Helen Mary Burt

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Copper-based therapeutics : creating a formulation platform to facilitate development of an emerging drug class (2017)

Copper-based therapeutics (CBTs) are a promising class of drug candidates that have been shown to have anti-cancer activity. One of the major challenges associated with developing this emerging class of medicines, however, is their poor aqueous solubility (
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The effect of P-glycoprotein inhibition and ultrasound exposure on the cytotoxicity of taxane loaded diblock copolymer nanoparticles in multidrug resistant cells (2014)

One of the major mechanisms of multidrug resistance involves an efflux protein, P-glycoprotein (Pgp), which pumps commonly used anticancer drugs such as taxanes out of cells, leading to a decrease in cellular drug accumulation. The overall goal of this project was to develop strategies to enhance intracellular drug accumulation and cytotoxicity of nanoparticulate taxanes in multi-drug resistant (MDR) cell lines.Paclitaxel (PTX) loaded nanoparticles fabricated from micelle forming MePEG₁₁₄-b-PCL₁₉ and nanosphere forming MePEG₁₁₄-b-PCL₁₀₄ were compared for drug and block copolymer uptake, and cytotoxicity in drug sensitive MDCKII and drug resistant MDCKII-MDR1 cell lines. PTX loaded micelles were more cytotoxic than PTX loaded nanospheres. Co-administration of the known Pgp inhibitor, MePEG₁₇-b-PCL₅, with PTX loaded micelles or nanospheres significantly increased drug cytotoxicity in MDCKII-MDR1 cells. Mixed molecular weight (MW) PCL₂₀₀/PCL₅ nanoparticles composed of long hydrophobic block, MePEG₁₁₄-b-PCL₂₀₀, and MePEG₁₇-b-PCL₅, were developed and characterized for the co-delivery of taxanes and Pgp inhibitor. Both PTX and docetaxel (DTX) loaded mixed MW PCL₂₀₀/PCL₅ nanoparticles were demonstrated to release MePEG₁₇-b-PCL₅ in a controlled release manner and increase drug cytotoxicity in MDR cells as compared to the drug loaded MePEG₁₁₄-b-PCL₂₀₀ nanoparticles in the absence of MePEG₁₇-b-PCL₅. The mixed MW nanoparticles remained in the plasma for longer than the drugs with approximately 3% of the injected dose remaining 24 hrs post injection. Ultrasound irradiation was investigated as a potential strategy to enhance the cytotoxicity of PTX loaded MePEG-b-PDLLA micelles in MDR cells. Using an ultrasound regime of a single 10-second burst of high frequency (4 MHz) and high intensity (32 W/cm²) ultrasound, it was shown that ultrasound irradiation resulted in a two-fold increase in intracellular uptake of PTX in drug sensitive MDCKII and MCF-7 cell lines and their respective Pgp-overexpressing MDCKII-MDR1 and NCI-ADR counterparts as compared to untreated cells (no ultrasound). The enhanced accumulation and retention of PTX resulting from ultrasound treatment translated into greater cytotoxicity in both drug sensitive and resistant cell lines.In conclusion, we have demonstrated two promising strategies for enhancing MDR cellular drug accumulation and effectiveness: the use of mixed molecular weight taxane loaded nanoparticles and ultrasound irradiation.

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Development of co-loaded fusidic acid and rifampicin polymeric microspheres and nanofibers : phase characterization and in vivo evaluation in a rodent model of orthopaedic infection (2012)

The overall goal of this project was to develop and characterize a biodegradable, polymeric formulation using poly (D,L-lactic acid-co-glycolic acid) (PLGA) for the controlled delivery of fusidic acid (FA) and rifampicin (RIF) to orthopaedic surgical sites to achieve high localized concentrations above the MIC of potential microorganisms to prevent implant-associated infections. Our primary formulation strategy using PLGA microspheres was inappropriate for the controlled delivery of either agent due to a phase separation phenomena of the solid drugs from PLGA. The phase separation, lead us to investigate the solid state properties of FA and the phase behavior of FA and RIF in microspheres. Four distinct solid forms of FA (Form I-IV), and an amorphate were identified. Form IV and amorphous FA had significantly greater intrinsic dissolution rates, and the interconversion of both Form IV and amorphous FA to Form III in aqueous mileau suggests a risk of interconversion upon exposure to moisture if FA is formulated in the solid state. The phase separation of FA and RIF from PLGA microspheres was characterized by drug microdomains localized on the microsphere surface (for FA), or a dimpled microsphere surface (for RIF). Novel micromanipulation techniques allowed the visualization of the phase separation events for FA and RIF, which was correlated with the compatibility between each drug and PLGA. When co-loaded, FA and RIF phase separate in a single event, intermediate to each drug alone. Suface distribution of drug microdomains, and drug release, was dependent on the weight fraction of FA. A more suitable controlled release formulation was PLGA nanofibers prepared using electrospinning. The drug-loaded formulations were defect-free and had a biphasic drug release profile. All dual-loaded formulations showed direct antimicrobial activity in vitro against 4 Gram positive microorganisms. Furthermore, lead formulations containing 10% (w/w) FA/SF and 5% (w/w) RIF were able to prevent the adherence of methicillin resistant S. aureus to a titanium implant in an in vivo rodent model of implant-associated infection. The data in this thesis contributes to the understanding of drug phase separation from PLGA, and drug-loaded electrospun nanofibers provide a preclinical proof-of-principle for the prevention of implant-associated infections.

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Development and characterization of intravesical mucoadhesive nanoparticulate formulations of paclitaxel and docetaxel for postoperative chemotherapy in non-muscle-invasive bladder cancer (2011)

The present work describes the development, in vitro and in vivo evaluation of mucoadhesive nanoparticulate formulations of paclitaxel (PTX) and docetaxel (DTX) for intravesical bladder cancer therapy. The nanoparticles developed for PTX and DTX delivery consisted of hyperbranched polyglycerol (HPG), which were hydrophobically derivatized with alkyl chains (C₈/₁₀) in the core to allow drug loading and were further derivatized with methoxy polyethylene glycol (MePEG) and primary amine groups in the shell (HPG-C₈/₁₀-MePEG-NH₂) to increase their mucoadhesiveness. Human urothelial carcinoma cell lines were treated with various concentrations of PTX and DTX formulations in vitro. Mice with established KU7-luc tumors were intravesically treated with various concentrations of PTX and DTX formulations or control vehicles. Drug uptake was conducted using LC/MS/MS and tumor microenvironment and uptake of rhodamine labeled HPGs was assessed by immunohistochemistry-based tumor mapping and fluorescence microscopy. The effects of HPG nanoparticles on the urinary bladder wall were studied on isolated porcine bladder tissues as well as in live mouse bladders. To investigate the toxicity and tolerability, histopathological evaluations were preformed following a single intravesical instillation with empty HPG nanoparticles in healthy female nude mice. In vitro, PTX and DTX loaded HPG nanoparticles were found to have equivalent cytotoxicities as their commercial formulations of Taxol® and Taxotere®, respectively. The surface modification of HPG-C₈/₁₀-MePEG with amine groups resulted in highly positive charged nanoparticles (HPG-C₈/₁₀-MePEG-NH₂) with improved mucoadhesive properties. A single intravesical instillation with DTX loaded HPG-C₈/₁₀-MePEG-NH₂ at a lower dose significantly inhibited tumor growth and increased drug uptake in mouse bladder tissues. HPG-C₈/₁₀-MePEG-NH₂ nanoparticles were found to significantly increase the uptake of DTX in both isolated pig bladder as well as in live mouse bladder tissues likely resulting from changes to the urothelial barrier function and morphology through opening of tight junctions and exfoliation of the superficial umbrella cells. Our preliminary studies suggest that this exfoliation was triggered by an apoptosis mechanism followed by a rapid recovery of the urothelium within 24 h post-instillation. Overall, our data show promising in vivo anti-tumor efficacy and provide preclinical proof-of-principle for intravesical application of these nanoparticulate formulations in the treatment of non-muscle-invasive bladder cancer.

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Parathyroid hormone effects on marrow stromal cells for potantial bone regeneration applications : delivery systems development and biological characterization (2010)

Despite the inherent ability of bone tissue to regenerate upon damage, there are incidences such as ‘critical’ defects where the damaged and lost bone will not repair or regenerate itself. Thus, the natural bone regeneration process must be augmented with the application of therapeutic agents (growth factors, hormones, cells). In addition, in every possible scenariowhere surgical interventions are performed, there exists the risk of infections that must be minimized and managed with the administration of antibiotics. The overall goals of this thesis were to engineer, develop, and characterize biodegradable andbioresorbable polymeric microsphere and porous scaffold delivery systems for parathyroid hormone (PTH) and marrow stromal cells (MSC5), respectively, for enhancing the innate regenerative capacity of bone, and to investigate the effects of continuous and pulsatile PTH treatments on MSCs to better understand its regulatory actions on MSC differentiation, proliferation and clonogenicity. In addition, the development and characterization of biodegradable and bioresorbable polymeric microsphere delivery systems for the antibiotic, fusidic acid (FA) for potential localized application in bone infection were also undertaken.PTH-loaded poly(lactic-co-glycolic acid) (PLGA) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) microspheres were developed. However, these initial formulations did not achieve the precise level of controlled release of PTH required for MSCs. Osteogenic differentiation of MSCs was found to increase with continuous PTH treatment, and decrease with pulsatile PTH exposure. The observed effects of PTH were strongly dependent on the presence of dexamethasone. PTH treatments did not influence MSC proliferation but was found to increase the colony forming unit-fibroblast (CFU-F) content within MSC cultures.Biocompatible, biodegradable and bioresorbable porous gelatin-alginate scaffolds produced by microwave vacuum drying were found to support MSC attachment, proliferation and differentiation. However, MSC differentiation (osteogenic, chondrogenic, adipogenic) were suppressed in vivo compared to in vitro when seeded on these scaffolds. In the process of formulating FA-loaded PLGA and PHBV microspheres, an interesting phase separation phenomenon of FA in PLGA but not in PHBV polymer was observed. Phase separated FA formed distinct, large, completely amorphous, spherical FA-rich solidmicrodomains throughout the PLGA microsphere, and on the microsphere surface. FA release kinetics from the microsphere formulations were controlled by selective formulation factors determined from factorial design experiments. Thus, the data presented in this thesis contribute to our understanding of PTH effects on MSCs, the responses of MSCs on porous gelatin-alginate scaffolds as well as the solid-statecharacteristics and release of FA loaded in PLGA and PHBV microspheres.

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Development of methoxy poly(ethylene glycol)-block-poly(caprolactone) amphiphilic diblock copolymer nanoparticulate formulations for the delivery of paclitaxel (2008)

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres.Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior.Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization.The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction.In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively.

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Master's Student Supervision (2010 - 2018)
Development of a nanoparticulate formulation of docetaxel for the treatment of non-muscle-invasive bladder cancer (2014)

Approximately 70-85% of bladder cancer patients present with non-muscle invasive bladder cancer (NMIBC). These patients are usually treated by surgical resection of bladder tumours followed by the intravesical administration of anticancer drugs such as mitomycin C (MMC), doxorubicin or gemcitabine. However the recurrence rate after 5 years remains high (70%) so that the development of more effective chemotherapeutic strategies is essential. We have previously shown that hyperbranched polyglycerol (HPG-C₈/₁₀-MePEG-NH₂) nanoparticulate carriers of docetaxel (DTX) offered an improved and effective formulation of the drug for intravesical delivery in mice. The present work describes the effect of concentration and exposure times of three HPG-C₈/₁₀-MePEG-NH₂’s with increasing degrees of amineation on ex vivo porcine bladder tissue morphology and the tissue depth uptake of DTX. The results demonstrated the exfoliation of porcine bladder tissues in a time and concentration-dependent manner. Exfoliation and DTX uptake was significantly enhanced upon treatment with medium or high-density HPG-C₈/₁₀-MePEG-NH₂’s, as compared to a commercially available DTX/polysorbate 80 formulation. Further studies on the local effect of the chemotherapeutic agents MMC, doxorubicin and gemcitabine, on ex vivo porcine bladder tissue showed that these drugs all caused exfoliation of urothelium and were well taken up by the bladder tissue with no additional effect of HPG-C₈/₁₀-MePEG-NH₂ pre-treatment. The exfoliating effect of these three drugs was shown to enhance the bladder tissue uptake of paclitaxel (PTX) or DTX when the bladder was exposed to combinations of taxanes with either MMC, doxorubicin or gemcitabine. Generally, the exfoliation effect of HPG-C₈/₁₀-MePEG-NH₂’s, MMC, doxorubicin and gemcitabine is attributed to an interaction of the positively charged amine groups on all these agents with the negatively charged mucosal surface. This binding may modulate tight junction protein function followed by exfoliation of the protective urothelial layer so that drugs may penetrate the exposed underlying tissue. In conclusion this thesis supports a novel role of DTX loaded-HPG-C₈/₁₀-MePEG-NH₂ nanoparticles as an improved drug delivery vehicle for the potential chemotherapeutic treatment of bladder cancer. Additionally, data suggests promising strategies for intravesical combination drug therapies, to enhance the uptake of taxanes with potential additive therapeutic effects for improved efficacy in the treatment of NMIBC.

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The development and characterization of nanocomposite films for the controlled release and localized delivery of tetracycline and alendronate for periodontal application (2011)

It has been proposed that localized and controlled delivery of alendronate and tetracycline to periodontal pocket fluids via guided tissue regeneration (GTR) membranes may be a valuable adjunctive treatment for advanced periodontitis. The objectives of this work were to develop a co-loaded, controlled release tetracycline and alendronate nanocomposite plasticized poly(lactic-co-glycolic acid) (PLGA) film that would form a suitable matrix supporting osteoblast proliferation and differentiation.Alendronate release was successfully controlled, with complete suppression of the burst phase of release by intercalation of alendronate anions in magnesium/aluminum layered double hydroxide (LDH) clay nanoparticles and dispersed in the PLGA film matrix. Tetracycline, loaded as free drug into the film together with alendronate-LDH clay complex released more rapidly than alendronate, but showed evidence of intercalation in the LDH clay particles. The dual drug loaded nanocomposite films were biocompatible with osteoblasts and after five week incubations, significant increase in alkaline phosphatase activity and bone nodule formation were observed.

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Bladder tissue distribution of paclitaxel and docetaxel from polymeric nanoparticles (2010)

Taxane based drugs are commonly used second-line adjuvant therapies for the treatment of superficial bladder cancer. However, the effectiveness of these drugs following intravesical instillation is limited, because of a short drug residence time and poor drug penetration into the bladder wall. The goals of this project were to use PTX and DTX loaded nanoparticulate formulations and investigate their bladder mucosal permeability and distribution characteristics in ex vivo isolated porcine bladders. PTX and DTX were loaded into nanoparticles of methoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) (MePEG-PDLLA), methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-PCL) and hydrophobically derivatized hyperbranched polyglycerol, HPG-C10-MePEG. In vitro drug release profiles of PTX and DTX loaded MePEG-PDLLA micelles (approx. 20 nm in size) demonstrated controlled and complete release of the drugs over 7 days. The penetration and distribution characteristics of PTX and DTX from nanoparticulate formulations (spiked with tritium labelled drugs) into freshly excised porcine bladder tissue were evaluated using Franz diffusion cells. Nanoparticle dispersions were instilled onto bladder tissue for 2 h. The tissues were frozen, sectioned and drugs quantified using liquid scintillation counting. The drug tissue levels were highest in the urothelium and then decreased exponentially with increasing tissue depth. Micellar formulations of MePEG-PDLLA showed higher bladder tissue uptake levels of PTX and DTX compared to control commercial formulations. Drug uptake levels did not differ significantly between concentrations of 0.5 and 1 mg/mL. However, DTX was present in bladder tissue at significantly higher concentrations than PTX. Micellar formulations of MePEG-PDLLA showed higher bladder tissue uptake levels of PTX compared to both MePEG-PCL copolymer formulations. The rapid and high levels of PTX and DTX penetration observed in bladder tissue using the MePEG-PDLLA micellar formulation of these drugs was likely due to the use of high concentrations of the drugs with these polymeric micelles, greater free drug released from the MePEG-PDLLA micelles and the ability to increase the contact of the formulation at the bladder wall surface allowing for improved partitioning of the drug into the tissues.

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