浙江大学 文本 Skin electroporation for transdermal and topical delivery_生理学

Aaableonlineatmwnesciencedlretcon SCIENCE@DIRECT” DRUG DELVERY .om/ Skin electroporation for transdermal and topical delivery Anne-Rose Denet,Rita Vanbever,Veronique Preat* AhsirS aehsehgo2a日wiaautoame

Skin electroporation for transdermal and topical delivery Anne-Rose Denet, Rita Vanbever, Ve´ronique Pre´at* Unite´ de Pharmacie Gale´nique, Universite´ Catholique de Louvain, Avenue E. Mounier, 73 UCL 7320, 1200 Brussels, Belgium Received 9 September 2003; accepted 13 October 2003 Abstract 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. D 2004 Elsevier B.V. All rights reserved. Keywords: Electroporation; Skin permeability; Transdermal delivery; Topical delivery; Mechanism; Safety Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2. Mechanism of transdermal drug transport by electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2.1. Electroporation of the skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2.2. Pathways of transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2.2.1. New aqueous pathways or electropores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2.2.2. Localization of transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 2.3. Mechanisms of molecular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662 2.3.1. Electrophoretic movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 2.3.2. Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 2.3.3. Electroosmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 3. Parameters controlling drug delivery by electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 3.1. Electrical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 3.1.1. Pulse waveform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663 3.1.2. Pulse voltage, duration, number and rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664 3.1.3. Pulsing protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664 3.1.4. Electrode design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2003.10.027 * Corresponding author. Tel.: +32-2-764-7309; fax: +32-2-764-7398. E-mail address: preat@farg.ucl.ac.be (V. Pre´at). www.elsevier.com/locate/addr Advanced Drug Delivery Reviews 56 (2004) 659 – 674

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3.2. Physicochemical properties of the drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664 3.2.1. Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 3.2.2. Lipophilicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 3.2.3. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 3.2.4. Formulation of drug reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 4. Potential clinical applications of skin electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666 4.1. Transdermal drug delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666 4.2. Topical drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667 5. Combinations of enhancing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668 5.1. Electroporation and chemical enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668 5.2. Electroporation and ultrasound. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668 5.3. Electroporation and iontophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668 6. Safety issues associated with skin electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670 7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671 1. Introduction Transdermal drug delivery offers several advan￾tages over conventional routes [1,2]. It avoids the first-pass metabolism and the gastrointestinal tract. Transdermal delivery has the potential for sustained and controlled drug release. Moreover, it is a non￾invasive mode of drug delivery with no trauma or risk of infection. Patient compliance may be improved by this user-friendly method. In spite of the advantages of the transdermal deliv￾ery, only a small percentage of drugs can be delivered transdermally due to the barrier properties of the skin: only small potent lipophilic drugs can be delivered at therapeutic rates by passive diffusion [3]. Moreover, transport of most drugs across the skin is very slow and lag-times to reach steady state fluxes are in hours. Achievement of a therapeutically effective drug level, is therefore, difficult without enhancing skin perme￾ation. A number of approaches have been developed to enhance and control transport across the skin, and expand the range of drugs delivered. These involve chemical and physical methods, based on two strate￾gies: increasing skin permeability and/or providing a driving force acting on the drug [4,5]. Electroporation or electropermeabilization is the transitory structural perturbation of lipid bilayer mem￾branes due to the application of high voltage pulses. This phenomenon occurs in different kinds of lipid bilayer membranes: artificial (liposomes), cellular (bacteria, yeast, plant, mammalian cell) or in a more complex structure (stratum corneum). Hence, electro￾poration has been used for different applications. Elec￾trical exposures typically involve electric field pulses that generate transmembrane potentials of 0.5– 1.0 V and last for 10 As to 10 ms. Reversible electrical breakdown and high molecular transport are observed, resulting from structural rearrangements of the cell membrane [6]. It has been hypothesized that these rearrangements consist of temporary aqueous path￾ways, with the electric field inducing pore formation and providing a local driving force for molecular transport. The first use of electroporation was to introduce some DNA materials into cells in vitro. Due to its application as a method of DNA transfection, electroporation has been applied in tissues (e.g. for gene therapy) and shown to reversibly permeabilize them. One interesting application of tissue electropora￾tion is electrochemotherapy, which consists of apply￾ing high voltage pulses to permeabilize tumor cells to an impermeable cytotoxic drug [7]. Electrochemother￾apy has been shown to be more efficient than the chemotherapy alone in eliminating local tumors, e.g. in the skin [8]. About 10 years ago, the use of electroporation for transdermal delivery was suggested (Fig. 1) [9]. Al￾though the technique is normally used on the unila￾mellar phospholipid bilayers of cell membranes, it has been demonstrated that electroporation of skin is fea￾sible, even though the stratum corneum contains multi￾lamellar, intracellular lipid bilayers with few phos￾pholipids [9 – 12]. Hence, electroporation has taken its place among the physical techniques of transdermal drug delivery, like ultrasound and iontophoresis. 660 A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674

A-Rm时W/franred Drag Prinery见nw5矫050-674 66 102 crease in transport has becn observed for transdermal voltages of 30-100 V (100-1500 V applied vol- 10 tages),which well corresponds to the range of vol- 0 tages used for electroporation in cells,i.e.0.3-1.0 V per bilayer [9.15]. 10 New aqueous pathways would be created within 10 the stratum corneum due electroporation of its lipid 10 bilayers [6.12.16].Molocular transport through tran- 击金金血金 siently permeahilized skin then occurs due to different 100 280300 400 500 mechanisms,mainly by eloctrophoresis and enhanced Volraga [volts) diffusion [10,16,17].Thermal effects may be involved [11,181. Fig.I.Aerge tronsderal fuves of calcein due to exposue of hunan skn to dfferent elctrical condior:application ot torward. 2.2.Pathw3a时transport polariry pelses (and I h atter pulsing in the peverse direction (A).Doror solutionc cakein I mM in PBS barter.Froms Prausniz et al.91. 2.2.1.New agucous patinmys or electropores Electroporation of lipid bilayers induces a dramatic and reversible increase in tmansmembrane transport The cancept of skin electroparation and the sup and structural changes in membrane barrier 9.Mod- porting preliminary data have motivated a number of els to explain these dramatic changes in memhrane suhsequent studies,mainly in vitro hut nlso a few in properties associaled with high voltage application vivo in animals and in humans.The combination of invol小e the creation of“pocs“oraq0 oous pathways. electroporation with ocher enhancement methads open Evidence for the creation of these pores is still entirely new perspectives (5.13.14]. indireet.The pores are believed to be small (<10 nm). In this paper,studies on transdermal and topical sparse (0.1%of surface area),and genemlly short- drug delivery using electroporation are reviewed with lived (us to s)[12,19,20]. emphasis on potential clinical applications.Efficacy Cousistent with the electroporation features of of tmnsport hy skin electroporation alone or in com- single lipid bilayers,experimental and theoretical bination with other methods and safety issues are data support the hypothesis that applicmtion of high discussed voltage pukses to skin also induces the creation of new and/or the enlargement of existing aqueous pathwnys in the stratum corneum [12].The skin 2.Mechanism of transdermal drug transport by resistance drops by several orders of magnitude clectroporation during pulsing and is partly reversible.In vitro transport increases by up to four orders of magnitude 2.1.Electroporation of the skin compared to passive diflusion.The eflective fraction- al area for small ian transport during electroporation Because the stratum comeum is the main bamer to is approximately 0.1%[21-231 For the same amount transdermal transport,the disnuption of the stratum of transferred charges,the dng transport is higher for corneum can dramatically influence overall skin per electroporation than iontophoresis,suggesting that meability and it has been suggested that clectopora- clcctropboresis alone cannot cxplain drug transport tion of its intercellular lipid hilayers might enhance by skin electroporation and that skin stncture must percutaneous drug delivery.The biological composi- be altered. tion and structure of the stratum comeum.the outer- most layer of the skin,make it particularly attractive 2.2.2.Loculizution of framnspor! for electroporation.The stratum comeum contains Whatever the electroporation protocol used,the approximately 100 bilayer membranes in series,and transdermal transport by high voltage pulses has been clectrical breakdown associated with dramatic in- shown to occur through highly localized transport

The concept of skin electroporation and the sup￾porting preliminary data have motivated a number of subsequent studies, mainly in vitro but also a few in vivo in animals and in humans. The combination of electroporation with other enhancement methods open new perspectives [5,13,14]. In this paper, studies on transdermal and topical drug delivery using electroporation are reviewed with emphasis on potential clinical applications. Efficacy of transport by skin electroporation alone or in com￾bination with other methods and safety issues are discussed. 2. Mechanism of transdermal drug transport by electroporation 2.1. Electroporation of the skin Because the stratum corneum is the main barrier to transdermal transport, the disruption of the stratum corneum can dramatically influence overall skin per￾meability and it has been suggested that electropora￾tion of its intercellular lipid bilayers might enhance percutaneous drug delivery. The biological composi￾tion and structure of the stratum corneum, the outer￾most layer of the skin, make it particularly attractive for electroporation. The stratum corneum contains approximately 100 bilayer membranes in series, and electrical breakdown associated with dramatic in￾crease in transport has been observed for transdermal voltages of 30 – 100 V (100 – 1500 V applied vol￾tages), which well corresponds to the range of vol￾tages used for electroporation in cells, i.e. 0.3– 1.0 V per bilayer [9,15]. New aqueous pathways would be created within the stratum corneum due electroporation of its lipid bilayers [6,12,16]. Molecular transport through tran￾siently permeabilized skin then occurs due to different mechanisms, mainly by electrophoresis and enhanced diffusion [10,16,17]. Thermal effects may be involved [11,18]. 2.2. Pathways of transport 2.2.1. New aqueous pathways or electropores Electroporation of lipid bilayers induces a dramatic and reversible increase in transmembrane transport and structural changes in membrane barrier [9]. Mod￾els to explain these dramatic changes in membrane properties associated with high voltage application involve the creation of ‘‘pores’’ or aqueous pathways. Evidence for the creation of these pores is still entirely indirect. The pores are believed to be small (<10 nm), sparse (0.1% of surface area), and generally short￾lived (As to s) [12,19,20]. Consistent with the electroporation features of single lipid bilayers, experimental and theoretical data support the hypothesis that application of high voltage pulses to skin also induces the creation of new and/or the enlargement of existing aqueous pathways in the stratum corneum [12]. The skin resistance drops by several orders of magnitude during pulsing and is partly reversible. In vitro transport increases by up to four orders of magnitude compared to passive diffusion. The effective fraction￾al area for small ion transport during electroporation is approximately 0.1% [21 – 23]. For the same amount of transferred charges, the drug transport is higher for electroporation than iontophoresis, suggesting that electrophoresis alone cannot explain drug transport by skin electroporation and that skin structure must be altered. 2.2.2. Localization of transport Whatever the electroporation protocol used, the transdermal transport by high voltage pulses has been shown to occur through highly localized transport Fig. 1. Average transdermal fluxes of calcein due to exposure of human skin to different electrical conditions: application of forward￾polarity pulses (n) and 1 h after pulsing in the reverse direction (E). Donor solution: calcein 1 mM in PBS buffer. From Prausnitz et al. [9]. A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674 661

662 L-几Dener e!a./4ae0 r Deliwery Bevins 5②uS级6T4 regions (LTR)of the stratum comeum,covering pares are possihle pathways for electrical current at hetween 0.02%nnd 25%of the skin surface [21,24]. moderate voltage [27.28].The permeahilizing effect of The permeabilization of the stratum comeum is not electroporation on epidermal cells could be potentially homogenoous within a LTR The current density and interesting for a targeted topical treatment [29,31). hence dnig transport is maximal at the center of the The molecular weight is another parameter influ- LTR.Moreover,it has been reported that LTR are encing the route of transport:the smaller the molec- surounded by localized dissipation regions (LDR) ular weight.the more intraccllular the penetration. nresenting a low resistivity where transport of only Lamhry et al.[30]showed that FITC molecules (low small ions can occur (Fig.2)25].The sice and the molecular weight)penetrated in the keratinocytes. number of the LTR are dependent on the pulsing while FITC-dextran molecules of 38 kDa was mainly protocol.The size of LTR increasod with the duration around the keratinceytes with only a small fraction and the mumber of pulses,and it ranged between 0.1 entering the cell and 0.2-2.5 mm in diameter for short and long pulses, Electroporation of skin is associated with a tem- respectively.In contrast,the number of LTR increased perature rise within the highly LDR.When an electric with the pulse voltage:while LTR number ranged field is applied to the skin,it dissipates energy and between 2 and 10 per 0.1 cm'for long duration beats up (Joule heating).This local temperature rise medium voltage pulses,it increasod to 20-100 per may affect the barrier function of the skin if the cm2 when short duration high voltage pulses were temperature rise induces a phase change in the applied [24-26] sphingo-lipids of the stratum comeum,increasing Within a LTR,molecular transport appears to occur the permeability of'skin.Indeed,dramatic decrease through intercellular andor transccllular pathways of the skin resistance occurs when 65-70 C are 21,24,25].The contributian of each transport is de. reached [18,32].As revealed hy temperature sensitive pendent on the pulse voltage.While molecular trans- crystals,during the pulse,the temperature did not rise port seems to he transcellular in the case of short high instantaneously over the entire skin surface but started voltage pulses,it seems to be intercellular with possi at small spots,resulting in a propagating heat front ble implication of appendages when decreasing the [11,18).Hence,the interior of the LTR was heated voltage and lengthening the pulse duration [25,26]. above the phase transition temperanre,while the I.DR Theoretical models suggest that appendageal macro- was also hemted but did not reach the phase transition temperature.As the transdermal voltage and pulse time constant increased the temperature tended to a LTR plateau but did not reach the phase transition of the water [33].During cooling,the multilamellar system LDR could not re-establish.leaving the existence of water- rich domains,which provided aqueous pathways even long after the pulse [18.33J. The biological significance of heating of the stra- tum comeum above the phase transition temperalure of sphingo-lipids is not yet understood.However,in vivo experiments using hairless rats showed no sig 2红* nificant skin irritation for short and long pulses [341 2 2.3.Mechanisms of molecular transport nig.2.lealod dawing of the ouer byers of the skin.showing the hypothetical stmcure of a LTk cotained within a LDR.The LTR is Molecular trasport through transiently permeahi- mepeeserted hy a cylindrical region.with a thickness of the SC d-0-2泊m)ard a rdfus价m-l0-1 which is rny lized skin by electroporation results from different dependemt en high veltage pulee duration.The larger concemric mechanisms at different times.Enhanced diffusion, ylinder npnes an ideli1D设，whoe in alo depend on during and after pulses,and clectrically driven trans- pube length From Pliguett ct al [25] port during pulses,i.e.electmophoretic movement and

regions (LTR) of the stratum corneum, covering between 0.02% and 25% of the skin surface [21,24]. The permeabilization of the stratum corneum is not homogeneous within a LTR. The current density and hence drug transport is maximal at the center of the LTR. Moreover, it has been reported that LTR are surrounded by localized dissipation regions (LDR), presenting a low resistivity where transport of only small ions can occur (Fig. 2) [25]. The size and the number of the LTR are dependent on the pulsing protocol. The size of LTR increased with the duration and the number of pulses, and it ranged between 0.1 and 0.2 –2.5 mm in diameter for short and long pulses, respectively. In contrast, the number of LTR increased with the pulse voltage: while LTR number ranged between 2 and 10 per 0.1 cm2 for long duration medium voltage pulses, it increased to 20– 100 per cm2 when short duration high voltage pulses were applied [24 – 26]. Within a LTR, molecular transport appears to occur through intercellular and/or transcellular pathways [21,24,25]. The contribution of each transport is de￾pendent on the pulse voltage. While molecular trans￾port seems to be transcellular in the case of short high voltage pulses, it seems to be intercellular with possi￾ble implication of appendages when decreasing the voltage and lengthening the pulse duration [25,26]. Theoretical models suggest that appendageal macro￾pores are possible pathways for electrical current at moderate voltage [27,28]. The permeabilizing effect of electroporation on epidermal cells could be potentially interesting for a targeted topical treatment [29,31]. The molecular weight is another parameter influ￾encing the route of transport: the smaller the molec￾ular weight, the more intracellular the penetration. Lombry et al. [30] showed that FITC molecules (low molecular weight) penetrated in the keratinocytes, while FITC – dextran molecules of 38 kDa was mainly around the keratinocytes with only a small fraction entering the cell. Electroporation of skin is associated with a tem￾perature rise within the highly LDR. When an electric field is applied to the skin, it dissipates energy and heats up (Joule heating). This local temperature rise may affect the barrier function of the skin if the temperature rise induces a phase change in the sphingo-lipids of the stratum corneum, increasing the permeability of skin. Indeed, dramatic decrease of the skin resistance occurs when 65 – 70 jC are reached [18,32]. As revealed by temperature sensitive crystals, during the pulse, the temperature did not rise instantaneously over the entire skin surface but started at small spots, resulting in a propagating heat front [11,18]. Hence, the interior of the LTR was heated above the phase transition temperature, while the LDR was also heated but did not reach the phase transition temperature. As the transdermal voltage and pulse time constant increased, the temperature tended to a plateau but did not reach the phase transition of the water [33]. During cooling, the multilamellar system could not re-establish, leaving the existence of water￾rich domains, which provided aqueous pathways even long after the pulse [18,33]. The biological significance of heating of the stra￾tum corneum above the phase transition temperature of sphingo-lipids is not yet understood. However, in vivo experiments using hairless rats showed no sig￾nificant skin irritation for short and long pulses [34]. 2.3. Mechanisms of molecular transport Molecular transport through transiently permeabi￾lized skin by electroporation results from different mechanisms at different times. Enhanced diffusion, during and after pulses, and electrically driven trans￾port during pulses, i.e. electrophoretic movement and Fig. 2. Idealized drawing of the outer layers of the skin, showing the hypothetical structure of a LTR contained within a LDR. The LTR is represented by a cylindrical region, with a thickness of the SC (dsc=10 – 20 Am) and a radius (rLTR=10 – 100 Am), which is strongly dependent on high voltage pulse duration. The larger concentric cylinder represents an idealized LDR, whose sizes also depend on pulse length. From Pliquett et al. [25]. 662 A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674

A.-R Dener e al.Adnanced Drg Delivery Bevtews 16 (2004)639-674 663 very slightly electroosmosis.are the main mechanisms of transpart.The contribution of electrophoresis and Panenclers afclig drug Inmspurt by skin clectnpuratitet (alapod diffusion are dependent on the physicocbemical prop- from Priat and Vurbever [8) erties of the molecule. Paramneters Increase in Elecmrical Pelse volage + 2.3.1.Electrophoretic movemenr parameers Pelse number Pelse lengh During high voltage pulses,the main driving force Phosicochemcal Charee for trarsport of charged molecules is electrophoresis pmperties of drug Molecelar weight [9.10,35,36].Evidence for this major contribution of Confoemation electrophoresis is the drop in the drug transport with Lpop山cy reverse electrode polarity oppasing electropboresis Focraul山rked Compeutive ions of drug reservoir 16a2ha通pd Vicosry 2.3.2.Dse t.Positive effect:-regative effect Molecular transport through skin highly permeahi- lized by electroporation is also due to enhanced passive diffusion.Although much higher skin perme- ability is achieved during the pulse,prolonged per square wave),voltage (50-1500 V),duration (few meabilization and thereby transport occur after us to ms).and interval between pulses (few s to min). pulsing.lasting for hours inin vitro studses.Evidences These electricnl parmmeters can be optimized,depend- for the contribution of enhanced post-pulse diffusion ing on experiment requirements and clinical applica- arise from the increased transport seen (i)with reverse tion of clectroporation [35]. coctrode polarity,(u)with neutral molecules.and (ii) when the drug is ndded afier applicntion of the pulses 3.1.1.Pukse waveform 35-37J Two kinds of pulse generators are usually em ployed to transport molecules by clectroporation: 23.3.0Hs there are generators which can deliver exponentially In contrast with iontophoresis,the contribution of decaying pulses [9,10.39]or square wave pulses electroosmosis during high voltage pulses is low.The [7.8.40].Both are used for different applications. short time of current application (fw s)limits the role ie.drug delivery,electrochemotherapy and gene of electmosmosis in dnag transport hy skin electmo- therapy.Due to its long voltage tail profile,the main poration.Further evidence comes from the similar potential advantages of exponentially decaying anodic and cathodic transport of neutral molocules pulses are to maintain or expand the high permeabil- 371 ity state of the skin induced by electroporation and to promote the electrophoretic movement.However, because the duration of exponential decay pulses 3.Parameters controlling drug delivery by depends on the resistance of the skin and the electro clectroporation poration system (electrodes,conducting modium),the reproducibility of the pulse conditions for clinical use Electrical parameters of the pulses.physicochem- could be a problem.In contrast,voltage and duration ical properties of the drug and formlation of the drug of square wave pulses remain constant whatever the reservoir can aflect and allow control of transdermal skin or drug reservoir.Hence.square wave pulses drug delivery by electroporation.These parameters should be used to have a better control and repro- are summarized in Tahle 1. ducibility of drug transport.Until recently,the squre wave pulses were exclusively used for electrochemo- 3.1.Electrical parameters therapy and DNA electrotransfer.whereas exponen- tially decaying pukes were restricted to transdermal Electrical pulses are characterized hy their electri. drug delivery to take advantage of the long voltage cal parameters:waveform (exponential decay or tail

very slightly electroosmosis, are the main mechanisms of transport. The contribution of electrophoresis and diffusion are dependent on the physicochemical prop￾erties of the molecule. 2.3.1. Electrophoretic movement During high voltage pulses, the main driving force for transport of charged molecules is electrophoresis [9,10,35,36]. Evidence for this major contribution of electrophoresis is the drop in the drug transport with reverse electrode polarity opposing electrophoresis. 2.3.2. Diffusion Molecular transport through skin highly permeabi￾lized by electroporation is also due to enhanced passive diffusion. Although much higher skin perme￾ability is achieved during the pulse, prolonged per￾meabilization and thereby transport occur after pulsing, lasting for hours in in vitro studies. Evidences for the contribution of enhanced post-pulse diffusion arise from the increased transport seen (i) with reverse electrode polarity, (ii) with neutral molecules, and (iii) when the drug is added after application of the pulses [35 – 37]. 2.3.3. Electroosmosis In contrast with iontophoresis, the contribution of electroosmosis during high voltage pulses is low. The short time of current application (few s) limits the role of electroosmosis in drug transport by skin electro￾poration. Further evidence comes from the similar anodic and cathodic transport of neutral molecules [37]. 3. Parameters controlling drug delivery by electroporation Electrical parameters of the pulses, physicochem￾ical properties of the drug and formulation of the drug reservoir can affect and allow control of transdermal drug delivery by electroporation. These parameters are summarized in Table 1. 3.1. Electrical parameters Electrical pulses are characterized by their electri￾cal parameters: waveform (exponential decay or square wave), voltage (50 –1500 V), duration (few As to ms), and interval between pulses (few s to min). These electrical parameters can be optimized, depend￾ing on experiment requirements and clinical applica￾tion of electroporation [35]. 3.1.1. Pulse waveform Two kinds of pulse generators are usually em￾ployed to transport molecules by electroporation: there are generators which can deliver exponentially decaying pulses [9,10,39] or square wave pulses [7,8,40]. Both are used for different applications, i.e. drug delivery, electrochemotherapy and gene therapy. Due to its long voltage tail profile, the main potential advantages of exponentially decaying pulses are to maintain or expand the high permeabil￾ity state of the skin induced by electroporation and to promote the electrophoretic movement. However, because the duration of exponential decay pulses depends on the resistance of the skin and the electro￾poration system (electrodes, conducting medium), the reproducibility of the pulse conditions for clinical use could be a problem. In contrast, voltage and duration of square wave pulses remain constant whatever the skin or drug reservoir. Hence, square wave pulses should be used to have a better control and repro￾ducibility of drug transport. Until recently, the square wave pulses were exclusively used for electrochemo￾therapy and DNA electrotransfer, whereas exponen￾tially decaying pulses were restricted to transdermal drug delivery to take advantage of the long voltage tail. Table 1 Parameters affecting drug transport by skin electroporation (adapted from Pre´at and Vanbever [38]) Parameters Increase in Effect Electrical Pulse voltage + parameters Pulse number + Pulse length + Physicochemical Charge + properties of drug Molecular weight
Conformation ? Lipophilicity
Formulation Competitive ions
of drug reservoir Ionization (pH) + Viscosity
+, Positive effect;
, negative effect. A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674 663

66科 A-:Dent r ml Adianred Tirvg Delwry Bruinx 56 (M0)550-674 3.1.2.Pulse vollage.duration.namber and rale 3.1.3.Pulsing protocol Control on drug transport by skin electro- Two dillerent t少pes of pulsing protocols are usual山y poration can be achieved by controlling the pulse reported in the literature.They can mainly he distin- voltage,duration,number and rate.The effect of guished by the pulse duration:(i)numerous (>100), varying these clectrical parameters on transdermal short duration (1-2 ms),high voltage pulses:and (ii) transport has heen extensively studied in vitro a low numher (<20),long duration (70-1000 ms) 9,10.2935,41-431 medium voltage pulses.In the case of exponentially Because significant voltage drop occurs within docaying pulses,at the same total electrical charge the electroporation system,the transdermal voltage transported through the skin,a few long pulses is only a fraction (ca.10-50%)of the voltage allowed generally higber molecular transport than applied across the electrodes,depending of the many sbort pulses [26]. relative resistance of the skin and the drug reservoir. Flx rate always enhances when electrical pulse 3.1.4.Elecmode dexign conditions strengthen:when the number,the voltage The design of'the electrodes is still a critical issue. or the duration of the pulses increase,the transder- both in terms of efficacy of drug transport and mal drug transport increased (see Figs.I and 3). tolerance.The early research on skin electroporation With inxcreasing voltage of pulses,the transdermal was performed in vitro with electrodes placed on both flux increases but less steeply at high voltages sides of the skin.If insights on transdermnl dmg [9.35].When the pulse duration and pulse numbers delivery were gained,the position and design of the increases the drg transport often linearly increases electrodes were not representative of in vivo condi- (10J.Increasing the pulse rate increases transdermal tions.Various electrodes and reservoir systems,e.g. flux as well [10,26,41]. plate clectrodes with a skin fold [31.44].meander The electrical parameters influence transdermal electrodes [44],have been designed for in vivo ux but also onset time for transport,which decreases applications.The efficacy of drug transport is inllu- with increasing pulse duration and rate,but is inde- eneed by the electrode design because the distnhution pendent of voltage [26,41]. and intensity of the electrical field in the skin are affocted [45,461.The simplest configuration to gener- ate a more or less uniform electric field is parallel plate electrodes in the form of calipers.However. umderlying nerves and muscles could he subjected to clectrical stimalus and superficial skin burning could 14 he observed.The meander electrodes oonsist of nn 14 1210 130 array of interweaving electrode fingers,allowing the electric field to be mostly kcalizod within the super- U 60 ficial layers of the skin,thereby avoiding undesirahle effects in underlying tissues. 400 As the reaction is extremely fast,inert electrodes, e.g.platinum,are preferred to active electrodes,e.g. silver/silver chloride electmdes.As oxydoreduction occurs at the electrodes,hydrogen and hydroxyl ions 150 Palse vohage CV) 10 are produced and could lead to a pH shift in the Pulse Time (ns reservoir. 10 pulves 3.2.Physicochemical properties of the drug Fig.3.Effict of pube vohage and tine on fenmonyl tramdemal tarsport by electroporatioa.The plot presents values calculated 0maep0gu的xeGq12n0a0 btzined by13a1dcs知 In addition to the electrical pammeters of the pulses, Donoc solutio:北ay40μel■a cmrate butt红Q01MNl51. the physico-chemical properties of drug can affect the From Varbever et aL.35L transdermal drug delivery by clectroporation

3.1.2. Pulse voltage, duration, number and rate Control on drug transport by skin electro￾poration can be achieved by controlling the pulse voltage, duration, number and rate. The effect of varying these electrical parameters on transdermal transport has been extensively studied in vitro [9,10,29,35,41 –43]. Because significant voltage drop occurs within the electroporation system, the transdermal voltage is only a fraction (ca. 10 – 50%) of the voltage applied across the electrodes, depending of the relative resistance of the skin and the drug reservoir. Flux rate always enhances when electrical pulse conditions strengthen: when the number, the voltage or the duration of the pulses increase, the transder￾mal drug transport increased (see Figs. 1 and 3). With increasing voltage of pulses, the transdermal flux increases but less steeply at high voltages [9,35]. When the pulse duration and pulse numbers increases the drug transport often linearly increases [10]. Increasing the pulse rate increases transdermal flux as well [10,26,41]. The electrical parameters influence transdermal flux but also onset time for transport, which decreases with increasing pulse duration and rate, but is inde￾pendent of voltage [26,41]. 3.1.3. Pulsing protocol Two different types of pulsing protocols are usually reported in the literature. They can mainly be distin￾guished by the pulse duration: (i) numerous (>100), short duration (1 – 2 ms), high voltage pulses; and (ii) a low number (<20), long duration (70 – 1000 ms), medium voltage pulses. In the case of exponentially decaying pulses, at the same total electrical charge transported through the skin, a few long pulses allowed generally higher molecular transport than many short pulses [26]. 3.1.4. Electrode design The design of the electrodes is still a critical issue, both in terms of efficacy of drug transport and tolerance. The early research on skin electroporation was performed in vitro with electrodes placed on both sides of the skin. If insights on transdermal drug delivery were gained, the position and design of the electrodes were not representative of in vivo condi￾tions. Various electrodes and reservoir systems, e.g. plate electrodes with a skin fold [31,44], meander electrodes [44], have been designed for in vivo applications. The efficacy of drug transport is influ￾enced by the electrode design because the distribution and intensity of the electrical field in the skin are affected [45,46]. The simplest configuration to gener￾ate a more or less uniform electric field is parallel plate electrodes in the form of calipers. However, underlying nerves and muscles could be subjected to electrical stimulus and superficial skin burning could be observed. The meander electrodes consist of an array of interweaving electrode fingers, allowing the electric field to be mostly localized within the super￾ficial layers of the skin, thereby avoiding undesirable effects in underlying tissues. As the reaction is extremely fast, inert electrodes, e.g. platinum, are preferred to active electrodes, e.g. silver/silver chloride electrodes. As oxydoreduction occurs at the electrodes, hydrogen and hydroxyl ions are produced and could lead to a pH shift in the reservoir. 3.2. Physicochemical properties of the drug In addition to the electrical parameters of the pulses, the physico-chemical properties of drug can affect the transdermal drug delivery by electroporation. Fig. 3. Effect of pulse voltage and time on fentanyl transdermal transport by electroporation. The plot presents values calculated from a response surface equation obtained by factorial design. Donor solution: fentanyl 40 Ag/ml in a citrate buffer (0.01 M, pH 5). From Vanbever et al. [35]. 664 A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674

A-R Dmef ef al.Asanced Drug Deivery Peviens 56 (2004)659-674 665 32.1.Charge nalbuphine and its prodmugs were similar but the Becmuise the electrophoretic movement is the main enhancement ratio decreased as the lipophilicity in- mechanism of transport for charged molecules creased [43]. through a highly permeabilized skin by eloctropora- tion,the pa of the drug and the pH of the delivery 3.2.3.Moiecular weighr solution are essential parameters influencing the elec- Another physicochemical property of the drug tric charges of the molecule to deliver.Increasing the influencing the transdemmal transport by skin electro- charge of the permeant enhances its tramsport.Hence, poration is its molecular weight.Using FITC-dextran the pH of the drug reservoir,which afliects drug of'increasing molecular weight,Lombry et al.[30 ionization,will influence the efficscy of drug delivery. showed that significant transport and intracellular The transport of nutral molecules is also enhanced by penetration in the skin were detected after high electroporation,due to passive diffusion through the voltage pulse application (Fig.4).The greater the permeabilized skin [37J.This transport of neutral molecular size,the lower the transdermal transport. molccules is lower,compared to the transport of The absence of molecular weight cut off (at least up to charged molecules during pulses. 40 kDa)suggested that electroporation could be useful At physiological conditions,the skin is negatively for macromolecule delivery. charged,presenting a better permselectivity to the cations.However,due to the short duration of current 3.2.4.Formulation of drug reservoir application,the contrihution of electroosmosis is lim- Because the drug concentration affects the trans. ited [37].suggesting that the skin permselectivity is dermal transport of drug by electroporation.the choice not as important as for iontophoretic transport. of drug concentration in the reservoir could allow control an drug delivery.The higher the drug concen. 3.2.2.Lipophilicity tration,the higher the transport.However,a pon-linear The influence of the partition coefficient of the relationship between the quantity of drug delivered in permeant has not been systematically investigated.In the skin and the drug concentration of the reservoir contrast to passive diffusion.increasing the lipophi- has been reported [29,47). licity of the dnig tends to decrease the enhancement The ions present in the dnig reservoir (hufter inns, ratio.The transdermal fluxes of nalbuphine and lipo- counter ions.ions from the skin)competc with the philic prodrugs were enhanced hy electroporation,as delivered drug for the electrophoretic movement. compared to passive diffusion.The total amount of Hence,the seloction and optimization of the reservoir FIIC 11D44 Molecules Flus (pmctcazh) FITC 5,6±491 Fm4.4 108.25±1668 FD12 18,30±330 FD 38 20.52±063 (h) Fig 4.Camelative and of FITC and FITC-dextran (FT)nf increasing malecubr uright (44.12 and 38 kDal in he cepur martmsa fincti ofme,afer exporetially deraying eleimpurticn (1ples of 150V.150 m)doner ltinn )50 pM Fwm【nmhry ct al B0g

3.2.1. Charge Because the electrophoretic movement is the main mechanism of transport for charged molecules through a highly permeabilized skin by electropora￾tion, the pKa of the drug and the pH of the delivery solution are essential parameters influencing the elec￾tric charges of the molecule to deliver. Increasing the charge of the permeant enhances its transport. Hence, the pH of the drug reservoir, which affects drug ionization, will influence the efficacy of drug delivery. The transport of neutral molecules is also enhanced by electroporation, due to passive diffusion through the permeabilized skin [37]. This transport of neutral molecules is lower, compared to the transport of charged molecules during pulses. At physiological conditions, the skin is negatively charged, presenting a better permselectivity to the cations. However, due to the short duration of current application, the contribution of electroosmosis is lim￾ited [37], suggesting that the skin permselectivity is not as important as for iontophoretic transport. 3.2.2. Lipophilicity The influence of the partition coefficient of the permeant has not been systematically investigated. In contrast to passive diffusion, increasing the lipophi￾licity of the drug tends to decrease the enhancement ratio. The transdermal fluxes of nalbuphine and lipo￾philic prodrugs were enhanced by electroporation, as compared to passive diffusion. The total amount of nalbuphine and its prodrugs were similar but the enhancement ratio decreased as the lipophilicity in￾creased [43]. 3.2.3. Molecular weight Another physicochemical property of the drug influencing the transdermal transport by skin electro￾poration is its molecular weight. Using FITC – dextran of increasing molecular weight, Lombry et al. [30] showed that significant transport and intracellular penetration in the skin were detected after high voltage pulse application (Fig. 4). The greater the molecular size, the lower the transdermal transport. The absence of molecular weight cut off (at least up to 40 kDa) suggested that electroporation could be useful for macromolecule delivery. 3.2.4. Formulation of drug reservoir Because the drug concentration affects the trans￾dermal transport of drug by electroporation, the choice of drug concentration in the reservoir could allow control on drug delivery. The higher the drug concen￾tration, the higher the transport. However, a non-linear relationship between the quantity of drug delivered in the skin and the drug concentration of the reservoir has been reported [29,47]. The ions present in the drug reservoir (buffer ions, counter ions, ions from the skin) compete with the delivered drug for the electrophoretic movement. Hence, the selection and optimization of the reservoir Fig. 4. Cumulative quantities and fluxes of FITC and FITC – dextran (FD) of increasing molecular weight (4.4, 12 and 38 kDa) detected in the receptor compartment as a function of time, after exponentially decaying electroporation (10 pulses of 150 V, 150 ms), donor solution: 250 AM. From Lombry et al. [30]. A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674 665

666 L-R.Deer ea./A小ed dirvg Delvery Reve4s塔200s5人-6T4 composition must take into account the drug joniza- molecules.for charged (e.g.heparin 50)and neutral tion (pl),the pl shift induced with inert electrodes (e.g.mannitol [37])malecules (Table 2). (presence of a buffer required).the presence of com- The in vitro transport of'drugs by skin electro- petitive ions (ionic strength and composition of the porntion has heen shown to increase by up to four solution)and the conductivity (high conductivity orders of magnitude with lag-times of'only seconds to compared to the skin).Hence,the composition is a minutes,indicating that molccules rapidly respond to critical parameter to enhance drug transport.Even electric pulses.The enhancement ratio and the onset though theoretical inputs help in selecting appeopriate time for transport deperd on the electncal parameters pH (drug ionization)buffer composition,buffer ca- of the pulses and the physicochemienl properties of pacity (number of charged transferred)and buffer the drug as well as on the experimental model (eg. conductivity,the optimization of the reservoir formu- skin model).Hence,the control of drug trnsport by lation remains a“trials and errors”optimixation skin electroporation can be obtained by the selection [1035.401 of these parameters. An increase of the viscosity of the drug solution The in vitro features of transport have been shown has been reported to decrease drug transport by skin to translate in vivo,in support of the potential thera- electroporation[4刀 peutic applications.In vivo data have especially Tabke 2 4.Potential clinical applications of skin In vitro smdies on transdemal drug delvery by electroporanion clectroporation adapted from Preat and Vnhever [38]1 Comnpnd Mulecube Chanpe 1a呢 Refirenoes The dranaie and reversible increase in skin per- 陆口UH cluromait rlis meahility caused by clectroporation indicates that drugs might be delivered transdermally at significant. Water 18 3 Manninol 182 0 [37 ly enhanced rates.Especially for macromolecules. Atrnolnl 66 2 [51 such as proteins and gene-hased dngs,electropora- Metoprolnl 67 + [1047] tion-mediated transdermal drug delivery could be a Tdncane 301 -1 [52☒ promising route of administration.Electroporation can Alnisan 302 ++2 [4☒ Timndlal 316 +l 2 [4可 also be used for topical delivery. Mahy lne blu: 320 +L+2 [5可 Extensive work on molecular transport by skin Fentityl 336 *1 2 [173匀 electroporation has been performed in vitro.Essential N自yaik 350 -1 [54 features of transport include (i)high fluxes for many L气1u0g different compoun点，(mpidly responsive mole. Nalhaphine t57 +l 4 FITC 0 - [间 lar transport and (iii)modulation of transport by Dmperidone 42 +1 [5句 controlling the electrical parameters and the physico- 【nrifer yellw 457 -2 [5句 chemical praperties of drug and reservoir. Tenocain 460 [575周 s04 [5 4.1.Transdermal dnrg delivery Sulfeehuamnin 607 -1 222656 Calein 623 -4 4 9,22,s6 Erythnoin 1025 -1 4 明 Application of high voltnge pulses has heen shown dervatie to increase trarsport across andor into the skin for Cyclosporine A 1201 0 60 compounds ranging in size (i)from small.e.g.fen- Salmon 3600 +1 49 tanyl,timolol [35,40];(i)to moderated-sized mole. calo0▣ 000 cules,e.g.calcein 9]:(ii)to macromolecules,e.g. Dextren suirne Highby 2 61 Hepann 12.00 Highby 2 150 LHRH,calcitonin,heparin,FITC-dextran up to 40 Delisnioe 50 Hihly [6] kDa [30,48-50).Orders of magnitude increase in FITC-dexinn 4-38.000 30U transport has also heen reported for lipophilic (e.g. Nuo- 10am- Hiahly- 216364 timolol [401)and bydrophilic (e.g.metoprolol [10]) n起ophc4S山m

composition must take into account the drug ioniza￾tion (pH), the pH shift induced with inert electrodes (presence of a buffer required), the presence of com￾petitive ions (ionic strength and composition of the solution) and the conductivity (high conductivity compared to the skin). Hence, the composition is a critical parameter to enhance drug transport. Even though theoretical inputs help in selecting appropriate pH (drug ionization), buffer composition, buffer ca￾pacity (number of charged transferred) and buffer conductivity, the optimization of the reservoir formu￾lation remains a ‘‘trials and errors’’ optimization [10,35,40]. An increase of the viscosity of the drug solution has been reported to decrease drug transport by skin electroporation [47]. 4. Potential clinical applications of skin electroporation The dramatic and reversible increase in skin per￾meability caused by electroporation indicates that drugs might be delivered transdermally at significant￾ly enhanced rates. Especially for macromolecules, such as proteins and gene-based drugs, electropora￾tion-mediated transdermal drug delivery could be a promising route of administration. Electroporation can also be used for topical delivery. Extensive work on molecular transport by skin electroporation has been performed in vitro. Essential features of transport include (i) high fluxes for many different compounds, (ii) rapidly responsive molecu￾lar transport and (iii) modulation of transport by controlling the electrical parameters and the physico￾chemical properties of drug and reservoir. 4.1. Transdermal drug delivery Application of high voltage pulses has been shown to increase transport across and/or into the skin for compounds ranging in size (i) from small, e.g. fen￾tanyl, timolol [35,40]; (ii) to moderated-sized mole￾cules, e.g. calcein [9]; (iii) to macromolecules, e.g. LHRH, calcitonin, heparin, FITC – dextran up to 40 kDa [30,48 – 50]. Orders of magnitude increase in transport has also been reported for lipophilic (e.g. timolol [40]) and hydrophilic (e.g. metoprolol [10]) molecules, for charged (e.g. heparin [50]) and neutral (e.g. mannitol [37]) molecules (Table 2). The in vitro transport of drugs by skin electro￾poration has been shown to increase by up to four orders of magnitude with lag-times of only seconds to minutes, indicating that molecules rapidly respond to electric pulses. The enhancement ratio and the onset time for transport depend on the electrical parameters of the pulses and the physicochemical properties of the drug as well as on the experimental model (e.g. skin model). Hence, the control of drug transport by skin electroporation can be obtained by the selection of these parameters. The in vitro features of transport have been shown to translate in vivo, in support of the potential thera￾peutic applications. In vivo data have especially Table 2 In vitro studies on transdermal drug delivery by electroporation (adapted from Pre´at and Vanbever [38]) Compound Molecular weight Charge Log enhancement ratio References Water 18 0 1 [37] Mannitol 182 0 2 [37] Atenolol 266 +1 2 [51] Metoprolol 267 +1 3 [10,47] Tetracaine 301
1 1 [52] Alnitidan 302 +1/+2 2 [42] Timolol 316 +1 2 [40] Methylene blue 320 +1/+2 [53] Fentanyl 336 +1 2 [17,35] Na nonivamide acetate 350
1 1 [54] Nalbuphine 357 +1 1 [43] FITC 390
1 [30] Domperidone 426 +1 2 [55] Lucifer yellow 457
2 4 [56] Terazosin 460 [57,58] Buprenorphine 504 +1 1 [59] Sulforhodamine 607
1 3 [22,26,56] Calcein 623
4 4 [9,22,56] Erythrosin derivative 1025
1 4 [9] Cyclosporine A 1201 0 1 [60] Salmon calcitonin 3600 +1 2 [49] Dextran sulfate 5000 Highly
2 [61] Heparin 12,000 Highly
2 [50] Defibrase 36,000 Highly
[62] FITC – dextran 4 – 38,000
[30] Nano￾microspheres 10 nm – 45 Am Highly

[21,63,64] 666 A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674

A-Rm时w/franed Drag Pviner是nw5矫050-674 66 underlined the enhanced transport and the rapid onset Table 3 for transport.In the first in vivo study,calcein Topical delivery of drags and mocmomnolsales ato the skin tsing clectroporarion transport by skin electroporation was assessed in rats serum:fluxes of calcein were greater than two arders Penan Mudd Skin Refinens of magnitude than the control (Fig.1)[9].Another in Vianin C In virro Hirman [6同 vivo stuady using hairless rats showed that fentanyl Lidecaine In vin n [6 rapidly responded to electric pulses:very rapid trans- Cycnsorine A In vimo Hairies r [6 Nliprudatide In viro Hairies r [369,0 dermal delivery of fentanyl (within 15 min)at thera- Amigens In vin Mire 7 peutic level were obtained by skin electroporation, Ovalbumin In vin Mire 72 inducing a deep analgesia lasting for about an hour DNA (plaamid In vho Mice.rat 31.73] (g.5)[65]- The in vitro and preclinical in vivo data inxdicate that electroporation could he used to enhance trans- skin can also enhance the permeahility of the cells dermal drug delivery and expand the range of com- underlying the stratum comneum.The first evidence pounds delivered to hydrophilic,charged and even for this cellular permeabilization came from eloctro- macromolecular substances.Compared to other trans- chemotherapy that consists in treating subcutaneous dermal delivery methods,electroporation could be tumor by the injection of a noa-permeant cyloloxic more adapted for a rapid and pulsed delivery andor bleomycin followed hy the local application of high macromolecule delivery. voltage pulses to permeabilize the oells exposod to the electrical field [7].Hence,to target impermeable 4.2.Topical drg delivery substances to the keratinocytes or to dermal cell. electroporation could be particularly promising.spe- The rationale for using electroporatian for topical cially for macramolecules (see Table 3).Enhancement delivery is based on the permeabilizing effect of high of topical delivery of both lipophalic and hydrophilic voltage pulses on lipid bilayers:besides the perme- dnugs by electroporation has been nchieved.Electro- abilization of the main skin barrier,the stratum poration enhanced by one order of magnitude the corneum.application of high voltage pulses to the topical delivery of cyclosporine A formulated as a coevaporate to increase its water solubility 68 It also increased by the quantity vitamin C in the skin with an efficacy depending on the formulation [66]. The topical delivery of macromolecules can be enhanced by electroporntion.Electrical parameters of pulses and oligonucleotide concentration control the amount of oligonucleotide delivered by clectropora- tion in the viahle tissue of the skin 36,69].Unlike ionlophoresis,which does not permeabilize keratino- cytes,electroporation induces a rapid delivery of oligonucleotide in the keratinocytes [69).Electropora- tion also enhances the topical delivery of DNA in the epidermis,inducing an intracellular delivery of the plasmid within several hours and an enhanced gene Time (h) expression (see Fig.6)[31].The expression of the reported gene in the epidermis lasted for 7 days. Fig.5.Featiny plasma concenrations as a tunetion of time after As the skin is an immuno-competent organ,im- trasdemal delvery usig clectroporadion.Electropornou was munizalion through intraderal or topical route are camed out using 15 expouentially docaying pulses of 250 Vand 200 ms.applied ttem time 0 to 5 ma in hairless rat skin n vivo.Foams at under investigation.Passive diffusion of an antigen the cathode and anode were soaked with a soltion of fentanyl (400 applied topically elicits an immune response when an ug'ml in a citrabe buffer 001 M at pll 5)From Varbever et aL [651. adjuvant such as cholera toxin is used 74.Eloctro-

underlined the enhanced transport and the rapid onset for transport. In the first in vivo study, calcein transport by skin electroporation was assessed in rats serum: fluxes of calcein were greater than two orders of magnitude than the control (Fig. 1) [9]. Another in vivo study using hairless rats showed that fentanyl rapidly responded to electric pulses: very rapid trans￾dermal delivery of fentanyl (within 15 min) at thera￾peutic level were obtained by skin electroporation, inducing a deep analgesia lasting for about an hour (Fig. 5) [65]. The in vitro and preclinical in vivo data indicate that electroporation could be used to enhance trans￾dermal drug delivery and expand the range of com￾pounds delivered to hydrophilic, charged and even macromolecular substances. Compared to other trans￾dermal delivery methods, electroporation could be more adapted for a rapid and pulsed delivery and/or macromolecule delivery. 4.2. Topical drug delivery The rationale for using electroporation for topical delivery is based on the permeabilizing effect of high voltage pulses on lipid bilayers: besides the perme￾abilization of the main skin barrier, the stratum corneum, application of high voltage pulses to the skin can also enhance the permeability of the cells underlying the stratum corneum. The first evidence for this cellular permeabilization came from electro￾chemotherapy that consists in treating subcutaneous tumor by the injection of a non-permeant cytotoxic bleomycin followed by the local application of high voltage pulses to permeabilize the cells exposed to the electrical field [7]. Hence, to target impermeable substances to the keratinocytes or to dermal cells, electroporation could be particularly promising, spe￾cially for macromolecules (see Table 3). Enhancement of topical delivery of both lipophilic and hydrophilic drugs by electroporation has been achieved. Electro￾poration enhanced by one order of magnitude the topical delivery of cyclosporine A formulated as a coevaporate to increase its water solubility [68]. It also increased by the quantity vitamin C in the skin with an efficacy depending on the formulation [66]. The topical delivery of macromolecules can be enhanced by electroporation. Electrical parameters of pulses and oligonucleotide concentration control the amount of oligonucleotide delivered by electropora￾tion in the viable tissue of the skin [36,69]. Unlike iontophoresis, which does not permeabilize keratino￾cytes, electroporation induces a rapid delivery of oligonucleotide in the keratinocytes [69]. Electropora￾tion also enhances the topical delivery of DNA in the epidermis, inducing an intracellular delivery of the plasmid within several hours and an enhanced gene expression (see Fig. 6) [31]. The expression of the reported gene in the epidermis lasted for 7 days. As the skin is an immuno-competent organ, im￾munization through intradermal or topical route are under investigation. Passive diffusion of an antigen applied topically elicits an immune response when an adjuvant such as cholera toxin is used [74]. Electro￾Fig. 5. Fentanyl plasma concentrations as a function of time after transdermal delivery using electroporation. Electroporation was carried out using 15 exponentially decaying pulses of 250 V and 200 ms, applied from time 0 to 5 min in hairless rat skin in vivo. Foams at the cathode and anode were soaked with a solution of fentanyl (400 Ag/ml in a citrate buffer 0.01 M at pH 5). From Vanbever et al. [65]. Table 3 Topical delivery of drugs and macromolecules into the skin using electroporation Permeant Model Skin References Vitamin C In vitro Human [66] Lidocaine In vivo Human [67] Cyclosporine A In vitro Hairless rat [68] Oligonucleotide In vitro Hairless rat [36,69,70] Antigens In vivo Mice [71] Ovalbumin In vivo Mice [72] DNA (plasmid) In vivo Mice, rat [31,73] A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674 667

66s A-R.Dener et al.Adhanced Drag Delivery Revies 56 (2004)559-674 dermal delivery,an effective chemical enhancers for electroporation does not need to dismpt lipids.but should stahilize the transient disruptions created by electroporation The hypothesis for the combination of these twa methods is to create enlarged aqueous path- ways and/or to prolong the lifetime of'the electropores. Vanbever et al.76]and Weaver et al.78]showed that macromolecules (heparin or dextran-sulfate)increxsed transdermal transport of mannitol by electroporation. No enhancement was observed during passive diffu- sion or iontophoresis,suggesting that macromolecules F.五灯plane CLSM on shwing the lasalicalion of the interact specifically with transport pathways created at florescent bbelled plagnid in the epidermis 8 h following skin high voltage.The persistent low post-pulse electrical ekctmeomtion (10x1 V.10 s)The imoge uas acquired at resistance could result from the insertion of these linear dph12lmth脚s年r6 e of gripped skin Scale hir50m macromolecules in the aqueous pathways.Zewert et From Duipinlin et al.B1]. aL.75 showed that significant macromolecule trans- dermal fluxes occurred when sodium thiosulfate was poration was tested to achieve a needle free,adjuvant present,supporting the hypothesis that enlarged aque free and non-invasive immunization with antigen.As ous pathways or microconduits were created allowing it increases the penetration of antigens into the skin, large quantities of macromolecules to be transported an clectrically-assisted delivery of antigens,e.g.myr- through human skin.Anionic phospholipids were istilated peptide,dipheleria toxoid and ovalhumin, found to enhance the transdermal transport of FITC- elicites a higher antigen specifie IgG response in the dextran up to 40 kDa by electroporation,probably by plasma,mainly through a Th2 response [71.72]. interacting with the lipids of stmatum comeum [77]. 5.2.Electruporulion and truyo S.Comhinations of enhancing methods Due to their similar mechanisms of action,the In addition to electroporation,various physical and combination of ultrasourd and electroporation could chemical methods have been used for enhancing be less promising.However,synergy between ultra- transdermal drug transpont hy different mechanisms: sound and electroporation has heen reported hy Kost (i)increasing skin permeability (chemical enhancers, et al.[79].A simaltaneous application of ultrasound ultrasound and electroporation)andor (ii)providing a and clectroporation cnhanced transdermal calcein driving force (ultrasound,iontophoresis and electro- transport.The applicntion of ultrasound also reduced poration)[14].While all these enhancers have been the threshold voltage for electroporation. shown to increase drug transport.their combinations have heen hypochesized to he more effective com- 5.3.Electroporation and ionluphoresis pared to each of them alone and to increase the safery. The rationale for the combination of iontophoresis 5.1.Electruporation and chemical enhuncers and electroporation is basod on the differenee between the mechanisms of action of these enhancers.Specif- The synergistic effects between electroporation and ically,electroporation may disorder the lipid bilayers pre or co-treatment with chemicals enhancers have of the skin and ereate new transport pathways into the heen reported [14,75-77].These chemicals are differ. skin,thus facilitating passage of current during sub. ent than those mentioned commonly in the literalure sequent iontophoresis and resulting in increased and include polysaocharides (heparin and dextmn). transdermal transport [14].The application of electro urca,sodium thiosulfate and pbospholipids.In contrast porution before iontophoresis bas been reportod cither with traditional chemical enhancers for passive trns- to increase drug transport andor shorten the lag-time

poration was tested to achieve a needle free, adjuvant free and non-invasive immunization with antigen. As it increases the penetration of antigens into the skin, an electrically-assisted delivery of antigens, e.g. myr￾istilated peptide, dipheleria toxoid and ovalbumin, elicites a higher antigen specific IgG response in the plasma, mainly through a Th2 response [71,72]. 5. Combinations of enhancing methods In addition to electroporation, various physical and chemical methods have been used for enhancing transdermal drug transport by different mechanisms: (i) increasing skin permeability (chemical enhancers, ultrasound and electroporation) and/or (ii) providing a driving force (ultrasound, iontophoresis and electro￾poration) [14]. While all these enhancers have been shown to increase drug transport, their combinations have been hypothesized to be more effective com￾pared to each of them alone and to increase the safety. 5.1. Electroporation and chemical enhancers The synergistic effects between electroporation and pre or co-treatment with chemicals enhancers have been reported [14,75 – 77]. These chemicals are differ￾ent than those mentioned commonly in the literature and include polysaccharides (heparin and dextran), urea, sodium thiosulfate and phospholipids. In contrast with traditional chemical enhancers for passive trans￾dermal delivery, an effective chemical enhancers for electroporation does not need to disrupt lipids, but should stabilize the transient disruptions created by electroporation. The hypothesis for the combination of these two methods is to create enlarged aqueous path￾ways and/or to prolong the lifetime of the electropores. Vanbever et al. [76] and Weaver et al. [78] showed that macromolecules (heparin or dextran-sulfate) increased transdermal transport of mannitol by electroporation. No enhancement was observed during passive diffu￾sion or iontophoresis, suggesting that macromolecules interact specifically with transport pathways created at high voltage. The persistent low post-pulse electrical resistance could result from the insertion of these linear macromolecules in the aqueous pathways. Zewert et al. [75] showed that significant macromolecule trans￾dermal fluxes occurred when sodium thiosulfate was present, supporting the hypothesis that enlarged aque￾ous pathways or microconduits were created allowing large quantities of macromolecules to be transported through human skin. Anionic phospholipids were found to enhance the transdermal transport of FITC – dextran up to 40 kDa by electroporation, probably by interacting with the lipids of stratum corneum [77]. 5.2. Electroporation and ultrasound Due to their similar mechanisms of action, the combination of ultrasound and electroporation could be less promising. However, synergy between ultra￾sound and electroporation has been reported by Kost et al. [79]. A simultaneous application of ultrasound and electroporation enhanced transdermal calcein transport. The application of ultrasound also reduced the threshold voltage for electroporation. 5.3. Electroporation and iontophoresis The rationale for the combination of iontophoresis and electroporation is based on the difference between the mechanisms of action of these enhancers. Specif￾ically, electroporation may disorder the lipid bilayers of the skin and create new transport pathways into the skin, thus facilitating passage of current during sub￾sequent iontophoresis and resulting in increased transdermal transport [14]. The application of electro￾poration before iontophoresis has been reported either to increase drug transport and/or shorten the lag-time, Fig. 6. XY-planar CLSM section showing the localization of the fluorescent labelled plasmid in the epidermis 8 h following skin electroporation (101000 V, 100 As). The image was acquired at depth 12 Am below the surface of stripped skin. Scale bar: 50 Am. From Dujardin et al. [31]. 668 A.-R. Denet et al. / Advanced Drug Delivery Reviews 56 (2004) 659–674