Caged Molecular Glues as Photoactivatable Tags for Nuclear

Article Cite This: J. Am. Chem. Soc. 2018, 140, 2687−2692

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Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells Akio Arisaka,†,§ Rina Mogaki,†,§ Kou Okuro,*,† and Takuzo Aida*,†,‡ †

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Riken Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan W Web-Enhanced Feature * S Supporting Information *

ABSTRACT: We developed dendritic caged molecular glues (CagedGlue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Negatively charged CagedGlue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of CagedGlueR to UV light or near-infrared (NIR) light, the BANVOC groups of CagedGlue-R are rapidly detached to yield an uncaged molecular glue (UncagedGlue-R) that carries multiple Gu+ pendants. Because Gu+ forms a salt bridge with PO4−, Uncaged Glue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with CagedGlue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the CagedGlue-R tag is photochemically uncaged to form UncagedGlue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with Caged Glue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.

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adhere 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. CagedGlue-R is designed not to interact with cell membranes by protecting its Gu+ pendants with an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC).13 Thus, guests such as drugs, when tagged with CagedGlue-R, are supposed to be taken up into living cells mostly via endocytosis, as commonly observed for negatively charged nanoparticles,14 and hide in endosomes (Figure 2). However, upon photoirradiation, the CagedGlue-R tag is converted into UncagedGlue-R, carrying multiple Gu+ pendants (Figure 1), which in turn interacts strongly with the endosomal membrane. Hence, guest/UncagedGlue-R conjugates can migrate from endosomes into the cytoplasm (Figure 2). Subsequently, they are possibly translocated into the nucleus (Figure 2) by the action of the Gu+-rich UncagedGlue-R tag. Unless photouncaged, guests remain dormant in endosomes; therefore, point-wise photoirradiation enables their spatiotemporal nuclear translocation. Translocation of guest/CagedGlue-R conjugates, triggered by two-photon near-infrared (NIR) light, also took place.

INTRODUCTION The cell nucleus is one of the most important organelles as a subcellular drug-delivery target.1 Anticancer drugs such as cisplatin, doxorubicin, and camptothecin exert their efficacy mostly in the nucleus by inhibiting DNA replication or transcription.2 In gene therapy, nucleic acids delivered to the nucleus compensate dysfunctional and/or missing genes.3 For nucleus-targeted drug delivery, drugs must be designed to overcome several cellular barriers: the plasma, endosomal, and nuclear membranes.1b A promising strategy to achieve efficient nuclear translocation of drugs is to attach guanidinium ion (Gu+)-rich tags,4 because Gu+-rich molecules are known to bind and activate nuclear transporter proteins such as importinβ.5 Because they can also interact strongly with and efficiently permeate through the plasma and endosomal membranes,6 such Gu+-tag/drug conjugates could certainly be delivered to the nucleus without suffering from cellular barriers. However, the conjugates can also translocate into the nucleus of undesired cells, leading to serious side effects. Therefore, they need to implement a particular mechanism to realize the siteselective operation. Herein, we report caged molecular glues, CagedGlue-R (Figure 1), as conceptually new dendritic tags for nucleus-targeted drug delivery, which can be site-selectively activated by photoirradiation. Previously, we developed water-soluble molecular glues7−11 bearing multiple Gu+ pendants,12 which tightly © 2018 American Chemical Society

Received: December 23, 2017 Published: January 30, 2018 2687

DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692

Article

Journal of the American Chemical Society

Figure 1. Schematic structures of CagedGlue-R, caged molecular glue that bears 9 protected guanidinium ion (Gu+) pendants by a butyratesubstituted nitroveratryloxycarbonyl (BANVOC) group; UncagedGlue-R, photouncaged molecular glue that bears 9 Gu+ pendants; and Glue-R, molecular glues carrying no cage. The focal core of Glue-R is functionalized with either nitrobenzoxadiazole (NBD) or dibenzocyclooctyne (DBCO).

Figure 2. Schematic illustration of light-triggered nuclear translocation of guests (drugs) conjugated with a CagedGlue tag. The guest/CagedGlue conjugate is taken up into living cells via endocytosis. Upon photoirradiation, the CagedGlue tag is uncaged to yield an UncagedGlue tag, which strongly interacts with the endosomal membrane and therefore facilitates endosomal escape of the tagged guest. Subsequently, the tagged guest is delivered into the nucleus.

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RESULTS AND DISCUSSION Glue-NBD (Figure 1) was synthesized by the following procedures (see Supporting Information). First, a dendron carrying both a nitrobenzoxadiazole (NBD)-substituted focal core and three azide (N3) pendants was conjugated by an azide−alkyne “click” reaction with a dendron carrying both an alkyne-substituted focal core and three Gu+ pendants protected by a BANVOC tert-butyl ester (tBu-BANVOC) unit, affording a dendron carrying nine tBu-BANVOC-protected Gu+ pendants in 68% yield. Then, the resultant dendron underwent acid hydrolysis of the tBu esters to give CagedGlue-NBD in 85% yield. We also synthesized dibenzocyclooctyne (DBCO)-appended caged molecular glue CagedGlue-DBCO (Figure 1), so that N3appended guests could be conjugated by a copper-free click reaction. A dendron carrying both a BocNH-substituted focal core and three N3-functionalized pendants was conjugated with a dendron carrying both an alkyne-substituted focal core and three tBu-BANVOC-protected Gu+ pendants by a click reaction, affording a dendron carrying both a BocNH-substituted focal core and nine tBu-BANVOC-protected Gu+ pendants in 43% yield. Then, the tBu and Boc groups of the resultant dendron were removed by the treatment with HCl, followed by condensation with DBCO to give CagedGlue-DBCO in 81% yield (see Supporting Information). Although both CagedGlueNBD and CagedGlue-DBCO exhibited complicated 1H and 13C NMR spectra (Figures S1, S2, S5, and S6) due to tautomerization of the protected Gu+ units,15 they were unambiguously characterized by referring to a model compound carrying a tBu-BANVOC-protected Gu+ unit

(Figures S7−S9). Neither CagedGlue-NBD nor CagedGlueDBCO showed characteristic signals due to tBu groups (1H NMR, δ 1.43−1.44 ppm; 13C NMR, δ 28.1−28.2 and 80.5− 80.7 ppm), indicating complete removal of the tBu esters and Boc groups. As a reference for CagedGlue-NBD, noncaged molecular glue Glue-NBD (Figure 1) was likewise synthesized using a dendron carrying three Boc-protected Gu+ pendants instead of the dendron carrying three tBu-BANVOC-protected Gu+ pendants (see Supporting Information). At first, we investigated the uncaging reaction of CagedGlueNBD with UV light. As shown in Figure 3a (blue), CagedGlueNBD (50 μM) in Tris-HCl buffer (20 mM, pH 7.2) exhibited a negative zeta potential (−68 ± 8 mV) due to its negatively charged CO2− groups. However, after 60 min exposure of the Caged Glue-NBD solution to UV light at 365 nm, the zeta potential became positive (32 ± 5 mV; Figure 3a, red), indicating successful deprotection of the Gu+ groups of Caged Glue-NBD to give UncagedGlue-NBD. The zeta potential observed for UncagedGlue-NBD was smaller than that expected from noncaged Glue-NBD in Tris-HCl buffer (53 ± 6 mV; Figure 3a, green), suggesting that the liberated BANVOC ions are salt-bridged tightly with the Gu+ pendants in the buffer medium and attenuate their net charge. Complete uncaging of Caged Glue-NBD with UV light was also confirmed by MALDITOF mass spectrometry (Figure S10). Caged Glue-NBD in situ rapidly exerts its affinity for anionic biomolecules upon exposure to UV light. We used a doublestranded DNA as a model, because it bears multiple phosphate

Caged

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DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692

Article

Journal of the American Chemical Society

was substantially identical to that of l-pUC19 without CagedGlue-NBD (Figure 3b, 0 s). Notably, when the mixture was exposed to UV light at 365 nm for 30 s, the band due to lpUC19 completely disappeared (Figure 3b, 30−60 s). This is most likely due to the charge neutralization of l-pUC19 by Uncaged Glue-NBD generated from CagedGlue-NBD upon UV exposure. We evaluated the association constants (Kassoc) of CagedGlueNBD, UncagedGlue-NBD, and Glue-NBD with DNA by fluorescence resonance energy transfer (FRET) using doublestranded DNA-TAMRA (17 bp) fluorescently labeled with 5carboxytetramethylrhodamine (TAMRA) as a FRET acceptor for NBD.16 If a NBD-appended molecular glue binds to DNATAMRA, a FRET between NBD and TAMRA occurs. Thus, DNA-TAMRA (100 nM) was titrated with CagedGlue-NBD (0− 160 nM) at 20 °C in Tris-HCl (20 mM, pH 7.2) buffer. As shown in Figure 3c, the fluorescence intensity at 570 nm (λex = 480 nm), assignable to the emission of TAMRA, did not increase substantially. In sharp contrast, upon titration with Uncaged Glue-NBD (0−320 nM), generated by 2 min of UV exposure of CagedGlue-NBD at 365 nm, the fluorescence intensity at 570 nm significantly increased (Figure 3d), indicating the FRET between NBD and TAMRA. An analogous fluorescence spectral change profile was observed for noncaged Glue-NBD (0−160 nM; Figure 3e). According to the reported method,17 the fractions of DNA-TAMRA bound to CagedGlueNBD, UncagedGlue-NBD, and Glue-NBD (Figure 3f) were estimated from their FRET efficiencies (see Supporting Information). By fitting the profiles thus observed for Uncaged Glue-NBD and Glue-NBD (Figure 3f, red and green, respectively) to the Hill equation,18 their Kassoc values for DNATAMRA were evaluated. Importantly, the Kassoc value of Uncaged Glue-NBD (1.6 × 107 M−1; half-maximal effective concentration, EC50 = 63 nM) was comparable to that of Glue-NBD (2.3 × 107 M−1; EC50 = 43 nM), indicating that Caged Glue-NBD was uncaged almost quantitatively by 2 min of UV exposure. The Kassoc value of CagedGlue-NBD was negligibly small compared to that of UncagedGlue-NBD (Figure 3f, blue), most likely due to an electrostatic repulsion between the BA NVOC groups in CagedGlue-NBD and the PO4− units in DNA. Caged Glue-NBD is taken up into cells via endocytosis and, upon UV irradiation, migrates into the cytoplasm and, more importantly, the cell nucleus eventually. Previously, a few cellpenetrating peptides having lysine residues caged with onitrobenzyl groups have been reported.19 They can penetrate the plasma membrane upon photoirradiation but do not migrate into the nucleus.19 We incubated human hepatocellular carcinoma Hep3B cells (5.0 × 103 cells/well) in a serum-free Eagle’s minimal essential medium (EMEM, 200 μL) containing Caged Glue-NBD (10 μM) for 3 h at 37 °C. After being rinsed with Dulbecco’s phosphate-buffered saline (D-PBS, 100 μL × 2), the cell sample was incubated at 37 °C for 20 min in EMEM containing a mixture of 10% fetal bovine serum (FBS) and LysoTracker Red (100 nM) as an endosome-labeling reagent, and then subjected to confocal laser scanning microscopy. Upon excitation at 488 nm, a strong fluorescence emission, assignable to CagedGlue-NBD, was observed punctately from the cell interior (Figure 4a and c, green). An analogous micrograph was obtained upon excitation at 543 nm for LysoTracker Red in endosomes (Figure 4b and c, red), indicating that CagedGlueNBD is localized in endosomes. Interestingly, when an

Figure 3. (a) Zeta potential profiles at 25 °C of CagedGlue-NBD (50 μM) in Tris-HCl (20 mM, pH 7.2) buffer before (blue) and after UV exposure at 365 nm for 60 min (UncagedGlue-NBD, red) with GlueNBD (50 μM, green) as a reference. (b) Agarose gel electrophoresis profiles (λex = 310 nm) of l-pUC19 (0.06 nM) in the absence and presence of CagedGlue-NBD (1 μM) before and after UV exposure at 365 nm for 15−60 s, developed by staining with ethidium bromide. (c−e) Fluorescence spectra (λex = 480 nm) of DNA-TAMRA (100 nM) at 25 °C in Tris-HCl (20 mM, pH 7.2) buffer upon titration with (c) CagedGlue-NBD (0−160 nM), (d) UncagedGlue-NBD (0−320 nM), and (e) Glue-NBD (0−160 nM) as a reference. (f) Binding profiles of Caged Glue-NBD (0−320 nM, blue), UncagedGlue-NBD (0−320 nM, red), and Glue-NBD (0−160 nM, green) as a reference to DNATAMRA (0.06 nM) in Tris-HCl (20 mM, pH 7.2) buffer.

anions (PO4−) just like cell membranes. When a Tris-HCl (20 mM, pH 7.2) buffer solution of a mixture of CagedGlue-NBD (1 μM) and linearized pUC19 DNA (l-pUC19; 2 686 bp, 0.06 nM) was subjected to agarose gel electrophoresis, the profile developed by staining with ethidium bromide (λex = 310 nm) 2689

DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692

Article

Journal of the American Chemical Society

NBD, rapidly migrates into the cytoplasm followed by the nucleus in a manner similar to noncaged Glue-NBD (Figure S12). No significant cytotoxicity was observed for the cells treated with CagedGlue-NBD before and even after the UV exposure (Figure S14). Of particular interest, such a light-triggered migration of Caged Glue-NBD into the cell nucleus is achieved by two-photon excitation with NIR light. This is advantageous for in vivo applications because NIR light can penetrate deep tissues.20 Analogous to the previous experiment (Figure 4d), CagedGlueNBD was allowed to be taken up into Hep3B cells via endocytosis to stay in endosomes (Figure 4f). When the cells were irradiated with a two-photon NIR laser (λext = 710 nm; Figure 4f, white dashed circle) for 2 min (30 s × 4), CagedGlueNBD, located in the irradiated area, rapidly migrated into the cytoplasm and nucleus subsequently (Figure 4g). In sharp contrast, CagedGlue-NBD in nonirradiated areas did not undergo nuclear translocation (Figure 4g). These observations prompted us to investigate the applicability of CagedGlue-R as a nuclear targeting tag for guest delivery. We used quantum dots (QDs; DH = 15−20 nm) as the guest. Nuclear delivery of QDs remains a challenge because many of the reported QDs, even when conjugated with Gu+rich tags, do not migrate into the cell nucleus.6p,21 The QD/CagedGlue-R conjugate (CagedGlue-QDs) was prepared by a copper-free click reaction between N3-functionalized QDs and Caged Glue-DBCO (see Supporting Information). The employed QDs were larger in diameter (15−20 nm) than the nuclear pores (∼5 nm)22 and could hardly diffuse into the nucleus. Therefore, unless the CagedGlue-R tags are uncaged to activate nuclear transporter proteins,5 the tagged QDs would not be delivered into the nucleus. Thus, Hep3B cells were incubated in EMEM (200 μL) containing CagedGlue-QDs (10 nM) at 37 °C for 3 h, whereupon CagedGlue-QDs were taken up into the cells (Figure 5a, red; λext = 488 nm). Although CagedGlue-QDs did not migrate into the nucleus (Figure 5a, blue; λext = 405 nm), a fluorescence emission assignable to QDs emerged in the nucleus (Figure 5b and c) when the sample was incubated for 10 min at 37 °C after being exposed to UV light for 2 min at 365 nm. This observation indicates the successful uncaging of Caged Glue-QDs and subsequent nuclear translocation of the resulting UncagedGlue-QDs. Accordingly, a cross-sectional image of the cells showed that the nucleus indeed emitted the fluorescence (Figure 5c and d).

Figure 4. Confocal laser scanning micrographs of Hep3B cells after 3h incubation at 37 °C in EMEM containing CagedGlue-NBD (10 μM) followed by rinsing with D-PBS. (a, b) Micrographs recorded upon excitation at (a) 488 nm (λobs = 500−530 nm) and (b) 543 nm (λobs = 565−620 nm) after 20 min incubation in EMEM (10% FBS) containing LysoTracker Red (100 nM). (c) Merged image of (a) and (b). (d, e) Micrographs recorded upon excitation at 488 nm (λobs = 500−530 nm, green) and 710 nm (two-photon; λobs = 390−465 nm, blue). The Hep3B cells, treated with CagedGlue-NBD, were incubated at 37 °C in EMEM (10% FBS) containing Hoechst 33342 (5 μg/mL) before (d) and after (e) 2 min UV exposure at 365 nm. (f, g) Micrographs recorded upon excitation at 488 nm (λobs = 500−530 nm) before (f) and after (g) two-photon irradiation at 710 nm for 2 min (30 s × 4). The white dashed circle in (f) represents the irradiated area. Scale bars = 20 μm. See also Movie 1.

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CONCLUSIONS In conclusion, we developed caged molecular glues CagedGlue-R (Figure 1) as photoactivatable tags for nucleus-targeted guest delivery. We demonstrated that QDs tagged with CagedGlue-R are efficiently delivered to the cell nucleus upon photouncaging of their CagedGlue-R tags (Figure 2), suggesting the potential applicability of the CagedGlue-R tag to tissue-selective drug delivery. The CagedGlue-R tag is advantageous for in vivo practical applications, because it can be uncaged by two-photon NIR light as well as UV light and does not show serious phototoxicity before and even after being uncaged. As exemplified by the use of QDs, CagedGlue-R could deliver macromolecular guests with large dimensions into the cell nucleus. The nuclear translocation using the CagedGlue-R tags is likely more promising in its high site-selectivity and low toxicity than previous systems comprising Gu+-rich noncaged tags in combination with singlet O2 generators for photochemical

analogous cell sample without LysoTracker Red was exposed to UV light at 365 nm for 2 min, followed by 10 min incubation in EMEM (10% FBS) containing Hoechst 33342 (5 μg/mL) as a nuclear labeling reagent, both the cytoplasm and nucleus interior (Figure 4e, blue) were fluorescent due to NBD (Figure 4e, green), whereas only endosomes were fluorescent unless exposed to UV light (Figure 4d). These results indicate that Uncaged Glue-NBD, photochemically generated from CagedGlue2690

DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692

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Journal of the American Chemical Society Author Contributions §

A.A. and R.M. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Center for NanoBio Integration, the University of Tokyo. This work was supported by Grant-in-Aid for Young Scientists (B) (26810046) to K.O. and partially supported by Grant-in-Aid for Specially Promoted Research (25000005) to T.A. 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).

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Figure 5. Confocal laser scanning micrographs of Hep3B cells upon excitation at 405 nm (λobs = 430−520 nm, blue) and 488 nm (λobs = 625−680 nm, red). Hep3B cells were incubated at 37 °C for 3 h in EMEM containing CagedGlue-QD (10 nM), rinsed with D-PBS, and then incubated at 37 °C for 10 min in EMEM (10% FBS) containing Hoechst 33342 (5 μg/mL) before (a) and after (b, c) 2 min exposure to UV light at 365 nm. (d) Cross-sectional image along the yellow line in (c). Scale bars = 20 μm.

deterioration of the cell membranes.4 Along this line, spatiotemporal nuclear delivery of proteins and nucleic acids using the CagedGlue-R tag for the regulation of gene expression is an interesting subject for addressing several issues of genetic disorders.3

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b13614. Synthesis of CagedGlue-NBD, Glue-NBD, and CagedGlueDBCO; 1H NMR, 13C NMR, and MALDI-TOF-MS spectral data; cytotoxicity profiles; and related experimental procedures (PDF) W Web-Enhanced Feature *

Cells undergoing on/off cycles of irradiation are available in a video file in the HTML version of the paper.

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Kou Okuro: 0000-0003-0445-6358 Takuzo Aida: 0000-0002-0002-8017 2691

DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692

Article

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DOI: 10.1021/jacs.7b13614 J. Am. Chem. Soc. 2018, 140, 2687−2692