Overcoming Resistance of Caco-2 Cells to 5-Fluorouracil through Diruthenium Complex Encapsulation in PMMA Nanoparticles

Abstract

Drug resistance, one of the main drawbacks in cancer chemotherapy, can be tackled by employing a combination of drugs that target different biological processes in the cell, enhancing the therapeutic efficacy. Herein, we report the synthesis and characterization of a new paddlewheel diruthenium complex that includes 5-fluorouracil (5-FU), a commonly used anticancer drug. This drug was functionalized with a carboxylate group to take advantage of the previously demonstrated release capacity of carboxylate ligands from the diruthenium core. The resulting hydrophobic complex, [Ru₂Cl(DPhF)₃(5-FUA)] (Ru-5-FUA) (DPhF = N,N'-diphenylformamidinate; 5-FUA = 5-fluorouracil-1-acetate) was subsequently entrapped in poly(methyl methacrylate) (PMMA) nanoparticles (PMMA@Ru-5-FUA) via a reprecipitation method to be transported in biological media. The optimized encapsulation procedure yielded particles with an average size of 81.2 nm, a PDI of 0.11, and a zeta potential of 29.2 mV. The cytotoxicity of the particles was tested in vitro using the human colon carcinoma cell line Caco-2. The IC₅₀ (half maximal inhibitory concentration) of PMMA@Ru-5-FUA (6.08 μM) was just slightly lower than that found for the drug 5-FU (7.64 μM). Most importantly, while cells seemed to have developed drug resistance against 5-FU, PMMA@Ru-5-FUA showed an almost complete lethality at ∼30 μM. Conversely, an analogous diruthenium complex devoid of the 5-FU moiety, [Ru₂Cl(DPhF)₃(O₂CCH₃)] (PMMA@RuA), displayed a reduced cytotoxicity at equivalent concentrations. These findings highlight the effect of combining the anticancer properties of 5-FU with those of diruthenium species. This suggests that the distinct modes of action of the two chemical species are crucial for overcoming drug resistance.

Scheme 1. Schematic Synthesis of [Ru₂Cl(DPhF)₃(5-FUA)] (Ru-5-FUA) (DPhF = N,N′-Diphenylformamidinate; 5-FUA = 5-Fluorouracil-1-acetate)

Figure 1

View of the asymmetric unit of Ru-5-FUA·0.5THF with selected atoms labeled. Ru atoms are shown in pink, N atoms in blue, O atoms in red, Cl atom in green, C atoms in gray, F atoms in yellow, and H atoms in white. Crystallization solvent molecules have been omitted for clarity.

Figure 2

Intermolecular interactions found in the crystal structure of Ru-5-FUA·0.5THF.

Figure 3

DLS analysis for PMMA@Ru-5-FUA particles obtained via method 1 under (a) droplet stabilization at 20 °C and a fast THF evaporation process, (b) droplet stabilization at 20 °C and a slow THF evaporation process, and (c) droplet stabilization at 4 °C and a slow THF evaporation process.

Figure 4

(a) DLS number size distributions for the PMMA@Ru-5-FUA particles prepared via method 1 (squares) under different conditions and method 2 (triangles). (b) DLS spectra registered for nonloaded PMMA and PMMA@Ru-5-FUA particles obtained via method 1 under droplet stabilization at 20 °C, followed by slow THF evaporation at room temperature. The inset displays images of both aqueous dispersions, showing the Tyndall effect.

Figure 5

DLS analysis for the aqueous dispersion of PMMA@Ru-5-FUA nanoparticles prepared in ultrapure Milli-Q water.

Figure 6

(a, b) TEM microphotographs showing the PMMA@Ru-5-FUA nanoparticles. (c) Particle size distribution histogram. The average diameter (D_m), standard deviation (S_D) and number of particles (N) used for TEM analysis are also indicated.

Figure 7

Semilogarithmic representation of Caco-2 cell viability after 72 h of incubation in the presence of the indicated compounds. IC₅₀ values are indicated. Values corresponding to 5-FU and PMMA@Ru-5-FUA are the average ± SD of 5 different assays whereas PMMA and PMMA@RuA was only assayed twice.

Figure 8

Confocal images of Caco-2 cells exposed to PMMA nanoparticles containing coumarin 6 after 1, 4, and 24 h.