Bioconjugate Chem. 2006, 17, 189−194
189
Cellular Delivery of Polyheteroaromate-Peptide Nucleic Acid Conjugates Mediated by Cationic Lipids Takehiko Shiraishi, Nadia Bendifallah, and Peter E. Nielsen* Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark. Received August 11, 2005; Revised Manuscript Received November 8, 2005
In the search of facile and efficient methods for PNA cellular delivery, we have tested a series of PNA conjugates based on (hetero) aromatic, lipophilic compounds such as 9-aminoacridine, benzimidazoles, carbazole, anthraquinone, porphyrine, psoralen, pyrene, and phenyl-bis-benzimidazole (“Hoechst”). These chemically modified PNAs were delivered to cultured pLuc705HeLa cells mediated by cationic liposomes (LipofectAMINE or LiofectAMINE2000), and their nuclear delivery was inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. PNAs modified with 9-aminoacridine, “Hoechst”, or acetyl-“Hoechst” showed highest antisense activities (while unmodified PNA failed to show any significant antisense activity). In particular, bis-acridine-conjugated PNA showed nearly 60% splicing correction at 250 nM concentration in combination with LipofectAMINE2000. Interestingly, relative differences between the derivatives were observed when LipofectAMINE was used as compared to LipofectAMINE2000, but in general the latter yielded the higher antisense activity. The most active modifications of these PNA constructs were further tested for antisense downregulation of luciferase in p53R cells in order to evaluate the cytoplasmic activity (uptake) of the PNAs. A dosedependent down regulation of luciferase was demonstrated also in this system. The PNA conjugated to acetylHoechst caused a reduction of luciferase activity to less than 40% of the control at a concentration of 1 µM. These results indicate that conjugation of (hetero) polyaromatic compounds to PNA can dramatically improve liposome-mediated cellular delivery both to cytoplasm as well as to the nucleus. However, no clear structure/ activity relations are apparent from the present results, except that both 9-aminoacridine and “Hoechst” are also nucleic acid binding ligands.
INTRODUCTION Peptide nucleic acids (PNA) (1) are potentially attractive candidates for discovery and development of gene therapeutic antisense and antigene drugs (2-4). However, rapid progress has been difficult because of a lack of facile and robust cellular delivery methods (5, 6). Several methods have been devised, to improve the poor cellular uptake of the unmodified PNA. These methods involve conjugation of PNA to cell-penetrating peptides (7-11) (such as penetratin (12), transportan (13), oligo arginine (14), Tat-peptide (15), or a combination of these (16)) or the employment of cationic liposome carriers (17-19). In the former case, nontrivial synthesis of the conjugates is often encountered as well as relatively low efficacy of the conjugates due to endosomal entrapment (11). In the latter case, PNA modifications are required such as covalent (PNA-DNA chimera) (20) or noncovalent conjugation (PNA/DNA complexation by hybridization (17)) to DNA phosphodiester oligomers, or covalent conjugation to lipophilic compounds (18, 19), all with the purpose of increasing the affinity of the PNAs for the cationic liposome carriers. Synthesis of PNA-DNA chimera requires tedious synthesis, and PNA/DNA hybrid transfection requires multistep transfection and careful optimization of the complementary DNA sequences for each PNA although this method is relatively efficient (9, 21). Conjugation of PNAs to fatty acids is straightforward and allows cationic liposome delivery of the conjugates (18, 19), but unfortunately the conjugates have poor aqueous solubility and a high propensity for aggregation. However, we recently discovered that PNAs conjugated to the tricyclic, heteroaromatic * Corresponding author. Tel: +45-35327762; fax: +45-35396042; e-mail:
[email protected].
DNA intercalator 9-aminoacridine can be effectively delivered to eucaryotic cells in culture by cationic lipids (22). We tentatively ascribed this effect of the acridine to its lipophilic character (of the nonprotonated form), combined with the reasonable aqueous solubility of the protonated form. These results would hold promise that other polycyclic heteroaromatic ligands could be identified that (more) effectively promote cationic lipid assisted PNA delivery. To elucidate this possibility, we have synthesized eight new PNA conjugates with different lipophilic, polycyclic heteroaromatic ligands (including nucleic acid binding ligands) and tested these for lipid-mediated antisense activities both in HeLa pLuc 705 cells (splicing correction) and p53R cells (translational inhibition). These results show that simple modification of the PNAs with lipophilic compounds dramatically improved lipidmediated delivery of the PNA both to cytoplasm as well as to the nucleus.
EXPERIMENTAL PROCEDURES Synthesis of PNAs. The sequences and targets of the PNAs are listed in Table 1. PNA synthesis was carried out as reported previously (23). A variety of (hetero) polyaromatic ligands (Chart 1) obtained as carboxylic acid derivatives were covalently linked to PNA at the N-terminal on the solid support through a glycine, ethylene glycol linker (eg1, 8-amino-2,6-dioxaoctanoic acid) or 6-aminohexanoic acid (-aha-) via continuous synthesis (Table 1). All conjugates except those with benzimidazole and anthraquinone showed one major peak by reverse phase HPLC analysis of the raw product, and this was readily purified by HPLC and corresponded to the desired conjugates as characterized by MALDI-TOF mass spectrometry. The PNAs were lyophilized and stored at 4 °C until use, at which time they were dissolved in water and thereafter stored at -20 °C.
10.1021/bc050246z CCC: $33.50 © 2006 American Chemical Society Published on Web 12/24/2005
190 Bioconjugate Chem., Vol. 17, No. 1, 2006
Shiraishi et al.
Table 1. PNAs Used in This Study target splicingb
translationd
PNA no.
sequence
mass found (calcd)
purity (%)a
2389 2390 2391 2490 2846 2847 2848 2849 2850 2851 2858 2861 2862 2863 2870 2840 2841 2842 2882 2883
H-CCTCTTACCTCAGTTACA-NH2 adamantyl-CCTCTTACCTCAGTTACA-NH2 Acr-eg1-CCTCTTACCTCAGTTACA-NH2 Acr-(Acraeg)-eg1-CCTCTTACCTCAGTTACA-NH2c porphyrin-aha-CCTCTTACCTCAGTTACA-NH2 pyrene-CCTCTTACCTCAGTTACA-NH2 Hoechst-aha-CCTCTTACCTCAGTTACA-NH2 Ac-Hoechst-aha-CCTCTTACCTCAGTTACA-NH2 carbazole-CCTCTTACCTCAGTTACA-NH2 psoralen-aha-CCTCTTACCTCAGTTACA-NH2 anthraquinone-aha-CCTCTTACCTCAGTTACA-NH2 benzimidazole-Lys(benzimidazole)-CCTCTTACCTCAGTTACA-NH2 Ac-Lys(benzimidazole)-CCTCTTACCTCAGTTACA-NH2 perimidine-aha-CCTCTTACCTCAGTTACA-NH2 Ac-CCTCTTACCTCAGTTACA-NH2 Acr-eg1-CCT TTC TTTATG TTT-NH2 Acr-eg1-GCG TCT TCC ATG GTG-NH2 Acr-eg1-TGG TGG CTT TAC CAA-NH2 Hoechst-aha- GCG TCT TCC ATG GTG-NH2 Ac-Hoechst-aha- GCG TCT TCC ATG GTG-NH2
4767 (4765) 4941 (4942) 5301 (5301) 5593 (5591) 5518 (5519) 5034 (5036) 5243 (5243) 5288 (5286) 4987 (4987) 5120 (5121) 5113 (5113) 5239 (5238) 5107 (5108) 5136 (5137) 4953 (4953) 4435 (4435) 4520 (4520) 4528 (4528) 4564 (4563) 4608 (4695)
95+ 95+ 98+ 98+ 98+ 95+ 98+ 98+ 98+ 95+ 90+ 90+ 90+ 95+ 98+ 98+ 98+ 95+ 95+ 95+
a 98+ corresponds to single HPLC peak; 95+ corresponds to single peak with slight foot. b Targeting aberrant splice site of luciferase pre-mRNA in HeLa pLuc705 cells. c Acraeg is a 9-aminoacridine monomer based on the aminoethylglycine PNA backbone (27). d Targeting translational initiation site of luciferase mRNA in p53R cells.
Chart 1. Chemical Structures of the Ligands Conjugated to PNA on the Solid Supporta
a The carboxylic acid derivatives of the ligands were obtained from commercial sources (Sigma-Aldrich: adamantly, anthraquinone, benzimidazole, carbazole, perimidine, pyrene; Porhyrin Systems, Germany: porphyrin) or synthesized as described (acridine (27); Hoecshst (28); psoralen (29)).
Cell Culture. HeLa pLuc705 cells and p53R cells were purchased from Gene Tools and ATCC, respectively. The cells were cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 1.5% glutamax (Gibco), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in humidified air with 5% CO2. For the transfection studies in 96-well plate format, cells were trypsinized and seeded (1.2 × 104 cells/well) 16-24 h before transfection. For the 24-well plate, 7.2 × 104 cells/well were used. Polyheteroaromate-PNA Conjugate Transfection. Cells, seeded in 96-well plates the day before transfections, were treated with 100 µL/well of OPTI-MEM (Gibco) containing the PNA/lipid complex at the desired concentration for 4 h. The PNA-lipid complex was formed by incubating 10 µL of PNA
solution (in water) with LipofectAMINE (LFA, Invitrogen) or LipofectAMINE2000 (LFA2K, Invitrogen) in 10 µL OPTIMEM for 5 min at room temperature. Following addition of 80 µL of OPTI-MEM, this mixture was added to the cells. (Concentrations of LFA or LFA2K were 12 µL/ml or 8 µL/ml (final concentration), respectively, unless otherwise stated). After 4 h incubation, 100 µL/well of growth medium (RPMI-1640 containing 20% FBS and 1.5% glutamax) was added to the transfection mixture and incubated further for 24 h. For the transfection in 24-well plate format, 0.25 mL/well of OPTIMEM and growth medium were used. PNA/DNA Heteroduplex Transfection. Cells, seeded in 24well plates the day before transfections, were treated with 250 µL/well of OPTI-MEM (Gibco) containing the PNA/DNA/
Polyheteroaromate−PNA Conjugates
Bioconjugate Chem., Vol. 17, No. 1, 2006 191
Figure 1. Screening of the PNAs conjugated with heteroaromatic compounds for the transfection in combination with cationic lipids. PNAs, targeting splicing correction, were listed in Table 1. PNAs were complexed with LipofectAMINE (LFA (A)) or LipofectAMINE2000 (LFA2K, (B)) and used for the transfection. HeLa pLuc705 cells were transfected with PNA/LFA or PNA LFA2K complex for 4 h in the absence of serum and incubated for an additional 24 h in the presence of serum. After PNA transfections, the cells were subjected to the luciferase assay with Bright-Glo assay (Promega). The values represent the mean ( SD of three experiments.
LFA2K complex at the desired concentration for 4 h. For the formation of PNA/DNA heteroduplex, PNA2389 was hybridized with DNA2389 (5′-AATATGTAACTGAGGTA-3′) as reported previously (22). The PNA/DNA heteroduplex was incubated with LFA2K in 50 µL of OPTI-MEM for 10 min and added to the cells after addition of 200 µL of OPTI-MEM. After 4 h transfection, 250 µL/well of growth medium (RPMI-1640 containing 20% FBS and 1.5% glutamax) was added to the transfection mixture and incubated further for 24 h. Luciferase Assay. After PNA transfections, the cells were subjected to the luciferase analysis. Luciferase activity of the cells in 96-well plate format or 24-well plate format were measured by using Bright-Glo Luciferase assay system (Promega) or Luciferase Assay System (Promega), respectively, according to the manufacturer’s instructions. Luminescent readings from 96-well plate formats (with background subtracted) are presented as relative light units (RLU). For the cells in 24-well plate formats, cell were lysed with Passive Lysis buffer (Promega) and subjected to the luciferase assay. Obtained RLU values were normalized by protein concentrations and are presented as RLU/mg protein. Protein concentrations were measured using BCA protein assay (Pierce) according to the manufacturer’s instruction. RT-PCR. Total RNA was extracted from the samples in Passive Lysis buffer using RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction. Three ng of total RNA was used for RT-PCR reaction (20 µL/reaction) by using OneStep RT-PCR kit (Qiagen). Primers for the RT-PCR were as follows: forward primer, 5′-TTGATATGTGGATTTCGAGTCGTC-3′; reverse primer, 5′-TGTCAAT-CAGAGTGCTTTTGGCG-3′. 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 products were analyzed on 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 AND DISCUSSION The PNA conjugates listed in Table 1 (splicing) were synthesized. These all contain the same PNA oligomer targeting the cryptic splice site within the luciferase gene in HeLa pLuc705 cells and previously shown to effectively correct missplicing of this modified luciferase gene and induce the production of normal luciferase protein (22, 24). Therefore, the antisense effect in this cellular system provides a very good measure of the cellular and nuclear uptake of the PNA conjugates. The PNAs were tested in combination with two types of cationic liposomes, LipofectAMINE (LFA) or LipofectAMINE 2000 (LFA2K) (Figure 1,parts A and B, respectively). In this series of PNA conjugates, the PNA conjugated to the acetylated minor groove binder, “Ac-Hoechst” (Chart 1), showed the highest activity in combination with LFA (Figure 1A), being approximately 10 times more active than all other conjugates. As expected, unmodified PNA did not show any significant activities neither in the presence nor in the absence of cationic liposomes, but a few of the other new conjugates (apart from the already described adamantyl and acridine conjugates) were also active, in particular the Hoechst and the perimidine conjugates. We also tested these PNAs in combination with a more recently introduced lipofection reagent, LFA2K (Figure 1B).
192 Bioconjugate Chem., Vol. 17, No. 1, 2006
Shiraishi et al.
Figure 2. Transfection of the heteroaromate-PNAs (conjugated with Hoechst, acetyl-Hoechst (Ac-Hoechst), 9-aminoacridine (Acr), or bis-acridine (bisAcr)) or PNA/DNA heteroduplex in combination with LipofectAMINE (LFA) or LipofectAMINE2000 (LFA2K). (A) HeLa pLuc705 cells were transfected with Hoechst, Ac-Hoechst, Acr, and bisAcr-PNA in complex with LFA(a) or LFA2K (b) as mentioned in Figure 1. After 28 h transfection of the PNAs, cells were subjected to the luciferase analysis. Luciferase activities were measured using Luciferase Assay System (Promega) and normalized by protein concentration (shown as RLU/mg protein). The values represent the mean ( SD of three experiments. (B) RT-PCR analysis of the missplicing correction of luciferase pre-mRNAs by bisAcrPNA/LFA2k complex. Total RNA was extracted from the same samples in (A) and subjected to RT-PCR. U, fragments without mis-splicing 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. (C) Comparison of cationic lipid mediated delivery between bisAcr-PNA and PNA/DNA heteroduplex. For the bisAcr-PNA transfection, PNA2490 was transfected to the cells in complex with LFA2K (8 µL/ml). For the PNA/DNA heteroduplex transfection, PNA2389 was hybridized with its complementary DNA (DNA2389) and transfected to the cells in complex with LFA2K (8 µL/mL). After 28 h transfection, cells were subjected to the luciferase analysis using Luciferase Assay System (Promega). Luciferase activity was normalized by protein concentration and shown as RLU/mg protein. The values represent the mean ( SD of three experiments.
Most of the PNA conjugates showed a higher effect than with LFA, and with this agent even unmodified and acetylated PNAs were somewhat active, but the best activity was observed with the acridine and in particular with a bis-acridine PNA conjugate. In contrast, Ac-Hoechst showed significantly lower activity than with LFA, while Hoechst PNA showed higher activity with LFA2K. These results clearly indicate the complexity of the lipid-mediated PNA delivery in terms of the combination of cationic liposome and auxiliary ligand. Thus, we decided to focus on the four seemingly most active PNA constructs (namely, Acr, bisAcr, Hoechst, and Ac-Hoechst) for further studies. A more elaborate dose response experiment was performed with these four PNAs in combination with LFA and LFA2K (Figure 2A). These results confirm the results obtained from the initial screening (Figure 1) but provide a much more detailed picture of the activities. In particular, the superiority of the AcHoechst conjugate in combination with LFA and the both relative and absolute superiority of the bis-acridine conjugate in combination with LFA2K is noteworthy. To further support that the increment of luciferase activity is indeed the result of splicing correction, we performed an RNA analysis by RT-PCR. Total RNA was extracted from the same samples as used for luciferase assay and subjected to RT-PCR analysis (Figure 2B). These results fully corroborate the enzyme activity data showing
57% splicing correction at 250 nM and essentially full correction at 2 µM. However, it is important to realize that transfection experiments are not quantitatively reproducible from one set of experiments to the next due to differences in the exact state of the cell culture, subtle differences in the liposome preparation, etc. (Rasmussen, Nielsen, and Koppelhus et al., manuscript in preparation). Therefore, only results within the same experiment should be directly compared. In this context we performed a direct comparison of the antisense effect of the bis-acr-PNA conjugate delivered by LFA2K with that of the same unmodified PNA delivered by LFA2K as a sequence and length optimized DNA-PNA hybrid (Rasmussen, Nielsen, Koppelhus et al., manuscript in preparation). This experiment (Figure 2C) indicates that DNA mediated delivery is slightly (2-3-fold) more effective than “direct” delivery of the conjugate. Furthermore, the relative difference in efficacy most certainly will vary with cell type and gene target. The antisense activity in the HeLa pLuc 705 cellular system only reflects the PNA concentrations in the nucleus although the size of the PNA molecules (
