Plasmid DNA pAC-GFP-N1 was kindly provided by the College of Life Sciences at the Nanjing Agricultural University (Nanjing, China). Plasmid DNA pGN was kept in our laboratory. Information regarding the vector map and basic description is shown in Fig. 1. Plasmid DNA was amplified using Escherichia coli DH5a and purified using the E.Z.N.A.Â^® Endo-Free Plasmid Mini kit I (Omega, Norcross, GA, USA).

The Bcap-37 human breast cancer cell line (ER⁻, p53 mutated) was purchased from the Shanghai Cell Collection, CAS (Shanghai, China). Bcap-37 cells were cultured in DMEM (Gibco) medium, supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere of 5% CO2.

Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB) was purchased from Pierce. Peptide derivative SPB-NLS was synthesized by Sangon Biotech (Shanghai, China) with the following sequences: SPB-PKKKRKV.

The cytotoxic effects of DMSO (J&K) and menthol (J&K) on the Bcap-37 cell line were evaluated by MTT assays. Approximately 5,000 Bcap-37 cells per population were plated in a flat-bottomed 96-well plate and incubated at 37°C for 24 h before the assays. Preliminary studies were performed to define the optimal concentration of DMSO and menthol which was suitable for testing. DMSO, ranging from 0.5 to 10% (v/v), was added into the Bcap-37 cells to evaluate the toxicity. After incubation with DMSO for 48 h at 37°C in complete medium, the medium was removed and the cells were rinsed with PBS 3 times, then 75 μl complete medium and 25 μl MTT (2 mg/ml in PBS) were added for another 4 h. After 4 h, the medium was aspirated and replaced with 100 μl DMSO to dissolve formazan crystals. Absorbance was measured at 540 nm, with untreated cells serving as the control. The same experiments were carried out to define the safe working concentration of menthol. The concentration of menthol that was added into the cells to evaluate the toxicity ranged from 12.5 to 400 μM.

Bcap-37 cells were incubated at 37°C for 48 h before transfection. Transfection was performed with the pGN plasmid (Lipofectamine^® 2000 Reagent; Invitrogen™) following the instructions of the manufacturer. The primers used for detecting growth hormone (GH) mRNA expression are listed in Table I. The cells were divided into 12 groups (Tables II and III). Two groups were separately treated with 12.5 μM menthol and 2% DMSO 48 h before transfection. SPB-NLS/DNA complexes were prepared 30 min before transfection. Another 2 groups were separately treated with 2% DMSO and 12.5 μM menthol 2 h after transfection. DMEM medium was replaced with complete medium 6 h after transfection. GH mRNA expression was analyzed by quantitative RT-PCR 48 h after transfection. All transfection experiments were performed in triplicate.

After detecting the mRNA expression levels, we evaluated protein expression efficiency in the DMSO- and menthol-treated groups. The pAC-GFP-N1 plasmid was used to evaluate the transgene expression efficiency. Bcap-37 cells were seeded at 10,000/well in a flat-bottomed 12-well plate 24 h prior to the experiments. Transfection was performed as mentioned above. After transfection, the cells were incubated for 48 h at 37°C for green fluorescent protein (GFP) expression. After incubation, the cells were washed twice with phosphate-buffered saline (PBS). Images were obtained by standard fluorescence microscopy (Olympus).

As the results of fluorescence microscopy showed improved GFP expression efficiency in the DMSO- and menthol-treated groups, we aimed to calculate the increased percentage of transfected cells in the different groups. The capacity of flow cytometry for the rapid, individual analysis of a large number of cells make it ideally adapted for the study of transfection efficiency. Transfection was performed as mentioned above with the pAC-GFP-N1 plasmid. Cells were harvested and washed 3 times by PBS 48 h after transfection. Finally, the harvested cells were suspended in 500 μl cold PBS and examined by a FACSCalibur flow cytometer, which was equipped with an argon laser (488 nm).

Previous studies have suggested that DMSO and menthol treatments significantly increase gene delivery efficiency (14,16). In this study, in order to determine the mechanism by which the chemicals, DMSO and menthol, improve gene transfer efficiency, we examined the cell cycle changes in the different groups by flow cytometry. Bcap-37 cells were treated with DMSO and menthol 48 h before the assays. The cells were harvested and washed 3 times with PBS. The cells were then treated with RNase A (5 μg/ml) for 10 min at room temperature, followed by staining with propidium iodide (PI; 5 μg/ml), a DNA-binding dye for 2 h at room temperature. Subsequently, the cells were subjected to flow cytometry to analyze the cell cycle changes.

Statistical analyses of transfection efficiency and cell cycle changes were carried out using one-way analysis of variance (ANOVA). A P-value <0.05 denoted statistically significant differences. All data are expressed as the means ± standard error of the mean.

One of the major requirements for gene delivery is low cytotoxicity. In order to define the safe working concentration of DMSO and menthol for the Bcap-37 cells, we performed MTT assays. The viability of Bcap-37 cells in the absence or presence of various concentrations of DMSO is shown in Fig. 2A. The results showed that cell viability was surpassed by >90% when the concentration of DMSO was <1% (v/v). However, cell viability only exceeded 80% when the concentration of DMSO was <2% (v/v), and significantly decreased at higher concentrations of DMSO [2.5 and 5% (v/v)]. The minimum cell viability of Bcap-37 cells was 17.2 in 10% DMSO. As shown in Fig. 2A, among 8 tested concentrations of DMSO, 2% (v/v) DMSO was the optimum working concentration.

We also detected the working concentration of menthol. The viabilities in the absence or presence of various concentrations of menthol are shown in Fig. 2B. We observed a strange phenomenon: cell viability was surpassed by >80% when the concentration of menthol was <50 μM. Once the concentration of menthol was up to 100 μM, cell viability sharply decreased to <10% (Fig. 2B). According to the literature, cell proliferation speed significantly decreases when cells are treated with high concentrations of menthol (17). According to the afore-mentioned results, we selected 2% DMSO and 12.5 μM menthol for our experiments.

Quantitative RT-PCR was conducted to evaluate the transfection efficiency of the different treatments (DMSO and menthol). The results revealed that the expression of GH mRNA in the DMSO- and menthol-treated groups was significantly higher than that in the liposome-mediated group. This result was consistent with the study by Jain and Gewirtz (16). Our results clearly demonstrated that the post-DMSO treatment group expressed much more GH mRNA than the pre-DMSO treatment group, which suggests, as also shown by a previous study, that DMSO affects the nuclear pore in gene transfer (23). Taken together, our results show that post-DMSO treatment is most effective in enhancing gene delivery. Of note, the DMSO/SPB-NLS co-treatment was less efficient in enhancing gene transfer compared to the post-DMSO treatment group. Although the exact mechanism of the DMSO-mediated gene delivery remains unknown, our results clearly demonstrate that the addition of DMSO influences the transfection efficiency. We speculated, as also shown in a previous study, that pre-DMSO treatment may increase transfection efficiency by regulating the gene controlled cell cycle and cell hydrophobic properties (26), while post-DMSO treatment may improve transfection efficiency by influencing the formation of the nuclear pore.

Similar results were also found in the menthol-treated groups. What was different from the DMSO-treated groups was that pre-menthol treatment had more significant results compared to post-menthol treatment concerning GH mRNA expression. Another difference was that the menthol/SPB-NLS co-treatment group achieved supreme transfer efficiency. As for the reason why menthol/SPB-NLS co-treatment had such an impact on transfection efficiency, it remains to be further investigated.

After we investigated the function of DMSO and menthol in improving GH mRNA expression, we evaluated protein (GFP) expression efficiency in the DMSO- and menthol-treated groups by fluorescence microscopy and flow cytometry. Flow cytometry measurements made every accurate determination of fluorescence intensities feasible. The results from both analyses showed that DMSO and menthol treatments increased GFP expression efficiency. DMSO and menthol treatments only slightly improved the expression of GFP. These results are completely different from those obtained by qRT-PCR. The reason behind this phenomenon may be the following: the slight improvement in GFP expression in the DMSO- and menthol-treated groups implied that DMSO and menthol treatments may affect cell viability, which in turn limits gene expression.

We also found that the post-DMSO/SPB-NLS co-treatment group achieved the highest GFP expression efficiency (increased by 90%; flow cytometry). Similarly, the pre-menthol/SPB-NLS co-treatment group also obtained a high efficiency in GFP expression (increased by 52%; flow cytometry). Notably, high-level transcription efficiency (DMSO- and menthol-treated group) did not always mean high-level expression efficiency. Among all treatments, the post-DMSO/SPB-NLS and the pre-menthol/SPB-NLS co-treatment groups not only showed high transcription levels (GH mRNA expression levels), but also displayed high translation levels (GFP expression levels).

Apart from some isolated reports (27,28), the majority of studies have aimed at increasing DNA nuclear import by adding NLS-containing proteins or using novel polymers. There has been relatively little success transforming information gathered into more effective methods for DNA delivery in vitro. We aimed to search for versatile solutions to improve transfection efficiency. DMSO and menthol seemed to be a suitable way to solve this problem. Previous studies using DMSO have shown that this amphiphilic molecule affects transfection by influencing the formation of the nuclear pore (6). DMSO also alters the cell cycle to accomodate gene transfer. Cell cycle synchronization has been confirmed to be a crucial part in gene delivery (29,30). Previous studies using synchronized cells have shown that cells express more gene product when transfected in the G2 or G2-M phase (8,18). DNA microinjected into the nucleus produces robust gene expression, while cytoplasm injection results in low expression levels. Thus, if plasmids were presented in the cytoplasm, they could enter into the nucleus when the envelope was disrupted (30). It was well known that non-dividing or growth-arrested cells cannot be easily transfected. By contrast, cells undergoing mitosis are much more receptive to transfection (31). The results from the present study demonstrate that DMSO stops the cell cycle during mitosis, which explains the high gene delivery efficiency in the DMSO-treated groups.

Menthol is a naturally-occurring cyclic terpene alcohol of plant origin, which has been frequently used since antiquity for medicinal purposes. However, as a penetration enhancer, the ability of menthol in improving gene delivery efficiency has not been reported. Menthol may be used to increase the permeability of cell membranes or to affect the integrity of the nucleus (23). As detected in previous experiments, the mechanism by which menthol enhances transmembrane permeation may include the possible reversible disruption of the intercellular lipid domain (20,21).

Although high transfection efficiency has been demonstrated, issues related to the effects of DMSO or menthol on transfection efficiency and the mechanisms have not been fully addressed. The present investigation aimed to generate versatile solutions to further improve transfection efficiency. It would be hopeful to provide impetus for searching for more versatile solutions in transferring plasmid DNA.

In conclusion, DMSO can increase the transfection efficiency by influencing the pore integrity of the nucleus or regulating the gene controlled cell cycle and cell hydrophobic properties. Additionally, it is likely that menthol facilitates the gene transfer efficiency by increasing the permeability of cell membranes. Our results clearly demonstrate that pre-menthol and post-DMSO treatments both improve gene delivery efficiency.

The pre-menthol and post-DMSO treatments are developing techniques. We stronlgy believe that only versatile transfection techniques would aid in the development of a controlled release gene transfer method, unlike many of the conventional nucleic acid carriers that are purely liposome-based. Hopefully, our research will lead to a safer alternative to viral systems for gene delivery.