Redox-Responsive Tumor Targeted Dual-Drug Loaded Biocompatible Metal-Organic Frameworks for Enhancing Anticancer Cytotoxicity

Metal-organic frameworks (MOFs) have proven to be a promising class of drug carriers due to their high porosity, crystalline properties with dened structure information, and their potential for further functionalization. However, to date, no extensive research has been conducted on MOF-based drug carriers with stimuli-responsive, dual-drug delivery, and tumor targeting functions. Here, we demonstrate the strategy of constructing a redox responsive and tumor-targeted MOF, as dual-drug carrier, by anchoring functional disulde anhydride and folic acid (FA) molecules to the organic links of MOFs, respectively. The MOF composites show the controlled release of loaded 5-uorouracil (5-FU) entrapped within UiO-66-NH 2 nanostructures modied by dichloroacetic acid (DCA). Moreover, the MOF building block DCA acts as a synergistical drug to 5-FU in cancer cells inhibition. Through disulde bonds, the gated MOF has redox-responsive drugs release. The confocal laser scanning microscopy further proved that conjugation of folic acid to the MOF surface can signicantly enhance the targeting ability to cancer cells and the cancer cell uptake of FA-MOFs. The synthesis of redox-responsive dual-drug delivery MOF hybrids paves the way to assemble of other MOF hybrids that respond to other triggering factors such as light, temperature, pH, or biomarkers. The properties and functions of such materials are expected to inuence the development of sensors, new catalysts, photonic devices, and drug delivery carriers. NHS were added to activate the carboxyl groups of DCA-UiO-DTDP. After 6 hours, 5 mg of aminated FA was added to the solution. 5-FU-DCA-UiO-DTDP-FA were collected by centrifugation (12000 r/min for 20 min) and washed with MeOH (x2) by dispersive centrifugation cycles to ensure that there is no residual 5-FU on the particle surfaces. The resulting MOFs were dried under vacuum at least 24 hours before analysis. The amount of 5-FU adsorption in the porous solids was estimated by UV-Vis. The wavelengths used for 5-FU quantication was 266 nm. different periods up to 24 hours. After incubation at 37 ℃ cells suspension was diluted with 2 mL of PBS and centrifuged at 800 × g. The cell pellet was washed twice with PBS by centrifugation and resuspended in 500 µL of serum-free media. Fluorescence analysis was performed with BD LSRFORTESSA using Alexa Fluor 700-A channels and a 640 nm emission lter. MOFs: metal-organic frameworks; PSM: post-synthetic modication; TEM: transmission electron microscopy; FT-IR: Fourier-transform infrared spectroscopy; PXRD: powder X-ray diffraction; NMR: nuclear magnetic resonance spectroscopy; CLSM: confocal laser scanning microscopy; MFI: mean uorescent intensity; FA: folic acid; DCA: dichloroacetic acid; 5-FU: 5-uorouracil; DTDPA: 3,3’-dithiodipropionic acid anhydride.

nanoparticle-based therapy illustrates the power of this approach. Currently, many promising nanoparticles-drug combinations are at different stages of clinical development.
Metal-organic frameworks (MOFs), as a new type of porous and crystalline materials, assembled from metal ions (or metal clusters) and organic linkers, have shown various potential applications, such as adsorption of gas or water [19], catalysis [20], sensing [21], proton conduction [22], and drug delivery [23,24]. In the past few decades, nanoscale metal-organic frameworks have attracted great attention in the eld of particle-mediated cancer drug delivery [25]. They have shown advantages over conventional drug carrier materials, for example, they have various morphologies with well-de ned crystal structure, high surface area, large drug loading capacity, and intrinsic biodegradability caused by relatively unstable coordination bonds. Due to its hybrid nature related to organic ligands and inorganic building blocks, MOFs have tremendous opportunities for further customization and functionalization. In general, the organic components of MOFs are more concerned with the subsequent post-synthetic modi cation (PSM) [26], and organic molecules are covalently connected through various organic reactions, thereof providing new chemical functions for grafting of drug molecules. Through reasonable PSM, MOFs drug carriers can co-deliver two different chemical anticancer drugs and potentially stimuli-responsive release drugs to tumor cells because cancer cells have higher overexpressing reducing components than normal cells [27]. The fabrication of stimuli-responsive surfaces on MOFs for environmentally sensitive sitespeci c drug delivery can also reduce the risk of premature drug leakage. Interestingly, although some PSMs of MOFs have been reported, the combination of stimuli-responsive and targeting components to MOFs has not been explored. In addition to introducing stimuli-responsive/targeting MOF hybrids as a new functional material for targeting tumor and controlled drug release, the use of MOFs also has important advantages over other stimuli-responsive porous carriers. Furthermore, the formation of the MOFs determines a uniform pore size, and therefore, it is expected that the released loads will reveal a more controlled release pro le. The conjugation of anhydride acids with MOFs as stimuli-responsive capping units is particularly interesting because biological components can be used to unlock the chemical bonds of caps to achieve rapid drug release. Due to the high concentration of folic acid receptors in tumor tissues, it also binds to folic acid (FA) to build a targeting drug-delivery system [28].
In this study, we report the design of redox-responsive dual-drug delivery tumor-targeted MOFs nanocarriers (Scheme 1). Here, UiO-66-NH 2 was selected as the model as a drug carrier [29]. During the synthesis of nanoparticles, we introduced dichloroacetic acid (DCA) as part of the organic links fused in the framework to control the size and as a second anticancer drug to enhance the cancer cell cytotoxicity of the dual-drug delivery system. The amino groups on the surface of the nanoparticles were then modi ed with the 3,3'-dithiodipropionic acid anhydride (DTDPA). By entrapping 5-uorouracil (5-FU) in the DTDPA-modi ed MOFs, the resulting motif was then coordinated by aminated folic acid molecules to prevent drug leakage. The obtained dual-drug delivery MOFs is redox-responsive and can target cancer cells via folic acid. Furthermore, DCA can stimulate the 5-FU effect through synergistic.

Results And Discussion
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 [30]. In order to promote cytoplasmic release through passive diffusion, UiO-66-NH 2 (DCA-UiO-66-NH 2 ) doped with dichloroacetic acid (DCA) was synthesized by heating N, N'-dimethylformamide (DMF) solution in the oven at 120 °C for 24 hours, containing Zr 4+ , 2-amino-1,4-benzenedicarboxylicacid (NH 2 -H 2 BDC) ligands, and dichloroacetic acid, respectively, with gently stirring. In the synthesis of the DCA-UiO-66-NH 2 , different ratios of metal ions and ligands were used to obtain the optimal nanoscale products. As a regulator of UiO-66-NH 2 solvothermal synthesis, it showed that DCA was incorporated into the defects of UiO-66-NH 2 nanoparticles and their surfaces, as shown in Fig. S1. For DCA-UiO-66-NH 2 , when the molar ratio of Zr 4+ , NH 2 -H 2 BDC, 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 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-NH 2 , Fourier-transform infrared spectroscopy (FT-IR) and powder X-ray diffraction (PXRD) analysis were performed. The FT-IR spectra of DCA-UiO-66-NH 2 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 Zr 6 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 (. 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-NH 2 (Fig. 2).
The covalent post-synthetic modi cation of DCA-UiO-66-NH 2 was explored by using an alkyl anhydride to produce carboxylic group terminal frameworks designated DCA-UiO-DTDP (in Fig. S3). According to the previous literature in Fig. S4, 3,3'-dithiodipropionic acid anhydride (DTDPA) was obtained by acylation of 3,3'-dithiodipropionic (DTDP) acid with acetyl chloride [31]. The resonance at δ = 12.35 ppm corresponded to the carboxyl proton of the DTDP, and the peak disappeared after re uxing in acetyl chloride, which indicated the successful formation of DTDPA in 1 H NMR in Fig. S5. In a typical post-synthetic modi cation reaction, after the synthesis of DCA-UiO-66-NH 2 , 1:1 molar ratio of DTDPA was placed in the above DMF solution. After standing at room temperature for 24 hours, the sample was rinsed with methanol to extract byproducts from the porous solids. The modi cation was con rmed by nuclear magnetic resonance (NMR) spectroscopy. The sample of post-synthetic modi cation treated DCA-UiO-66-NH 2 materials was digested with D 2 SO 4 and DMSO-d 6 for examination by 1 H NMR spectroscopy. The digestion of unmodi ed DCA-UiO-66-NH 2 mainly showed resonances related to 2-amino-1,4-benzene dicarboxylic acid, and the resonance at δ = 6.2 ppm corresponded to the -CCl 2 H proton of DCA in 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 1 H NMR standard curve of DCA, from the 1 H NMR spectra of acid digested DCA-UiO-66-NH 2 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 (Fig. S7). The TEM in Fig. 1b and XRD in Fig. 2 con rmed that there was no signi cant change in the morphology and crystallinity of DCA-UiO-DTDP nanoparticles after modi cation with anhydrate substance.
Next, we tried to establish an active targeting model on the MOFs platform by performing post-synthetic modi cation on the modi ed 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 (NH 2 -FA) has a great chance to be coordinated with the ligands on the surface of DCA-UiO-DTDP. Fig. S8 shows the synthesis procedure of aminated folic acid between folic acid and ethylenediamine [32]. The obtained NH 2  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-uorouracil (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 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 Fig. S13. The results of the 5-FU loading experiments con rmed that the loading e ciency of DCA-UiO-DTDP-FA reached 31.6 wt % after 24 hours.
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 disul de bonds. DTDPA modi ed DCA-UiO-66-NH2 nanoparticles can form disul de bonds with organic ligands on the surface of the nanoparticles, and then coordinate with NH 2 -FA to limit drug leakage as a gate. GSH and its oxidized form (GSSG) are responsible for the formation of intracellular redox buffer [33]. Intracellular GSH attacks the thiolate moiety and is oxidized in the process as it cleaves the existing disul de 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 hours in the presence of 10 mM DTT. This result was attributed to the higher number of disul de 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 hours. 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 signi cance to anticancer drug delivery systems based on post-synthetic modi ed stimuli-responsive MOFs nanoparticles.
Free 5-FU itself has signi cant dose-responsive cytotoxic behavior not only on cancer cells but also on normal cells (in Fig. 3b and 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 e cient cell internalization of the nanoparticles with FA conjugation, or the DCA in the MOF structure had synergistic effect with 5-FU. To con rm 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 e ciency 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 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 hours at 37 ℃ respectively, as shown in Figs. 4 and 6. For each gure, the cell nucleus is stained with 4',6-diamidino-2-phenylindole (DAPI), emits blue uorescence, the DCA-UiO-DTDP-FA and DCA-UiO-DTDP was labeled with Sulfo-Cyanine5.5 NHS ester (Sulfo-Cy5.5) that has red uorescence. In Fig. 4a, almost no red emission was observed in the rst 1 to 6 hours, 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 hours, compared with the same time in Fig. 4b, much stronger Sulfo-Cy5.5 red uorescence 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 NH 2 -FA-modi ed nanoparticles were uptaken by MDA-MB-231 cancer cells. The same results can also be seen from the mean uorescent intensity (MFI) of the different cellular populations treated with different groups (Fig. 5). The MFI of the DCA-UiO-DTDP-FA group increased signi cantly, which was much higher than that of the DCA-UiO-DTDP group. Meanwhile, no signi cant 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 con rm that the prepared nanoparticles can be effectively phagocytized by MDA-MB-231 cancer cells through receptor-mediated endocytosis, thereby achieving the tumor cells targeting.

Conclusion
In this work, by incorporating DCA at defect sites of UiO-66-NH 2 , we have produced a MOF with high drug loading capacity. Then, we carried out two post-synthetic modi cation steps to functionalize the MOF with redox stimuli-responsive and tumor targeting through anchoring functional FA molecules to the disul de bond anhydrate modi ed surface of MOFs. By using the anticancer drug 5-FU as the model drug, the drug showed redox responsive release, which is relevant to cancer microenvironment. In addition, cell culture experiments have proved that the building block DCA in MOF structure can enhance the effect of 5-FU on cancer cell inhibition, moreover the 5-FU-DCA-UiO-DTDP-FA nanocomposite had enhanced cytotoxicity comparing to pure 5-FU + DCA mixture. More interestingly, the folic acid-modi cation on MOF had enhanced the drug uptake in cancer cells and the nanoparticles had clear selection towards cancer cells and they did not inhibit healthy cells. The results presented here indicate that MOFs are exible in building blocks and surface functionalization, which had great potential in different types of tailored drug delivery systems, thereby the biocompatible MOFs are promising in future clinical applications.
Biological studies. Cell proliferation assay Roche diagnostics reagent (WST-1), phosphate-buffered saline (PBS), Penicillin-Streptomycin, and dimethylsulfoxide (DMSO) for cell culture solution were purchased from Sigma Aldrich. Dulbecco's modi ed eagle's medium (DMEM), trypsin-EDTA (1X), and fetal bovine serum (FBS) were purchased from Lonza. Minimum essential medium non-essential amino acids (MEM NEAA, 100X) was purchased from Gibco. L-Glutamine 100X was purchased from Biowest. Breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were used for in vitro studies. The cells were cultured in high-glucose DMEM containing 10% heat-inactivated fetal bovine serum, 0.5% penicillin-streptomycin, 1% MEM NEAA, and 1% L-glutamine at 37 °C in the sterile condition of 5% CO 2 incubator with the humidi ed atmosphere. Cells are exponentially cultured as monolayer up to 70-80% con uency. After optimal growth, cells were detached using trypsin-EDTA. All the cell culture work was performed under sterile conditions. Synthesis of DCA-UiO-66-NH 2 . Following these general steps to prepare DCA-UiO-66-NH 2 nanoparticles: in separate vessels, ZrCl 4 (1.0 g, 4.2 mmol) and H 2 BDC-NH 2 (761.0 mg, 4.2 mmol) were dissolved in 150 mL of DMF. After mixing both precursor solutions transferring into a 250 mL vial, dichloroacetic acid (DCA, 3.0 mL, 36.0 mmol) was added to the reaction mixture, after gently stirring, it was placed in the oven at 120 ℃ for 24 hours. After cooling to room temperature, the powders were collected by centrifugation and washed 3 times with DMF and 3 times with ethanol by dispersion centrifugation cycles. The resulting MOFs were dried under vacuum for at least 12 hours before analysis.
Synthesis of 3,3′-dithiodipropionic anhydride (DTDPA). 1.0 g of 3,3′-dithiodipropionic acid (DTDP) was added to 5.0 mL of acetyl chloride and re uxed at 65 ℃ for 2 hours. The resulting solution was cooled to room temperature and the solvent was evaporated. The residue was then precipitated in diethyl ether and washed repeatedly with diethyl ether. The product 3,3′-dithiodipropionic anhydride (DTDPA) was dried in a vacuum desiccator overnight and used directly in the next step. was reacted at 50 °C for 6 hours. The obtained folic acid-NHS was mixed with ethylenediamine (10.0 mmol) and 500.0 µg pyridine and allowed to react at room temperature overnight. The crude product was precipitated by adding an excess of acetonitrile, ltered and washed three times with diethyl ether, and then dried under vacuum.
Preparation of 5-FU-loaded DCA-UiO-DTDP. Fresh DCA-UiO-DTDP was immersed in methanol for 24 hours, and then the extract was discarded. Fresh methanol was then added, and the sample was immersed for another 24 h to remove H 2 O and DMF. The sample was then treated with dichloromethane to remove methanol solvates using the same procedures. After dichloromethane was removed by decantation, the sample was activated by using a dynamic vacuum at 100 ℃ for 12 hours. To load 5-FU into the pores of DCA-UiO-DTDP, dehydrated DCA-UiO-DTDP (5 mg) was dispersed in a 5-FU containing MeOH solution (5 mL) and stirred for 1 day to yield a uniform light-yellow solution. The 5-FU-DCA-UiO-DTDP was collected by centrifugation (13000 rpm for 10 min) and washed with MeOH (x2) by dispersive centrifugation cycles to ensure that no residual 5-FU remained on the particle surfaces. The resulting MOFs were dried under vacuum at least 24 hours before analysis. The amount of 5-FU adsorption in the porous solids was estimated by UV-Vis. The wavelength used for 5-FU quanti cation was 266 nm. In vitro cellular assay Cell viability assay. 5-FU acts as a thymidylate synthase (TS) inhibitor, so it needs to reach the nucleus of cancer cells to be effective, while DCA inhibits pyruvate kinase and therefore acts on mitochondria. The cytotoxicity of the free DCA, 5-FU, 5 were rst seeded in confocal dishes and grown for 12 hours before the uptake study, and then incubated with the prepared DCA-UiO-DTDP and DCA-UiO-DTDP-FA suspensions (2 mL, 500 mg·mL − 1 ) at 37 ℃ for different times (1, 6 and 16 hours). At different time points, the cells were washed three times with PBS. Thereafter, the cells were xed with 4% paraformaldehyde (2 mL/well) at 37 ℃ for 10 min and further rinsed three times with PBS. For nucleus labeling, the nucleus were stained with DAPI solution (20 mg·mL − 1 in PBS, 1 mL/well) for 5 minutes, and the cells were then washed three times with PBS again, and the samples were examined with a Zeiss LSM880 with air scan instrument.
Flow cytometric detection of cellular uptake and kinetics. The cellular uptake kinetics was studied using ow cytometer. 1-5 × 10 5 density of breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were detached using trypsin and resuspended in 2.00 mL cell culture serum-free media containing DCA-UiO-DTDP and DCA-UiO-DTDP-FA at the concentration 500 mg·mL − 1 at different periods up to 24 hours. After incubation at 37 ℃ cells suspension was diluted with 2 mL of PBS and centrifuged at 800 × g. The cell pellet was washed twice with PBS by centrifugation and resuspended in 500 µL of serum-free media. Fluorescence analysis was performed with BD LSRFORTESSA using Alexa Fluor 700-A channels and a 640 nm emission lter. strategic guidance during development of idea, wrote and revised the manuscript. All authors read and approved the nal manuscript.