pH-responsive berberine release from metal-organic framework based nanocarriers by regulating oxidative stress for targeted therapy of oral squamous cell carcinoma

2.1 Materials

FeCl₃·6H₂O, 2-Aminoterephtalic acid (NH₂-BDC), N-hydrox-ysuccinimide (NHS), 1-(3-dimethylaminoproply)-3-ethylcarbodiimide hydrochloride (EDC), Methylene blue (MB), Glutathione (GSH), Berberine (BER), Folic acid (FA), and NH₂—PEG—COOH were purchased from Aladdin (Shanghai, China). Ethanol absolute, N, N- dimethylformamide (DMF), and Dimethyl sulfoxide (DMSO) were purchased from Concord (Tianjin, China). Reduced Glutathione Content Assay Kit, Hydrogen Peroxide (H₂O₂) assay kit, Total Superoxide Dismutase (T-SOD) assay kit, and Malondialdehyde (MDA) assay kit were purchased from Nanjing Jiancheng (Nanjing, China). Cell Counting Kit-8 (CCK-8), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), Mitochondrial Membrane Potential Assay Kit with JC-1, and Calcein AM/PI live/dead cell staining kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were purchased from Jiangsu Meimian Industrial Co., Ltd. RNAsimple Total RNA Kit, FastQuant RT Kit (with gDNase), and SuperReal fluorescence quantitative premixing kit were purchased from TIANGEN (Beijing, China).

2.2 Preparation of materials

2.3 Optimization of MIL-101@BER

2.4 Characterization

The particle size and potential of nanoparticles were detected by DLS (Thermo Fisher, USA); The surface morphology of nanoparticles was observed by TEM; UV–vis spectrophotometer (Cary 60, USA) recorded the ultraviolet spectra of the samples; The structural composition of the nanoparticles was detected by FT-IR; The temperature-mass relationship of nanoparticles in the range of 0 − 800 °C was detected by TGA; XRD was used to analyze the crystal structure of nanoparticles.

2.5 Drug loading and release characteristic

The feasibility of MIL-101 as a nano drug carrier was evaluated by investigating the release of BER loaded material in buffer solutions with different pH values. In this experiment, MIL-101@BER and MIL-101@BER/PEG-FA were dissolved in PBS buffer solutions with pH 5.0 and 7.4, and solutions with different pH values were added to dialysis bags (MW 3500 Da), which were placed in PBS buffer solutions with the same pH value and placed in a shaker at 37 °C with a speed of 110 rpm to avoid light shock. At the predetermined time points of 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, and 96 h, 3 mL of buffer solution was taken and the corresponding pH buffer solution was made up. The absorbance of BER was detected at 345 nm, and the concentration of BER was calculated. The cumulative release was calculated according to the following formula: $${\text{D}}_{\text{n}}\left(\%\right)={\text{C}}_{\text{n}}\times \text{V}\times \text{X}/\text{M}\times \text{1}00$$ $$\text{D}\left(\%\right)={\text{D}}_{\text{n}}+\left({\text{D}}_{\text{1}}+{\text{D}}_{\text{2}}+{\text{D}}_{\text{n}}-\text{1}\right)\times \text{3}/\text{8}0\times \text{1}00$$ D_n is the release degree of the samples taken at different time points, Cn is the concentration of the samples taken at different time points, V is the volume of the release medium, X is the dilution factor, M is the total weight of the initial drug, D is the cumulative release degree.

2.6 ·OH generation ability

·OH generation ability of MIL-101 and MIL-101@BER/PEG-FA at pH 6.5 was detected by MB degradation experiment. ·OH generated by the material was analyzed according to the fading degree of MB, and ·OH could make the blue color of MB solution become lighter. MIL-101@BER and MIL-101@BER/PEG-FA (100 µg/mL) were incubated with PBS (pH 6.5) containing GSH (10 mM), MB (10 µg/mL) and H₂O₂ (10 mM) at 37 °C for 30 min, and the absorbance of the solution at 665 nm was determined by UV–vis.

2.7 Cell culture

CAL27 cells and KB cells were cultured in 10 % heat-inactivated fetal bovine serum (FBS), 1 % penicillin and streptomycin (PS), and 90 % Dulbecco's Modified Eagle's Medium (DMEM) solution, and the cells were cultured in a cell incubator at 37 °C and 5 % CO₂.

2.8 Cytotoxicity study

CCK8 method was used to detect the cytotoxicity of MIL-101, MIL-101/PEG-FA and the anti-tumor activity of MIL-101@BER and MIL-101@BER/PEG-FA. CAL27 and KB cells were seeded in 96-well plates at a density of 1 × 10⁴/mL for 24 h. Incubated with different concentrations of drugs for 24 and 48 h. 100 μL of CCK-8 solution was added and incubated at 37 °C for 2 h. The absorbance of each group at 450 nm wavelength was detected by using Microplate reader.

2.9 Cellular uptake study

Due to the weak fluorescence of BER, it was substituted with the fluorescent agent rhodamine 6G (R6G), and the drug loading method remained consistent with that described in “2.2″ section.

The cellular internalization of MIL-101@R6G/PEG and MIL-101@R6G/PEG-FA was employed to assess the targeting efficacy of FA. CAL27 and KB cells were seeded in 12-well plates at a density of 2 × 10⁵/mL for 24 h, and then were treated with MIL-101@R6G/PEG and MIL-101@R6G/PEG-FA for 1, 2, 4, and 6 h. The cells were fixed with 4 % paraformaldehyde for 30 min, the nuclei were positioned with 10 µg/mL of Hoechst 33342 solution for 30 min. The fluorescence in each group of cells was detected using an automated cell imaging system. The average fluorescence intensity was quantified utilizing Image J software.

2.10 Detection of oxidative stress indicators

2.11 Enzyme-linked immunosorbent assay (ELISA)

CAL27 cells were seeded in 6-well plates (1 × 10⁶/mL) and co-cultured with PBS, 5-FU, BER, MIL-101@BER, and MIL-101@BER/PEG-FA solutions for 24 h. After trypsin digestion and centrifugation, the cells were subjected to ultrasonic cell disruption following PBS washing. After centrifugation at 14,000 r/min for 15 min, the supernatant was collected and the levels of TNF-α and IL-1β in the cells were quantified following the instructions provided by the Elisa kit.

2.12 Mitochondrial membrane potential (MMP) measurement

To analyze the effect of MIL-101@BER/PEG-FA on mitochondrial function of CAL27 cells, Mitochondrial Membrane Potential (MMP) Assay Kit with JC-1 was used to determine the MMP. The cell culture and grouping conditions were consistent with those described in “2.10.2″ section. Cells in each group were incubated with JC-1 staining solution for 30 min, followed by two washes using JC-1 staining buffer (1 × ). The MMP within each group of cells was assessed utilizing a fully automated cell imaging system.

2.13 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

The expression of LKB1, AMPK, mTOR, Caspase 3, and Caspase 9 in CAL27 cells was detected by qRT-PCR. Total mRNA of CAL27 cells was extracted and purified according to the RNAsimple Total RNA Kit. The mRNA concentration was detected by a microprotein nucleic acid analyzer. The mRNA was reversed to cDNA according to the FastQuant RT Kit (with gDNase). The expression of each gene in the cells was detected according to the SuperReal fluorescence quantitative premix kit. The data were processed by relative quantitative method. The primer sequence was designed by Primer 5.0 software (Table 1).

2.14 Live/Dead staining in vitro

The cell culture and grouping conditions were consistent with those described in section “2.10.2″. Cells in each group were incubated with Calcein AM (2 μM) for 30 min, PI (5 μM) were incubated for 15 min in the absence of light. The fluorescence ratio of live/dead cells was detected by a fully automated cell imaging system.

2.15 Hemolysis experiment

Fresh blood was collected from black mice, followed by centrifugation at 4 °C °C and 3000 rpm for 10 min. The resulting supernatant was then discarded. The red blood cells were washed with PBS for 3 times, and 5 mL of PBS was added to make a 2 % red blood cell suspension. MIL-101 and MIL-101/PEG-FA were accurately weighed and dissolved in ultrasonic solution to prepare a 1 mg/mL stock solution. Subsequently, the stock solutions of both groups were further diluted to obtain working solutions with concentrations of 25, 50, 100, 200, 400, and 1000 μg/mL. Deionized water was used as positive control group, and PBS was used as negative control group. 200 µL erythrocyte suspension containing working solution shaken at 37 °C (100 rpm) for 8 h, centrifuged at 4 °C at 3000 rpm for 10 min, and 200 µL supernatant was absorbed and added into a 96-well plate. The absorbance at 541 nm was detected by a microplate reader. The experiment was repeated 3 times, and the HP was calculated as follows: HP (%) = (A_s-A_c2) / (A_c1-A_c2) × 100.

HP was hemolysis percentage; A_s represented the absorbance value of supernatant in each experimental group, A_c1 and A_c2 represented the absorbance value at 541 nm in positive control group and negative control group, respectively.

2.16 Construction of tumor model

BALB/c male nude mice (4–6 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd. All animal experiments were approved by the Laboratory Animal Ethics Review Committee of Tianjin University of Traditional Chinese Medicine. For the mouse oral cancer model, the BALB/c male nude mice were subcutaneously injected 100 μL of CAL27 cells (1 × 10⁷/mL) to construct the model. The mice were experimented until the tumor volume reached 80–100 mm³. The tumor-bearing mice were randomly divided into control, BER, and MIL-101@BER/PEG-FA groups for in vivo experiments.

2.17 Statistical analysis

All data were expressed as mean ± SD of at least three independent experiments. ANOVA test was used to compare the significance of the difference between the two groups (P<0.05).

3.1 Single factor and response surface analysis

The results of single factor experiment were shown in Fig. S1. The drug loading efficiency (Y) of MIL-101 were analyzed by using Design-Expert12 software. The regression equation model and the significance test results of each factor were shown in Table S1. The quadratic multiple regression equation was Y (drug loading efficiency) = 33.33–3.97X₁ + 0.3014X₂ + 1.82X₃ + 0.7084X₁X₂ − 0.5038X₁X₃ + 2.16X₂X₃ − 5.52X₁² − 4.11X₂² − 2.80X₃² (P<0.01, R² = 0.9039). (X₁: drug loading ratio, X₂: time, X₃: temperature). The predictive fitting degree R² was −0.3257 and the corrected fitting degree R² was 0.7803, the predictive fitting degree was ≤ the corrected fitting degree, indicating that the model was accurate and could be used for the analysis and prediction of drug loading results. The results of Tab. S1 showed that X₁, X₁², and X₂² had significant differences (P<0.05). As shown in Fig. S2, the three-dimensional response surface had a large radian, which was consistent with the results of variance analysis of the model.

3.2 Synthesis and characterization of the nanocomposites

MIL-101 was observed under TEM as a regular octahedron with an average particle size of 162.09 nm (Fig. 1A). After the surface modification of MIL-101 with PEG-FA, MIL-101/PEG-FA retained its octahedral structure identical to that of MIL-101 by TEM, while exhibiting an increased average particle size of 185.16 nm (Fig. 1B). In addition, the hydrodynamic particle sizes of MIL-101 and MIL-101/PEG-FA detected by DLS were 189.68 and 219.04 nm, respectively (Fig. 1C). Due to the high scattering intensity of DLS on larger particles, the disparity between TEM and DLS results arises from variations in particle solubility in water, leading to disparate dispersion characteristics. Moreover, the potential of MIL-101 particles was 34.81 mV, and the potential of MIL-101/PEG-FA decreased to −8.58 mV after PEG-FA surface modification (Fig. 1D). The potential of FA and BER was −27.95 and −0.15 mV, respectively (Fig. S3).

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Fig. 1

Characterization of MIL-101/PEG-FA. (A) and (B) TEM images of MIL-101 NPs and MIL-101/PEG-FA NPs. (C) Particle size and (D) Surface charge of NPs detected by DLS. (E) XRD pattern, (F) FT-IR spectra, (G) TGA curves, and (H) UV–vis of nanomaterial drug loading of nanocomposite. (I) Drug release characteristic of MIL-101@BER and MIL-101@BER/PEG-FA at pH 5.0 and 7.4.

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As shown in Fig. 1E, after surface modification of PEG-FA, the characteristic diffraction peaks of MIL-101/PEG-FA are basically the same as those of MIL-101, indicating that modification of PEG-FA on the surface of MIL-101 does not affect the crystal structure.

The spectral changes of FA, NH₂—PEG—COOH, MIL-101-NH₂, FA-PEG-COOH and MIL-101/PEG-FA were detected by FT-IR. Fig. 1F showed that the C—N and C—O stretching vibration of carboxylate appeared at 1295, 1405 and 1511 cm⁻¹, indicating the presence of 2-aminoterephthalate anion in the skeleton. N—H stretching vibration appeared at 3237 and 3455 cm⁻¹, indicating the presence of primary amine groups (Liu et al., 2018; Liu et al., 2022). 1623, 1070, and 1529 cm⁻¹ correspond to the —COOH, C—O—C, and —NH₂ of FA, respectively. 1743, 1068, and 1614 cm⁻¹ correspond to the —COOH, C—O—C and —NH₂ of NH₂—PEG—COOH, respectively. MIL-101/PEG-FA had a C—O—C characteristic peak corresponding to PEG and FA near 1070 cm⁻¹, a carbonyl characteristic peak at 1635 cm⁻¹, and an enhanced amide I band. These spectral performances indicated that FA-PEG-COOH was successfully connected to the surface of MIL-101-NH₂ through amide bonds. Meanwhile, C—O—C characteristic peaks and C—N stretching vibration peaks of BER appear at MIL-101@BER at 1074 and 1353 cm⁻¹, indicating that MIL-101 was successfully loaded BER (Fig. S4).

TGA was used to detect the thermogravimetric changes of MIL-101. Fig. 1G showed that the weight of MIL-101 changed significantly in the range of 0 − 200 °C °C and 250 − 500 °C, and the decrease may be related to the loss of solvent and skeleton dissociation in the nanoparticles. After PEG-FA modification, the TG of MIL-101/PEG-FA decreased by 18.8 % and 29.05 %, respectively. At 550 − 600 °C, the TG of MIL-101/PEG-FA decreased by 10.61 %, which may be related to the decomposition of PEG-FA.

The ultraviolet absorbance of MIL-101, MIL-101@BER and MIL-101@BER/PEG-FA were detected by UV–vis. MIL-101 showed a broad peak in the range of 300–400 nm, and a strong peak at 345 nm after MIL-101 and MIL-101/PEG-FA loaded BER, indicating successful embedding of BER (Fig. 1H). Furthermore, we detected the ultraviolet absorbance of MIL-101, FA and MIL-101/PEG-FA to analysis whether FA was successfully modified on the surface of MIL-101. Fig. S5 showed that FA and MIL-101/PEG-FA had a strong peak at 283 nm, MIL-101 showed no peak.

In order to analyze the effect of modified PEG-FA on the release and pH responsiveness of MIL-101 loaded BER, the release of BER in MIL-101@BER and MIL-101@BER/PEG-FA was detected in PBS solutions (pH 7.4 and pH 5.0), respectively. As shown in Fig. 1I, the coating of PEG-FA on MIL-101 could prevent premature drug release. At pH 5.0, the release rate of MIL-101@BER was 7.08 % lower than that of MIL-101@BER/PEG-FA within 12 h, and the release rate of MIL-101@BER/PEG-FA reached a relative equilibrium state (70.22 %) in 96 h. The release rate of MIL-101@BER in PBS (pH 7.4 and pH 5.0) was 31.80 % and 54.35 %, respectively. The release amount of MIL-101@BER was higher than that of MIL-101@BER/PEG-FA (pH 7.4), which was related to the pH responsiveness of PEG-FA. The above results demonstrated the potential application value of MIL-101@BER/PEG-FA as a pH-responsive nanocarrier platform.

3.3 Cell experiments

3.3.1

3.3.1 Cytotoxicity and cellular uptake

The cytotoxicity of nanocarriers MIL-101 and MIL-101/PEG-FA in CAL27 and KB cells was evaluated by CCK-8 method. As shown in Fig. 2 (A, B, C and D), all nanocarriers in the concentration of 0–200 μg/mL, the survival rates of CAL27 and KB cells were above 80 % at 24 and 48 h, indicating that MIL-101/PEG-FA had good biocompatibility and low toxicity.

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Fig. 2

Cell viability of CAL27 and KB cells cultured with different concentrations of NPs for (A C) 24 h and (B D) 48 h. (E) and (F) Cell viability of CAL27 cells cultured with different concentrations of MIL-101@Ber and MIL-101@Ber/PEG-FA (24 and 48 h).

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The anti-oral cancer effects of MIL-101@BER and MIL-101@BER/PEG-FA were examined. As shown in Fig. 2 (E and F), the activity of CAL27 was effectively inhibited in a concentration-dependent manner by MIL-101@BER and MIL-101@BER/PEG-FA, leading to a gradual decrease in the survival rate of CAL27 cells with increasing concentration. The cell survival rates of MIL-101@BER and MIL-101@BER/PEG-FA (100 μg/mL) were 70.30 % and 63.12 %, respectively, at 24 h; the survival rates decreased to 48.23 % and 38.94 %, respectively, at 48h. The anti-cancer efficacy of MIL-101@BER/PEG-FA was enhanced, attributed to its pH-responsive behavior that facilitated the expedited release of BER within the TME.

To investigate the targeting efficiency of MIL-101/PEG-FA as a nanocarrier, we substituted BER with the fluorescent probe “R6G” to assess the cellular uptake of MIL-101@R6G/PEG and MIL-101@R6G/PEG-FA in CAL27 and KB cells. As shown in Fig. 3 (A and B), the red fluorescence intensity in the cells of MIL-101@R6G/PEG and MIL-101@R6G/PEG-FA groups exhibited a time-dependent increase, indicating temporal dependence in the cellular uptake of the material by CAL27 and KB cells (Fig. S6). The intracellular red fluorescence intensity of MIL-101@R6G/PEG-FA group was significantly enhanced (P<0.05) compared to MIL-101@R6G/PEG, indicating the targeted effect of FA and its promotion on the uptake of MIL-101@R6G/PEG-FA by CAL27 and KB cells. Therefore, MIL-101/PEG-FA can serve as a promising targeted delivery system for OSCC treatment.

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Fig. 3

The cellular uptake of materials by CAL27 and KB cells. (A and B) Fluorescence images of CAL27 cells incubated with MIL-101@R6G/PEG and MIL-101@R6G/PEG-FA for 1, 2, 4, and 6 h. (C and D) Quantification of the mean fluorescence intensity of CAL27 and KB cells. (MIL-101@R6G/PEG vs. MIL-101@R6G/PEG-FA, *P<0.05). Scar bar = 100 μm.

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3.3.2

3.3.2 MIL-101@BER/PEG-FA promoted oxidative damage

GSH is the most important antioxidant to maintain tumor redox homeostasis. Excessive GSH in the TME can scavenge ·OH, reduce the level of oxidative stress, and protect the antioxidant capacity of tumor cells. Therefore, consuming GSH to promote ·OH production, aggravate oxidative stress and promote apoptosis of tumor cells is an effective method for the treatment of tumors. As shown in Fig. 4A, after coculture with MIL-101@BER/PEG-FA, the intracellular abundance of GSH in the tumor microenvironment effectively reduced Fe³⁺ to Fe²⁺, leading to significant consumption of GSH. This synergistic effect surpassed that of MIL-101@BER and free BER (P<0.001). Meanwhile, Fe²⁺ mediated Fenton reaction can induce the generation of ROS and Fe³⁺.

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Fig. 4

Oxidative stress indicators levels of CAL27 cells incubated with different groups. (A) GSH, (B) SOD, (C) MDA and (D) H₂O₂ levels of CAL27 cells. (E) The changes of MB concentration in MIL-101@BER and MIL-101@BER/PEG-FA. (F) and (G) ROS fluorescence image and average fluorescence degree. (H) and (I) TNF-α and IL-1β levels in CAL27 cells. Values are represented as mean ± standard ((Vs. Control, *P<0.05, ^**P<0.005, ^***P<0.001, Vs. 5-FU, ^#P<0.05, ^##P<0.005, ^###P<0.001). Scar bar = 100 μm.

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SOD can remove oxygen free radicals and reduce the damage caused by oxygen free radicals. As a biomarker for lipid peroxidation, MDA serves as an indicator of the extent of cellular damage caused by ROS. The cellular levels of SOD and MDA can serve as indirect indicators of in vivo oxygen free radical metabolism, enabling the assessment of cellular oxidative stress levels. It was observed a significant reduction of SOD in MIL-101@BER/PEG-FA-treated CAL27 cells (P<0.001) (Fig. 4B). Moreover, the content of lipid peroxidation index MDA was detected, the results showed that MIL-101@BER/PEG-FA promoted MDA accumulation with statistically significant differences (P<0.001) (Fig. 4C). The decreased concentration of SOD and the accumulation of MDA further exacerbated the intracellular oxidative imbalance.

H₂O₂, as the main component of ROS, has toxic effects on cells and can be used as the main target for tumor specific diagnosis and treatment·H₂O₂ concentration was significantly increased in CAL27 cells treated with 5-FU, BER, MIL-101@BER and MIL-101@BER/PEG-FA (P<0.05) (Fig. 4D), and MIL-101@BER/PEG-FA had the most significant effect, which effectively promoted the accumulation of H₂O₂, and Fe²⁺ converts H₂O₂ into more toxic ·OH. As shown in Fig. 4E, the concentration of MB in MIL-101@BER and MIL-101@BER/PEG-FA solution decreased by 8.97 % and 13.72 % respectively, which might be associated with the dissociation of Fe³⁺ from MIL-101 and subsequent reduction of Fe²⁺ facilitated by GSH. Thus, more ·OH was produced, which promoted the degradation of MB.

The TME exhibits a robust redox homeostasis, which, when disrupted, leads to tumor cell damage and apoptosis induction. Elevated levels of ROS in tumor tissues are essential for maintaining normal physiological processes. However, excessive ROS concentrations can disrupt redox homeostasis and facilitate tumor cell death (Kumari et al., 2018). MIL-101@BER/PEG-FA had effectively consumed high concentration of intracellular GSH, regulated SOD and MDA activities, promoted H₂O₂ production in CAL27 cells, and caused redox homeostasis imbalance. As shown in Fig. 4 (F, G), It was observed that co-culturing with MIL-101@BER/PEG-FA led to a significant and statistically meaningful increase in intracellular ROS levels (P<0.001). The production of ROS could also be promoted by MIL-101@BER; however, its impact was significantly inferior to that of MIL-101@BER/PEG-FA due to the absence of targeting and pH responsiveness. In the TME, MIL-101@BER/PEG-FA demonstrated pH responsiveness and targeting capabilities, thereby augmenting the therapeutic efficacy of BER. Therefore, we concluded that MIL-101@BER/PEG-FA consumed GSH and produced Fe²⁺ through the dissociated Fe³⁺, which down-regulated SOD level and induced the generation of peroxide product MDA. Fe²⁺ promoted the generation of ROS, significantly damaged the redox homeostasis in tumor cells, and greatly increased the level of intracellular oxidative stress, ultimately leading to tumor cell damage.

3.3.4

3.3.4 MIL-101@BER/PEG-FA aggravated mitochondrial damage

The mitochondria play a pivotal role in the generation of ROS, redox molecular metabolites, regulation of cellular signaling and apoptosis, as well as bioanabolism. Excessive ROS can damage DNA structure and cause mitochondrial dysfunction, thus inducing cell apoptosis (Xie et al., 2022). As a result, ROS-induced apoptosis is often accompanied by disruption of the linear membrane potential. High intracellular ROS levels can lead to oxidative stress, which can lead to mitochondrial damage. The normal growth of cancer cells is closely related to mitochondrial homeostasis, and the decrease of MMP means that the cell is about to die. We employed the JC-1 probe to localize within CAL27 cells for monitoring alterations in MMP under distinct treatments. Subsequently, a remarkable enhancement in green fluorescence was observed upon MMP reduction, facilitating the analysis of cellular mitochondrial damage. As shown in Fig. 5, after the co-incubation of 5-FU, BER, MIL-101@BER and MIL-101@BER/PEG-FA with CAL27 cells, the green fluorescence intensity increased, the intracellular red fluorescence intensity decreased. Consequently, the MMP underwent a reduction, leading to mitochondrial dysfunction. MIL-101@BER/PEG-FA effectively targeted CAL27 cells, facilitating the release of BER in the TME, which significantly exacerbated intracellular oxidative stress damage and induced mitochondrial dysfunction.

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Fig. 5

Effects of MIL-101@BER/PEG-FA on MMP of CAL27 cells. Values are represented as mean ± standard ((Vs. Control, *P<0.05, ^**P<0.005, ^***P<0.001, Vs. 5-FU, ^#P<0.05, ^##P<0.005, ^###P<0.001). Scar bar = 100 μm.

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3.3.5

3.3.5 MIL-101@BER/PEG-FA activated autophagy by regulating LKB1/AMPK pathway

To explore the mechanism of MIL-101@BER/PEG-FA in the treatment of OSCC, we detected the expressions of LKB1, AMPK, mTOR, ATG5, Beclin-1, Caspase 3 and Caspase 9 genes in CAL27 cells, and analyzed the role of LKB1/AMPK pathway in the treatment of OSCC. The LKB1/AMPK pathway plays a crucial role in the regulation of apoptosis and autophagy, with LKB1 promoting autophagy through AMPK activation (Park et al., 2023). Moreover, AMPK exerts its tumor-suppressive effects by enhancing the p53 pathway, inhibiting the mTOR pathway, and facilitating apoptosis among other mechanisms (Luo et al., 2005). The results demonstrated a significant upregulation of LKB1 and AMPK genes in CAL27 cells treated with 5-FU, BER, MIL-101@BER, and MIL-101@BER/PEG-FA (P<0.05) (Fig. 6). Additionally, the expression of mTOR gene was significantly inhibited (P<0.001), leading to an upregulation of autophagy-related genes (ATG5, Beclin-1) and apoptosis-related genes (Caspase 3, Caspase 9). Consequently, autophagy was activated to induce apoptosis in CAL27 cells. Meanwhile, it was evident that MIL-101@BER/PEG-FA effectively facilitated the targeted delivery of BER to CAL27 cells and augmented its anti-tumor efficacy by activating mitochondrial autophagy and inducing apoptosis in CAL27 cells through modulation of the LKB1/AMPK pathway.

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Fig. 6

Analysis of LKB1/AMPK pathway in MIL-101@BER/PEG-FA-treated CAL27 cells. Values are represented as mean ± standard ((Vs. Control, *P<0.05, ^**P<0.005, ^***P<0.001, Vs. 5-FU, ^#P<0.05, ^##P<0.005, ^###P<0.001).

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3.3.6

3.3.6 Effect of MIL-101@BER/PEG-FA on apoptosis of CAL27 cells

To further evaluate the antitumor effect of MIL-101@BER/PEG-FA. We analyzed the killing effect of MIL-101@BER/PEG-FA on CAL27 cells using Calcein AM/PI staining to assess cell viability and mortality rates. In Fig. 7, after treating CAL27 cells with 5-FU, BER, MIL-101@BER, MIL-101@BER/PEG-FA, the intracellular red fluorescence intensity significantly increased, indicative of an augmented incidence of cellular deaths. MIL-101@BER/PEG-FA increased the concentration of BER in tumor cells and induced the expression of apoptosis-related genes Caspase 3 and Caspase 9. The increase of dead cells indicated that MIL-101@BER/PEG-FA promoted cells death.

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Fig. 7

Calcein AM and propidium iodide (PI) staining of CAL27 cells in different groups. Scar bar = 100 μm.

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3.4 Biocompatibility of MIL-101/PEG-FA

In order to further validate the in vitro biosafety of MIL-101/PEG-FA nanoparticles, we conducted a hemolysis test to assess their potential toxicity. As shown in Fig. 8, the hemolysis rates of MIL-101 and MIL-101/PEG-FA nanoparticles were detected in the concentration range of 25–1000 µg/mL. The hemolysis rates of 25 µg/mL were 0.59 % and 1.94 %, respectively. The hemolysis rates of 1000 µg/mL were 4.29 % and 4.83 %, respectively, and the hemolysis rates were less than 5 %. Therefore, MIL-101 and MIL-101/PEG-FA exhibited favorable biocompatibility, thereby establishing a solid foundation for subsequent animal experimentation.

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Fig. 8

Hemolytic effects of MIL-101 and MIL-101/PEG-FA.

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3.5 Antitumor efficacy assays in vivo

To evaluate the in vivo anti-tumor effect of MIL-101@BER/PEG-FA, we constructed CAL27 tumor-bearing mice. Nude mice were divided into three groups of PBS, BER, and MIL-101@BER/PEG-FA, and the drug was injected around the tumors. The treatment effect was shown in Fig. 9, after 10 days of treatment, the tumor volume was 537.57 mm³ in the control group. BER and MIL-101@BER/PEG-FA were effective in inhibiting the tumor growth as compared to the control group, with the tumor volume being 204.54 and 152.66 mm³, respectively. Meanwhile, the nude mice treated with MIL-101@BER/PEG-FA showed slow tumor growth and the strongest inhibitory effect, which further determined that FA was effectively targeted to promote the release of BER in the tumor tissues, thus achieving the anti-tumor effect.

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Fig. 9

In vivo anticancer effect of the MIL-101@BER/PEG-FA in CAL27 tumor-bearing mice. (A) Tumor images of mice in control, BER, and MIL-101@BER/PEG-FA after 10 days of treatment. (B) Heart, liver, spleen, lungs, kidneys and tongue images of mice in control, BER, and MIL-101@BER/PEG-FA after 10 days of treatment. (C) Tumor growth curves in 10 days of treatment. (D) Changes of body weight of tumor-bearing mice during treatment.

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