Here, we combined lipid nanovesicles (ethosomes, liposomes) as the drug carrier systems with two physical methods (electroporation, sonoporation) to enhance transdermal delivery of a hydrophilic model molecule, calcein. First, using different formulations, ethosomes greatly enhanced calcein permeation by passive diffusion compared to liposomes and calcein in buffer, which is most likely due to a synergism between the ethanol action on the stratum corneum lipids and the penetration of the elastic vesicles. Liposomes permeated poorly through the skin and, as also suggested by other authors, seem to remain confined to the outer layers of the skin. By creating localized effects, liposomes would be better suited to topical dermal delivery than transdermal delivery. Using sonoporation as the physical enhancement method, sonication (5 min) showed improvement over passive diffusion; however, only for the ethosome formulation, and not for solution and liposomes. Similarly, electroporation greatly enhanced delivery of calcein, which was again more pronounced for ethosomes than liposomes and calcein in buffer. Finally, three different transdermal delivery enhancement methods were coupled, using ethosomes as carriers, along with both electroporation and sonoporation, to investigate the potential for synergistic effects. However, these combinations failed to achieve not only synergistic effects, but also additive effects. Nevertheless, combination of the ethosome formulation of calcein with either of electroporation or sonoporation achieves significant enhancement of transdermal molecular delivery being safe for potential clinical use.

Measurement of the electrical and viscoelastic properties of tissue is important in determining its physiological state. We present here a proof of principle study of a new technique that yields both sets of properties for soft tissue in vivo, with minimal discomfort. The variation of skin capacitance with time after the application of pressurized electrodes is used to determine the viscoelastic properties of the tissue. The mechanical compression of the tissue, measured by the capacitance increase with time, is related to the mechanical strain. Knowledge of the stress applied by the electrodes then yields the viscoelastic properties of the tissue corresponding to the standard, linear four-element viscoelastic model. Results are presented for three different subjects to show that the method is sensitive to individual variability. The series elasticity, the viscoelastic ( parallel) elasticity and viscosity, and the series viscosity are determined for six applied stresses in the MPa range. Significant non-linearity is observed with the viscoelastic parameters increasing with the applied stress. This new method provides values for the viscoelastic properties of the skin comparable to those reported in the literature and could be of significant value for clinical diagnostics and for surgical modeling.

Due to low permeability of the stratum corneum, different enhancement techniques are required to promote transport of larger molecules across the skin. In our present study we focused on skin electroporation with emphasis on the design of the experimental system and experimental protocols. With this approach the results and the conclusions drawn have greater relevance for in vivo use and later translation into clinical practice. Our results show a statistically significant enhancement of calcein delivery (after one hour of passive diffusion following treatment) already after only 6x100 short (100 μs) high voltage (200 V) electrical pulses.

We studied fractional Er:YAG laser to enhance transdermal drug delivery of compounds possessing different molecular weights: FITC-dextrans (or FD) with average molecular weights of 4, 10 and 20kDa. Vertical glass Franz diffusion cells were used to study molecular transport through dermatomed porcine skin and histological analysis of laser-treated skin was performed after treatment with different laser pulse protocols. We were comparing different pulse durations at constant or varying pulse energies. We found that the energy of the delivered pulses mostly dictates the size/depth of laser-created microchannels, while the duration of the pulses dictates the extent of thermally altered tissue. That is, tissue ablation threshold is lowered at shorter pulse durations with higher power, which is preferred as it lowers thermal effects on viable skin layers. Especially for smaller molecules, transdermal delivery is increased by increasing laser-created microchannel size, but also by making partitioning into tissue easier when less thermal damage is caused on tissue. For large molecules, molecular transport through the remainder of skin tissue becomes increasingly difficult regardless of laser pulse parameters.

Electroporation was used as an enhancement method for transdermal delivery of a model molecule, Patent Blue. The level of increase of stratum corneum permeability was assessed with a numerical model and experimentally on ex vivo full-thickness porcine ear skin. Green Skin Pore square wave pulse generator (Iskra Medical LLC, Slovenia) was used in the study. The pulses were delivered with an array of 7 pin electrodes arranged in a honeycomb configuration. Our results show increased delivery of Patent blue after pretreatment of skin with electric pulses. Also, the extent of the permeabilized stratum corneum is higher and more homogeneous when the electrode is moved in circular motion during pulse delivery. In this way, larger portion of the skin under the electrode is exposed to electric pulses, leading to increased efficiency.

In our present study we focus on two physical enhancement methods for transdermal drug delivery: ultrasound and electric pulses either alone or in combination. Great emphasis has been given on the design of the experimental system and protocols, so the results and the conclusions drawn from them would have greater relevance for in vivo use and later translation into clinical practice. Our results show a statistically significant enhancement of calcein delivery (after one hour of passive diffusion following treatment) already after 5 minutes of ultrasound application, or only 6 × 100 short high voltage electrical pulses. We also experimented with combinations of the two enhancement methods hoping for synergistic effects, however, the results showed no evident drastic improvement over single method. Looking closer at physics of both methods, this absence of synergy in our in vivo oriented experimental setting is not surprising. The mechanism of action of both methods is the creation of aqueous pathways in the stratum corneum leading to increased skin permeability. However, when used in combination (regardless of the order of methods), the second method was unsuccessful in adding many new aqueous pathways in the stratum corneum, as it acted preferentially near the sites of the existing ones.

In this paper, we discuss some of the primary experimental factors that should be considered when interpreting and implementing the published results of skin electroporation studies concerning measurements of mass transport across the stratum corneum (SC) in the Franz cell. It is explained that the pulse magnitude should always be considered in the context of pulse shape and that transport measurements should always be presented in the context of the trans-SC potential difference (instead of the voltage between the electrodes). The condition of the SC prior to the application of the long-duration pulse strongly influences the evolution of the local transport region (LTR). This is quantified in a simple analytical investigation of the conditions that affect the thermodynamic response of the skin.

In this study we consider the physics underlying electroporation which is administered to skin in order to radically increase transdermal drug delivery. The method involves the application of intense electric fields to alter the structure of the impermeable outer layer, the stratum corneum. A generally held view in the field of skin electroporation is that the skin’s drop in resistance (to transport) is proportional to the total power of the pulses (which may be inferred by the number of pulses administered). Contrary to this belief, experiments conducted in this study show that the application of high voltage pulses prior to the application of low voltage pulses result in lower transport than when low voltage pulses alone are applied (when less total pulse power is administered). In order to reconcile these unexpected experimental results, a computational model is used to conduct an analysis which shows that the high density distribution of very small aqueous pathways through the stratum corneum associated with high voltage pulses is detrimental to the evolution of larger pathways that are associated with low voltage pulses.

Electroporation can be used as an active enhancement method for intra- and transdermal drug delivery. Differences in response of skin to electric pulses depend on their amplitude, duration and number and have been a point of interest in the past. While protocols consisting of the same repetitive, mostly exponentially decaying pulses have been used before, this study is focused on comparing different combinations of square wave short high voltage (HV) and longer low voltage (LV) electroporation pulses. Our in vitro experimental results show that longer LV pulses significantly increase subsequent passive transport of calcein through dermatomed pig skin, while short HV pulses alone result in negligible calcein passive transdermal transport. Surprisingly, when the long LV pulses are preceded by short duration HV pulses, the total calcein transported is reduced significantly. This result is explained using a theoretical physics based model of individual local transport region (LTR) evolution during the applied LV pulse. The theoretical model shows that HV pulses alter the structure of the stratum corneum in such a way that when the LV pulses are applied, insufficient thermal energy is generated to initiate LTR expansion. Together, the experimental results and theoretical predictions show that the total pulse energy alone cannot account for total solute transport: that the order of the types of pulses administered must also be considered. Our findings open a direction for further improvement of the method using new protocols.

Many advantages of transdermal drug delivery have led to investigation into a range of methods that are capable of overcoming the considerable barrier properties of the skin. Different physical enhancement methods are used for this purpose, one of them being the use of fractional lasers to create transport pathways in the stratum corneum and enhance transdermal delivery of drugs [1, 2]. he aim of the study was to demonstrate in vitro that different types and energies of laser pulses greatly influence transdermal transport of FITC-dextran through pigs’ ear skin. Vertical glass Franz diffusion cells were used to study molecular transport through excised and dermatomed pigs’ ear skin. The donor compartment contained 10 kDa FITC-dextran solution (0.1 mM) in phosphate buffer at pH 7.4. The receiver solution was a phosphate buffer (pH 7.4) thermo-regulated at 37°C. The concentration of FITC-dextran in the receiver compartment after laser treatment was measured with a spectrofluorometer.

Transdermal route has some advantages over other drug administration routes. These include avoidance of first pass effect (hepatic metabolism), better pharmacokinetic profile, reduction of side effects and good patient compliance. The greatest obstacle for the drugs to be delivered through the skin is overcoming the impermeable outermost layer of the skin – the stratum corneum. Quite a few enhancement techniques can be used to overcome the stratum corneum barrier and facilitate transdermal drug delivery. These include various passive (penetration enhancers, liposomes) and active approaches (electroporation, iontophoresis, microneedles), which are of prime interest for transdermal drug delivery research area.

This study concerns the modeling and treatment of mass transport associated with electroporation of the skin. Electroporation involves the exposure of relatively impermeable phospholipid membranes to an intense electric field, resulting in order of magnitude increases in permeability to mass transport and increased electrical conductivity. Electroporation is currently conducted in clinical settings to increase the permeability of cells, as well as in the treatment of the skin's barrier layer. Skin electroporation is used to enhance the success of localized transdermal transport and is the focus of this chapter. This chapter reviews the current understanding of skin electroporation and the methods that have been developed to model the physical process of the alteration of the skin's barrier structure under the influence of an intense electric field.

Electroporation – the use of electric pulses to increase cell membrane permeability – has also been used on skin for enhanced transdermal molecular delivery or to deliver molecules into viable skin cells. We theoretically described skin electropermeabilization and the amount of heating in and around an electrically created pore in the stratum corneum (SC), using finite element modeling. Theoretical results obtained with the model show no significant further thermal expansion of the aqueous pore for electrode design and pulse protocols we used for gene electrotransfer in vivo (already published results), as well as no thermal damage to the tissue. With some modifications to the protocol, electroporation could be used to a) create pores in the SC through which to transport the DNA, and then b) introduce the DNA into viable skin cells.

The use of electric pulses to increase cell membrane permeability – electroporation – has, among other applications also been used on skin for (a) enhanced transdermal molecular delivery or (b) the delivery of drugs or DNA into viable skin cells. Based on finite element numerical method, we theoretically described skin electropermeabilization and the amount of heating in and around an electrically created pore in the stratum corneum (SC). With the model, we address both, electrical as well as thermal effects on skin tissue, specifically for electrode design and pulse protocols we used for gene electrotransfer in vivo (already published results), where plasmid DNA was injected intradermally with a syringe and external plate electrodes were used for pulse delivery. Theoretical results obtained with the model show no significant further thermal expansion of the aqueous pore for our specific pulse protocol (one short high voltage pulse: 400V, 100μs+one longer low voltage pulse: 80V, 400ms), as well as no thermal damage to the tissue. With some modifications to the protocol, electroporation could be used to (a) create pores in the SC through which to transport the DNA, and then (b) introduce the DNA into viable skin cells.

As a simple definition, a mathematical model is a representation of chosen essential aspects of a real system (may it be a living, engineering or social system), described by a set of variables and a set of equations that establish relationships between the variables. The aim of such a model is to gain knowledge about the represented system, explaining some of its phenomena, or help designing it. Mathematical modeling is a vast scientific and engineering field and is therefore not possible to explain in detail in one single book, let alone in one paper. It is used in countless scientific, engineering and even social studies and represents an important field in the study of the effects of the electromagnetic fields and accompanying coupled phenomena on cells, tissues and organs (Fear and Stuchly, 1998; Debruin and Krassowska, 1999; Debruin and Krassowska, 1999a).

The association of microneedles with electric pulses causing electroporation could result in an efficient and less painful delivery of drugs and DNA into the skin. Hollow conductive microneedles were used for (1) needle-free intradermal injection and (2) electric pulse application in order to achieve electric field in the superficial layers of the skin sufficient for electroporation. Microneedle array was used in combination with a vibratory inserter to disrupt the stratum corneum, thus piercing the skin. Effective injection of proteins into the skin was achieved, resulting in an immune response directed to the model antigen ovalbumin. However, when used both as microneedles to inject and as electrodes to apply the electric pulses, the setup showed several limitations for DNA electrotransfer. This could be due to the distribution of the electric field in the skin as shown by numerical calculations and/or the low dose of DNA injected. Further investigation of these parameters is needed in order to optimize minimally invasive DNA electrotransfer in the skin.

There is increased interest for the use of impedance spectroscopy to measure skin dielectric properties in vivo. The aim of such measurements can be either to evaluate the hydration state of the skin, to detect diseased states such as skin cancer, to follow the progress of transdermal drug delivery, or simply to gather data on skin tissue impedance to be used in theoretical studies. However, obtaining reliable data can be difficult. Namely, skin is a highly nonhomogeneous multi-layered structure whose composition and dimensions differ depending on the location on the body and interindividual variations. Also, impedance measurements on skin are accompanied by a number of artefacts. We performed a series of impedance measurements using an Agilent/HP 4284A precision LCR meter with parallel plate electrodes pressed on the skin, at different locations on the body. We observed substantial impedance changes over the course of the measurement. These changes can be mainly attributed to skin deformation caused by the electrodes pressing against skin. The analysis showed that skin mechanical properties and layer thicknesses can be inferred from these temporal changes. Such data on mechanical properties of skin tissue give valuable extra information, crucial for successful estimation of the impedance of different skin layers.

Most widely used electrodes in gene electrotransfer in the skin are external plate electrodes of different geometries where we first have to cause the electrical breakdown of the stratum corneum, in order to reach the viable layers underneath. Therefore, painless microneedle electrodes for transdermal drug delivery and electrogene transfection in skin were proposed and are under development. Their depth of penetration is small enough not to cause any pain for the patient. A preliminary comparison, by means of numerical models, between different settings of microelectrode arrays was made. Comparison with external plate electrodes showed that the volume of the tissue exposed to electric fields exceeding the irreversible electroporation threshold, is smaller when needle microelectrodes are used. Further, as the areas of high (irreversible) electric field coincide with the areas of DNA injection, (both near the electrode tips), it may be sensible to alternate the role of the microneedles, using half of them (every second) as electrodes and the other half as DNA delivery device.

Background. Numerous experiments have to be performed before a biomedical application is put to practical use in clinical environment. As a complementary work to in vitro, in vivo and medical experiments, we can use analytical and numerical models to represent, as realistically as possible, real biological phenomena of, in our case, electroporation. In this way we can evaluate different electrical parameters in advance, such as pulse amplitude, duration, number of pulses, or different electrode geometries. Such numerical models can contribute significantly to the understanding of an experiment and treatment planning as well as to the design of new electroporation devices and electrodes.Methods. We used commercially available modeling software, based on finite element method. We constructed a model of a subcutaneous tumor during electrochemotherapy (EMAS) and a model of skin during gene electrotransfer (COMSOL Multiphysics). Tissue-electrode geometries, pulse parameters and current-voltage measurements from in vivo experiments were used to develop and validate the models.Results. To describe adequately our in vivo observations, a tissue conductivity increase during electroporation was included in our numerical models. The output currents of the models were compared to the currents and the voltages measured during in vivo experiments and a good agreement was obtained. Also, when comparing the voltages needed for a successful electropermeabilization as suggested by the models, to voltages applied in experiments and achieving a successful electrochemotherapy or in vivo gene electrotransfer, good agreement can be observed.Conclusions. Modeling of electric current and electric field distribution during cell and tissue electroporation proves to be helpful in describing different aspects of the process and allowing us to design electrodes and electroporation protocols as a part of treatment planning.

The protective function of skin (its low permeability) presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the possibilities to temporarily breach the barrier function of skin is using electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. In addition, the application of electric pulses to biological cells causes the electroporation of cell membrane, increasing its permeability, thus enabling cell uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA. The electropermeabilization process in skin was described theoretically, by means of numerical modeling, leaning on data derived from our in vivo experiments previously published. The numerical models took into account the layered structure of skin, macroscopical changes of its bulk electrical properties during electroporation, as well as the presence of localized sites of increased molecular transport termed local transport regions. The output of the models was compared with the in vivo experiments, and a good agreement was obtained. In addition, a comparison of our results with already published findings on skin electropermeabilization showed that permeabilizing voltage amplitudes suggested by the model are also well in the range of the voltage amplitudes reported by other authors to cause skin permeabilization. The subject of tissue conductivity changes due to electroporation is still a rather unexplored field; however, we used the available data to describe the mechanism of the nonlinear process of the tissue electropermeabilization.