Dichloroacetic acid (DCA) is a pyruvate D-kinase inhibitor that is overexpressed in cancer cells. Its cytotoxic effect on cancer cells depends on effective cytoplasmic release and mitochondrial localization, making it an ideal probe molecule for cellular uptake mechanism . In order to promote cytoplasmic release through passive diffusion, UiO-66-NH2 (DCA-UiO-66-NH2) doped with dichloroacetic acid (DCA) was synthesized by heating N, N’-dimethylformamide (DMF) solution in the oven at 120 °C for 24 h, containing Zr4+, 2-amino-1,4-benzenedicarboxylicacid (NH2-H2BDC) ligands, and dichloroacetic acid, respectively, with gently stirring. In the synthesis of the DCA-UiO-66-NH2, different ratios of metal ions and ligands were used to obtain the optimal nanoscale products. As a regulator of UiO-66-NH2 solvothermal synthesis, it showed that DCA was incorporated into the defects of UiO-66-NH2 nanoparticles and their surfaces, as shown in Additional file 1: Fig. S1. For DCA-UiO-66-NH2, when the molar ratio of Zr4+, NH2-H2BDC, and DCA was 1:1:9, the particle size of the obtained product was around 50 nm, as characterized by transmission electron microscopy (TEM) (see Additional file 1: Fig. S1a), which is superior to most MOF-based targeted drug carriers reported in the literature. In order to prove the surface chemistry and structure of the synthesized DCA-UiO-66-NH2, Fourier-transform infrared spectroscopy (FT-IR) and powder X-ray diffraction (PXRD) analysis were performed. The FT-IR spectra of DCA-UiO-66-NH2 showed a new band in the carboxylic acid region but shifted compared to free DCA, which was characteristic of the carboxylic acid in DCA that connected to the Zr6 units. The presence of a new band related to the C–Cl stretching at around 800 cm−1 was appreciable, and no shifting was observed (Additional file 1: Fig. S2). Furthermore, their PXRD patterns closely matched the simulated patterns derived from the single-crystal X-ray diffraction data, which proved the successful synthesis of the DCA-UiO-66-NH2 (Fig. 2).
The covalent post-synthetic modification of DCA-UiO-66-NH2 was explored by using an alkyl anhydride to produce carboxylic group terminal frameworks designated DCA-UiO-DTDP (in Additional file 1: Fig. S3). According to the previous literature in Additional file 1: Fig. S4, 3,3′-dithiodipropionic acid anhydride (DTDPA) was obtained by acylation of 3,3′-dithiodipropionic (DTDP) acid with acetyl chloride . The resonance at δ = 12.35 ppm corresponded to the carboxyl proton of the DTDP, and the peak disappeared after refluxing in acetyl chloride, which indicated the successful formation of DTDPA in 1H NMR in Additional file 1: Fig. S5. In a typical post-synthetic modification reaction, after the synthesis of DCA-UiO-66-NH2, 1:1 molar ratio of DTDPA was placed in the above DMF solution. After standing at room temperature for 24 h, the sample was rinsed with methanol to extract byproducts from the porous solids. The modification was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The sample of post-synthetic modification treated DCA-UiO-66-NH2 materials was digested with D2SO4 and DMSO-d6 for examination by 1H NMR spectroscopy. The digestion of unmodified DCA-UiO-66-NH2 mainly showed resonances related to 2-amino-1,4-benzene dicarboxylic acid, and the resonance at δ = 6.2 ppm corresponded to the –CCl2H proton of DCA in Additional file 1: Fig. S6. It is not possible to quantitatively determine the DCA loading value from the NMR spectra alone. However, according to the external standard method of 1H NMR standard curve of DCA in Additional file 1: Fig. S6, from the 1H NMR spectra of acid digested DCA-UiO-66-NH2 samples, it was estimated that the incorporation amount of DCA was 17.96%. At the same time, some minor impurities were also observed in the aromatic region of DCA-UiO-DTDP, which seemed to be associated with the amino-functionalized benzenedicarboxylate (BDC) ligand (Additional file 1: Fig. S7). The TEM in Fig. 1b and XRD in Fig. 2 confirmed that there was no significant change in the morphology and crystallinity of DCA-UiO-DTDP nanoparticles after modification with anhydrate substance.
Next, we tried to establish an active targeting model on the MOFs platform by performing post-synthetic modification on the modified surfaces of DCA-UiO-DTDP. Since there are available binding sites on the surface of DCA-UiO-DTDP, the terminal amino group of aminated folic acid (NH2-FA) has a great chance to be coordinated with the ligands on the surface of DCA-UiO-DTDP. Additional file 1: Fig. S8 shows the synthesis procedure of aminated folic acid between folic acid and ethylenediamine . The obtained NH2-FA was analyzed in Additional file 1: Fig. S9 by using 1H NMR. The NH2-FA-modified DCA-UiO-DTDP was prepared by stirring a solution containing DCA-UiO-DTDP and aminated folic acid at room temperature for 6 h. The comparison of the FT-IR spectra of DCA-UiO-DTDP and DCA-UiO-DTDP-FA showed that the absorption peaks of FA had a strong contribution to the FT-IR spectra of DCA-UiO-DTDP-FA (Additional file 1: Fig. S10), which indicated that the reactions were successful. In contrast, the PXRD patterns of the DCA-UiO-DTDP-FA and its parent MOF did not show any differences, demonstrating the integrity of their frameworks after the reaction (Fig. 2). After the anchoring with the NH2-FA, a thin and uniform layer was formed on the surface of DCA-UiO-66-DTDP crystals, and its thickness was about several nanometers, as shown in Fig. 1c.
For biological applications, the biocompatibility and cytotoxicity of the anticancer drugs and prepared materials must be evaluated, and here we have used the cell proliferation assay Roche diagnostics reagent (WST-1). The cytotoxicity of the DCA-UiO-DTDP, DCA-UiO-DTDP-FA and free DCA (in the form of sodium dichloroacetate, NaDCA, eliminating cytotoxic effects caused by pH changes) were measured towards breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A. The WST-1 assay results for NaDCA are shown in Additional file 1: Fig. S11, confirming that DCA has little effect on cell proliferation due to its natural hydrophilic properties leading to poor internalization, and a decrease in cell viability was observed only at concentrations higher than 2 mg/mL, with 69.5 ± 9.9% and 91.0 ± 12.6% viabilities after incubation of NaDCA in MDA-MB-231 and MCF-10A, respectively. Then, the cell viability was measured by the WST-1 assay for 72 h with different concentrations of the DCA-UiO-DTDP and DCA-UiO-DTDP-FA (10, 20, 50, 100, 200, 500, and 1000 μg/mL). As can be seen from Additional file 1: Fig. S12, even at concentrations as high as 1000 μg/mL, the samples showed high viability over the entire concentration range. When MDA-MB-231 cancer cells were incubated with various concentrations of DCA-UiO-DTDP and DCA-UiO-DTDP-FA, the nanocomposites showed little toxicity to concentrations up to 500 μg/mL, and cell viability was about 80%, even with the active targeting capability of DCA-UiO-DTDP-FA. Moreover, the incubation of DCA-UiO-DTDP and DCA-UiO-DTDP-FA with MCF-10A cells revealed that the DCA-UiO-DTDP-FA had no effect on normal cell proliferation in vitro. These data demonstrate that DCA-UiO-DTDP-FA shows excellent biocompatibility in vitro.
According to the report, DCA can enhance the cytotoxic activity of anticancer drugs such as 5-fluorouracil (5-FU) and reducing the drug resistance of cancer cells. We selected 5-FU as a typical anticancer drug to evaluate drug loading and release behaviors and the enhanced cytotoxicity ability of 5-FU-DCA-UiO-DTDP-FA. DCA-UiO-DTDP-FA and 5-FU were used for drug loading experiments in an excess of 5-FU in methanol, and the concentration of 5-FU was measured after various concentration ratios between nanoparticles and drug (see Additional file 1: Table S1). The UV/Vis absorbance of the solution was measured at regular interval to determine the amount of loading of 5-FU on the DCA-UiO-DTDP-FA based on the UV/Vis absorbance and the standard absorbance curve of 5-FU in Additional file 1: Fig. S13. The results of the 5-FU loading experiments confirmed that the loading efficiency of DCA-UiO-DTDP-FA reached 31.6 wt% after 24 h.
The release of 5-FU from the 5-FU-DCA-UiO-DTDP-FA nanoparticles was investigated in the presence of dithiothreitol (DTT, a reducing agent that mimics the action of GSH, which is in the microenvironment of cancer cells provides a reducing environment.). It was assumed that the release of 5-FU from the nanoparticles occurs due to the breakage of disulfide bonds. DTDPA modified DCA-UiO-66-NH2 nanoparticles can form disulfide bonds with organic ligands on the surface of the nanoparticles, and then coordinate with NH2-FA to limit drug leakage as a gate. GSH and its oxidized form (GSSG) are responsible for the formation of intracellular redox buffer . Intracellular GSH attacks the thiolate moiety and is oxidized in the process as it cleaves the existing disulfide bonds. The encapsulation and release of the drug from the DCA-UiO-DTDP-FA were investigated at 37 °C with or without DTT. As shown in Fig. 3a, the release curve indicates that about 80% of 5-FU was released from the nanoparticles after 24 h in the presence of 10 mM DTT. This result was attributed to the higher number of disulfide bonds that were cleaved in the presence of high concentration of DTT. Compared to the control experiment without DTT, limited drug was released within 24 h. The results showed that 5-FU was rapidly released from the nanoparticles in excess of DTT, mimicking the GSH present in cancer cells and the stability in plasma. The above results are of great significance to anticancer drug delivery systems based on post-synthetic modified stimuli-responsive MOFs nanoparticles.
Free 5-FU itself has significant dose-responsive cytotoxic behavior not only on cancer cells but also on normal cells (in Fig. 3b and Additional file 1: S14), but the cytotoxicity of the 5-FU-DCA-UiO-DTDP-FA samples showed higher toxicity to MDA-MB-231 cancer cells compared to 5-FU at same concentration in Fig. 3b. This may be the result of more efficient cell internalization of the nanoparticles with FA conjugation, or the DCA in the MOF structure had synergistic effect with 5-FU. To confirm this, we incubated the cells with 5-FU and NaDCA pure drugs mixed solution. Interestingly, the addition of DCA had enhanced the 5-FU effect, while the pure drug combination was still less efficiency comparing to the nanoparticles. 5-FU-DCA-UiO-DTDP-FA showed a greater apparent cytotoxic effect in all tested concentrations, in comparison to free 5-FU and free 5-FU + DCA. In the case of MCF-10A cell, as shown in Additional file 1: Fig. S14, the nanoparticles had limited cytotoxicity, which proved its selectivity towards cancer cells to normal cells.
To access the cellular uptake of DCA-UiO-DTDP-FA and DCA-UiO-DTDP, confocal laser scanning microscopy (CLSM) images were recorded for MDA-MB-231 cancer cells and MCF-10A cells incubated with the nanocomposites for 1, 6, and 16 h at 37 °C respectively, as shown in Figs. 4 and 6. For each figure, the cell nucleus is stained with 4′,6-diamidino-2-phenylindole (DAPI), emits blue fluorescence, the DCA-UiO-DTDP-FA and DCA-UiO-DTDP was labeled with Sulfo-Cyanine5.5 NHS ester (Sulfo-Cy5.5) that has red fluorescence. In Fig. 4a, almost no red emission was observed in the first 1–6 h, which indicated that very few DCA-UiO-DTDP-FA nanoparticles were phagocytized by MDA-MB-231 cancer cells. The same results were also shown in the DCA-UiO-DTDP nanoparticles group in Fig. 4b. However, when the incubation time was extended to 16 h, compared with the same time in Fig. 4b, much stronger Sulfo-Cy5.5 red fluorescence emission was observed in the cytoplasm and cell nucleus in DCA-UiO-DTDP-FA group (Fig. 4a) comparing to DCA-UiO-DTDP group, which indicated that more NH2-FA-modified nanoparticles were uptaken by MDA-MB-231 cancer cells. The same results can also be seen from the mean fluorescent intensity (MFI) of the different cellular populations treated with different groups (Fig. 5). The MFI of the DCA-UiO-DTDP-FA group increased significantly, which was much higher than that of the DCA-UiO-DTDP group. Meanwhile, no significant red emission was observed during the entire uptaking process in MCF-10 cells (Fig. 6), which means that the two types of nanoparticles were indiscriminately consumed by normal cells and the passive uptake of normal cells was also less active than cancer cells. These results confirm that the prepared nanoparticles can be effectively phagocytized by MDA-MB-231 cancer cells through receptor-mediated endocytosis, thereby achieving the tumor cells targeting.