Enhancement of the transfection efficiency of DNA into Crocus sativus L. cells via PEI nanoparticles

Behnam Firoozi, Nasser Zare, Omid Sofalian, Parisa Sheikhzade-Mosadegh

Department of Agronomy and Plant Breeding, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili,Ardabil 56199-11367, Iran

Abstract Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine (PEI) was used for gene therapy successfully. The present study investigated the effect of PEI and ultrasound waves on ssDNA delivery into saffron cells. Gel retardation, dynamic light scattering (DLS) and scanning electron microscopy (SEM) assays were employed to determine the physicochemical properties of PEI/f-DNA polyplex (complex of PEI and fluorescently labeled DNA). Moreover, the cytotoxicity of PEI, PEI/f-DNA polyplex and ultrasound were investigated on saffron cells at different concentrations. The gel retardation results indicated that the formation and neutralization of the PEI/f-DNA polyplex were completed at N/P=5. The particle size distribution of the polyplexes was from 50 to 122 nm. The experimental results revealed that the cytotoxicity of the PEI/f-DNA polyplex was lower than that of PEI alone, hence the cells showed both dose- and exposure duration-dependent responses. Furthermore, the viability of saffron cells declined extremely after 5 and 10 min sonication but this reduction was not significant at 2 min exposure duration. The results also indicated that the combined utilization of ultrasound and PEI nanoparticles increased the transfection efficiency of saffron cells up to two times higher than those obtained by PEI or ultrasound separately.

Keywords: PEI/f-DNA polyplex, plant cell transfection, saffron, sonication

1. lntroduction

Plant biotechnology plays an essential role in modern agriculture, by improving the genetic composition of plants.Advances in plant genetic transformation methods have made it feasible to genetically modify a large number of plant species, especially agriculturally important crops, for better agronomical traits such as disease and insect resistance,nutritional values, environmental stresses resistance,production of medicinally valuable secondary metabolites,recombinant proteins, vaccines in plants and other favorable traits (Fischeret al.2004; Vain 2007; Pasupathyet al.2008).Compared to mammalian cell transformation, gene delivery into plant cells is much more challenging and difficult due to the presence of the cell wall barrier (Pasupathyet al.2008).Different physical, chemical and biological procedures have also been successfully applied to achieve direct or indirect DNA delivery into plants.Agrobacterium-mediated transformation has inherent advantages over direct DNA delivery systems including the ability to transfer large intact segments of DNA, simple transgene insertions with defined ends and low copy number, constant integration and inheritance, and consistent gene expression over the generations (Barampuram and Zhang 2011). But in the case of this method, the efficiency for monocots is still unsatisfactory (Rakoczy-Trojanowska 2002).

Other methods, such as electroporation of protoplasts,polyethylene glycol (PEG) treatment, biolistic particle bombardment (gene gun) and silicon carbide whiskermediated transformation of plants and plant cells with intact cell walls, have been used in a variety of plant species and shown to be partially successful. Nevertheless, they are often considered as, expensive, and cause a significant agitation on cell growth (Pasupathyet al.2008).

As a result of the exclusive physical and chemical properties, nanoparticle-mediated gene delivery have the potential to directly transfer of DNA into plant cells, thereby achieving transient or stable expression of the transgene and also targeted gene delivery to plant cell organelles including the chloroplast (Hamidiet al. 2008; Raiet al.2015). Nanoparticles, as a novel kind of non-viral gene delivery carriers, possess many advantages over traditional gene carriers in gene delivery. Firstly, these particles are applicable to both monocots and dicots and any types of organs. Secondly, these carriers can overcome barriers such as the cell wall and protect DNA molecules against nuclease enzymes degradation by preventing access to the cleavage sites due to enfolding and condensing nucleic acids (DNA/RNA). Thirdly, nanoparticles can be easily functionalized in order to enhance gene delivery efficiency to the cell organelles. Fourthly, nanoparticles can efficiently overcome transgenic silencing by controlling the copies of DNA conjugated to nanoparticles and the controlled release of DNA. Consequently, the DNA stability and duration of action are enhanced. Finally, nanoparticlemediated multigene transformation can be achieved without involving the traditional building method of a complex carrier(Vijayanathanet al. 2002; Fuet al. 2012). As gene carriers,nanoparticles have become popular in the field of gene and drug delivery into mammalian cells (Nguyenet al. 2009),while its application in plant cell studies are still very limited and in progress (Pasupathyet al.2008; Nairet al. 2010).

Nanomaterials and nanoparticles have been widely exploited in biomedicine for site-targeted delivery of drugs and macromolecules including DNA (Silvaet al. 2014;McDonaldet al. 2015). Sunet al. (2016) reported that PEG-CS/siRNA polyplexes efficiently facilitated take up of the siRNA than naked siRNA and significantly reduced the growth of xenograft tumors of 4T1 cellsin vivo. Despite of these developments, nanomaterials use in plant biology,agriculture and horticulture, is still in nascent stages (Singhet al. 2015). Fuet al. (2012), moreover, have been utilized ZnS (quantum dots) nanoparticles modified with positively charged poly-L-lysine(PLL) for successful GUS-encoding plasmid DNA delivery into tobacco cells using ultrasoundassisted method.

Polyethylenimines (PEIs) are among the most positively charged polymers (a cationic polymer) synthesized in different molecular weights and conformation, including branched (BPEI) and linear (LPEI) structures, which have high and moderate transfection activityin vitroandin vivo,respectively (Godbeyet al. 1999; McKenzieet al. 1999;Yuet al. 2016). It is assumed that, PEI enter cellsviaan endocytosis pathway due to the strong buffering capacity and the ability to swell when protonated (Amiji 2004). Once endocytosed, studies have demonstrated that PEI endures nuclear localization while retaining an ordered configuration.The transfection efficacy and amount of transgene depends on the PEI molecular weight and configuration, nature of the polymer used and the amount of pDNA (plasmid DNA)delivered (Amiji 2004). Fuet al. (2012) provided a new procedure for the use of ZnS nanoparticles as a gene carrier in plant gene engineering. Their study showed that ZnS nanoparticles modified with positively charged poly-L-lysine(PLL)viaultrasound treatment can successfully deliver GUS-encoding plasmid DNA into tobacco cells.

In recent years, a large number of studies have been conducted on PEI-mediated gene delivery to animal and human cells and several groups have reported improved transfection efficiencies with PEI. However, these researches have been mostly focused on animal cells. Cuiet al. (2012) employed polyethylenimine (PEI)-modified magnetic nanoparticles as vectors to deliver GFP (green fluorescent protein) gene to porcine kidney-15 cells (PK15).They found that the surface of the particles becomes rough with increased average diameter, which implied the effective conjugation of nanoparticles/DNA. Zhenget al. (2017)developed a cationic, α-helical polypeptide (PPABLG) which could efficiently transfect both isolatedArabidopsis thalianaprotoplasts and intact leaves by the GFP gene. Accordingly,the present study documents the synergistic effect of PEI nanopolymers, as a new nanocarrier, and ultrasound on DNA delivery into the intact cells ofCrocus sativusL.

2. Materials and methods

2.1. Plant materials

The saffron corms were obtained from Ghaen(33°43′N, 59°11′E), South Khorasan Province, Iran.After surface sterilization, corm segments (1–2 cm) were cultured on the MS (Murashige and Skoog 1962) basal solid medium supplemented with 1 mg L–1NAA and 1 mg L–1Kin.Thereafter, cultures were maintained in a growth chamber at (25±1)°C under 16 h/8 h photoperiod with 700–800 lx of cool-white light intensity and were subcultured at monthly intervals. After three months, the calli appeared on the explants. A total of 2 g of the friable calli were transferred into 150 mL Erlenmeyer flask containing 30 mL of liquid MS basal medium supplemented with 1 mg L–1NAA (Duchefa,Netherlands) and 0.5 mg L–1Kinetin (Duchefa, Netherlands)and sucrose (3%). Every 10 days, 10 mL of fresh medium was added to the culture flasks until it reached the final volume of 60 mL. The cultures were maintained at (25±1)°C on a rotary shaker (110 r min–1) and 16 h photoperiods with a light intensity of 700–800 lx. After about 45–60 days,suspended cells with active cell division and proliferation were achieved.

2.2. Preparation of PEl nanoparticles and PEl/f-DNA polyplex

Linear polyethylenimine (L-PEI) with MW of 25 kDa(Cat.#23966) was purchased from Polysciences Inc.(Warrington, PA, USA). L-PEI stock solution (1 mg mL–1)was prepared by dissolving in PBS (phosphate-buffered saline, pH=7.0) followed by sonication for 5 min and filtered (0.22 μm pore size; MCE membrane, Biofil?).A 6-carboxyfluorescein labeled random oligonucleotide(f-DNA), (6-FAM)-ACCGAACAGAACACGAAAGC-3′, was prepared from Bioneer Company (Daejeon, Republic of Korea). A 20-mer oligonucleotide was dissolved in sterile distilled water (SDW) at 100 pmol μL–1concentration and stored according to the manufacturer’s instructions until use.

The PEI/f-DNA polyplex was prepared by combining 1 μg of fluorescein labeled DNA (f-DNA) and L-PEI at N/P ratios of (1, 2, 3, 4, 5, 6 and 7) followed by vigorous pipetting for thorough mixing, then it was allowed to stand for 30 min to polyplex self-assembly (Ahnet al. 2008).

2.3. Gel retardation study

Gel retardation assay was used to evaluate the DNA binding ability of L-PEI and its capability to neutralize the surface charge of DNA. A total of 10 μL of freshly prepared polyplex at series of N/P ratio were electrophoresed on 1.5% (w/v)agarose gel at 100 V and kept for ～20 min. The results of electrophoresis and DNA banding pattern were revealed by gel documentation (ATP Co., Ltd., Tehran, Iran).

2.4. PEl/f-DNA polyplex characterization

The PEI/f-DNA polyplex was analyzed using LEO 1430VP scanning electron microscopy (SEM). For this purpose,the PEI/f-DNA polyplex was prepared at N/P=5, thereafter it was dropped on the clean glass slide, and dried at 25°C for 2 h in an aseptic cabinet (Sunet al. 2008). Before SEM observation, the samples were coated with gold nanoparticles followed by scanning. The size distribution and ζ-potential of the PEI/f-DNA polyplex at the N/P ratio of 5 were also determinedviadynamic light scattering (DLS,Malvern Zetasizer, Malvern Instruments, Malvern, UK) at 25°C.

2.5. PEl cytotoxicity assay

The viability of saffron cells exposed to L-PEI nanopolymers and PEI/f-DNA was assessed by both trypan blue exclusion assay (Louis and Siegel 2011) and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mosmann 1983). For trypan blue exclusion test,2 mL of suspension culture of saffron with a density of 2×105cells mL–1were transferred to 12-well plates. The cells were then exposed to different concentrations of L-PEI (2, 4, 8,20 and 40 μg mL–1) or PEI/f-DNA and incubated at (25±1)°C on a rotary incubator shaker (110 r min–1). Cell viability was determined at 6, 12, 24 and 48 h after exposure, using the trypan-blue exclusion method (Louis and Siegel 2011).The percentage of cell viability (%) was determined by a haemocytometer slide and calculated using the following equation:

For MTT assay, stock solution of MTT was prepared as 5 mg mL–1in PBS (pH=7.2) and filtered. 500 μL of suspended saffron cells with cell density of 2×105cell mL–1added to 96 well microplates. At the end of the treatment with PEI and polyplex (as mentioned in trypan blue assay),30 μL of MTT solution was added to each well. After incubation for 3 h in the dark at 37°C, MTT was removed completely and the cells washed with PBS buffer. Then,solubilizing buffer (DMSO) was added to wells and incubated for 2 h at room temperature. Finally, the absorbance was measured at 570 nm by Spectrophotometer (SmartSpec Plus, Bio-Rad Laboratories, Hercules, CA, USA). The percentage of the viable cells was calculated using the following formula:

2.6. The effects of ultrasound on saffron cells

The morphology of untreated and ultrasound-treated cells was evaluated using SEM scanning. For this, 1 mL of saffron suspension cells were transferred to 2 mL centrifuge tubes, sonicated by Bandelin sonicator (160–640 W, 35 kHz,Sonorex Digitec, Bandelin, Germany) for a short and a long duration (1 and 3 min, respectively) and promptly fixed using methanol according to Talbot and White’s (2013) method.The sample was dispersed on a clean glass slide and after drying, it was analysed by LEO 1430VP SEM. Also, the effects of ultrasound waves on the viability of saffron cells were evaluated using the trypan-blue exclusion assay (Louis and Siegel 2011). In this manner, 2 mL of the suspension culture of saffron was sonicated for 0, 2, 5 and 10 min at 25°C, using the Bandelin sonicator (160–640 W, 35 kHz,Sonorex Digitec, Bandelin, Germany). The percentage of cell viability was determined by a haemocytometer slide.

2.7. Ultrasound-assisted PEl/f-DNA polyplex transfection assay

Firstly, PEI/f-DNA polyplex at N/P=5 ratio was prepared and incubated at room temperature for 30 min in PBS(pH=7.4) buffer. Then, 20 mL of suspension cells of saffron(density of 2×105cells mL–1) was prepared and subjected to the hypertonic solution (equivalent volume of fresh PBS containing 9% mannitol) for 30 min and divided into 20 aliquots in 2 mL centrifuge tubes. The suspension tubes were allowed to stand for 15 min at room temperature,until sedimentation was complete. The supernatants were discarded and then PEI/f-DNA polyplex were added to each microcentrifuge tube and sonicated for 2 min. The tubes were incubated on a rotary shaker at 110 r min–1and 25°C for 10 h. To remove the free and excess f-DNA, PEI and PEI/f-DNA polyplex, the cells were washed several times with PBS buffer (pH=7.4). The transfection of intact saffron cells by PEI/f-DNA polyplex was analysed by flow cytometry (FACS calibur, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and observed by fluorescence microscopy.In order to conduct flow cytometry analysis, the cells were sedimented, fixed immediately with paraformaldehyde solution (4% w/v, Sigma-Aldrich) in PBS (pH=7.4) for 20 min at room temperature (Sanchez-Aguayoet al. 2004). The transfection efficiency was expressed as percentage of f-DNA positive cells.

In order to perform fluorescence microscopy analysis,20 μL of treated cells were transferred to a clean microscope glass slide and visualized by fluorescence microscopy Hund H 600 AFL (Helmut Hund GmbH, Wetzlar, Germany). The photographs were acquired digitally with a Coolpix S10 camera (Nikon, Tokyo, Japan).

2.8. Statistical analysis

All results were presented as mean±SE. Tests of significance were performed using one-way analysis of variance by SPSS ver. 16 (SPSS Inc, Chicago, IL.) software.The graphs were also produced using Microsoft Office Excel 2010,P≤0.05 considered to be statistically significant.

3. Results

3.1. Gel retardation analysis

The interaction of f-DNA with PEI at different N/P ratios and the formation of PEI/f-DNA was confirmed by gel retardation assay (Fig. 1). At lower N/P ratios (lower than 4), the interaction of f-DNA and PEI was not complete and the negative charge of DNA was not neutralized completely,whereas with increasing N/P ratios the efficiency of the DNA and PEI interaction and formation of polyplex were increased significantly so that at N/P=5 and higher ones, the PEI/f-DNA complex formation was completed and polyplex was immobilized at the well (Fig. 1).

3.2. PEl/f-DNA polyplex characterization

The compatibility of PEI surface charge and size distribution for gene delivery was confirmed by DLS and SEM microscopy(Fig. 2-A–D). The PEI/f-DNA polyplex characters are presented in Fig. 2-A. The polyplex size determined with number and volume distribution gave partly similar results.The DLS results of the PEI/f-DNA polyplexes showed only one peak in the number (Fig. 2-B) and three peaks in the volume distribution. Hence, based on the number and volume distributions, the hydrodynamic diameter mean was 88.9 nm (90.7% of total volume). Furthermore, DLS allowed measurement of the zeta potential of the polyplex. The last researches showed that only positively charged polyplexes were taken up through endocytosis due to the negative surface charge of the cell membrane. The following zeta potential of PEI/f-DNA polyplex at N/P=5 was obtained as+6.12 mV (Fig. 2-C).

The SEM images of PEI/f-DNA polyplexes (Fig. 2-D)revealed that PEI/f-DNA polyplexes have a spherical morphology (diameter ranged from 50.7 to 122 nm) with no noticeable aggregates. The images of ultrasound treated saffron cells (Fig. 3-A and B) indicated that perforation occurred on the cell wall after a low intensity of ultrasound treatment. As revealed in Fig. 3-C, the intact saffron cells are mostly spheroid in shape and the viability is also favorable.Moreover, saffron cells showed a rough surface with wrinkles and dimples.

Fig. 1 Analysis of PEI/f-DNA (complex of polyethylenimine and fluorescently labeled DNA) polyplex formation by gel retardation assay. f-DNA was complexed with PEI at different N/P ratio:lane 1, f-DNA only; lane 2, N/P=2; lane 3, N/P=3; lane 4, N/P=4; lane 5, N/P=5; lane 6, N/P=6; lane 7, N/P=7.

Fig. 2 Characterization of PEI/f-DNA (complex of polyethylenimine and fluorescently labeled DNA) polyplex by dynamic light scattering (DLS) technique. A, main characteristics of PEI/f-DNA polyplex (Dh, hydrodynamic diameter; PdI, polydispersity index;Z, zeta potential). B, size distribution. C, zeta potential. D, scanning electron microscopy (SEM) image of the PEI/f-DNA polyplex at N/P=5.

Fig. 3 Scanning electron microscopy (SEM) analysis of saffron cells exposed to ultrasonic waves. A, saffron cells exposed to 30 s. B, 3 m duration of ultrasound. C, intact saffron cells. D,PEI-treated saffron cells.

3.3. PEI in vitro cytocompatibility

The effect of PEI and PEI/f-DNA polyplex on the viability of the saffron cells were assessed by trypan blue exclusion assay and MTT tests. As shown in Fig. 4, the viable cells were characterized with a bright center (in trypan blue assay) and dark purple color (formazan product) (MTT assay).

Fig. 4 Image of the saffron cells stained with trypan blue and MTT. A, control cells in MTT assay. B, cells treated with 40 μg mL–1 PEI stained with MTT. C, control cells stained with trypan blue. D, cells treated with 40 μg mL–1 PEI (polyethylenimine)stained with trypan blue. In trypan blue and MTT tests, living cells appeared as yellow and dark purple, respectively. The scale for figures A and B is 10×, and for C and D is 4×.

Fig. 5 Relative saffron cells viability at 6 h (A), 12 h (B), 24 h (C), and 48 h (D) after treatment with different concentrations of PEI(polyethylenimine) and PEI/f-DNA (complex of PEI and fluorescently labeled DNA) polyplex measured by trypan blue and MTT assays. The values represent the mean of three replications. The bars show standard error (SE).

Both trypan blue exclusion test and MTT assay produced similar results. The cells incubated in the liquid MS medium were considered as control. Fig. 3-D shows the saffron cell surface after the treatment with PEI. The ANOVA analysis showed significant differences between the cytotoxicity of the PEI/f-DNA polyplex and PEI (P≤0.05) after 6, 12 and 24 hpt (hours post treatment). As clearly indicated in Fig. 5,the cytotoxicity of the PEI polymer increased with increasing the PEI concentration. The higher concentrations (20 and 40 μg mL–1) of PEI showed the highest cytotoxicity; such that the saffron cells treated with 40 μg mL–1PEI showed a 14.5% reduction (in trypan blue assay) in cell viability at 6 h after treatment, as compared to the ones treated with 2 μg mL–1PEI (Fig. 5-A). As shown in Fig. 5-A–D, the cytotoxicity of PEI/f-DNA polyplex at all concentrations and times after treatment were significantly lower than that of PEI. The cytotoxicity of PEI/f-DNA polyplexes also increased with increasing polyplex concentration and time after treatment.Among PEI/f-DNA treatments, the lowest cell viability was observed at a concentration of 40 μg mL–1and 48 h after the treatment in trypan blue assay (Fig. 5-D).

3.4. Effect of sonication on saffron cells viability

Fig. 6 The effect of different exposure durations of ultrasound(US) on saffron cells viability at 10 min, 24 and 48 h after treatment. The values represent the mean of three replications.The bars show standard error (SE).

Fig. 6 presents the percentage of saffron cell viability on 10 min–48 h after exposure to ultrasound for 2, 5 and 10 min. The cell viability was adversely affected by ultrasound and the responses of the saffron cells to different exposure duration of ultrasound (sonication) were different.As shown in Fig. 6, at 2 min ultrasound exposure duration,the cell viability decreased simultaneously and reached a plateau 24 h after treatment and no noticeable reduction was seen in the percentage of cell viability. Whereas, at 5 min exposure duration, the cell viability was significantly reduced continuously until it declined to 1% on 48 h after treatment. Sonication for 10 min caused severe cell damage and the cell viability dropped down to 1.5% just 10 min after treatment (Fig. 6).

3.5. Saffron cells transfection analysis

Flow cytometry quantitative analysis of intracellular f-DNA trafficking demonstrated the PEI ability for DNA delivery into saffron cells (Fig. 7). The ANOVA analysis of FACS(fluorescence-activated cell sorter) data indicated that transfection efficiency varied significantly among the treatments (Fig. 8). As shown in Fig. 8, the presence of PEI in the PEI/f-DNA polyplex resulted in about 3-fold higher cellular transfection compared to the cells treated by only f-DNA.

The percentages of f-DNA internalization by PEI/f-DNA polyplex and ultrasound were not significantly different while the combined utilization of PEI/f-DNA and ultrasound treatments noticeably enhanced the percentage of transfected saffron cells. The highest percentage of transfection (51.27%±3.58) was recorded in the cells treated with PEI/f-DNA polyplex and exposed to ultrasound.Furthermore, the cellular internalization of f-DNA was confirmed directly by fluorescence microscopy observation(Fig. 9).

4. Discussion

Surface charge and hydrodynamic diameter are two important parameters that determine the stability and dispersion of nanoparticles in an aqueous medium (Stumm and Morgan 1996). Although positively charged polyplex is able to associate more efficiently with negatively charged cell membrane surfaces, it still must be optimized to reduce its cytotoxic properties for any cell type. As demonstrated by the gel retardation assay (Fig. 1), due to the nature of the PEI, this nanopolymer was able to condense the anionic DNA. With increasing N/P ratio, the efficacy of polyplex formation and DNA condensation increased, so that at N/P=5 and higher, the whole DNA complexed as f-DNA/PEI polyplex was trapped at the well (Fig. 1).

PEI compacts the DNA molecule into globular nanostructures (Fig. 2-D), which then undergo cellular internalization through the process of endocytosis. Ogriset al. (2001) evaluated the effects of DNA/PEI complex size on DNA transfection in K562 cells (human erythromyeoloid leukemia, ATCC CCL-243) and reported that in large complexes, improper condensation of DNA occurred.Moreover, laser scanning microscopy confirmed that the large and aggregated complexes mainly adhered to the cell surface and were scantily internalized, whereas with the small DNA/Tf-PEI (transferrin) complexes, perfect internalization was observed.

Fig. 7 Flow cytometry analysis of f-DNA (fluorescently labeled DNA) uptake by saffron cells transfected with: f-DNA (A); PEI/f-DNA(complex of polyethylenimine and f-DNA) polyplex (B); f-DNA+Ultrasound (C); PEI/f-DNA polyplex+Ultrasound (D). Plots indicate population of the positive cells for fluorescently labeled DNA (green color).

Fig. 8 PEI/f-DNA (complex of polyethylenimine and fluorescently labeled DNA) polyplex transfection efficiency in saffron intact cells at N/P=5, as determined by fluorescenceactivated cell sorter (FACS). The values represent the mean of three replications. The bars show standard error (SE).Values followed by the same letter are not significantly different(P=0.05) using Duncan’s multiple range test.

Fig. 9 Fluorescence microscopy of saffron cells transfected by PEI/f-DNA (complex of polyethylenimine and fluorescently labeled DNA) polyplex at N/P=5. A1–A3, control cell. B1–B3, PEI/f-DNA polyplex treatment. C1–C3 and D1–D3, ultrasound+PEI/f-DNA.The transfected cells were observed as a bright green under fluorescence microscope.

The ideal transfection method should have some properties such as high transfection efficiency, low cell toxicity and minimal impact on cell/tissue physiology (Kim and Eberwine 2010). Accordingly, it is essential to measure thein vitrocytotoxicity of PEI polymer alone and PEI/f-DNA polyplexes to determine its favorable concentration and type of application. Strong electrostatic interactions between protonated amino groups of the cationic polymers and cellular compartments cause cell membrane destabilization.According to the results obtained from PEI cytotoxicity assay(Fig. 5-A–D), the cell viability declined significantly as the concentration and incubation time were extended from 2 to 40 μg mL–1and 6 to 48 hpt in both PEI alone and complexed with f-DNA, respectively. We observed a severe viability drop when the cells were treated with 40 μg mL–1of PEI alone (even below 25% after 48 h of incubation in trypan blue) as compared to 20 μg mL–1. The complexation of DNA with PEI and polyplex formation significantly reduced the cytotoxicity of the PEI polymer (Fig. 5-A–D) and it may be due to the masking of the extra cationic charges of PEI and therefore, reduction and even neutralization of the cationic charge of PEI in PEI/f-DNA polyplexes. The free form of PEI leftovers in the DNA/PEI mixture during the complexation process is one of the outstanding events in PEI-mediated transfection protocols. Two different mechanisms have been reported for the cytotoxicity of PEI; 1) the disruption of the cell membrane leading to necrotic cell death (immediate)and 2) apoptosis caused by interruption of the mitochondrial membrane after internalization (delayed) (Moghimiet al.2005). However, these studies mainly focused on animal cells and also the lethal dose of PEI varied over a range of concentrations and cell types. Our results are in complete accordance with previous studies and the hypothesis that argued the charge shielding effect of DNA and change in zeta potentials of PEI in polyplex may be ascribed to the decreased cytotoxicity of the PEI/f-DNA polyplex as compared to free PEI (Kimet al. 2005; Chung and Young 2010).

Despite the advantages of ultrasound in gene transfection,a high intensity and long duration of sonication could be harmful due to damage to the cells. Therefore, the intensity and duration of the ultrasound should be optimized to maintain cell viability. As shown in Fig. 6, the viability of saffron cells was adversely influenced by ultrasound and the cell viability was significantly reduced with increasing exposure duration. These results may be attributed to the capability of saffron cells to restore and repair the damage caused by a short duration (2 min) of ultrasound exposure;but at the higher exposure durations (5 and 10 min, in the current study), the mechanical and biochemical damage caused by ultrasound were irreparable.In vitrosonication has been extensively considered and it revealed that some factors, such as frequency (Milleret al. 1999), exposure duration (Guzmanet al. 2001), number of pulse and temperature (Zarnitsyn and Prausnitz 2004) influence the cells/tissues response to ultrasound. Cheonet al. (2009)evaluated the effect of sonication at 40 kHz and 419 W for 90 s on cell membrane permeability and cell viability of a transgenic rice (Oryza sativaL.cv. Dongjin) cell line and reported that exposure of the PEI/siRNA polyplex with a mass ratio of 1:10 and sonication of the cells for 90 s did not show a noticeable decline in cell viability.

The best complexation and transfection conditions vary among studies and depend on the cell type (animal or plant)and line, N/P ratios and complexes concentration, incubation duration, medium conditions and other additional agents and treatments. In the study of Lianget al. (2012), the highest transfection efficiency ((86.05±5.22)%) of PEG-PEI/siRNA nanoparticles to neural stem cells (NSCs) was achieved at a N/P=15. As shown in Fig. 8, the sonication of naked fluorescently labelled DNA improved transfection by up to 3.5-fold as compared to f-DNA alone. Moreover, f-DNA complexation with PEI led to internalization rate up to three times in comparison with naked f-DNA. On the other hand,there was no significant difference between transfection by the PEI/f-DNA complex and ultrasound, indicating that ultrasound treatment and PEI polymers are important factors in the transfection efficiency of saffron cells. Alternatively,the sonication of saffron cells in the presence of PEI/f-DNA polyplexes improved the f-DNA internalization up to ～12 and two-fold as compared to the naked f-DNA and either PEI or ultrasound (as separately) (Fig. 8). These results indicate the synergistic effects between ultrasound and PEI nanopolymers for DNA delivery into plant cells. As shown in Fig. 3-A, SEM micrographs indicated that sonication of the saffron cells led to the formation of several pores on the cell surface which could facilitate macro-molecules uptake and internalization. These synergistic effects could be attributed to the sonoporation and increased cell membrane permeability caused by cavitation and the physical effects of ultrasound (Karshafianet al. 2009; Phillipset al. 2010).Similarly, Chenet al. (2012) reported that the combined utilization of ultrasound and PEI for luciferase encoding gene delivery into the human MCF7 cells lines (human breast adenocarcinoma) significantly increased the transfection efficiency. Furthermore, Shihet al. (2015) showed that the cationic polymer-mediated cell transfection efficiency was significantly enhanced to 150% compared to the control by using low energy ultrasound in HEK-293 and COS-7 cell lines. Deshpande and Prausnitz (2007) showed improved transfection efficiency in human muscle cells by applying ultrasound or PEI/DNA polyplex up to 18 and 90-fold,respectively; while the combination of ultrasound and PEI,synergistically resulted to increased levels of internalization up to 200-fold.

5. Conclusion

Nanoparticles-mediated gene delivery to intact cells provides a new and efficient approach for the direct transfer of DNA to the plants and transgenic plant production. The results of the present study indicated that the PEI/f-DNA polyplex and sonication have a synergistic effect on f-DNA delivery into saffron cells. Moreover, the maximum DNA delivery occurred with the PEI/f-DNA polyplex formed at N/P=5 and 2 min sonication. Altogether, it is recommended that future works focus on studying the effect of other types of polyplex (polyplex with higher N/P ratio and other copolymers such as PEG/PEI and PEG-chitosan-PEI) and its surface modification in gene transfection efficiency in plant intact cells.

Acknowledgements

This study was supported by the University of Mohaghegh Ardabili, India under Grant (51-487).