(PNA) Cholic Acid - American Chemical Society

Jan 13, 2012 - Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA). Cholic Acid (“Umbrella”) and Cholesterol Conjugates Delivered ...
0 downloads 0 Views 379KB Size
Article pubs.acs.org/bc

Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid (“Umbrella”) and Cholesterol Conjugates Delivered by Cationic Lipids Takehiko Shiraishi# and Peter E. Nielsen*,#,‡ #

Department of Cellular and Molecular Medicine and ‡Department of Molecular Drug Research, Faculty of Health and Medical Sciences, University of Copenhagen, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark S Supporting Information *

ABSTRACT: Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid (“umbrella”) derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC50 ∼ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA−cholic acid conjugates which, in contrast to PNA−cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid “umbrella” derivatives as a delivery modality for antisense oligomers.



INTRODUCTION It is generally accepted that the full potential of antisense and RNA interference agents is still unleashed due to suboptimal delivery technologies resulting in poor bioavailability at the intracellular nucleic acid target.1 This is also the case concerning peptide nucleic acids (PNAs),2 despite the recent progress exploiting cell penetrating peptides3 and polyethylene imine conjugates.4 However, these methods typically result in only micromolar activity even when boosting the activity with endosomolytic agents, such as chloroquine5,6 or photodynamic dyes,7 whereas almost 1000-fold higher activity can be achieved via delivery of analogous anionic PNA−peptidephosphonate conjugates by cationic liposomes (Lipofectamine2000), constituting the most effective PNA delivery system at present.8 The ideal molecules for cellular uptake are overall hydrophilic but exhibit transient lipophilicity, i.e., they can exist in equilibrium between an overall hydrophilic and an overall lipophilic state. For small molecules, this is often achieved by amine protonation equilibrium, and for larger molecules, including oligonucleotides the amphipatic properties of cholic acid has been exploited in order to create “umbrella” molecules, in which the hydrophilic surface of the cholic acid ligands is facing the solvent in an aqueous environment, whereas the hydrophobic surface is facing the lipids when traversing the cellular membrane.9,10 In an attempt to exploit this approach, © 2012 American Chemical Society

we have synthesized cholic acid umbrella derivatives of active antisense PNAs. Furthermore, inspired by the progress in the oligonucleotide siRNA field using cholesterol conjugates,11−14 we also synthesized PNAs conjugated to a cholesterol moiety via two different linkers. The cellular activity of the PNA conjugates was determined by using the well established HeLa pLuc 705 cell culture assay,15 in which nuclear antisense activity via targeting a cryptic splice site of the engineered luciferase gene is sensitively measured. Unexpectedly, we find that the umbrella cholic acid PNA conjugates were practically inactive up to 1 μM. However, their activity was very dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar antisense activity (EC50 ∼ 25 nM). These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA−cholic acid conjugates which, in contrast to PNA− cholesterol conjugates, exhibit sufficient water solubility. Received: August 23, 2011 Revised: January 12, 2012 Published: January 13, 2012 196

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

Table 1. PNA Oligomers no.

name

sequencea

masse

2389 2964 3086 3087 3088 2974 2977 2978 3587 3588

Naked PNA Cholate-PNA (Cholate)2-PNA (Cholate)3-PNA (Cholate)4-PNA Cholate-PNA(Cholate)3 Chol-hs-PNA Chol-cbm-PNA Chol-hs-PNA-MMd Cholate-PNA-MM

H-CCTCTTACCTCAGTTACA-NH2 Cholic acid-CCTCTTACCTCAGTTACA-NH2 Cholic acid-Lys(Cholic acid)-CCTCTTACCTCAGTTACA-NH2 Cholic acid-[Lys(Cholic acid)]2-CCTCTTACCTCAGTTACA-NH2 Cholic acid-[Lys(Cholic acid)]3-CCTCTTACCTCAGTTACA-NH2 Cholic acid-eg1b-CCTCTLys(Chol)cTACCTLys(Chol)CA GTTLys(Chol)ACA-NH2 Cholesteryl hemisuccinate-CCTCTTACCTCAGTTACA-NH2 Cholesteryl carbamate-CCTCTTACCTCAGTTACA-NH2 Cholesteryl hemisuccinate-CCTCTGACCTCATTTACA-NH2 Cholic acid-CCTCTGACCTCATTTACA-NH2

4766 5156 5675 6190 6709 6341 5234 5177 5241 5153

(4765) (5158) (5674) (6192) (6710) (6337) (5234) (5178) (5235) (5157)

purity (%) 95 98 98 95 90 95 98 98 98 95

a

The sequences of the PNAs are written from N-terminal to C-terminal end. Cholic acid and Cholesterol derivatives were conjugated to N-terminal of the PNA (or Lys) and the ε-amino group of a lysine residue. beg1, ethylene glycol linker (8-amino-3,6-dioxaoctanoic acid). cCholic acid was conjugated to the ε-amino group of the lysine backbone thymine residue (TLys).17 dMM, mismatch PNA sequence (the two mismatches are indicated in bold). eFound (Calculated).



MATERIALS AND METHODS PNA Synthesis. The sequences of the PNAs are listed in Table 1. PNA synthesis was carried out by the tBoc method as reported previously.16 Cholic acid and cholesterol conjugates were obtained by solution HBTU coupling of the corresponding carboxylic acid derivatives to PNA at the N-terminal and/or the ε-amino group of lysine residues in the PNA backbone: 3 mg HBTU dissolved in 50 μL DMF was mixed with 5 mg cholic acid (or cholestrylhemisuccintate) dissolved in 200 μL DMSO and to this mixture was added 10 μL DIEA. After 30 min incubation at RT, 2 mg PNA2389 dissolved in 200 μL DMSO was added and the reaction is allowed to proceed overnight. The cholesterol carbamate conjugate was synthesized by reacting 1.2 mg PNA2389 dissolved in 50 μL DMSO in solution with 2.8 mg cholesteryl chloroformate dissolved in 50 μL DMSO and adding 6 μL DIEA in 100 μL DMF. The mixture was incubated at RT overnight. The resulting PNA conjugates were HPLC purified and characterized by MALDITOF mass spectrometry (see Supporting Information). The PNAs were lyophilized and stored at 4 °C until use. The PNAs containing thymines with lysine modified backbone were synthesized as previously described.17 Cell Culture. HeLa pLuc705 cells15 were purchased from GENE TOOLS (USA). Cells were grown in RPMI1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 1% glutamax (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C in humidified air with 5% CO2. The cells were seeded on the plate 16−24 h before treatment at a cell density of 1.2 × 104 cells/well or 7.2 × 104 cells/well for 96 well plates and 24 well plates, respectively. PNA Transfection. For the unaided delivery, the cells (replated in the plate the day before transfection) were incubated in OPTI-MEM (Invitrogen) containing PNA for 4 h. Then, the cells were incubated further for 20 h after supplementing an equal volume of growth medium containing 20% FBS and subjected to further analysis. For cationic lipid mediated transfection, the cells were incubated with the PNA/ lipid complex in RPMI (10% FBS, 1% glutamax) for 24 h and subjected to further analysis. The PNA−lipid complex was formed by incubating 30 μL of PNA solution (in water) with 30 μL of diluted LipofectAMINE2000 (LFA2000, Invitrogen) in OPTI-MEM for 10 min at room temperature. The resulting lipoplex solution was mixed with 0.24 mL of RPMI1640 (10% FBS, 1% glutamax) and used for transfection. For DNA-assisted

PNA transfection, PNA/DNA heteroduplexes were formed as published previously8 using PNA2389 and cDNA (5′AATATGTAACTGAGGTA-3′). Luciferase Assay. Luciferase activity of the cells was measured by using the Bright-Glo Luciferase assay system (Promega) according to the manufacturer’s instructions. Luminescent readings from 96 well plate formats were background subtracted and presented as relative light units (RLU/well). For the 24 well plate format, the cells were lyzed with Passive Lysis buffer (Promega) and subjected to the luciferase assay. Luminescent readings from the 24-well format were background-subtracted and normalized by protein concentrations, and are presented as RLU/mg protein. Protein concentrations were determined by the BCA protein assay (Pierce) according to the manufacturer’s instruction. Cytotoxicity Test. The cells in 96 well plates were transfected and assayed for viability by using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS-assay, Promega) according to the manufacturer’s instructions. Alternatively, cell lysates in passive lysis buffer (from the luciferase assay) were analyzed for ATP content by using the CellTiter-Glo Luminescent cell viability assay (Promega) according to the manufacturer’s instructions. The values are presented as relative cellular viability (the value from non-PNAtreated cells was set as 100%). RT-PCR. Total RNA was extracted from cell lysates in passive lysis buffer using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction. Four nanograms of total RNA was used for each RT-PCR reaction (20 μL/ reaction) by using the OneStep RT-PCR kit (Qiagen). Primers for the RT-PCR were as follows: 5′-TTGATATGTGGATTTCGAGTCGTC-3′ (forward primer) and 5′TGTCAATCAGAGTGC-TTTTGGCG-3′ (reverse primer). The RT-PCR program was as follows: [(55 °C, 35 min) × 1 cycle, (95 °C, 15 min) × 1 cycle, (94 °C, 0.5 min; 55 °C, 0.5 min; 72 °C, 0.5 min) × 26−28 cycles]. RT-PCR DNA products were analyzed on a 2% agarose gel with 1× TBE buffer and visualized by ethidium bromide staining. Gel images were captured by ImageMaster (Pharmacia Biotech) and analyzed by UN-SCAN-IT software (Silk Scientific Corporation).



RESULTS A series of cholic acid PNA conjugates were synthesized based on a PNA sequence that in a variety of delivery contexts has shown excellent splice correction antisense activity4−8 in the 197

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

Figure 1. Chemical structures of the lipophilic conjugates used in this study. Cholic acid was conjugated to the N-terminal amine and to the ε-amino groups of PNA backbone lysines (at T-residues), and both “lipo-face in” or “lipo-face out” conformations are shown.

pLuc705 HeLa cell culture assay.15 PNA−cholate conjugates containing one to four cholate moieties attached to the amino end of the PNA via a lysine scaffold or four cholate moieties attached along the PNA oligomer via lysine modifications in the PNA backbone (Table 1, Figure 1). Furthermore, PNA cholesterol conjugates via a hemisuccinate or a carbamate linkage at the amino terminal of the PNA were synthesized (Table 1, Figure 1). None of the PNAs showed any significant antisense activity without cationic lipids (unaided delivery) even at the highest concentration tested (1 μM) (Figure 2a), and they did not show any cellular toxicity either (Figure 3a). This very low antisense activity and by inference poor cellular uptake of the cholic acid conjugates is unexpected in view of previous reports in other systems9,10 (see Discussion). In contrast to the unaided delivery, these PNA conjugates showed excellent antisense activity upon cationic lipid-mediated transfection (Figure 2b). This is in full analogy to our previous findings with PNA conjugated to lipophilic, polyheteroaromatic ligands,19 but the present conjugates exhibit more than 10-fold higher efficiency. It is noteworthy that increasing the number of

cholate ligands at the N-terminal had little or no effect on the activity, and the monocholate-PNA (PNA2964)) showed similar activity to the PNA with four cholate modifications distributed along the PNA (PNA2974) (having one cholic acid at the N-terminal and three cholic acids attached to in the PNA backbone). In addition, the tri- and tetra-cholate PNA conjugates showed increased cell toxicity upon lipofection (Figure 3b). Interestingly, the two simplest PNA conjugates containing a single cholic acid (PNA3588) or a cholesterol with a hemisuccinate linker (PNA3587) showed the highest antisense activity exhibiting EC50 ∼ 25 nM, which was confirmed by directly measuring the relative amount of corrected luciferase mRNA by an RT-PCR assay (Figure 4). Finally, the antisense activity obtained with the cholate−PNA and cholesterol−PNA conjugates is several-fold higher than that obtained by PNA delivery via a cDNA carrier18 (Figure 4). In order to ascertain that the observed effects are indeed caused by a bona fide antisense mechanism, we tested double mismatched (nucleobase interchange) PNA oliogomers of the 198

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

Figure 2. Cellular uptake of the PNAs conjugated with different ligands (cholesterol or a different number of cholic acids) for the transfection. HeLa pLuc705 cells were treated with the conjugates in the absence (a) or with the aid (b) of cationic lipids LipofectAMINE2000 for 24 h and subjected to the luciferase assay. The values represent the mean ± SD of three experiments.

Figure 3. Comparison of the cellular viability of the cells treated with PNAs conjugated with different ligands (cholesterol or a different number of cholic acids). HeLa pLuc705 cells were treated with the conjugates in the absence (a) or with the aid (b) of cationic lipids LipofectAMINE2000 for 24 h and subjected to the cellular viability test using MTS-assay (Promega). The values were normalized to the value of nontreated samples and represented as mean ± SD of three experiments.

cholate and cholesterol hemisuccinate PNAs, and these showed practically no luciferase activating effect (Figure 5). Finally, we also tested a cholesterol derivative conjugated through a carbamate linker instead of the hemisuccinate linker, and this showed significantly lower activity (aprrox. 4-fold; Supporting Information Figure 1). Whether this difference is due to the stability of the linker (the ester of the hemisuccinate is susceptible to esterase cleavage) remains to be seen. Cellular delivery via cationic lipid carriers involves the formation of lipoplexes in the form of liposomes, aggregates, or nanoparticles with positive zeta potential which have affinity for the negatively charged cell surface. Thus, the lipoplexes could up-concentrate on the cells, and thus, dose−response dependence relations would correlate with the amount of lipoplexes (in a proportionally larger volume) rather than with concentration per se. In order to address this issue, we tested the effect of different volumes of the transfection solution in a 10-fold range at different PNA concentrations (3−300 nM by varying the amount of the PNA while using a fixed concentration of cationic lipids) (Figure 6). As only the volume is changed and the PNAs have no charge, the lipoplex concentration and structure are expected to be virtually unchanged in these experiments. The results clearly showed that PNA antisense activity increased in a PNA dose-dependent manner for all transfection volumes (Figure 6a), but also showed that increased volume (hence increasing the total amount of PNA lipoplex/well) resulted in higher antisense activity with an almost linear relationship up to 4 relative volumes (the leveling off could be due to surface saturation), while an increase of toxicity with increasing volume was only significant at the highest PNA concentration (300 nM). These results indicate that a large fraction of the PNA lipoplexes

accumulated onto the cell membrane surface (analogous observations have been reported for PNA−CPP conjugates20). Thus, with cationic transfection reagents the efficacy of the reagent will be a relative measure dependent on the specific transfection conditions. Nonetheless, the present method appears far superior to using, e.g., cell penetrating peptides for delivery of PNA oligomers to eukaryotic cells in culture, for which in the same pLuc705 HeLa cell system under comparable conditions more than 10-fold higher PNA-CPP concentrations (EC50 ∼ 1 μM) are required to yield similar antisense effects.5,7,21−23



DISCUSSION AND CONCLUSIONS

The present results show that PNA oligomers conjugated to a cholic acid or cholesterol moiety and complexed with cationic lipids are very potent antisense agents in HeLa cells in culture, thereby implying that this method efficiently delivers PNA to (the nucleus of) eukaryotic cells. Notably (and disappointingly), we also find that PNA-cholic acid “umbrella” conjugates did not show any significant antisense effect in the very sensitive pLucHeLa system without lipoplexation. PNAs are charge neutral molecules and thus would be expected to constitute much less challenging cargos than, for instance, polyanionic oligonucleotides for which significantly improved penetration of synthetic phopholipid bilayers has been reported using cholic acid umbrella conjugates.24 The pLucHeLa system directly reports biological (nuclear) effects, and we therefore consider this a much more reliable assay in terms of predicting 199

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

Figure 4. Comparison of the antisense activity of two PNA conjugates (PNA2977 (Chol-hs-) and PNA2964 (Cholate-)) and that of unmodified PNA delivered as PNA/DNA heteroduplex by lipofection. PNA conjugates and PNA/DNA heteroduplex were complexed with LipofectAMINE2000 and delivered to the HeLa pLuc 705 cells for 24 h. After transfection, the cells were subjected to the further analysis: (a) Luciferase activity. The values were normalized to protein concentration and represented as mean of two experiments. (b) RTPCR analysis of the mis-splicing correction of luciferase pre-mRNA by PNA. U: fragments without correction (268 bp). C: Fragments with mis-splicing correction (142 bp). The numbers under the figure indicate the relative amount (%) of the corrected form to the sum of corrected form and uncorrected form.

Figure 6. Effect of transfection volume for the transfection of PNA cholesterol hemisuccinate conjugate (PNA2977). The PNA conjugate (at the concentrations indicated) was complexed with fixed concentration of LipofectAMINE2000 and then mixed with growth medium for transfection. Different volumes of the growth medium containing PNA/LFA2000 complex (0.25 mL/well for a relative volume 1) was added to the HeLa pLuc 705 cells in a 24 well plate and incubated for 24 h. After transfection, the cells were subjected to the further analysis. The values represent the mean ± SD of three experiments. (a) Luciferase analysis. (b) Cellular viability measured using CellTiter-Glo Luminescent cell viability assay (Promega). The values were normalized to the value of non-PNA-treated sample of relative volume 2 (v2).

thus presenting the hydrophobic face of the cholic acid to the environment (see Figure 1), and thereby giving the conjugate a hydrophobic surface that should allow membrane penetration by diffusion. While this principle seems effective for small molecule cargoes,10 examples of cellular target effects of umbrella oligonucleotides are still lacking. However, it is more challenging both structurally and kinetically to cover a larger linear oligomer like an oligonucleotide or a PNA sufficiently with cholic acid moieties to make this conjugate migrate through the robust cellular membrane lipid bilayer. In the present study, we designed two tetra-cholate PNAs with one cholate per 4.5 nucleobases and having the cholates either concentrated at the “head” of the PNA (PNA3088) or evenly distributed along the PNA chain (PNA2974). Clearly, neither of these showed significant biological antisense activity, indicating poor intracellular bioavailability and thus membrane penetration. Of course, these specific umbrella conjugates have not been optimized, and further studies are clearly warranted, but the total lack of cellular activity of these PNAs do raise questions concerning the general utility of umbrella type compounds for cellular (and in vivo) delivery of antisense agents, or at least show that major chemical optimization will be required for obtaining high efficacy. It has previously been found that conjugation of lipophilic, polyaromatic ligands to antisense PNA oligomers very significantly enhances cationic lipid mediated cellular delivery.19 However, the present cholesterol and cholic acid conjugates show 10-fold higher activity. Although they are still around 10fold less active than the most active PNA−peptidephosphonate

Figure 5. Comparison of two antisense PNA conjugates (PNA2977 (Chol-hs-) and PNA2964 (Cholate-)) with mismatch PNAs (PNA3587 (Chol-hs-MM) and PNA3588 (Cholate-MM)). HeLa pLuc705 cells were treated with the conjugates with the aid (a) or in the absence (b) of cationic lipids LipofectAMINE2000 for 24 h. After transfection, the cells were subjected to the luciferase analysis.

effective cellular delivery than solely relying on fluorescence techniques and synthetic lipid vesicles. Mechanistically, the umbrella effect of cholic acid should shield hydrophilic cargos through interactions with the hydrophilic face of the cholic acid, 200

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

conjugates,8 because of easy chemical accessibility and charge neutrality they could well be the conjugates of choice for cell culture studies and for future in vivo drug discovery approaches, and maybe could also be used in combination with novel peptide delivery strategies such as Pepfect26 and CADY27 technologies. In particular, we favor the cholic acid conjugates, as these do not have the solubility challenges posed by cholesterol conjugates. The high efficiency of the cholic acid conjugates may seem somewhat surprising because cholic acid is significantly less hydrophobic than cholesterol and therefore could be expected to form less stable lipoplexes. On the other hand, the lower hydrophobicity could facilitate dissociation from the lipids once inside the cell and provide better intracellular bioavailability. The structure of the lipoplexes is not known at present. However, because the PNAs are charge neutral, in contrast to oligonucleotides they do not affect the overall charge of the lipoplexes, and they cannot engage in charge dependent aggregation and nanoparticle formation. Therefore, most likely the cholate−PNA lipoplexes have liposome rather than nanoparticle character, but further studies are warranted to elucidate the detailed mechanism of the delivery. Clearly, the present approach should also be useful more generally for delivery of other charge neutral antisense (and antigene) agents including, for example, morpholino oligomers (PMOs), and possibly also for therapeutic peptides. Finally, cholesterol and cholic acid PNA conjugates may be of particular interest for liver specific therapy in vivo as indicated by the successful liver targeting of cholesterol12,13,24 or cholic acid28conjugates.



polyethyleneimine via disulfide linkers. Bioconjugate Chem. 21, 1933− 8. (5) Shiraishi, T., Pankratova, S., and Nielsen, P. E. (2005) Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides. Chem. Biol. 12, 923−9. (6) Abes, S., Williams, D., Prevot, P., Thierry, A., Gait, M. J., and Lebleu, B. (2006) Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J. Controlled Release 110, 595−604. (7) Shiraishi, T., and Nielsen, P. E. (2006) Photochemically enhanced cellular delivery of cell penetrating peptide-PNA conjugates. FEBS Lett. 580, 1451−6. (8) Shiraishi, T., Hamzavi, R., and Nielsen, P. E. (2008) Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells. Nucleic Acids Res. 36, 4424−32. (9) Janout, V., Staina, I. V., Bandyopadhyay, P., and Regen, S. L. (2001) Evidence for an umbrella mechanism of bilayer transport. J. Am. Chem. Soc. 123, 9926−7. (10) Ge, D., Wu, D., Wang, Z., Shi, W., Wu, T., Zhang, A., Hong, S., Wang, J., Zhang, Y., and Ren, L. (2009) Cellular uptake mechanism of molecular umbrella. Bioconjugate Chem. 20, 2311−6. (11) Bijsterbosch, M. K., Rump, E. T., De Vrueh, R. L., Dorland, R., van Veghel, R., Tivel, K. L., Biessen, E. A., van Berkel, T. J., and Manoharan, M. (2000) Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation. Nucleic Acids Res. 28, 2717−25. (12) Joshi, R., Mishra, R., Pohmann, R., and Engelmann, J. (2010) MR contrast agent composed of cholesterol and peptide nucleic acids: design, synthesis and cellular uptake. Bioorg. Med. Chem. Lett. 20, 2238−41. (13) Bijsterbosch, M. K., Manoharan, M., Dorland, R., Van Veghel, R., Biessen, E. A., and Van Berkel, T. J. (2002) bis-Cholesterylconjugated phosphorothioate oligodeoxynucleotides are highly selectively taken up by the liver. J. Pharmacol. Exp. Ther. 302, 619−26. (14) Soutschek, J, Akinc, A, Bramlage, B, Charisse, K, Constien, R, Donoghue, M, Elbashir, S, Geick, A, Hadwiger, P, Harborth, J, John, M, Kesavan, V, Lavine, G, Pandey, R. K., Racie, T, Rajeev, K. G., Röhl, I, Toudjarska, I, Wang, G, Wuschko, S, Bumcrot, D, Koteliansky, V, Limmer, S, Manoharan, M, and Vornlocher, H. P. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173−8. (15) Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37, 6235−9. (16) Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P. E., Coull, J., and Berg, R. H. (1995) Solid-phase synthesis of peptide nucleic acids. J. Pept. Sci. 1, 175−83. (17) Haaima, G., Lohse, A., Buchardt, O., and Nielsen, P. E. (1996) Peptide nucleic acids (PNAs) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of DLysine PNA. Angew. Chem. 35, 1939−1941. (18) Doyle, D. F., Braasch, D. A., Simmons, C. G., Janowski, B. A., and Corey, D. R. (2001) Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length. Biochemistry 40, 53−64. (19) Shiraishi, T., Bendifallah, N., and Nielsen, P. E. (2006) Cellular delivery of polyheteroaromate-peptide nucleic acid conjugates mediated by cationic lipids. Bioconjugate Chem. 17, 189−94. (20) Hallbrink, M., Oehlke, J., Papsdorf, G., and Bienert, M. (2004) Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration. Biochim. Biophys. Acta 1667, 222−8. (21) Shiraishi, T., and Nielsen, P. E. (2006) Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption. Nat. Protoc. 1, 633−6.

ASSOCIATED CONTENT

S Supporting Information *

Additional figure and spectra as described. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel: +45 35 32 77 62. Fax: +45 35 32 60 42. E-mail: ptrn@ sund.ku.dk. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Lundbeck Foundation. The expert technical assistance of Ms. Jolanta Ludvigsen for PNA synthesis and characterization is gratefully acknowledged.



REFERENCES

(1) De Rosa, G., and La Rotonda, M. I. (2009) Nano and microtechnologies for the delivery of oligonucleotides with gene silencing properties. Molecules 14, 2801−23. (2) Shiraishi, T., and Nielsen, P. E. (2009) Cellular Biovailability of Peptide Nucleic Acids (PNAs) Conjugated to Cell Penetrating Peptides. In Delivery Technologies for Biopharmaceuticals, Peptides, Proteins, Nucleic Acids and Vaccines (Jørgensen, L., and Mørck Nielsen, H., Eds.) pp 305−338, Wiley, Chichester, UK. (3) Mae, M., Andaloussi, S. E., Lehto, T., and Langel, U. (2009) Chemically modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opin. Drug Delivery 6, 1195−205. (4) Berthold, P. R., Shiraishi, T., and Nielsen, P. E. (2010) Cellular delivery and antisense effects of peptide nucleic acid conjugated to 201

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202

Bioconjugate Chemistry

Article

(22) Bendifallah, N., Rasmussen, F. W., Zachar, V., Ebbesen, P., Nielsen, P. E., and Koppelhus, U. (2006) Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA). Bioconjugate Chem. 17, 750−758. (23) Koppelhus, U., Shiraishi, T., Zachar, V., Pankratova, S., and Nielsen, P. E. (2008) Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain. Bioconjugate Chem. 19, 1526−1534. (24) Janout, V., Jing, B., and Regen, S. L. (2005) Molecular umbrellaassisted transport of an oligonucleotide across cholesterol-rich phospholipid bilayers. J. Am. Chem. Soc. 127, 15862−70. (25) Cline, L. L., Janout, V, Fisher, M, Juliano, R. L., and Regen, S. L. (2011) A molecular umbrella approach to the intracellular delivery of small interfering RNA. Bioconjugate Chem. 22, 2210−6. (26) Ezzat, K., El Andaloussi, S., Zaghloul, E. M., Lehto, T., Lindberg, S., Moreno, P. M., Viola, J. R., Magdy, T., Abdo, R., Guterstam, P., Sillard, R., Hammond, S. M., Wood, M. J., Arzumanov, A. A., Gait, M. J., Smith, C. I., Hallbrink, M., and Langel, U. (2011) PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Res. 39, 5284−98. (27) Crombez, L., and Divita, G. (2011) A non-covalent peptidebased strategy for siRNA delivery. Methods Mol. Biol. 683, 349−60. (28) Betebenner, D. A., Carney, P. L., Zimmer, A. M., Kazikiewicz, J. M., Brücher, E, Sherry, A. D., and Johnson, D. K. (1991) Hepatobiliary delivery of polyaminopolycarboxylate chelates: synthesis and characterization of a cholic acid conjugate of EDTA and biodistribution and imaging studies with its indium-111 chelate. Bioconjugate Chem. 2, 117−23.

202

dx.doi.org/10.1021/bc200460t | Bioconjugate Chem. 2012, 23, 196−202