Cellular Uptake of Adamantyl Conjugated Peptide Nucleic Acids

Oct 29, 1999 - It is found that adamantyl-PNAs show greatly improved (endosomal) cellular uptake, but that this uptake is dependent on the cell line. ...
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Bioconjugate Chem. 1999, 10, 965−972

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Cellular Uptake of Adamantyl Conjugated Peptide Nucleic Acids Trine Ljungstrøm, Helle Knudsen, and Peter E. Nielsen* Center for Biomolecular Recognition, Department of Medical Biochemistry & Genetics, Biochemistry Laboratory B, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark. Received April 30, 1999; Revised Manuscript Received August 30, 1999

Peptide nucleic acids (PNA) (15-mers) conjugated to adamantyl acetic acid and labeled with fluorescein have been prepared, and their (liposome mediated) uptake in human cells in culture (HeLa, IMR-90 and MDA-MB-453) has been studied by confocal fluorescence microscopy. It is found that adamantylPNAs show greatly improved (endosomal) cellular uptake, but that this uptake is dependent on the cell line. Cellular uptake of such lipophilic PNAs is further mediated by cationic liposomes, and in some cases, the intracellular localization is diffuse cytoplasmic or nuclear, again cell-type dependent. The results show that this simple PNA modification can indeed greatly improve cellular uptake, but the effect appears strongly cell-type as well as PNA-sequence dependent.

INTRODUCTION

Table 1: List of the PNAs Used in the Studya

Short synthetic oligonucleotides as highly specific inhibitors of translation or transcription have shown great promise as antisense (and antigene) gene therapeutic drugs (Agrawal, 1996; Altmann et al., 1996; Sharma and Narayanan 1995; Wagner and Flanagan, 1997; Bennet, 1998). The quest for oligonucleotide analogues and mimics with improved properties in terms of nuclease resistance and target affinity have led to the development of a large number of DNA analogues (Agrawal and Iyer, 1995; De Mesmaeker et al., 1995; Freier and Altmann, 1997).One of the more promising of these mimics is peptide nucleic acid (PNA), which contains a pseudo-peptide backbone composed of uncharged N-(2-aminoethyl)glycine units to which the nucleobases are attached via methylene carbonyl linkers (Nielsen et al., 1991; Egholm et al., 1992a,b; Egholm et al., 1993; Hyrup and Nielsen, 1996; Good and Nielsen, 1997). PNA hybridizes with high affinity and sequence selectivity to complementary DNA or RNA oligomers by normal Watson-Crick base pairing (Egholm et al., 1993; Leijon et al., 1994; Brown et al., 1994; Jensen et al., 1997; Eriksson and Nielsen, 1996a,b). Notably, homopyrimidine PNAs binds to sequence complementary targets in double-stranded DNA by forming a strand displacement complex where two PNAs bind to the target strand, forming a triplex, and the nontarget strand is looped out (Cherny et al., 1993; Nielsen et al., 1994a). Such complexes are extremely stable and can efficiently block transcription and replication elongation (Hanvey et al., 1992; Nielsen et al., 1994b; Taylor et al., 1997). Likewise, homopyrimidine PNAs form thermally extremly stable PNA2-RNA triplexes with targets in RNA (Egholm et al., 1992a,b; Kim et al., 1993). PNA is resistant to degradation by nucleases and proteases and show high stability in cellular extracts (Demidov et al., 1994). They are conveniently synthesized by solid-phase peptide chemistry (Christensen et al., 1995). Taken together, these properties make PNA a promising candidate for development of gene-specific drugs. Indeed, PNA has

PNA or S-ON

sequence

target

960 1118 1196 1252 1253 1401 1402 1662 S-ON

Fl-TTT AGC TTC CTT AGC-LysNH2 Ada-Fl-TTT AGC TTC CTT AGC-LysNH2 Ada-Fl-TTT GGG CGG CAT GAC-LysNH2 Fl-GGT GCT CAC TGC GGC-LysNH2 Ada-Fl-GGT GCT CAC TGC GGC-LysNH2 Ada-Fl-GGTLys GCTLys CAC TLysGC GGC-LysNH2 Fl-GGTLysGCTLysCAC TLysGC GGC-LysNH2 Ada-Fl-ACT TGC GGC TCC GGC C-LysNH2 Fl-GGT GCT CAC TGC GGC

CAT CAT Rb Her-2 Her-2 Her-2 Her-2 Her-2 Her-2

* To whom correspondence should be addressed. Phone: (+45) 35327762. Fax: (+45) 35396042. E-mail: [email protected].

a Ada is adamantyl acetic acid, Fl is fluoresceinated PNA monomer, and TLys is a thymine PNA monomer with a lysine residue.

been shown in vitro to have antisense activity against interleukin-2 receptor R (IL-2RR) (Hanvey et al., 1992), chloramphenicol acetyltransferase (CAT) (Knudsen and Nielsen, 1996) and promyelocytic leukemia/retinoic acid receptor R (PML/RARR) (Gambacorti-Passerini et al., 1996; Mologni et al., 1998). However, simple PNAs show very poor cellular uptake when studied in cell culture (Bonham et al., 1995; Gray et al., 1997). Previous experiments with anionic oligonucleotides have shown that their cellular uptake can be greatly improved by covalent attachment of lipophilic groups, such as cholesterol (Letsinger et al., 1989; Stein et al., 1991; Boutorine et al., 1993) or aliphatic chains of different lengths (Kabanov et al., 1990; Saison-Behmoaras et al., 1991; Svinarchuk et al., 1993; Hubus et al., 1995; Mishra et al., 1995). However, the only general solution for delivery of oligonucleotides to cells in culture has been employment of cationic liposomes as vehicles (Bennett et al., 1992; Thierry and Dritschillo, 1992; Zelphati and Szoka, 1996; Wielbo et al., 1997). Recently, various small peptides have been used to transport PNA into cultured cells. For instance, PNA conjugated to the insulin-like growth factor 1 peptide was reported to be taken up if the cognate receptor was expressed on the cell surface (BALB/c 3T3 cells) (Basu and Wickstrom, 1997). Hybrids between PNAs and cationic peptides, such as the third helix of the homeodomain of Antennapedia, were reported to cross through cell membranes and enter immortal human prostate

10.1021/bc990053+ CCC: $18.00 © 1999 American Chemical Society Published on Web 10/29/1999

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Figure 1. HPLC chromatographs of raw and purified PNA 1253 (A and B, respectively) and 1401 (C and D, respectively). The gradient (35 min) was 0 to 100% (0-30 min, 0 to 50%; 30-33 min, 50%; 33-35 min, 50 to 100%) CH3CN in H2O, 0.1% CF3COOH, and the PNA oligomers eluted at 28 min. MALDI-TOF mass spectra (Kratos Maldi-II) of PNAs 1253 (E) and 1401 (D). The found (and calculated) masses are 4933.7 (4928.8) (PNA1253) and 5133.1 (5141) (PNA1401), respectively. The signal from PNA 1401 was weak and, thus, rather broad and less accurate.

tumor-derived D4145 cells (Simmons et al., 1997). This peptide (or its retro-inverso analogue) was also recently used as a vehicle to carry PNA into mammalian cells (Pooga et al., 1998; Aldrian-Herrada et al., 1998), and in one case, down regulation of an antisense targeted neural receptor was reported using a PNA conjugated to “penetratin” (Pooga et al., 1998). As PNAs are inherently charge neutral oligomers, these will not spontaneously associate with cationic liposomes as do the anionic oligonucleotides, and liposome delivery of PNAs is therefore impeded by inefficient liposome loading. Others have used cationic oligonucleotides either conjugated to the PNA (Uhlmann et al., 1996) or just complexed by hybridization (Hamilton et al., 1999) to increase the efficiency of liposomal loading. We reasoned that supplying PNAs with a lipophilic tail

Figure 2. Chemical structure of adamantyl conjugated, fluoresceinated PNA. For regular PNA units R ) H and B ) adenine, cytosine, guanine or thymine; for Tlys modified PNA units R ) -(CH2)4-NH2 and B ) thymine.

Cellular Uptake of Peptide Nucleic Acids

Figure 3. Fluorescence microscopic images of HeLa cells incubated with PNA 1118/DOPE/DDAB (A) or PNA 1118 (B).

should greatly increase their affinity toward liposomes and could even confer spontaneous incorporation upon liposome assembly. Furthermore, such PNA-lipid conjugates could by themselves exhibit improved cellular uptake by initial membrane association and subsequent internalization. We therefore set out to synthesize lipidPNA conjugates and study their (liposome mediated) cellular uptake. The cellular uptake of five PNAs was investigated in three different cell lines, HeLa, IMR-90, and MDA-MB453. Fluorescein-labeled adamantyl-conjugated PNAs were added to cell cultures either alone or in combination with cationic DOPE/DDAB liposomes, and the uptake was observed by fluorescence (confocal) microscopy. We find that the pattern of uptake varies with the type of cells used. Furthermore, the behavior of the PNAs in the cell culture is highly dependent on their sequence as well as the chemical modification. EXPERIMENTAL PROCEDURES

Synthesis of PNAs and S-ON. PNAs were synthesized and purified as described previously (Christensen et al., 1995). The sequences of the employed PNAs and their gene targets are outlined in Table 1. Conjugation of fatty acids was done using the normal coupling procedure (Christensen et al., 1995). TLys monomer (Dform) was synthesized and incorporated into PNA oligomers as described (Haaima et al., 1996). As for standard PNA Boc-synthesis, the procedure yielded PNA oligomers of good purity that were further purified by reversed-

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phase HPLC and characterized by MALDI-TOF mass spectrometry (Figure 1). Typically, the yield of purified PNA was 1-2 µmol from a 10 µmol (50 mg of resin) synthesis. Phosphorothioate oligonucleotides were synthesized by standard procedures and were kindly provided by Dr. Otto Dahl, University of Copenhagen. Preparation of DOPE/DDAB Liposomes. DOPE/ DDAB liposomes were prepared by a modification of the ethanol injection method described by Campbell (Campbell, 1995). Briefly, 13.4 µmol of DOPE (dioleyl-L-Rphosphatidylethanolamine) and 6.6 µmol of DDAB (dimethyldioctadecylammonium bromide) were dissolved in 1 mL of 96% ethanol. Then 40 µL of this solution was combined with 10 µL of a 2.5 mM PNA solution in DMSO. The resulting 50 µL of PNA/lipid mix was added rapidly to 950 µL of sterile distilled water while vortex mixing. The PNA concentration in the liposome solution was thus 25 µM. Uptake Experiments. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The human breast cancer cell line MDA-MB-453 and HeLa cells were grown in RPMI 1640 medium containing Glutamax, penicillin/streptomycin (10 µg/mL), and 10% fetal calf serum. The human fibroblast cell line IMR-90 was grown in DMEM containing the same supplements. On day 0, the cells were plated at the appropriate density in 35 mm dishes containing a glass coverslip. On day 1, the semiconfluent cells were washed twice with OptiMEM (Life Technologies: modified Eagle medium with L-glutamine, NaHCO3, Hepes, sodium-pyruvate, hypoxanthine, thymidine, trace elements, and growth factors) and given 1 mL of OptiMEM containing either PNA or DNA alone or PNA combined with DOPE/DDAB liposomes or DNA combined with LipofectAMINE. On day 3, the cells were washed three times with PBS and fixed for 15 min on ice in 3% formaldehyde/0.2% glutaraldehyde. The coverslips were then mounted on objective glasses and the cells examined by fluorescence microscopy on a Leitz Diaplan microscope. Micrographs were taken using a Kodak Ektachrome 1600 ASA film. Confocal microscopy was performed on a Nicon DIAPHOT 200 controlled by a Silicon Graphics Computer. RESULTS AND DISCUSSION

Initially, we synthesized PNAs conjugated to various fatty acids (heptanoic acid, decanoic acid, dodecanoic acid, and adamantyl acetic acid) by coupling of this acid to the terminal amine of a PNA on the solid support using conventional in situ activation, followed by deprotection and cleavage from the solid support by the low/high TFMSA procedure routinely used for PNA synthesis (Christensen et al., 1995). However, upon purification by RP-HPLC, only the heptyl and adamantyl derivatives eluted off the column as sharp peaks, and since these in initial cellular experiments appeared to behave identical, we focused on the adamantyl derivatives which could be purified and handled without special precautions. Four different 15-mer PNAs (1118, 1196, 1253, and 1662) as listed in Table 1 were conjugated to adamantyl and a fluorescein monomer (Lohse et al., 1997) was included as well as a marker for cellular uptake. Furthermore, a derivative of PNA 1253 containing three TLysmonomers (having a lysine replacing the glycine in the backbone) was made (PNA 1401) to increase the aqueous solubility of the PNA (Haaima et al., 1996). Finally, control PNAs without adamantyl were made (PNAs 960, 1252, and 1402) as well as a phosphorothioate corresponding to PNAs 1253, 1401, and 1402.

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Figure 4. Confocal images of cells incubated overnight with 1 µM fluorescein labeled adamantyl-PNA. (a) HeLa cells incubated with PNA 1118. (b) IMR-90 cells incubated with PNA 1118. (c) HeLa cells incubated with PNA 1196. (d) IMR-90 cells incubated with PNA 1196. The scale-bar is 10 µm. The contour of the cells are indicated by white lines.

Uptake of Adamantyl-Conjugated PNAs. To avoid possible interfering interactions between PNA and serum proteins, the PNAs were added to the cells in serum-free medium. Generally, the uptake was evaluated after an overnight incubation (16-18 h). Three different monolayer cell lines were used for the uptake studies (HeLa, IMR-90, and MDA-MB-453) and the PNA was delivered either in pure water (added to the serum free medium) or as a liposome preparation (in serum free medium). Selected and representative fluorescence images are presented in Figures3-6 and schematically the results are compiled in Table 2. At first sight, these data may appear confusing since the uptake apart from being strongly influenced by the presence of the lipophilic adamantyl group as well as the use of a cationic liposome vehicle (Figure 3), is also strongly dependent both on the cell type and on the PNA (sequence). Nonetheless, some trends can easily be extracted.

First of all, no significant cellular uptake was found for any of the simple PNAs (960 and 1252) even with the help of liposomes. Furthermore, the attachment of the adamantyl group clearly increased the spontaneous uptake (PNA 1118 and 1196), although PNA 1118 was not taken up by the MDA-MB-453 cells. This uptake appeared, however, exclusively endosomal as concluded from a distinctly punctuated appearance by (confocal) fluorescence microscopy (Figure 4). Employment of cationic liposomes to mediate the uptake of the adamantyl PNAs 1118 and 1196 clearly increased the uptake (Figure 5). However, more interestingly a qualitatively different intracellular distribution of the PNA was obtained at least with some cells. In HeLa cells PNA 1118 showed clear nuclear as well as diffuse cytoplasmic localization (Figure 6a), whereas diffuse cytoplasmic localization was obtained with PNA 1196 in IMR-90 cells (Figure 5b). PNAs 1253 and 1662, which are both relatively purine (and especially guanine) rich, aggregated easily and,

Cellular Uptake of Peptide Nucleic Acids

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Figure 5. Confocal images of cells incubated overnight with 1 µM fluorescein labeled ada-PNA in DOPE/DDAB complexes. (a) HeLa cells incubated with PNA 1118/DOPE/DDAB. (b) IMR-90 cells incubated with PNA 1196/DOPE/DDAB. The contour of the cells are indicated by white lines and that of the nuclei by red lines.

Figure 6. Confocal images of cells incubated overnight with 1 µM PNA 1401. (a) HeLa cells. (b) IMR-90 cells. The contour of the cells are indicated by white lines and that of the nuclei by red lines.

therefore, could not be handled in terms of cellular uptake experiments. To produce a more water-soluble derivative of PNA 1253, we included three TLys monomers in the PNA (1401), which introduces three positive charges and which we have previously found to confer significantly increased aqueous solubility (Haaima et al., 1996). As a control, we also tested such a PNA (1402) lacking the adamantyl group. Quite interestingly, membrane association as well as some endosomal uptake was observed with this PNAsin contrast to the normal PNA (1252)sindicating that simply equipping the PNA with positive charges may significantly facilitate cellular uptake. This mechanism could be analogous to that responsible for the effect of, e.g. the Antennapedia peptide which is also cationic, as well as the use as polyethylenimine as carrier in DNA transfection. The introduction of the lysine backbone modification also significantly

increased the aqueous solubility of the corresponding adamantyl conjugated PNA (1401) which showed spontaneous endosomal uptake (Figure 6). This uptake was, however, not facilitated further by liposomes. Instead aggregation was observed. Possibly, the positive charges of the PNA are interfering with the incorporation into the also cationic liposomes. Finally, uptake of the PNAs were compared with the uptake of a phosphorothioate having the same sequence as PNA 1253 (and 1401 and 1402). The phosphorothioate was added to HeLa and IMR-90 cells either alone or combined with commercial liposomes (LipofectAMINE) and to MDA-MB-453 cells combined with LipofectAMINE. The S-ON alone showed clear endosomal uptake in HeLa and IMR-90 cells, whereas in combination with LipofectAMINE strongly fluorescent nuclei were observed in all three cell lines (Figure 7).

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Table 2: Summary of Uptake Patterns of the PNAs in the Three Cell Lines Studied PNA or S-ON 960 1118 1196 1252 1253 1662 1401 1402 S-ON a

treatment

HeLa

IMR-90

MDA-MB-453

alone DOPE + DDAB alone DOPE + DDAB alone DOPE + DDAB alone DOPE + DDAB alone DOPE + DDAB alone DOPE + DDAB alone DOPE/DDAB alone DOPE/DDAB alone LipofectAMINE

no uptake no uptake endosomal nuclear endosomal endosomal nd nd aggregation aggregation aggregation aggregation endosomal aggregation nd nd endosomal nuclear

nda nd endosomal no uptake endosomal/aggregation diffuse, cytoplasmic no uptake no uptake aggregation aggregation aggregation aggregation endosomal aggregation membrane-association and endosomal endosomal endosomal nuclear

nd nd no uptake no uptake endosomal endosomal nd nd aggregation aggregation aggregaton aggregation membrane-association aggregation membrane-association and endosomal endosomal nd nuclear

nd, not done.

mantyl and backbone lysine modifications represent significant improvements toward more efficient PNA antisense reagents. Naturally, the real test of such PNAs will be antisense efficacy and such experiments are now in progress. ACKNOWLEDGMENT

This work was supported by the Danish Cancer Society, the European Commission (contract no BMH4-CT960848) and the Danish National Research Foundation. We thank Peter Walmod from the Protein Laboratory, University of Copenhagen, for doing the confocal microscopy. LITERATURE CITED

Figure 7. Fluorescence images of IMR-90 cells incubated overnight with 1 µM fluorescein labeled S-ON. (A) Alone or (B) in combination with LipofectAMINE. CONCLUSION

The present results clearly demonstrate that conjugation of PNAs to a simple lipophilic ligand, such as an adamantyl group, can confer dramatically increased cellular uptake properties, especially when mediated by cationic liposomes. The results also show that increased cellular uptake may be attained by using a lysine backbone modification. However, the results just as clearly show that the cellular uptake by any of these schemes is highly dependent on the cell-type as well as on the PNA sequence. The limited data, unfortunately, does not yet allow us to deduce guidelines as to which modification to be used with which cells and PNA sequences, but it should eventually be possible to arrive at such more general guidelines. Nonetheless, the ada-

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