Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats

A previous study indicated that the parameters governing the performance of electroporative delivery to the skin, are voltage, pulse length, number of pulses and electrode area.1 This article describes a study in which the reversibility of the electroporation technique is evaluated with in vitro methods. The skin's reversal from an enhanced permeation mode as a result of electroporation to the base level was used as an index to understand the mechanism of drug delivery and also as a preliminary indicator of safety. Maximum delivery of the model drug, terazosin hydrochloride, occurred during the pulsing. Electroporative delivery with a wire electrode (small-area electrode, 0.56 cm(2)) using 20 pulses at U(skin,0) 88 V, and pulse length 20 ms, did not cause any damage to the skin. Increasing the pulse length to 60 ms, while keeping the rest of the parameters fixed, caused a visible change in the external appearance of the skin. However, with the use of a spiral electrode (large-area electrode, 2.74 cm(2)) at 60-ms pulse length, there was minimal damage to the skin. This may be attributed to the more uniform flow of current over the whole skin area. The large-area electrode required a smaller electrode voltage, U(electrode,0) for any given U(skin,0) and also delivered nearly double the instantaneous power density compared with the small-area electrode. These findings indicate that using shorter pulses and large-area electrodes is a safer technique than large pulses and small-area electrodes when electroporation is used to enhance skin's permeability for drug delivery.     

Electrically enhanced transdermal delivery of a macromolecule

The purpose of this study was to establish the delivery parameters for the enhanced transdermal delivery of dextran sulfate (MW 5000 Da). Full-thickness pig skin or epidermis separated from human cadaver skin was used. Silver-silver chloride electrodes were used to deliver the current (0.5 mA cm-2). For electroporation experiments, one or more pulses were given using an exponential decay pulse generator. The correct polarity for iontophoresis and pulsing was first established as cathode in the donor. The amount of drug delivered increased with increasing donor concentration up to a point, but not any further. The amount delivered also increased with pulse voltage, the delivery being twice as much as with iontophoresis alone (144.5+/-10.35 microg cm(-2)), when 6 pulses of 500 V were applied at time zero before iontophoresis (276+/-45.2 microg cm(-2)). It was observed that the amount delivered was a function of increasing pulse length when the apparent charge delivered was kept constant. Transport through pig skin (107.4+/-24.4 microg cm(-2)) was found to be comparable with that through human epidermis (84.9+/-18.4 microg cm(-2)). In conclusion, we have demonstrated the transdermal delivery of a 5000 Da molecular weight dextran sulfate using iontophoresis. It was also seen that iontophoretic delivery could be enhanced by simultaneous electroporation.     

Skin electroporation for transdermal and topical delivery

Electroporation is the transitory structural perturbation of lipid bilayer membranes due to the application of high voltage pulses. Its application to the skin has been shown to increase transdermal drug delivery by several orders of magnitude. Moreover, electroporation, used alone or in combination with other enhancement methods, expands the range of drugs (small to macromolecules, lipophilic or hydrophilic, charged or neutral molecules) which can be delivered transdermally. Molecular transport through transiently permeabilized skin by electroporation results mainly from enhanced diffusion and electrophoresis. The efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. The in vivo application of high voltage pulses is well tolerated but muscle contractions are usually induced. The electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans.     

A practical assessment of transdermal drug delivery by skin electroporation

Transdermal drug delivery has many potential advantages, but the skin's poorly-permeable stratum corneum blocks delivery of most drugs at therapeutic levels. Short high-voltage pulses have been used to electroporate the skin's lipid bilayer barriers and thereby deliver compounds at rates increased by as much as four orders of magnitude. Evidence that the observed flux enhancement is due to physical alteration of the skin by electroporation, as opposed to only providing an iontophoretic driving force, is supported by a number of different transport, electrical and microscopy studies. Practical applications of electroporation's unique effects on skin are motivated by large flux increases for many different compounds, rapidly responsive delivery profiles, and efficient use of skin area and electrical charge. Greater enhancement can be achieved by combining skin electroporation with iontophoresis, ultrasound, and macromolecules. Sensation due to electroporation can be avoided by using appropriate electrical protocols and electrode design. To develop skin electroporation as a successful transdermal drug delivery technology, the strong set of existing in vitro mechanistic studies must be supplemented with studies addressing in vivo/clinical issues and device design.     

Skin targeted DNA vaccine delivery using electroporation in rabbits II. Safety

The Achilles heel of gene-based therapy is gene delivery into the target cells efficiently with minimal toxic effects. Viral vectors for gene/DNA vaccine delivery are limited by the safety and immunological problems. Recently, nonviral gene delivery mediated by electroporation has been shown to be efficient in different tissues including skin. There are no detailed reports about the effects of electroporation on skin tissue, when used for gene/DNA vaccine delivery. In a previous study we demonstrated the efficacy of skin targeted DNA vaccine delivery using electroporation in rabbits [Medi, B.M., Hoselton, S., Marepalli, B.R., Singh, J., 2005. Skin targeted DNA vaccine delivery using electroporation in rabbits. I. Efficacy. Int. J. Pharm. 294, 53-63]. In the present study, we investigated the safety aspects of the electroporation technique in vivo in rabbits. Different electroporation parameters (100-300 V) were tested for their effects on skin viability, macroscopic barrier property, irritation and microscopic changes in the skin. Skin viability was not affected by the electroporation protocols tested. The electroporation pulses induced skin barrier perturbation and irritation as indicated by elevated transepidermal water loss (TEWL) and erythema/edema, respectively. Microscopic studies revealed inflammatory responses in the epidermis following electroporation using 200 and 300 V pulses. However, these changes due to electroporation were reversible within a week. The results suggest that the electroporation does not induce any irreversible changes in the skin and can be a useful technique for skin targeted DNA vaccine delivery.     

Parameters Controlling Topical Delivery of Oligonucleotides by Electroporation

Abstract

Electroporation, using high voltage electrical pulses has been recognized as a powerful method for delivering macromolecules such as DNA and proteins in cells, or smaller molecules through the skin. Transdermal electroporation could combine targeted delivery of drugs to the skin and permeabilization of skin cells, suggesting that electroporation could be an interesting alternative for topical delivery of oligonucleotides. This work is devoted to the determination of the electroporation parameters that allow optimal delivery of oligonucleotides to the viable tissues of hairless rat skin in vitro. Phosphorothioate derivatives were preferred to the phosphodiester congeners as the former were found to be much less degraded when extracted from the tissues. Long duration (100-500ms)—medium voltage (100-200V)—exponentially decaying pulses appeared to be the best conditions for delivering oligonucleotides to the skin. The oligonucleotide quantity permeating the viable tissues of the skin was controlled by the selection of the electrical parameters of the pulses (voltage, pulse time and number of pulses) or by the ON concentration in the donor compartment. After delivery by electroporation, therapeutic levels of oligonucleotides were reached in the viable tissues of the skin (above 1 μM or 10 μM in intact or stripped skin respectively). Taken together, our results show that electroporation could be an interesting method for the delivery of oligonucleotides to the skin.     

Drug and gene delivery using electrotransfer

The use of a low intensity current (iontophoresis) and high voltage pulses (electroporation which permeabilizes lipid bilayers) has a potential for the administration of conventional and biotechnology-produced drugs. Iontophoresis and electroporation enhance transdermal delivery of drugs, including peptides and oligonucleotides. Electrochemotherapy, i.e., combination of a systemic or local delivery of a non-permeant cytostatic drug with electroporation, kills locally tumor cells. Recently, it has been shown that the local injection of a plasmid before electroporation increases significantly gene transfection. Hence, electrotransfer is a promising alternative for drug and gene delivery.     

Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for beta-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of beta-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p<0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.     

Transdermal Delivery of Fentanyl by Electroporation II. Mechanisms Involved in Drug Transport

Purpose. The aim of the present report was to systematically analyze the mechanisms involved in fentanyl transdermal transport by skin electroporation. Methods. The study was performed in vitro with full-thickness hairless rat skin, skin electroporation being carried out with five exponentially-decaying pulses of 100 V applied voltage and around 600 ms pulse duration. Results. Transport during and after pulsing are both important in transdermal delivery of fentanyl by skin electroporation. Rapid transport occurred during pulsing due to electrophoresis and diffusion through highly permeabilized skin. No electroosmosis was observed. The slow post-pulse passive transport was explained by lasting changes in skin permeability. Measurements of fentanyl quantities in the skin demonstrated that pulses rapidly loaded the viable part of the skin with fentanyl and hence rapidly overcame skin barrier. Conclusions. The different contributions of the transport mechanisms appear to depend on the physicochemical parameters of the transported molecule as well as the solution, suggesting that mechanistic analysis and careful consideration of formulation variables are essential for the development and optimization of drug delivery by skin electroporation.     

Transdermal Delivery of Metoprolol by Electroporation

Electroporation, i.e., the creation of transient pores in lipid membranes leading to increased permeability, could be used to promote transdermal drug delivery. We have evaluated metoprolol permeation through full thickness hairless rat skin in vitro following electroporation with an exponentially decaying pulse. Application of electric pulses increased metoprolol permeation as compared to diffusion through untreated skin. Raising the number of twin pulses (300 V, 3 ms; followed after 1 s by 100 V, 620 ms) from 1 to 20 increased drug transport. Single pulse (100 V, 620 ms) was as effective as twin pulse application (2200 V, 1100 V or 300 V, 3 ms; followed after 1 s by 100 V, 620 ms). In order to investigate the effect of pulse voltage on metoprolol permeation, 5 single pulses (each separated by 1 min) were applied at varying voltages from 24 to 450 V (pulse time 620 ms). A linear correlation between pulse voltage and cumulative metoprolol transported after 4 h suggested that voltage controls the quantity of drug delivered. Then, the effect of pulse time on metoprolol permeation was studied by varying pulse duration of 5 single 100 V pulses from 80 to 710 ms (each pulse also separated by 1 min). Cumulative metoprolol transported after 4 h increased linearly with the pulse time. Therefore, pulse time was also a control factor of the quantity of drug delivered but to a lesser extent than the voltage at least at 100 V. The mechanisms behind improved transdermal drug delivery by electroporation involved reversible increased skin permeability, electrophoretic movement of drug into the skin during pulse application, and drug release from the skin reservoir formed by electroporation. Thus, electroporation did occur as shown by the increased transdermal permeation, on indicator of structural skin changes and their reversibility. Electroporation has potential for enhancing transdermal drug delivery.     

Electroporation-mediated delivery of molecules to model intestinal epithelia

This study was conducted to determine if electroporation can deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. In addition, the long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. Caco-2 and T84 cells were electroporated as monolayers using calcein and fluorescein-labeled bovine serum albumin as marker molecules for measuring delivery into cells. Confocal microscopy and flow cytometry were used, respectively, to visualize and quantify uptake of these molecules. Transepithelial resistance was used as a measure of physiologic barrier function. We found that intracellular uptake of calcein and bovine serum albumin occurred uniformly throughout both types of model epithelia and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. We also observed that monolayers exposed to electroporation that induced uptake of up to 10⁶ molecules/cell were able to recover normal barrier function within one day. These findings suggest that electroporation may be useful for intracellular delivery into monolayers to study epithelial biology and, possibly, for drug delivery to intestinal epithelium.     

Transdermal delivery of nalbuphine and its prodrugs by electroporation.

The aim of this study was to assess the effects of electroporation on transdermal permeation of nalbuphine (NA) and its prodrugs. The permeation characteristics were investigated under various electrical factors and skin barriers to elucidate the mechanisms involved in transdermal delivery of NA and its prodrugs by skin electroporation. The in vitro permeation studies were performed using side-by-side diffusion cells. The various electrical factors investigated were pulse voltage, pulse duration and pulse number; the different skin barriers studied were intact hairless mouse skin, stratum corneum (SC)-stripped skin, delipid skin as well as furry Wistar rat skin. The prodrugs were fully converted to parent drug after skin permeation. Application of electroporation significantly enhanced transdermal permeation of NA and its prodrugs. The enhancement ratios were highest for NA and the four prodrugs showed the similar permeability after electroporation. The permeation amounts of NA and its prodrugs may be increased by application of higher pulse voltage, pulse duration as well as pulse number. Various kinetics and mechanisms were observed for the permeation of the hydrophilic NA and lipophilic nalbuphine enanthate through different skin barriers by applying electroporation. This study demonstrated that electroporation may enhance and control transdermal permeation of NA and its prodrugs. The results also indicated that the physicochemical properties of prodrug had significant effects on kinetics as well as mechanisms of transdermal permeation by electroporation.     

Antitumor drug delivery by tissue electroporation

Tissue electroporation has been explored to enhance the local delivery of chemotherapueutic agents to solid tumors. The technique, known as electrochemotherapy (ECT), uses high-voltage pulses to deliver drugs across cancerous tissues. ECT has been demonstrated to be an effective treatment for cutaneous malignancies. Recent studies also indicate that the applications of ECT can be extended from the treatment of cutaneous cancers to the treatment of tumors of vital organs such as brain, liver, lungs and others. This review also discusses electrogene antitumor therapy.     

The effects of iontophoresis and electroporation on transdermal delivery of buprenorphine from solutions and hydrogels

The in-vitro permeation of buprenorphine across skin was investigated to assess the effects of iontophoresis and electroporation on drug permeation from solutions as well as from hydrogels. Iontophoresis (0.3 mA cm(-2)) increased the buprenorphine permeation from solution by a factor of 14.27 as compared with passive diffusion; the application of electroporation increased the buprenorphine permeation from solutions by a factor of 8.45. The permeation experiments using cellulose membrane and stratum corneum (SC)-stripped skin as permeation barriers suggested that the enhancement with iontophoresis was primarily due to strong electrophoretic drift of buprenorphine molecules, whereas the enhancement seen with electroporation was mainly attributed to the creation of transient aqueous pores in the SC layer. Application of high-voltage pulses followed by iontophoresis resulted in a shorter permeation onset time from both solutions and hydrogels as compared with iontophoresis or electroporation alone. The charge repulsion between buprenorphine and chitosan vehicles as well as the competition effects of counter-ions for carboxymethylcellulose (CMC)-based polymers may account for the different permeation rates under electrical field. This study demonstrates the feasibility of using hydrogels for delivery of buprenorphine under the application of iontophoresis or electroporation, separately or together.     

Transdermal Delivery of Fentanyl by Electroporation I. Influence of Electrical Factors

Purpose. Electroporation, a method of reversibly permeabilizing lipid bilayers by the application of an electric pulse, has been shown to induce increased transdermal passage of molecules. The aim of the present report was to study in vitro with hairless rat skin the potential of electroporation for transdermal delivery of fentanyl. Results. The application of electric pulses can strongly promote transdermal delivery of fentanyl compared to passive diffusion through untreated skin. We also point out that the choice of the waveform of the electric pulses is important: at the same applied energy, a few exponentially-decaying (ED) pulses increased fentanyl permeation more than a few square-wave pulses and to the same extent as the repeated application of higher voltage-shorter duration ED pulses. A factorial design showed that the voltage, duration, and number of ED pulses allowed control of the quantity of drug transported through the skin. Conclusions. Skin electroporation could be a good way to improve the transdermal diffusion of fentanyl.     

Iontophoresis and electroporation: comparisons and contrasts

The techniques of iontophoresis and electroporation can be used to enhance topical and transdermal drug delivery. Iontophoresis applies a small low voltage (typically 10 V or less) continuous constant current (typically 0.5 mA/cm2 or less) to push a charged drug into skin or other tissue. In contrast, electroporation applies a high voltage (typically, ?100 V) pulse for a very short (micros-ms) duration to permeabilize the skin. This electric assistance of drug delivery across skin will expand the scope of transdermal delivery to hydrophilic macromolecules such as the drugs of biotechnology. These two techniques differ in several aspects such as the mode of application and pathways of transport but can be used together for effective drug delivery. Iontophoresis is already used clinically in physical therapy clinics and is close to commercialization for development of a systemic delivery patch with miniaturized circuits and similar in overall size to a passive patch. The use of electroporation for drug delivery is relatively new and is being actively researched.     

In vivo efficacy and safety of skin electroporation

This article reviews the studies on skin electroporation carried out in vivo in animals and emphasizes its potential therapeutic applications for transdermal and topical drug delivery. In agreement with in vitro studies, transport across skin due to high-voltage pulses in vivo was shown to increase by orders of magnitude on a timescale of minutes. Increased transdermal transport was measured by systemic blood uptake and/or pharmacological response, and demonstrated for calcein, a fluorescent tracer, fentanyl, a potent analgesic and flurbiprofen, an antiinflammatory drug. Combined electroporation with iontophoresis was shown to provide rapidly responsive transdermal transport of luteinizing hormone releasing hormone ex vivo as well. These data underline the potential of skin electroporation for improving the delivery profile of existing conventional transdermal patches, but also for replacing the injectable route.High-voltage pulses can increase drug permeation within and across skin but are also an efficient tool to permeabilize the membrane of cells of the cutaneous or subcutaneous tissue. This was shown beneficial for targeting cutaneous cells with oligonucleotides or genes and might open new opportunities for gene therapy and DNA vaccination.The safety of the application of high-voltage pulses on skin was assessed in vivo, using histological and visual scores, and bioengineering methods. While changes in skin barrier and function were observed, the irritation was mild and short-lived. Further optimization of the electrode configuration for improved targeting of the stratum corneum should still improve tolerance and levels of sensation.     

Drug delivery across the skin

Since the introduction of the first through the skin (TTS) therapeutic in 1980, a total of 34 TTS products have been marketed and numerous drugs have been tested by more than 50 commercial organisations for their suitability for TTS delivery. Most of the agents which have been tested have had low molecular weights, due to the impermeability of the skin barrier. This barrier resides in the outermost skin layer, the stratum corneum. It is mechanical, anatomical, as well as chemical in nature; laterally overlapping cell multi-layers are sealed by tightly packed, intercellular, lipid multi-lamellae. Chemical skin permeation enhancers increase the transport across the barrier by partly solubilising or extracting the skin lipids and by creating hydrophobic pores. This is often irritating and not always well-tolerated. The TTS approach allows drugs (< 400 kDa in size) to permeate through the resulting pores in the skin, with a short lag-time and subsequent steady-state period. Drug bioavailability for TTS delivery is typically below 50%, avoiding the first pass effect. Wider, hydrophilic channels can be generated by skin poration, with the aid of a small electrical current (> 0.4 mA/cm2) across the skin (iontophoresis) or therapeutic ultrasound (few W/cm2; sonoporation). High-voltage (> 150 V, electroporation) widens the pores even more and often irreversibly. These standard poration methods require experience and equipment and are therefore, not practical; at best, charged/small molecules (≤ 4000 kDa in size) can be delivered efficiently across the skin. In spite of the potential harm of gadget-driven skin poration, this method is used to deliver molecules which conventional TTS patches are unable to deliver, especially polypeptides. Lipid-based drug carriers (liposomes, niosomes, nanoparticle microemulsions, etc.) were proposed as alternative, low-risk delivery vehicles. Such suspensions provide an improved drug reservoir on the skin, but the aggregates remain confined to the surface. Conventional carrier suspensions increase skin hydration and/or behave as skin permeation enhancers. The recently developed carriers; Transferomes, comprise pharmaceutically-acceptable, established compounds and are thought to penetrate the skin barrier along the naturally occurring transcutaneous moisture gradient. Transfersomes are believed to penetrate the hydrophilic (virtual) channels in the skin and widen the former after non-occlusive administration. Both small and large hydrophobic and hydrophilic molecules are deliverable across the stratum after conjugation with Transfersomes. Drug distribution after transdermal delivery probably proceeds via the lymph. This results in quasi-zero order kinetics with significant systemic drug levels reached after a lag-time of up to a few hours. The relative efficiency of TTS drug delivery with Transfersomes is typically above 50 %; with the added possibility of regional drug targeting.     

Drug delivery across the skin

Since the introduction of the first through the skin (TTS) therapeutic in 1980, a total of 34 TTS products have been marketed and numerous drugs have been tested by more than 50 commercial organisations for their suitability for TTS delivery. Most of the agents which have been tested have had low molecular weights, due to the impermeability of the skin barrier. This barrier resides in the outermost skin layer, the stratum corneum. It is mechanical, anatomical, as well as chemical in nature; laterally overlapping cell multi-layers are sealed by tightly packed, intercellular, lipid multi-lamellae. Chemical skin permeation enhancers increase the transport across the barrier by partly solubilising or extracting the skin lipids and by creating hydrophobic pores. This is often irritating and not always well-tolerated. The TTS approach allows drugs (< 400 kDa in size) to permeate through the resulting pores in the skin, with a short lag-time and subsequent steady-state period. Drug bioavailability for TTS delivery is typically below 50%, avoiding the first pass effect. Wider, hydrophilic channels can be generated by skin poration, with the aid of a small electrical current (> 0.4 mA/cm2) across the skin (iontophoresis) or therapeutic ultrasound (few W/cm2; sonoporation). High-voltage (> 150 V, electroporation) widens the pores even more and often irreversibly. These standard poration methods require experience and equipment and are therefore, not practical; at best, charged/small molecules (< or = 4000 kDa in size) can be delivered efficiently across the skin. In spite of the potential harm of gadget-driven skin poration, this method is used to deliver molecules which conventional TTS patches are unable to deliver, especially polypeptides. Lipid-based drug carriers (liposomes, niosomes, nanoparticle microemulsions, etc.) were proposed as alternative, low-risk delivery vehicles. Such suspensions provide an improved drug reservoir on the skin, but the aggregates remain confined to the surface. Conventional carrier suspensions increase skin hydration and/or behave as skin permeation enhancers. The recently developed carriers; Transferomes, comprise pharmaceutically-acceptable, established compounds and are thought to penetrate the skin barrier along the naturally occurring transcutaneous moisture gradient. Transfersomes are believed to penetrate the hydrophilic (virtual) channels in the skin and widen the former after non-occlusive administration. Both small and large hydrophobic and hydrophilic molecules are deliverable across the stratum after conjugation with Transfersomes. Drug distribution after transdermal delivery probably proceeds via the lymph. This results in quasi-zero order kinetics with significant systemic drug levels reached after a lag-time of up to a few hours. The relative efficiency of TTS drug delivery with Transfersomes is typically above 50 %; with the added possibility of regional drug targeting.     

Investigation into the potential of electroporation facilitated topical delivery of cyclosporin a

Purpose: The potential for electroporation-facilitated topical transport of cyclosporin A (CysA) was investigated using rat skin. Methods: Studies of various electrical factors acting on the deposition of CysA into the stratum corneum and deeper skin of in vitro electroporation were performed. We also tested the synergistic effect of electroporation and other approaches such as chemical enhancers and low-frequency ultrasound on topical drug delivery of CysA. Results: Electroporation increased the amount of CysA retained in the skin by only 3 times that of passive diffusion. We found that the efficacy of electroporation in enhancing topical delivery can be further increased by pretreatment of skin with chemical enhancers, such as Azone and menthol. Meanwhile, only a small amount was seen to transport across the full skin into the receiver compartment. Trimodality treatment comprised of pretreatment with Azone and ultrasound in combination followed by electroporation was not effective in enhancing the topical delivery of CysA. However, this combination strategy increased the penetration of CysA through rat skin by an order of 15. Conclusion: In general, the enhanced skin accumulation of CysA by the combination of electroporation and chemical enhancers could help significantly to optimize the targeting of the drug without a concomitant increase in systemic side effects.