HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process.

The onset of cell fusion mediated by HIV-1 IIIB Env is preceded by a lag phase of 15-20 min. Fusion mediated by the CD4-independent HIV-1 Env 8x, which is capable of interacting directly with CXCR4, proceeds with a greatly reduced lag phase. We probed the intermediate steps during the lag phase in HIV-1 IIIB Env-mediated fusion with Leu3-a, an inhibitor of attachment of gp120 to CD4, AMD3100, an inhibitor of attachment of gp120 to CXCR4, and C34, a synthetic peptide that interferes with the transition of gp41 to the fusion active state. Inhibitions of fusion as a function of time of addition of C34 and of AMD3100 were equivalent, indicating that engagement of gp120 by CXCR4 and formation of the gp41 six-helix bundle follow similar kinetics. The initial steps in fusion mediated by the CD4-independent Env 8x are too rapid for these inhibitors to interfere with. However, when 8x Env-expressing cells were incubated with target cells at 25 degrees C in the presence of AMD3100 or C34, prior to incubation at 37 degrees C, these inhibitors were capable of inhibiting 8x Env-mediated fusion. To further examine engagement of gp120 by CXCR4 and exposure of binding sites for C34, we have reversibly arrested the fusion reaction at 37 degrees C by adding cytochalasin B to the medium. We show that CXCR4 engagement and six-helix bundle formation only occur after the release of the cytochalasin arrest, indicating that a high degree of cooperativity is required to trigger the initial steps in HIV-1 Env-mediated fusion.

Mode of action of an antiviral peptide from HIV-1. Inhibition at a post-lipid mixing stage.

DP178, a synthetic peptide corresponding to a segment of the transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus, type 1 (HIV-1), is a potent inhibitor of viral infection and virus-mediated cell-cell fusion. Nevertheless, DP178 does not contain gp41 coiled-coil cavity binding residues postulated to be essential for inhibiting HIV-1 entry. We find that DP178 inhibits phospholipid redistribution mediated by the HIV-1 envelope glycoprotein at a concentration 8 times greater than that of solute redistribution (the IC(50) values are 43 and 335 nm, respectively). In contrast, C34, a synthetic peptide which overlaps with DP178 but contains the cavity binding residues, did not show this phenomenon (11 and 25 nm, respectively). The ability of DP178 to inhibit membrane fusion at a post-lipid mixing stage correlates with its ability to bind and oligomerize on the surface of membranes. Furthermore, our results are consistent with a model in which DP178 inhibits the formation of gp41 viral hairpin structure at low affinity, whereas C34 inhibits its formation at high affinity: the failure to form the viral hairpin prevents both lipid and solute from redistributing between cells. However, our data also suggest an additional membrane-bound inhibitory site for DP178 in the ectodomain of gp41 within a region immediately adjacent to the membrane-spanning domain. By binding to this higher affinity site, DP178 inhibits the recruitment of several gp41-membrane complexes, thus inhibiting fusion pore formation.

Using surface electrodes to monitor the electric-pulse-induced permeabilization of porcine skin.

The stratum corneum, the outermost layer of the skin, acts as a barrier between the skin and the outside world, preventing evaporation of water from underlying tissues while impeding the diffusion of foreign molecules into the body (1,2). Densely packed layers of flattened, dead, keratinized cells (2,3) are incorporated into a lipid lamellae matrix consisting primarily of ceramides, cholesterol, and fatty acids (2,4), forming an impermeable, hydrophobic partition. The stratum corneum represents the main obstacle to efficient transdermal drug delivery (1,2). If the stratum corneum is disrupted, the barrier to molecular transport is greatly reduced.

Transdermal delivery using surface electrodes in porcine skin.

The main barrier to cutaneous or transcutaneous drug and gene delivery is the impermeability of the stratum corneum (SC), the outermost layer of the skin (1). If the integrity of the SC is disrupted, the barrier to molecular transit may be greatly reduced. Cutaneous absorption can be increased by removal of the SC by tape-stripping or dermabrasion, by vehicle (solvent-carrier) optimization, or by the use of penetration enhancers like DMSO (dimethylsulfoxide), oleic acid, and alcohols (2,3). An electric field can also be used to enhance delivery. Disruption of the SC can be achieved by electroporation, which is the creation of penetration sites by an electric pulse. Ions and molecules move through induced gaps of the SC by diffusion and electromotive or electroosmotic transport (4-6). Electroporation differs from iontophoresis, in which there is an increased migration of ions or charged molecules through the skin when an electrical potential gradient is applied. The primary transdermal route for iontophoresis seems to be appendageal or intercellular through preexisting pathways (5,7), or as a result of low-voltage (<5 V) induced permeabilization of appendageal bilayers (8). A third form of electroenhanced drug delivery, electrochemotherapy (9), refers to localized delivery of electric pulses across a tumor following systemic or intratumor drug administration, and usually does not involve cutaneous or transcutaneous delivery.

Time-dependent ultrastructural changes to porcine stratum corneum following an electric pulse.

The morphological changes to heat-stripped porcine stratum corneum following an electroporating pulse were studied by time-resolved freeze fracture electron microscopy. Pulses at a supra-electroporation threshold of 80 volts and 300 microseconds were applied across the stratum corneum with a pair of copper plate electrodes, which also served as cooling contacts. Multilamellar vesicles of 0.1-5.5 mm in diameter in the intercellular lipid bilayers of the stratum corneum appeared in less than milliseconds after pulsing. Pulsed samples exhibited aggregations of vesicles, whereas only occasional single vesicles were seen in the unpulsed samples. Aggregates form in less than a millisecond and disappear within minutes after the pulse. Their size ranged from 0.3 to 700 mm2. The size of individual vesicles, aggregate density, and size were analyzed as functions of postpulse time. These aggregate formations seem to be a secondary reaction to the pulse-induced skin permeabilization, determined by the resistance drop and recovery after the pulse. Heating the samples to 65 degrees C also caused vesicle aggregates of similar appearance to form, suggesting that these aggregations are related to the heating effect of the pulse. Hydration is thought to play an important role in aggregate formation.

Electrorheological modeling of the permeabilization of the stratum corneum: theory and experiment.

Experimentally observed changes in the conductivity of skin under the influence of a pulsing electric field were theoretically analyzed on the basis of a proposed electrorheological model of the stratum corneum (SC). The dependence of relative changes in conductivity on the amplitude of electric field and timelike parameters of applied pulses or pulse trains have been mathematically described. Statistical characteristics of phenomena of transient and long-term electroporation of SC were taken into consideration. The time-dependent decreases of skin resistance depicted by the models were fitted to experimental data for transient and long-term skin permeabilization by electric pulses. The results show two characteristic times and two spectra of characteristic energies for transient and long-term permeabilizations. The rheological parameters derived from the fittings agreed with those reported elsewhere for biological membranes.

A pulsed electric field enhances cutaneous delivery of methylene blue in excised full-thickness porcine skin.

We used electric pulses to permeabilize porcine stratum corneum and demonstrate enhanced epidermal transport of methylene blue, a water-soluble cationic dye. Electrodes were placed on the outer surface of excised full-thickness porcine skin, and methylene blue was applied to the skin beneath the positive electrode; 1 ms pulses of up to 240 V were delivered at frequencies of 20-100 Hz for up to 30 min. The amount of dye in a skin sample was determined from absorbance spectra of dissolved punch biopsy sections. Penetration depth and concentration of the dye were measured with light and fluorescence microscopy of cryosections. At an electric exposure dose VT (applied voltage x frequency x pulse width x treatment duration) of about 4700 Vs, there is a threshold for efficient drug delivery. Increasing the applied voltage or field application time resulted in increased dye penetration. Transport induced by electric pulses was more than an order of magnitude greater than that seen following iontophoresis. We believe that the enhanced cutaneous delivery of methylene blue is due to a combination of de novo permeabilization of the stratum corneum by electric pulses, passive diffusion through the permeabilization sites, and electrophoretic and electroosmotic transport by the electric pulses. Pulsed electric fields may have important applications for drug delivery in a variety of fields where topical drug delivery is a goal.

Characterization of electric-pulse-induced permeabilization of porcine skin using surface electrodes.

We measured the transient and long-term changes of permeability of full-thickness porcine skin after the application of a single or a train of electric pulses, as the basis for optimization of the electrical parameters for enhancing transdermal drug or gene delivery by electroporation. Two electrodes were attached to the stratum corneum of excised skin for transdermal electric pulse delivery and impedance measurement. Both transient and long-term permeabilization were found to be dependent on the electrical exposure dose, i.e., the product of pulse voltage and cumulative pulsing (exposure) time. Skin resistance dropped to about 20% of its prepulsing value when pulsed beyond a critical dosage of 0.4 V-s (with 20-40 V across each skin path), but recovered rapidly within seconds after the pulse. Long-term permeabilization of the skin required repeated pulsing with a minimum potential of 160 V (80 V across each skin path). The maximum long-term resistance drop, to 35% of the initial value, required a dose greater than 200 V-s, recovering slowly and seldom completely in tens of minutes to hours. The decrease and recovery of the resistance were dependent on the frequency and pulse length only for low-dose electrical exposure.