Comparing organic and metallo-organic hydrazone molecular cages as potential carriers for doxorubicin delivery

Abstract

Molecular cages are three-dimensional supramolecular structures that completely wrap guest molecules by encapsulation. We describe a rare comparative study between a metallo-organic cage and a fully organic analogous system, obtained by hydrazone bond formation self-assembly. Both cages are able to encapsulate the anticancer drug doxorubicin, with the organic cage forming a 1 : 1 inclusion complex with μM affinity, whereas the metallo-organic host experiences disassembly by interaction with the drug. Stability experiments reveal that the ligands of the metallo-organic cage are displaced in buffer at neutral, acidic, and basic pH, while the organic cage only disassembles under acidic conditions. Notably, the organic cage also shows minimal cell toxicity, even at high doses, whilst the doxorubicin-cage complex shows in vitro anti-cancer activity. Collectively, these results show that the attributes of the pure organic molecular cage are suitable for the future challenges of in vivo drug delivery using molecular cages.

Fig. 1. Synthesis of molecular cages containing hydrazone bonds showing the main objective of the work focused on the comparison of properties of a metallo-cage with a fully organic cage.

Fig. 2. Self-assembly of molecular cages C1 and C2 by hydrazone bond formation. The figure shows X-ray crystal structure of C1·NO₃ cage (counteranions removed for clarity) while the C2 cage structure was obtained from Spartan MMFF molecular modelling (see ESI for X-ray and molecular modelling details).

Fig. 3. Fluorescence titration experiments. (a) Schematic representation of drug encapsulation; (b) binding data for the encapsulation of DOXO by cage C2 obtained from the fluorescence spectra for the addition of C2 to DOXO (50 μM) in 100 μM phosphate buffer, pH 7.2. The solid points are experimental data, and the continuous lines is the fitted binding isotherm for a 1 : 1 host–guest model, obtained as the average of 4 independent titrations (vertical bars show the standard error for each point). Fluorescence measured at 590 nm with λ_exc = 470 nm (phosphate buffer 100 μM/DMSO 0.25–2.0%, pH 7.2); (c) MMFF molecular model of the supramolecular complex [DOXO⊂C2] obtained with the Spartan'20 software.

Fig. 4. DOXO release experiments. (a) Schematic representation of DOXO release by competition with DMSO molecules. (b) Plot of recovery of non-bound DOXO versus percentage of added DMSO (6–55%) to the solution containing C2 (C_initial = 50 μM, C_final = 25 μM) and DOXO (C_initial = 50 μM, C_final = 25 μM) in phosphate buffer (1 mM, pH 7.2). The green dot indicates the release at 2% DMSO as determined in the binding experiments of Fig. 3. The blue line indicates the expected release changes by dilution from 50 μM to 25 μM in a solution containing 2% DMSO.

Fig. 5. Stability of cages C1·NO₃ (10 μM, (a–c) 0, 1, 2, 4 days) and C2 (10 μM, (d–f) 0, 1, 2, and 3 days) at different pH values over time in a buffered solution with phosphate (100 μM) and 1% DMSO. Absorbance spectra of building block components (pH 7.1 buffered solution with phosphate (100 μM) with 5% DMSO): (g) linker 1 (25 μM). (h) Pd(ii) complex 2·NO₃ (50 μM). (i) Cavitand 3 (25 μM). (j) Ligand S2 (20 μM).

Fig. 6. ¹H NMR disassembly experiment of C1·NO₃ in DMSO-d₆ (0.5 mM, 550 μL) with phosphate buffer (pH 7.2, 100 mM, 50 μL). (a) ¹H NMR spectra of cage C1·NO₃. (b) ¹H NMR spectrum after the addition of phosphate buffer (ca. 5 min), causing cage disassembly. (c) ¹H NMR spectra of free ligand (see structure S2 in ESI†).

Fig. 7. Cell viability measured by WST-1. Data represented as mean ± SEM (n = 3). Toxicity assay with organic cage C2 (yellow) and palladium metallo-organic cage C1·NO₃ (blue) on: (a) 4T1 cells and (b) SK-Mel-103 cells. Treatment with DOXO⊂C2 complex (black) and free doxorubicin (red) on: (c) 4T1 cells and (d) SK-Mel-103 cells. Increasing concentrations of DOXO and a fixed concentration of organic cage C2 (25 μM) was used to obtain 95% encapsulation of DOXO. (e) Time-lapse confocal images of SK-Mel-103 cells incubated with Hoechst (blue nuclei marker), WGA (green membrane marker) and treated with the DOXO⊂C2 complex (fluorescent red) at 5 μM up to 10 min. Scale bar represents 20 μm.