilized the concentration and manipulation ability of the device for deriving high efficiency membrane degradation. By generating strong microstreaming and microstreaming derived shear stress, the acoustofluidic devices can generate strong vertex flow fields in channel that can capture and lyse mammal cells. Since the acoustofluidic cell lysis is totally a physical process without participation of any chemical reagent and demonstrates a high lysis efficiency, this acoustofluidics application has potential for achieving high efficiency cell analysis. Since the acoustofluidic technology has demonstrated potential for concentration and lysis effect by generating high flow rate microstreaming vertex, we further investigated whether similar effect can derive exosome concentration and lysis. By generating acoustofluidic vertex in droplet containing exosome, nanoparticles, and small molecule drugs, exosome concentration and lysis effects were utilized for high efficiency drug loading and carrier encapsulation. Derived by the acoustofluidic concentration effect, the porous nanoparticles and drug molecules are concentrated in small area of the fluid system and this active concentration increasing induces a high drug loading rate. Simultaneously, the acoustofluidic vertex disrupts exosome membrane and concentrates exosomes with the nanoparticles, which induces exosome encapsulation. These exosome encapsulated drug-loaded nanoparticles demonstrate high intake rate of cells and derive more efficient drug delivery rate. Since the drug loading and exosome encapsulation are physical processes, the acoustofluidic technology derived particle manipulation has potential for deriving loading and encapsulation for large varieties of drugs, particles, and vesicles, which significantly expand the technology’s application.

Bone is a dynamic tissue which continuously undergoes remodeling primarily through osteoblast-mediated bone formation and osteoclast-mediated bone resorption. This balance is vital in maintaining both bone homeostasis and bone regeneration. With the increase in the global elderly population, the two most prominent bone disorders of fracture and osteoporosis pose a tremendous burden to the healthcare system. While these bone disorders are increasing in prevalence, treatment options remain stagnant, demonstrating the unmet need for new clinical solutions. Strategies that induce innate repair yet eliminate the need for expensive cellular or recombinant protein-based therapies are appealing. Adenosine, a naturally occurring nucleoside, has emerged as a part of key metabolic pathway that regulates bone tissue formation, function, and homeostasis. In this dissertation, I investigate the therapeutic potential of adenosine delivery to mitigate bone disorders. Despite the regenerative capacity of bone, age-associated changes result in injuries that suffer delayed healing. Therapeutic interventions that circumvent the age-associated impairments in bone tissue and promote healing are attractive options for geriatric fracture repair. Herein, I examined the changes in extracellular adenosine signaling with aging and the potential of local delivery of adenosine to promote fracture healing in aged mice. My results showed a concomitant reduction of CD73 expression in the bone and marrow of aged mice. Local delivery of adenosine using injectable microgel building blocks and drug carriers yielded a pro-regenerative environment and promoted fracture healing in aged mice. This study provides new understandings of age-related physiological changes in adenosine levels and demonstrates the therapeutic potential of local delivery of adenosine at the fracture site to circumvent the impaired healing capacity of aged fractures. Given the multi-functionality of adenosine signaling, it is possible that extracellular adenosine delivery influences various phases of bone healing. Towards this, I examined the potential immunomodulatory effect of adenosine delivery on both the local and systemic immune system for fracture repair. My results indicated that the immune cell populations of neutrophils and macrophages did not change with adenosine treatment in the fractured callus at either 3-, 7-, or 14-days post fracture. Additionally in the peripheral blood, CD8+ and CD4+ T cell populations did not change at any of the timepoints following adenosine treatment. This study provides potential insight into the role of exogenous adenosine in the inflammatory stage of fracture healing in young animals. Aging not only poses a risk for delayed fracture healing, but also for the development of osteoporosis. Osteoporosis results in bone fragility and subsequently a higher risk for fracture incidence. This disease is characterized by an imbalance in the coupled bone remodeling process with enhanced osteoclastic activity can lead to excessive bone resorption, resulting in bone thinning. Once activated, osteoclasts bind to the bone surface and acidify the local niche. This acidic environment could serve as a potential trigger for the delivery of therapeutic agents into the osteoporotic bone tissue. To this end, I developed a pH-responsive nanocarrier-based drug delivery system that binds to the bone tissue and delivers the osteoanabolic molecule, adenosine. Adenosine is incorporated into a hyaluronic acid (HA)-based nanocarrier through a pH-sensitive ketal group. The HA-nanocarrier was further functionalized with alendronate moieties to improve binding to the bone tissues. Systemic administration of the nanocarrier containing adenosine attenuated bone loss in an ovariectomized mice model of osteoporosis and showed comparable bone qualities to that of healthy mice. Delivery of osteoanabolic small molecules, such as adenosine, that can contribute to bone formation and inhibit excessive osteoclast activity by leveraging the tissue-specific milieu could serve as viable therapeutics for osteoporosis. Overall, this dissertation offers novel findings regarding adenosine as a therapeutic to treat both fractures and osteoporosis. These findings, along with the biomaterial delivery systems developed, further advance the potential of using adenosine as a therapeutic molecule to treat bone disorders.

Antivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.

ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.

Phase separation of macromolecules is a critical phenomenon for the human condition. This phenomenon has also been exploited for biotechnological development to improve human morbidity and mortality. However, there is still much more to learn regarding how this behavior is encoded within a protein sequence. Thus, this thesis seeks to 1) further explore the sequence space to understand how phase separation is encoded, with an emphasis on polypeptides with upper critical solution temperature (UCST) transitions and 2) use this phase separation to control availability of macromolecules at various length scales.

Using traditional molecular biology techniques, we will recombinantly express and purify a large number of polypeptides with variable sequence composition and sequence architecture. Then, using traditional polymer science and material science techniques combined with microscopic techniques that span the macro-scale and nano-scale, we will characterize their phase separation behavior and the interaction of these materials with biological systems.

We developed a practical mutation strategy that allows for complete control of the UCST binodal line in physiologic conditions that is useful for de novo design of artificial IDPs with UCST phase behavior. We evaluated the interaction of these polypeptides and their phase separation in the presence of bacterial, eukaryotic cells and in mice demonstrating how this binodal line fused to biological active partners can control biologic functions.

In bacteria, we made artificial phase separated puncta, akin to naturally occurring phase separated droplets, that have non-canonical function, demonstrating how primary features of the polypeptide chain affect enzymatic function. We created block co-polypeptides comprised of UCST and LCST protein sequences that exhibit remarkably tunable and robust nanoscale self-assembly into spherical micelles, worm-like micelles and vesicular structures capable of displaying large targeting domains on their surface. In the presence of eukaryotic cells, these nanomaterials can dramatically increase polypeptide uptake, increasing the avidity of the targeting molecule by over 1000-fold.

Finally, we demonstrated that phase separated polypeptides can sequester an active peptide GLP-1 from systemic circulation, controlling the peptide’s bioactivity through control of the phase diagram. Taken together, we demonstrate the universal power of the phase diagram, across many length scales, where the transducing agent for controlling biological activity is an engineered, repetitive polypeptide sequence.

Traditional small molecule chemotherapeutics show limited effectiveness in the clinic as their poor pharmacokinetics lead to rapid clearance from circulation and their exposure to off-target tissues results in dose-limiting toxicity. The objective of this dissertation is to exploit a class of recombinant chimeric polypeptides (CPs) to actively target drugs to tumors as conjugation to macromolecular carriers has demonstrated improved efficacy by increasing plasma retention time, reducing uptake by healthy tissues, and enhancing tumor accumulation by exploiting the leaky vasculature and impaired lymphatic drainage characteristic of solid tumors. CPs consist of two principal components: (1) a thermally responsive elastin-like polypeptide (ELP) that displays a soluble-to-aggregate phase transition above a characteristic transition temperature (Tt); and (2) a cysteine-rich peptide fused to one end of the ELP to which small molecule therapeutics can be covalently attached (the conjugation domain). This work describes the development of CP drug-loaded nanoparticles that can be targeted to solid tumors by the external application of mild regional hyperthermia (39-43°C).

Highly repetitive ELP polymers were assembled by Plasmid Reconstruction Recursive Directional Ligation (PRe-RDL), in which two halves of a parent plasmid, each containing a copy of an oligomer, were ligated together to dimerize the oligomer and reconstitute the functional plasmid. Chimeric polypeptides were constructed by fusing the ELP sequence to a (CGG)8 conjugation domain, expressed in Escherichia coli, and loaded with small molecule hydrophobes through site specific attachment to the conjugation domain. Drug attachment induced the assembly of nanoparticles that retained the thermal responsiveness of the parent ELP in that they experienced a phase transition from soluble nanoparticles to an aggregated phase above their Tt. Importantly, the Tt of these nanoparticles was near-independent of the CP concentration and the structure of the conjugated molecule as long as it displayed an octanol-water distribution coefficient (LogD) > 1.5.

A series of CP nanoparticles with varying ratios of alanine and valine in the guest residue position was used to develop a quantitative model that described the CP transition temperature in terms of three variables - sequence, chain length, and concentration - and the model was used to identify CPs of varying molecular weights that displayed transition temperatures between 39°C and 43°C. A murine dorsal skin fold window chamber model using a human tumor xenograft was used to validate that only the thermoresponsive CP nanoparticles (and not the controls) exhibited a micelle-to-aggregate phase transition between 39-43°C in vivo. Furthermore, quantitative analysis of the biodistribution profile demonstrated that accumulation of these thermoresponsive CP nanoparticles was significantly enhanced by applying heat in a cyclical manner. It is hoped that this work will provide a helpful resource for the use of thermoresponsive CP nanoparticles in a variety of biomedical applications.

Recombinant technology has given us the powerful ability to imagine and create novel biological entities, from potent therapeutics to functionally active materials. By harnessing nature’s building blocks and reconfiguring these components, recombinant engineering unlocks the potential to tailor drug specificity and pharmacokinetics, rationally design biomaterials, understand and define protein structure, and probe cellular function with molecular precision. These technological feats are made possible with a few simple biological ingredients: nucleotides, sugars, and amino acids. These components, exquisitely crafted by evolution, are individually combined in useful ratios and precise sequences in living systems to synthesize DNA, RNA, polysaccharides, and proteins. These macromolecules collectively support organismal structure and function and give rise to the incredible diversity in Charles Darwin’s “great tree” of life. However, the seemingly infinite potential for new materials built from these components is, in fact, limited. The chemical identity of these building blocks – with a particular focus herein on the twenty naturally-occurring amino acids – limits the scope and functionality of the recombinant materials we can produce. In order to functionalize these products, to fundamentally change their chemical identity while preserving their biological functionality, we require the finesse of bioorthogonal chemistries and modification techniques.

Bioorthogonal reactions modify biological materials within living systems without perturbing function, much as two orthogonal lines can extend in different directions and intersect only at a single point. That point of intersection can be precisely defined through recombinant technology and gives us access to new classes of biomaterials. The term “bioorthogonal”, coined by Carolyn Bertozzi, importantly defines these unique chemistries, which inertly co-exist with biology until the exact moment when the desired reactions are initiated, to enhance – and even transform – biological systems.

Bioorthogonal modification of proteins will, by definition, require expansion of the biochemical toolbox; there are a variety of techniques used to achieve this goal. In these studies, we explore the use of genetic code expansion for incorporation of unnatural amino acids. This technology permits co-translational incorporation of amino acids with unique and non-canonical R-groups directly into the polypeptide backbone of a protein or biopolymer. These residues introduce unique chemical reactivity for further functionalization with desired moieties or chemical transformation.

We have used this technology to develop novel therapeutic and material platforms comprised of a unique biopolymer, elastin-like polypeptide (ELP). This thermally responsive biopolymer is easily recombinantly synthesized, though more biochemically complex ELPs require successful bioorthogonal modification. We designed the unnatural amino acid-containing ELPs necessary to enable our strategies for developing three distinct biomaterial platforms: 1) photoreactive ELPs which can generate stable hydrogel particles spanning four orders of magnitude in size; 2) a universal strategy for drug-loaded, targeted ELP nanoparticles by incorporation of a unique site for drug attachment; 3) a sustained-release therapeutic for treatment of brain tumors combining proteins of distinct cellular origin.

We have combined existing tools, technologies, and materials to generate these novel platforms with utility in biomaterials, drug delivery, and cancer therapeutics. The optimizations performed in developing each of these systems will inform future studies with similar goals; similarly, the reactions and strategies employed will contribute to furthering our understanding of the full potential of these important bioorthogonal chemistries.

Over 30 million people in the United States suffer from type 2 diabetes (T2D), and this figure is rapidly increasing. Currently available glucose-lowering drugs largely treat the symptoms of diabetes and not the underlying pathology, leaving one third of diabetes patients with improperly managed disease. Thus, there exists an urgent need for novel drugs that slow T2D progression while posing a minimal burden on the patient.

The metabolic regulatory factor fibroblast growth factor 21 (FGF21) is under investigation as a T2D therapeutic due to its favorable effects on glycemic control and body weight. However, the feasibility of native FGF21 as a drug candidate is impeded by its rapid in vivo clearance and by costly production methods associated with poor protein solubility. To address these issues, FGF21 was recombinantly expressed in E. coli as a fusion with an elastin-like polypeptide (ELP), a repetitive peptide polymer with reversible thermal phase behavior. Below their transition temperature (Tt), ELPs exist as soluble unimers, while above their Tt, they aggregate into an insoluble coacervate. The thermal responsiveness of the ELP was retained when genetically fused to FGF21, with several notably positive impacts for the synthesis and efficacy of this protein drug. First, the ELP fusion partner acted as a solubility enhancer, yielding 50 mg/L of active FGF21 protein from the soluble cell lysate fraction in shaker flask culture, and eliminating the need for protein refolding. Second, the phase transition behavior of the ELP was exploited for chromatography-free FGF21 purification. Third, the Tt of the ELP was tuned to below body temperature, such that the phase transition was initiated solely by body heat. Indeed, in vivo injection of the fusion resulted in an immiscible viscous phase in the subcutaneous (s.c.) space that dissolved at a steady rate, temporally releasing fusion unimers into circulation. The injectable FGF21 drug depot was tested in diabetic ob/ob mice, and conferred dose-dependent glucose-and weight-lowering effects that were sustained out to 5 days following a single s.c. injection.

Once an optimized ELP-based FGF21 delivery strategy was established, the fusion concept was applied to a combination therapy to afford even greater metabolic benefits, while providing controlled release properties exclusive to the ELP platform. Recent evidence supports the development of combination drug treatments that incorporate complementary mechanisms of action to more effectively treat T2D. Thus, we developed a unimolecular dual agonist by combining the incretin glucagon-like peptide-1 (GLP1) with FGF21, hypothesizing that this agent would merge the insulinotropic and anorectic effects of GLP1 with the enhanced insulin sensitivity and energy expenditure associated with FGF21 signaling. The dual agonist was designed as a single polypeptide fusion, with GLP1 located at the N terminus and FGF21 at the C terminus. This orientation allowed each peptide to activate its endogenous receptor, while the linear architecture enabled facile synthesis in a bacterial expression system. An ELP was fused between GLP1 and FGF21 to serve as both a flexible linker and a depot-forming delivery scaffold. Indeed, a single s.c. injection of GLP1-ELP-FGF21 into diabetic db/db mice resulted in potent metabolic effects that were sustained at least 7 days, indicating formation of an ELP depot with a highly controlled rate of drug release. Furthermore, dual agonist treatment outperformed a long-acting GLP1 analog in restoring glycemic control and inducing weight loss, supporting the rationale for a GLP1/FGF21 combination therapy.

With a significant proportion of T2D patients failing to properly manage their disease, there is an urgent need for novel drug and drug combinations that effectively target disease pathophysiology, while posing a minimal burden on the patient. Meanwhile, the vast – and global – prevalence of metabolic disease argues for cost-effective and scalable manufacturing methods for new drugs. An ELP-based approach to therapeutics precisely addresses these needs by providing a streamlined method for production, as well as an innovative strategy for drug delivery to reduce the frequency of administration and thereby promote patient compliance. Furthermore, the ELP platform can be utilized to unite distinct drugs into one multi-functioning molecule to more effectively treat diabetes, altogether simplifying and improving metabolic disease management.

Although two-thirds of the global cancer mortality burden is predicted to occur in low- and middle-income countries (LMICs), citizens of these countries have disproportionately less access to resources and facilities to provide effective care. Surgery, radiation therapy, and chemotherapy form the foundation of effective cancer care in high-income countries (HICs), but these modalities are largely unavailable in LMICs. Stemming from this disparity, long-term cancer survival rates are lower, and the mortality-to-incidence ratio is higher in LMICs. With limited healthcare spending and a large portion of expenditures out-of-pocket, non-communicable diseases such as cancer lead to financial catastrophe for millions of families annually and are a barrier to global development. To expand global access to cancer care and buttress the anti-cancer capabilities of overextended healthcare systems in LMICs, it is necessary to develop a therapy compatible with the constraints imposed by resource-limited settings.

To accomplish this goal, the work presented here describes a low-cost injectable ablative therapy suitable for widespread use in LMICs. This therapy is a modification of an existing technique entailing intratumoral injection of ethanol to induce necrosis of malignant cells (termed “ethanol ablation”) utilized to reduce tumor volume with either curative or palliative intent. Modifications are based on analysis of the mechanics of the injection process and entail the incorporation of the water-insoluble, ethanol-soluble polymer ethyl cellulose and reduction of the infusion rate and volume. Ethanol ablation is one of the original forms of tumor ablation, treatments in which the tumor microenvironment is altered via chemical or thermal means to destroy malignant tissue, and has achieved widespread clinical success in HICs. It is appealing for use in LMICs because it is low-cost, portable, electricity-independent, and minimally invasive. However, injected ethanol is highly pressurized and forms cracks within tissue leading to excessive leakage and an unpredictable distribution of injected ethanol, poor tumor coverage, and damage to adjacent organs. With the recognition of pressure-induced crack formation as a source of leakage, reducing the infusion rate and volume will improve localization. Further, the incorporation of ethyl cellulose is likely to reduce leakage because it forms a gel upon exposure to the aqueous tissue environment and reduces the permeability of fractured tissue. These innovations are poised to improve upon ethanol ablation while retaining its suitability for use in resource-limited settings.

Three specific aims were proposed to establish crack formation as a limiting factor for efficacy of ethanol ablation, characterize this novel tumor ablation technique and develop a framework for tailoring treatment protocols to specific lesion types and sizes. The first aim described the rheological properties of ethyl cellulose-ethanol and the gelling behavior upon exposure to water and found that reducing the infusion rate and incorporating ethyl cellulose decreased leakage in tissue-mimicking surrogates and improved ablative efficacy in chemically induced squamous cell carcinoma tumors in the hamster oral cavity. The viscosity of ethyl cellulose-ethanol solutions increases with the ethyl cellulose concentration, which has been found to improve localization of injected solutions. Further, as expected from a water-insoluble polymer, gel formation increases with higher ethyl cellulose concentrations and higher water-to-ethanol ratios as well. These findings motivate the use of higher ethyl cellulose concentrations and low infusion volumes, and indicate that gel forms upon injection as water diffuses into and ethanol diffuses away from the injection site.

Tissue-mimicking surrogates composed of agarose were utilized because they are transparent and poroelastic. This makes visualization of injected ethanol feasible in a material that replicates the dynamics of tissue’s mechanical response to infusion. In these surrogates, ethyl cellulose was demonstrated to reduce leakage and increase the distribution volume of injected ethanol, but only at moderate infusion rates. At infusion rates typically used in conventional ethanol ablation (approximately 100 mL/hr), excessive leakage was observed for pure ethanol and ethyl cellulose-ethanol alike. This result, taken in context with the established linear relationship between infusion pressure and rate, suggests that reducing the infusion rate is necessary to localize injected ethanol in addition to incorporating ethyl cellulose.

To demonstrate proof-of-concept of improved therapeutic efficacy, chemically induced oral squamous cell carcinoma tumors in the hamster oral cavity were utilized as they are similar to human primary tumors. Further, since they protrude from the surface of the oral cavity and injected fluid is not confined by adjacent tissue, they are susceptible to leakage and more difficult to treat. To evaluate conventional ethanol ablation in this model, high-rate (100 mL/hr) infusions were performed with an infusion volume 4x greater than the tumor volume. This protocol led to regression of only 4 of 13 treated tumors. However, with the reduction of the infusion rate to 10 mL/hr and infusion volume to a quarter of tumor volume, and the incorporation ethyl cellulose, 7 of 7 tumors regressed completely. In the absence of ethyl cellulose, reduction of infusion rate and volume led to regression of 0 of 5 tumors.

With the characterization of ethyl cellulose-ethanol and demonstration of proof-of-concept in Aim 1, the objective of Aim 2 was to investigate the role of infusion pressure in the mechanics of crack formation, as well as of ethyl cellulose in preventing leakage. Pressure-induced crack formation has been described to occur at a material-inherent critical pressure dictated by the fracture toughness and elasticity and can be quantified as the maximum pressure achieved during the infusion of air. In this aim, transparent tissue-mimicking surrogates were fabricated to match the critical pressure of ex vivo swine liver. To determine the relevance of the critical pressure, infusions were performed with two contrast agents dissolved in ethanol– one smaller than the surrogate pore size (fluorescein) and one larger (graphite). When the agarose pore structure was unfractured, only fluorescein was visible. After it was fractured, both contrast agents were visible. Using this system, fracture was observed to occur at the critical pressure and a modified technique to detect fractures via infusion pressure was established. While previous studies have demonstrated that fracture can be observed during the infusion, this is only possible with low-viscosity fluids unlike ethyl cellulose-ethanol. In these studies, it was demonstrated that unfractured agarose retains an elevated post-infusion pressure, but fractured agarose allows the pressure to dissipate rapidly. This result allows for non-invasive detection of crack formation in tissue during infusion of viscous fluids.

In ex vivo swine liver, as was the case in tissue-mimicking surrogates, crack formation was detected when the critical pressure was exceeded and increased leakage. In these studies, the injected ethanol distribution was determined by adding fluorescein to the injection solution, freezing tissue after the infusion, sectioning it, and imaging with a fluorescent microscope. Since the infusion pressure increases with rate and volume, this finding motivates the use of low rates and volumes when possible to improve localization. For low-volume infusions in which the pressure remained below the critical pressure, there was minimal leakage. While leakage, and the infusion pressure, increased with infusion rate (from 1 to 10 mL/hr) for pure ethanol, it did not increase for 6% ethyl cellulose-ethanol. The gel formation behavior of ethyl cellulose reduces leakage in the presence of infusion-induced cracks.

Having established proof-of-concept of ethyl cellulose-ethanol and its mechanism of action in localizing injected ethanol, the focus of Aim 3 was to characterize computed tomography (CT) imaging as rapid, non-destructive method to visualize injected ethanol, optimize the ethyl cellulose concentration, and investigate the relationship between the injected ethanol distribution and resultant extent of induced necrosis. Since ethanol is less attenuating of x-rays than water or tissue, it is readily visible with CT imaging. However, the accuracy of extraction of ethanol concentration from CT imaging has not yet been established. Utilizing ethanol-water mixtures as in vitro surrogates, the random and systematic components of measurement error were quantified, with the combined error defined as the root sum square of both components. The random error component arises from the variance of the radiodensity of a solution of fixed concentration. The systematic error component was quantified as the difference between the predicted and true radiodensity of ethanol-water mixtures, with the predicted value determined by a linear two-point calibration equation with pure water and ethanol at the extremes. The total measurement error was 13.4% with both components contributing approximately equal amounts. This error is low enough to confidently delineate between treated and untreated tissue.

Having established the utility of CT imaging to quantify the ethanol distribution volume, the ethyl cellulose concentration was optimized in ex vivo rat liver tissue submerged in buffer over a wider range of concentrations than has been feasible in previous models. The optimal ethyl cellulose concentration was defined as the formulation that maximized the volume of tissue infiltrated with a cytotoxic (> 20%) ethanol concentration. In these studies, 12% ethyl cellulose maximized the ethanol distribution volume by 8-fold in comparison to pure ethanol. It also led to the most spherical distributions as defined by the aspect ratio quantified as the ratio of the radius of gyration to the effective radius. These results were confirmed in in vivo rat liver in which 12% ethyl cellulose-ethanol yielded a distribution volume 3-times greater than pure ethanol.

In addition to improving localization of injected ethanol, 12% ethyl cellulose increased the extent of induced necrosis by 6-times in comparison to pure ethanol. Necrosis was quantified by excising treated tissue 24 hours post-ablation, cryopreserving, sectioning, and staining it with NADH-diaphorase. There was an approximate one-to-one equivalence of the ethanol distribution volume with the necrotic volume for 12% ethyl cellulose-ethanol. This validates the concentration-based thresholding strategy utilized to determine the ethanol distribution volume and confirms the utility of CT imaging. CT imaging is particularly appealing to assess the morphology of the ablative extent as three-dimensional reconstruction of the ablative extent from pathology is challenging. The equivalence between the distribution volume visualized with CT imaging and necrotic volume determined via pathology motivates further use of CT imaging in optimization of the ablation parameters. Pure ethanol had a necrotic volume of nearly half of the injected ethanol volume. While the comparison of this relationship between pure ethanol and 12% ethyl cellulose-ethanol was not statistically significant, it is indicative of prolonged exposure time achieved by ethyl cellulose that may be caused by delayed vascular clearance in vivo. This aim establishes CT imaging with concentration-based thresholding as a non-destructive, high-throughput method to optimize ablation parameters and tailor treatment to specific lesion types and sizes.

In conclusion, the objective of this work was to establish ethyl cellulose-ethanol ablation as an effective tumor ablation technique suitable for use in resource-limited settings with the goal of expanding global access to cancer treatment. In pursuit of this goal, aim 1 assessed the rheological and gelling behavior of ethyl cellulose-ethanol, established improved localization, and demonstrated proof-of-concept in treatment of chemically induced oral tumors. Aim 2 investigated the relationship between crack formation and infusion pressure, adapted an established model to detect crack formation by demonstrating that post-infusion pressure dissipation is characteristic of fractured tissue, and found that ethyl cellulose decreases leakage when cracks do form. Finally, aim 3 characterized the ethanol concentration measurement accuracy of CT imaging, optimized the ethyl cellulose concentration, and investigated the relationship between ethanol distribution volume and the resultant extent of induced necrosis. Ultimately, this work demonstrates that ethyl cellulose reduces leakage associated with ethanol ablation, improves therapeutic efficacy, and establishes a methodology for further optimization and to tailor treatment for specific applications.

Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials.

However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward.

This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.

The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models.

The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.

In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.

Neuroinflammation associated with lumbar radiculopathy and peripheral nerve injury is characterized by locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in local delivery of anti-inflammatory drugs to treat this pathology, as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation.

ELPs are biopolymers capable of thermally-triggered in situ depot formation and have been successfully employed as drug carriers and biomaterials in several applications. A library of ELP-curcumin conjugates were synthesized and characterized. One lead conjugate was shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα and NF-κB with near-equivalent potency compared to free curcumin. When injected into the perineural space via intramuscular (i.m.) injection proximal to the sciatic nerve in mice, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4 days post-injection and decreased systemic exposure of curcumin 3-fold.

The results of this dissertation support the use of ELP as a drug carrier for local perineural drug delivery, and the strategy presented here for drug conjugate development and use of depot-forming ELP-curcumin conjugates represents a novel means of providing sustained treatment of neuroinflammation and pain associated with radiculopathy and peripheral nerve injury.

The clinical utility of many peptide, protein and small molecule drugs is limited by their short in-vivo ¬half-life. To address this limitation, we report a new class of biomaterials that have a long plasma circulation time. In particular, taking inspiration and cues from natural proteins and synthetic polymers, we have worked to create polypeptide-based drug carriers that are biocompatible and biodegradable. These peptide polymers or polypeptides can be attached to therapeutics with molecular precision as they are designed from the gene level.

In the first part of this thesis (Chapter 3-4), we report on the development of a new class of biomaterials called zwitterionic polypeptides (ZIPPs) that exhibit “stealth” behavior, and when fused to therapeutics, improve their pharmacological efficacy. To identify an optimal polypeptide design, we first synthesized a library of ZIPPs by incorporating various oppositely charged amino acids within an intrinsically disordered polypeptide motif, (VPX1X2G)n, where X1 and X2 are cationic and anionic amino acids, respectively, and n is the number of repeats. The (VPX1X2G)n motif is derived from the disordered region of human tropo-elastin. By systematically varying the identity of the charged amino acids and the chain length of the polypeptide, we determined the optimal polypeptide sequence that maximizes the pharmacokinetics for intravenous and subcutaneous routes of administration. We show that a combination of lysine and glutamic acid in the ZIPP confer superior pharmacokinetics, for both intravenous and subcutaneous administration, compared to uncharged control polypeptides. We report detailed physicochemical characterization of this new class of polypeptide-based drug carriers and show its clinical utility for drug delivery by using it to deliver a peptide drug. The peptide drug used is Glucagon like peptide 1 (GLP1) – a therapeutic peptide that is approved for treatment of type 2 diabetes but has seen limited clinical utility because of its short two-minutes half-life. We find that the GLP1-ZIPP conjugate reduced blood glucose level for up to 3 days in a diet induced obesity model of type-2 diabetes in mice after a single s.c. injection. This is a 70-fold improvement over the injection of the unmodified drug and a 1.5-fold improvement over an uncharged polypeptide control.

To further demonstrate the clinical utility of ZIPPs, in the second part of this thesis (Chapter 5), we used ZIPPs to create a nanoparticle system that can package and deliver hydrophobic chemotherapeutic drugs to the tumor with higher efficacy and lower toxicity. Such nanoparticle drug carriers are attractive for systemic delivery of chemotherapeutics because they improve the half-life of the drug, protect the drug from early degradation, and increase selective accumulation of drugs in tumors via the enhanced permeation and retention effect (EPR). The EPR effect is a consequence of the leaky vasculature and poorly developed lymphatic drainage system present in the tumors. These attributes of nanoparticles are significant and desirable because drug delivery systems that can improve circulation time and tumor accumulation of chemotherapeutics have the ability to improve the patient prognosis and survival by controlling the tumors at their local sites. To that end, we conjugated paclitaxel a chemotherapy drug that is used to treat different types of cancer to ZIPPs and showed that it imparts sufficient amphiphilicity to the polypeptide chain to drive its self-assembly into sub-100 nm nanoparticles. We report that ZIPPs can increase the systemic exposure of paclitaxel by 17-fold compared to the free drug and 1.6-fold compared to uncharged recombinant control. Treatment of mice bearing highly aggressive prostate cancer or colon cancer with a single dose of ZIPP-Paclitaxel nanoparticles leads to a near complete-eradication of the tumors (5 out of 7 cures in prostate cancer) and (2 out of 7 cures in colon cancer) and it outperforms Abraxane, which is an FDA approved taxane nanoformulation and current gold standard for paclitaxel delivery.

In summary, this doctoral research is multidisciplinary, which integrates the field of protein engineering, molecular biology, bioconjugate chemistry, soft matter physics and cancer biology for rational design of biomaterials for drug delivery.

Although various proof-of-concept studies have demonstrated the eventual potential of a multifunctional SERS-active metallic nanostructures for biological applications such as single cell analysis/measurement and drug delivery, the actual development and testing of such a system in vitro has remained challenging. One key point at which many potentially useful biomethods encounter difficulty lies in the translation of early proof-of-concept experiments in a clean, aqueous solution to complex, crowded, biologically-active environments such as the interior of living cells. The research hypotheses for this work state that multifunctional nanoconstructs can be fabricated and used effectively in conjunction with surface-enhanced Raman scattering (SERS) spectroscopy and other photonics-based methods to make intracellular measurements in and deliver treatment to single cells. The results of experimental work address the specific research aims, to 1) establish temporal and spatial parameters of nanoprobe uptake and modulation, 2) demonstrate targeting of functionalized nanoparticles to the cytoplasm and nucleus of single cells, 3) deliver to and activate drug treatment in cells using a multifunctional nanosystem, and 4) make intracellular measurements in normal and disease cells using external nanoprobes,

Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate labeled silver nanoparticles in single cells using SERS detection. To study the efficiency of cellular uptake, silver nanoparticles were functionalized with three differently charged SERS/Raman labels and co-incubated with J774 mouse macrophage cell cultures for internalization via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake.

To demonstrate delivery of functionalized nanoparticles to specific locations within the cell, silver nanoparticles were co-functionalized with the HIV-1 TAT (49-57) peptide for cell-penetrating and nuclear-targeting ability and p-mercaptobenzoic acid (pMBA) molecules as a surface-enhanced Raman scattering (SERS) label for tracking and imaging. Two-dimensional SERS mapping was used to track the spatial and temporal progress of nanoparticle uptake in PC-3 human prostate cells and to characterize localization at various time points, demonstrating the potential for an intracellularly-targeted multiplexed nanosystem. Silver nanoparticles co-functionalized with the TAT peptide showed greatly enhanced cellular uptake and nuclear localization as compared with the control nanoparticles lacking the targeting moiety.

The efficacy of targeted nanoparticles as a drug delivery vehicle was demonstrated with development and testing of an anti-cancer treatment in which novel scintillating nanoparticles functionalized with HIV-1 TAT (49-57) for cell-penetrating and nuclear-targeting ability were loaded with tethered psoralen molecules as cargo. The experiments were designed to investigate a nanodrug system consisting of psoralen tethered to a nuclear targeting peptide anchored to UVA-emitting, X-ray luminescent yttrium oxide nanoparticles. Absorption of the emitted UVA photons by nanoparticle-tethered psoralen has the potential to cross-link adenine and thymine residues in DNA located in the nucleus. Such cross-linking by free psoralen following activation with UVA light has previously been shown to cause apoptosis in vitro and an immunogenic response in vivo. Experimental results using the PC-3 human prostate cancer cell line demonstrate that X-ray excitation of these psoralen-functionalized Y2O3 nanoscintillators yields concentration-dependent reductions in cell number density when compared to control cultures containing psoralen-free Y2O3 nanoscintillators.

The development and demonstration of a small molecule-sensitive SERS-active fiber-optic nanoprobe suitable for intracellular bioanalysis was demonstrated using pH measurements in single living human cells. The proof-of-concept for the SERS-based fiber-optic nanoprobes was illustrated by measurements of intracellular pH in MCF-7 human breast cancer, HMEC-15/hTERT immortalized normal human mammary epithelial, and PC-3 human prostate cancer cells. Clinical relevance was demonstrated by pH measurements in patient biopsy cell samples. The results indicated that that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically relevant small molecules at the single cell level.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

Adequate pain management within the first 3-5 days following a procedure is vital to the enhancement of patient healing and recovery. The current gold standard for relieving post operative pain consists of oral medication which, though effective, only offers temporary relief through frequent doses as pain persists, delivers drug systemically and inefficiently, and introduces an inherent potential of misuse and abuse of pain killers. A probable solution is the fabrication of a drug-load matrix to impart local, sustained release of analgesic compounds. Specifically, a novel class of polymers, poly(ester urea)s (PEUs) have been developed and analyzed for their ability to achieve controlled drug delivery. In recent literature, PEUs have displayed a limited inflammatory response, tunable mechanical properties, and degradation. Due to their versatility, PEUs have been applied to a variety of applications, ranging from sturdy bone implants to grafts for soft tissue repair. Moreover, PEUs have high flexibility in processability and in turn different matrix fabrications for drug delivery are feasible, such as drug-loaded implantable devices or injectable dosage forms (microparticles). Thus far, non-opioid analgesic compounds (i.e. bupivacaine, lidocaine, and etoricoxib) have successfully been incorporated into PEU films. The release profiles of the active pharmaceutical ingredients (APIs) from PEU films reveal sustained release over time that varies with chemical composition, film thickness, and drug-load. Additionally, drug diffusion from the polymer matrices follow Fickian diffusion as suggested through fitting to a Higuchi model. Similar release results have also been shown in vivo with etoricoxib and varying polymer composition. Preliminary results reveal integration of the film with local tissue while limiting exposure of drug systemically. Overall, our work highlights the adaptability of PEUs as an emerging biomaterial that’s potential has yet to be exhausted as well as providing a promising alternative to achieving sufficient post-operative pain management.

Albumin is emerging as a promising and versatile carrier to improve the pharmacokinetic and therapeutic profiles of drugs because of its unique physiological properties. The objective of this dissertation is to develop drug delivery systems that bind to and exploit the endogenous albumin in order to extend the plasma half-life of small molecule and peptide pharmaceutics for cancer and diabetes therapy. Three albumin binding drug delivery systems are developed and explored here: 1) albumin binding micelles, and 2) albumin binding peptide-drug conjugates (PDCs), both for cancer therapy, and 3) albumin binding peptide chimera for diabetes therapy. Elastin like polypeptide (ELP) is used as the recombinant expression and production platform and also as the carrier backbone for the micellar system.

For the albumin binding micelles, ELP was fused to the C-terminus of a protein-G derived albumin binding domain (ABD) and the ABD-ELP fusion was recombinantly expressed in and purified from Escherichia coli. Doxorubicin (Dox) was conjugated to the C-terminus of the ABD-ELP fusion, and conjugation of 4-5 copies of the drug to one end of the ABD-CP triggered its self-assembly into ~100 nm diameter spherical micelles. ABD-decorated micelles exhibited sub-micromolar binding affinity for albumin and also preserved their spherical morphology in the presence of albumin. In a murine model, albumin-binding micelles exhibited dose-independent pharmacokinetics, while naked micelles exhibited dose-dependent pharmacokinetics. In addition, in a canine model, albumin binding micelles resulted in a 3-fold increase in plasma half-life and 6-fold increase in plasma exposure as defined by the area under the curve (AUC) of the drug, compared with naked micelles. Furthermore, in a murine colon carcinoma model, albumin-binding nanoparticles demonstrated lower uptake by the reticuloendothelial system (RES) system organs —the liver and spleen— and higher uptake by the tumor than naked micelles. The increased uptake by s.c. C26 colon carcinoma tumors in mice translated to a wider therapeutic window of doses ranging from 20-60 mg equivalent of Dox per kg body weight (mg Dox Equiv.kg-1 BW) for albumin-binding CP-Dox micelles, as compared to naked micelles that were only effective at their maximum tolerated dose of 40 mg Dox Equiv.kg-1 BW.

For the albumin binding PDC system, 1 to 2 Dox molecules were conjugated to ABD via a pH-sensitive linker without the loss of aqueous solubility. ELP was used as a purification tag for the recombinant synthesis of ABD and was removed by an enzymatically-catalyzed reaction following drug conjugation. As with the albumin binging micells, albumin binding PDCs (ABD-Dox) showed strong nanomolar binding affinity for human and mouse serum albumin. Upon intravenous administration in mice, ABD-Dox showed an elimination half-life of about 26.5 h that is close to mouse albumin circulation time. In addition, within 2 h after administration, ABD-Dox distributed into tumor at approximately 4-fold higher concentration than free Dox and moreover while free Dox showed a quick clearance from the tumor site, ABD-Dox maintained a steady concentration in tumor for at least 72 h. The improved pharmacokinetic and pharmacodynamic profiles of ABD-Dox resulted in enhanced therapeutic efficacy in syngeneic C26 colon carcinoma and xenograft MIA-PaCa2 pancreatic tumor models compared to free Dox as well as aldoxorubicin, an albumin binding and the first-ever Dox prodrug to show superiority over Dox as a single agent in clinical trials.

Finally, for the albumin binding peptide chimera system, ABD was recombinantly fused to glucagon like peptide-1 (GLP1). In an obese and diabetic (db/db) mouse model, a single subcutaneous injection of the albumin binding GLP1 chimera provided glycemic control and effecting weight loss for over 7 days compared with PBS treated controls.

Collectively, the albumin binding technology developed here promises great potential for delivery of the traditional small molecule and peptide drugs as well as nanoparticulate therapeutics, whose clinical effectiveness is impaired by their poor pharmacokinetics and/or pharmacodynamics.

Peptide drugs are an exciting class of pharmaceuticals currently in development for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections. We have developed two novel peptide delivery approaches -Protease Operated Depots (PODs) and GLP-1-ELP depots- to provide sustained and tunable release of a peptide drug from an injectable s.c. depot.

We demonstrate proof-of-concept of these delivery systems, by fusion of monomer or protease cleavable oligomers of glucagon-like peptide-1 (GLP-1), a type-2 diabetes peptide drug, and a thermally responsive, depot-forming elastin-like-polypeptide (ELP) that undergoes thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. Utilizing a novel system we designed for repetitive gene synthesis, various GLP-1 polymers were designed and tested as potential therapeutic payload for PODs. By attachment to various ELPs, designed to transition above or below body temperature, we created both depot forming GLP-ELP fusions and soluble control. All fusion constructs maintained alpha helical content and were shown to be resistant to proteolytic degradation. In vitro activated PODs and GLP-ELP fusions were able to activate the GLP-1 receptor and remarkably, a single injection of both GLP-1 PODs and GLP-ELP fusions were able to reduce blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide drug. These findings suggest that ELP based peptide depots may offer a modular, genetically encoded alternative to various synthetic peptide delivery schemes for sustained delivery of peptide therapeutics.

Cytotoxic chemotherapy is a mainstay of cancer treatment, administered to patients who require systemic treatment based on the stage of their disease. Due to its dose-limiting side-effects, chemotherapy is commonly administered to avoid lethal toxicity rather than to maximize efficacy. Therefore, a great deal of effort has been focused on packaging drugs into nanoparticles, promoting its accumulation in tumors due to the presence of leaky neo-vasculature and re-directing the drug away from critical organs. While these drug-delivery technologies consistently improve treatment efficacy compared to conventional therapies in primary tumors of preclinical models, new treatment modalities are often not assessed for their ability to interfere with the metastatic process. Therefore, we studied the efficacy of our genetically encoded polypeptide nanoparticle for doxorubicin delivery (CP-Dox) in syngeneic metastatic murine models 4T1 and Lewis lung carcinoma. When our nanoparticle drug treatment was combined with primary tumor resection, greater than 60% of the mice were cured in both the 4T1 and Lewis lung carcinoma models as opposed to a 20% survival rate when treated with free drug. Mechanistic studies suggest that metastasis inhibition and survival increase were achieved by preventing the dissemination of viable tumor cells from the primary tumor.

While targeting metastatic disease directly is the standard goal of systemic chemotherapy, drugs that improve the treatment of primary tumors still hold promise for treating disseminated disease if they can stimulate a systemic host antitumoral immune response. Interestingly, certain cytotoxic drugs like doxorubicin can alter the host immune response to tumors by causing immunogenic cell death, revealing tumor-associated antigens and recruiting antitumoral leukocytes that can be further activated by co-treatment with immunostimulatory agents. However, the drug delivery field currently lacks an understanding of how packaging cytotoxic drugs into nanoparticles alters or potentially enhances these immunomodulatory phenomena. Previous studies have often administered doxorubicin intratumorally or studied the phenomenon in immunogenic tumor models. Whether the immunomodulatory properties of doxorubicin can be observed with systemic administration against a poorly immunogenic tumor model is unclear. Therefore, we have performed extensive studies of CP-Dox while interfering with aspects of the immune system to determine the role of the host antitumoral immune response in its efficacy. In this project, we show that a single intravenous (IV) administration of CP-Dox enhances the host anti-tumor immune response, enabling CD8+ cells to contribute to the prevention of metastasis in 4T1 mammary carcinoma. We show that IV CP-Dox increases the ratio of Th1 to Th2 cytokines in the tumor, and that IFN-y depletion reduces the efficacy of CP-Dox. We observed that the myeloid cell infiltrate was re-polarized to express markers associated with an anti-tumor phenotype. Importantly, these effects were not seen in mice treated with free doxorubicin. Our studies provide evidence that formulating cytotoxic chemotherapies as nanoparticles can better enable their in vivo immunomodulatory capabilities, demonstrating the potential of combining nanoparticle delivery strategies with immunotherapy to improve the treatment of cancer. 

Cytokine treatment was the first successful immunotherapy for cancer. Systemic administration of IL-2 leads to treatment responses in about 20% of patients with melanoma and renal cell carcinoma. However, the treatments have significant, life-threatening side-effects, leading to interest in local administration of IL-2. We have developed a bioactive ELP/IL-2 fusion which forms an insoluble coacervate upon in vivo injection. Intratumoral treatment of the fusion delayed the growth of B16 melanoma, and showed improved efficacy when combined with CP-Dox treatment. We also developed a bioactive ELP/GM-CSF fusion which was shown to recruit leukocytes to the site of subcutaneous injections. We show that the immunostimulatory activity of CpG is enhanced by condensation an ELP containing a lysine trailer. Thus we have developed several key ingredients for an in situ cancer vaccine which can generate antigen (CP-Dox), stimulate antigen presentation (GM-CSF and CpG), and stimulate T cells (IL-2).

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.

Cancer treatment regimens often include surgery, radiation, and chemotherapy. Though the World Health Organization (WHO) Essential Medicines List includes many globally accessible chemotherapies, surgery and radiation are inaccessible to 90% of patients in low- and middle-income countries (LMICs) due to lack of infrastructure, medical specialists, and funds. Novel treatment options, such as immune checkpoint inhibitors (ICIs), are increasing in use in high income countries (HICs), but can be prohibitively expensive for patients, especially in LMICs. Further, even when accessible in HICs, ICI therapies are not always effective. Breast cancers are especially non-responsive to ICIs. There is a compelling need to advance and/or enhance therapies in both HICs and LMICs. We have developed a novel ablation therapy that encases ethanol in a polymer local destruction of tumors. This proposal shows how we can adapt this for both scenarios as described in greater detail below.Ablation, the chemical or thermal destruction of tissue, is an alternative or adjunct to surgery and radiation because it is less expensive, less time intensive and minimally invasive. In HICs ablation is mainly used for local tumor control, but it can also induce immunomodulation that aids systemic response. When used in combination with chemotherapy or ICI therapy, it can target local and systemic responses. However, LMICs, which often lack access to surgery, also lack access to thermal ablation methods such as radiofrequency ablation (RFA), microwave ablation (MWA), and cryoablation due to cost, reliability of electricity. Further, they often lack trained physicians and personnel to maintain equipment. Even in HICs, thermal ablation is not always accessible or possible due to tumor location, exclusion criteria, or cost. Overall, to achieve clinical translation of this therapy it is essential to understand both: 1) the effect of delivery parameters on distribution and necrosis and 2) the potential for combination of novel ablative therapies with chemotherapy and ICI therapy to inform treatment and practice. Ethanol ablation is portable and low-cost, allowing it to overcome treatment barriers in LMICs. However, ethanol ablation has limited treatment efficacy due to poor ethanol localization and off-target leakage. Incorporating ethyl-cellulose (EC), an ethanol-soluble, water-insoluble polymer, to ethanol help mitigate these limitations. EC-ethanol (ECE) transitions from liquid to fibrous gel upon injection into tissue (in-situ gelation). This acts to sequester ethanol, reduces off-target leakage and, overall, can improve ablation efficacy. ECE has the potential to create a more predictable distribution of ablation, therefore I investigated the impact of key components affecting the delivery and therapeutic effect of ECE (Aim 1) and investigate the biological impact of ECE in combination with current clinical treatment paradigms and as a novel drug delivery agent (Aim 2). Pursuit of these aims was intended to elucidate the efficacy, safety, and predictability of ECE ablation for use in cancer treatment and inform eventual clinical translation of this technology. The outcome should demonstrate that ECE is safe for human use and exhibits pharmacological activity, bringing this technology steps closer to investigation in clinical trials. Research in this dissertation pursued a thorough understanding of key factors governing the therapeutic effects of ECE with goal of informing translation of ECE to a clinical setting. I assessed the effect of formulation and delivery parameters on the resultant distribution or leakage and on necrosis. This can lead to algorithms enabling clinicians to select optimal tools and delivery methods to maximize treatment efficacy. Further, adoption of ECE in the clinical setting cannot be achieved without a clear understanding of the healing response to ablation and the safety of the procedure. Thus, time course analysis of the wound healing response and treatment safety compared to traditional ethanol ablation is necessary. To assess these key components, we need a method for assessment that allows for real time visualization of the ablation. For optimization we can us a high resolution more expensive technology, such as computed tomography, with the intent of adapting methods for more accessible technologies like ultrasound in the future. I developed a method for utilizing CT and investigated delivery parameters in both small and large animal models. In addition to investigating larger scale models to inform clinical translation (Aim 1), I also assessed these key determinants of injections success in small animal models to inform the biological mechanisms of injection efficacy (Aim 2). This led to investigating the synergy of ECE with ICI therapy and modification of the ECE formulation as a cytotoxic drug carrier. In particular, ECE ablation has potential for synergy with combination therapeutics, specifically immunotherapies and chemotherapeutic agents. This could have high potential for impact in HICs where implementation of immunotherapies and intensive chemotherapy regimens is more common and accessible. ECE exposes tumor antigens to T cells, evoking an immune-stimulatory response. I hypothesized that ECE can prime “cold” tumors to enhance response to ICI, for which many breast cancers are non-responsive. Previous work demonstrated that low- dose cyclophosphamide enhances the therapeutic effect of ECE. Therefore the combination of ECE ablation and low-dose cyclophosphamide was a logical choice to investigate a neoadjuvant therapy to enhance response to ICIs in non-responsive tumors. I hypothesized that the in-situ gelation of EC can be implemented to improve intra-tumoral drug delivery. Ethanol is know for its cytotoxic effects on cells. vehicle compared to many inert polymer vehicles. Combining ECE with chemotherapy (often, small drug molecules) as a local treatment could synergize apoptotic and necrotic cell death induced by the drugs and ethanol, respectively, a therapeutic process absent in traditional drug carriers. Thus, I focused upon effect of ECE on small molecule transport, drug uptake and distribution throughout the body over time, and also assessed safety and efficacy of this novel combination treatment. The goal of this dissertation research was to improve efficacy, safety, and predictability of ECE ablation. I aimed to optimize delivery of ECE, working to understand the effects of salient injection parameters on distribution and necrosis. I also investigated ECE ablation in combination with chemotherapies or ICIs, helping to lay the groundwork for clinical translation, and informing the foundation for local and systemic treatment responses. To achieve this goal, I completed two parallel aims. Aim 1 focused on delivery of ECE, specifically development of real-time assessment methods, infusion parameter assessment at preclinical and clinical scales, and investigation of resultant necrosis and the wound healing response. Aim 2 focused on investigating the utility, efficacy, and safety of the ECE formulation as a cytotoxic drug carrier, and examined the synergy of ECE with ICIs.

We have developed a broadly applicable platform that harnesses the power of protein engineering and genetic screening to produce efficacious protein-drug combinations for cancer therapy. For proof-of-concept, we implemented this strategy to engineer targeted pro-apoptotic drug combinations that overcome cancer resistance to protein agonists of death receptor 5 (DR5), a key upregulated marker in colorectal cancer (CRC). Over the past decade, various DR5 agonists have shown poor clinical efficacy, including both engineered antibodies and TRAIL, the natural ligand for this receptor. Comprehensive studies suggest that there are three major obstacles to success of these agents: 1) potency, 2) delivery, and 3) resistance.

We have systematically addressed these challenges by engineering a sustained-release formulation of a highly potent, hexavalent death receptor 5 agonist (DRA), and administering the agonist as a sustained release depot, in combination with rationally nominated targeted drugs that overcome intrinsic resistance to DRAs. To address the need for sustained delivery of therapeutic proteins, we developed injectable depots of DRAs recombinantly fused to thermally responsive elastin-like polypeptide (ELP) biopolymers. The bioactive ELP-DRA fusions undergo temperature-driven phase transition upon subcutaneous injection in vivo, resulting in the formation of a gel-like depot suitable for sustained drug delivery. A single 30 mg/kg injection of the gel-like ELP-DRA depot induced significant tumor regression in Colo205 mouse xenografts. To pinpoint the genetic drivers of CRC resistance to the DRA, we used a gain-of-function ORF screen and a CRISPR/Cas9 knockout screen. The screens identified genes that confer sensitivity to the DRA in resistant CRC cell lines. Over twenty small molecule drugs targeting pathways and proteins identified from the screens were then tested in combination with the DRA to identify highly synergistic combinations using cytotoxicity assays. Clonogenic, time-to-progression, and cell viability assays showed that pharmacological blockade of XIAP, Bcl-XL, and CDK4/6 strongly enhances antitumor activity of DRA in established human CRC cell lines and patient-derived CRC cells. In vivo tumor regression studies demonstrated the potent anti-tumor efficacy of combining inhibitors of XIAP and Bcl-XL with the sustained release formulation of ELP-DRA.

By addressing both delivery and resistance issues with our protein engineering and genomics platform, we have overcome the key obstacles to DRA translation as a successful drug in the clinic. Our rational approach elegantly provides optimal protein-small molecule drug combinations that elicit a robust anticancer response, exhibit minimal toxicity, and combat drug resistance.