Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation.

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1-183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60-80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.

Doxorubicin (DOX) is a potent anticancer agent that binds both DNA and cardiolipin (CL). To investigate DOX binding to CL versus DNA, aqueous soluble, CL-enriched nanoparticles, termed nanodisks (ND), were employed. Upon incubation with CL-ND, but not with phosphatidylcholine ND, DOX binding was detected. DOX binding to CL-ND was sensitive to buffer pH and ionic strength. To investigate if a DOX binding preference for DNA versus CL-ND exists, an agarose gel-based dye binding assay was developed. Under conditions wherein the commercial fluorescent dye, GelRed, detects a 636 bp DNA template following electrophoresis, DOX staining failed to visualize this DNA band. Incubation of the template DNA with DOX prior to electrophoresis resulted in a DOX concentration-dependent attenuation of GelRed staining intensity. When the template DNA was pre-incubated with equivalent amounts of free DOX or DOX-CL-ND, no differences in the extent of GelRed staining intensity attenuation were noted. When DOX was incubated with DNA alone, or a mixture of DNA and CL-ND, the extent of DOX-induced GelRed staining intensity attenuation was equivalent. Thus, DOX has a binding preference for DNA versus CL and, moreover, DOX-CL-ND offer a potential strategy to prevent DOX-induced cardiotoxicity while not affecting its affinity for DNA.

Alkaline surface treatment and time-resolved reading of mn-doped nanocrystal signal transducer for enhanced bioassay sensitivity.

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer.

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. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle's lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin B as the hydrophobic bioactive agent.

Studies of the cardiolipin interactome.

Cardiolipin (CL) is a unique phospholipid that is fundamental to the structure and function of the highly curved cristae membranes of mitochondria. Given its distinctive cone-shaped molecular architecture, CL induces negative membrane curvature in a bilayer setting. Another key feature of CL is its intrinsic ability to interact with various ligands, including cytochrome c, the anti-neoplastic anthracycline, doxorubicin, and the divalent cation, calcium. Although these, and other, binding interactions exert profound effects on mitochondrial and cellular function, they are difficult to study in intact mitochondria. Whereas liposomes provide a potential model membrane system, their relatively large size, limited ability to accommodate CL and the presence of an inaccessible interior bilayer leaflet, make these structures suboptimal. The discovery that CL can be formulated into aqueous soluble, reconstituted high density lipoprotein particles, termed nanodisks (ND), provides an alternative model membrane system. Comprised solely of CL and an apolipoprotein scaffold, CL-ND exist as a disk-shaped phospholipid bilayer whose perimeter is stabilized by contact with the scaffold protein. In these nanoscale particles, both leaflets of the bilayer are solvent accessible, an advantage for studies of ligand interactions. Recent experiments employing CL-ND have yielded novel insight into apoptosis, cardiotoxicity and CL-dependent bilayer to non-bilayer transitions.

Foam fractionation of a recombinant biosurfactant apolipoprotein.

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.

Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity.

Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage.

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

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

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 CaCl2 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 CaCl2-induced effects. Centrifugation of CaCl2-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 CaCl2 at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl2 under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca2+ 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.

Reconstituted HDL as a therapeutic delivery device.

Studies of "pre ß" high density lipoprotein (HDL) and reconstituted HDL (rHDL) have contributed to our understanding of the Reverse Cholesterol Transport pathway. The relative ease with which discoidal rHDL can be generated in vitro has led to novel applications including a) infusion of rHDL into patients to promote regression of atherosclerosis; b) use of rHDL as a miniature membrane for integration of transmembrane proteins in a native-like conformation and c) incorporation of hydrophobic bioactive molecules into rHDL, creating a delivery device. The present review is focused on bioactive agent containing rHDL. The broad array of hydrophobic bioactive molecules successfully incorporated into these particles is discussed, as well as the use of natural lipids and synthetic lipid analogs to confer distinctive binding activity. This technology remains in its infancy with the full potential of these simple, yet elegant, nanoparticles still to be discovered.

Calcium-induced transformation of cardiolipin nanodisks.

Miniature membranes comprised of tetramyristoylcardiolipin (CL) and apolipoprotein (apo) A-I, termed nanodisks (ND), are stable, aqueous soluble, reconstituted high density lipoproteins. When CL ND, but not dimyristoylphosphatidylcholine (PC) ND, were incubated with CaCl2, a concentration dependent increase in sample turbidity occurred, consistent with CL undergoing a bilayer to non-bilayer transition. To assess the cation specificity of this reaction, CL ND were incubated with various mono- and divalent cations. Whereas monovalent cations had no discernable effect, MgCl2 and SrCl2 induced a response similar to CaCl2. When ND were formulated using different weight ratios of CL and PC, those possessing 100% CL or 75% CL remained susceptible to CaCl2 induced sample turbidity development while ND possessing 50% CL displayed reduced susceptibility. ND comprised of 25% CL and 75% PC were unaffected by CaCl2 under these conditions. SDS PAGE analysis of insoluble material generated by incubation of CL ND with CaCl2 revealed that nearly all apoA-I was recovered in the insoluble fraction along with CL. One h after addition of EDTA to CaCl2-treated CL ND, sample clarity was restored. Collectively, the data are consistent with a model wherein Ca2+ forms a bidentate interaction with anionic phosphates in the polar head group of CL. As phosphate group repositioning occurs to maximize Ca2+ binding, CL acyl chains reposition, accentuating the conical shape of CL to an extent that is incompatible with the ND bilayer structure.