Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats

The use of electroporation pulses as a physical means of enhancing the permeability of skin to deliver drugs is in the early stages of development. In this article, a systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to hairless rat skin are reported. It was found that voltage, pulse length (tau), and number of pulses were the three most important parameters, in that order. For creating a significant enhancement in drug delivery to the skin, without causing any apparent change in its external appearance, it was necessary to deliver five or more exponentially decaying electroporation pulses, at 88 +/- 2.5 V (voltage across the skin), with a decay time constant of 20 ms. Electrodes with larger area could attain the same voltages across the skin with a much lower applied voltage and possessed other advantages with regard to performance of the drug delivery system.     

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.     

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.     

Transdermal drug delivery by electroporation applied on the stratum corneum of rat using stamp-type electrode and frog-type electrode in vitro

Transdermal enhancement effects of electroporation applied only on the stratum corneum by two electrode types, the stamp-type electrode and the frog-type electrode, were investigated in vitro using excised rat skin. Carboxyfluorescein (CF) was selected as a model compound. The excised skin was set in a Franz type diffusion cell and a square wave electric pulse was applied to the stratum corneum under various electric pulse conditions. We determined the permeability of CF to the receptor compartment under these conditions. Voltage, electric pulse length, and number of electric pulses, were varied from 10 to 1000 V, 50 micros to 15 ms and 5 to 30 pulses, respectively. Flux rate was enhanced as the electric pulse condition strengthened. However, the maximum value was attained in the flux rate, above which no increase was observed despite strengthening of the electric pulse. Although at low electric pulses, the enhancement effect of the frog-type electrode was superior to that of the stamp-type electrode, the maximum flux rates were the same. These results indicate that electroporation on the stratum corneum using the stamp-type electrode or frog-type electrode, is useful for transdermal drug delivery.     

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.     

Methods for in Vivo Tissue Electroporation Using Surface Electrodes

Electroporation of tissues has many potential biomedical applications, including transdermal and targeted drug delivery. Although there are established protocols for electroporation of single cells, electroporation of tissues remains largely unexplored. Here we describe methods for in vivo tissue electroporation with surface electrodes, where either (a) molecules are provided at the outer surface of skin and electroporation is caused in order to transport molecules across the skin or (b) molecules are injected into an animal and internal tissue cells are electroporated to enhance up-take of the injected molecules. Factors considered in our experimental design include selection of electrode material and electrode/tissue geometry. Electrode materials were used which could accommodate a high instantaneous current density and were expected not to form harmful electrochemical products. Due to the complex heterogeneous nature of tissues, the choice of electrical pulse parameters was guided by, but not based solely on, results from single-cell systems. Multiple pulses appeared to be useful in causing electroporation of tissues located deeper than the skin's stratum corneum. Secondary problems which resulted from the direct muscle stimulation associated with pulsing were also considered. Developing methods for in vivo tissue electroporation required an interdisciplinary approach, involving both high-voltage electric fields and the basic properties of living tissue.     

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相似文献   

Enhanced transdermal delivery of tetracaine by electroporation

The effect of electroporation on the transport of tetracaine through skin in vitro was studied using side-by-side compartment diffusion cells method. After achieving steady state by passive diffusion, fluxes of tetracaine achieved with passive diffusion, electroporative pulse and iontophoresis were compared. Electroporation (square-wave pulse, voltage 130 V, pulse time 0.4 s, pulse frequency 40 pulses min(-1)) or iontophoresis (0.2.mA cm(-2), lasting for 4 h) increased the transport of tetracaine through skin. The flux of tetracaine at 0.25 h after electroporation (pulse number 400) was 54.6+/-6.0 microg.cm(-2).h(-1), that after iontophoresis was 17.4+/-5.8 microg.cm(-2).h(-1) and that after passive diffusion was 8.2+/-0.5 microg.cm(-2).h(-1). In addition, the fluxes of tetracaine increased with the increasing of pulse number. From these results, it is clear that electroporation is effective in enhancing transdermal delivery of tetracaine and its function is better than iontophoresis.     

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.     

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.     

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.     

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.     

Gene expression and antitumor effect following imelectroporation delivery of human interferon α2 gene

AIM: To investigate the gene expression and antitumor effect following im electroporation delivery of human interferon alpha 2 (hIFN-alpha 2) gene. METHODS: The pcD2/hIFN-alpha 2 was injected into the middle of the quadriceps muscle of female BALB/c mice or the leukemia-bearing female BALB/c nude mice, and then electroporation was given to the injection site. Optimal electrical parameters and the efficiency of gene transfer was studied with hIFN-alpha 2 ELISA kit. The HL-60 tumor model in BALB/c nude mice was used to investigate therapeutic effects of im electroporation delivery of pcD2/hIFN-alpha 2. RESULTS: The optimal conditions for the electric pulses were as follows: voltage at 200 V/cm; pulse duration at 40 ms per pulse; number of pulse at 6 pulses and frequency at 1 Hz. Under optimal conditions, the serum hIFN-alpha 2 levels in electroporation group (160 microg/L+/-31 microg/L) were 45-fold higher than those of nonelectroporation group (3.6 microg/L+/-1.6 microg/L, P<0.01). The growth of leukemia was inhibited more obviously and the survival time of the leukemia-bearing nude mice was prolonged after im electroporation delivery of pcD2/hIFN-alpha 2 100 microg or 200 microg. CONCLUSION: Electroporation was an efficient method for the delivery of plasmid DNA and im electroporation delivery of pcD2/hIFN-alpha 2 was effective in treating leukemia.     

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.     

Electronically facilitated transdermal delivery of human parathyroid hormone (1-34)

Electronically facilitated transdermal delivery of human parathyroid hormone (1-34), hPTH (1-34), was investigated in vitro, using dermatomed porcine skin. The effect of iontophoretic current density, electroporative pulse voltages and also electroporation followed by iontophoresis was investigated on the in vitro percutaneous absorption of hPTH (1-34). Iontophoresis at 0.5 mA/cm2 current density significantly enhanced (P<0.05) the flux of hPTH (1-34) in comparison to passive flux. Electroporation pulses of 100, 200 and 300 V significantly increased (P<0.05) the flux of hPTH (1-34) in comparison with the passive as well as iontophoretic flux at 0.5 mA/cm2. The electroporative flux of hPTH (1-34) was found to vary linearly (R2 = 0.97) with the pulse amplitude. The principal barrier of the skin, stratum corneum, was found perturbed following the pulses as evident by light microscopy studies. The application of electroporation pulses followed by iontophoresis further increased the flux by several fold. The flux of hPTH (1-34) with the electroporation pulses of 100 and 300 V followed by iontophoresis at 0.2 mA/cm2 was 10- and 5-fold higher, respectively, in comparison to the flux with corresponding pulses alone. This shows the synergistic effect of iontophoresis in combination with electroporation on skin permeability of hPTH (1-34). The results indicate the possibility of designing controlled transdermal delivery systems for hPTH (1-34) using electroporation followed by iontophoresis.     

Mechanisms of a phosphorothioate oligonucleotide delivery by skin electroporation

Skin electroporation has great potential for topical delivery of oligonucleotides. Controled therapeutic levels of an intact phosphorothioate oligonucleotide (PS) can be reached in the viable tissue of the skin. The aim of this work was to investigate the transport mechanisms of a PS in hairless rat skin by electroporation, and hence to allow optimization of oligonucleotides (ONs) topical delivery. The pulsing condition used was five exponentially-decaying pulses of 100 V and 500 ms pulse time. The main mechanism of PS transport in the skin viable tissues during pulsing was electrophoresis. The electroosmosis contribution was negligible. Electrophoresis created within minutes a reservoir of PS in the skin viable tissues, which persisted within a therapeutic range of hours. A strong PS/stratum corneum interaction occurred.     

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.     

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.     

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.     

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.