New insights into the mechanisms of gene electrotransfer--experimental and theoretical analysis

Abstract

Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.

Figure 1

(A) Effect of electric field strength on the percentage transfection of CHO cells (%TR), viability (%Survival) and electropermeabilization (%PI uptake) for 4 × 200 μs pulses (HV) and 1 Hz repetition frequency. Results are shown for plated cells (closed symbols) and cells in a suspension (open symbols) and are presented as mean ± standard error of at least three independent experiments. (B) Dependence of %TR on the fraction of permeabilized membrane area S_c/S₀ as given in Eq. 3, for plated cells and cells in a suspension. The bright shaded surface of the spherical cell represents the permeabilized cell membrane - S_c.

Figure 2. Effect of different HV-LV protocols on the electrotransfer efficiency for different plasmid concentrations.

Panels A and C plated cells, B and D cells in a suspension, % TR (A and B) and average maximal fluorescence intensity in A.U. Pulse parameters were: HV pulses (4 × 200 μs), 1 Hz and 1 × 100 ms LV E_HV = 1 kV/cm (400 V) and E_LV = 0.075 kV/cm. The results are presented as mean ± standard error of at least three independent experiments.

Figure 3. DNA – membrane interaction for different HV-LV protocols for c_DNA = 10 μg/ml and 2 μg/ml.

The average maximal fluorescence intensity FL^TOTO is presented as an average ± standard error; the representative images for HV, HV+LV, LV+HV and LV pulses are shown on top. Please note that the scale of fluorescence intensity in the micrographs is adjusted between Min = 100 and Max = 700 A.U. in images with c_DNA = 10 μg/μl, and between Min = 100 and Max = 300 A.U. for c_DNA = 2 μg/ml. Immediately after the labeled plasmid was added to the cells, different combinations of HV (4 × 200 μs, E_HV = 1.4 kV/cm, 1 Hz) and LV pulses (1 × 100 ms, E_LV = 0.137 kV/cm) were applied.

Figure 4. Theoretical analysis of DNA accumulation at the cell membrane due to electrophoretic force.

The number of DNA molecules (N_DNA) available for contact with the permeabilized part of the cell membrane for different plasmid c_DNA for plated cells (A) and cells in a suspension (B) are shown for HV and HV+LV pulses (corresponding transfection efficiencies are shown in Fig 1). The schematic representation shows the calculation of N_DNA, where L is the distance traveled due to electrophoresis and r_p is the radius of the permeabilized membrane (dotted, yellow area) for plated cells (A) and cells in a suspension (B). The gray shaded region represents the volume V from which DNA molecules are brought in contact with the cell membrane. The strength and length of pulses determine the distance L from which DNA can access the cell membrane (gray) and E determines the area of the membrane which is electropermeabilized.

Figure 5. Different steps of gene electrotransfer.

(A) DNA is added to the electroporation buffer, (B) electropermeabilization of the cell membrane and DNA contact/insertion with/into the membrane, (C) transfer across the membrane and into the nucleus, (D) gene expression. Corresponding fluorescence images below: electropermeabilization (PI), interaction of DNA with the membrane (TOTO), transfer into cytoplasm (RHODAMIN) and gene expression (GFP).

Figure 6. Schematic representation of electrotransfection of plated cells (A) and cells in a suspension (B).

Cells in a suspension are spherical and with a narrow distribution of sizes, while plated cells have large variations in size, orientation and shape. Consequently, for plated cells it is very difficult to achieve pulses that would enable optimal electropermeabilization (yellow) and electrotransfer of all cells, in contrast to cells in suspension where this is possible. Furthermore, cells in a suspension rotate and move during pulses, thus a higher N_DNA is needed and therefore a higher c_DNA must be used compared to plated cells.

Figure 7. Two possible hypotheses of DNA entry into the cytoplasm.

(A) DNA is inserted into the permeabilized cell membrane during electric pulses and is translocated inside the cell by a slow process after pulse delivery; (B) DNA interacts with the permeabilized membrane and is consequently endocytosed.