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Delivery of antisense oligonucleotides to PC12 cells
Neuroscience Research 43 (2002) 81 /86 www.elsevier.com/locate/neures
Technical Note
Delivery of antisense oligonucleotides to PC12 cells Rosalinda Acosta, Cecilia Montan˜ez, Pablo Go´mez, Bulmaro Cisneros * Department of Genetics and Molecular Biology, Centro de Investigacio´n y de Estudios Avanzados del IPN, Avenida Instituto Polite´cnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F., Mexico Received 8 November 2001; accepted 31 January 2002
Abstract Optimal experimental conditions for the delivery of phosphodiester or phosphorothioate antisense oligonucleotides (P-ASO/SASO) to PC12 cells were determined. Fluorescently labeled P-ASO or S-ASO were transfected to PC12 cells and the uptake of antisense, free or entrapped in liposomes, was monitored by confocal and fluorescent microscopy. Efficient delivery of fluorescently labeled ASO with low rates of cell death was obtained when PC12 cells were transfected with liposomes in Opti-MEM medium supplemented with sera, compared with control experiments where nonliposomal ASO were transfected to PC12 cells in sera-free media. Compared with P-ASO, the application of S-ASO for antisense studies in PC12 cells is more suitable due to the lower concentration required for an efficient antisense uptake and its higher intracellular stability. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: PC12 cells; Liposomes; Antisense oligonucleotides; Phosphorothioate oligonucleotides; Antisense stability
1. Introduction Antisense oligodeoxiribonucleotides (ASO) are short, synthetic, single-strand DNA, whose mode of action is thorough hybridization to complementary sequences in the target gene or its messenger RNA (mRNA). The latter result in the formation of an RNA/DNA duplex that disrupts the normal translation of the genes and/or leads to degradation of the mRNA by RNase H. Consequently, these molecular events result in a reduction in the level of the protein (Lebedeva and Stein, 2001). A key parameter for antisense inhibition by ASO is their intracellular concentration. At the present time, it is believed that naked oligonucleotides enter the cell via active processes of adsorptive endocytosis and pinocytosis, however, the penetration of the endosomal barrier is a pre-requisite event for antisense activity and the naked ASO do not appear to do this in great extent (Lebedeva and Stein, 2001; Hughes et al., 2001). It is possible that a rare, stochastic endosomal rupture could cause transfer of ASO from endosome to cytoplasm, * Corresponding author. Fax: /52-747-38-00, ext. 5356. E-mail address: [email protected] (B. Cisneros).
from where they will be rapidly translocated to the nucleus. Although complexes of ASO with cationic liposomes have significantly enhanced intracellular delivery, they have simultaneously introduced a new disadvantage, on its own, their cytotoxicity (Lappalainen et al., 1994; Weiss et al., 1997). Another problem with cellular uptake of ASO is the degradation of antisense molecules by DNases present in the growth medium and into the cell. A longer half-life of ASO ensures they reach and interact with their target. To increase the biological half-life and efficacy of the natural antisense phosphodiester oligodeoxynucleosides (P-ASO), several chemical modifications have been made in their structure. These include changes in the phosphodiester backbone, in the deoxyribose sugar and in the heterocyclic base moieties (Juliano and Yoo, 2000). Among them, the most widely used modified ASO are the phosphorothioates (S-ASO), in which one of the non-bridging oxygen atoms in the conventional phosphodiester bond has been replaced with a sulfur atom. The S-ASO are extremely resistant to the action of endo- and exonucleases (Juliano and Yoo, 2000; Lebedeva and Stein, 2001). Although the problems described above represent the two main parameters in ASO delivery, there are other relatively less critical factors to be considered, for instance, the type of cells
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being studied as well as the tissue culture conditions and concentration of ASO (Crooke et al., 1995). In neurobiology, the application of ASO strategies has improved the study of various aspects of neuronal proliferation and differentiation in several cell lines (Weiss et al., 1997). In this respect, PC12 cells represent a neuronal cell model widely used to study neuronal cell behavior (Greene and Tischler, 1976), however, there is no data in the literature dealing with the key parameters affecting the uptake of ASO by these cells. Therefore, in an attempt to successfully deliver ASO to PC12 cells, we determined the uptake efficiency and intracellular stability of fluorescently labeled P-ASO or S-ASO in these cells, under different tissue culture and transfection conditions.
2. Materials and methods 2.1. Cell culture PC12 cells were grown in RPMI-1640 medium (Gibco-BRL) supplemented with 10% heat-inactivated horse serum (HS), 5% heat-inactivated fetal calf serum (FCS), 25 U/ml of penicillin, 25 mg/ml of streptomycin and 100 mg/ml of glutamine. Cell cultures were maintained at 37 8C in a humidified incubator with 5% CO2 atmosphere. Medium was changed every 3 days. 2.2. Cell experiments A 20-mer antisense oligonucleotide with the sequence 5?FITC-GCT GTT CCC TCA TGG TTG TA-FITC and its analog containing five 5? and five 3?-end phosphorothioate linkages were purchased from Gibco-BRL. The oligonucleotide was complementary to the rat Dp71 mRNA, position 99 /119 (Accession No. X65468). Dystrophin Dp71 is the smallest product of the Duchenne muscular dystrophy gene. Cells were seeded into a 6-well culture plate at :/4 /105 cells/cm2. To obtain lipid /DNA complexes, 20 mg/ml of lipofectin (Gibco-BRL) was mixed with 1 mM of S-ASO or 300 nM of P-ASO, as recommended by the provider. Cellular transfection was performed as follows: typically, supplemented RPMI medium was changed for sera-free Opti-MEM medium and cells at 80% of confluence were incubated with either free or liposomal FITC-antisense oligonucleotide for 5 h at 37 8C. Variations of the transfection conditions described above are indicated through the text. To determine the percentage of FITC-positive cells, plates were washed with cold PBS and cells counted in a Neubauer chamber by using phase contrast and fluorescent microscopy. All other chemicals were of analytical grade.
2.3. Cell mortality The degree of cell damage was determined by the Trypan blue dye exclusion method. For this purpose, a 10-ml aliquot of cells in control and treated wells was incubated with 0.4% Trypan blue, a dye that stains nonviable cells, for 5 min and the stained and unstained cells were counted. 2.4. Confocal microscopy Cells treated with phosphodiester or phosphorothioate FITC-ASO were washed with cold PBS and incubated, for certain time intervals, in a defined medium N2 (Gibco-BRL) or supplemented RPMI1640 medium, respectively. Subsequently, cells were washed with PBS, fixed with paraformaldehyde and observed in an epifluorescence Nikon microscope coupled with a confocal system Bio-Rad MRC600.
3. Results To evaluate the uptake of antisense oligonucleotides by PC12 cells under different experimental conditions, fluorescently labeled P-ASO or S-ASO were transfected into these cells and 5 h after transfection, live cells were analyzed by fluorescent or confocal fluorescent microscopy. As a first step, we estimated the delivery of liposomal and non-liposomal ASO to PC12 cells grown in two different media. Compared with free S-ASO, liposomally entrapped S-ASO showed an improvement of 100 and 49% in their cellular uptake, when PC12 cell were cultured on RPMI-1640 and Opti-MEM, respectively (Fig. 1A). The latter medium substantially increased the percentage of fluorescent cells treated with any liposomal ASO (from 20 to 45%) or non-liposomal ASO (from 11 to 29%), (Fig. 1A). Similar results were obtained when PC12 cells were transfected with P-ASO (Fig. 1B). In this case, the highest percentage of fluorescent cells (33%) was obtained when cells, grown in Opti-MEM, were treated with oligonucleotide entrapped in liposomes. Under the transfection conditions described above, oligo-lipid complexes were prepared in a sera-free medium, which is practically deprived of nuclease activity. However, it was observed that the lack of sera in the cell culture caused cell mortality of 68% (Fig. 2A). Hence, in an attempt to diminish cell death and keeping optimal S-ASO delivery, different concentrations of HS and FCS sera were added to the transfection medium and liposomal S-ASO delivery and cellular mortality were evaluated (Fig. 2A). A noticeable reduction in cellular mortality was observed with the three different concentrations of sera employed, being the cell dishes cultured on Opti-MEM medium supplemented with
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Fig. 1. Effect of cationic liposomes and transfection medium on cellular uptake of FITC-antisense oligonucleotides. Some 300 nm of SASO (A) or 1 mM of P-ASO (B) were incubated with PC12 cells for 5 h at 37 8C in the indicated medium. The percentage of fluorescent cells was evaluated by fluorescent microscopy. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
10% of HS and 5% of FCS which presented the lowest mortality (10%). Nonetheless, the presence of sera in the culture medium seems to have an inhibitory effect on the S-ASO uptake. The higher the concentration of sera in the culture dishes, the lower the number of fluorescent cells obtained. Therefore, an Opti-MEM medium containing 2% of HS and FCS, which rendered a cell mortality of only 15% and allowed a transfection efficiency of 37% (Fig. 2A), was used in further experiments with S-ASO. Parallel experiments using liposomal P-ASO produced similar conclusions regarding the recovery of cell viability when sera was added to the Opti-MEM medium, though the negative effect of sera on antisense oligonucleotides delivery was more pronounced (Fig. 2B). In this case, the use of N2, a defined medium, was successful in increasing uptake of liposomal P-ASO and keeping relatively low rates of cell death (Fig. 2B). Incubation time of DNA /lipid complexes with cells varied from 5 to 10 h and its effect on the uptake of fluorescent-labeled S-ASO into PC12 cells was evaluated. Regarding the percentage of fluorescent cells, no significant difference between these times was observed. However, cell mortality increased in parallel with increments in the incubation time (data not shown). Hence, 5 h incubation time was selected for the next experiments with both S-ASO and P-ASO.
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Fig. 2. Effect of sera on FITC-ASOs delivery and cellular viability. PC12 cells were incubated for 5 h at 37 8C with 300 nm of liposomal S-ASO (A) or 1 mM of liposomal P-ASO (B). The medium and the percentage of sera employed are indicated at the bottom. The percentages of fluorescent and dead cells were evaluated by fluorescent microscopy and by the Trypan blue dye exclusion method, respectively. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
Next, to determine the optimal concentration of antisense molecules, an ASO concentration /response curve was generated. The uptake of lower concentrations of S-ASO was concentration-dependent, reaching a maximum value of 70% with oligonucleotide concentration of 1 mM, after that value, further increments of antisense molecules generated no response in the percentage of fluorescent cells (Fig. 3A). Instead, a slight augment in cell mortality was observed at higher ASO concentrations (data not shown). Higher doses of PASO (50 mM) were required to reach a percentage of fluorescent cells similar to that obtained with S-ASO (Fig. 3B). The stability of fluorescent P-ASO and S-ASO inside PC12 cells was assessed by confocal microscopy over a 72-h period. Fig. 4(A) shows that following transfection, fluorescent S-ASO rapidly accumulated in the cell nucleus. At 24 h post-transfection, besides the strong fluorescence staining which remained in the nucleus, punctuate labeling in the cytoplasm was also evident. Nuclear fluorescence started to disappear at 48 h posttransfection and following 72 h, a granular diffuse staining was observed in most of the transfected cells, which directly correlates with the dissipation of fluor-
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Fig. 3. Effect of ASO quantity in cellular uptake of FITC-ASO. PC12 cells were incubated for 5 h at 37 8C with different amounts of liposomal S-ASO (A) or P-ASO (B), as indicated in the figure. The percentage of fluorescent cells was evaluated by fluorescent microscopy. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
escent labeling from the nucleus. The fate of P-ASO inside PC12 cells was similar to that shown by S-ASO, however, the P-ASO-associated fluorescence remained shorter in the cell; in fact, nuclear fluorescence disappeared totally at 48 h post-transfection (data not shown). Quantitative analysis of the cellular persistence of fluorescent ASO, in which we measured the number of fluorescent cells still present after incubation with ASO /liposome complex, washing and continuing cell life for over a 72 h period, was performed. Results clearly showed that S-ASO has longer stability inside PC12 cells than P-ASO, with estimated half-lives of 64 and 34 h, respectively (Fig. 4B).
4. Discussion ASO technology is regarded as an effective means of lowering the levels of a specific gene product. It is based on the finding that the antisense sequences hybridize to specific RNA transcripts, disrupting normal RNA processing, stability and translation and thereby preventing the expression of a targeted gene. Antisense technology has been employed as a tool to study fundamental problems in neurobiology, using animal
Fig. 4. (A) Intracellular distribution and stability of FITC-ASO in PC12 cells. PC12 cells were incubated for 5 h at 37 8C with 1 mM of liposomal S-ASO. After transfection, cells were analyzed by confocal laser microscopy at the indicated time intervals, as described in Section 2. White bars, 50 mm. (B) PC12 cells were transfected with 1 mM of liposomal S-ASO (2) or with 50 mM of liposomal P-ASO (I). After transfection, the percentage of fluorescent cells was evaluated at the indicated time intervals by fluorescent microscopy. Numbers 34 and 64, indicated near black arrows correspond to the time at which 50% of the initial percentage of fluorescent cells are still labeled in each condition, respectively. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
and cell line models. In this regard, PC12 cell line has been successfully employed in the identification of genes involved in cell proliferation, cell /cell interaction and neuronal differentiation (Davidkova et al., 1996; Masse and Kelly, 1997; Dobashi et al., 1998; MacLennan et al., 2000; Mobarak et al., 2000). Nevertheless, there are no studies describing the key parameters governing the uptake of antisense oligodeoxinucleotides in PC12 cells. Performance of such studies could constitute a useful starting-point in further antisense studies with this cell line. Hence, in this work, using an indirect fluorescentbased assay, we describe the optimization of the experimental conditions affecting the delivery of antisense oligonucleotides in PC12 cells. We found that delivery of liposomal ASO to PC12 cells is more efficient than that of free ASO, which is in agreement with previous reports showing that cationic
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lipids improved the uptake of antisense molecules by cultured cells (Lappalainen et al., 1994; Skalko-Basnet et al., 2000). Although the underlying mechanism is not fully understood, it is generally believed that the positively charged liposomes form aggregates with the negatively charged DNA. The resulting aggregates carry a positive net charge and are attracted to the negatively polarized cell membranes and enter target cells via endocytosis. Finally, to become accessible to the cellular transcription and translation machinery, the liposomal ASO have to leave the endosomal/lysosomal compartment after cellular internalization. Our data also indicate that even the choice of culture medium and its composition can affect the uptake of antisense molecules in PC12 cells. The delivery of ASO was more efficient in cells cultured in Opti-MEM medium than that obtained in cells grown in RPMI1640 medium. The reasons for that are unknown, but it is likely that defined serum-free medium, such as OptiMEM, contain lower amounts of nucleases than serumcontaining medium, which ultimately can influence the stability of ASO in the culture dishes. Nevertheless, it was found that PC12 cell survival decreased dramatically in a sera-free environment. Such alteration may be due to the adverse effects caused by the transfection process on cell growth, function and viability. We determined that the addition of 2% of HS and FBS to the Opti-MEM medium during the transfection reaction allowed efficient ASO delivery in PC12 cells with minimal negative effects on cell viability. A prerequisite for ASO to exert their inhibitory effects is that they accumulate in sufficient amounts and for a sufficiently long time at their target sites in the cytoplasm and/or nucleus. Because natural P-ASO are rather sensitive to extracellular and intracellular exoand endonucleases, nuclease-resistant S-ASO have been widely used in recent years as potent antisense inhibitors of gene expression (Weiss et al., 1997). We compared in PC12 cells the behavior of a P-ASO and its S-ASO analog, which contains five 5? and five 3?-end phosphorothioate linkages. The fate inside the cells of P-ASO and S-ASO was quite similar. At earlier stages following transfection, they accumulated mainly in the nucleus, however, S-ASO remained longer in the nucleus. Later, antisense S-ASO and P-ASO-associated fluorescence was exported from the nucleus to the cytoplasm and finally, disappeared from the cell. Previous experiments using fluorescent metabolites of ASO indicated that nuclease degradation of ASO is the mechanism preceding fluorescent loss (Fisher et al., 1993). Therefore, it could be assumed that fluorescence signal reflect the presence of intact ASO. Compared with P-ASO, S-ASO remained longer in the nucleus and presented longer half-life. Additionally, we found that a 50-fold higher concentration of P-ASO was required to equal the uptake efficiency of S-ASO. These results are consistent
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with earlier reports showing that phosphorothioatemodified oligonucleotides present a longer half life than phosphodiester oligonucleotides, due to their resistance against nucleases found in serum and in the cytoplasm and nucleus of live cells (Shoeman et al., 1998; Skalko-Basnet et al., 2000). Our results suggest that very high amounts of P-ASO are necessary to overcome its instability in the culture medium and inside cells. The basis of the nucleases resistance of S-ASO remain to be clearly determined though, it may be related to their affinity for subcellular structures such as cytoplasmic membranes and nuclear matrix/chromatin complexes, which have been shown to be higher than that of their corresponding P-ASO (Shoeman et al., 1998). Such binding activity of S-ASO to solid supports may further protect them from nucleolytic degradation and thus increase their retention time in the cell. In conclusion, optimal experimental conditions to successfully deliver ASO to PC12 cells were achieved when cells, cultured in Opti-MEM medium supplemented with 2% HS and FCS, were incubated for 5 h with 1 mM of liposomal S-ASO.
Acknowledgements We would like to thank Dr Rau´l Mena for providing access to the confocal microscopy facilities. We also thank Victor Ceja for his technical assistance in cell culturing. This work was supported by CONACyT, Me´xico, Grant No. 26392-M.
References Crooke, R.M., Graham, M.J., Cooke, M.E., Crooke, S.T. 1995. In vitro pharmacokinetics of phosphorothioate antisense oligonucleotide. J. Pharma. Exp. Ther. 275, 462 /473. Davidkova, G., Zhang, S.P., Nichols, R.A., Weiss, B. 1996. Reduced levels of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth. Neuroscience 75, 1003 /1019. Dobashi, Y., Shoji, M., Wakata, Y., Kameya, T. 1998. Expression of HuD protein is essential for initial phase of neuronal differentiation in rat pheochromocytoma PC12 cells. Biochem. Biophys. Res. Commun. 244, 226 /229. Fisher, T.L., Terhorst, T., Cao, X. 1993. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 21, 3857 /3865. Greene, L.A., Tischler, A.S. 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424 /2428. Hughes, M.D., Hussain, M., Nawaz, Q., Sayyed, P., Akhtar, S. 2001. The cellular delivery of antisense oligonucleotides and ribozyme. Drug Discov. Today 6, 303 /315. Juliano, R.L., Yoo, H. 2000. Aspects of the transport and delivery of antisense oligonucleotides. Curr. Opin. Mol. Ther. 2, 297 /303. Lappalainen, K., Urtti, A., So¨derling, E., Jaaskelainen, I., Syrjanen, K., Syrjanen, S. 1994. Cationic liposomes improve stability and
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intracellular delivery of antisense oligonucleotides into CaSki cells. Biochim. Biophys. Acta 1196, 201 /208. Lebedeva, I., Stein, C. 2001. Antisense oligonucleotides: promise and reality. Annu. Rev. Pharmacol. Toxicol. 4, 403 /419. MacLennan, A.J., Devlin, B., Marks, L., Gaskin, A.A., Neitzel, K.L., Lee, N. 2000. Antisense studies in PC12 cells suggest a role for H218, a sphingosine 1-phosphate receptor, in growth-factorinduced cell /cell interaction and neurite outgrowth. Dev. Neurosci. 22, 283 /295. Masse, T., Kelly, P.T. 1997. Overexpression of Ca2//calmodulindependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation. J. Neurosci. 17, 924 /931. Mobarak, C.D., Anderson, K.D., Morin, M., Beckel-Mitchener, A., Rogers, R.L., Furneaux, H., King, P., Perrone-Bizzozero, N.I.
2000. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression and PKC-dependent neurite outgrowth in PC12 cells. Mol. Biol. Cell 11, 3191 / 3203. Shoeman, R., Hartig, R., Huang, Y., Grub, S., Traub, P. 1998. Fluorescence microscopic comparison of the binding of phosphodiester and phosphorothioate (antisense) oligodeoxyribonucleotides to subcellular structures, including intermediate filaments, the endoplasmic reticulum, and the nuclear interior. Antisen. Nucleic Acid Drug Dev. 7, 291 /308. Skalko-Basnet, N., Tohda, M., Watanabe, H. 2000. Delivery of antisense oligonucleotides to neuroblastoma cells. NeuroReport 11, 3117 /3121. Weiss, B., Davidkova, G., Zhang, S. 1997. Antisense strategies in neurobiology. Neurochem. Int. 31, 321 /348.
Technical Note
Delivery of antisense oligonucleotides to PC12 cells Rosalinda Acosta, Cecilia Montan˜ez, Pablo Go´mez, Bulmaro Cisneros * Department of Genetics and Molecular Biology, Centro de Investigacio´n y de Estudios Avanzados del IPN, Avenida Instituto Polite´cnico Nacional 2508, Apartado Postal 14-740, C.P. 07000, Mexico D.F., Mexico Received 8 November 2001; accepted 31 January 2002
Abstract Optimal experimental conditions for the delivery of phosphodiester or phosphorothioate antisense oligonucleotides (P-ASO/SASO) to PC12 cells were determined. Fluorescently labeled P-ASO or S-ASO were transfected to PC12 cells and the uptake of antisense, free or entrapped in liposomes, was monitored by confocal and fluorescent microscopy. Efficient delivery of fluorescently labeled ASO with low rates of cell death was obtained when PC12 cells were transfected with liposomes in Opti-MEM medium supplemented with sera, compared with control experiments where nonliposomal ASO were transfected to PC12 cells in sera-free media. Compared with P-ASO, the application of S-ASO for antisense studies in PC12 cells is more suitable due to the lower concentration required for an efficient antisense uptake and its higher intracellular stability. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: PC12 cells; Liposomes; Antisense oligonucleotides; Phosphorothioate oligonucleotides; Antisense stability
1. Introduction Antisense oligodeoxiribonucleotides (ASO) are short, synthetic, single-strand DNA, whose mode of action is thorough hybridization to complementary sequences in the target gene or its messenger RNA (mRNA). The latter result in the formation of an RNA/DNA duplex that disrupts the normal translation of the genes and/or leads to degradation of the mRNA by RNase H. Consequently, these molecular events result in a reduction in the level of the protein (Lebedeva and Stein, 2001). A key parameter for antisense inhibition by ASO is their intracellular concentration. At the present time, it is believed that naked oligonucleotides enter the cell via active processes of adsorptive endocytosis and pinocytosis, however, the penetration of the endosomal barrier is a pre-requisite event for antisense activity and the naked ASO do not appear to do this in great extent (Lebedeva and Stein, 2001; Hughes et al., 2001). It is possible that a rare, stochastic endosomal rupture could cause transfer of ASO from endosome to cytoplasm, * Corresponding author. Fax: /52-747-38-00, ext. 5356. E-mail address: [email protected] (B. Cisneros).
from where they will be rapidly translocated to the nucleus. Although complexes of ASO with cationic liposomes have significantly enhanced intracellular delivery, they have simultaneously introduced a new disadvantage, on its own, their cytotoxicity (Lappalainen et al., 1994; Weiss et al., 1997). Another problem with cellular uptake of ASO is the degradation of antisense molecules by DNases present in the growth medium and into the cell. A longer half-life of ASO ensures they reach and interact with their target. To increase the biological half-life and efficacy of the natural antisense phosphodiester oligodeoxynucleosides (P-ASO), several chemical modifications have been made in their structure. These include changes in the phosphodiester backbone, in the deoxyribose sugar and in the heterocyclic base moieties (Juliano and Yoo, 2000). Among them, the most widely used modified ASO are the phosphorothioates (S-ASO), in which one of the non-bridging oxygen atoms in the conventional phosphodiester bond has been replaced with a sulfur atom. The S-ASO are extremely resistant to the action of endo- and exonucleases (Juliano and Yoo, 2000; Lebedeva and Stein, 2001). Although the problems described above represent the two main parameters in ASO delivery, there are other relatively less critical factors to be considered, for instance, the type of cells
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 0 1 4 - 7
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being studied as well as the tissue culture conditions and concentration of ASO (Crooke et al., 1995). In neurobiology, the application of ASO strategies has improved the study of various aspects of neuronal proliferation and differentiation in several cell lines (Weiss et al., 1997). In this respect, PC12 cells represent a neuronal cell model widely used to study neuronal cell behavior (Greene and Tischler, 1976), however, there is no data in the literature dealing with the key parameters affecting the uptake of ASO by these cells. Therefore, in an attempt to successfully deliver ASO to PC12 cells, we determined the uptake efficiency and intracellular stability of fluorescently labeled P-ASO or S-ASO in these cells, under different tissue culture and transfection conditions.
2. Materials and methods 2.1. Cell culture PC12 cells were grown in RPMI-1640 medium (Gibco-BRL) supplemented with 10% heat-inactivated horse serum (HS), 5% heat-inactivated fetal calf serum (FCS), 25 U/ml of penicillin, 25 mg/ml of streptomycin and 100 mg/ml of glutamine. Cell cultures were maintained at 37 8C in a humidified incubator with 5% CO2 atmosphere. Medium was changed every 3 days. 2.2. Cell experiments A 20-mer antisense oligonucleotide with the sequence 5?FITC-GCT GTT CCC TCA TGG TTG TA-FITC and its analog containing five 5? and five 3?-end phosphorothioate linkages were purchased from Gibco-BRL. The oligonucleotide was complementary to the rat Dp71 mRNA, position 99 /119 (Accession No. X65468). Dystrophin Dp71 is the smallest product of the Duchenne muscular dystrophy gene. Cells were seeded into a 6-well culture plate at :/4 /105 cells/cm2. To obtain lipid /DNA complexes, 20 mg/ml of lipofectin (Gibco-BRL) was mixed with 1 mM of S-ASO or 300 nM of P-ASO, as recommended by the provider. Cellular transfection was performed as follows: typically, supplemented RPMI medium was changed for sera-free Opti-MEM medium and cells at 80% of confluence were incubated with either free or liposomal FITC-antisense oligonucleotide for 5 h at 37 8C. Variations of the transfection conditions described above are indicated through the text. To determine the percentage of FITC-positive cells, plates were washed with cold PBS and cells counted in a Neubauer chamber by using phase contrast and fluorescent microscopy. All other chemicals were of analytical grade.
2.3. Cell mortality The degree of cell damage was determined by the Trypan blue dye exclusion method. For this purpose, a 10-ml aliquot of cells in control and treated wells was incubated with 0.4% Trypan blue, a dye that stains nonviable cells, for 5 min and the stained and unstained cells were counted. 2.4. Confocal microscopy Cells treated with phosphodiester or phosphorothioate FITC-ASO were washed with cold PBS and incubated, for certain time intervals, in a defined medium N2 (Gibco-BRL) or supplemented RPMI1640 medium, respectively. Subsequently, cells were washed with PBS, fixed with paraformaldehyde and observed in an epifluorescence Nikon microscope coupled with a confocal system Bio-Rad MRC600.
3. Results To evaluate the uptake of antisense oligonucleotides by PC12 cells under different experimental conditions, fluorescently labeled P-ASO or S-ASO were transfected into these cells and 5 h after transfection, live cells were analyzed by fluorescent or confocal fluorescent microscopy. As a first step, we estimated the delivery of liposomal and non-liposomal ASO to PC12 cells grown in two different media. Compared with free S-ASO, liposomally entrapped S-ASO showed an improvement of 100 and 49% in their cellular uptake, when PC12 cell were cultured on RPMI-1640 and Opti-MEM, respectively (Fig. 1A). The latter medium substantially increased the percentage of fluorescent cells treated with any liposomal ASO (from 20 to 45%) or non-liposomal ASO (from 11 to 29%), (Fig. 1A). Similar results were obtained when PC12 cells were transfected with P-ASO (Fig. 1B). In this case, the highest percentage of fluorescent cells (33%) was obtained when cells, grown in Opti-MEM, were treated with oligonucleotide entrapped in liposomes. Under the transfection conditions described above, oligo-lipid complexes were prepared in a sera-free medium, which is practically deprived of nuclease activity. However, it was observed that the lack of sera in the cell culture caused cell mortality of 68% (Fig. 2A). Hence, in an attempt to diminish cell death and keeping optimal S-ASO delivery, different concentrations of HS and FCS sera were added to the transfection medium and liposomal S-ASO delivery and cellular mortality were evaluated (Fig. 2A). A noticeable reduction in cellular mortality was observed with the three different concentrations of sera employed, being the cell dishes cultured on Opti-MEM medium supplemented with
R. Acosta et al. / Neuroscience Research 43 (2002) 81 /86
Fig. 1. Effect of cationic liposomes and transfection medium on cellular uptake of FITC-antisense oligonucleotides. Some 300 nm of SASO (A) or 1 mM of P-ASO (B) were incubated with PC12 cells for 5 h at 37 8C in the indicated medium. The percentage of fluorescent cells was evaluated by fluorescent microscopy. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
10% of HS and 5% of FCS which presented the lowest mortality (10%). Nonetheless, the presence of sera in the culture medium seems to have an inhibitory effect on the S-ASO uptake. The higher the concentration of sera in the culture dishes, the lower the number of fluorescent cells obtained. Therefore, an Opti-MEM medium containing 2% of HS and FCS, which rendered a cell mortality of only 15% and allowed a transfection efficiency of 37% (Fig. 2A), was used in further experiments with S-ASO. Parallel experiments using liposomal P-ASO produced similar conclusions regarding the recovery of cell viability when sera was added to the Opti-MEM medium, though the negative effect of sera on antisense oligonucleotides delivery was more pronounced (Fig. 2B). In this case, the use of N2, a defined medium, was successful in increasing uptake of liposomal P-ASO and keeping relatively low rates of cell death (Fig. 2B). Incubation time of DNA /lipid complexes with cells varied from 5 to 10 h and its effect on the uptake of fluorescent-labeled S-ASO into PC12 cells was evaluated. Regarding the percentage of fluorescent cells, no significant difference between these times was observed. However, cell mortality increased in parallel with increments in the incubation time (data not shown). Hence, 5 h incubation time was selected for the next experiments with both S-ASO and P-ASO.
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Fig. 2. Effect of sera on FITC-ASOs delivery and cellular viability. PC12 cells were incubated for 5 h at 37 8C with 300 nm of liposomal S-ASO (A) or 1 mM of liposomal P-ASO (B). The medium and the percentage of sera employed are indicated at the bottom. The percentages of fluorescent and dead cells were evaluated by fluorescent microscopy and by the Trypan blue dye exclusion method, respectively. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
Next, to determine the optimal concentration of antisense molecules, an ASO concentration /response curve was generated. The uptake of lower concentrations of S-ASO was concentration-dependent, reaching a maximum value of 70% with oligonucleotide concentration of 1 mM, after that value, further increments of antisense molecules generated no response in the percentage of fluorescent cells (Fig. 3A). Instead, a slight augment in cell mortality was observed at higher ASO concentrations (data not shown). Higher doses of PASO (50 mM) were required to reach a percentage of fluorescent cells similar to that obtained with S-ASO (Fig. 3B). The stability of fluorescent P-ASO and S-ASO inside PC12 cells was assessed by confocal microscopy over a 72-h period. Fig. 4(A) shows that following transfection, fluorescent S-ASO rapidly accumulated in the cell nucleus. At 24 h post-transfection, besides the strong fluorescence staining which remained in the nucleus, punctuate labeling in the cytoplasm was also evident. Nuclear fluorescence started to disappear at 48 h posttransfection and following 72 h, a granular diffuse staining was observed in most of the transfected cells, which directly correlates with the dissipation of fluor-
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Fig. 3. Effect of ASO quantity in cellular uptake of FITC-ASO. PC12 cells were incubated for 5 h at 37 8C with different amounts of liposomal S-ASO (A) or P-ASO (B), as indicated in the figure. The percentage of fluorescent cells was evaluated by fluorescent microscopy. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
escent labeling from the nucleus. The fate of P-ASO inside PC12 cells was similar to that shown by S-ASO, however, the P-ASO-associated fluorescence remained shorter in the cell; in fact, nuclear fluorescence disappeared totally at 48 h post-transfection (data not shown). Quantitative analysis of the cellular persistence of fluorescent ASO, in which we measured the number of fluorescent cells still present after incubation with ASO /liposome complex, washing and continuing cell life for over a 72 h period, was performed. Results clearly showed that S-ASO has longer stability inside PC12 cells than P-ASO, with estimated half-lives of 64 and 34 h, respectively (Fig. 4B).
4. Discussion ASO technology is regarded as an effective means of lowering the levels of a specific gene product. It is based on the finding that the antisense sequences hybridize to specific RNA transcripts, disrupting normal RNA processing, stability and translation and thereby preventing the expression of a targeted gene. Antisense technology has been employed as a tool to study fundamental problems in neurobiology, using animal
Fig. 4. (A) Intracellular distribution and stability of FITC-ASO in PC12 cells. PC12 cells were incubated for 5 h at 37 8C with 1 mM of liposomal S-ASO. After transfection, cells were analyzed by confocal laser microscopy at the indicated time intervals, as described in Section 2. White bars, 50 mm. (B) PC12 cells were transfected with 1 mM of liposomal S-ASO (2) or with 50 mM of liposomal P-ASO (I). After transfection, the percentage of fluorescent cells was evaluated at the indicated time intervals by fluorescent microscopy. Numbers 34 and 64, indicated near black arrows correspond to the time at which 50% of the initial percentage of fluorescent cells are still labeled in each condition, respectively. The values denote the mean of three experiments: S.D. for all values was B/5% of the value and is therefore not included.
and cell line models. In this regard, PC12 cell line has been successfully employed in the identification of genes involved in cell proliferation, cell /cell interaction and neuronal differentiation (Davidkova et al., 1996; Masse and Kelly, 1997; Dobashi et al., 1998; MacLennan et al., 2000; Mobarak et al., 2000). Nevertheless, there are no studies describing the key parameters governing the uptake of antisense oligodeoxinucleotides in PC12 cells. Performance of such studies could constitute a useful starting-point in further antisense studies with this cell line. Hence, in this work, using an indirect fluorescentbased assay, we describe the optimization of the experimental conditions affecting the delivery of antisense oligonucleotides in PC12 cells. We found that delivery of liposomal ASO to PC12 cells is more efficient than that of free ASO, which is in agreement with previous reports showing that cationic
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lipids improved the uptake of antisense molecules by cultured cells (Lappalainen et al., 1994; Skalko-Basnet et al., 2000). Although the underlying mechanism is not fully understood, it is generally believed that the positively charged liposomes form aggregates with the negatively charged DNA. The resulting aggregates carry a positive net charge and are attracted to the negatively polarized cell membranes and enter target cells via endocytosis. Finally, to become accessible to the cellular transcription and translation machinery, the liposomal ASO have to leave the endosomal/lysosomal compartment after cellular internalization. Our data also indicate that even the choice of culture medium and its composition can affect the uptake of antisense molecules in PC12 cells. The delivery of ASO was more efficient in cells cultured in Opti-MEM medium than that obtained in cells grown in RPMI1640 medium. The reasons for that are unknown, but it is likely that defined serum-free medium, such as OptiMEM, contain lower amounts of nucleases than serumcontaining medium, which ultimately can influence the stability of ASO in the culture dishes. Nevertheless, it was found that PC12 cell survival decreased dramatically in a sera-free environment. Such alteration may be due to the adverse effects caused by the transfection process on cell growth, function and viability. We determined that the addition of 2% of HS and FBS to the Opti-MEM medium during the transfection reaction allowed efficient ASO delivery in PC12 cells with minimal negative effects on cell viability. A prerequisite for ASO to exert their inhibitory effects is that they accumulate in sufficient amounts and for a sufficiently long time at their target sites in the cytoplasm and/or nucleus. Because natural P-ASO are rather sensitive to extracellular and intracellular exoand endonucleases, nuclease-resistant S-ASO have been widely used in recent years as potent antisense inhibitors of gene expression (Weiss et al., 1997). We compared in PC12 cells the behavior of a P-ASO and its S-ASO analog, which contains five 5? and five 3?-end phosphorothioate linkages. The fate inside the cells of P-ASO and S-ASO was quite similar. At earlier stages following transfection, they accumulated mainly in the nucleus, however, S-ASO remained longer in the nucleus. Later, antisense S-ASO and P-ASO-associated fluorescence was exported from the nucleus to the cytoplasm and finally, disappeared from the cell. Previous experiments using fluorescent metabolites of ASO indicated that nuclease degradation of ASO is the mechanism preceding fluorescent loss (Fisher et al., 1993). Therefore, it could be assumed that fluorescence signal reflect the presence of intact ASO. Compared with P-ASO, S-ASO remained longer in the nucleus and presented longer half-life. Additionally, we found that a 50-fold higher concentration of P-ASO was required to equal the uptake efficiency of S-ASO. These results are consistent
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with earlier reports showing that phosphorothioatemodified oligonucleotides present a longer half life than phosphodiester oligonucleotides, due to their resistance against nucleases found in serum and in the cytoplasm and nucleus of live cells (Shoeman et al., 1998; Skalko-Basnet et al., 2000). Our results suggest that very high amounts of P-ASO are necessary to overcome its instability in the culture medium and inside cells. The basis of the nucleases resistance of S-ASO remain to be clearly determined though, it may be related to their affinity for subcellular structures such as cytoplasmic membranes and nuclear matrix/chromatin complexes, which have been shown to be higher than that of their corresponding P-ASO (Shoeman et al., 1998). Such binding activity of S-ASO to solid supports may further protect them from nucleolytic degradation and thus increase their retention time in the cell. In conclusion, optimal experimental conditions to successfully deliver ASO to PC12 cells were achieved when cells, cultured in Opti-MEM medium supplemented with 2% HS and FCS, were incubated for 5 h with 1 mM of liposomal S-ASO.
Acknowledgements We would like to thank Dr Rau´l Mena for providing access to the confocal microscopy facilities. We also thank Victor Ceja for his technical assistance in cell culturing. This work was supported by CONACyT, Me´xico, Grant No. 26392-M.
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