Departmental Affiliation and City, State, Zip for All Authors

Alabama State University, Montgomery, AL 36104; Institute of Cardiovascular and Metabolic Diseases, UNT Health Science Center, Fort Worth, TX 76107; Departments of Physiology/Anatomy and Pediatrics, University of North Texas Health Science Center, Fort Worth, Texas 76107; Department of Physiology and Anatomy, UNT Health Science Center, Fort Worth, TX 76107

Scientific Abstract

Purpose: Synthetic or reconstituted High Density Lipoproteins (rHDL) are increasingly becoming an attractive drug delivery platform for anticancer therapy. These nanoparticles are made up of a central hydrophobic core and hydrophilic shell of Apoprotein A-1 (Apo A-1). This protein can self-associate at different conditions of pH, ionic strength, and storage conditions. Aggregation of this protein may hamper the quality of the rHDL nanoparticles prepared and the antitumor efficacy as a consequence. We studied Apo A-1 aggregation and investigated different methods and conditions to disassociate the aggregates and maintain monomeric form of this protein. These studies are important to develop/establish a methodology that results in preparation of high quality nanoparticles from this relatively expensive protein component. Methods: We used Fast Protein Liquid Chromatography (FPLC) to monitor the condition of Apo-A1 when subjected to various storage temperatures as well as exposure to different concentrations of denaturing agents such as Guanidine HCL. Results: The Apo-A1 stock (30mg/mL) appears to be a mixture of various types of aggregates even though stored at -200C. Apo-A1 is stable at -200C when compared to 40C and room temperature storage conditions. Moreover, exposing Apo-A1 to various concentrations of guanidine hydrochloride resulted in monomeric form of the protein with about 19 minutes elution time. Apo A-1 solutions excluding GnHCl stored at different temperatures only produced a monomer form when stored at -20˚C. Various protein standards were ran on FPLC to estimate the size of Apo-A1 monomer and aggregates. Conclusions: Self association of Apo A-1 is inherent at high concentrations. This study shed light on appropriate storage conditions as well as conditions to use at which monomeric form can be maintained that can result in high quality rHDL nanoparticles. Future studies are planned to investigate the effect of Apo-A1 aggregation on rHDL nanoparticle physical characteristics.

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INVESTIGATION OF APO-A1 AGGREGATION AND ITS EFFECT ON HIGH-DENSITY LIPOPROTEIN (RHDL) NANOPARTICLES

Purpose: Synthetic or reconstituted High Density Lipoproteins (rHDL) are increasingly becoming an attractive drug delivery platform for anticancer therapy. These nanoparticles are made up of a central hydrophobic core and hydrophilic shell of Apoprotein A-1 (Apo A-1). This protein can self-associate at different conditions of pH, ionic strength, and storage conditions. Aggregation of this protein may hamper the quality of the rHDL nanoparticles prepared and the antitumor efficacy as a consequence. We studied Apo A-1 aggregation and investigated different methods and conditions to disassociate the aggregates and maintain monomeric form of this protein. These studies are important to develop/establish a methodology that results in preparation of high quality nanoparticles from this relatively expensive protein component. Methods: We used Fast Protein Liquid Chromatography (FPLC) to monitor the condition of Apo-A1 when subjected to various storage temperatures as well as exposure to different concentrations of denaturing agents such as Guanidine HCL. Results: The Apo-A1 stock (30mg/mL) appears to be a mixture of various types of aggregates even though stored at -200C. Apo-A1 is stable at -200C when compared to 40C and room temperature storage conditions. Moreover, exposing Apo-A1 to various concentrations of guanidine hydrochloride resulted in monomeric form of the protein with about 19 minutes elution time. Apo A-1 solutions excluding GnHCl stored at different temperatures only produced a monomer form when stored at -20˚C. Various protein standards were ran on FPLC to estimate the size of Apo-A1 monomer and aggregates. Conclusions: Self association of Apo A-1 is inherent at high concentrations. This study shed light on appropriate storage conditions as well as conditions to use at which monomeric form can be maintained that can result in high quality rHDL nanoparticles. Future studies are planned to investigate the effect of Apo-A1 aggregation on rHDL nanoparticle physical characteristics.

Manuscript Number

1038