Efficient gene transfer by pulse parameters for electrochemotherapy of cells in vitro and in muscle and melanoma tumors in mice

The murine melanoma B16F10, myoblast C2C12 and fibroblast L929 cell lines (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco′s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, USA), supplemented with 5% (v/v) fetal bovine serum (FBS; Gibco), 10 ml/L L-glutamine (GlutaMAX; Gibco) and 1% (v/v) penicillin–streptomycin (stock solution, 10,000 U/mL, Gibco). The cells were maintained in a 5% CO₂ humidified incubator at 37 °C. The cells were routinely tested and confirmed to be free from mycoplasma infection, using the MycoAlertTM PLUS Mycoplasma Detection Kit (Lonza Group Ltd., Basel, Switzerland).

The plasmid pEGFP-N1 (pGFP, Clontech, Takara Bio, Shiga, Japan), which encodes enhanced green fluorescent protein (EGFP), was amplified in competent E. coli, purified with the Endo Free Plasmid Mega Kit (Qiagen, Hilden, Germany) according to the manufacturer′s protocol and dissolved in endotoxin-free water at a concentration of 1 mg/mL. The concentration of the plasmid was measured with a Qubit 3.0 fluorometer (Thermo Fisher Scientific). The purity of the isolated plasmids was determined using the Take3™ Micro-Volume Plate (BioTek Instruments, Winooski, VT, USA) by measuring the 260/230 and 260/280 absorbance ratios. In addition, the identity of the individual plasmids was verified using restriction analysis.

The cells were collected and centrifuged and then resuspended in cold electroporation buffer (125 mM sucrose, 10 mM K₂HPO₄, 2.5 mM KH₂PO₄, 2 mM MgCl₂ × 6 H₂O) at a concentration of 25 × 10⁶ cells/mL. The cells were mixed with the plasmid mixture (1 mg/mL) at a ratio of 1:0.2, and 50 μL (1× 10⁶ cells) was electroporated by an electric pulse generator (GeneDrive, IGEA S.p.A., Carpi, Italy). The GeneDrive electroporator was a generous gift from IGEA S.p.A. The mixture was pipetted between two parallel stainless-steel plate electrodes with a distance of 2.5 mm. Four pulse protocols were used (Table 1). After electroporation, the cells were incubated for 5 min in 24-well low-attachment plates (Corning Incorporated, Corning, NY, USA), and then, 1 mL of complete DMEM was added without phenol red. The cells were seeded for particular assays as described below (cytotoxicity and transfection assays).

The cells were plated on a 96-well plate; 10000 cells/well from each experimental group and 5000 cells/well for control non-electroporated cells. The cells were imaged, and fluorescence was measured in real time with a Cytation 1 multimodal reader (BioTek Instruments) for 24 h every two hours. The cells were incubated at 37°C and 5% CO₂ throughout the experiment. On the basis of the preliminary transfection results, the first image was captured 2 h after GET.

The analysis was performed using Gen5 data analysis software (BioTek Instruments). Images were acquired using a 4′ objective and imaging filter cubes of GFP (excitation at 469/35 nm and emission at 525/39 nm) for the detection of GFP. Two high-contrast brightfield images were also acquired: an in-focus image for reference and a defocused image for cell counting. Each well was focused with a laser autofocus cube. The exposure settings of the camera were optimized and kept the same for each experiment. The defocused image from the brightfield channel was processed with a black background and a 20 μm rolling ball. This increased the contrast and reduced each cell to a single bright spot. The GFP images were also processed with a black background and a 100 μm rolling ball to remove background fluorescence. The images were analyzed by masking the high-contrast defocused brightfield image and extending the brightfield mask to capture the GFP signal. The transfection efficiency was calculated by dividing the number of GFP-positive cells by the total number of cells in the brightfield image. The fluorescence intensity was determined by the mean of the intensity of all GFP-positive pixels in a field of view.

B16F10, C2C12 and L929 cells from each experimental group were plated in 100 μL of medium in 96-well plates (Corning Inc.) at a density of 1500 cells per well. The cells were then incubated at 37°C and 5% CO₂ for 72 h. Ten microliters of Presto Blue® reagent (Thermo Fisher Scientific) was added to the wells, and the fluorescence intensity was measured one hour later with a Cytation 1 Multimodal Reader (BioTek Instruments). The results were plotted as the percentage of viable cells compared with that of the nonelectroporated control group.

Ten-week-old female C57Bl/6NCrl (Charles River, Lecco, Italy) were used for the animal experiments. For tumor formation 100 mL of B16F10 cells in saline were injected into the flanks of the mice at a concentration of 3 × 10⁵ cells/mL. The mice were housed under specific pathogen-free conditions in a carousel mouse IVC rack system (Animal Care Systems Inc., Revere Parkway, USA) at a relative humidity of 55 ± 10%, a temperature of 20–24°C and a 12 h light/dark cycle. Food and water were provided ad libitum. The experimental procedures were performed in compliance with the guidelines for animal experiments of the EU directive (2010/63/EU) and with permission from the Veterinary Administration of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission no. U34401-3/2022/17). The experimental procedures followed the PREPARE, OBSERVE and ARRIVE guidelines.^22–24

Electroporation of the tumor and muscle tissue was performed with an electric pulse generator Cliniporator™ (IGEA s.r.l.).

After 48 h, the tumors and muscle were excised and fixed in 4% paraformaldehyde (PFA; Alfa Aesar) overnight. The samples were then incubated in 30% sucrose for 24 h, embedded in optimal cutting temperature compound (OCT compound, VWR International, Radnor, PA, US) and snap frozen in liquid nitrogen. Fourteen-micron-thick tumor sections were cut using a Leica CM1850 cryostat, dried for 10 min at 37 °C and washed for 5 min in 1′ PBS. All the sections were stained for 10 min with Hoechst solution (3 μg/mL) and then washed for 5 min in 1′ PBS. The slides were then mounted with ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific) and covered with cover glass, and the edges were sealed with nail polish. The slides were then imaged using a Zeiss Axio Observer fluorescence microscope (Carl Zeiss, Oberkochen, Germay) equipped with Colibri 7 LED Light source (UV (385nm), Violet (430nm), Blue (475nm), Green (555nm), Yellow (590nm), Red (630nm), Far Red (735nm)) and a Hamamatsu Orca Flash 4.0 V3 camera. Emitted light was imaged through the following filters: Filter Set HE BFP shift free (Carl Zeiss) for Hoechst 33342 (nuclei), filter set 38 HE eGFP shift free (Carl Zeiss) for EGFP. Images were then visualized in Imaris software (Oxford Instruments, Abingdon, UK).

The comparison of means of more than two groups was statistically evaluated by one-way analysis of variance (one-way ANOVA) followed by Dunnett′s or Tukey′s multiple comparisons test. A p value of < 0.05 was considered to indicate statistical significance. The values on the graphs are presented as the mean (AM) ± standard error of the mean (SE). All in vitro experiments were performed three times with six technical replicates. In vivo experiments were performed in six muscles and six tumors for each experimental protocol. For statistical analysis and preparation of graphs, GraphPad Prism 10.4.1 (La Jolla, CA, USA) was used.

The transfection efficiency of three different cell lines, i.e., B16F10 melanoma, C2C12 myoblasts and L929 fibroblasts, was measured as the percentage of transfected cells as well as the fluorescence intensity. To better understand the dynamics of transfection, the transfection efficiency timeline was measured (Figure 1). After two hours, the transfection was detected by an increase in the fluorescence intensity signal. Thereafter, it steadily increased and reached a plateau approximately 6–10 hours later.

FIGURE 1.

The transfection efficiency timeline of different cell lines according to the gene electrotransfer (GET) protocol. Percent of transfected B16F10 (A), C2C12 (B) and L929 (C) cells and fluorescence intensity of B16F10 (D), C2C12 (E) and L929 (F) cells.

*p < 0.05, statistically significant difference in GET1/GET2 compared with GET4 (A); *p < 0.05, statistically significant difference in GET1/GET4 compared with GET3 and GET2 (B); **p < 0.05, statistically significant difference in GET2 compared with GET3 (B); *p < 0.05, statistically significant difference in GET1/GET2/GET4 compared with GET4 (D and E)

In two tested cell lines, B16F10 and C2C12, we confirmed previous observations that the transfection efficiency of a cell line depends on the GET protocol. In both cell lines, the use of GET1, GET2 and GET4 resulted in higher percentage of transfected cells as well as fluorescence intensity than did the use of GET3 (Figure 1A, D, B, E). The transfection efficiency differed between the cell lines. The highest level of transfection was observed in B16F10 melanoma cells, followed by C2C12 and L929 cells (Figure 1A, B, C). The fluorescence intensity patterns were similar for both B16F10 and C2C12; compared with those of GET3, the fluorescence intensities of GET1, GET2 and GET4 were significantly higher (Figure 1D, E). Thus, GET3 with bipolar electric pulses was significantly less effective than monopolar GET1, GET2 and GET4 pulses in B16F10 and C2C12 cells. In the L929 fibroblast line, the transfection efficiency did not depend on the GET protocol, either in terms of the percentage of transfected cells or the fluorescence intensity.

The percentage of transfected cells at 24 h was used to compare the transfection efficiency among the three cell lines using four different GET protocols (Figure 2). This approach was used to select the most efficient GET protocol for specific cell types. The pulse parameters of GET1 and GET2 transfected B16F10 tumor cells significantly better than normal cells, whereas after GET4, B16F10 tumor cells were again significantly better transfected than L929 cells, but the difference in C2C12 cells was not significant. Compared with C2C12 myoblast cells, B16F10 melanoma and L929 fibroblasts were significantly more effectively transfected with GET3 (Figure 2).

FIGURE 2.

Transfection efficiency of different pulse parameter protocols in three different cell lines. Percent of transfected tumor (B16F10) and normal (C2C12, L929) cells after GET1 (A), GET2 (B), GET3 (C) and GET4 (D) treatment.

*p < 0.05 indicates a statistically significant difference; GET = gene electrotransfer

The cytotoxicity of electric pulses (EPs) alone and the GET of cells in the presence of the plasmid were tested in all three cell lines (Figure 3). The pattern of cell survival, as measured by the percentage of viable cells, was similar among the three cell lines. The application of EPs alone did not cause any significant reduction in cell viability, and up to 15% of the cells died.

FIGURE 3.

Cell survival after different pulse parameters and gene electrotransfer (GET) in B16F10 (A), C2C12 (B) and L929 (C) cells.

* = p < 0.05 indicates a statistically significant difference; EP = electroporation only; pGFP = plasmid only, which encodes the green fluorescent protein

In contrast, GET1, GET2 and GET4 significantly reduced cell survival compared with the corresponding EP protocol in B16F10 and C2C12 cells. In L929 cells, only GET1 and GET2 significantly reduced cell survival, whereas GET4 did not. GET3 did not cause a cytotoxic effect in any of the cell lines (Figure 3 A, B, C).

For in vivo transfection efficiency, two GET protocols, GET2 and GET4, were selected on the basis of the in vitro results. They differ only in repetition frequency: GET2 has 1 Hz, and GET4 has 5 kHz. GET2 had the greatest effect on the transfection efficiency (approximately 35%) of B16F10 melanoma cells but had a lower effect (approximately 15%) on C2C12 myoblasts and L929 fibroblasts. The GET4 pulse protocol had an approximately 20% transfection efficiency in tumor cells but a similar transfection efficiency in muscle cells. For fibroblasts, however, the efficiency was significantly lower but was still ~ 15% (Figure 2).

Frozen tumor and muscle sections were first stained with Hoechst for nuclei staining to visualize the tissue better. GFP was more highly localized in the rims of the B16F10 tumors (Figure 4). Both the GET2 (1 Hz) and GET4 (5 kHz) protocols resulted in similar transfection efficiencies; only a small percentage of the tumor cells were transfected. Compared with these data, similar transfection rates (approximately 2%) were reported in our previous studies in which millisecond pulses (600 V/cm, 5 ms, 1 Hz) were used.²⁵

FIGURE 4.

Transfection of B16F10 tumors. Untreated control tumors (A), GET2 (B) and GET4 (C) tumors. Nuclei are stained blue with Hoechst. The transfected cells are presented in green, indicating transfection with the GFP plasmid.

Scale bar = 1000 μm

In contrast to tumors, efficient transfection was obtained in muscle only after the GET4 protocol. However, the transfection efficiency was not as effective as that of standard GET with the 1HV-4LV pulse protocol, which has been optimized for GET into muscle in previous studies and served as a positive control in the current study (Figure 5D).

FIGURE 5.

Transfection of muscle. Control (A), GET2 (B), GET4 (C) and 1HV-4LV (D). The nuclei are stained blue with Hoechst. The transfected cells are presented in green, indicating transfection with the GFP plasmid.

Scale bar = 1000 μm.