M E T H O D S I N M O L E C U L A R M E D I C I N E TM Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery Electrically Mediated Delivery of Molecules to Cells Edited by Mark J Jaroszeski Richard Heller Richard Gilbert Humana Press Principles of Electroporation and Transport 1 Principles of Membrane Electroporation and Transport of Macromolecules Eberhard Neumann, Sergej Kakorin, and Katja Toensing Introduction The phenomenon of membrane electroporation (ME) methodologically comprises an electric technique to render lipid and lipid–protein membranes porous and permeable, transiently and reversibly, by electric voltage pulses It is of great practical importance that the primary structural changes induced by ME, condition the electroporated membrane for a variety of secondary processes, such as, for instance, the permeation of otherwise impermeable substances Historically, the structural concept of ME was derived from functional changes, explicitly from the electrically induced permeability changes, which were indirectly judged from the partial release of intracellular components (1) or from the uptake of macromolecules such as DNA, as indicated by electrotransformation data (2–4) The electrically facilitated uptake of foreign genes is called the direct electroporative gene transfer or electrotransformation of cells Similarly, electrofusion of single cells to large syncytia (5) and electroinsertion of foreign proteins (6) into electroporated membranes are also based on ME, that is, electrically induced structural changes in the membrane phase For the time being, the method of ME is widely used to manipulate all kinds of cells, organelles, and even intact tissue ME is applied to enhance iontophoretic drug transport through skin—see, for example, Pliquett et al (7)—or to introduce chemotherapeutics into cancer tissue—an approach pioneered by L Mir (8) From: Methods in Molecular Medicine, Vol 37: Electrically Mediated Delivery of Molecules to Cells Edited by: M J Jaroszeski, R Heller, and R Gilbert © Humana Press, Inc., Totowa, NJ Neumann, Kakorin, and Toensing Medically, ME may be qualified as a novel microsurgery tool using electric pulses as a microscalpel, transiently opening the cell membrane of tissue for the penetration of foreign substances (4,9,10) The combination of ME with drugs and genes also includes genes that code for effector substances such as interleukin-2 or the apoptosis proteins p53 and p73 Therefore, the understanding of the electroporative DNA transport is of crucial importance for gene therapy in general and antitumor therapy in particular Clearly, goal-directed applications of ME to cells and tissue require knowledge not only of the molecular membrane mechanisms, but potential cell biological consequences of transient ME on cell regeneration must be also elucidated, for instance, adverse effects of loss of intracellular compounds such as Ca2+, ATP, and K+ Due to the enormous complexity of cellular membranes, many fundamental problems of ME have to be studied at first on model systems, such as lipid bilayer membranes or unilamellar lipid vesicles When the primary processes are physicochemically understood, the specific electroporative properties of cell membranes and living tissue may also be quantitatively rationalized Electrooptical and conductometrical data of unilamellar liposomes showed that the electric field causes not only membrane pores but also shape deformation of liposomes It appears that ME and shape deformation are strongly coupled, mutually affecting each other (4,11,12) The primary field effect of ME and cell deformation triggers a cascade of numerous secondary phenomena, such as pore enlargement and transport of small and large molecules across the electroporated membrane Here we limit the discussion to the chemical– structural aspects of ME and cell deformation and the fundamentals of transport through electroporated membrane patches The theoretical part is essentially confined to those physicochemical analytical approaches that have been quantitatively conceptualized in some molecular detail, yielding transport parameters, such as permeation coefficients, electroporation rate coefficients, and pore fractions Theory of Membrane Electroporation The various electroporative transport phenomena of release of cytosolic components and uptake of foreign substances, such DNA or drugs are indeed ultimately caused by the external voltage pulses It is stressed again that the transient permeability changes, however, result from field-induced structural changes in the membrane phase Remarkably, these structural changes comprise transient, yet long-lived permeation sites, pathways, channels, or pores (3,13–17) Principles of Electroporation and Transport 2.1 The Pore Concept Field-induced penetrations of small ions and ionic druglike dyes are also observed in the afterfield time period, that is, in the absence of the electrodiffusive driving force (Fig 1) Therefore, the electrically induced permeation sites must be polarized and specifically ordered, local structures which are potentially “open for diffusion” of permeants As indicated by the longevity of the permeable membrane state, these local structures of lipids are longlived (milliseconds to seconds) compared to the field pulse durations (typically, 10 µs to 10 ms) Thus, the local permeation structures may be safely called transient pores or electropores in model membranes as well as in the lipid part of cell membranes The special structural order of a long-lived, potential permeation site may be modeled by the so-called inverted or hydrophilic (HI) pore (Fig 2) (17–19) On the same line, the massive ion transport through planar membranes, as observed in the dramatic conductivity increase when a voltage (≥100–500 mV) is applied, can hardly be rationalized without field-induced open passages or pores (17) The afterfield uptake of substances like dyes or drug molecules, added over a time period of minutes after the pulse application, suggests a kind of interactive diffusion, probably involving the transient complex formation between the permeant and the lipids of the pore wall to yield leaky, but transiently occluded, pores (9) 2.1.1 Pore Visualization Up to now there is no visible evidence for small electropores such as electromicrographs But also the movement of a permeant through an electroporated membrane patch has also not been visualized The large porelike crater structures or volcano funnels of 50 nm to 0.1 µm diameter, observed in electroporated red blood cells, most probably result from specific osmotic enlargement of smaller primary pores, invisible in microscopy (14) Voltagesensitive fluorescence microscopy at the membrane level has shown that the transmembrane potential in the pole caps of sea urchin eggs goes to a saturation level or even decreases, both as a function of pulse duration and external field strength, respectively If the membrane conductivity would remain very low, the transmembrane potential linearly increases with the external field strength Leveling off and decrease of the transmembrane potential at higher fields indicate that the ionic conductivity of the membrane has increased, providing evidence for ion-conductive electropores (15) On the same line, in direct current (DC) electric fields the fluorescence images of the contour of Neumann, Kakorin, and Toensing Fig Pore resealing kinetics indicated by dye uptake The fraction fC of colored cells as a function of the time t = t add of dye addition after the pulse B-lymphoma cells (line IIA1.6) were exposed to one rectangular electric field pulse (E=1.49 kV cm–1; pulse duration tE =110 µs) in the presence of the dye SERVA blue G (Mr = 854) (From ref 9, with permission.) Fig Specific chemical state transition scheme for the molecular rearrangements of the lipids in the pore edges of the lipid vesicle membrane C denotes the closed bilayer state The external electric field causes ionic interfacial polarization of the membrane dielectrics analogous to condenser plates (+, −) Em = Eind is the induced membrane field, leading to water entrance in the membrane to produce pores (P); cylindrical hydrophobic (HO) pores or inverted hydrophilic (HI) pores In the pore edge of the HI pore state, the lipid molecules are turned to minimize the hydrophobic contact with water In the open condenser the ion density adjacent to the aqueous pore (εW) is larger than in the remaining part (εL) because of εW >> εL elongated and electroporated giant vesicle shows large openings in the pole caps opposite to the external electrodes (20) Apparently, these openings are appearing after coalescence of small primary pores invisible in microscopy Theoretical analysis of the membrane curvature in the vesicle pole caps suggests that vesicle elongation under Maxwell stress must facilitate both pore formation and enlargement of existing pores Principles of Electroporation and Transport 2.1.2 Born Energy and Ion Transport Membrane electropermeabilization for small ions and larger ionic molecules cannot be simply described by a permeation across the densely packed lipids of an electrically modified membrane (17) Theoretically, a small monovalent ion, such as Na+(aq) of radius ri = 0.22 nm and of charge zie, where e is the elementary charge and zi the charge number of the ion i (with sign), passing through a lipid membrane encounters the Born energy barrier of ∆GB = zi2 · e2 (1/εm − 1/εw)/(8 · π · ε0 · ri ), where ε0 the vacuum permittivity, εm ≈ and εw ≈ 80 are the dielectric constants of membrane and water, respectively At T = 298K (25°C), ∆GB = 68 · kT, where k is the Boltzmann constant and T is the absolute temperature To overcome this high barrier, the transmembrane voltage |∆ϕ| = ∆GB / |zi · e| has to be 1.75 V An even larger voltage of 3.5 V is needed for divalent ions such as Ca2+ or Mg2+ (z+ = 2, ri = 0.22 nm) Nevertheless, the transmembrane potential required to cause conductivity changes of the cell membrane usually does not exceed 0.5 V (16,17) The reduction of the energy barrier can be readily achieved by a transient aqueous pore Certainly, the stationary open electropores can only be small (about ≤1 nm diameter) to prevent discharging of the membrane interface by ion conduction (4,9,18) 2.2 Transmembrane Field In line with the Maxwell definition of the electric field strength as the negative electric potential gradient, we define the membrane field strength by Em = −∆ϕm / d, (1) where ∆ϕm is the intrinsic cross membrane potential difference and d ≈ nm the dielectric membrane thickness This inner-membrane potential difference may generally consist of several contributions 2.2.1 Natural Membrane Potential and Surface Potential All living cell membranes are associated with a natural, metabolically maintained, (diffusion) potential difference ∆ϕnat, defined by ∆ϕnat = ϕ(i) − ϕ(o) as the difference between cell inside (i) and outside (o) (see Fig 3) Typically, this resting potential amounts to ∆ϕnat ≈ −70 mV, where ϕ(o) = is taken as the reference potential (21) Biomembranes usually have an excess of negatively charged groups at the interfaces between membrane surfaces and aqueous media The contribution of these fixed charges and that of the screening small ions are covered by the (o) (i) surface potentials ϕs and ϕs If cells are exposed to low ionic strength, the (o) (i) inequality |ϕs | > | ϕs | may apply Therefore there will be a finite value for Neumann, Kakorin, and Toensing Fig Electric membrane polarization of a cell of radius a (A) Cross section of a spherical membrane in the external field E The profiles of (B) the electrical potential ϕ across the cell membranes of thickness d, where ∆ϕind is the drop in the induced membrane potential in the direction of E and (C) the surface potential ϕs at zero external field as a function of distance, respectively; (D) ∆ϕnat is the natural (diffusive) potential difference at zero external field, also called resting potential (o) (i) the surface potential difference ∆ϕs = ϕs −ϕs (defined analogous to ∆ϕnat), which in this case is positive and therefore opposite to the diffusion potential ∆ϕnat (see Fig 3) Provided that additivity holds the field-determining potential difference is ∆ϕm = ∆ϕnat + ∆ϕs At larger values of ∆ϕs, the term ∆ϕnat may be compensated by ∆ϕs and therefore ∆ϕm ≈ If lipid vesicles containing a Principles of Electroporation and Transport surplus of anionic lipids are salt-filled and suspended in low ionic strength medium, the surface potential difference ∆ϕs > is finite, but ∆ϕnat = Generally, even in the absence of an external field, there can be a finite membrane field Em = |∆ϕnat + ∆ϕs| / d (21) Here we may neglect the locally very limited, but high (150–600 mV) dipole potentials in the boundary between lipid head groups and hydrocarbon chains of the lipids (22,23) 2.2.2 Field Amplification by Interfacial Polarization In static fields and low-frequency alternating fields dielectric objects such as cells, organelles, and lipid vesicles in electrolyte solution experience ionic interfacial polarization (Fig 3A) leading to an induced cross-membrane potential difference ∆ϕind, resulting in a size-dependent amplification of the membrane field For spherical geometry with cell or vesicle radius a the induced field Eind = −∆ϕind / d at the angular position θ relative to the external electric field vector E (Fig 3B) is given by 3·a Eind = –—– · E · f(λm) · |cos θ|, 2·d (2) where the conductivity factor f (λm) can be expressed in terms of a and d and the conductivities λm , λi , λ0 of the membrane, the cell (vesicle) interior and the external solution, respectively (21) Commonly, d 130 V) and the pulse length is too long (longer than hundreds of milliseconds) (34) 482 Zhang Fig Maximum depth and amount of lacZ gene expression in the skin as the function of pulse length and/or duration of the pressure Conditions indicated in the graph are 120 V and three pulses for each case (A) The maximum depth of gene expression below the epidermis was determined Pressure-only: gene expression was found only in the hair follicles (B) The number of transfected cells in the dermis per square millimeter was counted Using the same method and similar electrical parameters, we have demonstrated gene expression from GFP plasmid DNA as the second marker gene in hairless mice Positive results were obtained with surface electrodes and caliper electrodes (data not shown) It is desirable to increase the efficacy of topical gene delivery Skin-Targeted Gene Delivery 483 Fig Recovery profile of the resistance after pulsing lacZ genes on hairless mice in vivo Total of three pulses was applied at 120 V and 10 ms a FORMULATION It is possible to encapsulate DNA into liposome or biodegradable particles prior to topical application with pulsed electric fields Our feasibility studies demonstrate that pulsed electric fields can be applied to different formulations of deliverable molecules (Fig 7) b VISCOSITY To prevent leakage of DNA solution (see Subheading 3.3., steps and 6), one can increase the solution’s viscosity by adding a suitable chemical agent that becomes relative gelatinous c TEMPERATURE Cool the skin tissue locally to 4°C before pulsing so that the electrically induced pore may stay open longer than at room temperature d CONFORMATION AND CONCENTRATION OF DNA These are also important factors to be considered e CHEMICAL ENHANCER Since the high resistance of the SC is favorable for skin electroporation (electrical field strength mostly located within the SC), it is preferable not to use a chemical enhancer (e.g., ethanol) prior to EP treatment f TIME DEPENDENCE It is reasonable to explore the time window of gene expression to find the maximum expression (generally, 1–3 d) It depends on the DNA constructs and property of the tissue g ANIMAL SPECIES The level and depth of gene expression depends on the animal species being used due to the variable thickness of skin layers h COMBINATION OF METHODS A synergistic effect of DNA delivery may be obtained by taking the advantage of combining EP and iontophoresis (IPH) After creating new pathways in the SC by EP, the electrophoretic driving force provided by IPH pushes more DNA through the new pathways as well as the existing ones (hair follicles and sweat ducts) i ELECTRODE Different electrode configurations play a role in the effectiveness of gene delivery 484 Zhang Fig Time track of the V(t) and I(t) curve during the pulsing monitored by a digital oscilloscope In both panels A and B, the upper curve is I(t) and the lower curve is V(t) (A) Breakdown of the SC during the first pulse (120 V, 10 ms) (B) Breakdown of the SC during the third pulse (same as the first one) The resistance of the SC was decreased (I(t) was increased compared with the first pulse) by electrical pore formation j PULSING PARAMETERS One should try to find an optimum voltage (transdermal threshold is 60–100 V), pulse length (microseconds to milliseconds), and the number of pulses It is useful to apply multipulses at certain frequencies for certain applications Skin-Targeted Gene Delivery 485 Fig Topical or transdermal delivery of different formulations of molecules by pulsed electric fields It is necessary to run the negative control sample (untreated skin) for X-Gal assay For a quick and qualitative study of the delivery method, one can run in situ tissue X-gal It is simpler than the histochemical method Repeat steps 1–3 in Subheading 3.4 Rinse the tissues with PBS three times and incubate in X-Gal staining solution at room temperature for at least h It is necessary to measure a transient and a long-term (if any) gene expression For instance, at d 1, 2, 3, 7, 14, 21, or longer Skin damage and sensation caused by electrical parameters were reviewed and discussed (35) This is a very important issue to be addressed Sensation is related to the design of the electrodes and the parameters of the pulse (voltage and pulse length) Muscle twitching occurs during the pulse One should be careful not to use voltages or pulse lengths that result in any skin damage; i.e., a burn effect, etc References Hoeben, R C., Fallaux, F J., Van Tilburg, N H., Cramer, S J., VanOrmondt, H., Briet, E., and Van Der Eb, A J (1993) Toward gene therapy for hemophilia A: Long-term persistence of factor VIII-secreting fibroblasts after transplantation into immunodeficient mice Hum Gene Ther 4, 179–186 486 Zhang Lu, B., Federoff, H J., Wang, Y., Goldsmith, L A., and Scott, G (1997) Topical application of viral vectors for epidermal gene transfer J Invest Dermatol 108, 803–808 Medalie, D A., Eming, S A., Tompkins, R G., Yarmush, M L., Krueger, G G., and Morgan, J R 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Drugs and the Pharmaceutical Sciences Marcel Dekker, New York 21 Kost, J., Levy, D., and Langer, R (1989) Ultrasound as a transdermal enhancer Percutaneous Absorption: Mechanisms—Methodology—Drug Delivery, 2nd ed (Bronaugh, R L and Maibach, H I., eds.), pp 595–601 22 Banga, A K and Chien, Y W (1988) Iontophoretic delivery of drugs: fundamentals, developments and biomedical applications J Controlled Release 7, 1–24 23 Chien, Y W and Banga, A K (1989) Iontophoretic (transdermal) delivery of drugs: Overview of historical development J Pharm Sci 78, 353–354 24 Prausnitz, M R., Bose, V G., Langer, R., and Weaver, J C (1993) Electroporation of mammalian skin: A mechanism to enhance transdermal drug delivery Proc Natl Acad Sci USA 90, 10,504–10,508 25 Hofmann, G A., Rustrum, W V., and Suder, K S (1995) Electro-incorporation of microcarriers as a method for the transdermal delivery of large molecules Bioelectrochem Bioenerg 38, 209–222 26 Hofmann, G A., Zhang, L., Bremer, U., and Spencer, T (1996) Investigation of electroincorporation phenomena [Abstract] Proc 13th Int Symp Bioecectrochem Bioenerg January 7–12, Israel, pp 132 27 Muramatsu, T., Nakamura, A., and Park, H-M (1997) In vivo electroporation: A powerful and convenient means of nonviral gene transfer to tissues of living animals [Review] Int J Mol Med 1, 55–62 28 Neumann E., Sowers A E., and Jordan C A (1989) Electroporation and Electrofusion in Cell Biology Plenum, New York 29 Nickoloff, J A (1995) Electroporation Protocols for Microorganisms; Animal Cell Electroporation and Electrofusion Protocols; Plant Cell Electroporation and Electrofusion Protocols; Vols 47, 48, and 55 in Methods in Molecular Biology, Humana Press, Totowa, NJ 30 Weaver, J C (1993) Electroporation: A general phenomenon for manipulating cells and tissues J Cell Biochem 51, 426–435 31 Hofmann, G A., Dev, S B., and Nanda, G S (1996) Electrochemotherapy: Transition from laboratory to the clinic IEEE Eng Med Biol Nov./Dec., 124–132 32 Prausnitz, M R., Bose, V G., Langer, R., and Weaver, J C (1993) Electroporation of mammalian skin: A mechanism to enhance transdermal drug delivery Proc Natl Acad Sci USA 90, 10,504–10,508 33 Zhang, L., Li, L., Hofmann, G A., and Hoffman R M (1996) Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: An 488 Zhang approach to gene therapy of skin aging and other diseases Biochem Biophys Res Commun 220, 633–636 34 Pliquett, U., Langer, R., and Weaver, J C (1995) Changes in the passive electrical properties of human stratum corneum due to electroporation Biochim Biophys Acta 1239, 111–121 35 Prausnitz, M R (1996) The effects of electric current applied to skin: A review for transdermal drug delivery Adv Drug Deliv Rev 18, 395–425 ... molecule to be delivered: drug versus genes In vivo EP requires techniques for the delivery of the drug/ gene to the tissue site, and techniques for the delivery of the field The delivery of the field... genes and drugs into 22 Neumann, Kakorin, and Toensing the cell interior has essential features in common Therefore a general formalism was developed for the electroporative uptake of drug and. .. of cell electrotransformation and cell coloring, can be used to specify conditions for the practical purposes of gene transfer and drug delivery into the cells In electrochemotherapy, for instance,