Therapeutic uses of compounds produced by biotechnology are presently limited by the lack of noninvasive methods for continuous administration of biologically-active macromolecules. Transdermal delivery would be an attractive solution, except macromolecules have not previously been delivered clinically across human skin at therapeutic rates. To increase transport of a highly-charged macromolecule (heparin), high-voltage pulses believed to cause electroporation were applied to skin. Using this approach, transdermal heparin transport across human skin in vitro occurred at therapeutic rates (100-500 micrograms/cm2h), reported to be sufficient for systemic anticoagulation. In contrast, fluxes caused by low-voltage iontophoresis having the same time-averaged current were an order of magnitude lower. Heparin transported across the skin was biologically active, but with only one eighth the anticoagulant activity of heparin in the donor compartment due to preferential transport of small (less active) heparin molecules. Flux, activity, and transport number data together suggest that high-voltage pulsing creates transient changes in skin microstructure which do not occur during iontophoresis. Safety issues are discussed.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.

Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.

0 Scanning confocal fluorescence microscopy was used to image localized regions of calcein transport across human stratum corneum during constant low-voltage (iontophoresis) and pulsed highvoltage exposures. Following an electrical protocol, imaging revealed regions of fluorescence which were interpreted as sites where transport of a fluorescent probe (calcein) into the stratum corneum had taken place. Electrically-assisted transport of calcein, whether enhanced by iontophoresis or high-voltage pulsing, appears to occur through intercellular and, to some extent, transcellular pathways into localized regions of stratum corneum that are not associated with appendages. Uniquely associated with the highest voltage pulses used (300 V across the skin) was the appearance of small, brightly fluorescent areas containing nonfluorescent interiors, i.e., fluorescent "rings". We present evidence which suggests that the dark interiors represent sites through which transport occurred during pulsing, but where calcein was no longer present at the time of imaging. Transport of charged microspheres into the stratum corneum was also observed.