Synthesis and biological evaluation of novel folic acid receptor-targeted, β-cyclodextrin-based drug complexes for cancer treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant Ka was 1,639 M-1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Nutritional and functional evaluation of wheat flour cookies supplemented with gram flour

Protein-enriched cookies were prepared by supplementing gram flour into wheat flour at levels of 0%, 10%, 20%, 30%, 40% and 50% and analysed for physicochemical properties. The protein quality of the cookies was assessed by feeding gram flour-supplemented cookies to albino rats for 10 days. The supplementation resulted in a significant increase in protein, fat, crude fibre and ash contents of the cookies. The thickness and spread factor of cookies differ significantly while non-significant effect was observed in the width of the cookies. The protein efficiency ratio, net protein utilization, biological value and true digestibility differed significantly among diets containing cookies with gram flour fed to rats. Cookies with 30% substitution of straight grade flour and gram flour produced acceptable cookies as compared to control. The cookies containing 40-50% gram flour were best regarded as protein bioavailability for rats.

Controlled release of bioactive chemicals

This project has been concerned with the development of controlled release formulations of the herbicide atrazine and the pesticide chlorpyrifos. The results of both chemical and biological evaluation show that these controlled release formulations can significantly reduce leaching and therefore ground water pollution of atrazine without losing herbicidal potency in the early stages after application.

Synthesis of a novel pyrrole oxazole analogue of the insecticide pirate

The pyrrole oxazole 4 is a novel analogue of the broad-spectrum insecticide and miticide pirate 2. The expedient synthesis of pyrrole oxazole 4 in six steps from pyrrole is reported using a synthetic route that could have potential for the solution-phase combinatorial synthesis of analogues. Preliminary biological evaluation of the protected pyrrole oxazole 4 (LD₅₀ 1.13 μg mL^–1) and the deprotected pyrrole oxazole 5 (LD₅₀ 1.06 μg mL^–1) for potential cytotoxicity was carried out.

Surface-stress-based microcantilever aptasensor

This thesis presents the design, modelling, fabrication, and biological evaluation of a microcantilever-based aptasensor. It is the first reported work on aptasensors with aptamer immobilized on a bare SU-8 surface. Aptasensor surface funtionalisation was achieved using gas plasma treatment. Label-free detection of thrombin molecules using the aptasensor was successfully demonstrated.

Preliminary investigations into triazole derived androgen receptor antagonists

A range of 1,4-substituted-1,2,3-N-phenyltriazoles were synthesized and evaluated as non-steroidal androgen receptor (AR) antagonists. The motivation for this study was to replace the N-phenyl amide portion of small molecule antiandrogens with a 1,2,3-triazole and determine effects, if any, on biological activity. The synthetic methodology presented herein is robust, high yielding and extremely rapid. Using this methodology a series of 17 N-aryl triazoles were synthesized from commercially available starting materials in less than 3h. After preliminary biological screening at 20 and 40 μM, the most promising three compounds were found to display IC50 values of 40-50 μM against androgen dependent (LNCaP) cells and serve as a starting point for further structure-activity investigations. All compounds in this work were the focus of an in silico study to dock the compounds into the human androgen receptor ligand binding domain (hARLBD) and compare their predicted binding affinity with known antiandrogens. A comparison of receptor-ligand interactions for the wild type and T877A mutant AR revealed two novel polar interactions. One with Q738 of the wild type site and the second with the mutated A877 residue.

Synthesis and preliminary investigations into novel 1,2,3-triazole-derived androgen receptor antagonists inspired by bicalutamide.

A versatile and high yielding synthesis of novel androgen receptor (AR) antagonists is presented. Using this methodology, six 1,4-substituted-1,2,3-triazole derived bicalutamide mimics were synthesised in five steps and in isolated overall yields from 41% to 85%. Evaluation of these compounds for their anti-proliferative properties against androgen dependent (LNCaP) and independent (PC-3) cells showed promising IC50 values of 34-45 μM and 29-151 μM, respectively. The data suggest that the latter compounds may be an excellent starting point for the development of prostate cancer therapeutics for both androgen dependent and independent forms of this disease. Docking of these compounds (each enantiomer) in silico into the T877A mutated androgen receptor, as possessed by LNCaP cells, was also undertaken.

Evaluation of weaning strategies for intensively reared Australian fish, Murray cod, Maccullochella peelii peelii

The Australian Murray cod supports a growing national industry. However, with regard to the process of weaning fry, there is a lack of information and optimal procedures need to be developed. The aim of the present investigation was to test the biological and economic efficacy of different weaning strategies for Murray cod. Three weaning strategies were tested on triplicate groups of fish: (1) only Artemia for 5 d, 7 d on Artemia plus starter diet, and 14 d on dry diet only; (2) 12 d on Artemia plus starter diet and 14 d on dry diet only; and (3) directly to dry diet for the entire experimental period. No significant differences were recorded in the growth and feed efficiency, while significantly higher mortality (38.4 ± 0.35%) was recorded in fish weaned directly onto dry diet. Fish subjected to the first 5 d on Artemia only showed a growth reduction during this period, which was compensated by a phase of enhanced growth during the dry-diet phase. No significant differences were noted in the proximate composition of fish under the different treatments. The economic evaluation suggested that the treatment with the simultaneous supply of Artemia and starter diet is preferable.

Evaluation of brief dietary questions to estimate vegetable and fruit consumption - using serum carotenoids and red-cell folate

Objective To evaluate responses to self-administered brief questions regarding consumption of vegetables and fruit by comparison with blood levels of serum carotenoids and red-cell folate.

Design A cross-sectional study in which participants reported their usual intake of fruit and vegetables in servings per day, and serum levels of five carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and lycopene) and red-cell folate were measured. Serum carotenoid levels were determined by high-performance liquid chromatography, and red-cell folate by an automated immunoassay system.

Settings and subjects Between October and December 2000, a sample of 1598 adults aged 25 years and over, from six randomly selected urban centres in Queensland, Australia, were examined as part of a national study conducted to determine the prevalence of diabetes and associated cardiovascular risk factors.

Results Statistically significant (P<0.01) associations with vegetable and fruit intake (categorised into groups: ≤1 serving, 2–3 servings and ≥4 servings per day) were observed for α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and red-cell folate. The mean level of these carotenoids and of red-cell folate increased with increasing frequency of reported servings of vegetables and fruit, both before and after adjusting for potential confounding factors. A significant association with lycopene was observed only for vegetable intake before adjusting for confounders.

Conclusions These data indicate that brief questions may be a simple and valuable tool for monitoring vegetable and fruit intake in this population.

Development and evaluation of an ELISA for the detection and 3D antibodies to foot and mouth disease virus

A group specific ELISA (enzyme-linked immunosorbent assay) was developed to detect virus infection associated antibodies in the serum of animals infected with any serotype of foot and mouth disease virus. The assay was developed from non-infectious sources, and is therefore suitable for use in countries where FMDV is exotic.

An evaluation of Sea Search as a citizen science programme in Marine Protected Areas

Citizen science involves collaboration between multi-sector agencies and the public to address a natural resource management issue. The Sea Search citizen science programme involves community groups in monitoring and collecting subtidal rocky reef and intertidal rocky shore data in Victorian Marine Protected Areas (MPAs), Australia. In this study we compared volunteer and scientifically collected data and the volunteer motivation for participation in the Sea Search programme. Intertidal rocky shore volunteer-collected data was found to be typically comparable to data collected by scientists for species richness and diversity measures. For subtidal monitoring there was also no significant difference for species richness recorded by scientists and volunteers. However, low statistical power suggest only large changes could be detected due to reduced data replication. Generally volunteers recorded lower species diversity for biological groups compared to scientists, albeit not significant. Species abundance measures for algae species were significantly different between volunteers and scientists. These results suggest difficulty in identification and abundance measurements by volunteers and the need for additional training requirements necessary for surveying algae assemblages. The subtidal monitoring results also highlight the difficulties of collecting data in exposed rocky reef habitats with weather conditions and volunteer diver availability constraining sampling effort. The prime motivation for volunteer participation in Sea Search was to assist with scientific research followed closely by wanting to work close to nature. This study revealed two important themes for volunteer engagement in Sea Search: 1) volunteer training and participation and, 2) usability of volunteer collected data for MPA managers. Volunteer-collected data through the Sea Search citizen science programme has the potential to provide useable data to assist in informed management practices of Victoria’s MPAs, but requires the support and commitment from all partners involved.

Comparison of automated classification techniques for predicting benthic biological communities using hydroacoustics and video observations

The effective management of our marine ecosystems requires the capability to identify, characterise and predict the distribution of benthic biological communities within the overall seascape architecture. The rapid expansion of seabed mapping studies has seen an increase in the application of automated classification techniques to efficiently map benthic habitats, and the need of techniques to assess confidence of model outputs. We use towed video observations and 11 seafloor complexity variables derived from multibeam echosounder (MBES) bathymetry and backscatter to predict the distribution of 8 dominant benthic biological communities in a 54 km² site, off the central coast of Victoria, Australia. The same training and evaluation datasets were used to compare the accuracies of a Maximum Likelihood Classifier (MLC) and two new generation decision tree methods, QUEST (Quick Unbiased Efficient Statistical Tree) and CRUISE (Classification Rule with Unbiased Interaction Selection and Estimation), for predicting dominant biological communities. The QUEST classifier produced significantly better results than CRUISE and MLC model runs, with an overall accuracy of 80% (Kappa 0.75). We found that the level of accuracy with the size of training set varies for different algorithms. The QUEST results generally increased in a linear fashion, CRUISE performed well with smaller training data sets, and MLC performed least favourably overall, generating anomalous results with changes to training size. We also demonstrate how predicted habitat maps can provide insights into habitat spatial complexity on the continental shelf. Significant variation between patch-size and habitat types and significant correlations between patch size and depth were also observed.

Field evaluation of a suite of biomarkers in an Australian Tropical Reef Species, Stripey Seaperch (Lutjanus carponotatus) : Assessment of Produced Formation water from the Harriet A Platform

There is paucity of data regarding hydrocarbon exposure of tropical fish species inhabiting the waters near oil and gas platforms on the Northwest Shelf of Australia. A comprehensive field study assessed the exposure and potential effects associated with the produced water (PW) plume from the Harriet A production platform on the northwest shelf in a local reef species, Stripey seaperch (Lutjanus carponotatus). This field study was a continuation of an earlier pilot study which concluded that there were “warning signs” of potential biological effects on fish populations exposed to PW. A 10-day field caging study was conducted deploying 15 individual fish into 6 separate steel cages set 1-m subsurface at 3 stations in a concentration gradient moving away from the platform. A battery of biomarkers were evaluated including hepatosomatic index (HSI), total cytochrome P450, bile metabolites, CYP1A-, CYP2K- and CYP2M-like proteins, cholinesterase (ChE) activity, and histopathology of liver and gill tissues. Water column and PW effluent samples was also collected. Results confirmed that PAH metabolites in bile, CYP1A-, CYP2K-, and CYP2M-like proteins and liver histopathology provided evidence of significant exposure and effects after 10 days at the near-field site (~200 m off the Harriet A platform). Hepatosomatic index, total cytochrome P450, and ChE did not provide site-specific differences by day 10 of exposure to PW. CYP proteins were shown by principal component analysis (PCA) to be the best diagnostic tool for determining exposure and associated biological effects of PW on L. carponotatus. Using a suite of biomarkers has been widely advocated as a vital component in environmental risk assessments worldwide. This study demonstrates the usefulness of biomarkers for assessing the Harriet A PW discharge into Australian waters with broader applications for other PW discharges. This approach has merit as a valuable addition to environmental management strategies for protecting Australia’s tropical environment and its rich biodiversity.