MECHANISM OF ELECTROPORATIVE DYE UPTAKE BY MOUSE B-CELLS

The color change of electroporated intact immunoglobulin G receptor(-) (Fc gamma R-) mouse B cells (line llA1.6) after direct electroporativ e transfer of the dye SERVA blue G (M-r 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law , where, as a novelty, time-integrated flow coefficients are introduce d. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C reversible arrow P-1 reversible arrow P-2 r eversible arrow P-3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration t(E). At E = 2.1 kV cm(-1) and t(E) = 200 mu s, k(p ) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-de pendent) flow coefficient and permeability coefficient are k(f)(0) = ( 1.0 +/- 0.1) x 10(-2) s(-1) and P-0 = 2 cm s(-1), respectively. The ma ximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is N-p = (1.5 +/- 0. 1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm(2) s(-1), is slightly smaller than that of free dye d iffusion, indicating transient interaction of the dye with the pore li pids during translocation. The mean radii of the three pore states are (r) over bar(P-1) = 0.7 +/- 0.1 nm, (r) over bar(P-2) = 1.0 +/- 0.1 n m, and (r) over bar(P-3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10(-3) s(-1), independent of E. At zero field, the e quilibrium constant of the pore states (P) relative to closed membrane states (C) is K-p(0) = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results o f SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative deli very of drugs that are similar in structure to SERVA blue G, for insta nce, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.