Evaluating PLGA Encapsulated Recombinant LRP as a Treatment for Cardiovascular Disease

Abstract

Cardiovascular diseases are a classification of heart disorders that affect the heart and/or blood vessels with an estimated 523 million prevalent cases and 18.56 million deaths worldwide. In South Africa, CVD is the leading cause of death and accounts for 17.3% of yearly deaths. A frequent cause of most cardiovascular diseases is atherosclerosis, a disease characterized by chronic inflammation and the accumulation of fats and cholesterol that lead to plaque formation. When the plaques rupture, this leads to life threatening coronary syndromes. The most prescribed therapeutic intervention for atherosclerosis is statins. However, mortality rates are still high, nationally, and globally. Therefore, we could benefit from investigating other potential therapeutic biomolecules such as the 37/67 kDa laminin receptor precursor/laminin receptor (LRP/LR). LRP/LR is a multi-localized and multi-functional receptor that has also been shown to mediate cardioprotective effects in atherosclerosis. Hence, this study focused on investigating LRP/LR delivery into cells as a potential treatment for atherosclerosis. Firstly, recombinant LRP was overexpressed, purified, and characterized. The LRP protein was successfully overexpressed using E. coli and purified using affinity chromatography. The secondary structure and folding of the protein were characterized using circular dichroism and intrinsic tryptophan fluorescence spectroscopy, respectively. The protein was confirmed to be predominantly α-helical and correctly folded. Thereafter, functional characterization of the protein was performed indicating that the purified protein was active. The second aim of this project was to encapsulate the protein into PLGA nanoparticles to develop an in vitro delivery system. Characterization of the nanoparticles was performed using scanning electron microscopy and dynamic light scattering which showed that the nanoparticles have spherical morphology with a narrow size distribution of 221 nm, respectively. The release profile of LRP from the nanoparticles showed sustained release. Therefore, the successful encapsulation and release of LRP indicated that the therapeutic was successfully developed and it could be assessed as a possible treatment for CVD. The atherosclerotic cell culture model was successfully produced using 25 nM and 100 µg/ml of oxidized LDL. Effective doses of PLGA encapsulated LRP were determined on U937 cells and atherosclerotic cell models using an MTT assay and 75 µg/ml of encapsulated LRP was shown to exhibit therapeutic effects on the cells possibly through reverse cholesterol transport which led to regression of foam cells.