Electroporation of Skin Stratum Corneum Lipid Bilayer and Molecular

Pore Radius Calculation. S6. Sustain Electric field after electroporation. S7. Effect of Bilayer Composition on. Electroporation of the bilayer. S8. P...
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Biological and Environmental Phenomena at the Interface

Electroporation of Skin Stratum Corneum Lipid Bilayer and Molecular Mechanism of Drug Transport: A Molecular Dynamics Study Rakesh Gupta, and Beena Rai Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00423 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on April 30, 2018

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Electroporation of Skin Stratum Corneum Lipid Bilayer and Molecular Mechanism of Drug Transport: A Molecular Dynamics Study Rakesh Gupta and Beena Rai* Physical Science Research Area, Tata Research Development & Design Centre, TCS Innovation labs, Pune India, 411013 *Corresponding author: [email protected] Fax:

91-20-66086399

Tel:

91-20-66086203

Abstract Electroporation technique has been used significantly to increase drug permeation through skin. In this technique, electric field (generally, order of 50-300 V) applied on skin for shorter time (microseconds to millisecond) which may create microscopic pore in the skin layers. However, molecular mechanism, which resulted in enhancement of flux through skin, is still not known. In this study, extensive atomistic molecular dynamics simulation of skin lipids (made up of ceramide (CER), cholesterol (CHOL) and free fatty acid (FFA)) have been performed at various external electric field. We show for the first time the pore formation in the skin lipid bilayer during electroporation. We show the effect of applied external electrical field (0.6-1.0 V/nm) on the pore formation dynamics in lipid bilayer of different size (154, 616, 2464 lipids) and compositions (CER: CHOL: FFA, 1:0:0, 1:0:1, 1:1:0, 1:1:1). The pore formation and resealing kinetics were different and was found to be highly dependent on the composition of skin lipid bilayer. The pore formation time decreased with increase in the bilayer size. The pore sustaining electric field was found to be in the range of 0.20-0.25 V/nm for equimolar CER, CHOL and FFA lipid bilayer. The skin lipid bilayer (1:1:1), sealed itself within 20 ns after the removal of external electric field. We also present the molecular mechanism of enhancement of drug permeation in the presence of external field as compared

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to the passive diffusion. The molecular level understanding obtained here could help in optimizing/designing the electroporation experiments for effective drug delivery. For a given skin composition and size of drug molecule, the combination of pore formation time and pore growth model can be used to know aproiri the desired electric field and time for application of electric field. Introduction The delivery of drugs through skin has been an interesting and challenging area of research. Transdermal drug delivery offers numerous advantages over conventional drug delivery system such as absence of gastric irritation, avoidance of erratic absorption, non-invasive in nature, ease of access due to high surface area of skin and as well as improvement in patient compliance.1,2 The bio-availability and half-life of the drugs can be increased since there is no pre-systemic metabolism involved in transdermal route. However, only very few molecules can be delivered across the skin because of the hindrance provided by the 15-20 µm thick upper layer of skin known as stratum corneum (SC).1-3 The SC is highly selective in nature and typically allows only relatively lipophilic compounds to diffuse into the inner layers of skin.3,4 The SC is made up of 15–20 layers of flat dead cell shells known as corneocytes, which are interconnected by a lipid lamellar bilayer structure in a crystalline-gel phase.3,4 The SC is highly hydrophobic in nature and the transport of molecules across skin occurs primarily by passive diffusion and mostly through lamellar lipid matrix.4 Most of the drugs and protein, which are hydrophilic in nature, cannot breach this barrier. In the past few decades, several techniques have been developed and used to enhance the drug permeation by breaching the barrier function.5 These are broadly classified in two categories based on their working principle (a) passive transport through chemical penetration enhancers and liposomes and (b) active methods such as electroporation, iontophoresis, sonophoresis and thermophoresis.5 Chemical penetration enhancers and liposomes interact with the SC’s constituents and change the morphology of skin barrier function at molecular scale.5,6 The active methods such as electroporation, iontophoresis, sonophoresis and thermophoresis use external energy source (electric current, ionic flux etc.) and create temporary nanometer sized pores in the SC which felicitates the permeation of molecules.6 The permeation enhances such as ethanol7 and di-methylsufroxide (DMSO)8 perturb the skin morphology significantly thus disturbing the barrier function to undesirable extent. Although new membrane based models such as bicelles provide alternate to DMSO and ethanol for delivery of molecules through skin.9 The passive methods are also slower in

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nature as compared to active methods because of slow kinetics of passive permeation. A comprehensive review on the passive and active methods for delivery of protein/drug molecules through skin can be found here.10 Electroporation, is referred to the temporary perturbation of the skin by applying high voltage in the form of pulses for short duration of time (µs to ms).10 Electroporation was originally used to breach cells with macromolecules (such as DNA) by altering their cell lipid membranes in a reversible manner. Later on, this technique was also used for transdermal drug delivery of macromolecules such as peptides and proteins.11-13 The electroporation of skin requires higher voltages ( >50-300 V) as compared to the electroporation of cell membranes.10,12,13 Electroporation pulses are broadly classified into two regimes namely short and long pulses. The physical effects of these two regimes are very much different in nature and choice of regime mostly depends upon the drug size.14 A low molecular weight drug molecule will require shorter pulses. In short pulse electroporation, the pulse durations are typically less than 100 µs and the electric potential across the skin reaches critical value of 30–100 V14,15 (100-1500 V applied voltages) and the skin resistance drops to several orders of magnitude.15 The shorter pulse creates temporary nanopores in lipid matrix, while longer pulse (> ms) leads to electrophoresis and joule heating of skin lipid matrix.14,16 The larger charged molecules face huge viscous resistance to transport through the tortuous path of SC, even with increased skin permeability. The charged molecules are transported through SC by additional electrophoretic forces provided by long pulse electric field.16 The second important effect known as joule heating increases the skin local temperature which in turn increases the skin permeability by melting the lipid matrix packing.17 The packing of lipid matrix plays important role in determining skin permeability. It has been shown, both by experiments18,19 and simulation20, that the SC lipid matrix undergoes phase transition from gel to liquid crystalline phase in the temperature range from 363-380 K. The liquid crystalline phase lipid bilayer has very high permeability as compared to tightly packed gel crystalline phase lipid matrix.21,22 Since the last two decades, researchers have carried out numerous electroporation experiments for delivery of both small23-32 and macromolecules33-38 through skin. The efforts includes, delivery of various ionic, non-ionic, neutral and charged drug molecules through skin (rat, mouse and human) using low to high varying electric field. 23-32 Prausnitz et al.33 reported the transport of heparin (molecular weight 5000–30,000 Da) across the SC of cadaver skin using the high-voltage pulsing electroporation. Petchsangsai et al.34 reported that ACS Paragon Plus Environment

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total cumulative amount of drug permeated through skin using three combined techniques (electroporation, sonophoresis and microneedles ) was greater than the amount observed using a single method or combination of any two methods. Electroporation has also been widely used in experimental settings for gene transfer into and through the skin.35-37 Spanggaard et al.38 reported for the first time the safety and tolerability of intratumoral plasmid antiangiogenic metargidin peptide electro transfer into cutaneous metastatic melanoma. Despite of so many experimental studies, the underlying molecular mechanism/s which induce the structural changes in the skin during electroporation are still not well understood.39 However, it has been suggested that water pores form in the skin on application of high voltage pulses.10,39 It is reported that skin permeability increased significantly (up to four orders of magnitude) when voltage drop across the skin SC layer crossed 30 V.40 It was hypothesized that modification of skin lipid structure occurred due to the interaction between the water dipole and the electric field. To understand the macroscopic mechanism of electroporation, some mathematical transport model of skin electroporation have been developed by combining passive transport models with electroporation models.41 However, not a single study unravelling the molecular level mechanisms of electroporation of skin SC lipid matrix is reported till now. Molecular simulations provide a convenient way to understand permeation and dynamics processes of skin and yield important physical insights at molecular level which are difficult to obtain from experiments due to the associated time and length scales.42-46 In this study, we first time report the molecular mechanism of electroporation of skin SC using molecular dynamics simulations. In the first part of the study, we performed extensive electroporation simulations of skin lipid bilayer of various size and composition (different ratio of CER, CHOL and FFA). The effect of both size and composition of bilayer, on bilayer threshold electric field and bilayer poration time, is reported. In second part of the study, we report the sustaining electric field at which pore remained stable for longer time. The effect of sustaining electric field on pore radius is also presented. We also show the resealing/healing of pores after removal of the external electric field. Finally, we have presented the molecular mechanism of enhancement of the permeation of a drug molecule in the presence of external electric field.

2. System, Models and Methods

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The SC is made up of 15–20 layers of flat dead cell shells known as corneocytes, which are interconnected by a lipid lamellar bilayer structure in a crystalline-gel phase.3,4 The corneocytes and lipid matrix are arranged in brick and mortar fashion, respectively.3 The skin barrier function is mostly determined by the the packing of lipid matrix because corneocytes are almost impermeable in nature. The lipid matrix is a heterogeneous mixture of long chain ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA) in certain ratio.20,47 Skin lipid matrix is very complex in nature and consists of 300 different types of CER. Atomistic simulation of skin lipid bilayer comprising all kind of CERs and FFAs along with CHOL is beyond the current computational power. In order to simulate a realistic lipid bilayer of SC, most abundant ceramide, CER-NS, and free fatty acid (chain length of 24) are used. Earlier, we have developed and exhaustively tested skin model comprising of equimolar mixture of CER, CHOL and FFA both at atomistic20-22 and coarse grained level.43-46 In this study we have used same equimolar model20,22 to mimic the skin SC.

Table 1. System name, size and corresponding number of individual lipid and water molecules used in the study. System

No. of

No. of

No. of

No. of

No. of

Name

CER

CHOL

FFA

total

water

lipids

molecules

System Size

S0

52

52

50

154

5120

4.9 nm x 4.9 nm x 11.62 nm

S1

208

208

200

616

20480

9.79 nm x 9.79 nm x 11.61 nm

S2

832

832

800

2464

81920

19.56 nm x 19.56 nm x 11.62 nm

S3

128

0

0

128

5120

4.9 nm x 4.9 nm x 12.34 nm

S4

64

64

0

128

5120

4.87 nm x 4.87 nm x 11.22 nm

S5

84

0

84

168

5120

4.98 nm x 4.98 nm x 12.15 nm

The force field parameters of CER, CHOL and FFA were taken from our earlier simulation studies.20-22 The CER was modelled using combination of GROMOS and Berger force field.48 The methyl groups of the CER tails were treated as a united atom with a zero net charge. Dihedrals of the hydrocarbon chains of the CER and FFA were represented with RyckaertBellemans potential. For studying polar effects of the CERs, headgroups were expressed in fully atomistic way, and the partial charges on the molecule were taken from the previous simulations.20-22 Previously, Guo et al.49 modified the CHARMM force field parameters for

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CER-NS and CER-NP and showed the comparison with combination of GROMOS and Berger force field.48 The structural properties calculated at skin temperature was similar by using both force field, noticeable difference only occurred at very high temperature. As the CHARMM force field is more computationally expensive as compared to (GROMOS + Berger), and both gives similar bilayer properties at skin temperature, the later one is used in this study. All simulations were carried out in NVT and NPT ensemble using the GROMACS molecular dynamics package.50

-52

The temperature was controlled at a skin temperature of ~ 310 K,

using the Berendsen (equilibration run) and Nose-Hover (production run) thermostat with a time constant of 2 ps. Pressure was controlled by Berendsen and Parrinello-Rahman barostat with a time constant of 6 and 12 ps, respectively and compressibility of 4.0 x 10-5 bar-1 in equilibrium and production run respectively with semi-isotropic coupling. The pressure was controlled in XY and Z direction independently in order to obtain the tension free bilayer. All the bonds in lipid and solute molecules were constrained using LINCS algorithm while SETTLE algorithm was used for water. A time step of 2 fs was used for all simulations. A cut off of 1.2 nm was used for Van der Waals and electrostatic interactions. The long range electrostatic interactions were computed using particle mesh Ewald (PME) method. The bilayer systems were periodic in all three directions. The configuration was sampled at every 10 ps in a production run. The neighbor list was updated at every 10 steps. It is postulated that the electroporation induces nano to micro sized pores in the lipid bilayer.10-14 To capture the effect properly, we have also used bigger bilayer patches. The smaller patch of skin lipid bilayer (154 lipids (S0), CER-52, CHOL-50 and FFA-52, 40 water molecules per lipid molecule), taken from our earlier simulation,20,22 was replicated in X and Y direction to obtain the bigger bilayer patches of 616 (S1) and 2464 lipids (S2). The initial size of each bilayer and number of lipid and water molecules in each bilayer system is shown in Table 1. Both of the bigger patches were subjected to NVT run for 20 ns and followed by 200 ns NPT run. Final equilibrated structures were used for further electroporation simulations. The MD simulation of electroporation of planer lipid membrane can be performed using two techniques a) direct application of electric field across the bilayer normal53 and b) charge imbalance method.54,55

In former method, an electric field of strength, E, is applied

perpendicular to the lipid bilayer plane (here it is XY plane). This is implemented by adding additional force (F = E.qi) to all the charged atoms “i”.53 In the charge imbalance method, a

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net trans membrane voltage difference across the lipid bilayer is created by imposing a net difference of charges between the two baths (each have water and vacuum) surrounding the bilayer. The charge imbalance is achieved by displacing the individual ions within the surrounding baths.54,55 A detailed description of these methods would be outside the scope of this article and for more details reader can refer to this review article.55 In this study we have implemented direct application of electric field along the lipid bilayer method to capture the electroporation of skin SC lipid bilayer. 3. Results & Discussion 3.1 Pore Formation in Skin Lipid Bilayer The bilayer systems used for electroporation simulations are shown in Table 1. The external electric field was applied in the Z direction (normal to the XY plane of bilayer). The pore formation occurs after a certain critical electric field, also known as threshold field. All the fields, above the threshold field, do form pore in the bilayer and are known as porating fields.53 Experimentally it has been shown earlier that the skin permeability decreased several order of magnitude once the trans SC voltage reached a critical value of 30-100 V (applied voltages 100–1500 V)39 Since SC is composed of 100’s of lamellar bilayer in series, the effective trans-membrane voltage for each bilayer corresponds to 1.0-15.0 V.14,39 Based on this background, we have started with electric field of 0.3 V/nm and reached to 1.0 V/nm. To determine the threshold of the electric field, the smallest bilayer system S0 (154 lipids), was used. Majhi et al.53 performed electroporation MD simulation of POPC membrane for 50 ns, while we have simulated bilayer at least for 200 ns. The point to be noted here is that POPC bilayer was in liquid phase53 while the skin lipid bilayers used here are in gel phase,20,22 which inherit the slower dynamics. Since the electroporation process is stochastic in nature at molecular scale, four separate electroporation simulations were performed at each electric field. Initially, the electric field of 0.3 V/nm was applied along the bilayer normal and the simulations were run for 200 ns. Subsequently, the electric field was increased by 0.1 V/nm in each steps and simulations were run for 200 ns at each electric field. The simulation run time at each applied electric field and pore formation time are shown in Table S1 (Please see supporting information). The pore formation was not observed till the electric field of 0.775 V/nm in any of the four systems. The pore formation was observed at electric field of 0.8 V/nm. The pore formation at 0.8 V/nm, in all the four simulations did not happen at same time (Table S1, Please see supporting information). The pore formation was observed in 84.24 ± 2.11 ns at 0.8 V/nm

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electric field. Vasilkoski et al.56 developed relation between pore formation time for a single pore in planer skin lipid bilayer and the trans-membrane voltage across the bilayer. The pore formation time changed from ~ few seconds to ~ some nanoseconds for a trans-membrane voltage of 0.2 to 1.2 V respectively. The skin lipid bilayer thickness is almost ~5 nm, so applied electric field of 0.8 V/nm correspond to trans-membrane voltage of ~ 4 V. Our observation of pore formation time in nano seconds regime is well in agreement with the proposed theories.56 Note that the pore formation time increased in order of magnitude with the decrease in the applied voltage, this might be a reason for which we have not observed pore formation at lower electric field (with in few hundreds nanoseconds of simulation time). Also, the possibility of pore formation below electric field of 0.8 V/nm cannot be fully discarded based on the findings from these MD simulations. Earlier, Tieleman et al. performed MD simulation of smaller (256 lipids) and bigger (more than 2000 lipids) DOPC bilayer in the presence of external electric field of different strengths.57,58 The threshold of 0.33 V/nm and poration time of 50 ns, for a smaller DOPC membrane (256 lipids) was reported.58 Majhi et al.53 reported threshold of 0.3 V/nm and poration time of ~ 10 ns for POPC bilayer (128 lipids). Fernández et al.59 reported threshold of 0.325 V/nm and poration time of 9.75 ns for DOPC bilayer (128 lipid). It is quite interesting to note that, threshold electric field of skin lipid membrane is very high (~0.8 V/nm) as compared to phospholipid membrane53,57-59 and pore formation occurred at very long time scale (~80 ns).

Figure 1. Electroporation of small skin lipid bilayer: Front view of small lipid bilayer (S0) of 154 lipids during one of the electroporation simulation at applied electric field of 1.0 V/nm. Both water and headgroups are shown in “VDW” style of VMD software.66 The chains

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of lipid molecules are shown in “line” style of VMD software. The CER, CHOL and FFA are shown in orange, green and blue color respectively. The water pore is marked by the red color circle. The potential is positive at the top leaflet of the bilayer relative to the bottom leaflet. All snapshots were made using VMD software. The time given in each snapshots do represent only the kinetics of the defects developed in the bilayer after application of electric field. The simulations were performed at higher electric field (>0.8 V/nm) as well. The snapshots of bilayer system at applied electric field of 0.8 V/nm to 1.0 V/nm are shown in Fig. S1 (please see supporting information). The snapshots of the bilayer system (S0, 154 lipids) at applied electric field of 1.0 V/nm are shown in Figure 1. The electroporation process is stochastic in nature and exact value of time represented here are not exactly reproducible. Four independent simulations were run and snapshots of one of them are shown here. The potential is positive at the top leaflet of the bilayer relative to the bottom leaflet. The time given in each snapshots do represent only the kinetics of the defects developed in the bilayer after application of electric field. The pore formation initiation started at ~5500 ps with the formation of single-file like water defects, which penetrated into the skin lipid bilayer. The continuous application of electric field after pore formation lead to the bilayer disruption. The water pore size got bigger and bigger in size with time and eventually covered the whole simulation box. The water pore is mostly surrounded by the CER and FFA molecules (Figure 1). Which implies that the CER and FFA lipids rearranged them self to surround the water pore and create hydrophilic pore, which can felicitate the permeation of hydrophilic or charged molecules. Experimentally, Prausnitz et al.60 used scanning confocal fluorescence microscopy to image the localized regions of calcein transport across human stratum corneum during constant pulsed high voltage exposures. It was shown that the electrically assisted transportation of calcein occurred through intercellular and, to some extent, transcellular pathways into localized regions of stratum corneum.

This experimental observation of

transportation of most of calcein through intercellular domain is well aligned with our observation of pore formation in the skin lipid bilayer. 3.2 Water Pore Stabilization The water pore size got bigger and bigger in each bilayer system (S0, S1 and S2) after the application of porating electric field (Figure S2-S5). To stabilize the water pore, the electric field were reduced significantly, once the pore radius almost reached to ~ 3-4 nm. The system S0 was very small in size (~ 5 nm x 5 nm) and pore size of ~3nm has disrupted the bilayer,

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while system S2 (2464 lipids) was very big in size and was computationally very challenging. Hence, the water pore stabilization simulations were only performed for system S1 (616 lipids, ~9.8 nm x 9.8 nm). The initial structure of bilayer (having pore size ~ 3-4 nm) was taken from the electroporation simulation of S1 system at 1.0 V/nm. The electroporation process is stochastic in nature, so multiple simulations were run at each applied electric field (lower than porating field). The bilayers (five independent S1 systems) were subjected to lower electric field (starting from 0.1 V/nm) and the sustainable electric field was chosen at which the pore survived at least for 50 ns. The applied electric field was increased in steps (0.025 V/nm per step) and simulation was run for 50 ns at each step. The minimum pore stabilization electric field was found to be 0.2 V/nm, which is much higher as compared to phospholipids bilayer.53,62 The porating electric field of 0.33 V/nm and sustaining field of 0.06 V/nm for POPC bilayer has been reported earlier.53 Fernández et al.62 performed electroporation of 40 % CHOL containing DOPC lipid bilayer ( 128 DOPC and 86 CHOL) and reported porating electric field and sustaining electric field of 0.75 V/nm and 0.125 V/nm respectively. The porating field was almost 5 to 10 times higher than the sustaining electric field. In our simulation, we observed similar phenomena and porating field was ~4 times higher than the sustaining electric field. It should be noted that, at electric field of 0.2 V/nm, although the water pore survived for 50 ns, but it happened only in two out of five systems. The pore remained stable at higher electric field up to 0.25 V/nm. Below, the 0.20 V/nm pore vanished and above the 0.25 V/nm, the bilayer got disrupted. The size of pores changed with the applied sustaining electric field. Figure 2 shows the snapshots of big lipid bilayer (S1) of 616 lipids during one of the electroporation simulation at lower sustain electric field of 0.2, 0.225 and 0.25 V/nm. The potential is positive at the top leaflet of the bilayer relative to the bottom leaflet. Five independent simulations were run for each applied electric field (0.2, 0.225 and 0.25 V/nm) and snapshots of the systems, in which pore remained stable, are shown here. The pore size was higher at higher sustaining electric field. The pore shape changed along the bilayer normal z significantly.

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Figure 2. Sustain Electric field for stable pore: Front view of big lipid bilayer (S1) of 616 lipids during one of the electroporation simulation at lower sustain electric field of 0.2, 0.225 and 0.25 V/nm. Both water and headgroups are shown in “VDW” style of VMD software.66 The chains of lipid molecules are removed for the purpose of clarity. The CER, CHOL and FFA are shown in orange, green and blue color respectively. The potential is positive at the top leaflet of the bilayer relative to the bottom leaflet. All snapshots were made using VMD software.66 Five independent simulations were run for each applied electric field (0.2, 0.225 and 0.25 V/nm) and snapshots of the system, in which pore remained stable, is shown here. The initial pore formed bilayer configuration for these simulation were taken from the electroporation simulations (system S1 at electric field of 1.0 v/nm). Here Es, R and d stands for sustaining electric field, pore radius and distance along the bilayer normal.

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To quantify the pore size and shape, the pore radius were also calculated and shown in Figure 2. The details of the pore radius calculation are given in the section S4 of the supporting information. The pore shape was not homogenous in both the directions - along the bilayer normal z and in XY plane. The pore radius R, was highest for sustaining electric field of 0.25 V/nm at any given z position. The pore size changed along the bilayer normal z. In the middle of the bilayer, the pore radius was ~1.5 nm, 1.75 nm and 2.0 nm at sustaining electric field of 0.2 V/nm, 0.225 V/nm and 0.25 V/nm, respectively. Earlier, Fernández et al.62 performed electroporation MD simulations of mixed DOPC and CHOL (60:40 mol %) bilayer and obtained a sustaining electric field range from 0.125 V/nm to 0.175 V/nm. The pore size in XZ and XY direction increased with increase in sustaining electric field. Additional simulation (on sustaining electric field of 0.20-0.25 V/nm) have been performed on bilayers obtained from electroporation simulation at 0.85 and 0.9 V/nm. The snapshots of the sustained pores are shown in Figure S8 (please see supporting information). These simulations were also performed in group of five. In the bilayer system obtained from 0.85 V/nm electroporation simulation, no pore sustained at electric field of 0.25 V/nm in any of five independent simulation. While, pores were stable at all three sustaining electric field in the bilayer system obtained from 0.9 V/nm electroporation system. Experimentally observed phenomena of enhancement of the flux of the drug through skin after the electroporation can be explained in terms of above stable pores. Blagus et al.23 reported that application of electric pulses (70 to 570 V) on the mouse skin resulted in increased in the topical delivery of both doxorubicin and fentanyl drug. Moreover, the flux of both of the drugs increased with the amplitude of the applied electric pulses. Medi et al.32 applied electric pulses at 100, 200, and 300 V on skin and increased the delivery of human parathyroid hormone by 6.9, 16.5, and 20.4 times respectively, as compared with the passive diffusion (control). These experiments shows that the flux increased with increase in the applied electric field. In our simulation, we have observed the pore size become large (~ 2-2.5 nm) at higher sustaining electric field. The bigger pore, results in more pore area and which intern could increase the flux of the molecules. The experimental observation23,32 of higher flux at higher electric field, directly correlates with our observation of large pore size at higher sustaining electric field. Experimentally, Pliquett et al.61 that the transport of negatively charged fluorescent molecules were highly localized. Also, the size and number of these localized regions increased with increase in the applied voltage across the skin. 3.3 Effect of Bilayer Composition on Electroporation of the bilayer.

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We have also performed simulations of mixed lipid bilayer of CER, FFA and CHOL at different molar ratio to check the effect of individual lipid on the electroporation dynamics of the bilayer. Three bilayer systems, only CER bilayer (S3), CER & CHOL (50:50 mol %, S4) and CER & FFA (50:50 mol %, S5), were studied. The details of the systems (S3, S4 and S5) have been given in Table 1. The electroporation MD simulation protocol was applied as mentioned earlier in section 3.1. The simulations were run in the group of four at each applied electric field. The simulation time, poration time and threshold electric field for each bilayer system are tabulated in Table S3 (Please see supporting information). The snapshots of the electroporation of the system S4, S4 and S5 have been shown in Figure S9, Figure S10 and Figure S11 respectively (Please see supporting information). The threshold electric field of 1.1 V/nm, 0.85 V/nm, 0.65 V/nm and 0.8 V/nm was obtained for only CER, CER & CHOL, CER & FFA and mixed (CER, CHOL & FFA) lipid bilayer (Table S1 and Table S3) respectively. In our earlier study of skin lipid mixture20, it was shown that the mixing of FFA in CER bilayer reduces the area compressibility (rigidity) of the bilayer while presence of CHOL increases the rigidity of the bilayer. The area compressibility of bilayer (CER: CHOL: FFA) increased in the order of 0:0:1, 1:0:1, 1:1:1 1:1:0, 1:0:0. The obtained threshold value of electric field for 1:0:1, 1:1:1 1:1:0, 1:0:0 bilayer is 0.65 V/nm, 0.8 V/nm, 0.85 V/nm and 1.1 V/nm respectively. Which shows that the threshold electric field have one to one relation with area compressibility or rigidity of the bilayer. The lipid bilayer composition plays a critical role in determining its threshold electric field. The CER, CHOL and FFA have very small head groups as compared to phospholipid lipid bilayer20 and consist of very few atoms having partial charge. The less charge and small headgroup could be one possible reason for high threshold electric field in skin lipid bilayer. Earlier, Tieleman et al.58 performed MD simulation of both DOPC bilayer and octane layer in the presence of external electric field of different strengths. It was shown that pore formation occurred both in lipid bilayer and octane layer but the threshold field was 0.33 V/nm and 0.8 V/nm in lipid bilayer and octane layer, respectively. It was also shown that the pore formation mechanism in both lipid and octane layer was same, the pore formation begun with formation of single water file defects.58 It was postulated that the pore formation was mainly driven by the local electric gradients near the lipid or octane/water interface and lipid headgroups increased the poration rate.58 Polak et al.63 performed MD simulation of dipalmitoyl-phosphatidylcholine (DPPC), diphytanoyl-phosphocholine-ester (DPhPC-ester) and diphytanoylphosphocholine-ether (DPhPC-ether) lipid bilayers in presence of external

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electric field. The threshold electroporation voltage was found to be headgroup depended and the value of threshold voltage for DPPC, DPhPC-ester and DPhPC-ether bilayers were 1.82.2 V, 2.3-2.7 V and between 3.0-3.4 V, respectively. Gurtovenko et al.64 performed atomic MD simulations of POPC and POPE in the presence of external electric field. The threshold electric field of 0.4 V/nm and 0.6 V/nm was reported for POPC and POPE lipid bilayer respectively.

Figure 3. Effect of electric field on poration time: The poration time is referred to the time after which water file linked both of the leaflet of the bilayer as shown in the snapshot. At each applied porating electric field, four independent simulations were run. The average of these four runs has been shown here. The simulations were performed for S0, S1, S3, S4 and S5 systems.

Figure 3 shows the effect of applied porating field on poration time of each lipid bilayer system. The poration time, for a given porating field, was higher in CER bilayer and lower in CER & FFA bilayer. This could be due to the lateral packing of the lipid bilayer. In our earlier study20 of skin lipid bilayer mixture it was shown that the lateral packing of chains of CER bilayer (at skin temperature ~310 K) remains in hexagonal phase while in presence of FFA and CHOL, the packing is not perfect hexagonal.20 Also, small sized CHOL sits in between the CER molecules and changes the packing significantly. It should be noted that,

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although in presence of CHOL (in CER bilayer) the gel to liquid phase transition increases as compared to CER bilayer, but the threshold electric field is lower. It implies that, local defect present in the packing near the head group and in the interior of the bilayer plays important role in determining the threshold electric field. Earlier, Fernández et al.59 performed electroporation MD simulations of both DOPC bilayer and mixed DOPC and CHOL (60:40 mol %) bilayer and reported threshold field of 0.325 V/nm and 0.775 V/nm respectively. There are two important points to be noted, first, the threshold electric field was minimum for system S5 (1:0:1) and maximum for S3 (1:0:0) and second, for a given applied electric field, the poration time was longer in S3 (1:0:0) system as compared to S4 (1:1:0) system.

3.4 Pore Growth Rate The pore formation in lipid bilayer occurs after a certain critical electric field (threshold field). All the applied fields, above this threshold field, are known as porating fields. The pore growth rate gives an estimation of lipid bilayer dynamics after the application of electric field. The details of calculation of pore growth rate has been provided in the section S8 (please supporting information). The pore growth rate refers to change in the pore radius within the given simulation time. In each electroporation simulation, the pore radius changed along the bilayer normal as shown in Fig. S7. The pore growth was also calculated along the bilayer normal z. Figure S12 and S13 shows the pore size along the bilayer normal z, at two different time interval in S0, S4 and S5 bilayer system at each porating electric field. (Please see supporting information). The pore growth rate, along the bilayer normal z, in each bilayer system at different porating electric field is shown in Figure S14 (please see supporting information). At each applied porating electric field, four independent simulations were run. The average of these four runs has been shown here. The pore growth rate at each applied porating electric field is the average of the pore growth rate (Figure S14) along the bilayer normal z. The change in pore growth rate, in bilayer system S0, S4 and S5, with applied porating electric field, has been shown in Figure 4. The pore growth depends both on the composition of the bilayer. Manjhi et al.53 reported pore growth rate of POPC lipid bilayer (128 lipids) of ~ 0.002 nm/ps, 0.014 nm/ps and 0.022 nm/ps at the temperature of 275 K (gel phase), 300 K and 350 K (liquid phase) respectively. Here, we have obtained a pore growth rate of ~0.01 nm/ps at 310 K at porating electric field of 0.8 V/nm. The obtained order of the pore growth rate are similar to that obtained in earlier simulation of POPC lipid bilayer.53 For a given porating electric field, the pore growth rate was higher in mixed CER, CHOL & FFA ( S0) bilayer as compared to ACS Paragon Plus Environment

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CER & CHOL (S4) and CER & FFA (S5) bilayer. We have not observed any clear difference between the pore growth rate of system S4 and S5.

Figure 4. Pore Growth rate in bilayer on application of porating electric field: All the fields, above the threshold field, which form pore in the bilayer are referred as porating fields. The pore growth rate were calculated based on the change in the pore radius within given simulation time. The pore growth were calculated for system S0, S4 and S5. At each applied porating electric field, four independent simulations were run. The average of these four runs has been shown here. 3.5 Bilayer Reformation After the application of porating electric fields, the pore formed in the bilayer system within ~ 2ns-100 ns, based on the applied electric field and bilayer composition. Additional simulations have been performed to see the effects when porating electric field is completely removed. These simulation were only performed for system S1 (616 lipids, ~9.8 nm x 9.8 nm). The initial structures (bilayer having pore ~ > 3 nm) were taken from the electroporation simulation at porating electric field of 0.8, 0.85, 0.9 and 1.0 V/nm. Three independent simulations (20 ns each) were run for each structure (3x4 = 12 simulations) without application of electric field. Figure 5 shows the snapshots of the one of the simulation run. The water pore disappeared completely with in first ~15-20 ns of the simulation run. The pore

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disappearance time were not same in all 12 simulations but pore disappearance were observed in each simulation. It is interesting to note that, at lower porating electric field, pore closing was much faster than pore opening. The pore closing dynamics was almost independent of applied electric field.

Figure 5. Reforming of skin lipid bilayer: Front view of big lipid bilayer (S1) of 616 lipids during simulation after removing the electric field. The initial pore formed bilayer configuration for these simulation were taken from the electroporation simulations (system S1 at electric field of 1.0 v/nm). Both water and headgroups are shown in “VDW” style of VMD software.66 The chains of lipid molecules are shown in “line” style of VMD software. The CER, CHOL and FFA are shown in orange, green and blue color respectively. The water pore is marked by the red color circle. All snapshots were made using VMD software.66 Tarek et al.65 performed MD simulation of DMPC lipid bilayer and DMPC lipid bilayer containing a peptide nanotube channel, under the external electric field of 0.5 V/nm and 1.0 V/nm. They have shown that in both of the cases, the water wires and water channels formed across the membrane and the internal structures of the peptide nanotube assembly remained unchanged. After switching the external electric field, the bilayer got resealed within few ACS Paragon Plus Environment

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nanoseconds in both of the cases.65 Manjhi et al.53 reported that at liquid crystalline phase, both POPC and DPPC bilayer got resealed in less than 10 ns, while at very low temperature ~ 230 K, pore closure did not happen even after 50 ns in both POPC and DPPC bilayer. In our simulations, the pore closure dynamics was slower as compared to previously reported53,65 pore closure dynamics of phospholipid bilayer. The reason could be that the phospholipids were in liquid crystalline phase,53,65 while mixed skin lipid bilayer was in gel phase. It has been shown earlier that gel phase pore closure dynamics are slow as compared to liquid crystalline phase dynamics.53 3.6 Drug delivery using Electroporation In past, the electroporation has been successfully used for the delivery of the drug through skin.6,10-15,24-34 We have also performed simulation of drug permeation in the presence of external electric field. The initial bilayer structure was taken from the final structure of 50 ns simulation performed at sustaining electric field of 0.25 V/nm. Four benzoic acid (drug) molecules were inserted manually in the upper part of the water layer. The overlapped water molecules, with drug molecule, were removed. The system was energy minimized and further subjected to NPT MD run for 25 ns at sustaining electric field of 0.25 V/nm. Four independent simulations were performed with different initial configuration of drug molecules. Figure 6 shows the snapshots of one of the system simulated with drug molecules at sustaining electric field of 0.25 V/nm. Three out of fours molecules remained on the head group of lipid bilayer. Only one molecules entered in the water pore. It should be noted that, permeation event through the pore are stochastic in nature at atomistic scale. In two, out of four simulations, the pore size increased and bilayer got disrupted. Only in two simulations, both bilayer and pore were stable. The drug molecule permeation though the water pore was observed in only one simulation, which is shown in Figure 6. The permeation of the drug molecule through water pore was instantaneous and occurred in ~ 20 ns. We have performed additional 200 ns MD simulation of small bilayer system (S0) in the presence of 4 benzoic acid molecules. During the simulation, not a single drug molecule penetrated in the lipid bilayer. All drug molecules were adsorbed on the lipid head group. It shows that passive permeation of the benzoic acid drug molecules is either not possible or it is very slow to be captured by these small time scale atomistic MD simulations. However, on application of the sustaining electric field, the permeation happened within ~20 ns.

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Figure 6. Delivery of drug through pore: Front view of big lipid bilayer (S1) of 616 lipids during the simulation at sustain electric field in presence of drug (benzoic acid) molecules. The initial pore formed bilayer configuration for the simulation was taken from the electroporation simulations (system S1 at electric field of 1.0 v/nm). Water, drug and headgroups are shown in “VDW” style of VMD software.66 The chains of lipid molecules are not shown for the purpose of clarity. The CER, CHOL, FFA and drug molecules are shown in orange, green, blue and while color respectively. The drug molecule is marked by the red color eclipse. All snapshots were made using VMD software.66 The time given in each snapshots, do represent the kinetics of permeation of the drug molecule through the hydrophilic pore of bilayer. The permeation process is stochastic in nature and exact value of time represented here are not exactly reproducible. Four independent simulations were run and snapshots of one of them are shown here. Our observations are also supported by some of the experimental findings as well. Blagus et al.23 performed electroporation of mouse skin with non-invasive multi array electrodes. The applied amplitudes of electric pulses was in the range of 70 to 570 V. The patches of

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doxorubicin and fentanyl were applied to the skin before and after electroporation.23 The topical delivery of both of the drug increases with the amplitude of the applied electric pulses. Denet et al.24 applied 400 V of electric pulses of duration of 1 ms for 10 hours on human skin and reported that the flux of timolol maleate (lipophilic drug) increased about five times with electroporation protocol as compared to obtained in passive diffusion case. Mori et al.25 studied the transport of ionic compound benzoic acid through hairless rat skin using electroporation protocol. The cumulative permeated drugs over 4 hours was 196.94 ± 10.80 nmol/cm2 in passive diffusion (control) while application of one hour of electroporation increased the flux to 439.26 ± 38.1, 517.26 ± 50.0, and 1230.26 ± 128.3 nmol/cm2 in case of needle–needle, ring–needle and plate–plate electrode systems respectively.25 Mori et al.26 reported that the electroporation protocol enhanced the flux of non-ionic compound mannitol through hairless rat skin and postulated that pore production in the skin membrane resulted in higher flux of mannitol. Vanbever et al.27 applied both passive diffusion and electroporation (with an exponentially decaying pulse) technique for the transdermal delivery of metoprolol through full thickness hairless rat skin. It was reported that application of electric pulses increased metoprolol permeation as compared to passive diffusion through untreated skin and the applied voltage controlled the quantity of drug delivered.27 Sharma et al.28 reported the effect of number of pulses, voltage and pulse length during electroporation on transdermal delivery of terazosin hydrochloride to hairless rat skin. Sammeta et al.29 used electroporation to increase percutaneous penetration of doxepin across porcine skin. Sung et al.30 implemented electroporation protocol (1 pulse/30 sec for 10 mins at pulse voltage of 300 V and pulse duration of 200 ms) on rat skin to enhance the transdermal flux of nalbuphine drug. The passive penetration of nalbuphine drug through intact hairless rat skin was 51.16 ±5.50 nmol/cm2 while electroporation increased flux to 164.13 ± 25.31 nmol/cm2.30 Hu et al.31 also reported that the application of electroporation increased the flux of tetracaine drug through rat skin significantly. The transdermal flux value for the drug after electroporation was 54.6 ± 6 µg/cm2/h while passive flux was 8.29 ± 5 µg/cm2/h.31 Medi et al.32 reported that application of pulses at 100, 200, and 300 V increased delivery of human parathyroid hormone by 6.9, 16.5, and 20.4 times respectively, as compared with the passive diffusion control. It is also shown that skin properties have been altered at such high voltages which have resulted in nonlinear dependence of flux on applied voltage.32 The obtained molecular mechanism of electroporation of skin could help in designing targeted experiments. The drug molecules which needs to be tested experimentally, first can be tested on the given model (CER: CHOL: FFA, 1:1:1) under sustaining electric field. But ACS Paragon Plus Environment

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there are inherit challenges, first the model is only comprising single kind of CER (CER-NS), and since skin lipid bilayer has multiple kind of CERs, the model is still very simple. Moreover, presence of other CERs, will change the structural organisation of the lipids and further the pore formation kinetics. Another challenge is the system size effect, which comes with the MD simulation technique presented here (Please see supporting information section 2). The pore growth rate and poration time depends upon the system size. Given these challenges, still these kind of simulations before or with experiments could provide some molecular level details which could be used for better design of experiments. For a given skin composition and size of drug molecule, the combination of pore formation time and pore growth rate can be used to know aproiri the desired electric field and time for application of electric field.

Conclusion In this study, we have performed extensive atomistic MD simulation of skin lipid bilayer in presence of varying external electric field. The effect of bilayer size and it composition on the bilayer poration time and threshold electric field has been shown. The threshold electric field was maximum for CER bilayer while minimum for CER & FFA bilayer. The poration time decreased with increase in the bilayer system size for mixed CER, FFA & CHOL bilayer. The poration time, for a given external electric field, was maximum for CER bilayer and minimum for CER & FFA bilayer. The sustaining electric field was in the range of 0.20-0.25 V/nm. The pore radius was inhomogeneous both in X and Y direction and also changed along the bilayer normal. The pore radius increased with increase in sustaining electric field. Experimentally observed phenomena of increase in the flux of drug with increase in electric field, was correlated with the pore size at sustaining electric field. The electroporation process is reversible. The pore opening dynamics highly depends upon the strength of applied electric field. While, the pore closing dynamics of the lipid bilayer was highly independent of applied electric field. The atomistic simulation shows that interfacial water played a key role in the electroporation of skin lipid bilayer. In this study, on application of porating electric field, the water defect generated at lipid-water interface. These defects then entered in the interior of the bilayer and finally formed the water pore by combining opposing bilayer leaflet. We have also shown the permeation mechanism of charged drug molecule in the presence of external electric field. The benzoic acid drug molecules penetrated through the water pore with in the 20 ns, while no permeation was

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observed in passive MD simulation in after 200 ns. The provided molecular mechanism could help in optimizing/designing the electroporation experiments for better drug delivery.

Supporting Information. S1. Electroporation of small bilayer (S0). S2. Effect of Bilayer Size of Electroporation Dynamics. S3. Electroporation of ultra-bilayer system (S2). S4. Multi pore formation in big bilayer system during Electroporation S5. Pore Radius Calculation. S6. Sustain Electric field after electroporation. S7. Effect of Bilayer Composition on Electroporation of the bilayer. S8. Port Growth Rate Calculation.

Acknowledgement



Authors would like to thank High Performance Computing at Tata Consultancy Services (TCS) for providing access to EKA Super computer.



We would also like to thank Mr. K Ananth Krishanan, CTO, TCS, for his constant encouragement and support during this project

ORCID

• •

Rakesh Gupta Beena Rai

0000-0003-4144-9886 0000-0002-8637-7778

References 1. Perumal, O.; Murthy, S. N.; Kalia, Y. N. Turning Theory into Practice: The Development of Modern Transdermal Drug Delivery Systems and Future Trends. Skin Pharmacol. Physiol. 2013, 26, 331-342. 2. Paudel, K. S.; Milewski, M.; Swadley, C. L.; Brogden, N. K.; Ghosh, P.; Stinchcomb, A. L. Challenges and Opportunities in Dermal/Transdermal Delivery. Therapeutic delivery 2010, 1, 109-131. 3. Elias, P. M. Epidermal Lipids, Barrier function, and Desquamation. J. Invest. Dermatol.

1983, 80, 44s-49s. 4. Michaels, A. S.; Chandrasekaran, S. K.; Shaw, J. E. Drug Permeation through Human Skin: Theory and In-Vitro Experimental Measurement. AIChE J. 1975, 21, 985−996. 5. Mathur, V.; Satrawala, Y.; Rajput, M. Physical and Chemical Penetration Enhancers in Transdermal Drug Delivery System. Asian J. Pharm. 2010, 4, 173.

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6. Schoellhammer, C. M.; Blankschtein, D.; Langer, R. Skin permeabilization for transdermal drug delivery: recent advances and future prospects. Expert Opin Drug Deliv. 2014,11,393-407. 7. Kwak, S.; Brief, E.; Langlais, D.; Kitson, N.; Lafleur, M.; Thewalt, J. Ethanol perturbs lipid organization in models of stratum corneum membranes: an investigation combining differential scanning calorimetry, infrared and 2 H NMR spectroscopy. BBA Biomembranes 2012, 1818, 1410-1419. 8. Karrie, M. Dimethyl Sulfoxide: An Effective Penetration Enhancer for Topical Administration of NSAIDs. The Physician and Sportsmedicine. 2011, 39, 75-82. 9. Barbosa-Barros, L.; Rodríguez, G.; Barba, C.; Cócera, M.; Rubio, L.; Estelrich, J.; LópezIglesias, C.; De La Maza, A.; López, O. Bicelles: Lipid Nanostructured Platforms with Potential Dermal Applications. Small 2012, 8, 807–818. 10. Kalluri, H.; Banga, A.K. Transdermal delivery of proteins. Aaps Pharmscitech. 2011, 12, 431-41. 11. Prausnitz, M.R.; Langer, R. Transdermal drug delivery. Nature biotechnology. 2008, 26, 1261-1268. 12. Prausnitz, M.R. Do high-voltage pulses cause changes in skin structure? J. Control. Release. 1996, 40, 321–326. 13. Edwards, D.A.; Prausnitz, M.R.; Langer, R.; Weaver, J.C. Analysis of enhanced transdermal transport by skin electroporation. J. Control. Release. 1995, 34,211-221. 14. Denet, A. R.; Vanbever, R.; and Préat, V. Skin Electroporation for Transdermal and Topical Delivery. Adv. Drug Delivery Rev. 2004, 56, 659–674. 15. Pliquett, U.; Langer, R.; and Weaver, J. C. Changes in the Passive Electrical Properties of Human Stratum Corneum Due to Electroporation. Biochim. Biophys. Acta. 1995, 12392, 111–121 16. Satkauskas, S.; Andre, F.; Bureau, M. F.; Scherman, D.; Miklavcic, D.; and Mir, L. M. Electrophoretic Component of Electric Pulses Determines the Efficacy of In Vivo DNA Electrotransfer. Hum. Gene Ther. 2005, 1610, 1194–1201 17. Pliquett, U.; Gallo, S.; Hui, S. W.; Gusbeth, C.; Neumann, E. Local and Transient Changes in Stratum Corneum at High Electric Fields: Contribution of Joule Heating. Bioelectrochemistry 2005, 671, 37–46. 18. Golden, G. M.; Guzek, D. B.; Kennedy, A. H.; Mckie, J. E.; and Potts, R. O. Stratum Corneum Lipid Phase-Transitions and Water Barrier Properties. Biochemistry 1987, 268, 2382–2388. ACS Paragon Plus Environment

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19. Potts, R. O.; Francoeur, M. L. Lipid Biophysics of Water Loss Through the Skin. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3871–3873. 20. Gupta, R.; Rai, B. Molecular Dynamics Simulation Study of Skin Lipids: Effects of the Molar Ratio of Individual Components over a Wide Temperature Range. J. Phys. Chem. B 2015, 119, 11643-11655. 21. Gupta, R.; Dwadasi, B.S.; Rai, B. Molecular Dynamics Simulation of Skin Lipids: Effect of Ceramide Chain Lengths on Bilayer Properties. J. Phys. Chem. B 2016, 120, 1253612546. 22. Gupta, R.; Sridhar, D.B; Rai, B. Molecular Dynamics Simulation Study of Permeation of Molecules through Skin Lipid Bilayer. J. Phys. Chem. B 2016, 120, 8987-8996. 23. Blagus, T.; Markelc, B.; Cemazar, M.; Kosjek, T.; Preat, V.; Miklavcic, D.; Sersa, G. In vivo real-time monitoring system of electroporation mediated control of transdermal and topical drug delivery. J. Control. Release 2013, 172, 862–871. 24. Denet, A. R.; Préat, V. Transdermal delivery of timolol by electroporation through human skin. J. Control. Release 2003, 88, 253–262. 25. Mori, K.; Hasegawa, T.; Sato, S.; Sugibayashi, K. Effect of electric field on the enhanced skin permeation of drugs by electroporation. J. Control. Release, 2003, 90, 171–179. 26. Mori, K.; Watanabe, T.; Hasegawa, T.; Sato, H.; Sugibayashi, K.; Morimoto, T. Electroporation on the in vitro skin permeation of mannitol. Drug Deliv. Syst. 1999, 14, 101–106 27. Vanbever, R.; Lecouturier, N.; Preat, V. Transdermal delivery of metoprolol by electroporation. Pharm. Res. 1994, 11, 1657–1662. 28. Sharma, A.; Kara, M.; Smith, F.R.; Krishnan, T.R. Transdermal drug delivery using electroporation. I. Factors influencing in vitro delivery of terazosin hydrochloride in hairless rats. J. Pharm. Sci. 2000, 89, 528–535. 29. Sammeta, S.M.; Vaka, S.R.; Murthy, S.N. Transcutaneous electroporation mediated delivery of doxepin-HPCD complex: A sustained release approach for treatment of postherpetic neuralgia. J. Control. Release 2010, 142,361–367. 30. Sung, K.C.; Fang, J.-Y.; Wang, J. J.; Hu, O.Y.P. Transdermal delivery of nalbuphine and its prodrugs by electroporation. Eur. J. Pharm. Sci. 2003, 18, 63–70. 31. Hu, Q.; Liang, W.; Bao, J.; Ping, Q. Enhanced transdermal delivery of tetracaine by electroporation. Int. J. Pharm. 2000, 202, 121–124. 32. Medi, B.M.; Singh, J. Electronically facilitated transdermal delivery of human parathyroid hormone (1–34). Int J Pharm. 2003, 263, 25–33 ACS Paragon Plus Environment

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33. Prausnitz, M.R.; Edelman, E.R.; Gimm, J.A.; Langer, R.; Weaver, J.C. Transdermal delivery of heparin by skin electroporation. Biotechnology 1995, 13, 1205–1209. 34. Petchsangsai, M.; Rojanarata, T.; Opanasopit, P.; Ngawhirunpat, T. The combination of microneedles

with

electroporation

and

sonophoresis

to

enhance

hydrophilic

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