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Multivalent comb-type aptamer–siRNA conjugates for efficient and selective intracellular delivery† Hyundong Yoo,‡ Hyosook Jung,‡ Seung An Kim and Hyejung Mok*
Received 4th March 2014, Accepted 7th May 2014 DOI: 10.1039/c4cc01620c www.rsc.org/chemcomm
In this study, a simple and efficient strategy for selective intracellular delivery of RNA therapeutics into target cancer cells was designed using direct complementary base pairing between chemically conjugated multimeric antisense strands and aptamer-incorporating sense strands.
RNA therapeutics have several advantages including high target selectivity, easy chemical synthesis, and wide application for undruggable targets.1 However, in contrast to conventional chemical drugs and protein/antibody-based drugs, therapeutic siRNAs need to be located in the cytoplasm for their incorporation into RNA-induced silencing complex (RISC) and subsequent suppression of target genes, which is a considerable challenge. To overcome this crucial barrier and increase intracellular delivery efficiency, complicated formulation steps might be necessary to adopt multifunctional moieties for the correct localization of siRNAs; however, this also hinders reproducibility and the ability to scale-up production. Thus, the development of simple, targeted intracellular delivery systems for RNA therapeutics is needed. Recently, simple, nanoscale, nucleic acid-based self-assemblies (e.g., DNA/RNA origami, micelles, and hydrogels) have been extensively studied with respect to their ability to improve the delivery of RNA therapeutics.2 Nucleic acid-based self-assemblies in particular are good for facile entrapment of drugs as well as incorporation of multivalency for excellent binding to target molecules and cells. RNA or DNA aptamers have been recognized not only for their potential as therapeutics, but also as promising ligands for targeted delivery because of high selectivity for targets and low immunogenicity.3 Since the first siRNA–aptamer chimeric structure reported in 2006,4 much effort has been made to improve the siRNA–aptamer structure with respect to cost, preferable chemical synthesis, synergistic therapeutic effect, and target specificities.5 However, to the best of our knowledge, multivalent nucleic acid-based structures/conjugates composed of Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. E-mail: [email protected]; Tel: +82-2-450-0448 † Electronic supplementary information (ESI) available: Detailed experimental methods, supporting data. See DOI: 10.1039/c4cc01620c ‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
only functional siRNAs and aptamers without any additional nucleic acids as frame structures for enhanced and selective intracellular delivery have not been reported previously. In this study, multivalent comb-type aptamer–siRNA conjugates (Comb-Apt–siR) with high aspect ratios were first designed for superior intracellular delivery in a target cell-selective manner via chemical conjugation and simple hybridization. It should be noted that only functional RNAs and DNAs were incorporated within Comb-Apt–siR, which could minimize nonspecific immune responses in target cells elicited by alien nucleic acids.6 For targeting cancer cells, a DNA aptamer against mucin 1 (MUC1) was selected as a targeting ligand because MUC1 is highly overexpressed in malignant adenocarcinomas.7 Scheme 1a illustrates the method used to prepare Comb-Apt–siR, which uses direct complementary base pairing between chemically conjugated multimeric antisense strands and aptamer-incorporating
Scheme 1 Illustrations for (a) the preparation of multivalent comb-type aptamer–siRNA conjugates (Comb-Apt–siR) and (b) cellular uptake of CombApt–siR for a mucin 1 (MUC1)-overexpressing cancer cell like MCF-7 cell.
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sense strands. Antisense siRNA strands were linearly connected via thiol–maleimide coupling using the chemical crosslinker dithiobis-maleimidoethane (DTME) to produce multimerized antisense siRNA strands (multimeric antisense). Both 30 - and 50 -end thiol modified antisense strands were allowed to self-crosslink for 24 h. To prepare dimeric aptamer–siRNA conjugates, only 30 -thiol modified antisense strands of siRNA were used to produce dimeric antisense strands. MUC1 aptamer-sense strands of siRNAs were complementary hybridized to multimeric and dimeric antisense for the fabrication of Comb-Apt–siR and dimeric aptamer–siRNA conjugates (Di-Apt–siR). For effective recognition by target receptors and following intracellular uptake into cells, several physicochemical properties of nanomaterials, such as aspect ratios, ligand–receptor interaction valency, orientation, and spacing of ligands need to be considered.8 In vitro, rod-like particles with high aspect ratios show much more efficient internalization within cells via specific receptor-mediated endocytosis than particles with low aspect ratios, such as spheres and cubes.9 In this study, we hypothesized that rod-shaped siRNA conjugates with multivalent aptamers might provide enhanced intracellular uptake in MUC1-overexpressing cancer cells, such as MCF-7 and A549, due to an increased affinity for target molecules on cell membranes and fast wrapping for endocytosis (Scheme 1b). The resulting Apt–siR conjugates were analyzed by gel electrophoresis (Fig. 1a and b). Multimeric antisense strands exhibited retarded and heterogeneous RNA bands compared to monomeric and dimeric antisense due to increased molecular weights (Fig. 1a). Fig. 1b shows that monomeric, dimeric, and multimeric antisense strands were successfully hybridized with aptamer-sense strands to fabricate common aptamer– siRNA conjugates (Apt–siR), Di-Apt–siR, and Comb-Apt–siR. The smear bands from Comb-Apt–siR might be attributed to dynamic hybridization and equilibrium base pairing.10 To clearly visualize the length of the multimeric antisense strand, a biotin-labeled sense strand instead of an aptamer-sense strand was annealed to the multimeric antisense strand, which was then decorated with streptavidin-coated gold nanoparticles with a diameter of 5 nm and visualized by transmission electron microscopy (TEM; Fig. 1c and Fig. S1, ESI†). Fig. 1c shows long rod-like structures representing multimeric antisense/sense-biotin with a length of 137.4 77.2 nm. Considering that the diameter of double stranded RNA is approximately 2.3 nm, the aspect ratio of multimeric antisense/ sense-biotin is approximately 59.7 33.6.11 Intracellular uptake of Comb-Apt–siR in MCF-7 and HepG2 cells was examined quantitatively and qualitatively using
Fig. 1 (a and b) Polyacrylamide gel electrophoresis of a single stranded antisense with a monomer, a dimer, and multimeric structures (a) before and (b) after hybridization to aptamer sense (S) strands. (c) TEM images of multimeric antisense/ biotin-S double-stranded siRNA coated with streptavidin–gold nanoparticles.
6766 | Chem. Commun., 2014, 50, 6765--6767
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Fig. 2 (a) Confocal microscopy images of cells treated with three types of POPO-3-labeled aptamer–siRNA structures. (b and c) Fluorescence intensities (FI) in MCF-7 cells after incubation with aptamer–siRNA structures with different (b) incubation times (*p o 0.05) and (c) RNA concentrations. (d) Inhibition assay of cellular uptake by chemical inhibitors (*p o 0.005, **p o 0.05).
confocal microscopy and spectrofluorophotometer, compared to Apt–siR. To visualize siRNAs, a red fluorescent dye, POPO-3, was intercalated within each Apt–siR conjugate. As shown in Fig. 2a, cells treated with Comb-Apt–siR exhibited the strongest red fluorescence, which means that the highest intracellular uptake occurred with Comb-Apt–siR among the three types of Apt–siR conjugates. It should also be noted that Comb-Apt–siR showed strong intracellular uptake only in MUC1-positive MCF-7 cells (human breast cancer). No fluorescence was observed for MUC1-negative HepG2 cells (human hepatocellular carcinoma) after incubation with Comb-Apt–siR. This elevated intracellular uptake of Comb-Apt–siR might be attributed to increased chance of contact with MUC1 or the synergistic effects of multivalent aptamers for endocytosis. To further investigate these possibilities, the cellular uptake rate of Comb-Apt–siR was also determined by quantitative evaluation of intracellular fluorescence intensity at different incubation times (Fig. 2b). Comb-Apt–siR exhibited higher fluorescence intensity (FI) than Apt–siR at 0.5 h. Interestingly, Apt–siR showed a slight change in FI from 0.5 h to 6 h, while Comb-Apt–siR exhibited continuous and significant increase of FI for 6 h. This result might indicate that the high uptake of Comb-Apt–siR might originate not only from favorable initial attachment but also from efficient endocytosis. According to a previous study, several ligands need to be bound to receptors and cluster together on cell membranes for endocytosis due to the required free energy to drive complete membrane wrapping.12 Thus, elongated, multivalent Comb-Apt–siR with a length of B137 nm might be able to enter cells much more preferentially than Apt–siR with a length of B6 nm. It is also conceivable that adoption of flexible chemical linkers between double-stranded RNA fragments confers favorable properties with respect to stable attachment of Comb-Apt–siR to receptors on cell membranes, as well as being better suited to follow the contours of the membrane curvature during wrapping time. Fig. 2c shows that the extent of cellular uptake was elevated with increasing amounts of Apt–siRNA. FI in cells treated with Comb-Apt– siR was approximately 2-fold higher than that of those treated with Apt–siR at an RNA concentration of 0.3 mM. To assess the mechanisms by which Comb-Apt–siR is internalized into cells, different chemicals that prevent intracellular
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Fig. 3 Quantitative analysis of the biological activity of Comb-Apt–siRs. (a) Target GFP gene suppression for A549 cells (*p o 0.01; ns = not significant), (b) suppression of cell proliferation for two types of cancer cells (*p o 0.001), and (c) caspase-3 activity after transfection with four types of siRNA structures in serum-containing medium for MCF-7 cells (*p o 0.005; ns = not significant).
trafficking were applied and uptake efficiency was observed. Chemical inhibitors such as wortmannin (Wort-), nystatin, and chlorpromazine (Chlor-) were used to inhibit lipid raft-, caveolin-, and clathrin-mediated endocytosis, respectively.13 Fig. 2d shows that wortmannin, nystatin, and chlorpromazine inhibited intracellular uptake of Comb-Apt–siR down to 92.3% 3.7%, 86.8% 1.8%, and 59.6% 8.0%, respectively. However, all three inhibitors showed negligible effects on cellular uptake of the POPO-3 dye alone (Fig. S2, ESI†). This result demonstrates that Comb-Apt–siR was mainly taken up by cells via clathrin-dependent endocytosis. Considering that free MUC1 endocytosis is mediated by a clathrin-dependent process, the attached MUC1 aptamers in Comb-Apt–siR could provide specific cellular uptake.14 Target gene inhibition and the biological effects of CombApt–siR were also quantitatively assessed in cancer cells. Three types of siRNAs against the green fluorescent protein (GFP) gene, including Apt–siR, Di-Apt–siR and Comb-Apt–siR, were applied to MUC1positive, GFP-expressing A549 cells (a human lung adenocarcinoma epithelial cell line). Unexpectedly, Comb-Apt–siR showed no noticeable gene inhibition despite superior intracellular uptake (Fig. S3a, ESI†). To facilitate endosome escape, four types of siRNAs including monomeric siRNA (Mono-siR), multimeric siRNA (Multi-siR), Apt–siR, and Comb-Apt–siR, were mixed with low-molecular-weight linear LPEI (2.5k) at a weight ratio of 1 before transfection. Fig. 3a shows that only Comb-Apt–siR significantly inhibited target GFP gene expression. Considering that addition of LPEI allowed negligible condensation of RNA conjugates and only B20% increase of cellular uptake (Fig. S3b–d, ESI†), enhanced suppression of target genes by the Comb-Apt–siR–LPEI mixture might be due to successful endosome escape. Using therapeutic Bcl-2 siRNAs, the suppression level of cancer cell proliferation was assessed using MCF-7 cells and HepG2 cells (Fig. 3b). Comb-Apt–siR noticeably reduced the viability of MCF7 cells while no significant effects were observed for HepG2 cells. However, control siRNA conjugates showed no effects on cell viability (Fig. S4, ESI†). This result may be attributed to the intracellular delivery and specific targeting of Bcl-2 siRNAs, which can trigger massive apoptosis.15 To assess whether Comb-Apt–siR could alter cellular signaling related to apoptosis, caspase-3 activity was quantitatively analyzed after transfection. Fig. 3c shows that caspase-3 activity was noticeably elevated in cells treated with Comb-Apt–siR.
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Communication
This indicates that decreased cell viability by Comb-Apt–siR is likely because of caspase-3-dependent cellular apoptosis.16 In this study, a novel, rod-shaped Comb-Apt–siR was developed, which showed greatly enhanced intracellular delivery for target cells compared to conventional Apt–siR chimeras. Adoption of multivalent ligands to the Comb-Apt–siR allowed efficient internalization via clustering effects. Furthermore, the delivered Comb-Apt–siR successfully inhibited target genes and reduced cancer cell proliferation. The newly designed Comb-Apt–siR has the potential to serve as a platform delivery system for improved, selective siRNA delivery in vivo without any toxicity. This study was supported by the Global Innovative Research Center (GiRC) program (NRF-2012K1A1A2A01056094), the Fusion Technology Project (2012K-001397), and Basic Science Research Program (NRF-2011-0023201) through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea.
Notes and references 1 D. Bumcrot, M. Manoharan, V. Koteliansky and D. W. Sah, Nat. Chem. Biol., 2006, 2, 711. 2 (a) H. Lee, A. K. Lytton-Jean, Y. Chen, K. T. Love, A. I. Park, E. D. Karagiannis, A. Sehgal, W. Querbes, C. S. Zurenko, M. Jayaraman, C. G. Peng, K. Charisse, A. Borodovsky, M. Manoharan, J. S. Donahoe, J. Truelove, M. Nahrendorf, R. Langer and D. G. Anderson, Nat. Nanotechnol., 2012, 7, 389; (b) S. H. Lee, B. H. Chung, T. G. Park, Y. S. Nam and H. Mok, Acc. Chem. Res., 2012, 45, 1014; (c) J. B. Lee, J. Hong, D. K. Bonner, Z. Poon and P. T. Hammond, Nat. Mater., 2012, 11, 316. 3 (a) A. D. Keefe, S. Pai and A. Ellington, Nat. Rev. Drug Discovery, 2010, 9, 537; (b) G. Z. Zhu, M. Ye, M. J. Donovan, E. Q. Song, Z. L. Zhao and W. H. Tan, Chem. Commun., 2012, 48, 10472. 4 J. O. McNamara 2nd, E. R. Andrechek, Y. Wang, K. D. Viles, R. E. Rempel, E. Gilboa, B. A. Sullenger and P. H. Giangrande, Nat. Biotechnol., 2006, 24, 1005. 5 (a) J. P. Dassie, X. Y. Liu, G. S. Thomas, R. M. Whitaker, K. W. Thiel, K. R. Stockdale, D. K. Meyerholz, A. P. McCaffrey, J. O. McNamara 2nd and P. H. Giangrande, Nat. Biotechnol., 2009, 27, 839; (b) W. Y. Lai, W. Y. Wang, Y. C. Chang, C. J. Chang, P. C. Yang and K. Peck, Biomaterials, 2014, 35, 2905; (c) T. C. Chu, K. Y. Twu, A. D. Ellington and M. Levy, Nucleic Acids Res., 2006, 34, e73. 6 S. Koyama, K. J. Ishii, C. Coban and S. Akira, Cytokine, 2008, 43, 336. 7 (a) C. Yu, Y. Hu, J. Duan, W. Yuan, C. Wang, H. Xu and X. D. Yang, PLoS One, 2011, 6, e24077; (b) M. A. Cheever, J. P. Allison, A. S. Ferris, O. J. Finn, B. M. Hastings, T. T. Hecht, I. Mellman, S. A. Prindiville, J. L. Viner, L. M. Weiner and L. M. Matrisian, Clin. Cancer Res., 2009, 15, 5323. 8 (a) J. Shi, J. L. Choi, B. Chou, R. N. Johnson, J. G. Schellinger and S. H. Pun, ACS Nano, 2013, 7, 10612; (b) P. C. Rensen, L. A. Sliedregt, M. Ferns, E. Kieviet, S. M. van Rossenberg, S. H. van Leeuwen, T. J. van Berkel and E. A. Biessen, J. Biol. Chem., 2001, 276, 37577. 9 (a) S. Barua, J. W. Yoo, P. Kolhar, A. Wakankar, Y. R. Gokarn and S. Mitragotri, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 3270; (b) S. E. Gratton, P. A. Ropp, P. D. Pohlhaus, J. C. Luft, V. J. Madden, M. E. Napier and J. M. DeSimone, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 11613. 10 F. H. Nelissen, A. J. van Gammeren, M. Tessari, F. C. Girard, H. A. Heus and S. S. Wijmenga, Nucleic Acids Res., 2008, 36, e89. 11 G. M. Skinner, M. van den Hout, O. Broekmans, C. Dekker and N. H. Dekker, Nano Lett., 2009, 9, 2953. 12 (a) B. D. Chithrani and W. C. Chan, Nano Lett., 2007, 7, 1542; (b) H. Gao, W. Shi and L. B. Freund, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 9469. 13 R. Rothe, L. Liguori, A. Villegas-Mendez, B. Marques, D. Grunwald, E. Drouet and J. L. Lenormand, J. Biol. Chem., 2010, 285, 20224. 14 Y. Altschuler, C. L. Kinlough, P. A. Poland, J. B. Bruns, G. Apodaca, O. A. Weisz and R. P. Hughey, Mol. Biol. Cell, 2000, 11, 819. 15 M. Jiang and J. Milner, Genes Dev., 2003, 17, 832. 16 D. W. Nicholson and N. A. Thornberry, Trends Biochem. Sci., 1997, 22, 299.
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Multivalent comb-type aptamer–siRNA conjugates for efficient and selective intracellular delivery† Hyundong Yoo,‡ Hyosook Jung,‡ Seung An Kim and Hyejung Mok*
Received 4th March 2014, Accepted 7th May 2014 DOI: 10.1039/c4cc01620c www.rsc.org/chemcomm
In this study, a simple and efficient strategy for selective intracellular delivery of RNA therapeutics into target cancer cells was designed using direct complementary base pairing between chemically conjugated multimeric antisense strands and aptamer-incorporating sense strands.
RNA therapeutics have several advantages including high target selectivity, easy chemical synthesis, and wide application for undruggable targets.1 However, in contrast to conventional chemical drugs and protein/antibody-based drugs, therapeutic siRNAs need to be located in the cytoplasm for their incorporation into RNA-induced silencing complex (RISC) and subsequent suppression of target genes, which is a considerable challenge. To overcome this crucial barrier and increase intracellular delivery efficiency, complicated formulation steps might be necessary to adopt multifunctional moieties for the correct localization of siRNAs; however, this also hinders reproducibility and the ability to scale-up production. Thus, the development of simple, targeted intracellular delivery systems for RNA therapeutics is needed. Recently, simple, nanoscale, nucleic acid-based self-assemblies (e.g., DNA/RNA origami, micelles, and hydrogels) have been extensively studied with respect to their ability to improve the delivery of RNA therapeutics.2 Nucleic acid-based self-assemblies in particular are good for facile entrapment of drugs as well as incorporation of multivalency for excellent binding to target molecules and cells. RNA or DNA aptamers have been recognized not only for their potential as therapeutics, but also as promising ligands for targeted delivery because of high selectivity for targets and low immunogenicity.3 Since the first siRNA–aptamer chimeric structure reported in 2006,4 much effort has been made to improve the siRNA–aptamer structure with respect to cost, preferable chemical synthesis, synergistic therapeutic effect, and target specificities.5 However, to the best of our knowledge, multivalent nucleic acid-based structures/conjugates composed of Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. E-mail: [email protected]; Tel: +82-2-450-0448 † Electronic supplementary information (ESI) available: Detailed experimental methods, supporting data. See DOI: 10.1039/c4cc01620c ‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2014
only functional siRNAs and aptamers without any additional nucleic acids as frame structures for enhanced and selective intracellular delivery have not been reported previously. In this study, multivalent comb-type aptamer–siRNA conjugates (Comb-Apt–siR) with high aspect ratios were first designed for superior intracellular delivery in a target cell-selective manner via chemical conjugation and simple hybridization. It should be noted that only functional RNAs and DNAs were incorporated within Comb-Apt–siR, which could minimize nonspecific immune responses in target cells elicited by alien nucleic acids.6 For targeting cancer cells, a DNA aptamer against mucin 1 (MUC1) was selected as a targeting ligand because MUC1 is highly overexpressed in malignant adenocarcinomas.7 Scheme 1a illustrates the method used to prepare Comb-Apt–siR, which uses direct complementary base pairing between chemically conjugated multimeric antisense strands and aptamer-incorporating
Scheme 1 Illustrations for (a) the preparation of multivalent comb-type aptamer–siRNA conjugates (Comb-Apt–siR) and (b) cellular uptake of CombApt–siR for a mucin 1 (MUC1)-overexpressing cancer cell like MCF-7 cell.
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sense strands. Antisense siRNA strands were linearly connected via thiol–maleimide coupling using the chemical crosslinker dithiobis-maleimidoethane (DTME) to produce multimerized antisense siRNA strands (multimeric antisense). Both 30 - and 50 -end thiol modified antisense strands were allowed to self-crosslink for 24 h. To prepare dimeric aptamer–siRNA conjugates, only 30 -thiol modified antisense strands of siRNA were used to produce dimeric antisense strands. MUC1 aptamer-sense strands of siRNAs were complementary hybridized to multimeric and dimeric antisense for the fabrication of Comb-Apt–siR and dimeric aptamer–siRNA conjugates (Di-Apt–siR). For effective recognition by target receptors and following intracellular uptake into cells, several physicochemical properties of nanomaterials, such as aspect ratios, ligand–receptor interaction valency, orientation, and spacing of ligands need to be considered.8 In vitro, rod-like particles with high aspect ratios show much more efficient internalization within cells via specific receptor-mediated endocytosis than particles with low aspect ratios, such as spheres and cubes.9 In this study, we hypothesized that rod-shaped siRNA conjugates with multivalent aptamers might provide enhanced intracellular uptake in MUC1-overexpressing cancer cells, such as MCF-7 and A549, due to an increased affinity for target molecules on cell membranes and fast wrapping for endocytosis (Scheme 1b). The resulting Apt–siR conjugates were analyzed by gel electrophoresis (Fig. 1a and b). Multimeric antisense strands exhibited retarded and heterogeneous RNA bands compared to monomeric and dimeric antisense due to increased molecular weights (Fig. 1a). Fig. 1b shows that monomeric, dimeric, and multimeric antisense strands were successfully hybridized with aptamer-sense strands to fabricate common aptamer– siRNA conjugates (Apt–siR), Di-Apt–siR, and Comb-Apt–siR. The smear bands from Comb-Apt–siR might be attributed to dynamic hybridization and equilibrium base pairing.10 To clearly visualize the length of the multimeric antisense strand, a biotin-labeled sense strand instead of an aptamer-sense strand was annealed to the multimeric antisense strand, which was then decorated with streptavidin-coated gold nanoparticles with a diameter of 5 nm and visualized by transmission electron microscopy (TEM; Fig. 1c and Fig. S1, ESI†). Fig. 1c shows long rod-like structures representing multimeric antisense/sense-biotin with a length of 137.4 77.2 nm. Considering that the diameter of double stranded RNA is approximately 2.3 nm, the aspect ratio of multimeric antisense/ sense-biotin is approximately 59.7 33.6.11 Intracellular uptake of Comb-Apt–siR in MCF-7 and HepG2 cells was examined quantitatively and qualitatively using
Fig. 1 (a and b) Polyacrylamide gel electrophoresis of a single stranded antisense with a monomer, a dimer, and multimeric structures (a) before and (b) after hybridization to aptamer sense (S) strands. (c) TEM images of multimeric antisense/ biotin-S double-stranded siRNA coated with streptavidin–gold nanoparticles.
6766 | Chem. Commun., 2014, 50, 6765--6767
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Fig. 2 (a) Confocal microscopy images of cells treated with three types of POPO-3-labeled aptamer–siRNA structures. (b and c) Fluorescence intensities (FI) in MCF-7 cells after incubation with aptamer–siRNA structures with different (b) incubation times (*p o 0.05) and (c) RNA concentrations. (d) Inhibition assay of cellular uptake by chemical inhibitors (*p o 0.005, **p o 0.05).
confocal microscopy and spectrofluorophotometer, compared to Apt–siR. To visualize siRNAs, a red fluorescent dye, POPO-3, was intercalated within each Apt–siR conjugate. As shown in Fig. 2a, cells treated with Comb-Apt–siR exhibited the strongest red fluorescence, which means that the highest intracellular uptake occurred with Comb-Apt–siR among the three types of Apt–siR conjugates. It should also be noted that Comb-Apt–siR showed strong intracellular uptake only in MUC1-positive MCF-7 cells (human breast cancer). No fluorescence was observed for MUC1-negative HepG2 cells (human hepatocellular carcinoma) after incubation with Comb-Apt–siR. This elevated intracellular uptake of Comb-Apt–siR might be attributed to increased chance of contact with MUC1 or the synergistic effects of multivalent aptamers for endocytosis. To further investigate these possibilities, the cellular uptake rate of Comb-Apt–siR was also determined by quantitative evaluation of intracellular fluorescence intensity at different incubation times (Fig. 2b). Comb-Apt–siR exhibited higher fluorescence intensity (FI) than Apt–siR at 0.5 h. Interestingly, Apt–siR showed a slight change in FI from 0.5 h to 6 h, while Comb-Apt–siR exhibited continuous and significant increase of FI for 6 h. This result might indicate that the high uptake of Comb-Apt–siR might originate not only from favorable initial attachment but also from efficient endocytosis. According to a previous study, several ligands need to be bound to receptors and cluster together on cell membranes for endocytosis due to the required free energy to drive complete membrane wrapping.12 Thus, elongated, multivalent Comb-Apt–siR with a length of B137 nm might be able to enter cells much more preferentially than Apt–siR with a length of B6 nm. It is also conceivable that adoption of flexible chemical linkers between double-stranded RNA fragments confers favorable properties with respect to stable attachment of Comb-Apt–siR to receptors on cell membranes, as well as being better suited to follow the contours of the membrane curvature during wrapping time. Fig. 2c shows that the extent of cellular uptake was elevated with increasing amounts of Apt–siRNA. FI in cells treated with Comb-Apt– siR was approximately 2-fold higher than that of those treated with Apt–siR at an RNA concentration of 0.3 mM. To assess the mechanisms by which Comb-Apt–siR is internalized into cells, different chemicals that prevent intracellular
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Fig. 3 Quantitative analysis of the biological activity of Comb-Apt–siRs. (a) Target GFP gene suppression for A549 cells (*p o 0.01; ns = not significant), (b) suppression of cell proliferation for two types of cancer cells (*p o 0.001), and (c) caspase-3 activity after transfection with four types of siRNA structures in serum-containing medium for MCF-7 cells (*p o 0.005; ns = not significant).
trafficking were applied and uptake efficiency was observed. Chemical inhibitors such as wortmannin (Wort-), nystatin, and chlorpromazine (Chlor-) were used to inhibit lipid raft-, caveolin-, and clathrin-mediated endocytosis, respectively.13 Fig. 2d shows that wortmannin, nystatin, and chlorpromazine inhibited intracellular uptake of Comb-Apt–siR down to 92.3% 3.7%, 86.8% 1.8%, and 59.6% 8.0%, respectively. However, all three inhibitors showed negligible effects on cellular uptake of the POPO-3 dye alone (Fig. S2, ESI†). This result demonstrates that Comb-Apt–siR was mainly taken up by cells via clathrin-dependent endocytosis. Considering that free MUC1 endocytosis is mediated by a clathrin-dependent process, the attached MUC1 aptamers in Comb-Apt–siR could provide specific cellular uptake.14 Target gene inhibition and the biological effects of CombApt–siR were also quantitatively assessed in cancer cells. Three types of siRNAs against the green fluorescent protein (GFP) gene, including Apt–siR, Di-Apt–siR and Comb-Apt–siR, were applied to MUC1positive, GFP-expressing A549 cells (a human lung adenocarcinoma epithelial cell line). Unexpectedly, Comb-Apt–siR showed no noticeable gene inhibition despite superior intracellular uptake (Fig. S3a, ESI†). To facilitate endosome escape, four types of siRNAs including monomeric siRNA (Mono-siR), multimeric siRNA (Multi-siR), Apt–siR, and Comb-Apt–siR, were mixed with low-molecular-weight linear LPEI (2.5k) at a weight ratio of 1 before transfection. Fig. 3a shows that only Comb-Apt–siR significantly inhibited target GFP gene expression. Considering that addition of LPEI allowed negligible condensation of RNA conjugates and only B20% increase of cellular uptake (Fig. S3b–d, ESI†), enhanced suppression of target genes by the Comb-Apt–siR–LPEI mixture might be due to successful endosome escape. Using therapeutic Bcl-2 siRNAs, the suppression level of cancer cell proliferation was assessed using MCF-7 cells and HepG2 cells (Fig. 3b). Comb-Apt–siR noticeably reduced the viability of MCF7 cells while no significant effects were observed for HepG2 cells. However, control siRNA conjugates showed no effects on cell viability (Fig. S4, ESI†). This result may be attributed to the intracellular delivery and specific targeting of Bcl-2 siRNAs, which can trigger massive apoptosis.15 To assess whether Comb-Apt–siR could alter cellular signaling related to apoptosis, caspase-3 activity was quantitatively analyzed after transfection. Fig. 3c shows that caspase-3 activity was noticeably elevated in cells treated with Comb-Apt–siR.
This journal is © The Royal Society of Chemistry 2014
Communication
This indicates that decreased cell viability by Comb-Apt–siR is likely because of caspase-3-dependent cellular apoptosis.16 In this study, a novel, rod-shaped Comb-Apt–siR was developed, which showed greatly enhanced intracellular delivery for target cells compared to conventional Apt–siR chimeras. Adoption of multivalent ligands to the Comb-Apt–siR allowed efficient internalization via clustering effects. Furthermore, the delivered Comb-Apt–siR successfully inhibited target genes and reduced cancer cell proliferation. The newly designed Comb-Apt–siR has the potential to serve as a platform delivery system for improved, selective siRNA delivery in vivo without any toxicity. This study was supported by the Global Innovative Research Center (GiRC) program (NRF-2012K1A1A2A01056094), the Fusion Technology Project (2012K-001397), and Basic Science Research Program (NRF-2011-0023201) through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea.
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