Metal-Organic Polyhedron Capped with Cucurbit[8]uril Delivers Doxorubicin to Cancer Cells

Abstract

Self-assembly of ligand 1 and Pd(NO₃)₂ delivers Fujita-type metal-organic polyhedron (MOP) 3 which bears 24 covalently attached methyl viologen units on its external surface, as evidenced by ¹H NMR, diffusion-ordered spectroscopy NMR, electrospray mass spectrometry, transmission electron microscopy, and atomic force microscopy measurements. MOP 3 undergoes noncovalent complexation with cucurbit[n]urils to yield MOPs 4-6 with diameter ≈5-6 nm. MOP 5 can be fully loaded with doxorubicin (DOX) prodrug 2 via hetero-ternary complex formation to yield 7. The MOPs exhibit excellent stability toward neutral to slightly acidic pH in 10 mM sodium phosphate buffer, mitigating the concern of disassembly during circulation. The results of MTS assays show that MOP 7 is 10-fold more cytotoxic toward HeLa cells than equimolar quantities of DOX prodrug 2. The enhanced cytotoxicity can be traced to a combination of enhanced cellular uptake of 7 and DOX release as demonstrated by flow cytometry and confocal fluorescence microscopy. The confluence of properties imparted by the polycationic MOP architecture and plug-and-play CB[n] complexation provides a potent new platform for drug delivery application.

Figure 1

a) Self-assembly of metal organic polyhedron 3 in DMSO and non-covalent capping with CB[7] to yield MOP 4 in D₂O. The depicted structures were rendered based on the MMFF minimized structures. ¹H NMR spectra recorded (600 MHz, DMSO-d₆, RT) for b) 1 and c) 3. 2D ¹H DOSY NMR spectra recorded (600 MHz, DMSO-d₆, RT) for: d) 3 and e) 1.

Figure 2

¹H NMR spectra recorded (600 MHz, D2O, RT) for: a) 3, b) 4 and c) 4 with 24 equiv. of ADA. 2D ¹H DOSY NMR spectra recorded (600 MHz, D2O, RT) for d) 3, and e) 4.

Figure 3

a) Sequential self-assembly of the methyl viologen units of MOP 3 as first guest with CB[8] to yield MOP 5 followed by hetero ternary complex formation with HN or 2 to yield MOP 6 and MOP 7. ¹H NMR recorded (600 MHz, D₂O, RT) for: b) 3, c) 5, d) 6, and e) 7.

Figure 4

a) TEM image of 5 and the statistical distribution of particle size of 5. b) AFM image of 5 and its height profile. c) UV-Vis titration of 5 (20 μM) with HN (0 – 500 μM) in water. d) Plot of change in fluorescence intensity at λ_max = 597 nm versus time after incubating 2 in buffer at pH 7.4, 6.5, 5.5, and 4.0.

Figure 5

(a) DOSY NMR of MOP 7 in sodium phosphate buffer (10 mM) at pH 7.4. (b) Zeta potential of MOP 3, MOP 4, MOP 5 & MOP 7. (c) TEM image of MOP 5 at pH 5.5. (c) AFM image of MOP 5 at pH 5.5 and its height profile (1 & 2).

Figure 6

a) Plot of normalized mean fluorescence intensity (MFI) versus incubation time derived from flow cytometry experiments (N = 4) for HeLa cells treated with 2 (1 μM, o) or 7 ([2] = 1 μM, ■) for 1, 3, 6 or 12 h. b) Confocal fluorescence microscopy of HeLa cells treated with 2 (5 μM) or 7 ([2] = 5 μM) at 40× magnification. Scale bar = 30 μm. c) Results of MTS assay for HeLa cells treated with 2 (o) or 7 (■). Curve fitting of cytotoxicity data for 2 (−••−) and 7 (•••) yielded EC₅₀ values of 500 ± 110 nM and 48 ± 8 nM, respectively (N = 15). *p < 0.05, **p < 0.01, ***p < 0.001.

Scheme 1

Synthesis of 1 and 2. Conditions: (a) ethyl-4-bromobutyrate, CH₃CN, 70 °C, 4 d, 40%; (b) 2 M HBr, H₂O, 2 d, NH₄PF₆, 80%; (c) Pd(PPh₃)₄, K₃PO₄, H₂O/1,4-dioxane (1:1), 80 °C, 4 d, 72%; (d) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, DMAP, DMF, 2 d, (n-Bu)₄NNO₃, CH₃CN, 30%; e) Ethyl-5-bromovalerate, K₂CO₃, CH₃CN, 70 °C, 24 h, 82%; f) LiOH, THF/H₂O (1:1), 70 °C, 24 h, 1 M HCl, 90%; g) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 1-hydroxybenzotriazole, CH₃CN, 3 h, N₂H₄ in CH₃CN 0‒10 °C, 3 h, 65%; h) Doxorubicin hydrochloride, trifluoroacetic acid, MeOH, 12 h, room temperature, 78%.

Chart 1

Chemical structures of compounds used in this study.