Dendritic Molecular Glues with Reductively Cleavable Guanidinium Ion Pendants: Highly Efficient Intracellular siRNA Delivery via Direct Translocation

Abstract

We developed a dendritic molecular glue (Glue^SS-BP) bearing multiple guanidinium ion (Gu⁺) pendants via a disulfide (SS) spacer as a carrier for intracellular siRNA delivery. Glue^SS-BP adheres tightly to siRNA and forms a stable conjugate through multivalent salt-bridge formation between the Gu⁺ pendants of Glue^SS-BP and phosphate ions (PO₄⁻) of siRNA. The Glue^SS-BP/siRNA conjugate enters living cells via direct translocation. In the cytoplasm, the SS spacers in Glue^SS-BP are reductively cleaved off by glutathione (GSH) to efficiently release siRNA, thereby inducing RNA interference (RNAi). Products, formed upon reductive cleavage of Glue^SS-BP in the cytoplasm, barely cause cytotoxicity.

Dendritic molecular glues, Intracellular siRNA delivery, Direct translocation

Intracellular delivery of small interfering RNA (siRNA), which selectively suppresses expression of a certain gene through RNA interference (RNAi),¹ is a promising approach to the curing of both inherited and acquired diseases caused by genetic disorders. A variety of carriers for siRNA have been reported, such as those based on lipids, polymers, and nanoparticles,² most of which, however, are taken up via endocytosis into living cells.² This is unfavorable, since siRNA trapped in endosomes is subsequently degraded in lysosomes, resulting in a poor gene knockdown efficiency. Because only cytoplasmic siRNA can induce RNAi,¹ carriers that can directly penetrate the cell membrane to deliver siRNA to the cytoplasm are advantageous. Nevertheless, only a few peptide-based carriers have been reported to translocate siRNA directly into cells,³ and it still remains a big challenge to design such carriers with other molecular motifs. A promising strategy is to employ guanidinium ion (Gu⁺)-rich molecules⁴ that can strongly bind to and destabilize negatively charged cell membranes, thereby enabling direct intracellular translocation of siRNA. However, it should be noted that polycations, which possess a high efficacy for cellular uptake,⁵ often cause serious cytotoxicity.⁶

Herein, we report a dendritic molecular glue Glue^SS-BP (Figure 1) that can efficiently translocate siRNA directly into the cytoplasm (Figure 2) with minimal cytotoxicity. Previously, we developed water-soluble molecular glues^7–11 bearing multiple Gu⁺ pendants that adhere tightly to proteins,⁸ nucleic acids,⁹ phospholipid membranes,¹⁰ and clay nanosheets¹¹ via the formation of multiple salt bridges between their Gu⁺ pendants and oxyanionic groups on the targets.¹² In our previous communication,^9a we reported that a siRNA motif can efficiently template oxidative polymerization of oligomeric linear molecular glues with thiol (SH) groups at both termini, affording stable conjugates called “siRNA-nanocaplets”. Although the siRNA-nanocaplets are taken up into living cells via endocytosis, RNAi is successfully induced because the nanocaplets release siRNA by reductive cleavage of their disulfide (SS) bonds in the cytoplasm that contains glutathione (GSH) abundantly. On the basis of this finding, we designed, as a reductively cleavable dendritic analogue, Glue^SS-BP (Figure 1) that bears Gu⁺ pendants via a SS spacer. Unlike siRNA-nanocaplets, the Glue^SS-BP/siRNA conjugate can enter living cells via direct translocation (Figure 2). In the cytoplasm, the SS spacers in Glue^SS-BP are supposedly cleaved off to release siRNA, thereby suppressing target gene expression by RNAi (Figure 2) with minimal cytotoxicity.¹³ We also demonstrated the gene knockdown using the Glue^SS-BP/siRNA conjugate for different cell lines.

Figure 1.

Schematic structures of dendritic molecular glues Glue-BP, carrying 9 guanidinium ion (Gu⁺) pendants, and Glue^SS-BP, carrying 9 Gu⁺ pendants via a disulfide (SS) spacer. The SS spacer can be cleaved off reductively by glutathione (GSH).

Figure 2.

Schematic illustration of the intracellular delivery of a Glue^SS-BP/siRNA conjugate for the induction of RNA interference (RNAi). The Glue^SS-BP/siRNA conjugate is taken up into the cytoplasm via direct translocation or endocytosis followed by endosomal escape. The SS spacers in Glue^SS-BP are reductively cleaved off in the cytoplasm by glutathione (GSH), resulting in efficient release of siRNA and RNAi.

Glue^SS-BP and Glue-BP (Figure 1), a reference dendrimer without SS spacers, were synthesized according to the procedures described in the Supporting Information and characterized unambiguously using several analytical methods.¹⁴ In agarose gel electrophoresis, siRNA (1.5 µM) is known to migrate according to its net negative charges (Figure 3a, [Gu⁺]/[PO₄⁻] = 0). However, siRNA did not migrate when mixed with Glue^SS-BP ([Gu⁺]/[PO₄⁻] > 20), indicating that Glue^SS-BP adheres to siRNA and neutralizes its negative charges. Meanwhile, when the Glue^SS-BP/siRNA conjugates, prepared at [Gu⁺]/[PO₄⁻] = 0–60, were incubated with GSH (10 mM) at 37 °C for 2 h in HEPES buffer (10 mM, pH 7.4), the resulting electrophoresis profiles were all identical to that of the intact siRNA (Figure 3b), indicating the release of siRNA by reductive cleavage of the SS spacers in Glue^SS-BP. Dynamic light scattering (DLS) and zeta potential measurements also supported formation of the Glue^SS-BP/siRNA conjugate (D_h = 19.9 ± 4.6 nm, ζ = 23 ± 9 mV; Figures S4 and S5, respectively).¹⁴

Figure 3.

Agarose gel electrophoresis profiles of siRNA (1.5 µM) upon mixing with Glue^SS-BP (0–420 µM; [Gu⁺]/[PO₄⁻] = 0–60) before (a) and after (b) 2-h incubation with glutathione (GSH, 10 mM) at 37 °C in HEPES buffer (10 mM, pH 7.4), developed by staining with ethidium bromide.

We found that Glue^SS-BP carries siRNA into the cytoplasm of living cells. For visualization, we used siRNA fluorescently labeled with Cy3 (^Cy3siRNA). Human epithelial carcinoma HeLa cells were incubated at 37 °C for 1 h in a fetal bovine serum (FBS)-free Dulbecco's modified Eagle's medium (DMEM) containing ^Cy3siRNA (100 nM) and Glue^SS-BP (5 µM). Then, the cell sample was rinsed with Dulbecco's phosphate buffered saline (D-PBS) and subjected to confocal laser scanning microscopy (λ_ext = 435 nm). The cells displayed a fluorescence emission assignable to ^Cy3siRNA from their interior (Figure 4a). The diffuse fluorescence profile (Figure 4a) indicates the release of siRNA in the cytoplasm. In contrast, an analogous cell sample prepared using Glue-BP (5 µM) instead of Glue^SS-BP displayed a punctate fluorescence emission (Figure 4b). HeLa cells, pre-incubated for 24 h at 37 °C in DMEM (10% FBS) containing buthionine sulfoximine (BSO, 100 µM) as an inhibitor for the GSH biosynthesis before the addition of Glue^SS-BP and ^Cy3siRNA,¹⁵ also showed a punctate fluorescence profile (Figure 4c). These observations clearly indicate that cleavage of the SS spacers in Glue^SS-BP by GSH in the cytoplasm is essential for the liberation of siRNA.

Figure 4.

(a, b) Confocal laser scanning microscopy (CLSM, λ_ext = 435 nm) images of HeLa cells after 1-h incubation at 37 °C in a fetal bovine serum (FBS)-free Dulbecco's modified Eagle's medium (DMEM) containing ^Cy3siRNA (100 nM) and (a) Glue^SS-BP (5 µM) or (b) Glue-BP (5 µM). (c) A CLSM (λ_ext = 435 nm) image of HeLa cells after 24-h incubation at 37 °C in DMEM (10% FBS) containing buthionine sulfoximine (BSO, 100 µM) followed by 1-h incubation in a FBS-free DMEM containing a mixture of BSO (100 µM), ^Cy3siRNA (100 nM), and Glue^SS-BP (5 µM). (d) A CLSM (λ_ext = 435 nm) image of HeLa cells after 1-h incubation at 4 °C in an FBS-free DMEM containing a mixture of ^Cy3siRNA (100 nM) and Glue^SS-BP (5 µM). The insets show magnified images. Scale bars = 20 µm.

To our surprise, HeLa cells incubated with Glue^SS-BP (5 µM) and ^Cy3siRNA (100 nM) for 1 h at 4 °C also emitted fluorescence from their interior (Figure 4d). In contrast, no cellular uptake of ^Cy3siRNA took place at 4 °C using commercial transfection reagent Lipofectamine 2000 (LF2000, Figure S7),¹⁴ which is known to carry nucleic acids inside cells via endocytosis.¹⁶ Since energy-dependent cellular processes such as endocytosis are suppressed at 4 °C,¹⁷ these results indicate that the Glue^SS-BP/^Cy3siRNA conjugate enters the cells by direct penetration of the cell membrane (Figure 2). The punctate fluorescence emission observed in Figure 4d is most likely due to the aggregates of the Glue^SS-BP/^Cy3siRNA located in the cytoplasm. Taking into account that the interaction between Gu⁺ and anionic moieties on the cell surface is essential for direct translocation of Gu⁺-rich molecules,⁵ Glue^SS-BP, even after the conjugation with ^Cy3siRNA, possibly carries a certain number of unbound Gu⁺ pendants. This seems typical of dendritic molecular glues as carriers, considering that siRNA-nanocaplets consisting of oligomeric linear molecular glues do not migrate directly into the cytoplasm.^9a

We then examined the possibility of gene knockdown. Mutant HeLa cells stably expressing luciferase (HeLa-luc) were incubated at 37 °C in a FBS-free DMEM containing a mixture of siRNA for luciferase (siRNA^luc; 100 nM) and Glue^SS-BP (10 µM) for 4 h, followed by DMEM (10% FBS) for 24 h. The cell sample was then subjected to a luciferase activity assay using a Luciferase Assay System (Promega). As a result, the cells exhibited smaller luciferase activity (34%; Figure 5a, ii, green) than that of untreated HeLa-luc cells. Meanwhile, the suppression did not occur with mismatch siRNA (mis-siRNA; 100 nM) incapable of inducing RNAi for the luciferase gene (Figure 5a, ii, blue). These results suggest the occurrence of gene knockdown by RNAi with the Glue^SS-BP/siRNA^luc conjugate. When Glue-BP (10 µM) was used instead of Glue^SS-BP, not only siRNA^luc but also mis-siRNA suppressed the luciferase activity (19% and 29%; Figure 5a, i, green and blue, respectively), indicating that Glue-BP (10 µM) causes acute cell death. In fact, Glue-BP exhibited a LC₅₀ (median lethal concentration) value of 3.9 µM (Figure 6, blue), which is smaller than that of Glue^SS-BP (LC₅₀ = 11 µM, Figure 6, red), as evaluated by Cell Counting kit-8 (CCK-8).¹⁴ As expected, LF2000/siRNA^luc efficiently suppressed the luciferase activity (15%; Figure 5a, iii, green) with minimal cytotoxicity (Figure 5a, iii, blue). Even in the presence of FBS (10%), the Glue^SS-BP/siRNA^luc conjugate knocked down the luciferase gene (73%; Figure 5b, i), whereas no gene knockdown occurred with LF2000/siRNA^luc (Figure 5b, ii). Namely, the Glue^SS-BP/siRNA conjugate is more stable than LF2000/siRNA under physiological conditions. Similar gene knockdown experiments with Glue^SS-BP (10 µM) and siRNA^luc (100 nM) were carried out using luciferase-expressing mutants of mouse melanoma B16F10 cells (B16F10-luc), human hepatocellular carcinoma Huh-7 cells (Huh-7-luc), and human lung adenocarcinoma A549 cells (A549-luc). As shown in Figure 5c, all the examined cell lines showed suppressed luciferase activity without serious cytotoxicity.

Figure 5.

Normalized luciferase activities of cells using Luciferase Assay System (Promega). (a) HeLa-luc cells were incubated at 37 °C in a FBS-free DMEM containing a mixture of siRNA (100 nM; siRNA^luc and mis-siRNA, green and blue bars, respectively) and (i) Glue-BP (10 µM), (ii) Glue^SS-BP (10 µM), or (iii) Lipofectamine 2000 (LF2000) for 4 h, followed by incubation for 24 h in DMEM (10% FBS). LF2000 was used according to the procedure provided by the supplier (Invitrogen). (b) HeLa-luc cells were incubated at 37 °C in DMEM (10% FBS) containing a mixture of siRNA (100 nM; siRNA^luc and mis-siRNA, green and blue bars, respectively) and (i) Glue^SS-BP (10 µM) or (ii) LF2000 for 4 h, followed by incubation for 24 h in DMEM (10% FBS). (c) (i) HeLa-luc, (ii) B16F10-luc, (iii) Huh-7-luc, and (iv) A549-luc cells were incubated at 37 °C in a FBS-free DMEM containing a mixture of siRNA (100 nM; siRNA^luc and mis-siRNA, green and blue bars, respectively) and Glue^SS-BP (10 µM) for 4 h, followed by incubation for 24 h in DMEM (10% FBS).

Figure 6.

Cell viability of HeLa cells after 4-h incubation at 37 °C in a FBS-free DMEM containing Glue^SS-BP (0.05–50 µM, red) or Glue-BP (0.05–10 µM, blue), evaluated by Cell Counting kit-8 (CCK-8).

In conclusion, we developed a reductively cleavable dendritic molecular glue Glue^SS-BP (Figure 1) as a carrier for intracellular siRNA delivery. We demonstrated that Glue^SS-BP delivers siRNA into the cytoplasm of living cells via direct translocation (Figure 2), which is favorable for efficient induction of RNAi. Glue^SS-BP is promising as a carrier for siRNA owing to its high gene knockdown efficiency, low toxicity, and applicability to a wide range of cell lines. Incorporating a ligand moiety to the Glue^SS-BP/siRNA conjugate for site-selective delivery is an interesting subject worthy of further investigation.

Acknowledgment

We acknowledge the Center for NanoBio Integration, the University of Tokyo. This work was supported by Grant-in-Aid for Young Scientist (B) (26810046) to K.O. We appreciate Dr. T. Suzuki and Prof. T. Suzuki (the University of Tokyo) for electrophoresis experiments. We appreciate Prof. K. Miyata and K. Kataoka (the University of Tokyo) for gene knockdown assays. We appreciate Mr. N. Yoshinaga and Prof. H. Cabral (the University of Tokyo) for DLS and zeta potential measurements. R.M. thanks the Research Fellowships of Japan Society for the Promotion of Science (JSPS) for Young Scientists and the Program for Leading Graduate Schools (GPLLI).

Supporting Information is available on http://dx.doi.org/10.1246/cl.180551.

K. Okuro

T. Aida

H. Nemoto

R. Mogaki