A Pulsed Electric Field Enhances Cutaneous Delivery of Methylene Blue in Excised Full- Thickness Porcine Skin  Patricia G. Johnson, Stephen A. Gallo, Sek Wen Hui  Journal of Investigative Dermatology  Volume 111, Issue 3, Pages 457-463 (September 1998) DOI: 10.1046/j.1523-1747.1998.00301.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic diagram of the system used to monitor skin resistance and to deliver electric pulses. Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Visualization of MB penetration. Photomicrographs (light microscopy, 40×) of frozen section biopsies of porcine skin following MB application: (a) Control, MB applied to the skin surface for 30 min, no pulse; (b) 200 V, 10 Hz, τ = 1 ms, 30 min, 3600 Vs; (c) 100 V, 40 Hz, τ = 1 ms, 30 min, 7200 Vs; (d) 200 V, 40 Hz, τ = 1 ms, 30 min, 14,400 Vs. Scale bar: 50 μm. Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Total penetration of MB in a 5 mm punch biopsy, measured as the absorbance of drug in Solvable supernatant. Penetration as a function of the following. (a) Field application time: all samples were pulsed at a constant voltage (240 V), frequency (40 Hz), and pulse width (1 ms) for 1–30 min. Various symbols represent individual experiments; for each experiment, all absorbance values were normalized to the maximum optical density recorded for that experiment;○, mean values;----, sigmoidal fit. (b) Voltage: all samples (8640 Vs) were pulsed at a constant frequency (40 Hz) and pulse width (1 ms) at 60–240 V for 60–15 min; plotted as natural log of absorbance versus voltage;----, linear fit of exponential relationship. (c) Frequency: samples were pulsed at a constant voltage and pulse width at frequencies of 20–100 Hz for 6–30 min; mean value (± SD) Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Concentration of MB versus penetration depth. (a) Concentration as measured by image analyses of transmitted light (>615 nm): . . ., no pulse;––, 1800 Vs, 10 Hz, 100 V;–––, 18,000 Vs, 100 V, 100 Hz; ▀, 45,000 Vs, 250 V, 100 Hz. All at τ = 1 ms for 30 min. (b) Concentration as measured by fluorescence emission (630 nm excitation, detected using a 660 nm long-pass filter): . . ., no pulse;––, 360 Vs, 20 V, 10 Hz;–––, 900 Vs, 50 V, 10 Hz; ▀, 1800 Vs, 100 V, 10 Hz. All at τ = 1 ms for 30 min. Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Total penetration of MB in biopsy sections as a function of log VT (Vs). Penetration as measured by the integrated intensity (area under the curve) from image analyses of fluorescence (▪) and light transmittance (○). Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Total amount of MB penetration as a function of electrical exposure dose VT, as determined by the concentration (via absorbance) in Solvable supernatant (□) and light transmittance of scanned images (○). Journal of Investigative Dermatology 1998 111, 457-463DOI: (10.1046/j.1523-1747.1998.00301.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions