Overcoming Transport Barriers To Nanoparticle-Based Chemotherapy

University of Minnesota Ph.D. dissertation. August 2015. Major: Pharmaceutics. Advisor: Jayanth Panyam. 1 computer file (PDF); xix, 227 pages.Targeted delivery of drugs to tumors is critical for optimal implementation of chemotherapy. Targeted drug delivery can be achieved by encapsulating drugs in nanometer-sized drug carriers. Consequently, a significant amount of preclinical research is focused on the development of nanoparticle-based chemotherapeutics. In spite of many key advances, these systems still have some major limitations. These stem from extensive distribution of nanoparticles to off-target sites, and the presence of several resistance mechanisms within the tumor. The goal of this thesis was to investigate, and potentially improve, the various steps involved in the delivery of nanoparticles to solid tumors. Using a pharmacokinetic modeling approach, the effect of physicochemical properties of nanoparticles and vascular permeability of the tumor on the systemic distribution of nanoparticles was studied. Transport across compartments was calculated using equations for transvascular convection and diffusion of nanoparticles. The results of the modeling studies suggested that optimal drug targeting would be observed only in an intermediate particle size range. At a particle size below the vascular pore size of the toxicity compartment, nanoparticles extensively accumulated in that compartment. This reduced the fraction available for deposition into the tumor. At a particle size above the vascular pore size of the tumor, accumulation of nanoparticles in the tumor was not possible. Most of the drug was either eliminated in the form of nanoparticles or released in the central compartment. Hence, the lower and upper bounds of the optimal particle size range were determined by the vascular pore size of the toxicity and target compartments, respectively. With a decrease in drug release rate, the average number of visits of the drug in the target compartment increased. Hence, decreasing the rate of drug release could favorably affect the drug targeting index. Maximal benefit with this strategy was also experienced in an intermediate particle size range, which was dictated by vascular pore sizes of the toxicity and target compartments. Due to leaky tumor blood vessels and prothrombogenic tumor microenvironment, fibrinogen gets deposited in the tumor and is converted to cross-linked fibrin. Using a series of immunohistochemistry analysis and in vitro diffusion studies, it was established that most human solid tumors are characterized by the deposition of a significant amount of fibrin and that the presence of fibrin retarded the mobility of nanoparticles. However, no previous studies have analyzed the influence of fibrin on the intratumoral distribution of nanoparticles. The central hypothesis of this chapter was that degrading fibrin using a fibrinolytic enzyme [tissue plasminogen activator (tPA)] will improve the intratumoral distribution of nanoparticles and their chemotherapeutic efficacy. In an A549 orthotopic lung cancer model, co-administration of tPA improved the anti-cancer efficacy of paclitaxel nanoparticles (p2 months. In a B16F10 mouse melanoma model the combination therapy reduced the rate of tumor growth by ~1.5 fold relative to that with just paclitaxel nanoparticles (p=0.08). Further, immunohistochemistry revealed that administration of tPA resulted in the decompression of tumor blood vessels. Using Power Doppler ultrasound, we established that treatment with tPA led to a 3-4 fold improvement in tumor perfusion (p<0.05, after second dose). All these results suggest that fibrinolytic therapy may lead to an enhanced intratumoral distribution of nanoparticles. Tylocrebrine is a potent anti-cancer drug but has a narrow therapeutic index. Extensive distribution to the central nervous system and reduced cell uptake in the acidic tumor microenvironment limited the clinical translation of the drug. To address both issues, tylocrebrine was encapsulated in polymeric nanoparticles targeted to the epidermal growth factor receptor (EGFR). In vitro studies in human cancer cell lines showed that decrease in the extracellular pH led to a 3-fold decrease in drug uptake (p<0.05) and ~2-10 fold reduction in drug potency. However, when encapsulated in targeted nanoparticles, its potency was less affected by extracellular pH. Pharmacokinetic studies in mice revealed that the drug rapidly accumulated in the brain, and brain accumulation could be reduced by encapsulation in nanoparticles (~2-fold; p<0.05, compared to free drug at 0.5 h post dose). When delivered in targeted nanoparticles, the tumor accumulation and retention of the drug was also improved (p<0.05, compared to free drug at 4h post dose). In a xenograft mouse model of human epidermoid cancer, treatment with tylocrebrine solution retarded tumor growth. However, tumor inhibition was found to be more significant with EGFR targeted nanoparticles (p<0.05, compared to saline and non-targeted nanoparticle treated animals). This effect was likely due to the improved tumor accumulation and higher potency of targeted nanoparticles. In conclusion, this thesis presents an analysis of the three steps of nanoparticle transport from the site of administration to the site of action. These steps include systemic distribution, intratumoral distribution and cell uptake. Improving the efficiency of each of these steps can improve the overall efficacy of chemotherapy

Triggerable tough hydrogels for gastric resident dosage forms

Systems capable of residing for prolonged periods of time in the gastric cavity have transformed our ability to diagnose and treat patients. Gastric resident systems for drug delivery, ideally need to be: ingestible, be able to change shape or swell to ensure prolonged gastric residence, have the mechanical integrity to withstand the forces associated with gastrointestinal motility, be triggerable to address any side effects, and be drug loadable and release drug over a prolonged period of time. Materials that have been primarily utilized for these applications have been largely restricted to thermoplastics and thermosets. Here we describe a novel set of materials, triggerable tough hydrogels, meeting all these requirement, supported by evaluation in a large animal model and ultimately demonstrate the potential of triggerable tough hydrogels to serve as prolonged gastric resident drug depots. Triggerable tough hydrogels may be applied in myriad of applications, including bariatric interventions, drug delivery, and tissue engineering.Bill & Melinda Gates Foundation (Grant OPP1096734)Bill & Melinda Gates Foundation (Grant OPP1139927)National Institutes of Health (U.S.) (Grant EB000244

An Underground Coal Mine Fire Preparedness And Response Checklist: The Instrument

Preparedness is an important element of any underground mine's strategic plan in dealing with an unexpected event, such as a fire. A fully implemented fire preparedness and response plan is essential in reducing the probability and seriousness of a mine fire. This report describes the development of an underground coal mine fire preparedness and response checklist (MFPRC). The checklist is a data collection instrument for profiling both the fire prevention and response capabilities of a mine site and usually requires 3 to 4 days to complete. The checklist encompasses conditions, procedures, and equipment that have frequently been identified as the primary or contributing causes of underground coal mine fires. At least 1 day is needed underground to evaluate the water system. This entails measurements of water flows and pressures at fire hydrants, and water throw distances of fire hose and nozzles at several locations (mains and branch lines). A few of the other topics that are discussed with mine personnel include detection and suppression systems, combustible materials, mine rescue and fire brigades, and firefighting equipment. The MFPRC was developed by the National Institute for Occupational Safety and Health (NIOSH),Pittsburgh Research Laboratory. Under a Cooperative Research and Development Agreement (CRADA) with Cyprus Amax, Twenty mile Coal Co. (Oak Creek, CO), the checklist was field tested and further refined. Additional field tests were conducted at several other operating coal mines

Past, Present, and Future Drug Delivery Systems for Antiretrovirals

The human immunodeficiency virus has infected millions of people and the epidemic continues to grow rapidly in some parts of the world. Antiretroviral (ARV) therapy has provided improved treatment and prolonged the life expectancy of patients. Moreover, there is growing interest in using ARVs to protect against new infections. Hence, ARVs have emerged as our primary strategy in combating the virus. Unfortunately, several challenges limit the optimal performance of these drugs. First, ARVs often require life-long use and complex dosing regimens. This results in low patient adherence and periods of lapsed treatment manifesting in drug resistance. This has prompted the development of alternate dosage forms such as vaginal rings and long-acting injectables that stand to improve patient adherence. Another problem central to therapeutic failure is the inadequate penetration of drugs into infected tissues. This can lead to incomplete treatment, development of resistance, and viral rebound. Several strategies have been developed to improve drug penetration into these drug-free sanctuaries. These include encapsulation of drugs in nanoparticles, use of pharmacokinetic enhancers, and cell-based drug delivery platforms. In this review, we discuss issues surrounding ARV therapy and their impact on drug efficacy. We also describe various drug delivery–based approaches developed to overcome these issues.Bill & Melinda Gates Foundation (Grant OPP1139937)National Institutes of Health (U.S.) (Grant EB-000244

Machine Learning Uncovers Food- and Excipient-Drug Interactions

Inactive ingredients and generally recognized as safe compounds are regarded by the US Food and Drug Administration (FDA) as benign for human consumption within specified dose ranges, but a growing body of research has revealed that many inactive ingredients might have unknown biological effects at these concentrations and might alter treatment outcomes. To speed up such discoveries, we apply state-of-the-art machine learning to delineate currently unknown biological effects of inactive ingredients—focusing on P-glycoprotein (P-gp) and uridine diphosphate-glucuronosyltransferase-2B7 (UGT2B7), two proteins that impact the pharmacokinetics of approximately 20% of FDA-approved drugs. Our platform identifies vitamin A palmitate and abietic acid as inhibitors of P-gp and UGT2B7, respectively; in silico, in vitro, ex vivo, and in vivo validations support these interactions. Our predictive framework can elucidate biological effects of commonly consumed chemical matter with implications on food- and excipient-drug interactions and functional drug formulation development

Gastrointestinal Delivery of an mRNA Vaccine Using Immunostimulatory Polymeric Nanoparticles

Abstract mRNA vaccines can be translated into protein antigens, in vivo, to effectively induce humoral and cellular immunity against these proteins. While current mRNA vaccines have generated potent immune responses, the need for ultracold storage conditions (− 80 °C) and healthcare professionals to administer the vaccine through the parenteral route has somewhat limited their distribution in rural areas and developing countries. Overcoming these challenges stands to transform future deployment of mRNA vaccines. In this study, we developed an mRNA vaccine that can trigger a systemic immune response through administration via the gastrointestinal (GI) tract and is stable at 4 °C. A library of cationic branched poly(β-amino ester) (PBAE) polymers was synthesized and characterized, from which a polymer with high intracellular mRNA delivery efficiency and immune stimulation capacity was down-selected. mRNA vaccines made with the lead polymer-elicited cellular and humoral immunity in mice. Furthermore, lyophilization conditions of the formulation were optimized to enable storage under refrigeration. Our results suggest that PBAE nanoparticles are potent mRNA delivery platforms that can elicit B cell and T cell activation, including antigen-specific cellular and humoral responses. This system can serve as an easily administrable, potent oral mRNA vaccine. Graphical Abstrac

Local Targeting of NAD + Salvage Pathway Alters the Immune Tumor Microenvironment and Enhances Checkpoint Immunotherapy in Glioblastoma

© 2020 American Association for Cancer Research. The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced byNAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. Significance: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM

Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug Delivery

The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly­(d,l-lactide-<i>co</i>-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepared by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (<i>P</i>_app) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability