Painless skin electroporation as a novel way for insulin delivery.

BACKGROUND: Rigorous research efforts have been undertaken worldwide to develop a needle-free insulin delivery for many decades with limited success. This translational study aims to deliver insulin through skin with painless electroporation. METHODS: A recently designed microelectrode array was used to deliver insulin in mice with diabetes under electroporation conditions that are painless and harmless on human skin. RESULTS: Under such condition, a therapeutic amount of insulin was delivered successfully through mouse skin. Electroporation alone increased insulin transport around 100-fold compared with passive diffusion. Increased skin temperature to 40°C for 20 min augmented insulin transport to 237-fold more than the control value. Repeated electroporation showed no harm on human skin. CONCLUSION: The results indicate the potential of painless delivery of insulin through human skin in future clinical practice.

Enhancing transdermal drug delivery with electroporation.

The application of electroporation to enhance transdermal delivery has opened up a new possibility to introduce larger molecules such as peptide hormones and vaccines as well as minigenes and RNAi etc. through the transdermal route. Many devices have been developed to deliver the pulse electric field needed to permeate the skin. These devices include both non-puncturing surface electrodes as well as puncturing electrodes of different geometrical arrangements. The latter type uses electroporation only to increase uptake of molecules injected through the puncturing electrode or syringe. Different electroporation protocols have been developed to maximize transport, uptake and minimizing pain. Synergistic effect of chemical enhancers and physical (sonic, vibrational and thermal) treatments are used to enhance the transport. This article reviews the patents pertaining to the instrumentation as well as application protocols of transdermal delivery, uptake enhancement and interstitial fluid sampling by electroporation.

Overview of drug delivery and alternative methods to electroporation.

This chapter provides an overview of the application of electroporation to areas other than gene delivery. These areas include the delivery of drugs and vaccines to tissues and tumors as well as into and through the skin. Achievements and limitations of electroporation in these areas are presented. Alternative physical methods for gene and drug delivery besides electroporation are described. The advantages and drawbacks of electroporation, compared with these methods, are also discussed.

Influence of DMPS on the water retention capacity of electroporated stratum corneum: ATR-FTIR study.

Anionic lipids like phosphatidylserine are known to significantly enhance electroporation mediated transepidermal transport of polar solutes of molecular weights up to 10kDa. The underlying mechanism of the effect of anionic lipids on transdermal transport is not fully understood. The main barrier to transdermal transport lies within the intercellular lipid matrix (ILM) of the stratum corneum (SC) and our previous studies indicate that dimyristoyl phosphatidylserine (DMPS) can perturb the packing of this lipid matrix. Here we report on our investigation on water retention in the SC following electroporation in the presence and the absence of DMPS. The water content in the outer most layers of the SC of full thickness porcine skin was determined using ATR-FTIR-spectroscopy. The results show that in the presence of DMPS, the SC remains in a state of enhanced hydration for longer periods after electroporation. This increase in water retention in the SC by DMPS is likely to play an important role in trans-epidermal transport, since improved hydration of the skin barrier can be expected to increase the partitioning of polar solutes and possibly the permeability.

Resealing of electroporation of porcine epidermis using phospholipids and poloxamers.

The resealing of porcine epidermis after electroporation is investigated. Porcine epidermis was subjected to electroporation (30 pulses at 100 V, 1 ms and at 1 Hz) in a vertical diffusion apparatus, in the presence of 2 mg/ml dimyristoylphosphatidylserine, to produce a long lasting permeable state. Resealing treatments include incubation in 0.0625-0.25 mM poloxamer 188 (P188), or incorporation of phosphatidylcholines (PC) and/or cationic lipids with additional pulses. The recovery of electric resistance of the epidermis samples after electroporation with or without resealing treatments was monitored. The transports of carboxyfluorescein and glucose were measured during the recovery process. Both P188 and PC were effective in resealing in terms of electric conductance and transport, with P188 reacting more rapidly and completely. P188 mediated lipid exchange between stratum corneum lipid particles was measured by fluorescence resonance energy transfer (FRET). Lipid reorganization facilitated by P188 and PC is suggested to be a major resealing mechanism of electroporation damage.

Synergistic effect of anionic lipid enhancer and electroosmosis for transcutaneous delivery of insulin.

A lipid formulation consisting of 1,2-dimyristoyl-sn3-phosphatidylserine (DMPS) in a 0.2% sodium dodecylsulfate (SDS) solution was tested as an in vivo enhancer for the transcutaneous delivery of insulin. The formulation when applied to for 15 min was found to permeabilize porcine epidermis and prolong the permeable state as evidenced by electric resistance measurement. The formulation enhanced the transport of insulin through the epidermis by 40- to 100-fold, as compared to epidermis that was treated with SDS or DMPS alone. Application of electroosmosis across the formulation-treated epidermis enhanced the transport of insulin by an additional 10-fold. Pharmacokinetic studies were carried out in Sprague-Dawley rats. Transcutaneous delivery of insulin with formulation treatment and electroosmosis increased the plasma level of insulin by approximately 10-fold over delivery by formulation treatment alone. With the above protocol plasma insulin concentration remained relatively constant for up to 4h. The synergistic application of anionic lipid formulation and electroosmosis offers a promising non-invasive technique to deliver insulin transcutaneously.

Lipid and electroosmosis enhanced transdermal delivery of insulin by electroporation.

Transdermal transport of insulin and extraction of interstitial glucose under anodal iontophoresis (electroosmosis) following electroporation in the presence of 1,2-dimyristoylphophatidylserine (DMPS) was studied. An earlier study showed that DMPS increased the transport of insulin across porcine epidermis under electroporation by approximately fourfold. It was suggested that DMPS increased the lifetime of electropores in the epidermis resulting in an enhanced transport of permeants. When electroosmosis was applied across the epidermis following electroporation with DMPS, the enhancement of insulin transport was approximately 18-fold over electroporation alone. When the same strategy was applied to extract interstitial glucose, the enhancement was approximately 23-fold over electroporation alone. Real-time transdermal insulin transport kinetics was measured using FITC-labeled insulin and a custom-made vertical diffusion apparatus that had a fluorescence cuvette as the receiver compartment. Insulin transport by electroporation alone showed a nonlinear kinetics that is most likely due to the resealing of the electropores with time. The transport kinetics when electroporation was carried out in the presence of DMPS was more linear, confirming earlier studies that suggested the DMPS stabilizes transport paths formed by electroporation. The data suggests that in vivo, noninvasive insulin delivery to therapeutic levels and glucose extraction may be achieved by combining electroporation with anionic lipids and electroosmosis.

Painless electroporation with a new needle-free microelectrode array to enhance transdermal drug delivery.

A microelectrode array was designed to minimize the pain sensation of electroporation for enhancing transdermal drug delivery. The influence of the size of the electrode-skin contact area and of the distance between electrodes on the pain sensation was tested on human volunteers. The pain level decreased with the dimension of electrode-skin contact area and with inter-electrode distance. When both reached about 0.5 mm, the pain level was not perceptible even at the threshold of transdermal electroporation level of sixty electric pulses at 150 V, 1 ms at 1-10 Hz. An array of 11 x 11 alternately connected electrodes with 0.6 x 0.6 mm dimension was fabricated. The electric thresholds for effective drug delivery, using toluidine blue O as a marker on mouse skin, was found to be the same for microelectrode arrays as for larger electrodes and wider inter-electrode distances. In vivo transdermal electroporation using microelectrode array with 180 pulses of 150 V, 0.2 ms at 1 Hz, followed by 30 min methotrexate (MTX) occlusion increased more than 4 fold the systemic MTX level in mice. The results demonstrated the potential of painless delivery of significant amounts of chemotherapeutic agents through skin with the new electrode arrays in a clinical setting.

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of beta-amyloid.

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimer's patients measured by the Alzheimer's disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimer's disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.

Electroporation and transcutaneous extraction (ETE) for pharmacokinetic studies of drugs.

The therapeutic activity and toxicity of drugs often depends on the accumulation of drugs in the peripheral anatomical compartment rather than the central compartment. In the routine practice of therapeutic drug monitoring (TDM) and pharmacokinetic studies, drug concentration determined by intermittent blood sampling is used as a surrogate for calculating the drug concentration in the peripheral compartment tissues. Microdialysis, a relatively less invasive procedure, has been used for estimation of free drug levels in dermal, subcutaneous and muscle tissues. Transcutaneous extraction of drugs from the dermal tissue is a good noninvasive alternative to phlebotomy and microdialysis. This requires a technique, which can facilitate the extraction of significant and reproducible amounts of drugs from the dermal extracellular fluid (ECF) within a short sampling duration. In the present work, we assessed the feasibility of electroporation and transcutaneous extraction (ETE) method for determining the time course of drugs in dermal ECF, using salicylic acid (SA) as a test drug. Electroporation protocol was optimized based on the in vitro diffusion studies of salicylic acid across rat skin. The concentration-time profile of total SA was determined in rats after a single i.v. bolus administration. The in vivo permeability coefficient (P(in vivo)) of rat skin was determined under steady state plasma concentration of drug created by i.v. bolus followed by constant rate infusion of SA. The pharmacokinetic parameters of the drug were determined using a two-compartment pharmacokinetic model. The theoretical predicted time course of free SA in the dermal ECF after a single i.v. bolus administration was calculated using standard formulae. The concentration of free SA determined by ETE is in good agreement with that calculated using two-compartment pharmacokinetic model. This study thus provides a credible evidence for the validity of ETE technique for determining the concentration of SA in the dermal ECF.

Cyclodextrin enhanced transdermal delivery of piroxicam and carboxyfluorescein by electroporation.

The transdermal transport of cyclodextrins (CD) across porcine epidermis by electroporation was studied. Electroporation increased the permeation of beta-cyclodextrin (BCD) and hydroxy propyl beta-cyclodextrin (HPCD) by several orders of magnitude, relative to passive transport. The presence of BCD and HPCD enhanced the total transport of the test permeants piroxicam and carboxyfluorescein (CF), respectively, from both permeant solutions and suspensions. BCD enhanced the fraction of piroxicam transported across the epidermis into the receiver compartment medium. This was most likely due to the prolonged post-pulse permeability state of the epidermis. The fraction of CF retained in the epidermis was increased by HPCD. The rate of diffusion of CF from epidermis into the receiver compartment was decreased by the presence of HPCD, apparently due to the aggregate forming tendency of HPCD. The in vivo delivery of CF by electroporation in mice demonstrated the potential of HPCD for sustaining the transdermal absorption rate of hydrophilic molecules.

Surfactant-enhanced transdermal delivery by electroporation.

The objective of the experiment was to study the influence of sodium dodecyl sulfate (SDS) on transdermal transport of diffusants by electroporation. The resistance of porcine epidermis in contact with SDS solution (0.2% w/v) dropped by 40% within 24 h. SDS improved the efficiency of transdermal delivery of glucose, dextrans of molecular weight (MW) 4 kDa (FD4K) and 10 kDa (FD10K) by electroporation. However, the transport of dextran MW 35 kDa (FD35K) was not influenced significantly. Pretreatment of epidermis with SDS solution reduced its electroporation threshold from 80 to 60 V. It appears that presence of SDS during electroporation helps in achieving the desired transport with less electrical exposure dose. SDS enhanced the transdermal delivery of molecules by electroporation most likely by facilitating the barrier disruption during pulse application and also by prolonging the lifetime of electropores created by the pulse.

Temperature influences the postelectroporation permeability state of the skin.

The influence of temperature on the electrical conductance and transport of macromolecules across porcine epidermis during and after electroporation were studied. The passive diffusion of fluorescein isothiocyanate labeled dextran (molecular weight 10 kDa, FD10K), across the epidermis did not differ much at temperatures below 37 degrees C, but became significantly higher above 40 degrees C. The resistance drop during pulse application was less sensitive to temperature within the temperature range (10-50 degrees C) of this study. The kinetics of decrease in postpulse conductance of the electroporated epidermis was fit to a monoexponential function. The rate of decrease in postpulse conductance was significantly less and FD10K transport was markedly high at temperature over 40 degrees C relative to those observed at temperatures less than 37 degrees C. This jump in transport cannot be explained by electrophoresis induced by the pulse, or by the increased diffusion kinesis of the molecules. The enhanced transport is most likely due to the prolonged postpulse permeable state of the skin. Electroporation at mild hyperthermia temperatures resulted in delivering much higher quantities of macromolecules.

pH influences the postpulse permeability state of skin after electroporation.

The pH dependence of porcine epidermis permeability and the influence of pH on the electroporation transport of molecules were studied. The resistance was maximum at pH 5 and decreased with an increase or decrease in the pH of the donor medium. The permeability coefficient of glucose was significantly higher at pH 7.5, compared to pH 5. On electroporation, the resistance recovery rate of porcine epidermis was rapid below pH 5 and slower at above pH 7.5. The transport studies revealed that a donor medium pH above 7.5 helps to maintain the postpulse permeability state of the skin. By changing the donor medium pH from 5 to 7.5, the postpulse transport of glucose and fluorescein isothiocyanate (FITC)-labeled dextran (MW 10 kDa) (FD10K) was enhanced by about threefold. The lipids extracted from porcine epidermis showed pI values of 4.3 and 5.9. Vesicles of these lipids fused more rapidly at pH 5 than at pH 3, 7, and 10. The results imply that pH-sensitive postpulse resistance recovery and molecular transport are due to the charge states of epidermal lipids.

Localized delivery to CT-26 tumors in mice using thermosensitive liposomes.

A heat-sensitive liposomal drug delivery system was tested using Colon-26 (CT-26) cultured cells and tumors in mice. Lucifer yellow iodoacetamide (LY) was used as a fluorescence marker. The heat-sensitive liposomes exploit the temperature-dependence of critical micellar concentrations of the poloxamer, F127. LY release from unilamellar liposomes at different temperatures was measured. Onset of LY release occurred near 33 degrees C, and reached plateau above 42 degrees C when 90% of the LY was released. Temperature-treated liposomes were mixed with CT-26 cells to measure the binding of the released LY to cell surface. Temperature-dependency of cell-bound LY corresponds to the release curve. CT-26 tumors were grown subcutaneously in both hind legs of Balb/c mice. Mice received heat-sensitive or plain liposomes via tail vein injections, or no liposomes. For each mouse, one tumor was kept at 31.5 degrees C, while the counterlateral tumor was heated to 42 degrees C during injection and for 30min after. LY released in tumors was determined from fluorescence intensity. Tumors receiving heat-sensitive liposomes plus heat treatment showed 2.5-fold greater fluorescence than all other tumors, which were at the background level. This study demonstrates the possible use of poloxamer-containing liposomes as a heat-sensitive drug delivery system in vivo.

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 microg/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.

Enhanced transdermal transport by electroporation using anionic lipids.

Transdermal drug delivery is an attractive approach for either local or systemic treatment in medicine. In the last decade, different active transdermal delivery methods have been further investigated such as cationic liposomal delivery and electroporation-enhanced delivery. In light of gaining a synergistic effect of lipid and electroporation, a new method of using anionic lipids to enhance the transdermal transport of molecules under electroporation is reported here. Heat-stripped porcine epidermis was used for measurement of transdermal transport using an in vitro vertical diffusion apparatus. Lipid vesicles were prepared using a 1:1 mole ratio mixture of 1,2-dioleoyl-3-phosphatidylglycerol (DOPG) and 1,2-dioleoyl-3-phosphatidylcholine (DOPC). When the lipids were mixed with (but not encapsulating) the transport target molecule, the electroporation-induced transport through porcine epidermis was increased as compared to that without the lipids. The enhancement in transport was dependent upon the size and the charge of the transported molecule. Methylene blue (MB), protoporphyrin IX (PpIX) and dimethyl-protoporphyrin IX (DM-PpIX) were used as small target molecules, and FITC-dextrans (4 to 155 kDa) were used as large target molecules in our studies. Enhancement of transport, to varying degree, was observed for all three small molecules (molecular weights <1 kDa), in the presence of DOPG:DOPC vesicles. In the case of large molecules, lipid-enhanced transport was only observed for the 4 kDa dextran, and not for the larger ones (M(w)>10 kDa). Neutral or cationic lipids alone did not enhance the transdermal transport under the electroporation conditions we used.

Transdermal insulin delivery using lipid enhanced electroporation.

Transdermal insulin transport by electroporation was measured using porcine epidermis and fluorescein-labeled insulin. Previous studies have shown that anionic lipids can enhance the electroporative transport of molecules up to 10 kDa in size. It was also shown that it is the charge and not the type of the phospholipid head group that influences transdermal transport under electroporation. Moreover, phospholipids with saturated acyl chains enhance the transport of larger molecules more as compared to those with unsaturated chains. In the current study, based on those earlier findings, the effect of 1,2-dimyristoyl-3-phosphatidylserine (DMPS) on the transdermal transport of insulin by electroporation was examined. Porcine epidermis was used as a model for skin. Transport was measured using glass vertical diffusion apparatus in which the epidermis separated the donor and receiver compartments. Negative pulses were applied across the epidermis using platinum electrodes. Results show that when electroporation was carried out in the presence of DMPS, there was greater than 20-fold enhancement of insulin transport. Furthermore, while in the presence of the phospholipid, almost all the transported insulin was detected in the receiver compartment; in the absence of added lipids, only about half the insulin transported was in the receiver compartment and an almost equal amount of insulin remained in the epidermis. Fluorescence microscopy revealed that the insulin transport was mainly through the lipid multilayer regions that surround the corneocytes.

Electrically enhanced percutaneous delivery of delta-aminolevulinic acid using electric pulses and a DC potential.

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the stratum corneum. We used electric pulses to increase transdermal transport of delta-aminolevulinic acid (ALA), a precursor to the photosensitizer protoporphyrin IX (PpIX). ALA-filled electrodes were attached to the surface of excised porcine skin or the dorsal surface of mice. Pulses were administered and, in some in vivo cases, a continuous DC potential (6 V) was concomitantly applied. For in vitro 14C ALA penetration, 10 microm layers parallel to the stratum corneum were assayed by liquid scintillation analysis, and 10 microm cross sections were examined autoradiographically. As the electrical dose (voltage x frequency x pulse width x treatment duration) increased, there was an increase in penetration depth. In vivo delivery was assayed by measuring the fluorescence of PpIX in skin samples. A greater than two-fold enhancement of PpIX production with electroporative delivery was seen versus that obtained with passive delivery. Superimposition of a DC potential resulted in a nearly three-fold enhancement of PpIX production versus passive delivery. Levels were higher than the sum of PpIX detected after pulse-alone and DC-alone delivery. Electroporation and electrophoresis are likely factors in electrically enhanced delivery.

Utilizing temperature-sensitive association of Pluronic F-127 with lipid bilayers to control liposome-cell adhesion.

The temperature sensitive properties of Pluronic F-127 (MW approximately 12600, PEO(98)-PPO(67)-PEO(98)), a block co-polymer or poloxamer, was used to control liposome-cell adhesion. When associated with liposomes, the PEO moiety of the block co-polymer is expected to inhibit liposome-cell adhesion. Liposomes were made using egg phosphatidylcholine and different mole% of Pluronic F-127. Size measurement of the liposomes at different temperatures, in the presence and absence of Pluronic F-127, shows significant reduction in the size of multilamellar vesicles, at higher temperatures, by the Pluronic molecules. Negative stain electron microscopy study showed the presence of individual molecules and micelles of Pluronic, respectively at temperatures below and above the critical micellar temperature (CMT). Measurement of the surface associated Pluronics indicated that they associated with liposomes when the sample was heated above the Pluronic CMT, and dissociated from liposomes when cooled below the CMT. Attachment of the Pluronic containing liposomes to CHO cells was inhibited at temperatures above the CMT, but not at temperatures below CMT, indicating that temperature-sensitive control of liposome-cell adhesion is achieved.