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
In the last years, the application of electric pulses to induce biological effects proved to be an effective tool in different fields (e.g., cancer treatment, gene transfer, and electrofusion) [
Recently, the authors proposed a technique combining the application of nanosecond pulsed electric fields
The application to cells of one or several
However, the choice of a 10 ns pulse, due to its frequency content (first lobe at around 80–100 MHz depending on the rise and fall times), allows comfortably selecting the cuvette as applicator, even if the matching conditions of the structure need to be evaluated. Conversely, for shorter pulse durations (i.e., 3 ns and 1 ns), the complication of a broadband matching, up to GHz region, becomes necessary otherwise the cuvette will cause a significant distortion of the trapezoidal signal.
The use of a standard cuvette is also advantageous in the case of nanometer liposomes exposure related to the necessity to detect a fluorescent release. Indeed, due to their small dimensions, liposomes visibility under a microscope cannot be possible. In this case the fluorescent measurement is done with a spectrofluorimeter reader able to detect fluorescent molecule concentrations. This technique requires a minimum volume of solution, easily collected from the electroporation cuvette [
In this paper, we propose a methodology for the exposure of liposomes with dimension in the nanometer scale (in particular 200 and 400 nm liposomes) in terms of design of the exposure system combined with theoretical evaluation of the electric field threshold needed to porate the liposomes. As a first step, we propose an integrated approach to deal with experimental and modeling aspects, with the fundamental role of understanding and interpreting biological results. Once the specifications needed by our application were clarified, with particular care to the connection with the generator, the cuvette holder and the transition to the coaxial connector have been designed. A complete frequency characterization of the final structure, both with numerical modeling and measurements, has been done in order to understand the cuvette response under different experimental conditions. After the characterization of the exposure system, a microdosimetry model of the liposomes was also developed to approach the electric field needed for the poration considering the limitations imposed by the real experimental setup.
The researchers agree that, in biological issues resolution, the cooperation between experiments and modeling must be considered as a fundamental step [
Schematic of the integrated approach necessary to understand the biological results: the cycle points out the importance of a continuous exchange between the experimental and modeling aspects.
The basic concept is the possibility of beginning the cycle at different points of the experimental and model parts. Wherever there is an output from the cycle, an improvement in our biological knowledge is attained. Two different strategies exist:
The starting points for electroporation applications regard the feature of the pulse and the target of the stimulation.
The study of liposomes poration can be the first step for the use of this kind of nanocarriers in drug delivery applications driven by nanoelectroporation. In [
Electrical stimulus (ideal and measured pulses) and target for nanoelectroporation of liposomes.
The selected stimulus and target (coming from the prediction in [
In case of the 10 ns pulse, the spectral content is mainly contained in the first lobe (from 80 to 100 MHz) [
In this paragraph, the design of the structure for the connection with the 50 Ω generator has been reported. In particular, the final proposed structure is shown in Figure
Modeling of the cuvette connection to the 50 Ω generator.
The device consists of two brass electrodes with area of 33 × 10 mm2 and a thickness of 2 mm (Figure
To evaluate the performance both in frequency and time domains, the complete structure has been numerically simulated with finite element software ANSYS HFSS 2015. A wave port, applied to feed the coaxial cable, is used as input in the frequency domain; conversely a lumped port with the pulse as excitation is applied in the time domain.
After the design and characterization of the structure by numerical simulations, the fabrication process has been performed following the specifications obtained from the model results.
The electrodes are made in brass; the one connected with the pin of the coaxial cable has been perforated to allow the insertion of the pin and an easy weld. The second one has a hole of larger dimension for the insertion of the dielectric. The connection with the external sock is made wrapping a cylinder of brass previously welded to the second electrode in proximity of the hole (Figure
Realized structure.
Because the cuvette is disposable, the use of a structure as the one here proposed can be very useful instead of a direct connection of the coaxial cable with the electrodes of the cuvette to be renewed at any cuvette usage [
In order to characterize the structure, measurements in the frequency domain have been carried out with an Agilent Technologies PNA Network Analyzer E8363C (10 MHz–40 GHz) with the cuvette filled with solutions at different conductivity values (0.25, 0.55, and 1.6 S/m); hence it is possible to obtain the efficiency of the structure under different experimental conditions. In this first analysis, the solutions are just made of sodium chloride 0.9% with a different dilution of DI water to obtain the desired electrical conductivity of the solution. The conductivity values of the solutions were confirmed with measurement with a Precision LCR Meter E4980A from Agilent.
Due to the different experimental conditions, selectable when using liposomes (e.g., dimensions, inner conductivity, and external conductivity), a microdosimetry model has been proposed in order to understand the voltage needed at the generator for liposomes nanoporation. 2D numerical simulations have been carried out using the software Comsol Multiphysics v. 5.0. The 2D models consist of a rectangular box with dimensions of 70
All the electrical and geometrical parameters of the model are reported in Table
Electric and geometrical parameters.
Electrical and geometrical properties | |||
---|---|---|---|
|
|
Dimension ( | |
External medium | 67 [ |
0.05–1.6 | 100–70 |
Liposome membrane | 11.7 [ |
1.1 × 10−7 [ |
0.005 [ |
Inner liposome | 67 [ |
0.05–1.6 | 0.2/0.4 [ |
Denzi et al. 2016 [
As a first step in the analysis of the impedance behavior of the applicator, the real and the imaginary parts have been numerically evaluated. The results are reported in Figure
Impedance in terms of real and imaginary part of the holder with the cuvette placed and filled with a solution 0.25 S/m of conductivity.
In the same frequency range the
Measured (dashed lines) and simulated (solid lines)
At frequencies higher than 100 MHz the mismatch of the impedance cuvette is more evident and this limits the use of this kind of applicator only to pulses with duration ≥ 10 ns. The results achieved are comparable with the ones reported in [
Starting from the results obtained in [
The measurements and the simulations are in perfect agreement for all the considered solutions. As expected, the performances of the applicator strongly depend on the solution parameters, with best behavior for the less conductivity value (the best value according to the formula reported in [
In order to better understand how this parameter affects the pulse transmission inside the cuvette, in the next paragraph the analysis of response in time domain of the cuvette holder with the cuvette placed and filled has been numerically performed.
The 10 ns pulse has been applied to the coaxial cable of the structure (Figure
Electric field amplitude in the center of the cuvette gap. The efficiency value of the structure,
As expected from the frequency results, for the conductivity of 0.25 S/m, the amplitude of the transmitted pulse is 1000 V/m for 1 V input pulse. This is the maximum achievable value of electric field for 1 V applied to the 1 mm gap. The efficiency in transmission (
Once the behavior of our applicator is completely characterized, a microdosimetry model of the liposomes is needed to derive all the necessary parameters to drive a final experiment of liposome solution exposed to a 10 ns pulse. In particular, microdosimetry should provide values of liposome poration for different experimental conditions, as vesicles dimensions or inner and outer conductivity, and with regard to the efficacy of the real structure that will be used for the experiments. Hence, the results of the model have to be combined with the information about the efficiency of the real applicator developed here.
The microdosimetry model of the liposomes has been used to evaluate the electric field threshold value necessary to obtain the membrane liposome poration. In particular, 1 V has been considered as the threshold value for the electroporating transmembrane potential [
In Figure
Electric field threshold for 200 nm and 400 nm liposomes poration changing the internal and external liposome conductivity.
A dual situation occurs when the inner liposome conductivity is fixed and the external medium conductivity values change (i.e., a vertical line parallel to the
As expected, comparing the two different liposome dimensions (200 nm and 400 nm), the influence of the conductivities on the electric field threshold is the same, but the larger the dimension, the lower the field necessary for liposome membrane poration.
Considering from Figure
However, as previously reported in this paper, the efficiency of our applicator in the delivering of pulse to the sample is inversely proportional to the outer conductivity (i.e., 1, 0.62, and 0.27 (kV/m)/V for 0.25, 0.55, and 1.6 S/m conductivity, respectively, black horizontal lines reported in Figure
Hence, it is necessary to combine the data of Figures
To weight the results of the microdosimetry model with the real experimental setup characteristics, we have taken into account not only the efficiency of the designed structure but also the capability of our generator in terms of the maximum output voltage it is able to generate. For the High Voltage Pulse Generator FPG 10-1NM10, we are able to provide a pulse with amplitude in the range of 2–10 kV, fixing our maximum capability to obtain 10 MV/m in 1 mm gap distance applying the highest intensity available.
In Figure
Input voltage needed at the generator weighted with the effective efficacy of the structure.
It can be noticed how the highest conductivity value (
The design and characterization of an exposure system with the capability to porate liposomes with nanometer dimensions using a 10 ns pulsed electric field have been carried out. This has been performed with an accurate system characterization taking into account both theoretical and technological issues. It represents the base of the integrated approach proposed in Figure
In the design of the structure the first step was to consider the features of the excitation and of the target. Since the excitation is a 10 ns pulsed electric field and the target is liposomes with 200 or 400 nm diameter, as suggested by the authors in [
The structure has been completely characterized in frequency domain to understand its capability and performances in different experimental conditions. In particular, the analysis has been performed for different conductivity values of the solution inside the cuvette both with numerical models and measurements. A perfect agreement has been found between them. The higher the solution conductivity, the lower the efficacy of the structure and hence the lower the amplitude of the transmitted pulse to the cuvette.
In order to analyze the impact of the real performances of the device on the liposomes electroporation, a microdosimetry model of liposomes with 200 or 400 nm dimension has been reported. The simulations have been carried out considering different inner and outer liposome conductivities and the results have been combined with technological outcomes. Combining microdosimetry and technological information permits predicting that the optimal condition for a 400 nm liposome is with 0.25 S/m and 1.6 S/m for external and internal liposome conductivities, respectively, differently from the indications that one would follow on the basis of the microdosimetry alone (highest internal and external conductivity values). This discrepancy is due to the higher efficiency of the structure for the conductivity of 0.25 S/m. If during the experiments a change of some parameters becomes necessary, for example, different volumes of solution (keeping particular attention to avoid sparks in air) or a different value of inner or outer conductivities, a characterization with new simulations and measurements has to be performed in order to understand the real transmitted signal.
The authors declare that they have no conflicts of interest regarding the publication of this paper.
This project was supported by COST Action TD1104 Short Term Scientific Mission (25007) and COST Action BM1309 Short Term Scientific Mission (27189) to Elena della Valle. This project was performed within the framework of the Joint IIT-Sapienza LAB on Life-NanoScience Project (81/13 16-04-2013). The authors would like to thank Simone Chiccarella for his contribution in the device realization.