Localized Transport Regions Research Articles

Saturated Anionic Phospholipids Enhance Transdermal Transport by Electroporation

Anionic phospholipids, but not cationic or neutral phospholipids, were found to enhance the transdermal transport of molecules by electroporation. When added as liposomes to the milieus of water-soluble molecules to be delivered through the epidermis of porcine skin by electroporation, these phospholipids enhance, by one to two orders of magnitude, the transdermal flux. Encapsulation of molecules in liposomes is not necessary. Dimyristoylphosphatidylserine (DMPS), phosphatidylserine from bovine brain (brain-PS), dioleoylphosphatidylserine (DOPS), and dioleoylphosphatidylglycerol (DOPG) were used to test factors affecting the potency of anionic lipid transport enhancers. DMPS with saturated acyl chains was found to be a much more potent transport enhancer than those with unsaturated acyl chains (DOPS and DOPG). There was no headgroup preference. Saturated DMPS was also more effective in delaying resistance recovery after pulsing, and with a greater affinity in the epidermis after pulsing. Using fluorescent carboxyl fluorescein and fluorescein isothiocyanate (FITC)-labeled Dextrans as test water-soluble molecules for transport, and rhodamine-labeled phospholipids to track anionic phospholipids, we found, by conventional and confocal fluorescence microscopy, that transport of water-soluble molecules was localized in local transport spots or regions (LTRs) created by the electroporation pulses. Anionic phospholipids, especially DMPS, were located at the center of the LTRs and spanned the entire thickness of the stratum corneum (SC). The degree of saturation of anionic phospholipids made no difference in the densities of LTRs created. We deduce that, after being driven into the epidermis by negative electric pulses, saturated anionic phospholipids mix and are retained better by the SC lipids. Anionic lipids prefer loose layers or vesicular rather than multilamellar forms, thereby prolonging the structural recovery of SC lipids to the native multilamellar form. In the presence of 1 mg/ml DMPS in the transport milieu, the flux of FITC-Dextran-4k was enhanced by 80-fold and reached 175 μg/cm 2/min. Thus, the use of proper lipid enhancers greatly extends the upper size limit of transportable chemicals. Understanding the mechanism of lipid enhancers enables one to rationally design better enhancers for transdermal drug and vaccine delivery by electroporation.

Changes in skin structure and electrical properties following high voltage exposure.

Human skin experiences extensive alterations when exposed to a strong electric field. In high voltage electric shocks, tissues in the current path undergo large-scale changes; the stratum corneum (SC), the outermost layer of the skin, loses its barrier function against ionic transport. The primary mechanism of electric field interaction with the skin is the creation of aqueous pathways, which increase the permeability of the skin to ions and macromolecules. Changes in skin structure and electrical properties were characterized by measuring the transport of fluorescent molecules in an in vitro preparation of human cadaver skin in a permeation chamber. Application of high voltage pulses (Uskia > 150 V, tau pulse = 1 ms) decreased the skin resistance per unit area by three orders of magnitude from its initial value of 100 k omega-cm-2. The resistance reached a quasi-steady state after about 20 pulses. After the cessation of pulsing, Rskin recovered immediately to about 4 k omega-cm-2 and remained low for 2 h thereafter. Transport of fluorescent molecules by high voltage pulses occurs in highly localized transport regions (LTRs), with diameters not exceeding 100 microns for short pulses (tau pulse < 5 ms).

Creation of transdermal pathways for macromolecule transport by skin electroporation and a low toxicity, pathway-enlarging molecule

A combined electrical (HV, “high voltage”, pulsing) and chemical (topical sodium thiosulfate) intervention is hypothesized to create enlarged aqueous pathways that allow large quantities of macromolecules to be transported through human skin's stratum corneum (SC), the dominant barrier for transdermal drug delivery and biochemical analyte extraction. This expectation is based on the known structure and composition of the SC, and previous models and experiments for local transport regions (LTRs) due to transdermal HV pulsing. In vitro experiments demonstrated that transdermal macromolecule fluxes of 10 −9 to 10 −8 mol h −1 cm −2 (10 to 100 μg h −1 cm −2) or greater are possible for lactalbumin and an antibody (IgG), which are potentially therapeutic values for peptides, proteins and nucleic acids. In the absence of sodium thiosulfate, only a small molecule (sulforhodamine) flux increased significantly, consistent with many previous studies. Significant macromolecule transdermal fluxes occurred only if a pathway enlarging molecule (sodium thiosulfate) was present. Our results also provide support for the mechanism hypothesis that HV pulses leading to transdermal voltages U skin>50 V create straight-through aqueous pathways that penetrate multilamellar bilayer membranes, corneocyte envelopes and corneocyte interiors within the SC.

Comparison of the effects of short, high-voltage and long, medium-voltage pulses on skin electrical and transport properties

High-voltage pulses have been shown to increase rates of transport across skin by several orders of magnitude on a time scale of minutes to seconds. Two main pulse protocols have been employed to promote transport: the intermittent application of short (∼1 ms) high-voltage (∼100 V across skin) pulses and a few applications of long (=100 ms) medium-voltage (>30 V across skin) pulses. In order to better evaluate the benefits of each protocol for transdermal drug delivery, we compared these two protocols in vitro in terms of changes in skin electrical properties and transport of sulforhodamine, a fluorescent polar molecule of 607 g/mol and a charge of −1. Whereas both protocols induced similar alterations and recovery processes of skin electrical resistance, long pulses of medium-voltage appeared to be more efficient in transporting molecules across skin. Skin resistance decreased by three (short pulses) and two (long pulses) orders of magnitude, followed by incomplete recovery in both cases. For the same total transported charge, long pulses induced faster and greater molecular transport across skin than short pulses. In addition, a greater fraction of the aqueous pathways created by the electric field was involved in molecular transport when using long pulse protocols. Transport was concentrated in localized transport regions (LTRs) for both protocols but LTRs created by long pulses were an order of magnitude larger than those formed by short pulses and the short pulses created an order of magnitude more LTRs. Overall, this study is consistent with the creation of fewer, but larger aqueous pathways by long, medium-voltage pulses in comparison to short, high-voltage pulses.

Spatially constrained localized transport regions due to skin electroporation

Rapid, controlled molecular transport across human skin is of great interest for transdermal drug delivery and minimally invasive chemical sensing. Short, high-voltage pulses have been shown previously to create localized transport regions in the skin. Here, we show that these regions can be constrained to occur at specific sites using electrically insulating masks that restrict the field lines. The increase in total ionic and molecular transport per area was comparable to the levels observed in unconstrained electroporation of human skin. Constraining the area of intervention to encompass small areas of interest, a primary feature in the design of microdevices for transdermal drug delivery, can provide the same levels of flux as the unconstrained case.

Theory of electrical creation of aqueous pathways across skin transport barriers

Experimental studies have shown that application of electrical pulses to human skin that result in U skin>30 V for durations of about 1 ms or longer causes a large decrease in electrical resistance within microseconds, followed in seconds by an increase in molecular transport of water-soluble molecules. Local transport regions (LTRs), within which molecular transport is concentrated, mostly form away from the skin's appendages and rete pegs. Theoretical attempts to explain this behavior involve electrically created aqueous pathways (“pores”). For short (about 1 ms) “high voltage” (HV) pulses leading to about U skin>50 V, it was hypothesized that such pulses cause electroporation of the multilamellar lipid bilayer membranes of the skin's stratum corneum (SC). Much of the present experimental evidence supports the more specific hypothesis that such pulses create “straight through aqueous pathways”, mostly within LTRs, that perforate the SC lipid bilayers and pass through the interiors of hydrated corneocytes. Theoretical estimates of the localized heating within LTRs predict relatively small temperature rises. The theory of LTR formation is incomplete, with both stochastic and deterministic models under consideration. Moderate voltage (MV) pulses leading to about 5< U skin<50 V, are consistent with appendageal activation and electroporation. The largest molecular fluxes occur for HV pulses, for which theory predicts large numbers of straight-through aqueous pathways. Both appendageal and stratum corneum electroporation are different from iontophoresis, which occurs at U skin<5 V.

Local transport regions (LTRs) in human stratum corneum due to long and short `high voltage' pulses

Application of `high voltage' (HV) pulses (transdermal voltage U skin>50 V) to preparations of human skin have been previously hypothesized to cause electroporation of multilamellar lipid barriers within the stratum corneum (SC). Such pulses cause large increases in molecular transport and decrease in the skin's electrical resistance. Here we describe the local transport regions (LTRs) and the surrounding local dissipiation regions (LDRs) that dominate the skin's response to both `long' and `short' HV pulses. The number of LTR/LDRs depends on U skin, but their size depends on pulse duration, so that LDRs can merge to form large regions containing several LTRs. LTRs themselves are not spatially homogeneous, as they have a ringlike structure, which is interpreted as involving different transport behavior viz. aqueous pathways which are either predominantly perpendicular or parallel to the SC. Our observations are consistent with the hypothesis that localized aqueous pathway formation (electroporation) occurs first, followed by secondary processes involving the entry of water into the SC and also localized heating.

Theory of skin electroporation: implications of straight-through aqueous pathway segments that connect adjacent corneocytes.

Previous in vitro experiments have shown that transdermal high-voltage pulses (Uskin approximately 100 V; duration approximately 1 ms) create local transport regions (LTR) away from appendages in human skin. Quantitative interpretation of the associated ionic and molecular transport led to the view that a large number of aqueous pathways were created, and these connect the corneocytes within an LTR. Here we use the "brick wall" model of the stratum corneum, modified so that morphology important to understanding electrical behavior is emphasized. In this model a minimum-size LTR is regarded as an idealized stack of corneocytes in which the 5-6 multilamellar lipid bilayer membranes between adjacent corneocytes are electroporated. As in artificial planar bilayer and cell membrane electroporation, a distribution of pathway sizes is expected during pulsing, and during recovery after pulsing individual pathway segments are expected to shrink and close randomly, with a time constant tau(seg) that depends on temperature and on lipid composition. Numerical simulations based on stochastic closure of individual segments were used to predict the electrical conductance G(LTR)(t) of a minimum-size LTR after pulsing stops. These theoretical results show that simple exponential decay, G(LTR)(t) = G(LTR)(0)exp(-t/tau(seg)), occurs with minimal fluctuations if the number of pathways is large (np > 10(2)), but for much smaller values the conduction decreases erratically. A "stochastic bottleneck" leading to complete closure is reached only at about np < 3. Thus, for the same number of electrically created pathways, the stratum corneum will remain "open" longer if the pathways are located within an LTR than if the same number of pathways are distributed sparsely over the skin. These predictions are relevant to postpulse transport, including the trapping of linear macromolecules that can hold pathway segments open for prolonged intervals.

Skin electroporation causes molecular transport across the stratum corneum through localized transport regions.

High voltage pulsing of human skin (approximately 100 V across the skin, 1 ms pulses) has been hypothesized to cause electroporation of the stratum corneum, and to cause large fluxes of drugs and other molecules across the skin, through newly created aqueous pathways. In contrast, iontophoresis (<0.5 mA per cm2, <1 V across the skin) has long been used in transdermal drug delivery, and is believed to involve pre-existing pathways associated with hair follicles and sweat ducts. Either high voltage pulsing or iontophoresis was applied to human, hairless rat, or black rat snake skin. Hairless rat skin contains more hair follicles than human skin, and snake skin does not contain any hair follicles. All three types of skin had comparable electrical resistances at low voltages; however, the iontophoretic transport of charged fluorescent molecules was significant for human and hairless rat skin, but no transport occured across snake skin, indicating that hair follicles and sweat ducts play a major role in iontophoresis. Electroporation caused large molecular transport for all three types of skin, and involved spontaneously forming localized transport regions, not associated with appendages. These experiments thus provide further support for the hypothesis that high voltage pulsing causes electroporation in the stratum corneum, and that this transport mechanism is fundamentally different from iontophoresis.

Imaging of fluorescent molecule and small ion transport through human stratum corneum during high voltage pulsing: localized transport regions are involved

During the application of high-voltage pulses across the skin, transport of two negatively charged fluorescent molecules through the stratum corneum is highly localized. The apparent size of these localized transport regions (LTR's) is initially 10 μm in diameter for both calcein and sulforhodamine. Appearance of LTR's occurred at or above transdermal voltages of 75 V. In the range of 75 to 160 V, the number of LTR's increases with voltage, but their initial size is the same at all voltages; with additional pulses LTR's increase in size, reaching diameters of approximately 40–80 μm. Small ion currents across the skin are also localized and include the LTR's; however, the areas of current flux appeared to be larger. There was no visible damage to the structure of the skin seen at 100 × magnification for any of the voltages used (< 170 V across the skin). Significantly, LTR's are not sweat ducts or hair follicles.

Transdermal Transport of DNA Antisense Oligonucleotides by Electroporation

Fluorescein-labeled antisense oligodeoxynucleotides (ODNs) corresponding to the promoters of the protooncogene c-myb (24-mer) and the oncogene c-myc (15-mer) were transported through the human skin in vitro by electroporation. Fluxes of 6.4±2.1 pM/cm2 hr and 11.5±3.5 pM/cm2 hr, respectively, were achieved during exponential pulsing [τpulse = 1.1ms, transdermal voltage (Uskin) = 80V] every five seconds. The flux for Uskin <70V was undetectable, rapidly increased at 80V, but plateaued at higher values. Fluorescence imaging demonstrated that transport of the ODNs is concentrated in localized transport regions (LTRs)1 approximately 30μm in diameter.