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J C Weaver

Publications and source records attributed to J C Weaver.

At least 37 records · Page 2Linked to original sources
PubMed1998

Skin electroporation causes molecular transport across the stratum corneum through localized transport regions.

High voltage pulsing of human skin (approximately 100 V across the skin, 1 ms pulses) has been hypothesized to cause electroporation of the stratum corneum, and to cause large fluxes of drugs and other molecules across the skin, through newly created aqueous pathways. In contrast, iontophoresis (<0.5 mA per cm2, <1 V across the skin) has long been used in transdermal drug delivery, and is believed to involve pre-existing pathways associated with hair follicles and sweat ducts. Either high voltage pulsing or iontophoresis was applied to human, hairless rat, or black rat snake skin. Hairless rat skin contains more hair follicles than human skin, and snake skin does not contain any hair follicles. All three types of skin had comparable electrical resistances at low voltages; however, the iontophoretic transport of charged fluorescent molecules was significant for human and hairless rat skin, but no transport occured across snake skin, indicating that hair follicles and sweat ducts play a major role in iontophoresis. Electroporation caused large molecular transport for all three types of skin, and involved spontaneously forming localized transport regions, not associated with appendages. These experiments thus provide further support for the hypothesis that high voltage pulsing causes electroporation in the stratum corneum, and that this transport mechanism is fundamentally different from iontophoresis.

Animals↗
PubMed1997

Heparin alters transdermal transport associated with electroporation.

Short, high-voltage (HV; U(skin,max) approximately 100 V) pulses have been shown to increase rates of transdermal transport by several orders of magnitude via a mechanism hypothesized to involve electroporation. We show that heparin, a linear, highly charged macromolecule, significantly alters the molecular transport capacity and lifetime of aqueous pathways across human stratum corneum (SC) created by such pulses. If co-transported during pulsing, heparin molecules can interact with the SC and other molecules, thereby altering ionic and molecular transport. We also observed an increase in post-pulse skin permeability and persistent lower skin resistance. Because most heparin molecules are long enough to span the five to six lipid bilayer membranes that separate corneocytes within the SC, these results can be explained by the hypothesis that heparin molecules were trapped within the skin, holding open pathway segments connecting adjacent corneocytes. These results support the skin electroporation hypothesis and provide the first demonstration of a chemical enhancer effect for transdermal transport by HV pulsing.

Electroporation↗
PubMed1997

Gel microdrop technology for rapid isolation of rare and high producer cells.

Secreted proteins are a therapeutic cornerstone of the biotechnology industry, and numerous recombinant products, including human growth hormone, human erythropoietin and granulocyte-colony stimulating factor, are now widely prescribed. In addition to bioprocessing applications, GMD technology should benefit the emerging fields of cellular and gene therapy. The ability to assess rapidly and precisely the productivity of cells for ex vivo cell screening and expansion is a unique approach with numerous research and therapeutic uses.

Antibodies, Monoclonal↗
PubMed1996

Imaging of fluorescent molecule and small ion transport through human stratum corneum during high voltage pulsing: localized transport regions are involved.

During the application of high-voltage pulses across the skin, transport of two negatively charged fluorescent molecules through the stratum corneum is highly localized. The apparent size of these localized transport regions (LTR's) is initially 10 microns in diameter for both calcein and sulforhodamine. Appearance of LTR's occurred at or above transdermal voltages of 75 V. In the range of 75 to 160 V, the number of LTR's increases with voltage, but their initial size is the same at all voltages; with additional pulses LTR's increase in size, reaching diameters of approximately 40-80 microns. Small ion currents across the skin are also localized and include the LTR's; however, the areas of current flux appeared to be larger. There was no visible damage to the structure of the skin seen at 100 x magnification for any of the voltages used (< 170 V across the skin). Significantly, LTR's are not sweat ducts or hair follicles.

Adult↗
PubMed1996

Energetic constraints on the creation of cell membrane pores by magnetic particles.

Naturally occurring and contaminant ferromagnetic and ferrimagnetic particles have been found within or near cells, and might allow pulsed magnetic fields to create transient cell membrane opening ("pores"). We show that this possibility is significantly constrained by the maximum rotational energy that can be transferred to the cell membrane. For single biologically synthesized magnetosomes (radius rmag approximately 10(-7) m, magnetic moment mu approximately 2 x 10(-15) A m2) and typical cell membranes, the estimated pulse magnitude must exceed Bo approximately 6 x 10(-3) to 7 x 10(-2) T, and the optimal pulse durations are in the range 10(-5) s < tpulse < 10(-1) s. For larger contaminant particles with larger net magnetic moments, the pulse magnitudes could be only somewhat smaller, and the optimal durations are about the same. Very large pulses that exceed the coercive force of a particle are predicted to have a smaller effective magnitude and shorter effective duration.

Cell Membrane↗
PubMed1996

Imaging regions of transport across human stratum corneum during high-voltage and low-voltage exposures.

Scanning confocal fluorescence microscopy was used to image localized regions of calcein transport across human stratum corneum during constant low-voltage (iontophoresis) and pulsed high-voltage exposures. Following an electrical protocol, imaging revealed regions of fluorescence which were interpreted as sites where transport of a fluorescent probe (calcein) into the stratum corneum had taken place. Electrically-assisted transport of calcein, whether enhanced by iontophoresis or high-voltage pulsing, appears to occur through intercellular and, to some extent, transcellular pathways into localized regions of stratum corneum that are not associated with appendages. Uniquely associated with the highest voltage pulses used (300 V across the skin) was the appearance of small, brightly fluorescent areas containing nonfluorescent interiors, i.e., fluorescent "rings". We present evidence which suggests that the dark interiors represent sites through which transport occurred during pulsing, but where calcein was no longer present at the time of imaging. Transport of charged microspheres into the stratum corneum was also observed.

Biological Transport↗
PubMed1996

FACS-based isolation of slowly growing cells: double encapsulation of yeast in gel microdrops.

Isolating hyperproducing cells is important in biotechnology, but these cells usually grow slowly and can be overgrown by poorly producing cells. We describe a new method of isolating slowly growing cells from among rapidly growing cells, which has the potential for automation and high throughput (e.g., 100,000 cells/h). A model system is presented consisting of a mixed population of slowly growing mutant and rapidly growing wild-type yeast, which were encapsulated in double agarose gel microdrops (dGMDs); with most dGMDs initially containing single cells. Double encapsulation locates parent cells near dGMD centers, making microcolony measurement more accurate. After a 15-h incubation, fluorescent activated cell sorting was used to analyze and sort dGMDs with small microcolonies (slow growers) from dGMDs with large microcolonies (rapid growers). Successful isolation of slow growers from a mixed population of predominantly rapidly growing Saccharomyces cerevisiae cells was achieved.

Cell Division↗
PubMed1995

Changes in the passive electrical properties of human stratum corneum due to electroporation.

The stratum corneum (SC) is the main barrier to molecular and ionic transport across mammalian skin and has been extensively studied by others at low voltages (U(skin)(t) < 10 V) in order to partially characterize the skin. Here we use one or more exponential pulses (tau pulse = 1 ms) and a temperature of 25 +/- 2 degrees C and found that the low voltage passive electrical properties (impedance) change rapidly and significantly if these pulse result in U(skin),0 > 40 V. In contrast, the dynamic resistance (describing passive electrical behavior in a nonlinear range) changes dramatically by application of pulses between 40 V and 80 V and then it settles at levels between 50 omega and 100 omega. We also found that recovery of the low voltage electrical parameters after pulsing depends mainly on the voltage, and, for multiple pulse protocols, on the number of pulses. For single pulses of U(skin),0 approximately 90 V or less the electrical recovery was almost complete, returning to within 0.90 of the pre-pulse value. In contrast, larger pulses result progressively in decreased recovery. The recovery for pulses > 90 V revealed several characteristic times, suggesting the involvement of different processes. For multiple pulses with U(skin),0 > 130 V almost no recovery of the transdermal resistance, R(skin), was evident (returning to < 0.10 of pre-pulse values), i.e., essentially permanent changes in the stratum corneum occurred. This is similar to that of single bilayer membrane electroporation, for which a transition from reversible to irreversible behavior occurs as transmembrane voltage is increased. Thus, these results are consistent with the hypothesis that 'high-voltage' pulses cause electroporation within the SC, i.e., that elevated transmembrane voltage result in creation of new aqueous pathways ('pores') across SC lipid regions.

Electric Conductivity↗
PubMed1995

Transdermal transport of DNA antisense oligonucleotides by electroporation.

Fluorescein-labeled antisense oligodeoxynucleotides (ODNs) corresponding to the promoters of the protooncogene c-myb (24-mer) and the oncogene c-myc (15-mer) were transported through the human skin in vitro by electroporation. Fluxes of 6.4 +/- 2.1 pM/cm2* hr and 11.5 +/- 3.5 pM/cm2* hr, respectively, were achieved during exponential pulsing [tau pulse = 1.1ms, transdermal voltage (Uskin) = 80V] every five seconds. The flux for Uskin < 70V was undetectable, rapidly increased at 80V, but plateaued at higher values. Fluorescence imaging demonstrated that transport of the ODNs is concentrated in localized transport regions (LTRs)1 approximately 30 microns in diameter.

Base Sequence↗
PubMed1995

Rectification and signal averaging of weak electric fields by biological cells.

Oscillating electric fields can be rectified by proteins in cell membranes to give rise to a dc transport of a substance across the membrane or a net conversion of a substrate to a product. This provides a basis for signal averaging and may be important for understanding the effects of weak extremely low frequency (ELF) electric fields on cellular systems. We consider the limits imposed by thermal and "excess" biological noise on the magnitude and exposure duration of such electric field-induced membrane activity. Under certain circumstances, the excess noise leads to an increase in the signal-to-noise ratio in a manner similar to processes labeled "stochastic resonance." Numerical results indicate that it is difficult to reconcile biological effects with low field strengths.

Animals↗
PubMed1995

Observation of extremely heterogeneous electroporative molecular uptake by Saccharomyces cerevisiae which changes with electric field pulse amplitude.

Molecular uptake of a charged fluorescent molecule (calcein; 623 Da, z = -4) was quantitatively determined at the single cell level using flow cytometry. Dilutely suspended cells were exposed to one exponential pulse (tau p approximately 300 microseconds) for different field strength values. For an asymmetric cell such as the yeast Saccharomyces cerevisiae a significant variation in the number of molecules taken up by individual cells was expected for physical reasons. By carrying out several thousand individual cell measurements for each pulse condition, we found that the number of molecules per cell varies significantly within the cell population, and that this population distribution changes markedly as the field strength is varied. Surprisingly, in spite of significant changes in this distribution with field strength, the average uptake per cell reaches a non-equilibrium plateau for which the uptake per cell is much smaller than the product of the mean cell volume and the supplied extracellular concentration. These observations of different field-dependent cell population distributions of uptake support the hypotheses that (1) electroporation is a transmembrane voltage-responsive phenomenon, so that cells of different sizes, shapes and orientation, respond differently to even a spatially uniform applied field, (2) population average measurements of electroporation behavior can be incomplete and misleading, and (3) transport of small charged molecules is due to electrophoresis through the pores of a dynamically changing pore population.

Cell Membrane↗
PubMed1995

Mechanism of electroinduced ionic species transport through a multilamellar lipid system.

A theoretical model for electroporation of multilamellar lipid system due to a series of large electrical pulses is presented and then used to predict the functional dependence of the transport of charged molecules. Previously, electroporation has been considered only for single bilayer systems such as artificial planar bilayer membranes and cell membranes. The former have been extensively studied with respect to electrical and mechanical behavior, and the latter with respect to molecular transport. Recent experimental results for both molecular transport and electrical resistance changes in the stratum corneum (SC) suggest that electroporation also occurs in the multilamellar lipid membranes of the SC. In addition, there is the possibility that other skin structures (the "appendages") also experience electroporation. A compartment model is introduced to describe the transport of charged species across the SC, and the predicted dependence is compared with available data. In this model, the SC is assumed to contain many hydrophilic compartments in series separated by boundary bilayers, so that these compartments become connected only upon electroporation. Two limiting cases for the transport of charged molecules are considered: (1) transport along tortuous inter-bilayer pathways in each compartment, followed by transport across individual boundary bilayers due to electroporation, and (2) transport along straight-through pathways in the boundary bilayers with fast mixing in each compartment, which includes the interior space of corneocytes. Both models were fitted to the experimental data. The large electropore radius (rt approximately 200 A) and porated fractional area (ft approximately 10(-3) obtained from the fitting for the tortuous model relative to the more reasonable values obtained for the straight-through model (rs approximately 4 A, fs approximately 10(-6) suggest that the latter is a more realistic description of electroinduced transport of ionized species through the skin.

Biophysical Phenomena↗
PubMed1995

Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.

Biological Transport↗
PubMed1995

Measurement of rapid release kinetics for drug delivery.

A fluorescence measurement system and methods of data analysis were developed to measure rapid kinetics of transdermal transport in vitro. Three variations on the technique were demonstrated, where the receptor compartment concentration was determined by: 1) fluorescence measurements of aliquots removed at discrete time points, 2) continuous fluorescence measurements made directly in the receptor compartment using a custom-made fluorimeter cuvette as a permeation chamber, and 3) continuous fluorescence measurements made in a flow-through cuvette containing receptor solution continuously pumped from a flow-through permeation chamber. In each case, the measured signal was a convolution of the time-dependent molecular flux (the desired information) and the characteristic response of the measurement system. Algorithms for deconvolution of the signal were derived theoretically. For the most complicated case, (3), the experimental confirmation is shown here, proving a time resolution on the order of half a minute.

Animals↗
PubMed1995

Transdermal delivery of heparin by skin electroporation.

Therapeutic uses of compounds produced by biotechnology are presently limited by the lack of noninvasive methods for continuous administration of biologically-active macromolecules. Transdermal delivery would be an attractive solution, except macromolecules have not previously been delivered clinically across human skin at therapeutic rates. To increase transport of a highly-charged macromolecule (heparin), high-voltage pulses believed to cause electroporation were applied to skin. Using this approach, transdermal heparin transport across human skin in vitro occurred at therapeutic rates (100-500 micrograms/cm2h), reported to be sufficient for systemic anticoagulation. In contrast, fluxes caused by low-voltage iontophoresis having the same time-averaged current were an order of magnitude lower. Heparin transported across the skin was biologically active, but with only one eighth the anticoagulant activity of heparin in the donor compartment due to preferential transport of small (less active) heparin molecules. Flux, activity, and transport number data together suggest that high-voltage pulsing creates transient changes in skin microstructure which do not occur during iontophoresis. Safety issues are discussed.

Administration, Cutaneous↗
PubMed1995

Electroporation theory. Concepts and mechanisms.

The basic features of electrical and mechanical behavior of electroporated cell membranes are reasonably well established experimentally. Overall, the electrical and mechanical features of electroporation are consistent with a transient aqueous pore hypothesis, and several features, such as membrane rupture and reversible electrical breakdown, are reasonably well described quantitatively. This gives confidence that "electroporation" is an attractive hypothesis, and that the appearance of temporary pores owing to the simultaneous contributions of thermal fluctuations ("kT energy") and an elevated transmembrane voltage ("electric field energy") is the microscopic basis of electroporation.

Animals↗