Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses.

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane.

Drug resistance-associated pfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH.

Elucidating the altered physiology of various chloroquine resistant (CQR) strains of Plasmodium falciparum is essential for understanding the molecular basis of CQR. In this study, we have devised several new methods for analyzing digestive vacuolar (DV) pH for individual intraerythrocytic parasites under continuous perfusion. These use controlled illumination power and novel data acquisition software, and are based on either acridine orange (AO) emission spectra or ratiometric 5-(and 6-)carboxy-2',7'-dimethyl-3'-hydroxy-6'-N-ethylaminospiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one (DM NERF) excitation. Results show that DV pH is more acidic for laboratory strains of CQR parasites relative to chloroquine sensitive (CQS). Using mutant pfcrt allelic exchange clones not previously exposed to chloroquine (CQ), we now show a direct association between acid DV pH, CQ resistance and mutation of pfcrt to either South American (7G8) or South East Asian (Dd2) CQR-associated alleles. Surprisingly, these alleles confer a similar degree of DV acidification. Verapamil (VPL) reversed acid DV pH for the Dd2 mutant C3(Dd2) clone, in a surprisingly rapid fashion, but did not reverse acid DV pH for the 7G8 mutant C6(7G8) clone. Thus, there is a direct link between expression of two major CQR-associated pfcrt alleles and altered parasite DV physiology. The data also support models that envision direct but allele-specific interaction between PfCRT and VPL.

Highly efficient, large volume flow electroporation.

Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.