<p>  </p> <p>Cancer immunotherapies have revolutionized anticancer treatment, saving lives by utilizing patients’ immune systems. Immunogenic cell death inducing chemotherapies have recently gained interest as they can not only inhibit the growth of the tumor but also allows the patient to develop a long-lasting immune response to the tumor. However, due to the poor retention of chemotherapies in the tumor and immunosuppressive tumor microenvironment, the activity of immunogenic cell death inducing chemotherapy is limited. To overcome the limitations, I have developed immunofunctional poly(lactic-co-glycolic acid) nanoparticles to enhance the retention of immunogenic cell death inducers at the tumor and increase the recruitment of antigen-presenting cells to the tumor.</p> <p><br></p> <p>In our study, paclitaxel and carfilzomib were determined as immunogenic cell death inducers, supported by in vitro screening of damage-associated molecular patterns and in vivo vaccination study. Both drugs were identified as immunogenic cell death inducing chemotherapy agents. To deliver immunogenic cell death inducers, immunofunctional poly(lactic-co-glycolic acid) nanoparticles were developed by modifying the surface with adenosine triphosphate. The coating of adenosine triphosphate attracted dendritic cells in a concentration gradient manner and improved the stability of adenosine triphosphate against its degrading enzyme. Both paclitaxel and carfilzomib were successfully encapsulated into the developed nanoparticle formulation. Paclitaxel encapsulated nanoparticles were chosen as a lead candidate due to the inherent immunotoxicity of carfilzomib.</p> <p>Paclitaxel encapsulated nanoparticles coated with ATP effectively suppressed tumor growth in CT26 murine carcinoma and B16F10 murine melanoma. The formulation also increased the immune cell infiltration into the tumor, which may explain the enhanced efficacy of the nanoparticle formulation. Combinational therapy of nanoparticles with anti-PD-1 antibodies significantly increased the complete regression rate in tumor-bearing mice by invigorating the immunosuppressive environment. </p> <p><br></p> <p>In summary, paclitaxel (an immunogenic cell death inducer) encapsulated in adenosine triphosphate-coated poly(lactic-co-glycolic acid) nanoparticles attracted dendritic cells in a concentration gradient manner and effectively suppressed tumor. Additional anti-PD-1 antibodies further improved the antitumor effect, inducing complete tumor regression in 75% of CT26-bearing mice, by inhibiting the interactions between T cells and immunosuppressive cells (regulatory T cells and myeloid-derived suppressor cells). </p> <p><br></p> <p>Chapter 1 discusses the current understanding of immunotherapy and delivery systems to enhance immunotherapy. Chapter 2 describes the determination of immunogenic cell death inducers and the development of immunofunctional nanocarrier. The in vivo antitumor efficacy of the nanocarrier was tested in Chapter 3. </p>

<p dir="ltr">The success of an amorphous solid dispersion (ASD) lies in its ability to generate a supersaturated solution and maintain it for a physiologically relevant duration. Both these processes could be influenced by the presence of crystalline drug in ASDs. Even a low level of residual drug crystals in a freshly prepared ASD can act as seeds resulting in rapid recrystallization from solution if the drug is a fast crystallizer. Detecting crystallinity in an ASD at such low levels becomes limited by the detection technique. Residual crystallinity in posaconazole-hydroxypropyl methylcellulose acetate succinate (HPMCAS) ASDs was quantified using second harmonic generation based nonlinear optical imaging and its effect on the sustenance of supersaturation was evaluated in real time using orthogonal analytical methods including focused beam reflectance measurement.</p><p dir="ltr">Drug release and crystallization are both highly influenced by the polymer present in ASDs. Understanding polymer chemistry is especially important for ionic polymers like HPMCAS which is one of the commonly used polymers and is present in several commercially available ASD-based products. Acetate-to-succinate ratio in HPMCAS and buffer pH determine the polymer hydrophobicity and strength of interaction with a weakly basic drug. The more hydrophilic grade L released the drug at a faster rate but was weaker in its ability to inhibit crystallization as compared to the more hydrophobic grade H which released poorly, whereas M was found to be optimum. Polymer hydrophobicity correlated with the strength of its interactions with the drug as measured by fluorescence spectroscopy.</p><p dir="ltr">A major factor determining the rate of drug release from ASDs is its glass transition temperature (<i>T</i>_<em>g</em>). High-<i>T</i>_<em>g</em> drugs release poorly from ASDs making them challenging candidates unless formulated at very low drug loading resulting in pill burden. The effect of glyceryl tributyrate as a plasticizer was evaluated on the release rates of three high-<i>T</i>_<em>g</em> drugs, ivacaftor, cyclosporine, and ARV-825 from their ASDs with HPMCAS. Ternary ASDs containing glyceryl tributyrate had noticeably lower <i>T</i>_<em>g</em>s than their binary counterparts and a significant improvement in the release rates was observed. This enables formulation of high-<i>T</i>_<em>g</em> drugs as ASDs with higher drug loading. This work has helped expand the knowledge of dissolution of ASDs containing HPMCAS and its role in preventing drug crystallization.</p>

<p>Immunotherapy has significantly improved cancer treatment. However, many tumors are resistant to current immunotherapy due to the highly immunosuppressive tumor microenvironment (TME). Tumor cells can evade immune activation by externalizing phosphatidylserine (PS) on cell surface to trigger anti-inflammatory signals and induce immune tolerance. Recent studies show that PS is upregulated in TME and further increased after chemotherapy. For effective immunotherapy of tumors, the exposed PS needs to be blocked to relieve immunosuppressive TME and sensitize tumors to immune stimulants. </p> <p><br></p> <p>In this study, PS exposure level increased after the chemotherapy Doxil treatment on B16F10 melanoma cells, and the PS exposure reduced the response of antigen-presenting cells (APCs) to immune stimulants such as lipopolysaccharide. Dipicolylamine-Zn (DPA-Zn) shielded the PS exposure resulting from doxorubicin (DOX) treatment and reduced immunosuppressive interaction between tumors and APCs. The PS blockade by DPA-Zn improves the tumor response rate immune stimulants such as GM-CSF, STING agonist cyclic dinucleotides (CDN), anti-PD-L1 antibody. Among the combination at the tested doses, Doxil + DPA-Zn + CDN was the optimal combination that enhanced anti-tumor effect most significantly and prolonged the survival time in immune-cold B16F10 melanoma model. However, the anti-tumor efficacy was limited, which is attributed to poor tumor retention of CDN and DPA-Zn. </p> <p><br></p> <p>To prolong the intratumoral release of DPA-Zn and CDN and maximize the anti-tumor immunity, CDN was formulated as liposomes (CDN@lip), which significantly delayed the release of CDN in vitro and improved anti-tumor efficacy compared with free CDN formulation. Alginate hydrogel showed the potential to sustain release of DPA-Zn. DPA-Zn was loaded in the alginate hydrogel via electrostatic interaction, and the release rate was controlled by additional zinc gluconate. However, zinc caused detrimental effects on skin and can cause mice death at a high dose. To avoid the side effect of subcutaneously administered Zn, the dose of DPA-Zn in alginate hydrogel was readjusted based on the maximum tolerated dose study, and zinc gluconate was replaced with CaSO4.</p>

<p dir="ltr">Opioid use disorder (OUD) presents a challenging and nuanced condition with potential for debilitating social and physical consequences. Patients with OUD have access to treatment options, but they may encounter issues such as diversion, invasiveness, or poor adherence. With over 2.5 million adults in the US experiencing OUD as of 2021, the need for an OUD treatment that overcomes these challenges is clear. One available treatment method is Sublocade®, a PLGA-based in situ forming implant (ISFI) that releases buprenorphine. This treatment shows promise due to its physician administered extended release design, which addresses many current issues in OUD treatment. However, the practicality of this treatment remains a challenge due to its monthly injection requirement. To address this, we investigated how altering ISFI components impacts the timeframe of buprenorphine release from a PLGA-based ISFI. Our focus was on evaluating factors that lead to extended buprenorphine release while maintaining zero-order release. We varied polymer-to-solvent ratios, drug percentage, and solvent composition, assessing their effects through drug release studies. We also conducted SEM imaging and swelling/erosion studies to evaluate polymer behavior and implant microstructure, gaining further insights into drug release mechanisms. Our drug release studies revealed that higher buprenorphine content in the implant significantly reduced total drug release and linearized drug release patterns. Decreasing the polymer-to-solvent ratio similarly linearized drug release and reduced drug burst, although the overall amount of drug released over time remained similar. Introducing Triacetin (TA) as a solvent helped reduce drug burst and maintain release linearity in lower drug content implants. In higher drug content implants, TA appeared to increase drug release over time, likely due to degradation processes indicated by high swelling and increased degradation observed in SEM imaging. Erosion studies showed less implant erosion with higher drug loading, aligning with release study observations. In conclusion, solvent type and drug content significantly influence buprenorphine release in ISFI systems and should be carefully considered when designing extended release systems similar to Sublocade®.</p>

<p dir="ltr">Nucleic acids are promising drug candidates as they can address diseases with few “druggable” targets. Nevertheless, nucleic acids are challenging to deliver because of their large molecular weights, dense negative charges, proinflammatory activities, and short half-lives in biological fluids. Synthetic gene carriers based on cationic polymers or lipids have been used to overcome these challenges; however, their cationic nature results in dose-limiting toxicities and accelerated removal by the filtering MPS organs after systemic administration. In the past six years, several nucleic acid-based therapeutics have been approved by the FDA, formulated as lipid nanoparticles (LNPs). Nonetheless, LNPs show extensive liver accumulation after intravenous administration and, hence, are only indicated for hepatic or local vaccine delivery applications. Therefore, there is a critical unmet need for a nanocarrier that delivers nucleic acids to the extrahepatic organs without significant toxicities. To address this need, we developed Nanosac, a deformable and non-cationic nanocarrier, to deliver siRNA to solid tumors. Deformability can improve multiple aspects of the nanoparticle biotransport, ranging from circulation time and protein corona composition to biodistribution and interactions with the target cells. Meanwhile, a non-cationic carrier avoids proinflammatory complications and rapid clearance of cationic nanoparticles. For this application, we used siRNAs targeting CD47/SIRPa and PD-l/PD-L1 immune checkpoints due to their critical roles as “don't-eat-me” and “don't-find-me” signals to immune cells, respectively, which interfere with the development of innate and adaptive antitumor immune responses.</p><p dir="ltr">In the same context of enhancing the tumor delivery of nanomedicine, we developed two formulations for the small-molecule chemo drug, carfilzomib (CFZ). A nanocrystal formulation with optimized particle size had high CFZ loading, adequate colloidal stability in circulation and better antitumor activity in mice than the FDA-approved CFZ formulation. Despite its improved efficacy, the stiff nanocrystals aggravated CFZ immunotoxicity due to its excessive accumulation in mice spleens. To address this issue, we employed Nanosac technology for CFZ delivery, exploiting its deformability to reduce the non-specific spleen distribution and enhance CFZ tolerability.</p><p dir="ltr">Our results showed that Nanosac delivered siRNA to tumor cells and silenced the target protein expression better than LNPs. <i>In vivo</i>, Nanosac reduced siRNA accumulation in the MPS organs and achieved greater siRNA-mediated tumor suppression than LNPs in two murine tumor models. Moreover, Nanosac achieved greater checkpoint protein silencing in tumors, but less silencing in the MPS organs than LNPs, highlighting their differential biodistribution. The superior Nanosac performance relative to LNPs after systemic delivery is likely due to the difference in their protein coronas and cellular delivery capabilities. In addition, CFZ loading in Nanosac ameliorated CFZ immune cell toxicity <i>in vitro</i> and improved its tolerability in mice while maintaining similar therapeutic efficacy compared to the stiff nanocrystal formulation. Collectively, these findings highlight nanocarrier deformability and corona composition as viable strategies to improve the extrahepatic delivery of nucleic acids as well as to minimize toxicities related to extensive NP distribution to the off-target MPS organs.</p>

<p>Deep vein thrombosis (DVT) and Pulmonary embolism (PE) are responsible for over 900,000 cases and 100,000 deaths each year in the US. Direct fibrinolytic agents such as plasmin are being investigated for their treatment. However, plasmin administration is not widely studied as low plasmin concentrations are rapidly inactivated by antiplasmin in vivo, whereas high plasmin doses would deplete endogenous antiplasmin and impose bleeding risks. Thus, a plasmin delivery system that can achieve efficient clot lysis while minimizing inactivation by antiplasmin and has reduced bleeding risks is needed. To address this, we propose using reversible inhibitors of plasmin that can sequester plasmin from antiplasmin and release it on the surface of a fibrin clot to achieve clot lysis. The inhibition must be tuned such that it is strong enough to protect plasmin from antiplasmin and weak enough to release plasmin at the clot for lysis. To achieve this, we utilize principles of multivalency to synthesize three classes of inhibitors with varying potencies and mechanisms of inhibition: (i) Multivalent benzamidines (ii) Multivalent tranexamic acids (TXA), and (iii) Hetero-multivalent inhibitors having both benzamidine and TXA. Benzamidine is a competitive inhibitor of plasmin’s active site. TXA, on the other hand, is an FDA-approved weak active site inhibitor that is primarily used to disrupt plasmin(ogen) from binding to fibrin on the clot by inhibiting plasmin’s kringle domains. Multivalent inhibitors were synthesized using amine-reactive chemistry, purified using RP-HPLC and confirmed with Mass Spectrometry. Inhibition assays were performed to assess inhibition potency by determining Ki values (inhibition constants). Lower Ki values indicate stronger inhibition. With multivalent benzamidine derivatives, it was observed that changing valency and linker length substantially impacted inhibition and resulted in Ki values ranging from 2.1 to 1,395 μM. Inhibitors of higher valencies and shorter linker lengths exhibited stronger inhibition. Multivalent TXAs of valencies 1 to 16 were also tested and they exhibited Ki values varying from 2.5 to 21,370 μM indicating up to 8,548-fold improvement in inhibition due to valency. It was found that monovalent TXA, primarily a kringle inhibitor, can be converted into a stronger active site inhibitor by multivalency. With hetero-bivalent TXA-dPEG36-AMB, simultaneous binding of benzamidine to the active site and TXA to the kringle domains was achieved to attain improved inhibition. These results indicate that multivalency can significantly alter the potency of inhibitors and can modulate plasmin inhibition for drug delivery.</p>

<p>Biotherapeutics, such as monoclonal antibodies (mAbs), represent a primary mechanism for treatment of human disease, and there has been a steady increase in Food and Drug Administration approvals since the first one in 1982. Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method that increases the convenience and reduces cost compared to other delivery methods. However, progress is needed to optimize bioavailability via this route. This dissertation describes the methods for evaluation of mass transport of high molecular weight proteins, such as mAbs, following SC injection using <em>in vitro</em> and <em>ex vivo</em> modeling developed to describe the factors relevant for optimal distribution prior to uptake into systemic circulation. The first chapter describes a novel collagen and hyaluronic acid (HA) based hydrogel for investigation of macromolecule transport based on the physiochemical properties of the diffusing molecule and the tissue matrix. This initial study demonstrated that, in collagen alone, collagen combined with HA, and HA alone, the molecules demonstrated different transport paradigms dependent primarily on molecule size, matrix viscosity, and electrostatic charge, respectively. This showed that the local tissue heterogeneity and therapeutic properties could be determining factors for molecule transport and bioavailability. The second, third, and fourth chapters describe two novel platforms for the investigation of injection plume formation in SC tissue utilizing three-dimensional X-ray tomography. Injection plume analysis has been studied comprehensively in the context of insulin transport using co-injection of radiopaque dyes to track the protein distribution. However high molecular weight therapeutics have vastly different physiochemical properties than insulin and are injected under different rates, concentrations, volumes, and viscosities due to dosing considerations. To address the gap mAb distribution, we first developed a novel protein conjugated to an x-ray contrast agent to directly track injection plume formation and investigated the effects of injection rate and tissue location through injections into ex vivo porcine tissue, described in chapters three and four. Ex vivo tissue analysis showed that the rate did not influence the distribution, however, plume volume was lower in porcine belly compared to neck tissue. Whereas porcine tissue is an excellent model to represent the structural features of human injection, the large heterogeneity between animal subjects and collected samples is a disadvantage. Therefore, the fourth chapter describes the fabrication of a gelatin hydrogel-based injection platform representing the dermal and subcutaneous tissue layers for controlled injection plume analysis. In summary, all three models represent useful platforms for the assessment of macromolecular mass transport, pharmaceutical autoinjector performance, as well as the potential impact of tissue properties and intersubject heterogeneity on plume formation. Overall, the findings in these studies might better inform drug designers and clinicians on how to most optimally engineer an injection to deliver the most efficient patient outcomes through better dosing and increased cost savings. </p>

<p>Biotherapeutic compounds such as monoclonal antibodies help millions of people worldwide.  Currently, one of the most popular ways to deliver these compounds is via subcutaneous (SC)  injection. While it is understood that SC drug delivery does change with respect to injection  location, it is not understood why, as how the composition of SC changes as a function of location  is unknown. In this study, liquid chromatography mass spectrometry was used to understand and  describe how the SC tissue space changes on a molecular level. SC tissue from three different  locations, belly, breast, and behind the ear, of Yucatan minipigs was harvested and analyzed to  understand if and how SC tissue changes when anatomical location changes. It was determined  that there were distinct differences between the proteins identified in the three anatomical  locations. These differences included differences in relative cell populations, indicating that  different anatomical locations of SC tissue have different functions. Additionally, an ex vivo human SC tissue model was used to identify a core human proteome, as well as determine  compositional differences between female and male SC tissues. This model was also compared to  the Yucatan minipig model to determine compositional similarities between all groups. Finally,  proteomics were also used to ascertain whether the mass of SC tissue used affected the proteomic  results of the sample. These results indicated that human SC identifies the same number of proteins  down to samples of 10mg. This information can be used to design a proteomic experiment that  uses core needle biopsies to determine what gauge needle should be used in a wide scale clinical  study characterizing the human SC proteome. </p>