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. 2012 May 22;20(8):1590–1598. doi: 10.1038/mt.2012.95

LNA-based Oligonucleotide Electrotransfer for miRNA Inhibition

Sophie Chabot 1,2, Julie Orio 1,2, Romain Castanier 1,2, Elisabeth Bellard 1,2, Søren J Nielsen 3, Muriel Golzio 1,2,*, Justin Teissié 1,2,*
PMCID: PMC3412486  PMID: 22617110

Abstract

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases.

Introduction

Micro-RNAs (miRNAs) are small (~22 nt) noncoding RNAs that posttranscriptionally regulate gene expression by repressing translation or accelerating mRNA decay.1 MiRNAs play crucial roles in the control of critical biological processes, including development, cell differentiation, proliferation, and apoptosis.2 An increasing body of research suggests that miRNAs are involved in a wide variety of human diseases, such as cancer3 and, thus, have rapidly emerged as a new class of potential therapeutic targets.4 The binding of miRNA to its target mRNA by Watson–Crick base-pairing is essential for its biological function.5 Therefore, an obvious miRNA inhibitor is an oligonucleotide that is complementary to its target miRNA and binds to it with high affinity and specificity.6 However, due to its small size, miRNA-based therapy requires robust and improved antisense oligonucleotide technology. The locked nucleic acid (LNA) is a new generation of chemically modified oligonucleotides. LNA contains a methylene bridge that connects the 2′-oxygen with the 4′-carbon of ribose.7,8 The constraint on the sugar moiety results in a locked C3′-endo/N-type conformation that preorganizes the base for hybridization.9 LNA oligonucleotide exhibits unprecedented thermal stability when hybridized with its RNA target molecule.10 Oligomer that contains LNA bases (LNA/DNA oligomer) has significant improved mismatch discrimination compared with unmodified reference oligomer.11 Furthermore, LNA oligonucleotide is highly resistant to nuclease degradation and displays low toxicity in biological systems.12 Therefore, LNA-based molecules appear to be promising therapeutic tools for developing miRNA mimics or inhibitors.13 This applicability is illustrated by the evaluation of subcutaneous injections of the LNA-modified oligonucleotide SPC3649, which prevents Hepatitis C viral infection by inhibiting miR-122, into phase II trials.14 In addition, there are more than 10 clinical trials (listed on http://www.clinicaltrials.gov/) using either LNA or miRNA inhibitors.

Extensive application of miRNA-based therapeutics requires effective LNA/DNA oligomer delivery to target cells and tissues. Because of its size and charge, LNA/DNA oligomer can barely cross the cell membrane. Delivery is therefore one of the major challenges for miRNA-based therapeutics.15 We propose to deliver LNA/DNA oligomer using electropulsation (EP).16 EP is a physical method that applies electric pulses to target cells or tissues to transiently permeabilize the membranes, allowing molecules to enter these targets.17,18 This technique is currently used in the clinic by applying a direct field to the patient following injection of a cytotoxic drug (electrochemotherapy)19,20,21 and appears promising for clinical applications involving plasmid DNA (electrogenotherapy).22 Currently, there are more than 25 clinical trials (listed on www.clinicaltrials.gov) using EP to perform gene delivery to several tissues, including both therapeutic and vaccine approaches. In fact, EP offers a number of advantages, including specific targeting of the delivery, short delay between injection, and delivery, ease and low cost of the procedure. EP appears to be a promising technique for LNA/DNA oligomer delivery. However, the mechanism of LNA/DNA oligomer electrotransfer is unknown, as it has never been visualized or described.

In the present paper, a LNA/DNA oligomer has been 5′-conjugated with the cyanine 5 (Cy5) fluorochrome, allowing us to analyze delivery efficiency by flow cytometry and single cell confocal fluorescence microscopy. We demonstrated that EP is a powerful technique for LNA/DNA oligomer delivery. By analyzing the LNA/DNA oligomer electrotransfer mechanism, we also showed that the oligonucleotide has direct access to the cytoplasm and the nucleus where its miRNA target and/or precursor miRNA target, such as pri-, pre-miRNA, or miRNA gene, are located. Finally, we demonstrated that electrotransferred LNA/DNA oligomer is biologically functional. In summary, our results clear the way for using EP in LNA/DNA oligomer delivery for miRNA therapeutic target validation and oligonucleotide-based therapeutics for disease-associated miRNA.

Results

Efficiency of LNA/DNA oligomer electrotransfer

To test whether LNA/DNA oligomer can be efficiently electrotransferred, we used a Cy5-labeled oligomer and analyzed Cy5-positive cells by flow cytometry after EP. Electro-induced cell permeability depends on the pulse parameters used. To optimize LNA/DNA oligomer electrodelivery, cells in suspension were subjected to 5 milliseconds pulses at a 1-Hz frequency and two electrical parameters were modulated; pulse number and field intensity. We observed that, without pulse application (not pulsed (NP)), the addition of the Cy5-LNA/DNA oligomer led to a weak and nonsignificant cell-associated Cy5 fluorescence intensity (Figure 1a,b). However, when cells were submitted to electric field pulses (EP), a strong increase in the Cy5 mean fluorescence intensity was observed (9.3 ± 1.3- and 15.4 ± 1.6- fold higher with 8 pulses of 700 V/cm and 800 V/cm field strengths, respectively, compared to nonpulsed cells, Figure 1a,b). Furthermore, when pulses were applied, a Cy5high cell population (M2) appeared and depended on the field intensity (Figure 1c). Three conditions, which were 8 pulses at 700 V/cm and 4 or 8 pulses at 800 V/cm, allowed very efficient (>55%) LNA/DNA oligomer delivery (Figure 1c). Efficient LNA/DNA oligomer uptake occurred regardless of cell type (Supplementary Figure S1 online). Collectively, our data demonstrated that EP could efficiently increase LNA/DNA oligomer cellular uptake.

Figure 1.

Figure 1

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The LNA/DNA oligomer is efficiently delivered by EP. (a) Representative FACS patterns of HCT116 cells pulsed (P) or not pulsed (NP) in the presence of the Cy5-LNA/DNA oligomer (LNA) (250 nmol/l). (b) Cy5 mean fluorescence intensity (MFI) of the Cy5low to high cells (M1 population). (c) Percentage of Cy5high cells (M2 population). Pulse duration was 5 milliseconds, the delay between pulses was 1 second, the field intensity (E in V/cm) and number of pulses varied as indicated. Values are means + SEM of three data sets. EP, electropulsation; LNA, locked nucleic acid.

Optimization of LNA/DNA oligomer delivery by EP

Extreme electrical parameters can lead to cell death. In fact, cell viability decreased as the number of pulses or the field intensity increased (Figure 2a). When 8 pulses at 700 V/cm or 4 pulses at 800 V/cm were applied, cell viability remained ~50% (46% ± 3 and 53.3% ± 4.5, respectively), whereas with 8 pulses at 800 V/cm, cell survival dropped to only 22.2% ± 2.3 (Figure 2a). Thus, efficient electrical parameters for LNA/DNA oligomer delivery to cells in suspension were either 4 pulses of 800 V/cm or 8 pulses of 700 V/cm.

Figure 2.

Figure 2

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Determination of the optimum parameters for LNA/DNA oligomer electrodelivery. (a) Cell viability 24 hours after electropulsation as a function of the electrical parameters used. Pulse duration was 5 milliseconds and the delay between pulses was 1 second 0, 4, or 8 pulses with increasing field intensity (E in V/cm) were applied as indicated. (b) HCT116 cells were pulsed (8 pulses of 5 milliseconds duration at 700 V/cm, 1 Hz) in the presence of increasing LNA/DNA oligomer concentrations. Cell viability was measured 24 hours after electrotransfer by crystal violet. Values are means + SEM of three datasets. LNA, locked nucleic acid; NS, nonsignificant result (one-way ANOVA).

We then evaluated the concentration dependence of toxicity from electrotransferred LNA/DNA oligomer under these two pulsing conditions. No significant change in cell viability was detected at the oligonucleotide concentrations used, except a slight decrease at the highest concentrations (500 nmol/l and 1,000 nmol/l) (Figure 2b). The working LNA/DNA oligomer concentration was therefore kept at 250 nmol/l for our experiments.

Visualization and mechanism of LNA/DNA oligomer electrotransfer at the single cell level

Adherent CHO cells were pulsed in the presence of Cy5-LNA/DNA oligomer directly under a confocal fluorescence microscope to study the mechanism of LNA/DNA oligomer electrotransfer. This approach allowed us to visualize in real time the first steps of LNA/DNA oligomer electrotransfer (Figure 3a). Due to the difference in cell geometry of plated cells compared to cells in suspension and their increased sensitivity, the electric field must be lowered to preserve viability.16 Therefore, field intensity for this experiment was decreased to 300 V/cm (10 pulses of 5 milliseconds duration)23 and the oligonucleotide concentration was kept at 250 nmol/l.

Figure 3.

Figure 3

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Mechanism of LNA/DNA oligomer electrotransfer visualized by direct confocal fluorescence microscopy. (a) Plated CHO cells were pulsed (P) (300 V/cm, 10 pulses of 5 milliseconds, 1 Hz) or not pulsed (NP) in the presence of Cy5-LNA/DNA oligomer (250 nmol/l), and scan series were simultaneous carried out to detect oligonucleotide entrance during pulse train application. The anode was located at the top of the picture. Scale bare = 20 µm. An image differential treatment was applied to remove extracellular fluorescence (see Materials and Methods section). Cy5 fluorescence is in white (right pictures) and light transmission acquisitions are on the left. (b,c) The mean fluorescence intensity of intracellular Cy5-LNA/DNA oligomer was quantified across the cytoplasm facing the anode or the cathode (c) during (1 pulse, 5 pulses, and 10 pulses) and after (10 pulses + 1 second) pulse application. Data are means + SEM of six independent experiments; ***P ≤ 0.001; NS, nonsignificant result (two-way ANOVA). (d) Cy5-LNA/DNA oligomer was added 1 second (1 second post-pulse) or 10 seconds (10 seconds post-pulse) after pulse train application (300 V/cm, 10 pulses of 5 milliseconds, 1 Hz), and the cells were re-pulsed a second time (10 seconds post-pulse + pulse) using the same electrical parameters. Cy5 mean fluorescence intensity was quantified across the cytoplasm at the cathode facing side. Data are means + SEM of five independent experiments; *P ≤ 0.05 and ***P ≤ 0.001; NS, nonsignificant result (two-way ANOVA). (e) A representative light plot profile parallel to the electric field direction was plotted after 1 (1 pulse, straight line) and 2 (2 pulse, dashed line) pulses using bipolar condition (300 V/cm, 5 milliseconds, 1 Hz). Scale bar = 10 µm. Pictures of fluorescence are in pseudo-color, and gray pictures are light transmission acquisitions. LNA, locked nucleic acid.

We observed that LNA/DNA oligomer directly reached the cytoplasm of pulsed cells through the permeabilized membrane only at the side of the cell facing the negative electrode (cathode) (Figure 3a–c). No Cy5 fluorescence was detected in the cytoplasm on the side of the cell facing the anode (Figure 3b), although propidium iodide (PI) experiments showed that this side was also permeabilized (Supplementary Figure S2 online). This localized entry was not due to the electrical pulse conditions as similar observations were obtained using two classical types of electrical settings, i.e., pulse of high voltage and short duration (electrochemotherapy) and pulse of low voltage and long duration (electrogenotherapy), regardless of the cell type used (Supplementary Figure S3 online).

LNA/DNA oligomer cellular uptake occurred as soon as the first pulse was applied and increased with the number of pulses (Figure 3a,c, 1 pulse, 5 pulse, and 10 pulse), whereas no LNA/DNA oligomer entry was observed in the absence of an electrical field pulse (Figure 3a, NP). Less than 1 second after pulse application, no further increase in Cy5 mean fluorescence was observed (Figure 3c, 10 pulse + 1 second). When the Cy5-LNA/DNA oligomer was added after pulse application, no oligonucleotide entry was detected (Figure 3d, 1 second post-pulse and 10 seconds post-pulse), whereas cells were still permeabilized to PI (Supplementary Figure S4 online). When the same cells (in the presence of the Cy5-LNA/DNA oligomer) were pulsed again, the Cy5 mean fluorescence increased (Figure 3d, 10 seconds post-pulse + pulse). Collectively, these data show that LNA/DNA oligomer entry occurred solely during pulse application.

The polarity of the electrodes was changed between each pulse (“bipolar” condition) to evaluate if LNA/DNA oligomer entry was controlled by the direction of the electric field. We observed that the Cy5 fluorescence intensity alternatively increased at the two sides of the cell and always corresponded to the side of the membrane facing the cathode (Figure 3e). LNA/DNA oligomer delivery was driven by the electrical field direction.

Our findings show that (i) the Cy5 fluorescence intensity increased during the pulse train, (ii) LNA/DNA oligomer electrotransfer occurred solely during pulse application, and (iii) LNA/DNA oligomer electrotransfer depended on the electric field polarity. These observations directly support the model that the electrophoretic forces drove the negatively charged oligonucleotides into the cell.24

Intracellular localization of electrotransferred LNA/DNA oligomers

After studying the dynamics of the cellular uptake of electrotransferred LNA/DNA oligomers, we focused on its intracellular distribution. We observed that after its rapid cytoplasmic entry, the Cy5-labeled LNA/DNA oligomer progressively diffused in the cytoplasm and localized to the nucleus starting as soon as 30 seconds after pulse application (Figure 4a,b). No nuclear staining was detected when a Cy5-labeled siRNA or Cy5-LNA-modified siRNA (siLNA) were electrotransferred (Figure 4b). The siRNA and siLNA remained dispersed in the cytoplasm of the electrotransfected cells. These phenomenon were independent of cell type as a similar observation was obtained with CHO cells (Figure 4c).

Figure 4.

Figure 4

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Intracellular localization of LNA/DNA oligomers after electrotransfer. Plated Hela cells were pulsed (300 V/cm, 10 pulses of 5 milliseconds, 1 Hz) on coverslips under a confocal microscope in the presence of 250 nmol/l of the Cy5-LNA/DNA oligomer and scan series were conducted every 30 seconds after the electrotransfer. (a) Representative images of the intracellular localization of LNA/DNA oligomers after pulse application as a function of time. (b) Cy5-LNA/DNA oligomer (250 nmol/l), Cy5-siRNA (250 nmol/l) and Cy5-siLNA (250 nmol/l) biodistribution 10 minutes after electrotransfer. (c) Plated CHO cells were pulsed (300 V/cm, 10 pulses of 5 milliseconds, 1 Hz) on a coverslip under a confocal microscope in presence of either Cy5-LNA/DNA oligomer (LNA) or Cy5-siRNA (siRNA) and scan series were carried out each minute after the electrotransfer (1–6 minutes). Scale bar = 10 µm. Pictures of fluorescence are in pseudo-color, and gray pictures are light transmission acquisitions. LNA, locked nucleic acid.

Electrotransferred LNA/DNA oligomers are functional

We used the complementary sequence of miR-34a to verify the ability of the LNA/DNA oligomer to block miR-34a function (anti-34a); a corresponding scrambled LNA/DNA oligomer served as a control. HCT116 cells were treated with adriamycin to activate expression of the targeted miR-34a,25 which induced apoptosis26 and G1 cell-cycle arrest.27 We first evaluated miR34a inhibition by electrotransferring anti-34a in adriamycin-treated cells. Electrotransfer of the LNA/DNA oligomer anti-34a resulted in a significant decrease in miR34a levels (25% ± 4.8), as measured by quantitative reverse transcriptase polymerase chain reaction, compared with the scrambled LNA/DNA oligomer (Figure 5a). Consistent with this finding, anti-34a electrotransfer significantly reduced adriamycin-induced apoptosis (20% ± 5.6) compared to the control oligonucleotide (Figure 5b). In addition, when cells are pulsed with anti-34a, the number of cells in G1 arrest strongly decreased (20% ± 4.3) (Figure 5c). These three results demonstrated that electrotransferred anti-miR LNA/DNA oligomer downregulated the targeted miRNA and inhibited its physiological function. No effect was detected when anti-34a was added to the cells without electrotransfer (Figure 5a,c; NP).

Figure 5.

Figure 5

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Electrotransferred LNA/DNA oligomer is functional. HCT116 cells were pulsed (P) (700 V/cm, 8 pulses of 5 milliseconds, 1 Hz) or not pulsed (NP) with 250 nmol/l LNA/DNA oligomer anti-miR34a (anti-34a) or scrambled LNA/DNA oligomer (cont.) and treated or not treated (NT) with adriamycin. (a) qRT-PCR analysis of miR34a levels 48 hours after adriamycin treatment. Bars represent relative miR34a levels normalized to U6 and snoRNA134 in the same samples. RU, relative units. Values are means + SEM of four independent experiments; ***P = 0.0002; NS, nonsignificant result (Student's t-test). (b) After 3 days, cells were subjected to FACS for apoptosis analysis (annexin V+ cells). Values are means + SEM of six independent transfections; *P = 0.0147; NS, nonsignificant result (Student's t-test). (c) Percentage of cells in G1 cell-cycle phase after 2 days of treatment with adriamycin as determined with Modfit 3.0 (Verity Software House, Topsham, ME). Values are means + SEM of triplicate experiments; *P = 0.00438; NS = nonsignificant result (Student's t-test with Welch correction analysis). LNA, locked nucleic acid; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.

Discussion

Since the discovery that miRNAs are associated with many disease processes, there has been an explosion of interest in using this pathway for clinical applications. Although highly attractive as a therapeutic approach, several hurdles must be overcome to successfully introduce miRNA-based oligonucleotides into the clinic. To overcome the problems of biostability, off-target effects and toxicity, chemically modified oligonucleotides have been developed28 including LNA, which has an O-methylene linkage between the second and fourth positions of the ribose.11 This modification leads to exceptionally high-affinity binding and base-pairing specificity to complementary sequences and high resistance to nucleases.29 Thus, LNA-based oligonucleotide appears well-suited for the development of novel therapeutic approaches that target disease-associated miRNA.30 However, one limitation in its use is the lack of efficient, safe, and localized delivery to diseased cells and tissues. In this paper, we studied the LNA/DNA oligomer delivery potential of a clinically approved physical method; the EP technique.31

We first demonstrated by flow cytometry that EP efficiently transfers LNA/DNA oligomer into cells regardless of the cell type (Figure 1, Supplementary Figure S1 online). With the goal of high transfection level and cell viability, we determined efficient electrical parameters for cells in suspension as follows; 4 pulses of 5 milliseconds duration with an intensity of 800 V/cm or 8 pulses of 5 milliseconds duration with an intensity of 700 V/cm (Figures 1 and 2a). Using these conditions, we also demonstrated the absence of LNA/DNA oligomer toxicity even at high concentrations (Figure 2b).

Biophysical approaches have established that electrotransfer mechanisms depend on the chemical properties of the transferred molecules, but nothing is known about the basic processes underlying LNA-based oligonucleotide electrotransfer. Therefore, we examined the LNA/DNA oligomer electrotransfer mechanism. We used confocal fluorescence microscopy to give us access to topological distribution at the single cell level.32 We observed that, although both sides of the pulsed cell were permeabilized (Supplementary Figure S2 online), the LNA/DNA oligomer penetrated into the cytoplasm only on the side of the cell facing the negative electrode (cathode) and moved towards the anode (Figure 3a–c). LNA/DNA oligomer electrotransfer occurred solely during pulse application (Figure 3c,d) even when the membrane was not completely resealed (Supplementary Figure S4 online). Electrotransfer depended on the electric field polarity (Figure 3e). This behavior is experimental evidence that electrophoretic forces drive entry of the LNA/DNA oligomer which has a negatively charged backbone.

The mechanism described here is not limited to a certain cell type as similar results were observed with three different cell lines and therefore is not dependent on cell-specific membrane components or lipid compositions (Supplementary Figure S3 online). Electrotransferred LNA/DNA oligomers enter the cell directly through the (permeabilized) membrane without any surface aggregation. The LNA/DNA oligomer electrotransfer mechanism appears equivalent to that of siRNA24 and, therefore, is very different from that of plasmid DNA.32 These results suggest that the electrotransfer mechanism depends more on the size and the conformation of the molecule than on its chemical nature although electrotransfer efficacy is depending of the charge of the molecule.33 Insertion of LNA bases into DNA oligomer does not modified the negatively charged phosphodiester backbone.

Knowledge about oligonucleotide dynamics and distribution in live cells is important for their transfer optimization. Thus, we studied the intracellular localization of the electrotransferred LNA/DNA oligomer. As fixation protocols may influence the visual appearance of oligonucleotide intracellular localization compared to nonfixed cells,34 we decided to work with living cells. After electrotransfer, LNA/DNA oligomer was homogeneously distributed in the cytoplasm of pulsed cells, indicating direct and free access to the cytoplasm. By contrast, the few previous studies describing LNA-modified oligonucleotide delivery used a cationic lipid carrier and showed punctate labeling in cytosolic vesicular compartments that corresponded to endosomal transfer.35,36,37 Thus, with these lipid carriers, LNA-based oligonucleotide must undergo endosomal escape to reach its target before acidification by lysosomes. Retention in endosomes must be avoided as oligonucleotides could activate an immune response through the Toll Like Receptors that are present at these sites.38,39 Consequently, by bypassing the endocytic pathway, the EP technique provides a more effective transfer in comparison with other nonviral chemical delivery methods.

Rapidly after Cy5-LNA/DNA cytoplasmic electrodelivery, we observed localization of Cy5 fluorescence in the nucleus (Figure 4). We are aware that a fluorescent agent will lead to a strong nuclear staining of dead cells.40 In fact, the cells used for this type of experiment could be more vulnerable to a toxic effect due to special growth conditions (such as in chamber slides). However, no nuclear staining was detected when Cy5-siRNA or -siLNA were electrotransferred using a similar protocol (Figure 4). This last result demonstrates that (i) our cells remained healthy after the electrotransfer,23 (ii) the Cy5 fluorochrome did not induce nuclear localization, and (iii) EP by itself did not cause damage to the nuclear membrane. Together, these data demonstrate that, contrary to siRNA and siLNA, there is a rapid accumulation of electrotransferred LNA/DNA oligomer in the nucleus of intact cells. This difference may in part be due to the small size discrepancy of the two types of molecules although size is not the only limitation. A structural analysis showed that the double strand siRNA was bulkier than double strand DNA and therefore than the single strand LNA/DNA oligomer even if internal folding may be present due to base-pairing and reduced flexibility.41 Thus, contrary to double strand siRNA, single strand LNA/DNA can use nuclear pores to access the nucleus.

The LNA/DNA oligomer migrates within a few minutes to the nucleus (Figure 4a,c), contrasting with electrotransferred plasmid DNA that takes a long time to pass from the cytosol to the nucleus.42 Moreover, LNA/DNA oligomer migration is not due to LNA nucleotides, as siLNA does not penetrate into the nucleus (Figure 4b). It has been clearly established that small DNA oligonucleotide once introduced into the cytosol migrates to the nucleus regardless of the method of transfection used.43,44 This behavior has been described with LNA-modified oligonucleotide delivered using cationic lipid carriers,35,36,37 although the kinetic of nuclear localization is more rapid with EP than with lipid carriers (2 minutes versus 2 hour) (Figure 4a).45 It is believed that small oligonucleotides move passively through nuclear pores because neither chilling nor ATP depletion inhibit their accumulation in the nucleus.46 However, passive diffusion is not sufficient to explain the LNA/DNA oligomer nuclear accumulation that we observed, unless diffusion is followed by nuclear binding. An association between LNA/DNA oligomer and nuclear structures would compete with its target and decrease its activity. On the other hand, a strong correlation between LNA/DNA oligomer nuclear localization and its specific efficiency (as previously observed in cases of non-LNA-modified oligodeoxynucleotide)45 has been recently demonstrated.47 In fact, LNA/DNA oligomer decreases not only the level of its cytoplasmic mature miRNA target but also the level of its respective nuclear precursor miRNA, suggesting that LNA may affect the regulatory loop in miRNA processing.48 Considering these data, EP-induced LNA/DNA oligomer cytoplasmic delivery and strong nuclear accumulation should led to high inhibition of its cytoplasmic miRNA target and its nuclear precursor miRNA target.

The electrotransferred LNA/DNA oligomer is functional. We used, as a proof of concept, a LNA/DNA oligomer antisense to block the well-described effects of miR-34a,26 although this target is not of therapeutic interest. MiR-34a is directly transactivated by p53 inducers (such as adriamycin) and mediates cell apoptosis and G1 cell-cycle arrest.49 We observed that electrotransfer of LNA/DNA oligomer anti-34a significantly reduced adriamycin-increased miR34a levels (Figure 5a). In agreement with this result, electrotransferred LNA/DNA oligomer anti-34a strongly attenuated both adriamycin-induced apoptosis and G1 cell-cycle arrest compared to scrambled LNA/DNA oligomer (Figure 5b,c). These inhibitory effects are as interesting as the miRNA's redundancy renders difficult to have any physiological effect with antisense oligonucleotides.50 It is of note that in the absence of pulse application, no functional effect was observed with the LNA/DNA oligomer anti-34a (Figure 5a,c). Our findings support the notion that electrotransfer of LNA/DNA oligomer antisense may be used as a therapeutic tool for blocking deleterious miRNAs overexpressed in diseases.

In conclusion, EP is a promising nonvector strategy to increase LNA/DNA oligomer cellular uptake and provide the oligomer direct and rapid access to their cytoplasmic or/and nuclear targets, leading to functional inhibition of specific miRNA. Our data support that LNA-based oligonucleotide electrotransfer is a powerful tool for further studies of miRNA function in vitro and possibly in animal models and facilitates the validation of miRNA inhibitors as potential therapeutics.

Material and Methods

Cell lines and reagents. The HCT116 cell line, derived from a human colorectal carcinoma, was grown in Dulbecco's Modified Eagle Medium (Gibco-Invitrogen, Carlsbad, CA). CHO (WTT Chinese hamster ovary) cells were cultured in Eagle medium (Eurobio, Les Ulis, France) supplemented with 3.5 g/l glucose (Sigma-Aldrich, St-Louis, MO), 2.95 g/l tryptose phosphate (Sigma-Aldrich), 0.8 mg/ml L-glutamine (Eurobio) and 1X BME vitamins (Sigma-Aldrich). B16F10 mouse melanoma and Hela (human cervical adenocarcinoma cell line) cells were routinely maintained in Dulbecco's Modified Eagle Medium (Gibco-Invitrogen). All media were supplemented with penicillin/streptomycin (100 U/ml) (Gibco-Invitrogen) and 10% heat inactivated fetal calf serum (Gibco-Invitrogen). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.

Single-strand LNA/DNA oligomers, which were either unlabeled or labeled with Cy5, were supplied by Exiqon (Copenhagen, Denmark). Cy5-siRNA and siLNA were purchased from Qiagen Xeragon (Germantown, MD). The siRNA and siLNA sequences were: sense: 5′-gcaagcugaccc ugaaguucatt-3′ and antisense: 5′-gaacuucagggucagcuugccg-3′. The non- relevant LNA/DNA oligomer sequence was 5′-cagctcctcgcccttgctca-3′, and the anti-miR34a LNA/DNA oligomer sequence was 5′-acaaccagc taagacactgcc-3′. PI was obtained from Sigma Aldrich.

EP of cell suspensions. Cells (5 × 105) were harvested and resuspended in 100 µl of pulsing buffer (10 mmol/l K2HPO4/KH2PO4 buffer, 1 mmol/l MgCl2, 250 mmol/l sucrose, and pH 7.4) in the presence or absence of LNA/DNA oligomer. EP was conducted using a S20u electropulsator (Betatech, l'Union, France), which delivered square-wave pulses with independently adjustable electric parameters (voltage, number of pulses, duration, and frequency). Pulse parameters were monitored in real time by the electropulsator LCD screen. The cell suspension was placed between 4 mm-wide, stainless steel, flat, parallel electrodes (IGEA, Carpi, Italy) in a culture dish (Becton Dickinson, Rungis, France), and pulses lasting 5 milliseconds at a frequency of 1 Hz were applied. The cells were then incubated for 5 minutes at room temperature and cultured in a Petri dish (Becton Dickinson) with 1 ml of complete culture medium at 37 °C in a 5% CO2 incubator.

Flow cytometry analysis. The cells (5 × 106/ml) were pulsed or unpulsed in the presence of 250 nmol/l Cy5-labeled LNA/DNA oligomers. After electropulsation, the cells were resuspended in 300 µl of PBS and Cy5 fluorescence was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). A minimum of 104 events were acquired on the FL-4 channel and analyzed with CellQuest software (Becton Dickinson).

Determination of cell viability. Cell viability was measured by quantifying cellular growth over 24 hour (more than one generation) by crystal violet staining. Briefly, the cells were stained with 1 ml crystal violet (0.1% in pulsing buffer) for 20 minutes and then lysed with 500 µl acetic acid (10%) for 5 minutes. Cell density was evaluated by 595 nm OD measurements.

Confocal fluorescence microscopy. For fluorescence microscopy observations, the cells (8 × 104) were seeded on a glass coverslip chamber (Nalge Nunc International, Illkirch, France) overnight at 37 °C in a humidified atmosphere with 5% CO2. The electropulsation chamber was designed using two stainless steel parallel rods (1 mm diameter, 10 mm length, and 5 mm interelectrode distance) that were connected to the electropulsator. The chamber was set on the stage of an inverted confocal microscope (Zeiss LSM510, Carl Zeiss, MicroImaging GmbH, Göttingen, Germany) equipped with the 40X Zeiss objective (1.3 numerical aperture, oil immersion). Adherent cells were then electrotransfected in 500 µl of pulsing buffer in the presence of 250 nmol/l (final) Cy5-labeled LNA/DNA oligomer, Cy5-labeled siRNA, or Cy5-labeled siLNA using the following electrical parameters: 300 V/cm, 10 pulses of 5 milliseconds, 1 Hz. For bipolar conditions, we used a polarity inverter built in our laboratory, which allows triggering pulses with alternating polarities. Cy5 was visualized using a 633 nm laser (emission filter: 640–710 nm). Laser power and photomultiplier settings were kept identical for all samples to make the results comparable. Successive scans of less than 1 second were acquired to observe the transfer of the LNA/DNA oligomer into cells before, during and after electric pulse delivery. Eight-bit images were recorded with Zeiss LSM510 software (EMBL, Heidelberg, Germany). The laser scan was unidirectional and was perpendicular to the electric field direction to eliminate temporal delay during image acquisition.

Image analysis. LSM images were processed with ImageJ software (NIH, MD) as previously described.24 The fluorescence signal of the Cy5-labeled LNA/DNA oligomer in the cytoplasm of cells was analyzed by subtracting the first image (before EP) from each image of the scans series. This process eliminated the extracellular Cy5-labeled LNA/DNA oligomer signal (that was not affected by the electropulsation) and thus selectively discriminated the signal coming from inside the cells. A light-plot oriented parallel to the electric field direction was obtained across the cell to determine the pattern of Cy5-labeled LNA/DNA oligomer entrance (using ImageJ software).

Isolation of miRNA and quantitative real time reverse transcriptase polymerase chain reaction analysis. HCT116 cells in suspension were electrotransfected (8 pulses of 700 V/cm) with 250 nmol/l LNA/DNA oligomer anti-34a or scrambled LNA/DNA oligomer as a control, and 0.2 µg/ml of adriamycin (Sigma Aldrich) was added. After 48 hours of incubation, RNAs, including miRNAs, were extracted, using a miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions, and quantified with a Nano-Drop spectrophotometer. A total of 10 ng of extracted RNA was subjected to RT using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), and miR34a level was quantified using the TaqMan MicroRNA Assays (Applied Biosystems) with a ABI PRISM 7300 Sequence Detection system (Applied Biosystems). Each polymerase chain reaction was performed in triplicate. The miR34a expression was defined from the threshold cycle (Ct), and the relative expression levels were calculated by using the 2−ΔΔCt method after normalization to the expression levels of U6 and snoRNA165 small nuclear RNA using geNorm 3.5 software.

Apoptosis and cell-cycle analysis. HCT116 cells in suspension were electrotransfected (8 pulses of 700 V/cm) with 250 nmol/l LNA/DNA oligomer anti-34a or scrambled LNA/DNA oligomers as a control, and 0.05 µg/ml of adriamycin was then added. After 72 hours of incubation, the cells were trypsinized, combined with the supernatant, washed and stained for 10 minutes on ice with PI and annexin V-FITC antibody (BioVision, CA) according to manufacturer instructions. Analysis was performed on a FACSCalibur (Becton Dickinson) with CellQuest software (Becton Dickinson).

For DNA content analysis, the cells were electrotransfected and treated as described above. After 48 hours of incubation, the cells were harvested, washed with cold PBS and incubated on ice for 30 minutes with PI (25 µg/ml), 10% RNAse (Euromedex, France) and 0.1% Triton X-100 (Sigma). Cell-cycle distribution was determined on a FACSCalibur (Becton Dickinson) with Modfit 3.0 software (Verity Software House, ME, USA).

Statistical analyses. Quantitative data (represented as the means + SEM) were analyzed with Prism 4 software (GraphPad, San-Diego, CA). Before performing statistical tests, we determined whether the data were normally distributed and evaluated their variance. We then conducted appropriate tests as indicated. We report the actual P value for each test. P < 0.05 was considered to be statistically significant.

SUPPLEMENTARY MATERIAL Figure S1. LNA/DNA oligomer electrotransfer is efficient regardless of the cell type. Figure S2. Electropermeabilization of both membrane sides visualized by PI entry. Figure S3. Direct visualization of Cy5-LNA/DNA oligomer electrotransfer by confocal fluorescence microscopy. Figure S4. CHO membrane resealing.

Acknowledgments

We thank Laurent Paquereau and Vincent Ecochard (Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale) for helpful discussions. This work was supported by grants from the Seventh Framework European Programme (FP7) OncomiR [grant number 201102]. This work has been performed in collaboration with the “Toulouse Réseau Imagerie” core IPBS facility (Genotoul, Toulouse, France), which is supported by the Association Recherche Cancer, Region Midi Pyrenees, the European union (FEDER) and Grand Toulouse cluster.

The authors declared no conflict of interest.

Supplementary Material

Figure S1.

LNA/DNA oligomer electrotransfer is efficient regardless of the cell type.

Click here for additional data file. (11MB, tif)
Figure S2.

Electropermeabilization of both membrane sides visualized by PI entry.

Click here for additional data file. (2.4MB, tif)
Figure S3.

Direct visualization of Cy5-LNA/DNA oligomer electrotransfer by confocal fluorescence microscopy.

Click here for additional data file. (14.8MB, tif)
Figure S4.

CHO membrane resealing.

Click here for additional data file. (22.4MB, tif)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

LNA/DNA oligomer electrotransfer is efficient regardless of the cell type.

Click here for additional data file. (11MB, tif)
Figure S2.

Electropermeabilization of both membrane sides visualized by PI entry.

Click here for additional data file. (2.4MB, tif)
Figure S3.

Direct visualization of Cy5-LNA/DNA oligomer electrotransfer by confocal fluorescence microscopy.

Click here for additional data file. (14.8MB, tif)
Figure S4.

CHO membrane resealing.

Click here for additional data file. (22.4MB, tif)

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