Construction of a Multifunctional Nano-Scale Metal-Organic Framework-Based Drug Delivery System for Targeted Cancer Therapy

The antitumor activity of triptolide (TP) has received widespread attention, although its toxicity severely limits its clinical application. Therefore, the design of a targeted drug delivery system (TDDS) has important application prospects in tumor treatment. Metal–organic frameworks (MOFs), with high drug-carrying capacity and good biocompatibility, have aroused widespread interest for drug delivery systems. Herein, folic acid (FA) and 5-carboxylic acid fluorescein (5-FAM) were used to modify Fe-MIL-101 to construct a functionalized nano-platform (5-FAM/FA/TP@Fe-MIL-101) for the targeted delivery of the anti-tumor drug triptolide and realize in vivo fluorescence imaging. Compared with Fe-MIL-101, functionalized nanoparticles not only showed better targeted therapy efficiency, but also reduced the systemic toxicity of triptolide. In addition, the modification of 5-FAM facilitated fluorescence imaging of the tumor site and realized the construction of an integrated nano-platform for fluorescence imaging and treatment. Both in vitro and in vivo studies of functionalized nanoparticles have demonstrated excellent fluorescence imaging and synergistic targeting anticancer activity with negligible systemic toxicity. The development of functional nano-platform provides new ideas for the design of MOF-based multifunctional nano-drug delivery system, which can be used for precise treatment of tumor.


Introduction
At present, most chemotherapeutic drugs have been proven to have good anti-tumor effects, but their uneven distribution in the body and side effects on normal tissues and organs often affect their clinical applications [1]. During treatment, an increase in dose is used to solve the problem of biodistribution, but it can cause dose-related side effects [2]. Therefore, there is an urgent need for a targeted drug delivery system with biocompatibility to overcome the above-mentioned problems [3]. The rapid development of nanotechnology provides an opportunity to overcome challenges faced by the biomedical field.
As a multifunctional porous nanomaterial, metal-organic frameworks (MOFs) have received extensive attention in various fields since they were proposed [4][5][6]. They have been widely applied in gas storage, adsorption, catalysis, sensing, biological imaging, energy storage, and drug delivery [7][8][9][10][11][12][13]. The adjustable structure of MOFs can achieve targeted drug delivery, which is currently a research hotspot in the field of biomedicine [14]. Zhang et al. utilized a folate-modified MOF as a carrier of photosensitizers to achieve precise tumor treatment [15]. Vandana  Fe-MIL-101 MOFs were synthesized using the solvothermal method, as previously reported [35,36]. The reaction conditions, including the reaction time, temperature, and the molar ratio of FeCl 3 ·6H 2 O to NH 2 -H 2 BDC, were carefully optimized. Amounts of 2 mmol of FeCl 3 ·6H 2 O and 1 mmol of 2-amino-terephthalic acid (NH 2 -H 2 BDC) were dissolved in 35 mL of DMF and then transferred to an autoclave and reacted at 110 • C for 24 h. After cooling to room temperature, the obtained precipitates were washed with DMF and vacuum-dried at 110 • C for 12 h.

Synthesis of TP@Fe-MIL-101
A total of 10 mg Fe-MIL-101 was dispersed in 2 mL methanol solution; then, TP was added to the above solution. The mixture was stirred at room temperature for 72 h. After centrifugation, the obtained product was washed with methanol three times to obtain TP@Fe-MIL-101. The supernatant was collected, and the free TP concentration was determined by HPLC. The drug loading capacity of TP@Fe-MIL-101 is defined as follows: drug loading capacity (%) = weight of TP in TP@Fe-MIL-101/weight of TP@Fe-MIL-101 × 100%. Masses of 10 mg of TP@Fe-MIL-101, 10 mg of FA, and 10 mg of 5-FAM were dissolved in 20 mL PBS solution (pH 7.4), followed by the addition of EDC (10 mg) and NHS (10 mg); then, the mixture was stirred for 20 h in the dark at room temperature. After the reaction, 5-FAM/FA/TP@Fe-MIL-101 MOFs were obtained by centrifugation.

Characterizations of NPs
The surface morphology of the NPs was investigated by high-resolution transmission electron microscopy (TEM, JEM-2100, JEOL Corporation, Tokyo, Japan) and field emission scanning electron microscopy (SEM, JSM-7500F, JEOL Corporation, Tokyo, Japan). X-ray diffraction (XRD) was recorded with a Rigaku Uitima IV X-ray diffractometer. Nitrogen adsorption-desorption isotherms were recorded with a nitrogen adsorption-desorption analyzer (Bel Japan Inc., Tokyo, Japan). The simulation atomic coordinates are freely available for download from the Cambridge Crystallographic Data Centre. The structures were evaluated with a Fourier-transform infrared spectrometer (FTIR, NICOLET 6700, Thermo Fisher, Waltham, MA, USA). Thermogravimetric analysis (TGA) was carried out on a Mettler-Toledo thermogravimetric analyzer under a nitrogen flow in the range of room temperature to 800 • C. A Malvin nanometer laser particle size analyzer (Zetasizer Nano S90, Malvern Company, Malvern, UK) was used to measure the particle size distribution and zeta potential.

In Vitro Drug Release Study
The drug-loaded NPs were placed in a dialysis bag (8)(9)(10)(11)(12)(13)(14) with 50 mL PBS (pH 7.4 and pH 5.5) at 37 • C. At different time points, 1 mL of solution was taken out, and the same amount of fresh buffer was added. The TP concentration was determined by HPLC. The mobile phase was a mixture of methanol and water (50:50), the column temperature was 25 • C, the flow rate was 1.0 mL/min, and the detection wavelength was 218 nm. The cumulative release percentage of TP from 5-FAM/FA/TP@Fe-MIL-101 was calculated.

Cell Culture
A HepG2 cell line was purchased from Guangzhou Jenniobio Biotechnology Co., Ltd., Guangzhou, China, and HL-7702 cells were obtained from the China Infrastructure of Cell Line Resources. Cells were cultured in high-glucose DMEM containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin. The cells were then placed in an incubator at 37 • C with 5% CO 2 .

In Vitro Cytotoxicity Assay
The MTT method was used to evaluate the toxicity of Fe-MIL-101 on HL-7702 cells and the antitumor effect of drug-loaded NPs on HepG2 cells. Cells were inoculated into 96-well plates (7 × 10 3 cells/well) and cultured in an incubator for 12 h. NPs with different concentrations (TP: 1, 0.5, 0.25, 0.125, 0.0625 µg/mL) were added. After a certain time, MTT was treated for another 4 h. Then, DMSO was added to dissolve the blue Formazan crystals by low-speed vibration for 5 min in a shaker. The absorbance was measured at 490 nm using a microplate analyzer (BioTek Instruments Inc., Winorsky, VT, USA).

Annexin V/PI Double-Staining Assay
HepG2 cells (4 × 10 5 cells/mL) were seeded in six-well plates and incubated overnight; then, functionalized NPs of different concentrations (0.25, 0.5, 1 µg/mL TP) were incubated for 12 h. After 40 s of trypsin digestion, the cells were collected by centrifugation, washed with PBS, and resuspended with binding buffer (295 µL). Annexin V-FITC (5 µL) and PI (10 µL) were added and incubated at room temperature for 15 min in the dark. Cell apoptosis was detected by flow cytometry after filtration with a nylon mesh (300 mesh). HepG2 cells were treated with functionalized NPs (0.5 µg/mL TP) for 12 h, and 1 mL DCFH-DA (10 µM) was added and incubated at room temperature for 20 min in the dark. The cells were washed twice with PBS, and the intracellular reactive oxygen species (ROS) were observed under the excitation wavelength of 488 nm using an inverted fluorescence microscope (ECLIPSE Ts2R, NIKON Instrument Co., Ltd., Tokyo, Japan).

Measurement of Mitochondrial Membrane Potential (MMP)
HepG2 cells were treated with drug-loaded NPs (0.5 µg/mL TP) for 12 h. Then, 1 mL of JC-1 (10 µM) was added and incubated at room temperature for 10 min in the dark. The cells were washed twice with PBS and observed under an inverted fluorescence microscope (ECLIPSE Ts2R, NIKON Instruments Co., Ltd).

Cell Uptake Study
Cells were seeded into six-well plates at a density of 4 × 10 5 cells/well and incubated overnight. HepG2 cells were treated with 5-FAM/FA/TP@Fe-MIL-101 and 5-FAM/TP@Fe-MIL-101 (1 µg/mL TP) for 4 h, and then the old medium was discarded. The cells were washed with PBS three times and observed with a laser confocal microscope under an excitation wavelength of 480 nm.

Western Blot Analysis
Western blotting was used to detect the effect of TP on apoptotic proteins in HepG2 cells. TP and functionalized NPs (1 µg/mL TP) were used to treated cells for 12 h. Then, the treated cells were washed twice with cold PBS and lysed with RIPA buffer for 30 min. The lysate was centrifuged at 4 • C (12,000 r/min, 10 min), and the total protein concentration was determined using a BCA protein assay kit. The mitochondria and cytoplasm were isolated using the Proteoextract ® Cytosol/Mitochondrial Isolation Kit. The protein lysates were separated by 10% SDS-PAGE and transferred to a PVDF membrane with a TBST buffer containing 5% skimmed milk. The membrane was sealed for 1 h and incubated overnight at 4 • C with primary antibody [CytC (1:5000), Bcl-2 (1:5000), Bax (1:2000), cleaved PARP (1:1000), cleaved Caspase 3 (1:500), cleaved Caspase 9 (1:2000), p53 (1:1000), COX-IV (1:5000), and β-actin (1:5000)]. Then, it was incubated with the horseradish peroxidaseconjugated secondary antibodies (1:5000 dilution) at room temperature for 1 h. The signal of the target protein was collected by an ECL detection system. All of the experimental results were repeated at least three times.

Animal and Tumor-Bearing Mouse Model
All the animal experiments were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. Balb/c nude mice (8-10 weeks, 15 g) were obtained from Sipeifu Biological Technology Co., Ltd. (Beijing, China), and all animal care and handing procedures were in accordance with the guidelines approved by the ethics committee of Beijing University of Chinese Medicine. A tumor-bearing mouse model was established initially. HepG2 cells were suspended in saline and subcutaneously injected in the right anterior armpit posterior of mice (1 × 10 7 cells/mL, 0.1 mL). Then, tumor volume was calculated as V = W 2 × L/2.

Distribution of Functionalized Nanoparticles In Vivo
When the tumor volumes reached 150 mm 3 , the HepG2 tumor-bearing mice were divided into two groups (n = 5). 5-FAM/FA/TP@Fe-MIL-101 and 5-FAM/TP@Fe-MIL-101 (100 µg/mL) were administered through the tail vein. After isoflurane anesthesia, the fluorescence images were recorded with a small animal in vivo imaging system (Ex = 490 nm, Em = 520 nm).

Antitumor Effect of Functionalized Nanoparticles In Vivo
Fifteen tumor-bearing mice were randomly divided into three groups, and each group was injected with saline, TP (0.2 mg/kg), or functional NPs (a TP concentration of 0.2 mg/kg) through the tail vein every 48 h. The body weights, tumor size, and tumor weights of the mice were measured. After 12 days of treatment, tumors were collected, and then subjected to DAPI staining and TUNEL assays. The major organs of the mice were harvested for H&E staining on the 12th day to determine the toxicity of the NPs.

Safety Evaluation of Functionalized NPs In Vivo
Blood of C57BL/6 mice treated with NPs (a TP concentration of 0.2 mg/kg) for 14 d was collected, and the samples were used to carry out blood biochemical assays. Blood from the mice without any treatments was defined as the control. The liver, spleen, and kidney (right kidney) were collected and stained with hematoxylin-eosin (H&E) to investigate the biotoxicity.

Statistical Methods
All experiments were performed with at least three independent replications, and data were expressed as means ± SDs. Comparisons were analyzed using one-way variance analysis (ANOVA). * p < 0.05 and ** p < 0.01 were considered to be statistically significant.

Preparation and Characterization of NPs
Fe-MIL-101 were synthesized using the solvothermal method [37]. As shown in Figure S1, the characteristic peaks of Fe-MIL-101 were obvious (2θ = 8.9 • , 9.5 • , 10.6 • ), and the crystallinity was good. Fe-MIL-101 NPs had a regular octahedral shape and monodisperse particles with a similar average size of 290.7 nm ( Figure S2). In addition, the XRD spectra of the synthesized MOFs were in good agreement with the simulated images of Fe-MIL-101 ( Figure S3a), indicating the successful synthesis of Fe-MIL-101. Simultaneously, N 2 adsorption-desorption isotherms were obtained to check the porosity of Fe-MIL-101 ( Figure S3b). The specific surface area of Fe-MIL-101 was 480.86 m 2 /g and the pore size was about 2.43 nm.
Additionally, TP was loaded on Fe-MIL-101 using the solvent adsorption method. In order to achieve active targeted drug delivery and fluorescence imaging, FAM/FA/TP@Fe-MIL-101 was prepared by post-synthesis modification [38][39][40]. The TP loading capacity in FAM/FA/TP@Fe-MIL-101 was 39.77 ± 1.24%. Scanning electron microscopy (SEM) showed that the morphology and size of NPs after modification did not change much ( Figure 1a). The successful modification of FA and FAM was confirmed via the following experiments. The infrared spectrum of Fe-MIL-101 showed a characteristic peak of a N-H bond at 1575 cm −1 ( Figure S4). The vibrational absorption peaks of carboxyl groups in FA and 5-FAM appeared at 1695 cm −1 and 1696 cm −1 , respectively. The appearance of the characteristic peak of the amide group at 1607 cm −1 and the disappearance of the characteristic peak of the carbonyl group in 5-FAM/FA/TP@Fe-MIL-101 indicated that FA and 5-FAM successfully combined with -NH 2 on the surface of the MOFs. In addition, FAM/FA/TP@Fe-MIL-101 had obvious green fluorescence from 5-FAM under a fluorescence microscope (Figure 1d-f). The particle size of functional NPs ranged from 300 to 400 nm, which was stable in PBS solution within 7 days (Figure 1b). When FA and 5-FAM were modified, the Zeta potential of FAM/FA/TP@Fe-MIL-101 changed from positive (Fe-MIL-101, 9.97 ± 1.02 mV; TP@Fe-MIL-101, 34.12 ± 0.76 mV) to negative (−13.14 ± 2.34 mV). According to the thermogravimetric curve (Figure 1c), there were two main weightlessness processes of Fe-MIL-101. The weight loss was about 15% at 30~150 • C, which indicated the weight loss of residual solvent and water molecules in the frame structure. In the range of 150~250 • C, the weight remained stable, whereas the skeleton of the NPs collapsed at 250~700 • C, indicating that the NPs exhibited good thermal stability within 250

Study on the Drug Release of Functionalized Nanoparticles In Vitro
The

In Vitro Cytotoxicity Assay
To evaluate the safety of 5-FAM/FA@Fe-MIL-101, an MTT experiment was carried out. The results revealed that even at higher concentrations (80 µg/mL), Fe-MIL-101 had no obvious cytotoxicity to HepG2 cells and HL-7702 cells and could be used as a safe nano-drug carrier (Figure 3a). In addition, the toxicity of TP to HepG2 and HL-7702 cells was also evaluated. TP had a strong killing effect on both types of cells, which proved the non-selective toxicity of TP ( Figure S5). However, 5-FAM/FA/TP@Fe-MIL-101 had a significantly enhanced cytotoxicity to HepG2 cells and almost no toxicity to HL-7702 cells, indicating that the functional nanoparticles could improve the antitumor activity of triptolide and reduce its toxicity (Figure 3b).

In Vitro Cytotoxicity Assay
To evaluate the safety of 5-FAM/FA@Fe-MIL-101, an MTT experiment was carried out. The results revealed that even at higher concentrations (80 µg/mL), Fe-MIL-101 had no obvious cytotoxicity to HepG2 cells and HL-7702 cells and could be used as a safe nanodrug carrier (Figure 3a). In addition, the toxicity of TP to HepG2 and HL-7702 cells was also evaluated. TP had a strong killing effect on both types of cells, which proved the nonselective toxicity of TP ( Figure S5). However, 5-FAM/FA/TP@Fe-MIL-101 had a significantly enhanced cytotoxicity to HepG2 cells and almost no toxicity to HL-7702 cells, indicating that the functional nanoparticles could improve the antitumor activity of triptolide and reduce its toxicity (Figure 3b). Annexin V-FITC/PI double staining showed that various concentrations of functionalized NPs could induce the apoptosis of HepG2 cells in a dose-dependent manner ( Figure  4a,b). The ROS levels in HepG2 cells were detected using the DCFH-DA fluorescent dye [41]. Compared with the control group, the HepG2 cells incubated with functionalized NPs (TP: 0.5 µg/mL) showed obvious green fluorescence, suggesting that the apoptosis of tumor cells induced by nanoparticles might be related to the damage of the cell redox state ( Figure S6). In addition, the mitochondrial membrane potential decreased significantly with the increase in the concentration of functionalized nanoparticles, presenting a significant dose-dependent relationship ( Figure S7).

Cell Uptake Study
The specific uptake of NPs by tumor cells is key to the effect of antitumor drugs. A laser confocal microscope was used to observe the specific uptake of NPs in vitro. As shown in Figure 5a (Figure 5c). These results indicated that FA-modified NPs had a high affinity to HepG2, and were preferentially internalized by HepG2 cells through FAreceptor-mediated endocytosis. Dong et al. successfully constructed tumor-targeted NMOF drug carriers by anchoring FA on NMOFs, which enhanced the specific uptake of tumor cells and was an efficient targeted drug delivery system [42]. Annexin V-FITC/PI double staining showed that various concentrations of functionalized NPs could induce the apoptosis of HepG2 cells in a dose-dependent manner (Figure 4a,b). The ROS levels in HepG2 cells were detected using the DCFH-DA fluorescent dye [41]. Compared with the control group, the HepG2 cells incubated with functionalized NPs (TP: 0.5 µg/mL) showed obvious green fluorescence, suggesting that the apoptosis of tumor cells induced by nanoparticles might be related to the damage of the cell redox state ( Figure S6). In addition, the mitochondrial membrane potential decreased significantly with the increase in the concentration of functionalized nanoparticles, presenting a significant dose-dependent relationship ( Figure S7).

Cell Uptake Study
The specific uptake of NPs by tumor cells is key to the effect of antitumor drugs. A laser confocal microscope was used to observe the specific uptake of NPs in vitro. As shown in Figure 5a (Figure 5c). These results indicated that FA-modified NPs had a high affinity to HepG2, and were preferentially internalized by HepG2 cells through FA-receptor-mediated endocytosis. Dong et al. successfully constructed tumor-targeted NMOF drug carriers by anchoring FA on NMOFs, which enhanced the specific uptake of tumor cells and was an efficient targeted drug delivery system [42].

Western Blot Analysis
Western blot results showed that protein expressions of Bax, cleaved Caspase-3, cleaved Caspase-9, p53, and PARP were significantly increased after 12 h treatment of HepG2 cells with TP and functionalized NPs, whereas the expression level of Bcl-2 was significantly decreased (Figure 6). This led to an increase in the Bax/Bcl-2 ratio. The levels of cytochrome c (Cytc) in the cytoplasm were significantly increased, whereas the level of Cytc in mitochondria was significantly decreased, indicating that TP could lead to mitochondrial dysfunction by increasing the permeability of the mitochondrial membrane of HepG2, thereby inducing cell apoptosis [43].

Western Blot Analysis
Western blot results showed that protein expressions of Bax, cleaved Caspase-3, cleaved Caspase-9, p53, and PARP were significantly increased after 12 h treatment of HepG2 cells with TP and functionalized NPs, whereas the expression level of Bcl-2 was significantly decreased (Figure 6). This led to an increase in the Bax/Bcl-2 ratio. The levels of cytochrome c (Cytc) in the cytoplasm were significantly increased, whereas the level of Cytc in mitochondria was significantly decreased, indicating that TP could lead to mitochondrial dysfunction by increasing the permeability of the mitochondrial membrane of HepG2, thereby inducing cell apoptosis [43].  Bax and Bcl-2 are typical apoptosis-related proteins [44,45], and the ratio of Bax/Bcl-2 is affected by ROS, which leads to the increased permeability of mitochondrial membranes and promotes the release of Cytc. Excessive production of ROS can lead to DNA damage [46]. The significantly increased ROS level in HepG2 indicated the existence of an oxidative stress response, which was also confirmed by the increased expression level of the p53 protein [47]. In summary, TP mainly induced the apoptosis of HepG2 through the mitochondrial endogenous pathway. Moreover, the effect of the functionalized NPs group on apoptotic proteins was better than that of the TP group. It was proven that the synthetic functionalized NPs could significantly improve the anti-tumor efficacy.

Distribution of Functionalized Nanoparticles In Vivo
The biological distribution of functional nanoparticles in tumor-bearing mice was evaluated by animal imaging in vivo. After 4 h of tail vein injection, the fluorescence intensity of the tumor in the 5-FAM/FA/TP@Fe-MIL-101 group was significantly higher than that in the 5-FAM/TP@Fe-MIL-101 group (Figure 7a). This might be related to the EPR effect and FA-mediated active targeting, which increase the accumulation of NPs at the tumor site [48]. After 12 h of tail vein injection, the tumors and main organs of each group were dissected and analyzed by in vitro fluorescence imaging. The results showed that the functionalized NP group had higher fluorescence intensity in tumor tissues, but lower fluorescence intensity in normal tissues such as the liver and kidney (Figure 7b). Upon modifying the folate ligand on Fe-MIL-101, functional NPs could specifically deliver anticancer drugs to tumors. Bax and Bcl-2 are typical apoptosis-related proteins [44,45], and the ratio of Bax/Bcl-2 is affected by ROS, which leads to the increased permeability of mitochondrial membranes and promotes the release of Cytc. Excessive production of ROS can lead to DNA damage [46]. The significantly increased ROS level in HepG2 indicated the existence of an oxidative stress response, which was also confirmed by the increased expression level of the p53 protein [47]. In summary, TP mainly induced the apoptosis of HepG2 through the mitochondrial endogenous pathway. Moreover, the effect of the functionalized NPs group on apoptotic proteins was better than that of the TP group. It was proven that the synthetic functionalized NPs could significantly improve the anti-tumor efficacy.

Distribution of Functionalized Nanoparticles In Vivo
The biological distribution of functional nanoparticles in tumor-bearing mice was evaluated by animal imaging in vivo. After 4 h of tail vein injection, the fluorescence intensity of the tumor in the 5-FAM/FA/TP@Fe-MIL-101 group was significantly higher than that in the 5-FAM/TP@Fe-MIL-101 group (Figure 7a). This might be related to the EPR effect and FA-mediated active targeting, which increase the accumulation of NPs at the tumor site [48]. After 12 h of tail vein injection, the tumors and main organs of each group were dissected and analyzed by in vitro fluorescence imaging. The results showed that the functionalized NP group had higher fluorescence intensity in tumor tissues, but lower fluorescence intensity in normal tissues such as the liver and kidney (Figure 7b). Upon modifying the folate ligand on Fe-MIL-101, functional NPs could specifically deliver anticancer drugs to tumors.

Antitumor Effect of Functionalized Nanoparticles In Vivo
The anti-cancer effect of functionalized NPs was further evaluated via tail vein injection at a dose of 0.2 mg/kg. After 12 days of administration, there was no significant difference in body weight between the different treatment groups and the control group, indicating that NPs did not increase systemic toxicity compared to direct TP administration (Figure 8a). There were significant differences in tumor volume between the groups. The average tumor volume in the saline group was 1538.50 ± 461.75 mm 3 ; in the TP group it was 841.38 ± 406.63 mm 3 ; and in the 5-FAM/FA/TP@Fe-MIL-101 group it was 511.76 ± 250.79 mm 3 (Figure 8b,c). Similarly, the tumor masses of the TP group and the 5-FAM/FA/TP@Fe-MIL-101 group were smaller than those of the saline group (Figure 8d). Compared with the control group and pure TP, 5-FAM/FA/TP@Fe-MIL-101 exhibited a superior anticancer effect, which might be related to the EPR effect of NPs and the active targeting effect mediated by FA. Histopathological results confirmed that tumors in the saline group were scattered in the liver, hepatocytes in the TP group had inflammation, and the functionalized nanoparticle group had no obvious side effects on the main organs (liver, spleen) of mice (Figure 8e). TUNEL and DAPI staining ( Figure S8) showed that the fluorescence intensity of the TP group and the functionalized NPs group was significantly increased, indicating a significant increase in the percentage of tumor cell apoptosis. The antitumor effect of functionalized NPs was more obvious, which proved that this new drug delivery system had better anti-tumor efficacy compared with TP direct administration.

Antitumor Effect of Functionalized Nanoparticles In Vivo
The anti-cancer effect of functionalized NPs was further evaluated via tail vein injection at a dose of 0.2 mg/kg. After 12 days of administration, there was no significant difference in body weight between the different treatment groups and the control group, indicating that NPs did not increase systemic toxicity compared to direct TP administration (Figure 8a). There were significant differences in tumor volume between the groups. The average tumor volume in the saline group was 1538.50 ± 461.75 mm 3 ; in the TP group it was 841.38 ± 406.63 mm 3 ; and in the 5-FAM/FA/TP@Fe-MIL-101 group it was 511.76 ± 250.79 mm 3 (Figure 8b,c). Similarly, the tumor masses of the TP group and the 5-FAM/FA/TP@Fe-MIL-101 group were smaller than those of the saline group (Figure 8d). Compared with the control group and pure TP, 5-FAM/FA/TP@Fe-MIL-101 exhibited a superior anticancer effect, which might be related to the EPR effect of NPs and the active targeting effect mediated by FA. Histopathological results confirmed that tumors in the saline group were scattered in the liver, hepatocytes in the TP group had inflammation, and the functionalized nanoparticle group had no obvious side effects on the main organs (liver, spleen) of mice (Figure 8e). TUNEL and DAPI staining ( Figure  S8) showed that the fluorescence intensity of the TP group and the functionalized NPs group was significantly increased, indicating a significant increase in the percentage of tumor cell apoptosis. The antitumor effect of functionalized NPs was more obvious, which proved that this new drug delivery system had better anti-tumor efficacy compared with TP direct administration.

Safety Evaluation of Functionalized NPs In Vivo
After 14 days of continuous administration, compared with the control group, the functionalized nanoparticle group had no significant effects on ALT, AST, ALP, or BUN ( Figure S9a). In addition, histopathological results showed that no abnormalities were found in the liver, spleen, or kidney tissues of mice in the functionalized NPs group, indicating that the prepared functionalized NPs have good biological safety ( Figure S9b).

Conclusions
In this research, we constructed a new multifunctional drug carrier, 5-FAM/FA/TP@Fe-MIL-101, to enhance antitumor cytotoxicity for cancer therapy. In vitro drug release results showed that TP exhibited good drug release performance in PBS. Cytotoxicity experimental results indicated that NPs have good antitumor activity. Through fluorescence imaging and flow cytometry analysis, NPs demonstrated good folate targeting properties. 5-FAM/FA/TP@Fe-MIL-101 has a strong tumor suppression rate in nude mice with liver cancer and possesses good antitumor effect. In conclusion, a drug delivery system based on MOFs with tumor targeting and fluorescence imaging was designed and constructed, providing new ideas for targeted drug delivery.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the cerresponding authors.