formulation-characterization-bioactive-agent-containing

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

University of Nevada

Coenzyme Q (CoQ ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ nanodisks (ND), contain 1.0 mg CoQ /5 mg PtdCho/2 mg apoA-I (97% CoQ solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ . Incubation of cultured cells with CoQ ND, but not empty ND, resulted in a significant increase in the CoQ content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ in lipid nanoparticles has been developed. The availability of CoQ ND provides a novel means to investigate biochemical aspects of CoQ uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.

Assembly and Characterization of Biocompatible Coenzyme Q -Enriched Lipid Nanoparticles.

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl-induced effects. Centrifugation of CaCl-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.

Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties.

Kyle Lethcoe

Department of Biochemistry and Molecular Biology

University of Nevada

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Kyle Lethcoe

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

University of Nevada

Formulation and Characterization of Bioactive Agent Containing Nanodisks

Department of Biochemistry and Molecular Biology