Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells

Akio Arisaka, Rina Mogaki, Kou Okuro, and Takuzo Aida. J. Am. Chem. Soc., 2018, 140, 2687–2692.

DOI: 10.1021/jacs.7b13614

 

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 ^CagedGlue-R 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^–, ^UncagedGlue-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 ^CagedGlue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.

 

Introduction

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target. ^[1] For nucleus-targeted drug delivery, drugs must be designed to overcome several cellular barriers such as the plasma, endosomal, and nuclear membranes. A promising strategy to achieve efficient nuclear translocation of drugs is to attach guanidinium ion (Gu⁺)-rich tags, because Gu⁺-rich molecules are known to bind and activate nuclear transporter proteins. T. Aita group at University of Tokyo report caged molecular glues, named Caged Glue-R (Figure 1), which is targeted to cell nucleus and can be site-selectively activated by photo-irradiation. They designed ^CagedGlue-R by incorporate their previously develoved Guanine dendrimer motif ^[2] with anionic photocleavable group ^[3] so that it does not to interact with cell membranes. As illustrated in Figure 2, ^CagedGlue-R can enter living cells via endocytosis and hide in endosome before it is converted to ^UncagedGlue-R by photo-irradiation. Subsequently, they are possibly translocated into the nucleus by the action of the Gu⁺-rich ^UncagedGlue-R tag.

 

Figure 1 shows the schematic structures of ^CagedGlue-R, caged molecular glue that bears 9 protected guanidinium ion (Gu⁺) pendants by a butyrate-substituted 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 shows the 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.

 

Details

Figure 3 (a) shows 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 Glue-NBD (50 μM, green) as a reference. (b) Agarose gel electrophoresis profiles (λ_ex = 310 nm) of l-pUC19 in the absence and presence of ^CagedGlue-NBD before and after UV exposure, with ethidium bromide stain. (c–e) Fluorescence spectra (λ_ex = 480 nm) of DNA-TAMRA (100 nM) at 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 ^CagedGlue-NBD (0–320 nM, blue), ^UncagedGlue-NBD (0–320 nM, red), and Glue-NBD (0–160 nM, green) as a reference to DNA-TAMRA (0.06 nM).

 

Figure 4 shows confocal laser scanning micrographs of Hep3B cells containing ^CagedGlue-NBD (10 μM). (a, b) Micrographs upon excitation at (a) 488 nm (λ_obs = 500–530 nm) and (b) 543 nm (λ_obs = 565–620 nm) containing LysoTracker Red. (c) Merged image of (a) and (b). (d, e) Micrographs recorded upon excitation at 488 nm (λ_obs = 500–530 nm, green) and 710 nm (two-photon; λ_obs = 390–465 nm, blue). The Hep3B cells, treated with ^CagedGlue-NBD, were incubated at 37 °C in EMEM containing Hoechst 33342 before (d) and after (e) 2 min UV exposure at 365 nm. (f, g) Micrographs recorded upon excitation at 488 nm (λ_obs = 500–530 nm) before (f) and after (g) two-photon irradiation at 710 nm. The white dashed circle in (f) represents the irradiated area. Scale bars = 20 μm.

 

Figure 5 shows confocal laser scanning micrographs of Hep3B cells upon excitation at 405 nm (λ_obs = 430–520 nm, blue) and 488 nm (λ_obs = 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 containing Hoechst 33342 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.

 

Reference

Okuro, K.; Kinbara, K.; Tsumoto, K.; Ishii, N.; Aida, T. J. Am. Chem. Soc. 2009, 131, 1626−1627.

DOI: 10.1021/ja800491v

Hansen, M. J.; Velema, W. A.; Lerch, M. M.; Szymanski, W.; Feringa, B. L. Chem. Soc. Rev. 2015, 44, 3358−3377.

DOI: http://10.1039/C5CS00118H