Advisor(s)

Mansoor M. Amiji

Contributor(s)

Shashi Mehta, M. V. Sitkovskiĭ

Date of Award

2010

Date Accepted

12-2010

Degree Grantor

Northeastern University

Degree Level

M.S.

Degree Name

Master of Science

Department or Academic Unit

Bouve College of Health Sciences. Department of Pharmaceutical Sciences

Keywords

pharmaceutical sciences, angiogenesis, gelatin nanoparticles, HIF1a, metastasis, siRNA, VEGF, hypoxia

Subject Categories

Tumors, Neovascularization, Metastasis

Disciplines

Pharmacy and Pharmaceutical Sciences

Abstract

Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy.

Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy.

Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC- conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression.

Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to increase with the increase in the HIF1α levels. Gelatin and PEG-modified gelatin nanoparticles were successfully prepared having a particle size in the range of 200 to 300 nm in diameter. Cell uptake studies showed that both types of nanoparticles could be efficiently internalized in tumor cells with a maximum intracellular concentrations reaching after 6 hours of incubation. Cytotoxicity analysis using MTS (formazan) assay showed that there was no significant change in cell viability upon treatment with any of the nanoparticle formulations relative to untreated control. Comparative analysis of gelatin nanoparticles and lipofectamine transfection of HIF1α-siRNA, shows a higher HIF1α knockdown for gelatin nanoparticles.

Conclusion: Use of HIF1α siRNA for the treatment of cancer cells that overly express HIF1α has shown great therapeutic potential as shown by the results. The expression of VEGF, MMP-2 and MMP-9 decreases with the decrease of HIF1α expression. This may indicate the reversal of the aggressive phenotype of the tumors with HIF1α knockdown. HIF1α-siRNA, hence shows great potential for the therapy of aggressive tumors, however in vivo studies need to be carried out to validate these findings.

Document Type

Master's Thesis

Rights Information

copyright 2010

Rights Holder

Shruti Shah

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