Reconstitution of microtubule into GTP-responsive nanocapsules

Abstract

Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 °C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.

Fig. 1. Strategy used to prepare THD-based GTP-responsive ^CLNC_GTP/GTP*.

a Schematic illustrations of tubulin heterodimers (THDs) hybridized with GTP (THD_GTP), its nonhydrolysable analogue GTP* (THD_GTP*), and GDP (THD_GDP) at its β-tubulin unit. b Schematic illustration of two self-assembling modes of THD into microtubules (MTs). MT_GTP depolymerizes into THD_GDP upon GTP hydrolysis. THD_GDP rehybridizes with GTP after a GTP treatment, facilitating the formation of MT_GTP. In contrast, MT_GTP* does not undergo depolymerization. c Molecular structures of photoreactive molecular glues (${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$, ${\mathrm{Glue}}^{{\mathrm{CO}}_2-\mathrm{Me}}$, and Glue^FITC) bearing three guanidinium ions (Gu⁺) and benzophenone (BP) groups at their periphery and ${\mathrm{CO}}_2-$, CO₂ Me, and FITC groups at the focal core. d The molecular glue covalently binds to the protein surface at its photoexcited BP groups after the noncovalent adhesion via a Gu⁺/oxyanion multivalent salt-bridge interaction. e Schematic illustration of the multistep procedure for the synthesis of crosslinked nanocapsules (^CLNC_GTP/GTP*) from MT_GTP. MT_GTP is depolymerized into THD_GDP, which is incubated with a mixture of GTP* (83 mol%) and GTP (17 mol%) to form nanosheet NS_GTP/GTP*. Upon treatment with ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$, NS_GTP/GTP* is transformed into spherical nanocapsules (NC_GTP/GTP*), which are further exposed to UV light, affording ^CLNC_GTP/GTP*. Upon addition of GTP, ^CLNC_GTP/GTP* collapses through the conformational change of the THD units induced by GTP hydrolysis.

Fig. 2. Reconstitution of MT into ^CLNC_GTP/GTP*.

a A typical synthetic procedure for the preparation of ^CLNC_GTP/GTP*. b DLS profiles of MT_GTP (gray), THD_GDP (blue), NS_GTP/GTP* (green), NC_GTP/GTP* (orange), and ^CLNC_GTP/GTP* (red) in PIPES buffer. c–e TEM images of MT_GTP (5.8 mg ml^–1; c), THD_GDP (0.3 mg ml^–1; d), and NS_GTP/GTP* (0.3 mg ml^–1; e). f AFM image of NS_GTP/GTP* (0.3 mg ml^–1) and its height profile. g, h TEM images of NC_GTP/GTP* (13 µg ml^–1; g) and ^CLNC_GTP/GTP* (13 µg ml^–1; h). All TEM samples were negatively stained with uranyl acetate. Inset scale bars, 250 nm.

Fig. 3. MD simulation of the adhesion events of Glue^CO2– onto the surface of THD_GTP*.

a Three laterally assembled THD_GTP* units ([THD_GTP*]₃) in MT_GTP* as a partial model of NS. b An equilibrated MD snapshot of ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$. c, d The outer (c) and inner (d) views of [THD_GTP*]₃ hybridized with 30 equivalents of ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$. e, f The outer (e) and inner (f) views of [THD_GTP*]₃ with its electrostatic surface potential in the absence (upper) and presence (lower) of 30 equivalents of hybridized ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$. Negative and positive potential areas are colored in red and blue, respectively. g The percentage of hydrophobic solvent-accessible surface area in the absence (47.5 ± 0.5; red) and presence (56.7 ± 2.0; blue) of 30 equivalents of hybridized ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$. Bars represent mean values ± SD from 2000 data points. h, i [THD_GTP*]₃ observed from the top view (h) and its angle distributions (i) in the absence (red) and presence (blue) of 30 equivalents of hybridized ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$. j Radial distribution functions g(r) of the Gu⁺ groups in ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$ with carboxylates (blue) and non-ionic hydroxyl groups (gray) on the [THD_GTP*]₃ surface, and the carboxylate at the focal core of ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$ (red). k Schematic illustration of a possible adhesion event of ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$ onto NS_GTP/GTP* and its effects on the features of NS_GTP/GTP*. The Gu⁺ groups in ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$ form a salt bridge with carboxylates on the NS_GTP/GTP* surface and at the focal core of ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$, and the ${\mathrm{Glue}}^{{\mathrm{CO}}_2-}$-based polymeric network thus formed through this process increases the hydrophobicity of the NS_GTP/GTP* surface, making NS_GTP/GTP* more flatten.

Fig. 4. GTP-triggered collapse of ^CLNC_GTP/GTP*.

a, b TEM images of ^CLNC_GTP/GTP* after a 100-min incubation with GTP at its concentrations of 0.2 mM (a) and 0.5 mM (b). c DLS profiles of ^CLNC_GTP/GTP* (8.7 µg ml^–1) in PIPES buffer after a 100-min incubation with GTP at its concentrations of 0 mM (red), 0.2 mM (orange), 0.5 mM (green), and 1 mM (blue). d GTPase activities of THD_GDP (left) and ^CLNC_GTP/GTP* (right) in PIPES buffer. The data was obtained from three biologically independent samples (n = 3). e DLS profiles of ^CLNC_GTP/GTP* (8.7 µg ml^–1) in PIPES buffer after a 100-min incubation with 1 mM of ATP (red), CTP (orange), and UTP (green). f TEM image of ^CLNC_GTP/GTP*⊃NP_Au ([^CLNC_GTP/GTP*] = 13 µg ml^–1, [NP_Au] = 13 pM). g CLSM images of FITC-labeled ^CLNC_GTP/GTP*⊃DOX ([^CLNC_GTP/GTP*] = 13 µg ml^–1, [DOX] = 10 µM) incubated without (upper panel) and with (lower panel) 1 mM GTP at 37 °C for 100 min. Micrographs display locations of FITC (i, green) and DOX (ii, red), and their merged images (iii). Scale bars, 2.0 µm. h Fluorescence intensities at 590 nm (λ_ext = 470 nm) of residual DOX obtained after 20, 50, and 100-min incubations of a PIPES solution of ^CLNC_GTP/GTP*⊃DOX with 1 mM GTP, followed by ultrafiltration. Red bars represent mean values ± SD from three different samples.

Fig. 5. Intracellular drug delivery using ^CLNC_GTP/GTP*.

a Schematic illustration of the uptake of FITC-labeled ^CLNC_GTP/GTP* into Hep3B cells. b Bright field (upper row) and CLSM images displaying FITC (middle row, green) in Hep3B cells and their merged images (lower row). The cells were incubated in EMEM containing ^CLNC_GTP/GTP* (0.5 µg ml^–1) for 2.5 h, rinsed with D-PBS, and further incubated in EMEM (10% FBS) for 1.5 h (i) and 21.5 h (ii). Scale bars, 20 µm. c Flow cytometry profiles showing FITC fluorescence of Hep3B cells (n > 660) incubated without (blue) and with FITC-labeled ^CLNC_GTP/GTP* for 2.5 h, rinsed with D-PBS, and further incubated in EMEM (10% FBS) for 1.5 h (i, orange) and 21.5 h (ii, green). d Schematic illustration of the cellular uptake of ^CLNC_GTP/GTP*⊃DOX. e Bright field (upper row) and CLSM images displaying DOX (middle row, red) in Hep3B cells and their merged images (lower row). The cells were incubated in EMEM containing ^CLNC_GTP/GTP*⊃DOX ([^CLNC_GTP/GTP*] = 2.6 µg ml^–1, [DOX] = 2 µM) for 2.5 h, rinsed with D-PBS, and further incubated in EMEM (10% FBS) for 1.5 h (iii) and 21.5 h (iv). Scale bars, 20 µm. f, g Flow cytometry profiles (f) showing DOX fluorescence of Hep3B cells (n > 390) and their normalized viabilities (g) determined using Cell Counting Kit-8 (n = 3). The cells were incubated without (blue) and with DOX (2 µM; orange), and ^CLNC_GTP/GTP*⊃DOX ([^CLNC_GTP/GTP*] = 2.6 µg ml^–1, [DOX] = 2 µM; red) for 2.5 h in EMEM, and then rinsed with D-PBS, followed by incubation in EMEM (10% FBS) for 21.5 h. Statistical significance was examined by two-sided Student’s t test (*p = 0.0094 < 0.01). Bars represent mean values ± SD from three different samples.