Resealing of electroporation of porcine epidermis using phospholipids and poloxamers

Abstract

The resealing of porcine epidermis after electroporation is investigated. Porcine epidermis was subjected to electroporation (30 pulses at 100 V, 1 ms and at 1 Hz) in a vertical diffusion apparatus, in the presence of 2 mg/ml dimyristoylphosphatidylserine, to produce a long lasting permeable state. Resealing treatments include incubation in 0.0625-0.25 mM poloxamer 188 (P188), or incorporation of phosphatidylcholines (PC) and/or cationic lipids with additional pulses. The recovery of electric resistance of the epidermis samples after electroporation with or without resealing treatments was monitored. The transports of carboxyfluorescein and glucose were measured during the recovery process. Both P188 and PC were effective in resealing in terms of electric conductance and transport, with P188 reacting more rapidly and completely. P188 mediated lipid exchange between stratum corneum lipid particles was measured by fluorescence resonance energy transfer (FRET). Lipid reorganization facilitated by P188 and PC is suggested to be a major resealing mechanism of electroporation damage.

Figure 1

Resistance recovery after 30 electroporation pulses in the presence of DMPS or DOPC, and 30 pulses of resealant treatment. The recovery is plotted as the ratio, R/R₀, of the time-dependent resistance value R relative to the resistance value R₀ measured immediately after the resealing treatment (at time zero). Pulse protocols are described in the legend block first as the number of electroporation pulses and the pulse media, followed by the number of treatment pulse and the treatment or control media. P188 (0.125 mM), when used, was added either during or after the treatment pulses in PBS, with similar results. 4 ≥n ≥3. Error bars represent standard deviation.

Figure 2

Typical recovery of resistance after 30 pulses in DMPS and 30 pulses in PBS, and treated with different concentrations of P188. The concentrations are: 0.25 mM (triangles), 0.025 mM (squares) and 0.0025 mM (diamonds). Difference between treatment concentrations are significant (p<0.005)

Figure 3

Plot of log (concentration) of CF transport against time. Pulse and treatment protocols described in the legend box have the same connotation as those in figure 1. 6>n>3. Standard deviations, typically less than half a log range, are left out for clarity.

Figure 4

Cumulative glucose transport in 30 minutes after various resealing treatments. 14 ≥n ≥3. Error bars represent standard deviation.

Figure 5

Lipid exchange between stratum corneum lipid particles suspended in pH 6.5 buffer solution with different concentrations of P188, measured as FRET intensity against time. Traces of repeated samples superimpose within noise range.