Mechanistic analysis of electroporation-induced cellular uptake of macromolecules

Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.     

Electroporation of abalone sperm enhances sperm-DNA association

The ability of sperm from the black-footed abalone Haliotis iris to take up foreign DNA in solution has been demonstrated. The efficiency of DNA uptake is related to the conditions of electroporation, including field strength (625 V cm⁻¹, 1000 V cm⁻¹), pulse length (18.6 ms, 27.4ms) and number of pulses (1, 2), and DNA concentration (20, 100 μg ml⁻¹). Sperm motility decreased with increased field strength and pulse number. At a field strength of 625 V cm⁻¹, neither the pulse length nor pulse number enhanced DNA uptake. A 40% enhancement in DNA uptake was observed when the sperm were shocked at 1000 V cm⁻¹ with two long pulses (27.4 ms each). Linear regression analysis revealed that pulse number ( p = 0.013) and field strength ( P =0.039) were the most important factors in sperm–DNA interaction. Higher DNA concentration enhanced sperm DNA uptake irrespective of field strength, pulse length and pulse number. The optimal electroporation conditions for DNA uptake were 1000 V cm', with two pulses of 27.4 ms each, and a DNA concentration of 100 μg ml⁻¹.     

The effect of pulse field strength on electric field stimulated biosorption of uranium by Kluyveromyces marxianus IMB3

Summary Improved biosorption of uranium by Kluyveromyces marxianus IMB3 biomass was achieved by increasing the electric field strength of delivered pulses from 1.25kV/cm to 2.5kV/cm. Although this had little or no effect on the maximum biosorption capacity (q_max), at low concentrations of uranium the amount bound to the biomass increased from 70 to 140mg uranium/g biomass. Significant increases in the maximum biosorption capacities (119–180 mg uranium/g biomass) were observed when the pulse field strength was increased from 2.5kV/cm to 3.25kV/cm.     

Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes

Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.     

The Effect of Pulsed Magnetic Field on the Molecular Uptake and Medium Conductivity of Leukemia Cell

The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Owing to the numerous applications, the introduction of non-permeate molecules into biologic cells has drawn considerable attention in the past years. The aim of our study was to investigate the effect of time-varying magnetic field on transmembrane molecular transport by measuring bleomycin cytotoxicity and conductivity modifying in K562 cells. The cells were exposed to magnetic pulses of 2.2 T strength peak and about 250-μs duration via Magstim stimulator and double 70-mm coil. Three different frequencies of 0.25, 1, and 10 Hz pulses for 56,112, and 28 numbers of pulses, respectively, were applied (nine experimental groups) and uptake and conductivity was measured in each group. Our results show that time-varying magnetic field increase transmembrane molecular transport and media conductivity; this enhancement is greater for 28 pulses with 1 Hz frequency. The observed uptake enhancement due to magnetic exposure is considerable.     

Effect of ion binding on protein transport through ultrafiltration membranes

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.     

Structural origins of the insulin-mimetic activity of bis(acetylacetonato)oxovanadium(IV)

We have investigated the interaction of bis(acetylacetonato)oxovanadium(IV) (VO(acac)(2)) with bovine serum albumin (BSA) by EPR and angle-selected electron nuclear double resonance, correlating results with assays of glucose uptake by 3T3-L1 adipocytes. EPR spectra of VO(acac)(2) showed no broadening in the presence of BSA; however, electron nuclear double resonance titrations of VO(acac)(2) in the presence of BSA were indicative of adduct formation of VO(acac)(2) with albumin of 1:1 stoichiometry. The influence of VO(acac)(2) on uptake of 2-deoxy-d-[1-(14)C]glucose by serum-starved 3T3-L1 adipocytes was measured in the presence and absence of BSA. Glucose uptake was stimulated 9-fold in the presence of 0.5 mm VO(acac)(2), 17-fold in the presence of 0.5 mm VO(acac)(2) plus 1 mm BSA, and 22-fold in the presence of 100 nm insulin. BSA had no influence on glucose uptake, on the action of insulin, or on glucose uptake in the presence of VOSO(4). The maximum insulin-mimetic effect of VO(acac)(2) was observed at VO(acac)(2):BSA ratios less than or equal to 1.0. Similar results were obtained also with bis(maltolato)oxovanadium(IV). These results suggest that the enhanced insulin-mimetic action of organic chelates of VO(2+) may be dependent on adduct formation with BSA and possibly other serum transport proteins.     

A quantitative study of electroporation showing a plateau in net molecular transport.

Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

The effects of intense submicrosecond electrical pulses on cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.     

Paradoxical loss of excitation with high intensity pulses during electric field stimulation of single cardiac cells

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

Conformational flexibility of membrane proteins in electric fields. I. Ultraviolet absorbance and light scattering of bacteriorhodopsin in purple membranes

Bacteriorhodopsin of halobacterial purple membranes exhibits conformational flexibility in high electric field pulses (1-30 x 10(5) V m(-1), 1-100 micros). High-field electric dichroism data of purple membrane suspensions indicate two kinetically different structural transitions within the protein; involving a rapid (approximately 1 micros) concerted change in the orientation of both retinal and tyrosine and/or tryptophan side chains concomitant with alterations in the local protein environment of these chromophores. as well as slower changes (approximately 100 micros) of the microenvironment of aromatic amino acid residues concomitant with pK changes in at least two types of proton-binding sites. Light scattering data are consistent with the maintenance of the random distribution of the membrane discs within the short duration of the applied electric fields. The kinetics of the electro-optic signals and the steep dependence of the relaxation amplitudes on the electric field strength suggest a saturable induced-dipole mechanism and a rather large reaction dipole moment of 1.1 x 10(-25) C m ( = 3.3 x 10(4) debye) per cooperative unit at E = 1.3 x 10(5) V m(-1), which is indicative of appreciable cooperativity in the probably unidirectional transversal displacement of ionic groups on the surfaces of and within the bacteriorhodopsin proteins of the membrane lattice. The electro-optic data of bacteriorhodopsin are suggestive of a possibly general, induced-dipole mechanism for electric field-dependent structural changes in membrane transport proteins such as the gating proteins in excitable membranes or the ATP synthetases.     

Development of a biomimetic nanoporous membrane for the selective transport of charged proteins

We report the use of a micrometer-thick platinum-coated nanoporous membrane for the separation of differently charged proteins. A high field strength of about 25 kV m(-1) was applied, using very low transmembrane potentials of +/-1.5 V between the platinum-coated membranes. The system mimics the cell membrane function of facilitated transport for specific solutes. The selectivity for Lys:BSA:Mb in a mixed protein solution could be tuned readily between the flux ratios of 2:2:1 and 96:1:12 respectively, by simple variation of the transmembrane potentials from +1.5 V to -1.5 V. The experimental fluxes agreed closely with calculated fluxes derived from a simple electrophoresis-potential shielding model at favourable transmembrane potentials.     

Transport of p-aminohippuric acid, uric acid and glucose in highly purified rabbit renal brush border membranes.

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%. Transport of D-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3--37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 muM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

水曲柳幼苗根系在不同浓度NH4NO3溶液中水流导度的变化

水分吸收过程是根系重要的生理过程。水孔蛋白在根系水分径向运输中起着重要的作用,根系水流导度(L_p)的测定是研究水孔蛋白的重要途径。该研究采用压力流的方法,对相同生长条件下的水曲柳(Fraxinus mandshurica)幼苗根系进行研究,测定了根系在去离子水和不同浓度NH₄NO₃溶液中的L_p。结果表明:未经处理的水曲柳幼苗根系,L_p随NH₄NO₃浓度的增加而上升,而且NH₄NO₃溶液中的L_p比去离子水中的L_p平均高77%;经HgCl₂处理后,水曲柳幼苗根系的L_p仍然随NH₄NO₃浓度的增加而增大,但是根系L_p在去离子水下降了22%,而在NH₄NO₃溶液中下降了68%,与以前的研究相比发现,经HgCl₂处理后,以营养液为吸水基质的根系L_p的降低值普遍高于以去离子水为基质的试验。因此,基质中养分离子的存在对根系中水孔蛋白活性产生了重要的影响,进而影响根系水分的吸收过程。     

Albumin extravasation rates in tissues of anesthetized and unanesthetized rats

Bovine serum albumin (BSA) labeled with 131I was injected intravenously in chronically prepared, unanesthetized rats and into pentobarbital-anesthetized rats that had received 2 ml 5% BSA to help sustain plasma volume. Initial uptake rates (clearances) in skin, skeletal muscles, diaphragm, and heart (left ventricle) were measured over 1 h. BSA labeled with 125I was injected terminally to correct for intravascular 131I-BSA. Observed clearances were in the following order in both groups of animals: heart much greater than diaphragm approximately equal to skin greater than resting skeletal muscles. Differences between unanesthetized and anesthetized animals were small and inconsistently directed. Our results suggest that the lower albumin clearances reported in the literature for anesthetized rats are not the result of their immobility or any direct effect of anesthesia on albumin transport in these tissues. The lower transport rates appear to result indirectly from changes produced by anesthesia and/or surgery in controllable parameters such as plasma volume and intravascular protein mass.     

Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility.

A simple micropipet technique was used to determine the critical electric field strength for membrane breakdown as a function of the applied membrane tension for three different reconstituted membranes: stearoyloleoylphosphatidylcholine (SOPC), red blood cell (RBC) lipid extract, and SOPC cholesterol (CHOL), 1:1. For these membranes the elastic area expansivity modulus increases from approximately 200 to 600 dyn/cm, and the tension at lysis increases from 5.7 to 13.2 dyn/cm, i.e., the membranes become more cohesive with increasing cholesterol content. The critical membrane voltage, Vc, required for breakdown was also found to increase with increasing cholesterol from 1.1 to 1.8 V at zero membrane tension. We have modeled the behavior in terms of the bilayer expansivity. Membrane area can be increased by either tensile or electrocompressive stresses. Both can store elastic energy in the membrane and eventually cause breakdown at a critical area dilation or critical energy. The model predicts a relation between tension and voltage at breakdown and this relation is verified experimentally for the three reconstituted membrane systems studied here.     

Studies of in vivo electropermeabilization by gamma camera measurements of (99m)Tc-DTPA

A protocol was developed to study the drug uptake from in vivo electropermeabilization at different settings of parameters influencing the uptake efficiency. Radiolabelled diethylenetriaminepentaacetic acid (DTPA) was used to trace the distribution and internalization of a hydrophilic drug after in vivo electropermeabilization. Skeletal muscle tissue in rat was treated with permeabilizing electric pulses before or after intravenous administration of (99m)Tc-DTPA. The drug accumulation in the treated volume was subsequently evaluated with a scintillation camera. The dependence of uptake on field strength and duration of the applied electric pulses was investigated for exponentially decaying pulses and square wave pulses. Further, the uptake dependence on time interval between injection and pulsation was studied as well as the uptake dependence on the number of pulses applied in a single electropermeabilization treatment. Dynamic gamma camera studies were performed to quantify the time scale of the drug uptake in electropermeabilized tissue.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.