Lluis M. Mir

Hollow Microneedle Arrays for Intradermal Drug Delivery and DNA Electroporation

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.

Demonstration of cell membrane permeabilization to medium-sized molecules caused by a single 10 ns electric pulse.

In our study, we used bleomycin to evaluate the permeabilization caused by nanosecond duration electric pulses (nanopulses). Bleomycin is a non permeant molecule which can be used both as a sensitive and quantitative marker to evaluate cell electropermeabilization. Indeed, the penetration of as few as 500 molecules is sufficient to entail a major biological effect: cell death. We show that one single nanopulse with a duration of 10 ns and a field strength of 40 kV/cm is sufficient to allow the uptake of at least 500 molecules of bleomycin in 20% of the cells when the external bleomycin concentration is 3 μM. When the external bleomycin concentration is reduced by a 100 fold, the same levels of cytotoxicity require an increase of about 25 times in the number of pulses. These results are in favor of the fact that each nanopulse creates new pores or defects on the cell membrane even if most of these pores can reseal between two consecutive pulses. Results also suggest that the cell permeability observed with classical markers when a large number of pulses are delivered results from the large number of nanopores or defects of the cell membrane created by the train of nanopulses.

Characterization of a 50-Ω Exposure Setup for High-Voltage Nanosecond Pulsed Electric Field Bioexperiments

An exposure system for a nanosecond pulsed electric field is presented and completely characterized in this paper. It is composed of a high-voltage generator and an applicator: the biological cuvette. The applied pulses have high intensities (up to 5 kV), short durations (3 and 10 ns), and different shapes (square, bipolar). A frequency characterization of the cuvette is carried out based on both an analytical model and experimental measurements (S11) in order to determine its matching bandwidth. High voltage measurements in the time domain are performed. Results show that the cuvette is well adapted to 10-ns pulses and limited to those of 3 ns. The rise/fall times of the pulses should not be less than 1.5 ns. In addition, numerical calculation providing voltage distribution within the cuvette is performed using an in-house finite-difference time-domain code. A good level of voltage homogeneity across the cuvette electrodes is obtained, as well as consistency with experimental data for all the applied pulses.

Ion fluxes, transmembrane potential, and osmotic stabilization: a new dynamic electrophysiological model for eukaryotic cells

Survival of mammalian cells is achieved by tight control of cell volume, while transmembrane potential has been known to control many cellular functions since the seminal work of Hodgkin and Huxley. Regulation of cell volume and transmembrane potential have a wide range of implications in physiology, from neurological and cardiac disorders to cancer and muscle fatigue. Therefore, understanding the relationship between transmembrane potential, ion fluxes, and cell volume regulation has become of great interest. In this paper we derive a system of differential equations that links transmembrane potential, ionic concentrations, and cell volume. In particular, we describe the dynamics of the cell within a few seconds after an osmotic stress, which cannot be done by the previous models in which either cell volume was constant or osmotic regulation instantaneous. This new model demonstrates that both membrane potential and cell volume stabilization occur within tens of seconds of changes in extracellular osmotic pressure. When the extracellular osmotic pressure is constant, the cell volume varies as a function of transmembrane potential and ion fluxes, thus providing an implicit link between transmembrane potential and cell volume. Experimental data provide results that corroborate the numerical simulations of the model in terms of time-related changes in cell volume and dynamics of the phenomena. This paper can be seen as a generalization of previous electrophysiological results, since under restrictive conditions they can be derived from our model.

Technological and Theoretical Aspects for Testing Electroporation on Liposomes

Recently, the use of nanometer liposomes as nanocarriers in drug delivery systems mediated by nanoelectroporation has been proposed. This technique takes advantage of the possibility of simultaneously electroporating liposomes and cell membrane with 10-nanosecond pulsed electric fields (nsPEF) facilitating the release of the drug from the liposomes and at the same time its uptake by the cells. In this paper the design and characterization of a 10 nsPEF exposure system is presented, for liposomes electroporation purposes. The design and the characterization of the applicator have been carried out choosing an electroporation cuvette with 1 mm gap between the electrodes. The structure efficiency has been evaluated at different experimental conditions by changing the solution conductivity from 0.25 to 1.6 S/m. With the aim to analyze the influence of device performances on the liposomes electroporation, microdosimetric simulations have been performed considering liposomes of 200 and 400 nm of dimension with different inner and outer conductivity (from 0.05 to 1.6 S/m) in order to identify the voltage needed for their poration.

Control of current intensity: Experimental proofs of the relevance of current density in biological cells permeabilisation caused by nanosecond electric pulses

Electropermeabilisation of cells can be obtained by electric pulses of very different durations. In this paper we compare the efficiency of pulses of 100 µs and 10 ns duration eventually in the presence of bleomycin, a cytotoxic drug in order to detect reversible permeabilisation of cells. Biological effects are evaluated using cloning efficiency tests. The impact of the external conductivity is studied for the two types of pulses. Experiments reveal that current density plays a critical role when 10 ns pulses are applied which is very different from the results achieved with more traditional pulses of 100 µs duration.

Real time electroporation control for accurate and safe in vivo electrogene therapy

In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested ex vivo. Controlled pulses resulted in protection of the tissue where uncorrected excessive applied voltages lead to intense tissue damage and consecutive loss of gene transfer expression.