Folic Acid Decorated Zeolitic Imidazolate Framework (ZIF-8) Loaded with Baicalin as a Nano-Drug Delivery System for Breast Cancer Therapy

Xiao Mi; Meigeng Hu; Mingran Dong; Zhihong Yang; Xia Zhan; Xinyue Chang; Juan Lu; Xi Chen

doi:10.2147/IJN.S340764

. 2021 Dec 24;16:8337–8352. doi: 10.2147/IJN.S340764

Folic Acid Decorated Zeolitic Imidazolate Framework (ZIF-8) Loaded with Baicalin as a Nano-Drug Delivery System for Breast Cancer Therapy

Xiao Mi ¹, Meigeng Hu ¹, Mingran Dong ¹, Zhihong Yang ¹, Xia Zhan ², Xinyue Chang ¹, Juan Lu ¹, Xi Chen ^1,^✉

PMCID: PMC8714011 PMID: 34992370

Abstract

Background

Baicalin (BAN) has attracted widespread attention due to its low-toxicity and efficient antitumor activity, but its poor water solubility and low bioavailability severely limit its clinical application. Development of a targeted drug delivery system is a good strategy to improve the antitumor activity of baicalin.

Methods

We prepared a BAN nano-drug delivery system PEG-FA@ZIF-8@BAN with a zeolite imidazole framework-8 (ZIF-8) as a carrier, which can achieve the response of folate receptor (FR). We characterized this system in terms of morphology, particle size, zeta-potential, infrared (IR), ultraviolet (UV), x-ray diffraction (XRD), and Brunel-Emmett-Teller (BET), and examined the in vitro cytotoxicity and cellular uptake properties of PEG-FA@ZIF-8@BAN using MCF-7 cells. Lastly, we established a 4T1 tumor-bearing mouse model and evaluated its in vivo anti-mammary cancer activity.

Results

The PEG-FA@ZIF-8@BAN nano-delivery system had good dispersion with a BAN loading efficiency of 41.45 ± 1.43%, hydrated particle size of 176 ± 8.1 nm, Zeta-potential of −23.83 ± 1.1 mV, and slow and massive drug release in an acidic environment (pH 5.0), whereas release was 11.03% in a neutral environment (pH 7.4). In vitro studies showed that PEG-FA@ZIF-8@BAN could significantly enhance the killing effect of BAN on MCF-7 cells, and the folic acid-mediated targeting could lead to better uptake of nanoparticles by tumor cells and thus better killing of cancer cells. In vivo studies also showed that PEG-FA@ZIF-8@BAN significantly increased the inhibition of the proliferation of solid breast cancer tumors (p < 0.01 or p < 0.001).

Conclusion

The PEG-FA@ZIF-8@BAN nano-drug delivery system significantly enhanced the anti-breast cancer effect of baicalin both in vivo and in vitro, providing a more promising drug delivery system for the clinical applications and tumor management.

Keywords: metal-organic framework, folic acid response, baicalin, breast cancer therapy

Introduction

Breast cancer, one of the most common cancers in women, is characterized by high morbidity, poor prognosis, and limited treatment options. Chemotherapy for breast cancer is usually administered in the form of a single antitumor agent, and side effects and drug resistance are major challenges.¹ Moreover, the high cost of most clinical anti-cancer drugs places a significant financial burden on patients and their families.² There is an urgent need for novel antitumor treatment options that have less toxicity, are more resistant to drug resistance, and are more affordable.³^,⁴ Targeted nano drug delivery systems have shown advantages over conventional drug treatment modalities due to their ability to enhance drug aggregation at the tumor site and specific drug delivery modalities. However, most of the drug carriers currently used suffer from inadequate long-term safety, poor biocompatibility, high manufacturing costs, and limited drug delivery efficiency,⁵ hence, developing stable drug delivery systems with targeted anticancer effects with low toxicity and good biocompatibility is key to improving the effectiveness of breast cancer treatment.

BAN is one of the main active ingredients in Scutellaria baicalensis Georgi, family Labiatae, with pharmacological effects such as antibacterial, anti-inflammatory, antihypertensive, and anti-allergic.⁶^,⁷ The literature reports the prominent antitumor activity of BAN against a variety of tumor types, such as colon cancer,⁸^,⁹ liver cancer,¹⁰ bladder cancer¹¹ and glioblastomas.¹² The anti-tumor effects are mainly reflected in inducing apoptosis, cell cycle arrest, and autophagy, and inhibiting angiogenesis and metastasis.¹³^,¹⁴ Studies have shown that BAN could induce tumor cell apoptosis through a variety of pathways.¹⁵ BAN could induce apoptosis and suppress migration/invasion of MDA-MB-231 cells via ROS-mediated activation of the p38/JNK signaling pathway.¹⁶ It also confirmed that BAN-induced apoptotic cell death in breast cancer cells involves the up-regulation of proapoptotic p53 and bax.¹⁷ However, the presence of sugar groups in the molecular structure of BAN leads to its low hydrophilicity (solubility 0.052 mg/mL in water) and lipophilicity (Papp = 0.037×10⁻⁶ cm/s),¹⁸ and its bioavailability after oral administration is only 2.2%,¹⁹ all of which limit its clinical application. However, the preparation of BAN into novel nano formulations can modify its physical properties and improve its bioavailability.

Metal-organic frameworks (MOFs) have high surface area, high porosity, tunable pore structure, and easy surface functionalization. They have great potential for applications in such areas as biosensing, bioimaging, and drug delivery.²⁰^,²¹ In recent years, zeolite imidazole framework-8 (ZIF-8) has received much attention as an emerging class of nano metal-organic frameworks (NMOFs). ZIF-8 is characterized by a simple synthetic strategy, easy functionalization, high loading capacity, and pH-responsive degradation.²² Due to these characteristics, nanoparticles of ZIF-8 have been widely used for DNA, protein, and drug delivery.^23–26 Therefore, the potential of ZIF-8-based nano drug delivery systems for hydrophobic drug-targeted intracellular delivery is worth exploring.

Folate is one of the most widely used small molecules in the study of targeted drug delivery systems and has an important role in the synthesis of DNA and RNA.²⁷ In normal tissue cells, folate enters the cell by transmembrane, while in cancer cells, folate receptors on the cell surface recognize folate and form folate complexes, at which point endocytic vesicles are formed to enter the cancer cell by internalization, which is the main way that folate molecules recognize and enter cancer cells.²⁸^,²⁹ Based on the fact that folate enters normal cells and tumor cells in different ways, it can be modified to the carrier surface as a targeting molecule, so that the drug delivery system can selectively enter tumor cells and release the loaded drug in the cytoplasm to play the role of anti-cancer agent in biomedicine and achieve targeted therapy for tumors.³⁰ The current research on folic acid focuses on folic acid polymer targeted drug delivery systems, such as the folic acid polyethylene glycol drug delivery system,³¹^,³² folic acid amphiphilic block copolymers³³^,³⁴ and so on. Many reports have shown that folic acid modified nanoparticles can target many kinds of cancer cells including breast cancer cells.^35–37 Human breast cancer cell MCF-7, as a FR overexpression cell line, has been used to evaluate various folate targeted nanoscale platforms.^38–41

Based on the idea suggested, we prepared a BAN nano delivery system using ZIF-8 as a carrier and Polyethylene glycol–folate (PEG-FA) as a target modification group. ZIF-8 was synthesized by coordination of 2-methylimidazole with Zn²⁺(molar ratio 4:1) and loaded with BAN by stirring at room temperature, followed by PEG-FA targeting modification on its surface to obtain PEG-FA@ZIF-8@BAN. Then the morphology, particle size, zeta-potential, IR, UV, XRD, and BET were characterized, and the release characteristics were investigated. In vitro cytotoxicity and cellular uptake studies of PEG-FA@ZIF-8@BAN were performed using MCF-7 cells, and their biocompatibility was evaluated. Finally, 4T1 tumor-bearing mouse model was established to study the in vivo anti-breast cancer activity of PEG-FA@ZIF-8@BAN. The nano drug delivery system can realize the response of folate receptor to increase the anti-tumor effect of the drug, which has a good development prospect.

Materials and Methods

Reagents and Instrument

Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O,99%) was purchased from Tianjin FuChen Chemical Reagent Co., Ltd, China. 2-methylimidazole (C₄H₆N₂,98%) was obtained from TCI Development Co., Ltd, China. BAN was purchased from Shanghai Winherb Medical Technology Co., Ltd,China. mPEG-FA was purchased from Shanghai Ponsure Biotechnology Co., Ltd, China. Fetal bovine serum was obtained from PAN Seratech GmbH, Germany. Anti-folate primary mouse monoclonal antibody (sc-515521) was purchased from Santa Cruz Biotechnology, Inc (Dallas, Texas). Goat Anti-Mouse secondary antibody conjugated to Alexa Fluor 488 was purchased from Shanghai Beyotime Biotechnology Co., Ltd, China. The absorbance was detected by TECAN spark multifunctional microplate reader. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Fluorescence images were taken using a Nikon Eclipse Ts2R fluorescence inverted microscope. Confocal laser scanning microscopy (CLSM) images were taken on Leica SP8. The loading efficiency and release rate were measured by Agilent Technologies Cary series UV-vis spectrophotometer. The blood biochemical indexes of the mice were detected by a Beckman Kurt AU480 automatic biochemical analyzer.

Cell Lines and Animals

Human breast cancer cell line MCF-7 cells and mouse epithelioid fibroblast L929 cells were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All cells were cultured at 37°C in 5% CO₂.

Healthy 20–22g BALB/c mice were provided by SBF (Beijing) experimental animal Technology Co., Ltd. All animals were housed in a facility with a barrier system under the conditions as follows: temperature, 17–25°C, humidity, 45–80%, and 12 hours light/dark cycle. All procedures were approved by the research animal ethics committee of the Institute of Medicinal Plant Development affiliated to the Chinese Academy of Medical Sciences (Beijing, China, Ethical review No. SLXD-20210621019). 2.5–3 kg New Zealand white rabbits were purchased from Beijing Fang Yuan farm and fed normally in the rabbit laboratory. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Synthesis of PEG-FA@ZIF-8@BAN

Synthesis of ZIF-8: 2.975 g of zinc nitrate hexahydrate and 3.284 g of 2-methylimidazole (molar ratio 1:4) were weighed precisely, dissolved in methanol, and stirred at 1000 rpm for 1.5 hours, and after standing for 24 hours, the precipitate was collected by centrifugation at 8000 rpm for 10 minutes, and then washed three times with methanol and dried under vacuum at 60°C to obtain ZIF-8.

Synthesis of ZIF-8@BAN: ZIF-8 was re-dispersed in methanol by ice bath sonication for 20 minutes, and then a certain amount of BAN was sonicated and dissolved in methanol; the two solutions were mixed and stirred at 600 rpm at room temperature for 12 hours. The precipitate was collected by centrifugation at 8000 rpm for 10 minutes, and then the methanol was washed three times and ZIF-8@BAN was obtained by vacuum drying at 60°C. The optimal loading ratio was investigated by controlling the feeding amount of BAN.

Synthesis of PEG-FA@ ZIF-8@BAN: 50 mg of PEG-FA was dissolved in 5 mL of deionized water solution; 50 mg of ZIF-8@BAN was added and stirred for 48 hours at room temperature and protected from light, and the resulting precipitate was washed three times with deionized water and centrifuged at 11,000 rpm. The resulting solid was dried under vacuum to obtain PEG-FA@ZIF-8@BAN.

Characterization of Nanoparticles

Transmission Electron Microscopy (TEM) micrographs were obtained from a FEI Talos F200X transmission electron microscope. Scanning electron microscopy (SEM) images were obtained using a ZEISS Sigma 500 scanning electron microscope. Size and zeta potential measurements of ZIF-8, ZIF-8@BAN and PEG-FA@ZIF-8@BAN were measured by Malvern Zetasizer Nano ZS90. Each nanoparticle system was measured three times. XRD were recorded on a Rigaku Smartlab SE diffractometer. The UV–vis absorption spectra were determined by a PE-Lambda 950 spectro photometer. FT-IR was collected on a Thermo Scientific Nicolet iN10 spectro photometer using KBr pellets. Nitrogen adsorption/desorption analysis was obtained from Micromeritics ASAP 2460.

Determination of Drug Loading and Drug Release

The drug loading efficiency (DLE%) and entrapment efficiency (EE%) was measured by UV spectrophotometer at 278 nm absorption wavelength, and the standard curve of the BAN solution was established in the concentration range of 5–40 μg/mL. The DLE% and EE% were calculated using the following equations:

Where: Inline graphic is the weight of the total drug added, is the weight of the drug loaded in the nanoparticles and is the total weight of the nanoparticles.

The in vitro release rate of the drug was also measured using a UV spectrophotometer. The dynamic membrane dialysis method was used to investigate the in vitro release characteristics of the nanosystem. ZIF-8@BAN and PEG-FA@ZIF-8@BAN were dispersed in phosphate-buffered saline (PBS) buffer solutions of different pH (7.4 and 5.0), and then placed in the processed dialysis bag (molecular weight cut off 3500 Da). The dialysis bag was placed in a release tube containing 10 mL of PBS with the corresponding pH, and then the tubes were placed in a water bath shaker at 37°C and gently shaken at 100 r/min; 0.5 mL of supernatant was removed at the specified time and supplemented with an equal volume of fresh PBS, diluted at appropriate multiples and assayed in the absorbance range, and finally, the cumulative release rate of the drug was calculated. The Korsmeyer-Peppas model was used to fit the drug release curve.

Immunofluorescence for FR Expression

Cells were seeded in a 6-well chamber slide at a density of 5×10⁵ cells/mL, 2 mL/well. After overnight incubation in a humidified chamber at 37°C, cells were washed with ice-cold 1 × PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After removing paraformaldehyde, the cells were washed three times with 1 × PBS for 3 min each, and blocked for 30 min with 1 × PBS containing 10% goat serum. Cells were then incubated 2 h with mouse monoclonal anti-FR antibody at 37°C. After incubation, cells were washed repeatedly with 1 × PBS (3 times, 3 min each) and incubated with the fluorochrome-conjugated secondary antibody for 30 min at room temperature. Cells were washed three times in 1 × PBS and incubated with 0.5 μg/mL DAPI for 5 min. Cell fluorescence images were taken using a fluorescent inverted microscope.

Cytotoxicity Assay

MCF-7 cells were inoculated in 96-well plates at a density of about 6×10³ per well, and then incubated in a constant-temperature cell culture incubator at 37°C for 24 hours. The culture medium was removed and re-added with BAN, ZIF-8@BAN, PEG-FA@ZIF-8@BAN cell culture medium containing the same BAN concentration to continue incubation for 12 hours or 24 hours. The control group was incubated with culture medium only. After incubation, the medium was aspirated and discarded; 100 μL of Cell Counting Kit-8 (CCK-8) dilution solution was added, and the absorbance value of each well was measured at 450 nm using an enzyme marker at the end of 1 hour incubation in the incubator. To examine the in vitro compatibility of the nanomaterials, a blank PEG-FA@ ZIF-8 group was set up and the toxic effects of the blank material on the cells were examined using the cancer cell line MCF-7 and the normal cell line L929 cells. Cell viability was calculated according to the formula:

where Inline graphic is the experimental well, is the control well, and is the blank well.

Live and Dead Cell Staining

MCF-7 cells were inoculated in 12-well culture dishes at a concentration of 5×10⁴ cells per well and cultured for 24 hours. After cell apposition, fresh media containing BAN, ZIF-8@BAN, and PEG-FA@ZIF-8@BAN were replaced with 40 μg/mL of BAN in each group. Cells were treated with blank nanomaterials containing the same amount of PEG-FA@ZIF-8 to examine the biocompatibility of the material, and cells treated with PEG-FA preincubation for 2 hours were continued to examine PEG-FA@ ZIF-8@BAN bio targeting. After the interaction with the drug, the residual drug was washed off with PBS and 0.5 mL Calcein – AM/PI solution was added to stain the cells for 30 minutes, and cell fluorescence images were taken using a fluorescent inverted microscope. In the control group, no drug was added.

Cellular Uptake Experiments

MCF-7 cells were used to examine the cellular uptake behavior of PEG-FA@ZIF-8@BAN. Approximately 1×10⁵ MCF-7 cells were inoculated in 6-well culture plates and incubated for 24 hours in a cell culture incubator after apposition. ZIF-8@BAN, PEG-FA@ZIF-8@BAN solution, and PEG-FA@ZIF-8@BAN were added to cells pre-incubated with free PEG-FA, followed by incubation for 0.5 hour or 3 hours. At the set time point, the cells were washed several times with PBS to remove the residual nanomaterials. After fixation with 4% paraformaldehyde, Hoechst 33342 dye for staining the nuclei and 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA) for measuring intracellular reactive oxygen species (ROS) production were added for observation under a laser scanning confocal microscope.

Hemolytic Test

Blood was extracted by puncturing the hearts of New Zealand rabbits, and 2% erythrocyte suspension was prepared by stirring with a glass rod to remove fibrin. 2% erythrocyte suspension, PBS, and test solution were added, as specified, using PBS as a negative and purified water as a positive control, mixed well, and immediately placed in a constant temperature water bath at 37°C ± 0.5°C for warming. The hemolysis and coagulation reactions were observed after 3 hours, and the absorbance was measured at 414 nm using an enzyme marker.

In vivo Experiments

In vivo experiments were performed using 4T1 inoculated female BALB/c mice. 4T1 cells were dispersed in physiological saline at a density of 1×10⁶ cells/mL, and 0.2 mL of cell suspension was subcutaneously inoculated into the axilla of the right forelimb of mice to establish a mouse breast cancer model. When the tumor-bearing mice reached a tumor volume of approximately 100 mm³, they were randomly divided into 4 groups of 7 mice per group. Each group of mice was injected with PBS, BAN, ZIF-8@BAN, PEG-FA@ZIF-8@BAN solution in tail vein every two days for 15 days, and the tumor volume was measured every two days, and the tumor volume was V = L ×W²/2 (where L and W refer to the longest and shortest diameter of the tumor, respectively). The relative tumor volume was V/V₀ (V₀ was the initial tumor size of the mice carrying the tumor). After in vivo drug efficacy experiments were completed, blood was removed from the eyes of the mice and serum was collected for determination of blood biochemical parameters. The mice were dissected and weighed, and the tumors and major organs (heart, liver, spleen, lungs, and kidneys) were collected, followed by hematoxylin and eosin stain (H&E) and terminal deoxynucleotide transfer modification dUTP-biotin end-labeling (TUNEL) staining tests.

Statistics

Data are expressed as mean ± SD. Statistical evaluation was performed by two-tailed Student’s t-test or one-way ANOVA. ns represents no statistical significance, * is p < 0.05, ** is p < 0.01, and *** is p < 0.001.

Results and Discussion

Synthesis and Characterization of Nanoparticles

Scheme 1 demonstrates a simple synthetic strategy for the PEG-FA@ZIF-8@BAN nano delivery system and its application in cancer therapy. 2-methylimidazole and Zn²⁺ formed ZIF-8 by coordination, and then BAN was loaded into the high porosity ZIF-8 carrier by ambient stirring method, and finally PEG-FA was modified on the surface of the drug delivery system to achieve tumor targeting. When administered intravenously, PEG-FA ensures that the nano delivery system is not degraded under normal physiological conditions and is intelligently targeted for rapid enrichment in tumor cells with high expression of the folate receptor. By degrading the outer layer of PEG-FA in the acidic environment of reduced pH in cancer cells, the ligand bond of ZIF-8 is also gradually broken in the acidic environment, thus releasing the drug slowly. Thus far, we have successfully constructed a tumor-specific response drug delivery system.

The materials were synthesized and morphologically observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As can be seen in Figures 1Aa–c and S1, the SEM images of ZIF-8, ZIF-8@BAN, PEG-FA@ZIF-8@BAN show that the average diameters of the synthesized uniformly dispersed nanoparticles are 89.53 nm, 90.85 nm, and 96.52 nm, respectively. As shown in TEM image Figure 1Ad–f, the synthesized ZIF-8 material has a rhombic dodecahedral three-dimensional structure with sharp edges that is consistent with the literature. Compared with the obvious stereoscopic structure of ZIF-8, the morphology of ZIF-8@BAN and PEG-FA@ZIF-8@BAN gradually become less regular and the angles become blurred, indicating the successful loading and surface modification of the guest molecule by ZIF-8.

(A) SEM images of ZIF-8 (a, 300 nm), ZIF-8@BAN (b, 300 nm), and PEG-FA@ZIF-8@BAN (c, 300 nm) and TEM images of ZIF-8 (d, 50 nm), ZIF-8@BAN (e, 50 nm), and PEG-FA@ZIF-8@BAN (f, 50 nm). (B) Size distribution of DLS measured by ZIF-8 (a), ZIF-8@BAN (b), and PEG-FA@ZIF-8@BAN (c) (means ± SD, n = 3). (C) Zeta-potential of ZIF-8, ZIF-8@BAN and PEG-FA@ZIF-8@BAN (means ± SD, n = 3).

The hydrated particle sizes of ZIF-8, ZIF-8@BAN and PEG-FA@ZIF-8@BAN were determined by a Malvern Mastersizer. As shown in Figure 1Ba–c, the particle size measurements were 155 ± 2.9 nm, 161.13 ± 1.19 nm and 176 ± 8.1 nm, respectively. Each had a narrow size distribution and good water dispersion. The size of PEG-FA@ZIF-8@BAN was significantly larger than that of ZIF-8, which could prove the successful loading of the drug and modification of PEG-FA. The hydrodynamic particle size of the nanoparticles measured by DLS was slightly larger than that observed by SEM, which may be caused by the presence of a hydration layer when the nanoparticles were dispersed in an aqueous solution. Figure 1C shows the zeta-potentials of ZIF-8, ZIF-8@BAN and PEG-FA@ZIF-8@BAN, with measurements of 25.47 ± 0.47 mV, −26.03 ± 0.58 mV, and −23.83 ± 1.1 mV, respectively. Due to the presence of a large number of phenolic hydroxyl groups on the surface of BAN, the zeta-potential turned negative after loading BAN, and remained negative after PEG-FA modification due to the presence of carboxyl groups in PEG-FA.

The crystal structures of each product were investigated by XRD. The XRD patterns of ZIF-8, ZIF-8@BAN, PEG-FA@ZIF-8@BAN and PEG-FA are shown in Figure 2A. The results showed that both blank ZIF-8 and drug-loaded ZIF-8 solids had a certain degree of crystallinity, and the XRD pattern data were more consistent with the reported crystal structure data of ZIF-8, which proved the successful synthesis of ZIF-8.⁴² Rietveld analysis was performed on ZIF-8, ZIF-8@BAN, and PEG-FA@ZIF-8@BAN using TOPAS software. As shown in Figure S2, the crystal structure of ZIF-8 after loading drugs and PEG-FA modification did not change much. However, the intensity of the diffraction peak of PEG-FA@ZIF-8@BAN at low angles was reduced after PEG-FA modification, which might be due to the existence of some masking effect on the crystal structure by the amorphous PEG-FA coating on the nanoparticle surface.

(A) XRD patterns of ZIF-8, ZIF-8@BAN, PEG-FA@ZIF-8@BAN and PEG-FA. (B) UV absorption patterns of ZIF-8, BAN, ZIF-8@BAN, and PEG-FA@ZIF-8@BAN. (C) FT-IR spectra of prepared ZIF-8, ZIF-8@BAN, PEG-FA@ZIF-8@BAN, BAN and PEG-FA. (D) N₂ absorption-desorption isotherms (h) of the prepared ZIF-8 and PEG-FA@ZIF-8@BAN.

The UV spectra of ZIF-8, BAN, ZIF-8@BAN, and PEG-FA@ZIF-8@BAN were examined, and the absorption sites at the position of the 278 nm characteristic absorption peak of BAN were decreasing, indicating the successful encapsulation of BAN by PEG-FA@ZIF-8@BAN (Figure 2B). In addition, the modification process of the nanoplatform was further determined qualitatively by Fourier transform infrared (FT-IR) spectroscopy, as shown in Figure 2C, with the appearance of PEG-FA characteristic peaks at 3436 cm⁻¹ (OH), 2920 cm⁻¹ (-CH₂), 1624 cm⁻¹ (C=O), and 1200 cm⁻¹ (C-O-C) in the PEG-FA@ZIF-8@BAN spectrum, indicating the successful modification of PEG-FA. A comparison of the spectra of BAN and ZIF-8@BAN reveals that the characteristic peak of BAN at 1062 cm⁻¹ does not appear in ZIF-8@BAN, indicating that the internal loading of ZIF-8 on BAN physically shields the characteristic peak. In addition, the imidazole ring of 2-methylimidazole has two double bonds and a lone pair of electrons from the nitrogen atom, which can interact in the plane of the imidazole ring. Thus, the imidazole ring can be considered as an aromatic compound and the BAN containing the benzene ring can also be loaded on ZIF-8 by π-π stacking.⁴³ In summary, BAN was successfully loaded in ZIF-8 and successfully modified PEG-FA on the surface to achieve the “targeting device” effect.

Brunel-Emmett-Teller (BET) measurements were performed to evaluate the porosity of PEG-FA@ZIF-8@BAN nanostructures, with ZIF-8 evaluated under similar conditions as a control variable. As shown in Figure 2D, they both exhibit typical type I N₂ absorption-desorption isotherms, which are one of the main features of microporous materials.⁴⁴ The BET surface area of ZIF-8 was 1267 m²/g, while the surface area of PEG-FA@ZIF-8@BAN was reduced to 120 m²/g. In addition, the corresponding pore volumes of the nanostructures of PEG-FA@ZIF-8@BAN were 0.85 cm³/g and 0.21 cm³/g, respectively. The pore size distribution results (Figure S3) shown that the pore width of ZIF-8 was mainly concentrated in micropores, and the pore width of PEG-FA@ZIF-8@BAN was concentrated in mesopores (2–50 nm) or macropores. The possible reason was that the drug occupied the micropores resulting in the nitrogen gas being able to enter only into the mesopores or macropores. Therefore, the pore size of PEG-FA@ZIF-8@BAN measured by BET was larger than ZIF-8. The above results indicate the successful loading of BAN and the successful modification of PEG-FA.

In vitro Release of Drugs

In order to evaluate the loading and release behavior of ZIF-8 on BAN, we first established a standard curve of BAN solution with good linear relationship. It can be seen from Figure 3A that the absorbance of BAN at 278 nm shows an increasing trend with increasing concentration. As shown in Figure S4, the highest loading efficiency of ZIF-8@BAN was achieved when the mass ratio of ZIF-8 to BAN was 1:1. In addition, we repeatedly measured the loading efficiency of PEG-FA@ZIF-8@BAN and obtained the result of 41.45% ± 1.43%. We measured the entrapment efficiency of ZIF-8 for BAN as 65.83 ± 3.35%. Compared with other BAN nano-delivery systems, PEG-FA@ZIF-8@BAN has greater drug loading efficiency.⁴⁵^,⁴⁶

(A) Standard curve of BAN solution detected by UV spectrophotometer at 278 nm. (B) Release curves of BAN in PBS buffer solution at pH 5.0 or 7.4 (means ± SD, n = 3).

In addition, we also measured the in vitro release behavior of PEG-FA@ZIF-8@BAN in PBS buffered solutions of different pH; as shown in Figure 3B, there was no significant release of the drug within 24 hours measured in neutral PBS solution, and the release amount was 11.03%, from which it can be inferred that PEG-FA@ZIF-8@BAN has good stability in a normal physiological environment. In contrast, the ZIF-8 metal-organic framework structure was broken relatively quickly in the acidic environment and released the drug, with a final assay of up to 78.6% drug release. ZIF-8@BAN finally released 37.72% of the drug in a pH 7.4 environment. This may be due to the lack of protection of the PEG shell, which led to the cleavage of part of the metal-organic framework and the drug was released. The release of ZIF-8@BAN drug in an acidic environment finally reached 89.66%, indicating that the PEG-FA-modified nanoparticles have a better sustained-release effect. The Korsmeyer-Peppas model was used to fit the drug release curve. We found that the fitting results have a good linear relationship (R² ≥ 0.950), indicating that the release of drugs under different pH environments conforms to the Korsmeyer-Peppas law. As shown in Table 1, the release constants (n) of ZIF-8@BAN in different pH environments were all greater than 0.89, indicating that the drug release mechanism of the nanosystem was mainly matrix erosion. The release mechanism of PEG-FA@ZIF-8@BAN at pH=5.0 was mainly skeletal dissolution, while the drug release mechanism of PEG-FA@ZIF-8@BAN at pH = 7.4 included Ficks diffusion and erosion mechanisms (0.45 < n < 0.89).⁴⁷ PEG-FA modified ZIF-8@BAN nanoparticles do not cause drug leakage at normal physiological pH = 7.4. These results suggest that PEG-FA@ZIF-8 can serve as a good drug delivery platform for BAN.

Table 1.

Correlation Coefficients (R²) and Release Exponent (n) According to the Korsmeyer-Peppas Model Used for Describing BAN Release Mechanisms

	ZIF-8@BAN		PEG-FA@ZIF-8@BAN
	pH=5.0	pH=7.4	pH=5.0	pH=7.4
R²	0.973	0.952	0.964	0.950
n	1.169	0.996	1.097	0.568

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FR Expression in MCF-7 Cells

The membrane surface of MCF-7 showed FR positive expression. Blue fluorescence represented the nucleus, while green fluorescence indicated FR on the MCF-7 cell membrane as pictured by the fluorochrome-conjugated (Alexa Fluor 488). The results are shown in Figure 4A. Green fluorescence was displayed on the corresponding cell membrane of each MCF-7 cell position. The results of this experiment shown that MCF-7 specifically expresses FR, and high expression of FR was expected to have more cellular uptake.

(A) MCF-7 cells for FR expression (scale bar: 5 μm). (B) Viability of MCF-7 cells and L929 cells after treatment with blank nanoparticles PEG-FA@ZIF-8 (means ± SD, n = 3). (C) Cell viability of MCF-7 cells after treatment with different samples containing the same BAN for 12 hours (means ± SD, n = 3). (D) Cell viability of MCF-7 cells after treatment with different samples containing the same BAN for 24 hours (means ± SD, n = 3). ** is p < 0.01, and ***is p < 0.001, compared with BAN group.

Cytotoxicity and Biocompatibility of Samples

The CCK-8 kit was used to detect the cytotoxicity of different concentrations of nanomaterials at different times of action. The cytotoxicity of the prepared PEG-FA@ZIF-8 blank material was firstly investigated. The results are shown in Figure 4B. The blank nanomaterial human breast cancer cells MCF-7 and normal mouse fibroblasts L929 did not show significant cytotoxicity in the dose range. Subsequently, as shown in Figure 4C and D, When MCF-7 cells were incubated with nanocomposites containing the same BAN concentration for different times, the cell survival showed a significant decreasing trend with increasing concentration, and the killing effect on tumor cells was stronger with increasing incubation time. The good tumor-killing effect of the nanoparticles was demonstrated by the cell survival rate of less than 10% after incubating MCF-7 cells with higher concentrations of ZIF-8@BAN and PEG-FA@ZIF-8@BAN for 24 hours.