Development and characterization of aptamer-amphiphiles against fractalkine for targeted drug delivery

University of Minnesota Ph.D. dissertation. December 2013. Major: Chemical Engineering. Advisor: Efrosini Kokkoli. 1 computer file (PDF); ix, 111 pages.A foundation of modern diagnostics and therapeutics is the ability to non-covalently bind to a molecule of interest. These affinity molecules are behind a broad array of products ranging from therapeutics to HIV tests. Currently, antibodies are used as the affinity molecule. Despite the success of antibodies, alternatives are needed due to high development and production costs, and issues with stability. Aptamers are an exciting alternative to antibodies. Aptamers are short sequences of single stranded DNA or RNA that bind molecular targets with high affinity and specificity. Aptamers are inexpensive to produce, are very stable, have long shelf lives, and could potentially replace antibodies in a number of applications. One potential application of aptamers is targeted drug delivery. The goal of targeted drug delivery is to selectively deliver a therapeutic payload to the site of action thereby increasing efficacy and decreasing side effects. Fractalkine is a cell surface protein expressed at sites of inflammation. It is expressed on several types of cancerous tissues and it is involved in the patheogenisis of arthritis, asthma, and atherosclerosis. This work describes the development and characterization of an aptamer that binds fractalkine with high affinity. The aptamer was modified with a hydrophobic tail, creating an aptamer-amphiphile, for use in a model drug delivery vesicle called a liposome. The aptamer-amphiphile was optimized for a high affinity interaction with fractalkine by adding a spacer molecule between the aptamer headgroup and the hydrophobic tail. The optimized amphiphile had high affinity for fractalkine and self-assembled into micelles and an interesting nanotape morphology. Finally, as a proof of concept, the optimized aptamer-amphiphile was incorporated into a liposome and targeted to fractalkine expressing cells. This work highlights the development of aptamers as affinity ligands, and demonstrates their use as potential drug delivery agents

The Role of Endothelial αvβ3 Integrin in Metastasis

The integrin αvβ3 is part of a family of membrane proteins with bi-directional signalling and activity. In this way αvβ3 plays an extensive and complex role in cancer biology. This study builds on results that show that knocking out αvβ3 in endothelial cells reduces cancer metastasis suggesting that endothelial αvβ3 plays a pro-metastatic role. In particular, there has been a focus on the pre-metastatic niche, a pro-metastatic environment set up by the primary tumour before metastatic growth. In this vein, changes to pre-metastatic environments have been studied via immune cell and cytokine profiling. To do this FACS and quantitative analysis of immunohistochemistry has been performed. While this study does not bring about a conclusive explanation of the pro-metastatic role of endothelial αvβ3, it opens up other areas of research. These include identifying changes in specific myeloid populations such as polarised neutrophils and identifying differences in the endothelial layer at metastatic sites

The effect of testosterone on factors associated with diabetes, atherosclerosis and obesity.

Obesity has recently become a major global health problem. Epidemiologic studies indicate that obesity is an important risk factor for type 2 diabetes (T2DM), atherosclerosis and low testosterone in men. Importantly, testosterone replacement treatment (TRT) can improve the condition of these diseases. According to human and animal studies testosterone can act as an anti-inflammatory and anti- atherogenic factor leading to inhibition of the risk factors and consequence of T2DM and atherosclerosis. Consequences of these diseases include dysregulation in atherogenic factors such as apolipoproteins or pro-inflammatory mediators such as cytokines and chemokines and their receptors. The effect of testosterone on these factors remains unclear. The objective of the present study was to demonstrate whether testosterone has antiinflammatory and anti-atherogenic action and by which mechanisms. This was achieved by using in vivo human and mice studies as well as in vitro models. The in vivo human study was conducted on short and long term studies, to determine the effect of TRT on anti and pro-inflammatory cytokines, HDL fractions and apolipoproteins in diabetic hypogonadal patients. Samples of liver tissue from testicular feminization mice (Tfm) were studied to investigate the effect of testosterone therapy on mRNA expression of adiponectin, PP ARb/o, PAI-1, apolipoproteins and pro-inflammatory chemokines with their receptors. Additionally, models of cell culture were studied including human macrophage THP-1 cells and mouse 3T3L1 cells to study the effect of testosterone with or without blocked androgen receptor (AR) on CX3CR1 and CCR2 and pro-inflammatory cytokines in macrophage cells and on adiponectin, PP ARB/o, PAI-1, leptin and chemokines in adipocyte cells, respectively.In the clinical studies, a reduction in adiponectin levels after 3 months was seen in the short-term study and an increase in HDL2 and HDL2/HDL3 ratio in the long-term study. No significant effect of testosterone was observed on body composition and atherogenic factors in either the short or long-term studies. In the animal study, testosterone increased hepatic expression of mRNA adiponectin, PP ARB/o and PAI-1 mRNA expression in Tfm. In the cell culture studies, testosterone treatment increased CCR2 mRNA expression and decreased secretion of IL-8 and TNF level in the supernatant of THP-1 macrophages. Testosterone decreased secretion of CCL2 and CX3CL1 from 3T3L1 adipocytes while increasing PAI-1 mRNA expression in these cells. The action of testosterone was based on the type of cells and time, route and dose size of treatment. In conclusion, although testosterone therapy showed a positive effect on some risk factors of obesity and its associated conditions, negative effects were also seen. However the exact mechanism of action of testosterone that influences risk factors of obesity and its associated conditions in men with low testosterone remain unclear, therefore further studies are needed to fully elucidate the above finding