pH-Responsive nanocarriers for drug delivery

© 2017 Dr. Adelene Sue Mei WongThe use of nanoparticles for drug delivery has been a burgeoning area of research in recent years. Nanoparticle carriers have the potential to improve therapeutic outcomes, offering advantages over the delivery of therapeutics alone such as longer circulation times and more targeted drug release. In this thesis, a modular, pH-responsive (pHlexi) nanoparticle system was engineered using a one-pot nanoprecipitation method combining two charge-shifting polymers, the homopolymer poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and the diblock copolymer poly(2-(diethylamino)ethyl methacrylate)-b-poly(ethylene glycol) (PDEAEMA-b-PEG). This particle system demonstrated suitability as a drug delivery carrier due to the successful encapsulation of a model cargo, as well as disassembly at a biologically relevant pH. These particles also demonstrated the ability to induce endosomal escape, chaperoning the delivery of calcein into the cytosol of cells. Following this, a series of experiments was carried out to further investigate the mechanism of endosomal escape induced by these pH-responsive particles. First, as the presence of hydrophobic groups in polymeric nanocarrier systems have been reported to play an important role in membrane destabilization, the effect of removing the hydrophobic component of PDEAEMA on cellular behaviour was assessed. Next, the ability of pHlexi particles to induce the escape of a range of molecular weights of dextran molecules was studied. The results of these investigations aided in the overall understanding of the endosomal escape behaviour of pHlexi particles, but importantly demonstrated that the process of assessing endosomal escape is varied and complex, requiring appropriate tools and experimental design. Finally, the incorporation of a model targeting protein, holo-transferrin (holo-Tf), to the pHlexi particles was investigated. Holo-Tf was conjugated to PDEAEMA and poly(2-(diethylamino)ethyl methacrylate)-ran-poly(2- (diisopropylamino)ethyl methacrylate (PDEAEMA-r-PDPAEMA), and the conjugates were observed to self-assemble, forming hybrid particles in an environment at a pH greater than the pKa of the polymer. The Tf-pHlexi particles demonstrated pH-tunability depending on the polymer constituent attached, and were capable of specific targeting to transferrin receptors on the surface of cells. The density of holo-Tf on the surface of the particles was also shown to be effectively tuned by the incorporation of a poly(ethylene glycol) (PEG) polymer. The ability to combine the properties of both polymeric and biological material provides the potential for overcoming challenges associated with therapeutic delivery such as targeting and biocompatibility. The collective work presented in this thesis offers the reader a thorough discussion of the design, synthesis, and challenges associated with the development of pH-responsive nanocarriers based on the charge-shifting polymers, PDEAEMA and PDPAEMA; and aids in our understanding of the behaviour of these pH-responsive nanoparticle systems in vitro

Association of Thoroughbred Racehorse Workloads and Rest Practices with Trainer Success

Understanding the relationship between the training practices of Thoroughbred racehorses and race performance is important to ensure advice given to trainers for injury prevention or management is practical and consistent. We assessed associations between intended volume and speed of gallop training (i.e., typical workloads for horses free of injury or other performance limiting conditions) and rest practices on official trainer career and previous season success rates (rate of wins and places, prizemoney per start). Sixty-six Australian Thoroughbred trainers were surveyed. Multivariable negative binomial regression models were employed for the outcomes career and previous season wins and places, and linear regression models for prizemoney per start. Intended training workload was not associated with prizemoney. Pre-trial total galloping distances (≥13.3 m/s) between 7500 m and 15,000 m were associated with a higher rate of career wins, and previous season wins and places per start (p < 0.05). Slow-speed (13.3–14.3 m/s) galloping distance to trial between 5000 m to 12,500 m was associated with higher rate of career placings per start, with reduced performance over 12,500 m (p = 0.003). Greater time between race starts was associated with a greater rate of previous season wins and prizemoney per start until three weeks between starts, with decline in performance thereafter (p < 0.05). Greater frequency of rest breaks was associated with greater prizemoney per start earnt in the previous season (p ≤ 0.01). These results suggest that modifications to training programs aimed at injury prevention, such as avoiding long galloping distances, should not adversely affect trainer success

Barriers to entering race training before 4 years of age for Thoroughbred horses born in the 2014 Australian foal crop.

Currently, there is a paucity of data on the barriers for Australian Thoroughbred horses transitioning from stud farm to racetrack. This paper reports the reasons why horses failed to enter race training and documents their exit destinations. Biographical records of Australian Thoroughbred horses born in 2014 were investigated to determine the number of horses that had not officially entered race training by the start of the 4-year old racing season (1 August 2018). Of the 13,677 foals born in 2014, 66% had commenced training and 51% had raced before the beginning of their 4-year-old season in Australia. A sampling frame based on the post code of the premises where foals were born and records from Racing Australia were used to select a geographically representative sample of the 2014 Australian Thoroughbred foal crop (n = 4,124). From the population eligible for sampling 1,275 horses that had not entered training were enrolled in the survey and their breeders were sent an online questionnaire with follow-up phone calls for those who had not responded. Of the 633 responses (50% of 1275) the most frequent outcomes for horses were: death (38%, n = 239), participation in the racing industry in their 4-year old racing season (24%, n = 154) and retirement (16%, n = 100) either as Australian Stud Book (ASB) bloodstock (n = 17), or as horses rehomed outside the Thoroughbred industry (n = 83). Illness or injury was the most frequent reason for horses not entering race training that were ASB bloodstock, rehomed or deceased. There was a loss of traceability at the point of sale with most horses sold at 1 year of age. This study provides important information on the reasons, alternative outcomes and gaps in traceability for horses not entering training prior to the 4-year-old racing season

The Regulatory Framework Across International Jurisdictions for Risks Associated with Consumption of Botanical Food Supplements

Dietary supplements, including those containing botanical ingredients and botanical-derived compounds, have been marketed to consumers globally for many decades. However, the legislative framework for such products remains inconsistent across jurisdictions internationally. This study aims to compare the regulatory framework of botanical food supplements in the EU, USA, Canada, Australia, New Zealand, India, Japan, and China. The study also aims to investigate and describe safety assessment criteria for botanical food supplements where they are present in the above said jurisdictions, and attempts to analyze whether these criteria are suitable for addressing the toxicological risks associated with the use of botanical food supplement products, based on the evaluation of reported adverse effects related to botanical food supplement use as examples. Finally, this study discusses some future issues that need further attention, such as the consideration of less than lifetime exposures, potential for misidentification, and adulteration of botanical supplements by pharmacologically active substances. It is concluded that the regulatory approaches towards botanical food supplements differ significantly across jurisdictions. In addition, national authorities are increasingly considering having more regulatory oversight for such products. Further consideration of the actual impact of adverse events arising from botanical food supplement usage will be helpful in guiding such decisions.</p

K-ras peptide mimotope induces antigen specific Th1 and B-cell immune responses against G12A-mutated K-Ras antigen in balb/c mice

KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses

The Regulatory Framework Across International Jurisdictions for Risks Associated with Consumption of Botanical Food Supplements

Dietary supplements, including those containing botanical ingredients and botanical-derived compounds, have been marketed to consumers globally for many decades. However, the legislative framework for such products remains inconsistent across jurisdictions internationally. This study aims to compare the regulatory framework of botanical food supplements in the EU, USA, Canada, Australia, New Zealand, India, Japan, and China. The study also aims to investigate and describe safety assessment criteria for botanical food supplements where they are present in the above said jurisdictions, and attempts to analyze whether these criteria are suitable for addressing the toxicological risks associated with the use of botanical food supplement products, based on the evaluation of reported adverse effects related to botanical food supplement use as examples. Finally, this study discusses some future issues that need further attention, such as the consideration of less than lifetime exposures, potential for misidentification, and adulteration of botanical supplements by pharmacologically active substances. It is concluded that the regulatory approaches towards botanical food supplements differ significantly across jurisdictions. In addition, national authorities are increasingly considering having more regulatory oversight for such products. Further consideration of the actual impact of adverse events arising from botanical food supplement usage will be helpful in guiding such decisions.</p

Endolysins against Streptococci as an antibiotic alternative

Multi-drug resistance has called for a race to uncover alternatives to existing antibiotics. Phage therapy is one of the explored alternatives, including the use of endolysins, which are phage-encoded peptidoglycan hydrolases responsible for bacterial lysis. Endolysins have been extensively researched in different fields, including medicine, food, and agricultural applications. While the target specificity of various endolysins varies greatly between species, this current review focuses specifically on streptococcal endolysins. Streptococcus spp. causes numerous infections, from the common strep throat to much more serious life-threatening infections such as pneumonia and meningitis. It is reported as a major crisis in various industries, causing systemic infections associated with high mortality and morbidity, as well as economic losses, especially in the agricultural industry. This review highlights the types of catalytic and cell wall-binding domains found in streptococcal endolysins and gives a comprehensive account of the lytic ability of both native and engineered streptococcal endolysins studied thus far, as well as its potential application across different industries. Finally, it gives an overview of the advantages and limitations of these enzyme-based antibiotics, which has caused the term enzybiotics to be conferred to it