Robert Blumenthal

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities, and purification of the dye

Abstract Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.

Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection.

ABSTRACT β-Defensins are small (3 to 5 kDa in size) secreted antimicrobial and antiviral proteins that are components of innate immunity. β-Defensins are secreted by epithelial cells, and they are expressed at high levels in several mucosae, including the mouth, where the concentration of these proteins can reach 100 μg/ml. Because of these properties, we wondered whether they could be part of the defenses that lower oral transmission of human immunodeficiency virus (HIV) compared to other mucosal sites. Our data show that select β-defensins, especially human β-defensin 2 (hBD2) and hBD3, inhibit R5 and X4 HIV infection in a dose-dependent manner at doses that are compatible with or below those measured in the oral cavity. We observed that β-defensin treatment inhibited accumulation of early products of reverse transcription, as detected by PCR. We could not, however, detect any reproducible inhibition of env-mediated fusion, and we did not observe any modulation of HIV coreceptors following treatment with hBD1 and hBD2, in both resting and phytohemagglutinin-activated cells. Our data instead suggest that, besides a direct inactivation of HIV virions, hBD2 inhibits HIV replication in the intracellular environment. Therefore, we speculate that β-defensins mediate a novel antiretroviral mechanism that contributes to prevention of oral HIV transmission in the oral cavity. Immunohistochemical data on hBD2 expression in oral mucosal tissue shows that hBD2 is constitutively expressed, forming a barrier layer across the epithelium in healthy subjects, while in HIV-positive subjects levels of hBD2 expression are dramatically diminished. This may predispose HIV-positive subjects to increased incidence of oral complications associated with HIV infection.

Conformational Changes in Cell Surface HIV-1 Envelope Glycoproteins Are Triggered by Cooperation between Cell Surface CD4 and Co-receptors

We have continuously measured CD4-induced conformational changes of cell surface-expressed human immunodeficiency virus type-1 envelope glycoprotein gp120-gp41 in situ using 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, a fluorescent probe that binds to hydrophobic groups. CD4-expressing human T cell lines induced significant and rapid conformational changes (<1 min delay) in gp120-gp41 from T cell-tropic strains, and little conformational changes in gp120-gp41 from macrophage-tropic strains, with equivalent levels of envelope expression. Conversely, CD4-expressing human macrophages induced significant and rapid conformational changes in gp120-gp41 from macrophage-tropic strains, and little conformational changes in gp120-gp41 from T cell-tropic strains. Thus, the conformational changes undergone by gp120-gp41, which lead to membrane fusion, are highly cooperative and require both receptor and co-receptor. We used a dye transfer assay to show that neither membrane lipid fusion or fusion pore formation can occur with host cells having different tropism from the envelope.

Phase transition release, a new approach to the interaction of proteins with lipid vesicles Application to lipoproteins

To study the interaction of proteins with lipid bilayers, we have developed a new experimental approach based on the release of a water-soluble fluorescent dye from liposomes during scans through the lipid phase transition temperature. The fluorescence of carboxyfluorescein is quenched at high dye concentrations inside the vesicles but appears when the dye is released and diluted into the external medium. This new approach, phase transition release, is here applied to the interaction of serum lipoproteins and apolipoproteins with liposomes (for the most part, small unilamellar vesicles of dipalmitoylphosphatidylcholine). The major findings are these: (i) All of the lipoproteins and apolipoproteins tested induce a smooth, rapid release of carboxyfluorescein, essentially complete within a few seconds. HDL apolipoprotein induces 50% carboxyfluorescein release at a lipid/protein molar ratio of 3 400 : 1, whereas a ratio of 160 : 1 is required for native HDL. (ii) The interaction is all-or-none and irreversible. It involves a sufficient perturbation of bilayer structure to permit equal release of carboxyfluorescein (Mr 373) and inulin (Mr 5 500). In the case of HDL apolipoprotein, this release accompanies formation of a relatively homogeneous population of vesicular recombinant structures. Only at much higher protein/lipid ratios are the often-studied small, disc-shaped recombinants formed. (iii) Much more dye is released if the transition temperature is approached from below than if it is approached from above. (iv) Phase transition release is seen with multilamellar and reverse phase evaporation vesicles, though with patterns different from those seen with small unilamellar vesicles. (v) A large number of proteins are found not to induce phase transition release, even at concentrations of at least 1000-times those required for the HDL apolipoprotein effect. These include trypsin, chymotrypsin, pronase, bovine serum albumin (crystalline), ovalbumin, rabbit immunoglobulin G (and its F(ab)′2 and Fc fragments), rabbit immunoglobulin M, hemoglobin, hen lysozyme, synexin, ankyrin, myosin, and microtubule-associated proteins. Tubulin and actin, on the other hand, do induce phase transition release. In addition to its use for analysis of protein-bilayer interaction, phase transition release provides a way of reconstituting relatively water-soluble proteins into vesicles, under quantitative control and without detergent.

Regulation of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Fusion by a Membrane-Interactive Domain in the gp41 Cytoplasmic Tail

ABSTRACT Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this “inside-out” regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.

Light-sensitive Lipid-based Nanoparticles for Drug Delivery: Design Principles and Future Considerations for Biological Applications

Abstract Radiation-based therapies aided by nanoparticles have been developed for decades, and can be primarily categorized into two main platforms. First, delivery of payload of photo-reactive drugs (photosensitizers) using the conventional nanoparticles, and second, design and development of photo-triggerable nanoparticles (primarily liposomes) to attain light-assisted on-demand drug delivery. The main focus of this review is to provide an update of the history, current status and future applications of photo-triggerable lipid-based nanoparticles (light-sensitive liposomes). We will begin with a brief overview on the applications of liposomes for delivery of photosensitizers, including the choice of photosensitizers for photodynamic therapy, as well as the currently available light sources (lasers) used for these applications. The main segment of this review will encompass the details of strategies used to develop photo-triggerable liposomes for their drug delivery function. The principles underlying the assembly of photoreactive lipids into nanoparticles (liposomes) and photo-triggering mechanisms will be presented. We will also discuss factors that limit the applications of these liposomes for in vivo triggered drug delivery and emerging concepts that may lead to the biologically viable photo-activation strategies. We will conclude with our view point on the future perspectives of light-sensitive liposomes in the clinic.

Charge clusters and the orientation of membrane proteins

SummaryAlthough hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though “electrophoresing” into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2Kb, HLA-A2, HLA-B7, and mouse Ig μ heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the “microscopic” membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.

HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (ZHER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were ∼70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm ∼0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.

Sizing and separation of liposomes, biological vesicles, and viruses by high-performance liquid chromatography

The ability of an HPLC gel exclusion column (TSK G6000PW) to separate lipid vesicles, viruses, and biological vesicles according to size was tested and compared with separations on Sephacryl S1000. The columns were calibrated using vesicular Stokes radii determined by quasielastic light scattering. The vesicles separated according to size on both types of column and remained intact during elution. Viruses of known diameters and clathrin-coated vesicles were also eluted as a function of size. The TSK G6000PW column was able to separate larger particles (greater than 500 nm) than the Sephacryl S1000, and, when used in combination with the TSK G5000PW column, gave more discrete separations of smaller particles (10 to 30 nm diameter). Moreover, the HPLC columns can be run significantly faster (10-20 min vs several hours) and give more precise results than Sephacryl S1000. Therefore, HPLC using a G6000PW column alone, or in combination with a G5000PW column, provides a rapid and accurate means of sizing and selecting specifically sized biological and artificial vesicles.

KINETICS OF THE LOW PH-INDUCED CONFORMATIONAL CHANGES AND FUSOGENIC ACTIVITY OF INFLUENZA HEMAGGLUTININ

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.