Larotrectinib sulfate (Lar), 2-aminoterephthalic acid (BDC-NH₂), ferrous acetate (FeAc₂), adipic acid dihydrazide (ADH), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N,N-dimethylformamide (DMF) were purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. An MTT assay kit was purchased from China Biyuntian Biotechnology Co., Ltd. Mice (three to four weeks old) were obtained from the Animal Experiment Center of Jinzhou Medical University, China. The animal study protocol was approved by the Animal Ethics Committee of the Jinzhou Medical University of China. All water used for experimentation was double-distilled water.

BDC-NH₂ (0.45 g) was weighed precisely and dissolved in 15 mL of DMF, then aqueous FeAc₂ solution (0.1 g/mL) was slowly added dropwise to the mixture while stirring, and the reaction was carried out at 65°C for 1–2 h. The solution was cooled naturally at room temperature. The supernatant was separated by centrifugation (6,000 rpm, 30 min) and discarded, and the precipitate was washed twice with DMF (centrifugation, 3,000 rpm, 15 min), followed by two more washes with anhydrous ethanol (centrifugation, 3,000 rpm, 15 min). Finally, the solid precipitate was dried in a vacuum drying oven, and Fe-MOF was obtained (Wan et al., 2019).

Lar (75 mg) was mixed with 50 mg of Fe-MOF in a 25 mL beaker. The mixture was shaken (200 rpm) for 12 h (Lar@Fe-MOF). The precipitated material was collected after centrifugation (6,000 rpm, 15 min) and dried.

The Lar content was determined by ultraviolet–visible (UV‒vis) spectroscopy. Lar was accurately weighed and added to 3% DMSO and anhydrous ethanol to obtain solutions of different concentrations, which were then filtered through a 0.45 μm nanoporous membrane. The absorbance of different samples was measured at 262 nm to calculate the Lar content and drug loading of Lar@Fe-MOF, and each experiment was repeated three times.

A small amount of Lar@Fe-MOF was weighed, added to anhydrous ethanol, sonicated, and dispersed for 5 min. A small amount of liquid was dropped onto a copper network, dried, observed by TEM, and imaged (Chen et al., 2020; Chen et al., 2021; Tan et al., 2022).

The prepared Lar API, blank Fe-MOF carrier, a Lar and Fe-MOF physical mixture, and Lar@Fe-MOF powder were subjected to DSC. The operating conditions were as follows: an empty aluminum crucible was used as the blank reference, and another crucible was used as the cuvette; 2–5 mg of samples were placed into the cuvette at the corresponding positions for the graphical scans; N₂ was used as the purge gas, and the heating rate was 10 °C/min. The DSC thermal characteristic curves of each sample were recorded and compared (Gaber et al., 2022).

The samples (Lar API, blank Fe-MOF carrier, a Lar and Fe-MOF physical mixture, and Lar@Fe-MOF) and an appropriate amount of potassium bromide were mixed in proportion and pressed into tablets. The tablets were then scanned by an FTIR spectroscopy instrument with a wavenumber range of 4,000–400 cm^-1 and a resolution of 4 cm^-1 (Lin et al., 2023).

Small amounts of Lar API, blank Fe-MOF carrier, and Lar@Fe-MOF samples were warmed up to 400°C under a protective N₂ atmosphere with a warming rate of 10°C/min to determine their weight loss curves.

Lar release from Lar@Fe-MOF was studied by performing dialysis in a constant-temperature shaker at 37°C and 100 rpm. Phosphate-buffered saline (PBS, pH 7.4) was chosen as the dialysis medium. Briefly, approximately 5 mg of Lar@Fe-MOF and Lar powder was placed in a dialysis bag, which were then soaked in PBS (100 mL) and tightened at the end. Then, 1 mL aliquots of release medium were removed at different time intervals, with the addition of 1 mL of new release medium to maintain a constant volume. Each group experiment was repeated 3 times. Drug release properties were assessed by evaluating the absorbance of the aliquots using UV‒vis spectroscopy.

Murine breast cancer cells (4T1) were used to assess the biosafety of Fe-MOF. Briefly, 4T1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator. Cells were inoculated into 96-well plates (density of 1×10⁴) and incubated for 24 h to induce wall attachment. The 96-well plate medium was discarded, and the cells were washed twice with PBS. Media containing different concentrations of Fe-MOF (0.5–50 μg/mL) were then added to each well and incubated for 24 h. Cell viability was determined using the MTT assay according to standard protocols (Javad Farhangi et al., 2021; Liu et al., 2021; Fang et al., 2022; Ji et al., 2022).

The hemocompatibility of the synthesized Fe-MOF was evaluated by analyzing the interaction of Fe-MOF with red blood cells according to a previously reported method (Nikam et al., 2022). Blood was removed from the eyes of mice and placed in tubes containing ethylenediaminetetraacetic acid (EDTA) solution. A certain amount of sodium chloride solution was added to wash the blood cells (centrifuged at 3,500 r/min for 10 min), and then the supernatant was discarded; this procedure was repeated 3 times. The obtained erythrocytes were prepared into a 2% (V/V) suspension with sodium chloride solution and refrigerated at 4°C for further experimentation. The specific experiments were as follows: 0.2 mL of erythrocyte suspension was mixed with 0.8 mL of Fe-MOF suspensions of different concentrations and then incubated at 37°C for 60 min. The supernatant was removed by centrifugation at 3,500 r/min for 10 min. To induce the oxidation of hemoglobin, the collected supernatant was left at room temperature for 10 min. The optical density of oxyhemoglobin was measured at 540 nm, the absorbance of the supernatant was measured, and the percentage of hemolysis was calculated.

Briefly, 4T1 cells were added to culture wells and incubated for 24 h to assess the in vitro antitumor activity of Lar@Fe-MOF (6.25–100 μg/mL). Follow up as in "2.10".

The hair around the mammary pads of 3- to 4-week-old female BALB/c mice was shaved off. A primary mammary cancer model was established by implanting 4T1 cells into the hair removal site (Yang et al., 2022). Lar@Fe-MOF preparation was administered orally to environmentally adapted mice at a daily dose of 50 mg/kg for 7 days, and the mice were weighed every other day. General conditions, such as coat color, mental and locomotor abilities, feeding and drinking, as well as signs of intoxication and death, were recorded. By measuring the length of the longest L) and shortest W) axes of the tumor, the tumor volume could be calculated by the formula "V = 1/2 (L × W²)". Tumor volumes were monitored every other day. After the last administration, mice in each group were sacrificed on day 8. Mammary tumors and major organs (heart, liver, spleen, lungs, and kidneys) of mice were dissected. The inhibitory effect and potential toxicity were assessed in vivo by tumor weighing and hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Zou et al., 2022; Qu et al., 2023).

All statistical tests (mean, standard deviation, and p-value) were performed in Excel software, and p < 0.05 was considered significant.

MOFs are a class of highly ordered crystalline porous coordination polymers (PCPs). MOFs are considered a promising class of drug nanocarriers due to their obvious structure, high specific surface area and porosity, tunable pore size, and easy chemical functionalization. In recent years, there has been much interest in the study of MOFs for biomedical applications (Sun et al., 2020). A schematic diagram of Fe-MOF synthesis is shown in Figure 1. At high temperatures, Fe²⁺ complexes with BDC-NH₂ organic ligands form complexes containing multiple coordination bonds (C₈H₄NO₄)nFe, which forms the spatial structure of nanoparticles. Since the Fe²⁺ complexes have multiple -COO- groups, a molecular arrangement with a regular pore structure is formed, conferring porosity to the synthesized organic framework.

The in vitro release curves (Figure 6) showed that the cumulative release of Lar was time dependent. The main release phase of the Lar solution last for 6 h, with Lar releases of 85.23% at 6 h and 90.91% at 12 h. In contrast, the Lar release of Lar@Fe-MOF reached 59.66% at 6 h, and the cumulative Lar release rate at 12 h was only 71.02%, indicating that Lar@Fe-MOF released Lar more slowly. Thus, the Lar release of Lar@Fe-MOF was much slower, indicating a possible slow-release effect in vivo. At present, slow-release controlled release materials are mainly used for drugs that have a short half-life or a low level of oral bioavailability, but which need to be used for a long period of time. The advantage is that the drug can be released at a certain rate over a few hours, weeks or months or even longer to maintain the effective blood concentration and improve bioavailability. Meanwhile, the number of drug administrations is reduced and the toxic side effects of the drug are reduced (Lazar et al., 2023; Shen et al., 2023). Therefore, using Fe-MOF carriers to protect drugs from gastric acid inactivation and to achieve uniform high-concentration drug distribution in various segments of the gastrointestinal tract by prolonged retention and slow release of the anticancer drug Lar through the gastrointestinal tract not only increases patient compliance and improves treatment efficacy, but also reduces the total amount of drug required (Fukumori et al., 2023; Yu et al., 2023).

To assess the cytotoxicity of the blank Fe-MOF carrier, 4T1 cells were incubated with Fe-MOF (0.5–50 μg/mL) for 24 h. The results are shown in Figure 7. After 24 h, blank Fe-MOF showed no cytotoxic effect and a negligible effect on cell viability. With increasing Fe-MOF concentration, the cell viability gradually decreased, but the cell viability was greater than 85%, indicating that the resulting Fe-MOF carriers had good cytocompatibility (Wang N. et al., 2022; Nikam et al., 2022).

Studying the interaction between erythrocytes and nanocarriers is essential for the in vivo application of nanocarriers (Wang D. et al., 2022; Gong et al., 2022). If the nanocarrier is toxic, it can cause the hemolysis of red blood cells. The results showed that the morphology of erythrocytes was not altered when Fe-MOF, within a mass concentration range of 5–500 μg/mL, was incubated with erythrocytes, and the percentage of hemolysis was less than 5% (Figure 8). Usually, a nanocarrier concentration of no higher than 2 mg/mL is considered a critical safety value (Nikam et al., 2022), which indicated that there was no interaction between the red blood cells and the nanocarrier and that the Fe-MOF carrier had good hemocompatibility and high biosafety.

As shown in Figure 9, the cell survival rate of the Lar@Fe-MOF group at 24 h was reduced compared with that of the Lar solution group, indicating that Lar@Fe-MOF enhanced the inhibitory effect of Lar on 4T1 cells. The half-inhibition concentration (IC50) curve was fitted with Graph Pad Prism 7.0. The IC50 values of the Lar solution group and Lar@Fe-MOF group were 14.98 μg/mL and 9.44 μg/mL, respectively. The inhibitory effect of Lar@Fe-MOF on the proliferation of 4T1 cells was stronger than that of the Lar solution group after 24 h of treatment, and the inhibitory effect of Lar@Fe-MOF on the proliferation of 4T1 cells showed a dose-dependent effect, indicating that the cytotoxicity was enhanced by the incorporation of Lar into the Fe-MOF nanoparticles. One reason for this could be the enhanced uptake of the drug by the cells after incorporation into the nanoparticles (Dong et al., 2022; Zhang et al., 2022).

The in vivo therapeutic effect of Lar@Fe-MOF was studied in a mouse 4T1 in situ breast cancer model. When the tumor volume reached ≈100 mm³, the mice were randomly divided into 3 treatment groups (n = 5) and treated with normal saline, Lar solution, and Lar@Fe-MOF for 7 days (Figure 10A). A plot of tumor growth showed that Lar@Fe-MOF resulted in the strongest tumor inhibition (Figure 10B). At day 8, the mean tumor volumes in the Lar and Lar@Fe-MOF groups were 67.5% and 52.1% of those in the saline group, respectively (Figure 10C). Apparently, the tumors were smaller in the Lar@Fe-MOF-treated mice. Cell proliferation in tumors was also significantly inhibited by Lar@Fe-MOF, as shown by the histological results (Figure 10D). TUNEL analysis revealed that Lar@Fe-MOF-treated tumors showed the highest percentage of apoptotic cells (Figure 10E). Thus, Lar@Fe-MOF treatment resulted in a significantly improved in vivo antitumor effect in tumor-bearing mice (Chen et al., 2023; Yan et al., 2023).

We evaluated the biosafety of Lar@Fe-MOF as this property is a crucial parameter for nanotherapeutic applications in cancer treatment (Ajaz et al., 2022; Jin et al., 2022). After oral administration of Lar@Fe-MOF, the mice showed no abnormal signs of survival and no abnormalities in coat color, diet, water intake, or urinary or fecal conditions. There was no significant difference in the body weight of the mice compared with that in the control group, which showed a normal growth trend (Figure 11A). The mice were dissected after drug administration, and their main organs were visually observed with the naked eye. The internal organs of the mice in each experimental group, including the heart, liver, spleen, lung, and kidney, did not show any apparent lesions. Histopathological sections of the organs, as shown in Figures 11B–F, also indicated that the cells of the tissues did not show noticeable microscopic damage or apparent necrosis and had intact tissue structures and that the organs could remain functionally intact at the administered dose. The above results suggest that Lar@Fe-MOF with excellent biocompatibility can be used as a highly effective oncological treatment strategy with promising applications (Gharehdaghi et al., 2023; Zhao et al., 2023).