Patricia G. Johnson

PHOTOSENSITIZATION OF MURINE TUMOR, VASCULATURE and SKIN BY 5‐AMINOLEVULINIC ACID‐INDUCED PORPHYRIN

Abstract— The effects of topical and systemic administration of 5‐aminolevulinic acid (ALA) were examined in several murine tumor systems with regard to porphyrin accumulation kinetics in tumor, skin and blood, vascular and tumor cell photosensitization and tumor response after light exposure. Marked, transient increases in porphyrin levels were observed in tumor and skin after systemic and topical ALA. Rapid, transient, dose‐dependent porphyrin increases were also observed in blood; these were pronounced after systemic ALA injection and mild after topical application. They were highest within 1 h after ALA injection, thereafter declining rapidly. This matched the clearing kinetics of injected exogenous protoporphyrin IX (PpIX). Initially, vascular photosensitivity changed inversely to blood porphyrin levels, increasing gradually up to 5 h post‐ALA, as porphyrin was clearing from the bloodstream. This pattern was again matched by injected, exogenous PpIX. After therapeutic tumor treatment vascular disruption of the tumor bed, while observed, was incomplete, especially at the tumor base. Minimal direct tumor cell kill was found at low photodynamic therapy (PDT) doses (250 mg/kg ALA, 135 J/cm2 light). Significant, but limited (<1 log) direct photodynamic tumor cell kill was obtained when the PDT dose was raised to 500 mg/kg systemic ALA, followed 3 h later by 270 J/cm2, a dose that was however toxic to the animals. The further reduction of clonogenic tumor cells over 24 h following treatment was moderate and probably limited by the incomplete disruption of the vasculature. Tumor responses were highest when light treatment was carried out at the time of highest tumor porphyrin content rather than at the time of highest vascular photosensitivity. Tumor destruction did not reach the tumor base, regardless of treatment conditions.

Characterization of electric-pulse-induced permeabilization of porcine skin using surface electrodes

We measured the transient and long-term changes of permeability of full-thickness porcine skin after the application of a single or a train of electric pulses, as the basis for optimization of the electrical parameters for enhancing transdermal drug or gene delivery by electroporation. Two electrodes were attached to the stratum corneum of excised skin for transdermal electric pulse delivery and impedance measurement. Both transient and long-term permeabilization were found to be dependent on the electrical exposure dose, i.e., the product of pulse voltage and cumulative pulsing (exposure) time. Skin resistance dropped to about 20% of its prepulsing value when pulsed beyond a critical dosage of 0.4 V-s (with 20-40 V across each skin path), but recovered rapidly within seconds after the pulse. Long-term permeabilization of the skin required repeated pulsing with a minimum potential of 160 V (80 V across each skin path). The maximum long-term resistance drop, to 35% of the initial value, required a dose greater than 200 V-s, recovering slowly and seldom completely in tens of minutes to hours. The decrease and recovery of the resistance were dependent on the frequency and pulse length only for low-dose electrical exposure.

Electrically Enhanced Percutaneous Delivery Of δ-Aminolevulinic Acid Using Electric Pulses and a DC Potential¶

Abstract Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the stratum corneum. We used electric pulses to increase transdermal transport of δ-aminolevulinic acid (ALA), a precursor to the photosensitizer protoporphyrin IX (PpIX). ALA-filled electrodes were attached to the surface of excised porcine skin or the dorsal surface of mice. Pulses were administered and, in some in vivo cases, a continuous DC potential (6 V) was concomitantly applied. For in vitro 14C ALA penetration, 10 μm layers parallel to the stratum corneum were assayed by liquid scintillation analysis, and 10 μm cross sections were examined autoradiographically. As the electrical dose (voltage × frequency × pulse width × treatment duration) increased, there was an increase in penetration depth. In vivo delivery was assayed by measuring the fluorescence of PpIX in skin samples. A greater than two-fold enhancement of PpIX production with electroporative delivery was seen versus that obtained with passive delivery. Superimposition of a DC potential resulted in a nearly three-fold enhancement of PpIX production versus passive delivery. Levels were higher than the sum of PpIX detected after pulse-alone and DC-alone delivery. Electroporation and electrophoresis are likely factors in electrically enhanced delivery.

THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS

Abstract— The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase‐locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators.

Increased DNA synthesis in INIT/10T1/2 cells after exposure to a 60 Hz magnetic field : A magnetic-field or a thermal effect?

This study was designed to test the hypothesis that a 0.1-0.8-mT 60 Hz magnetic field may act as a promoter of carcinogenesis. C3H 10T1/2 mouse fibroblasts initiated with the carcinogen methylcholanthrene (INIT/10T1/2 cells) were used; in these cells, expression of the carcinogenic phenotype is suppressed indefinitely by the presence of retinyl acetate in the culture medium. After withdrawal of retinyl acetate, expression of the carcinogenic phenotype may be observed as the loss of contact inhibition. Cells grown without retinyl acetate were exposed to 0.1-0.8-mT (rms) 60 Hz magnetic fields or to sham fields. Eight days after exposure, magnetic-field and sham-exposed cells showed the same levels of incorporation of [3H]thymidine, and both had counts significantly higher than those of unexposed cells. The rate of incorporation of [3H]thymidine was very sensitive to small (0.1-0.8 degrees C) and transient (60 min) increases in incubation temperature during the first few days of withdrawal of retinyl acetate. Exposure of Jurkat (human acute T-cell lymphoma) and GH3 (rat pituitary tumor) cells to magnetic fields and sham conditions yielded similar results. INIT/10T1/2 cells cultured in the presence of retinyl acetate showed no effect of exposure conditions. Both magnetic-field and sham exposures caused a slight increase in temperature within the exposure zone in the incubator. Thus the differences between rates of incorporation of [3H]thymidine in magnetic field-exposed, sham-exposed and unexposed cells seem to be attributable at least in part to a slight elevation in temperature during exposure. Since some cells appear to be extremely sensitive to small increases in temperature, measurements of magnetic-field effects must be made and interpreted with caution.

Electrorheological Modeling of the Permeabilization of the Stratum Corneum: Theory and Experiment

Experimentally observed changes in the conductivity of skin under the influence of a pulsing electric field were theoretically analyzed on the basis of a proposed electrorheological model of the stratum corneum (SC). The dependence of relative changes in conductivity on the amplitude of electric field and timelike parameters of applied pulses or pulse trains have been mathematically described. Statistical characteristics of phenomena of transient and long-term electroporation of SC were taken into consideration. The time-dependent decreases of skin resistance depicted by the models were fitted to experimental data for transient and long-term skin permeabilization by electric pulses. The results show two characteristic times and two spectra of characteristic energies for transient and long-term permeabilizations. The rheological parameters derived from the fittings agreed with those reported elsewhere for biological membranes.

Absence of 60-Hz, 0.1-mT magnetic field-induced changes in oncogene transcription rates or levels in CEM-CM3 cells.

Our objective was to assess the reproducibility of the 60-Hz magnetic field-induced, time-dependent transcription changes of c-fos, c-jun and c-myc oncogenes in CEM-CM3 cells reported by Phillips et al. (Biochim. Biophys. Acta, 1132 (1992) 140-144). Cells were exposed to a 60-Hz magnetic field (MF) at 0.1 mT (rms), generated by a pair of Helmholtz coils energized in a reinforcing (MF) mode, or to a null magnetic field when the coils were energized in a bucking (sham) mode. After MF or sham exposure for 15, 30, 60 or 120 min, nuclei and cytoplasmic RNA were extracted. Transcription rates were measured by a nuclear run-on assay, and values were normalized against either their zero-time exposure values, or against those of the c-G3PDH (housekeeping) gene at the same time points. There was no significant difference, at P=0.05, detected between MF and either sham-exposed or control cells at any time point. Transcript levels of the oncogenes were measured by Northern analysis and normalized as above. No significant difference (P=0.05) in transcript levels between MF and either sham-exposed or control cells was detected.

Using Surface Electrodes to Monitor the Electric-Pulse-Induced Permeabilization of Porcine Skin

The stratum corneum, the outermost layer of the skin, acts as a barrier between the skin and the outside world, preventing evaporation of water from underlying tissues while impeding the diffusion of foreign molecules into the body (1,2). Densely packed layers of flattened, dead, keratinized cells (2,3) are incorporated into a lipid lamellae matrix consisting primarily of ceramides, cholesterol, and fatty acids (2,4), forming an impermeable, hydrophobic partition. The stratum corneum represents the main obstacle to efficient transdermal drug delivery (1,2). If the stratum corneum is disrupted, the barrier to molecular transport is greatly reduced.

Transdermal Delivery Using Surface Electrodes in Porcine Skin

The main barrier to cutaneous or transcutaneous drug and gene delivery is the impermeability of the stratum corneum (SC), the outermost layer of the skin (1). If the integrity of the SC is disrupted, the barrier to molecular transit may be greatly reduced. Cutaneous absorption can be increased by removal of the SC by tape-stripping or dermabrasion, by vehicle (solvent-carrier) optimization, or by the use of penetration enhancers like DMSO (dimethylsulfoxide), oleic acid, and alcohols (2,3). An electric field can also be used to enhance delivery. Disruption of the SC can be achieved by electroporation, which is the creation of penetration sites by an electric pulse. Ions and molecules move through induced gaps of the SC by diffusion and electromotive or electroosmotic transport (4-6). Electroporation differs from iontophoresis, in which there is an increased migration of ions or charged molecules through the skin when an electrical potential gradient is applied. The primary transdermal route for iontophoresis seems to be appendageal or intercellular through preexisting pathways (5,7), or as a result of low-voltage (<5 V) induced permeabilization of appendageal bilayers (8). A third form of electroenhanced drug delivery, electrochemotherapy (9), refers to localized delivery of electric pulses across a tumor following systemic or intratumor drug administration, and usually does not involve cutaneous or transcutaneous delivery.

Electric-Pulse-Induced Permeabilization and Molecular Transport through Porcine Skin Using Surface Electrodes

The main barrier to the success of transdermal delivery is the impermeability of the stratum corneum (SC), the outermost layer of the skin.1,2 The SC is a 10 to 15 µm thick band composed of several layers of dead, flattened, dehydrated keratinocytes which, with their surrounding multilamellar lipids, provide the primary protective barrier of the skin. If the integrity of the SC is disrupted, the barrier to molecular transport is greatly reduced. Disruption of the SC can be achieved by electroporation (the creation of penetration sites by an electric field). If an electric field is imposed across the skin, most of the potential drop develops across the resistive SC3, where a breakdown is likely to occur when the imposed electric field rises beyond a certain critical strength. Cell membranes may be reversibly broken down by 30 psec pulses at about 0.8V of imposed transmembrane potential4, and membranes of cells in a centrifuged pellet break down at about 1V of imposed transmembrane potential5. Since the SC is equivalent to about 100 membrane layers6 we may expect that electroporation may occur at an imposed potential difference of about 100 volts.