Bioactive zeolitic imidazolate framework nanoconjugates as synergistic drug delivery agents for cancer nanotherapeutics

Abstract

The increasing effective, detectable, and targeted anticancer systems are driven by the growing cancer incidence and the side effects of current drugs. Natural products like saponin and apigenin have emerged as valuable compounds for precise treatment. Recent advancements in bioactive metal–organic frameworks (MOFs) have introduced multifunctional particles suitable for cellular imaging, targeted drug delivery, and early cancer treatment. In this study, bioactive ZIF-67 and ZIF-8 materials were synthesized, incorporating zinc nitrate and natural bioactive compounds such as saponin and apigenin to sensitize and deliver the material to damaged cancer tissue. The characterization of these bioactive nanostructures involved FT-IR, TEM, EDX, FESEM, and BET analysis. The study quantified the loading and release of natural products within the ZIF structure. Cytotoxicity assessments of drug-loaded MOFs were conducted on human oral cavity carcinoma cell lines OSCC, Hep-G2, Raji, MCF-7, and PDL under in vitro conditions. Flow cytometry analysis identified the combination of bioactive ZIF-67 and saponins as the most effective in inducing apoptosis. Finally, a novel synthesis of bioactive MOF compounds was developed with dual applications: drug delivery and cancer imaging, featuring a unique attribute that minimizes side effects on normal cells.

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1. Introduction

Cancer has been a persistent focus of medical research and efforts to conquer it for decades.¹ Since the use of derivatives of mustard gas to treat lymphoma in the 1940s, chemotherapy has advanced significantly² (see ESI† SI.1). Cisplatin, carboplatin, doxorubicin, and 5-fluorouracil are often used as systemic treatments; however, they have drawbacks. These drugs have serious adverse effects because they are highly toxic to both healthy and cancerous cells. Therefore, the administered dose is often limited by their toxicity.³ Additionally, many chemotherapeutic drugs have poor water solubility, which makes formulation and administration challenging.^4–7 The tumor microenvironment, with its disorganized blood supply, also hinders drug delivery to tumors.^8,9 Insufficient and inconsistent drug accumulation within tumors is a prominent cause of treatment ineffectiveness and failure. Consequently, it is imperative to explore alternative approaches that enhance the targeted transportation of drugs to tumors, while minimizing systemic drug absorption.⁵

Significant progress has been made in creating various nanomaterials, including organic polymers and inorganic porous materials, for treating cancer and biotechnological applications over the past several decades. However, limitations such as a lower loading capacity or unregulated release have hindered their practical utility. Researchers have recently explored novel solutions to address these challenges by using MOFs as carriers for biologically oriented purposes.¹⁰ Bioactive MOFs, consisting of nodes like metallic ions and binders like organic ligands, have emerged as extremely beneficial drug transporters, showcasing their versatility across numerous fields, including energy conversion, gas separation, magnetism, and fluorescent devices.^11,12 One specific type of bioactive MOF is the zeolite imidazole framework (ZIF), which exhibits hybrid characteristics. Bioactive ZIF structures can serve as effective drug carriers owing to their exceptional attributes including controlled diffusion capability, high adsorption capacity, and resistance to acidic conditions.¹³ Bioactive ZIF-8 is a well-known crystalline material with 2-methyl imidazolate binders coordinated with Zn²⁺ ions arranged in a quadrilateral formation. Noteworthy structural attributes confer distinctive qualities to this framework, granting it a natural aptitude for drug encapsulation throughout the delivery process.^14–17 Bioactive ZIF-67, another type of porous MOF, consists of Co²⁺ ions and the 2-methylimidazole organic binder. This MOF exhibits an exceptionally high surface area, making it a highly desirable material for diverse fields (see Fig. 1).^18–20 Bioactive ZIF-67 and ZIF-8 have been researched as possible delivery systems for various therapeutic agents, including anticancer substances. As bioactive ZIF-67 and ZIF-8 exhibit cage-like structures, they can host other bioactive species within their cavities, which boosts their anticancer properties.^21,22

Fig. 1 The bioactive ZIF-67 and ZIF-8 functionalized with saponin and apigenin for the proposed cellular apoptosis mechanism.

Saponin and apigenin are bioactive compounds that have been studied for their potential use in cancer therapy.^23,24 Apigenin and saponin have demonstrated promise in preclinical investigations, but their clinical use has been limited by their poor solubility and low bioavailability. The natural flavonoid apigenin has been studied as a potential chemotherapeutic agent for several malignancies, including breast, lung, and prostate cancers,²⁵ and has been shown to prevent cancer cell growth by triggering cell cycle arrest and death. Additionally, it inhibits tumor cell invasion and migration while enhancing the immune system²⁶ can change how cancer cells behave by modifying signaling pathways that are important in cellular processes like apoptosis, proliferation, and viability.²⁷ However, its clinical translation has been hampered by its poor solubility and low bioavailability, which limit its therapeutic efficacy.^28,29 Because of their potent effect on cancer cells, saponins, a type of glycoside found in plants, have attracted interest from the pharmaceutical industry for drug delivery. These compounds have demonstrated promising potential as anticancer agents by influencing cell cycle proteins.²³ The saponin molecule is formed once one or more oligosaccharide chains connect to a hydrophobic region called the aglycone. These chains form the hydrophilic region.³⁰ The majority of saponins only include two sugar chains, while some types have three or one. This distinctive arrangement renders saponins remarkably amphipathic, granting them the ability to generate foam and act as emulsifiers in aqueous solutions.^31,32 This study specifically used Quillaja bark saponin, which primarily contains a triterpenoid saponin called quillic acid-type aglycone, known for its anticancer properties. It has been demonstrated that saponins affect cancer cells through several mechanisms, including antioxidant activity, suppression of cellular invasion, activation of apoptosis, cell cycle arrest, and autophagy.^23,33 Saponins can obstruct signaling pathways that control the survival and metastasis of cancer cells, thereby slowing the spread of the disease.³⁴

Here, we report the excellent cell apoptosis properties of bioactive zeolite imidazole frameworks (ZIF-67 and ZIF-8). These materials are excellent platforms for the regulated release of natural product molecules (saponin and apigenin). Bioactive ZIF-67 and ZIF-8 were successfully synthesized, and their particle size, morphology, surface area, and pore size were investigated using various tests such as SEM, and BET. Then, the natural products saponin and apigenin were loaded into the cavities of the nanomaterials (see Fig. 1), and the release percentage was checked under acidic and neutral conditions. Two in vitro biological experiments were conducted to assess the potential anticancer properties of these compounds in OSCC, MCF-7, Hep-G2, Raji, and PDL normal dental cells using MTT assay and apoptotic evaluation. Additionally, flow cytometry was used to investigate apoptosis and necrosis, and the effect of the nanocomposites on cellular behavior. Finally, the fluorescent properties of nanocarriers were investigated to track cancer cells, these materials were placed in the vicinity of MCF-7 and PDL cells, and fluorescent imaging was performed. This study also investigated the side effects of simultaneous cancer treatment in normal cells. When chemotherapy is administered, normal cells can be affected, leading to toxicity. However, if normal cells are protected, the selectivity and potency of combinations of several drugs can be increased by adding synergistic drugs. Theoretically, this approach can eliminate the deadliest cancers with minimal side effects.

2. Experimental

2.1. Chemicals and materials

The materials used in the present research were obtained from Sigma-Aldrich and several other industrial vendors. These materials included all the necessary starting materials, solvents, and the required pure drugs. Analytical-grade chemicals and reagents were used in their original forms throughout the experiment. The Bioresource Collection and Research Center (Iran) provided human OSCC, Hep-G2, Raji (Michigan Cancer Foundation-7) MCF-7 cancer cells, and PDL in vitro for the study. Double distilled water was used as the aqueous solution. The cells were kept at 37 °C in an incubator containing 5% humidified CO₂.

Through the Sivan business in Shiraz, Iran, periodontal ligament fibroblasts (PDLs) were purchased. MCF-7, Raji, and Hep-G2 cells were purchased from the Pasteur Institute (Tehran, Iran). The Shayan Pars Cell Bank in Shiraz, Iran, provided the OSCC cells. Fetal bovine serum (FBS), trypsin, MTT solutions, Eagle's minimum essential medium (DMEM), Roswell Park Memorial Institute (RPMI), and minimum essential media (MEM) culture media used in this work, as well as trypsin, MTT reagents and phosphate buffer solution were all supplied by BIO-IDEA in Iran. The study used “Saponin from Quillaja Bark,” a commercially available saponin and apigenin, as the natural reagents.

2.2. Methods

The nanostructures were characterized using a variety of analytical techniques, such as Fourier transform infrared spectroscopy (FT-IR), which was registered for bioactive ZIF-67, ZIF-8, and their derivatives in the current study by Bruker (Berlin, Germany). Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), and dynamic light scattering (DLS) were used to assess the morphology and average particle size. For this purpose, a Mira 3 Tescan instrument, located in Kohoutovice, Czech Republic, was employed. Hitachi H-800 TEM (Tokyo, Japan) was used for the same analysis.

2.2.1. X-ray diffraction spectroscopy (XRD). A Philip-X’Pert X-ray diffractometer coupled with a CuKα X-ray source was used to record the X-ray diffraction patterns in order to examine the crystallographic structure.

2.2.2. UV-vis and fluorescence spectroscopy. A PerkinElmer Lambda 650S UV-vis spectrometer was used to measure the UV-vis absorption spectra.

2.2.3. Brunauer–Emmett–Teller (BET). A pore analyzer (Beckman Coulter, USA) and surface SA-3100 were utilized to measure the specific surface area and average pore size of the adsorbent.

2.2.4. Atomic force microscopy (AFM). The surface roughness of the membranes was evaluated by AFM. AFM measurements were conducted using a MULTIMODE8 instrument (Brook Technology Co., Ltd, NASDAQ, USA).

2.2.5. Raman spectroscopy (RS). Raman spectra of the samples were acquired using a Raman Micro 200 instrument (PerkinElmer, Waltham, MA, USA) equipped with a precise spectrometer (Spectrum software). A laser beam with an intensity of 5 mW was used for the measurements.

2.3. Preparation of bioactive ZIF-8 and ZIF-67

Bioactive ZIF-8 and ZIF-67 were prepared based on the literature with minor modifications^35,36 and characterized by analytical methods.

2.3.1. Bioactive ZIF-8. A facile solvothermal synthesis of nanosized bioactive ZIF-8 crystals was performed using a warm methanol solution (20 mL) of 0.12 g zinc nitrate hexahydrate [Zn (NO₃)₂·6H₂O] and 0.42 g 2-methylimidazole (Hmim). Having been filtered separately, both solutions were mixed and subjected to heat in a 25 mL Teflon-coated stainless-steel autoclave for 48 h at 95 °C. The resulting precipitate was centrifuged, washed with water and methanol repeatedly, and finally dried.

2.3.2. Bioactive ZIF-67. To synthesize bioactive ZIF-67, 1.436 g of Co(NO₃)₂·6H₂O and 3.244 g of 2-methylimidazole were dissolved separately in 100 mL methanol. After adding the metal solution, the ligand solution was stirred at room temperature until it turned dark purple in color. The solution was then agitated for 3 h at room temperature and allowed to stand for a further 24 h without stirring. To remove impurities, the product underwent 15 min of centrifugation at a rate of 5000 rpm before washing four times with methanol and once with chloroform. Ultimately, the product was dried entirely at 120 °C in a vacuum oven for three days.

The thermal stability of ZIF-67 and ZIF-8 showed the heat resistance up to around 300 and 350 °C, respectively.^37,38

2.4. Experimental and theoretical investigation of bioactive ZIFs with natural products

To assess the dispersion of bioactive ZIF-67 and ZIF-8 in phosphate-buffered saline (PBS) buffer (4 mg mL⁻¹, 4 mL, pH = 7.4), an aqueous solution of saponin (2 mg mL⁻¹) and apigenin (600 μg mL⁻¹) was added. For 24 h, the resultant mixture was agitated in the dark, after which the nanoparticles were collected by centrifugation (15 000 rpm, 5 min). To ascertain the quantity of loaded medicine, the solid material was eliminated and the supernatant was examined. We considered the factor separating the amount of primary drug remaining in the solution from the amount that could be loaded by the drug and then calculated the loading capacity of the drug. In Fig. S2 (ESI†), nanoparticles and their combinations with prepared natural products are shown.

The loading efficiency of apigenin and saponin in bioactive ZIF-67 and ZIF-8 was assessed using UV-vis spectroscopy. The loading efficiency and content were calculated using the following methodology:³⁹

2.5. In vitro release of saponin and apigenin

UV-vis spectrophotometry was used to analyze the release of saponin and apigenin from the bioactive ZIF-67 and ZIF-8. For this purpose, 4 mg of ZIF-67-saponin, ZIF-67-apigenin, ZIF-8-saponin, and ZIF-8-apigenin were dispersed in 4 mL of a buffer solution with pH values of 5.5 and 7.4. A temperature of 37 °C was used for the release experiments. An in vitro release experiment utilizing a dialysis membrane was performed to determine the release of saponin and apigenin from the ZIF-67 and ZIF-8 networks. The presence of 10 000 Da facilitated drug diffusion through the dialysis membrane, enabling the release of saponin and apigenin molecules.

In contrast, the structural size and rigid polymeric matrix of bioactive ZIF-67 and ZIF-8 restrict their permeation through the dialysis membrane. Four mL of the external solution outside the dialysis bag was withdrawn at regular intervals and its absorption was measured. The UV-vis spectroscopy instrument (UV 1800 PC) was used to monitor the absorption band at 480 nm. The quantity of released saponin and apigenin was determined based on the standard curve of UV-vis absorbance at 480 nm.

2.6. Luminescent properties and cellular uptake

The fluorescence properties of bioactive ZIF-8 and ZIF-67 samples were studied in water suspensions. Photoluminescence excitation (205 and 370 nm) and emission spectra (380 nm for the two MOFs) were recorded using a Hitachi F-7000 spectrophotometer.

In DMEM with 10% (v/v) FBS and 1% penicillin–streptomycin solution, MCF-7 and PDL cells were cultivated at 37 °C with 5% CO₂ to achieve 80% confluence in 24 h. Then, 1000 μg mL⁻¹ of bioactive ZIF-67 and ZIF-8 (saponin, apigenin) were added to the cells and incubated for zero moments, 4 and 24 h. Excess samples were eliminated by washing them three times with PBS. Fluorescent images were acquired using a fluorescence microscope.

2.7. Toxicity evaluation

This study assessed the cytotoxicity of bioactive ZIF-67 and ZIF-8 against OSCC, MCF-7, Hep-G2, Raji, and PDL cell lines using in vitro experiments. The experiments were conducted in triplicate. DMEM, MEM, and RPMI were used in this study. The medium was added to 1% penicillin–streptomycin and 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in a humidified environment containing 5% CO₂ before evaluation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to assess cytotoxic effects. Approximately 7000 cells per well were seeded in 96-well plates. In the initial phase, OSCC, MCF-7, Hep-G2, Raji, and PDL cells were incubated for 24 h. Subsequently, the cells were treated with bioactive ZIF-67 and ZIF-8, using saponin and apigenin as delivery agents at concentrations of 25, 100, 250, 500, and 1000 μg mL⁻¹. The cells initially incubated for 24 h at 37 °C with 5% CO₂, followed by incubation for a further 24 h. The MTT stock solution was made by dissolving 5 mg mL⁻¹ of MTT in PBS. Subsequently, the stock solution was diluted using phenol-free DMEM to achieve a 0.5 mg mL⁻¹ concentration. The MTT stock solution was diluted by mixing 1 mL of 5 mg mL⁻¹ MTT stock solution with 9 mL of phenol-free DMEM. After removing the medium from each well, 80 μL of diluted DMEM-MTT solution was added to each well. The plates were returned to the incubator at 37 °C and 5% CO₂ for 3 h. After incubation, 200 μL DMSO was added to the wells to dissolve the insoluble formazan crystals. The resulting solutions were subjected to absorbance measurements using a BioTEK PowerWave XS2 microplate reader. At 570 nm for the primary wavelength and 630 nm for the reference wavelength, measurements were made. During the data analysis, the readings obtained at the reference wavelength (A630) were subtracted from those obtained at the original wavelength (A570). This subtraction is necessary for factors such as cell debris or precipitated proteins in the wells. The formula for calculating cell viability is as follows:²⁰

The adsorption seen in every test sample is denoted by “A_t” in this context. “A_b” represents the adsorption observed in the blank well, which contains media, MTT, and DMSO but no cultured cells (negative control). Finally, cells that have not undergone any treatment serve as the positive control. “A_c” represents the adsorption followed in the control well. The results were calculated as the percentage of cell viability compared to the untreated control group.

2.8. Flow cytometric detection of necrosis/apoptosis

To assess the level of apoptosis in OSCC, MCF-7, Raji, and PDL cells after treatment, we used APC-annexin V and 7-AAD staining (BioLegend, London, UK). This choice of staining was based on previous findings, indicating that the nanoparticles used in the study did not affect these particular cellular pathways. After exposing the cells to the test compounds for 24 h, the medium and detached cells were stained with 50 μL of APC-annexin V (2.5 μL mL⁻¹ annexin binding buffer) for 30 min. The cells were labeled and washed three times with annexin-binding buffer. They were then returned to a staining solution that contained 2.5 μL mL⁻¹ 7-AAD. Using a FACSCanto II flow cytometer (BD Biosciences, San Jose, California, USA), 10 000 events per treatment were included in the cellular analysis. FlowJo 10 and GraphPad Prism 7 software were used for data analysis using a gating strategy. In this assay, cells treated with 10 μL of DMSO represented the control or untreated group.

2.9. Statistical analysis

Each biological study, including the MTT and apoptotic assays, was repeated three times for each study group. One-way analysis of variance (ANOVA) was performed on the collected data, followed by Tukey's post hoc test. SPSS software (IBM version 23, IBM Corp., Armonk, N.Y., USA) was used to conduct the analysis. The results’ statistical significance was evaluated using a p-value of <0.05.

3. Results and discussion

3.1. Fourier transform infrared spectroscopy (FT-IR)

The FT-IR spectra of (a) saponin, (b) apigenin, (c) ZIF-67, (d) ZIF-8, (e) ZIF-67-saponin, (f) ZIF-67-apigenin, (g) ZIF-8-saponin, and (h) ZIF-8-apigenin are shown in Fig. 2. Saponin's FT-IR spectrum, as shown in Fig. 2a, exhibits a prominent band at 1700 cm⁻¹, which is indicative of the carbonyl groups’ C=O stretching vibrations. In the alkene structure, the peak at 1600 cm⁻¹ is ascribed to C=C stretching. The C–O stretching vibrations that are characteristic of alcohol or ester functionalities are represented by the bands in the 1000–1300 cm⁻¹ range. Furthermore, the presence of alkyl and hydroxyl groups is indicated by the large peaks seen at 2800 and 3500 cm⁻¹, which correlate to C–H stretching and O–H vibrations. Apigenin displays distinctive peaks at 1600 cm⁻¹ for aromatic C=C stretching, which are indicative of its flavonoid structure, as seen in Fig. 2b. The C=O stretching of carbonyl groups is represented by the strong band at 1700 cm⁻¹. The broad band seen at 3400 cm⁻¹ is caused by OH groups, and a sharp peak at 1200 cm⁻¹ is ascribed to C–O stretching vibrations. The peaks at 1420 cm⁻¹ and 1580 cm⁻¹ in Fig. 2c, respectively, represent the C–N and C=C bonds of the imidazole ring. These peaks verify that ZIF-67 contains the 2-methylimidazole ligand coupled with Co²⁺ ions. The peaks in Fig. 2d at 1420 cm⁻¹ and 1580 cm⁻¹ reflect the imidazole ring's C=C and C–N bonds, respectively. The ZIF-67-saponin FT-IR spectrum is shown in Fig. 2e, where C=C stretching is responsible for peaks at 1600 cm⁻¹. The peak at 1200 cm⁻¹ is C–O stretching vibrations, which is a sign of saponin's glycosidic linkages. Fig. 2f shows the ZIF-67-apigenin FT-IR spectrum. The carbonyl (C=O) stretch is identified by the peak at about 1650 cm⁻¹, and the C–O–C bond in the pyran ring is shown by the peak at 1350 cm⁻¹. The C–N in the ZIF-67 structure is also visible near the peak at about 1420 cm⁻¹. The spectrum of ZIF-8-saponin exhibits distinctive peaks resembling those of ZIF-67-saponin, with C=C stretching vibrations at around 1600 cm⁻¹ and C–O stretching at about 1300 cm⁻¹, as seen in Fig. 2g. The FT-IR spectrum of ZIF-8-apigenin, which has distinctive peaks at 1653 cm⁻¹ (C=O stretch) and 1350 cm⁻¹ (C–O–C), is similar to that of ZIF-67-apigenin (Fig. 2h). The inclusion of apigenin in the ZIF-8 framework is validated by these peaks. Consequently, the FT-IR spectra verify that saponin and apigenin were successfully incorporated into the ZIF-67 and ZIF-8 frameworks.

Fig. 2 FT-IR evaluation of (a) saponin, (b) apigenin, (c) ZIF-67, (d) ZIF-8, (e) ZIF-67-saponin, (f) ZIF-67-apigenin, (g) ZIF-8-saponin and (h) ZIF-8-apigenin.

3.2. Raman spectroscopy (RS)

Fig. 3a and b show the Raman spectra, presenting prominent bands corresponding to specific vibrations of the methyl group and the imidazole ring. The band observed at 282 cm⁻¹ indicates Zn–N stretching, whereas the bands at 685, 1146, and 1460 cm⁻¹ are associated with imidazolium ring puckering, C5–N stretching, and methyl bending, respectively. Notably, the Raman modes exhibited by all samples showed identical positions and intensities, suggesting structural uniformity. In porous materials, the entry of guest molecules requires traversing the external crystal surface, which is equipped with terminating groups.

Fig. 3 Raman spectra of (a) ZIF-67 and (b) ZIF-8. XRD of the (c) ZIF-67, ZIF-67-saponin, ZIF-67-apigenin and (d) ZIF-8, ZIF-8-saponin, ZIF-8-apigenin.

Consequently, these groups have the potential to facilitate molecular sieving. On the other hand, bioactive ZIF-8 is a zinc-based metal–organic framework composed of Zn(II) ions coordinated with 2-methylimidazole ligands. The Raman spectrum of ZIF-8 also exhibited characteristic peaks that provided information about its structure and functional groups. The peak at approximately 400 cm⁻¹ corresponds to the Zn–N stretching mode, similar to that of bioactive ZIF-67. Moreover, the peak at approximately 1600 cm⁻¹ signifies the C=N stretching mode specific to the imidazole ring. Raman spectroscopy is a valuable tool for investigating the structural characteristics and chemical compositions of bioactive ZIF-67 and ZIF-8.

Additionally, the distinct peaks at 413, 469, 518, and 670 cm⁻¹ can be attributed to bioactive ZIF-67, encompassing the Co–N bond at 413 cm⁻¹ and the vibrational mode of the 2-methyl imidazolate ligand at 670 cm⁻¹. Furthermore, the vibrational mode of the Co–O bond is shown by the peak at 609 cm⁻¹. The Co–N bond strength was noticeably weakened as a result of the interaction with bioactive ZIF-67.

3.3. X-ray diffraction spectroscopy (XRD)

X-ray diffraction patterns of bioactive ZIF-8 and ZIF-67 nanoparticles were analyzed using the Rietveld method with Highscore-X’pert software. The X-ray diffraction pattern of ZIF-8 was identified according to the COD database code 7111971 and ZIF-67 according to the COD database code 4124528. All the XRD patterns exhibit sharp peaks, indicating their high crystallinity with the cubic crystal system and the I4̄3m space group. The most notable increase in bioactive ZIF-8 appears at approximately 2θ = 7.38°, while for ZIF-67, it is observed at approximately 2θ = 7.35°, both of which correspond to the (011) plane, indicating truncated rhombic dodecahedral crystal structures.⁴⁰ Using Rietveld refinement, the d-spacings [Å] were calculated to be 11.96419 and 12.053 for ZIF-8 and ZIF-67, respectively. In addition, the crystallite sizes obtained by the Williamson–Hall method in X’pert software for bioactive ZIF-8 and ZIF-67 are 320 Å and 370 Å, respectively^40–42 (see Fig. 3c and d).

3.4. Scanning electron microscopy (SEM) and dynamic light scattering (DLS)

Fig. 4a–f depicts the SEM images of the as-prepared bioactive ZIF-67 and ZIF-8 with a natural reagent, showing their cubic structures. The samples maintained their original morphology even after carbonization, and each structure exhibited a distinct hole in its shell. The effective synthesis of bioactive ZIF-8 and ZIF-67, which are distinguished by their regular hexagonal forms, was validated by a detailed SEM study. As depicted in Fig. 4g and h, the mean particle sizes for ZIF-67 and ZIF-8 are around 310 and 80 nm, respectively.

Fig. 4 SEM images of (a₁) and (a₂) ZIF-67, (b₁) and (b₂) ZIF-8, (c₁) and (c₂) ZIF-67-saponin, (d₁) and (d₂) ZIF-67-apigenin, (e₁) and (e₂) ZIF-8-saponin, and (f₁) and (f₂) ZIF-8-apigenin; DLS images of (g) ZIF-67, and (h) ZIF-8.

See Fig. S3 (ESI†) for more information regarding the EDX and mapping results.

3.5. Atomic force microscopy (AFM)

Atomic force microscopy (AFM) was used to assess the surface characteristics of the bioactive ZIF-67 and ZIF-8 channel films. The scan area was 5 μm × 5 μm, and the scan rate was 0.5 Hz. Accurate measurements were obtained by determining the surface thickness and roughness using the image processing and evaluation program XEI AFM. Fig. 5 shows a 3D AFM micrograph of the synthesized bioactive ZIF-67 and ZIF-8. The average surface roughness and thickness of bioactive ZIF-67 and ZIF-8 were determined to be 19.9 nm and 15.18 nm, respectively, confirming the crystallite properties previously estimated by XRD and SEM.

Fig. 5 The surface AFM 2D (a), surface AFM 3D (b), amplitude (c), and roughness (d) images of the ZIF-67 and the surface AFM 2D (e), surface AFM 3D (f), amplitude (g) and roughness (h) images of ZIF-8. TEM images of ZIF-67 (i₁)and (i₂) and ZIF-8 (j₁) and (j₂).

Analysis of AFM images of bioactive ZIF-67 and ZIF-8 nanocarriers can provide valuable insights into their surface morphology and properties. The size, shape, and distribution of the nanoparticles on the surface are shown by the 2D and 3D surface AFM images. Amplitude and roughness images can provide information on the height variations and roughness of the surface. Comparing the AFM images of bioactive ZIF-67 and ZIF-8 nanocarriers can help identify differences in their surface properties such as size, shape, and roughness. This information can be used to optimize the manufacturing process and improve the performance of nanocarriers (see Fig. 5a–h). According to the AFM study, the bioactive ZIF-67's nanoparticle size was marginally smaller than that of the bioactive ZIF-8's. The dimensions of the bioactive ZIF-67 and ZIF-8 particles were 34.63 nm and 36.29 nm, respectively. Although the two nanocarriers’ average roughness values were similar, the bioactive ZIF-67's surface roughness was almost rougher than ZIF-8's. The average roughness values of the ZIF-67 and ZIF-8 particles were 2.4284 × 10⁻⁶ nm and 1.4572 × 10⁻⁶ nm, respectively. The R_q value, which represents the root-mean-square roughness, was higher for bioactive ZIF-67 than for bioactive ZIF-8. The other roughness parameters, such as R_pm (average maximum profile peak height), R_vm (average maximum roughness valley depth), R_z (average maximum size of the profile), R_max (maximum peak-to-valley roughness), and R_q (root mean square roughness) are also higher for ZIF-67 than ZIF-8, indicating a more uneven surface. However, it should be noted that the standard deviation of the mean roughness values was higher for ZIF-67, indicating more significant variability in the surface properties (see Table 1).

Table 1 Morphological characteristics from section analysis of the AFM micrographs for the bioactive ZIF-67 and ZIF-8 thin films on the substrate

Sample	Nanoparticle size (nm)	Average roughness (nm)	R a (roughness)	R pm (nm)	R vm (nm)	R z (nm)	R max (nm)	R q (nm)
ZIF-67	34.63	Mean = 2.4284 × 10^−6	1.038 nm	4.123	−4.858	8.981	10.17	8.240
		SD = 2.6231
ZIF-8	36.29	Mean = 1.4572 × 10^−6	391.5 pm	3.689	−2.360	6.049	7.812	2.982
		SD = 1.9705

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3.6. Transmission electron microscopy (TEM)

TEM was used to examine the functionalization of the fabricated bioactive ZIF-67 and ZIF-8. TEM analysis showed that the bioactive ZIF-67 and ZIF-8 had similar crystalline structures with regular and uniform shapes. Nevertheless, there was a slight difference in the particle size between bioactive ZIF-67 and ZIF-8, with the former exhibiting a slightly smaller particle size than the latter. In addition, there were some differences between the lattice structures of the two nanotransporters. Bioactive ZIF-8 has a cubic structure with a smaller pore size, while bioactive ZIF-67 has a rhombic dodecahedral structure with larger pores. The difference in pore size may affect the loading and release of drug molecules in nanocarriers. As shown in Fig. 5(i₁, i₂) and (j₁, j₂), we successfully prepared nanoscale bioactive ZIF-67 and bioactive ZIF-8, which exhibited a spherical appearance with diameters of approximately 50 and 200 nm, respectively.

In contrast, the bare bioactive ZIF-67 and ZIF-8 nanoparticles without saponin and apigenin loading exhibited typical hexagonal structures [Fig. 5(i₁, i₂) ZIF-67 and Fig. 5(j₁, j₂) ZIF-8]. After functionalization, the TEM images show a rougher surface. Obtaining high-magnification TEM images of the mesopores proved to be challenging because of the high sensitivity of the samples to the electron beam. The observed presence of isolated and dispersed mesopores, characterized by irregular sizes and shapes, aligns with the double hysteresis loops observed in the N₂ adsorption–desorption isotherms. This observation implies that an ordered porous framework within bioactive ZIF-8 is formed by the disordered mesopores attached to the microporous walls.

Overall, the analysis confirmed the differences in the size and structure of bioactive ZIF-67 and ZIF-8, which may have implications for their potential applications in drug delivery and sensing.

3.7. Brunauer–Emmett–Teller (BET)

Nitrogen physisorption: The nitrogen isotherms are shown in Fig. S4 (ESI†); the bioactive ZIF-8 and ZIF-67 nitrogen isotherms agreed well with the reported isotherms for bioactive ZIF-8 and ZIF-67, which is a type I isotherm (see Fig. S4, ESI†). A hysteresis loop was observed at a higher relative pressure (P₀/P > 0.8), which was attributed to the large mesopores between the neighboring particles of the bioactive ZIF-8 and ZIF-67 MOFs. The BET surface area and micropore volume for the particles synthesized at 120 °C were 1546.6909 and 2142.2 m² g⁻¹, and the pore sizes were 20.0953 and 18.5 Å, respectively. See Table 2.

Table 2 BET surface areas and micropore area bioactive ZIF-8 and ZIF-67 MOFs

Sample name	BET surface area (m^2 g^−1)	Langmuir surface area (m^2 g^−1)	Pore size (Å)
ZIF-8	1546.6909	2038.9723	20.0953
ZIF-67	2142.2	2224.4	18.5

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3.8. Loading and release

The loading and release behavior of saponin and apigenin in bioactive ZIF-67 and ZIF-8 were evaluated using the standard curve of saponin and apigenin solutions at wavelengths of 210 and 240 nm, respectively. Drug loading was performed using the above ratios at a pH of 7.4. Table 3 shows that the ZIF-8 apigenin sample had the highest drug loading at 89.76%, and the ZIF-67 saponin sample had the lowest drug loading (52.89%). Then, the release behavior was measured at different pH values (pH = 5.5 or pH = 7.4). Based on the findings presented in Fig. 6, it can be observed that in a PBS solution simulating the pH of the normal physiological environment, the 24-hour release rates for ZIF-67-saponin, ZIF-67-apigenin, ZIF-8-saponin, and ZIF-8-apigenin were 45.61%, 45.19%, 49.48%, and 30.37%, respectively. Conversely, in a PBS solution simulating the slightly acidic environment of the tumor with a pH of 5.5, the final release rates were 64.4, 86.53, 72.96, and 61.72% for ZIF-67-saponin, ZIF-67-apigenin, ZIF-8-saponin, and ZIF-8-apigenin, respectively.

Table 3 Saponin and apigenin loading content and drug release at pH 5.5 and pH 7.4 after 24 h

MOF	Drugs	Loading capacity	Release capacity	Cell line	Ref.
ZIF-8	5-FU	21.2%	pH: 7.4: >70%	MCF-7	43
			pH: 5.8: 80%
3-MA@ZIF-8	3-Methyladenine	19.8%	pH: 7.4: 10%	HeLa	44
			pH: 5.0: 100%
RGD@CPT@ZIF-8	Camptothecin	15%	pH: 7.4: 19%	HeLa	45
			pH: 5.0: 76%
Ceftazidime@ZIF-8	Ceftazidime	10.8%	pH: 7.4: 60%	A542	46
			pH: 5.0: 75%	RAW 264.7
Fe3O4@Bio-MOF	5-FU	60%	pH: 7.4: 39%	NIH-3T3	47
			pH: 5.5: 92%	MDA-MB-231
DI@HMONs-PMOF	DOX, ICG	11.88% (DOX) 19.52% (ICG)	pH: 7.4: 15%	AT1	48
			pH: 5.0: 38%	MCF-7
HA/α-TOS@ZIF-8	HA, α-TOS	wt 43.03%	pH: 7.4: 5%	HeLa	49
			pH: 5.0: 76%
ZIF-90	5-FU, DOX	13.6% (5-FU)	pH: 7.4: 20%	—	50
		36.5% (DOX)	pH: 5.5: 95%
DOX/HMS@ZIF-8	DOX	wt 44%	pH: 7.4: 3%	HeLa	51
			pH: 5.0: 71.4%
MIL-100 NMOF	Topotecan	wt 33± 9%	pH: 7.4: 0%	MiaPaCa2, PANC1, A549	52
			pH: 5.0: 19%
MIL-100(Fe) and MIL-101(Fe)	CPT	wt 0.9 ± 18%	pH: 7.4: 9%	HeLa, Neuroblastoma (SY5Y), Fibroblast 3T3	53
			pH: 5.0: 38%
ZIF-67-saponin	Saponin	52.89%	pH: 7.4: 45.61%	PDL, OSCC, MCF-7, Hep-G2, Raji	Our study result
			pH: 5.5: 64.4%
ZIF-67-apigenin	Apigenin	89.59%	pH: 7.4: 45.19%	PDL, OSCC, MCF-7, Hep-G2, Raji	Our study result
			pH: 5.5: 86.53%
ZIF-8-saponin	Saponin	68.71%	pH: 7.4: 49.48%	PDL, OSCC, MCF-7, Hep-G2, Raji	Our study result
			pH: 5.5: 72.96%
ZIF-8-apigenin	Apigenin	89.76%	pH: 7.4: 30.37%	PDL, OSCC, MCF-7, Hep-G2, Raji	Our study result
			pH: 5.5: 61.72%

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Fig. 6 Release curves of (a) ZIF-67-saponin, (b) ZIF-67-apigenin, (c) ZIF-8-saponin, and (d) ZIF-8-apigenin in PBS buffered solution at pH: 5.5 and pH: 7.4 after 24 h. Data are representative of three independent experiments.

The highest release rate at pH 5.5 was 86.53% for the ZIF-67-apigenin sample. Cancer cells grow faster at acidic pH and can metastasize and spread even in a neutral environment. The results showed that bioactive ZIF-67 and ZIF-8 can release natural products in both acidic and neutral environments, with a higher release rate in acidic environments where cancer cell growth is more pronounced. Bioactive ZIF-67 and ZIF-8 nanocarriers provide a good platform for the release of natural anti-tumor agents in the body. Based on the release profiles at time zero, the release of natural compounds was less than 40%. Moreover, the release was continuous with a smooth increase over 24 h.

3.9. Cellular uptake study

Measurements of UV-Vis and fluorescence spectra were made in order to examine the fluorescent characteristics of bioactive ZIF-67 and ZIF-8. Fig. 7 displays the emission spectra of the bioactive ZIF-8 and ZIF-67.

Fig. 7 Illustration of the bioactive ZIF-8 fluorescence excitation spectrum (a) and emission spectrum (b), bioactive ZIF-67 fluorescence excitation spectrum (c) and emission spectrum (d). Fluorescence images of (e) ZIF-8 at (λ_ex = 325 nm) and (f) ZIF-67 at (λ_ex = 325 nm) (the emission of both samples was examined in the green channel).

The sharpest fluorescence peak of the bioactive ZIF-8 sample was observed at 443 nm with a lambda (λ) excitation of 205. In addition, the strongest fluorescence peak of the ZIF-67 sample was observed at 446 nm with a lambda (λ) excitation of 370 nm (Fig. 7a and c). Both the bioactive ZIF-67 and ZIF-8 samples exhibited fluorescence emission in the 500–570 nm region, corresponding to the green channel. Therefore, bioactive ZIF samples and treated cells were imaged using a fluorescence microscope with a lambda (λ) excitation of 325 nm (see Fig. 7e and f).

When comparing the treated PDL and MCF-7 cells, it is evident that the impact of ZIF treatment on MCF-7 cancer cells is quite pronounced, whereas normal PDL cells maintained a relatively stable form. Staining of cancer cells with saponin and apigenin incorporated into the ZIF-67 and ZIF-8 systems revealed a highly effective delivery of drugs to cancer cells. These results suggest that the developed nanocarrier can effectively approach MCF-7 cells via bioactive ZIF-67 and ZIF-8, facilitating the delivery of saponins and apigenin to cancer cells. Consequently, these nanocarriers are promising candidates for simultaneous pH-responsive drug delivery and cell imaging (see Fig. 8a₁–d₄). See Fig. S5 and S6 (ESI†) for more information on the cellular uptake results.

Fig. 8 White field and 2D-fluorescence images PDL (a₁) and (a₂) and MCF-7 (a₃) and (a₄) cell lines as a control. Intracellular uptake 2D-fluorescence images after zero moment, 4 h, and 24 h of treatment at 37 °C with 95% humidity. Excitation wavelength, 360 nm. Wavelength ranges, 500–570 nm for the green channel. Intracellular uptake MCF-7 Cell 2D-fluorescence images after zero moment: (b₁) and (b₂) ZIF-8-saponin, (b₃) and (b₄) ZIF-8-apigenin, after 4 h (c₁) and (c₂) ZIF-8-saponin, (c₃) and (c₄) ZIF-8-apigenin, and after 24 h (d₁) and (d₂) ZIF-8-saponin, (d₃) and (d₄) ZIF-8-apigenin.

We quantified the intracellular fluorescence intensity and found that the intracellular bioactive ZIF-67 and ZIF-8 concentrations increased monotonically over time in the two cell lines. The PDL cells had normal fluorescence emission, but the MCF-7 cells had no significant emission.

According to the results, the emission intensity of MCF-7 cells increased after 24 h, which was due to the internalization of bioactive ZIF-67 and ZIF-8 and the transfer of bioactive materials saponin and apigenin into the cell (see Fig. 9a and b).

Fig. 9 Quantification of intracellular fluorescence of (a) pdl and (b) MCF-7 cells at different incubation times (zero moment, 4 h and 24 h) at a concentration of 1000 μg mL⁻¹.

3.10. Biocompatibility studies

3.10.1. Natural product saponin. Comparison of nanocarrier and nanocarrier + drug toxicity in PDL, OSCC, Hep-G2, MCF-7, and Raji cells: the cytotoxicity of bioactive ZIF-67 and ZIF-8 nanocarriers alone and in combination with two natural anticancer agents, saponin and apigenin, was studied in five cell lines: PDL, OSCC, Hep-G2, MCF-7, and Raji. In addition, the toxicity of four common anticancer drugs, 5-fluorouracil-doxorubicin-cisplatin-carboplatin, was studied in these cell lines. Three duplicates of the MTT technique were used to investigate each sample, and the results are shown in the graph as the average ∓% of lifespan (see Fig. 10). Additionally, the inhibitory concentration (IC50) was determined to be the amount that decreased tumor cell proliferation by 50% as compared to untreated controls. See (Fig. S7 (ESI†) and Table 4) to obtain more information regarding the IC50 diagrams. The survival rates of PDL cells subjected to treatment with the bioactive ZIF-67 and ZIF-8 nanocarriers individually, at a concentration of 1000 μg mL⁻¹, were 15.21 ± 0.624% and 16.87 ± 1.079%, respectively. As the concentration decreased, the viability increased; at a concentration of 100 μg mL⁻¹, the viability of the cells was 84.53 ± 2.005% and 51.44 ± 0.521%, respectively. The lowest concentrations increased to 91 ± 0.33% and 97.01 ± 2.93%, respectively. The IC50 value of ZIF-8 (106 μg mL⁻¹) was substantially less than ZIF-67's (349 μg mL⁻¹). When bioactive ZIF-67 and ZIF-8 were combined with saponin, the cell viability rate of PDL at the highest concentration was only 12.51 ± 1.29% and 13.42 ± 0.448%, respectively. The viability rose dramatically as the concentration reduced; at a concentration of 250 μg mL⁻¹, the viability of cells from PDL was 67.23 ± 0.69% and 58.63 ± 0.9%, respectively. Viability increased to over 90% for both combinations at the lowest concentration. The viability rate of cells from PDL treated with saponin alone was only 54.24 ± 2.59% at the lowest concentration (IC50 = 47.5 μg mL⁻¹). This is because there were more drugs in the bioactive ZIF-67 and ZIF-8 cavities at lower concentrations.

Fig. 10 Comparison of drug toxicity and effect of ZIF-8 and ZIF-67 with saponin and apigenin on PDL cells and viability of OSCC, Hep-G2, MCF-7, and Raji. MTT assay results of anticancer drugs on (c) PDL cells, (f) OSCC, (j) Hep-G2, (s) MCF-7, and (z) Raji cells and nanoparticles with saponin on (a) PDL cells, (d) OSCC, (g) Hep-G2, (k) MCF-7, and (x) Raji cells. MTT assay results of nanoparticles with apigenin on (b) PDL cells, (e) OSCC, (h) Hep-G2, (m) MCF-7, and (o) Raji cells.

Table 4 IC₅₀ table of samples treated with PDL, OSCC, Hep-G2, MCF-7, and Raji cell lines

Samples	Concentration (μg mL^−1)	PDL	OSCC	Hep-G2	MCF-7	Raji
ZIF-67	1000–25	349	249.9	662	560	294
ZIF-8	1000–25	106	148	624	126	—
ZIF-67-saponin	1000–25	318	70	250.5	360	137
ZIF-8-saponin	1000–25	306	100	395	326.5	138.5
Saponin	1000–25	47.5	24.5	279	335	54.5
ZIF-67	300–18	207.5	149	219	196.2	90
ZIF-8	300–18	146	57.5	—	164.5	54
ZIF-67-apigenin	300–18	289	216	—	—	130
ZIF-8-apigenin	300–18	249	196	—	294	—

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The toxicity of nanocarriers and the above compounds in OSCC cells was also studied. The survival rate of OSCC cells treated with bioactive ZIF-67 and ZIF-8 nanocarriers at a concentration of 1000 μg mL⁻¹, was recorded as 22.1 ± 1.92% and 16.76 ± 1.83%, respectively. The IC50 values of the ZIF-8 and ZIF-67 samples were 148 μg mL⁻¹ and 249.9 μg mL⁻¹, respectively. However, when the concentration was reduced to 25 μg mL⁻¹, the viability rate significantly increased to 75.69 ± 2.31% and 80.96 ± 1.39%, respectively. Similar to the previous sample, the combination of bioactive ZIF-8 and saponin significantly affected OSCC cells. ± 0.899% of the cells maintained viability at a concentration of 250 μg mL⁻¹, whereas half were killed at 100 μg mL⁻¹.

The viability of Hep-G2 cells was examined after treatment with the bioactive ZIF-67 and ZIF-8 nanocarriers. At 1000 μg mL⁻¹, the viability rates recorded were 13.36 ± 0.51% and 10.75 ± 0.47%, respectively. However, as the nanocarrier concentration decreased, the cell viability increased significantly. At a concentration of 25 μg mL⁻¹, the viability rates reached 77.75 ± 1.16% and 80.70 ± 5.74% for the ZIF-67 and ZIF-8 nanocarriers, respectively. The impact of saponin compounds, bioactive ZIF-67 and ZIF-8, on this cell line at a concentration of 1000 μg mL⁻¹ was highly significant. At this concentration, only 6.34 ± 0.33% and 7.88 ± 0.26% viability rates were observed for bioactive ZIF-67 and ZIF-8, respectively. It is worth noting that for both compounds, the viability increased as the concentration decreased. At a concentration of 25 μg mL⁻¹, the viability exceeded 75%. The IC50 values of the ZIF-8 and ZIF-67 samples combined with saponin in the vicinity of the Hep-G2 cell line were 395 μg mL⁻¹ and 250.5 μg mL⁻¹, respectively, while the IC50 values of the ZIF-8 and ZIF-67 samples were 624 and 662 μg mL⁻¹, respectively.

MCF-7 cells treated with 1000 μg mL⁻¹ of bioactive ZIF-67 and ZIF-8 nanocarriers showed viability of 7.04 ± 0.56% and 11.99 ± 1.012%, respectively, and as with other cells, viability increased with decreasing concentration. The bioactive ZIF-67 and ZIF-8 saponin samples also greatly affected this cell line; therefore, only 28.77 ± 0.29% and 7.94 ± 1.59% viability was observed at a concentration of 500 μg mL⁻¹, respectively.

Based on the MTT assay, the bioactive ZIF-67 and ZIF-8 nanocarriers showed high toxicity to Raji cells. The lowest cell viability was associated with the ZIF-8 nanocarrier; therefore, only 23.45 ± 1.045% viability was observed up to a concentration of 100 mg mL⁻¹. The ZIF-67-saponin and ZIF-8-saponin compounds had similar toxicity to Raji cells at all concentrations, such that at a concentration of 250 μg mL⁻¹, viability of 10.74 ± 0.52% and 11.74 ± 0.61%, respectively, was observed. As the concentration decreased, viability showed an upward trend, demonstrating an inverse relationship. Specifically, when the concentration reached 25 μg mL⁻¹, both compounds exhibited toxicity levels of approximately 20%. The IC50 value of ZIF-67-saponin (137 μg mL⁻¹) was lower than that of ZIF-8-saponin (138.5 μg mL⁻¹).

3.10.2. Natural product apigenin. The following Fig. 10 shows the percentage viability of PDL, OSCC, Hep-G2, MCF-7, and Raji cells after 24-hour treatment with nanocarriers, nanocarriers, and apigenin at concentrations ranging from 18 to 300 μg mL⁻¹. The viability rate of PDL OSCC, Hep-G2, MCF-7, and Raji cells treated with bioactive nanocarrier ZIF-67 alone at a concentration of 300 μg mL⁻¹ was 22.38 ± 0.94%, 39.23 ± 0.27%, 51.93 ± 0.58%, 35.93 ± 0.766%, and 41.43 ± 0.32%, respectively. Cell viability increased with decreasing concentration. The highest viability rate at a concentration of 18 μg mL⁻¹ was achieved in PDL cells with 94.25 ± 0.25% viability (IC50 = 207.5 μg mL⁻¹). The viability rate increased to more than 80% in cancer cell lines. At a dose of 300 μg mL⁻¹, the cells' viability was 16.42 ± 1.34%, 26.8 ± 0.96%, 32.93 ± 0.44%, 15.09 ± 0.38%, and 19.79 ± 0.83%, respectively, after being treated with the bioactive nanocarrier ZIF-8. As with the previous sample, the viability increased with decreasing concentration. In this sample, the highest viability rate was at a concentration of 18 μg mL⁻¹ in PDL cells with 98.97 ± 0.76% (IC50 = 146 μg mL⁻¹).

3.10.3. Cell apoptosis studies. Flow cytometry analysis of apoptosis showed that the control group, which did not contain any drugs, did not have much effect on the apoptosis rate, either delayed or early, in any of the cell lines. Moreover, in the MCF-7 cell line more than 40% of the cells undergo apoptosis, most of which occurs in the early stages.

The effects of this anticancer compound on Raji, OSCC, and PDL cell lines was 25.53%, 26.64%, and 32.03%. In addition, saponin has moderate toxic effects on the normal PDL cell line, and deconjugation of ZIF-8 with saponin has yielded exciting results. This combination caused the MCF-7 cell line to undergo apoptosis by >55%. It also induced 38.45% apoptosis in the invasive Raji cell line. However, we observed the most effective combination of bioactive ZIF-67 and saponin. For MCF-7, OSCC, and Raji cells, this combination caused 66.77%, 34.94%, and 45.46% of cells to undergo apoptosis, respectively, which had the most significant impact in the early stages of apoptosis. Bioactive ZIF-67 also weakly affected the MCF-7, OSCC, and PDL cell lines. Interestingly, these substances had moderate effects on Raji cells, causing 26.52% of cells to undergo apoptosis. The same pattern was observed for bioactive ZIF-8. The effects of these two substances on normal PDL cells were insignificant, and this number was almost similar to that of the control group.

Interestingly, this arrangement reduced the cytotoxicity of saponin in the normal PDL cell line by 20%. In summary, the results of apoptosis showed that the combination of bioactive ZIF-8 with saponin had significant effects on apoptosis in cancer cell lines, especially aggressive cancers, while reducing the toxicity in normal cell lines compared to the natural product. These effects are due to the targeted delivery of MOFs to cancer tissues and the reduction in their presence in normal cell lines (see Fig. 11 and Table 5).

Fig. 11 After exposure to various nanostructures, the apoptotic percentage of PDL, OSCC, MCF-7, Raji, and Hep-G2 cell lines was evaluated through flow cytometry. The analysis focused on the distribution of annexin V+/7-AAD+ cells (indicative of late apoptotic cells), 7-AAD+ cells (indicative of necrotic cells), and annexin V/7-AAD cells (indicative of live cells) within each treatment group. The treatment groups included the control group (I), saponin-treated group (II), ZIF-8-treated group (III), ZIF-8–saponin-treated group (IV), ZIF-67-treated group (V), and ZIF-67–saponin-treated group (VI).

Table 5 The objective was to assess and compare the proportions of early apoptotic, late apoptotic, and cumulative apoptotic cell populations within each treatment group across PDL, OSCC, MCF-7, and Raji cell lines

Compound	Early apoptosis (%)				Late apoptosis (%)				Total apoptosis (%)
	PDL	OSCC	MCF-7	Raji	PDL	OSCC	MCF-7	Raji	PDL	OSCC	MCF-7	Raji
Control	1.02	0.25	1.92	0.32	0.14	0.46	1.68	0.59	1.15	0.70	3.60	0.91
Saponin	12.40	12.58	21.12	16.36	13.13	12.05	19.07	15.67	25.53	24.64	40.19	32.03
ZIF-67	4.95	11.43	11.44	14.86	2.40	8.97	9.62	11.66	7.35	20.40	21.05	26.52
ZIF-8	2.36	12.36	14.07	16.07	3.96	9.96	12.95	12.95	6.32	22.32	27.02	29.02
ZIF-8-saponin	11.56	16.64	28.93	21.63	9.81	12.94	26.65	16.82	21.37	29.58	55.57	38.45
ZIF-67-saponin	10.11	20.28	34.84	26.36	10.56	14.69	28.93	19.10	20.67	34.97	63.77	45.46

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PDL	ZIF-67	ZIF-8	ZIF-67-saponin	ZIF-8-saponin	Saponin
Concentration	25	25	250	250	100
OSCC	ZIF-67	ZIF-8	ZIF-67-saponin	ZIF-8-saponin	Saponin
Average	75.695	80.96066667	15.149	17.33133333	23.202
MCF-7	ZIF-67	ZIF-8	ZIF-67-saponin	ZIF-8-saponin	Saponin
Average	67.70966667	66.5608	5.796666667	7.940333333	5.588333333
Raji	ZIF-67	ZIF-8	ZIF-67-saponin	ZIF-8-saponin	Saponin
Average	66.04266667	47.23766667	10.749	11.74666667	15.14823333

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4. Discussion

Thus far, the anticancer properties of apigenin and saponin have been studied. One study used a new diosgenyl saponin derivative to prevent oral squamous cell carcinoma (OSCC) cell proliferation. MTT studies on the SCC-7 cell line showed that diosgenyl saponin at a concentration of 24 Mμ had toxicity of approximately 80% after 48 h.⁵⁴ In another study, the effects of apigenin and a nanoagent (apigenin in stearate–chitosan nanogel) on the viability of a K562 chronic myeloid leukemia cell line were investigated over different periods under laboratory conditions. An MTT toxicity assay showed that apigenin at 100 mg mL⁻¹ had a viability rate of approximately 40% after 24 h.⁵⁵ ZIFs are a class of MOFs that are isomorphous with zeolites and combine the advantages of zeolites and MOFs. Their excellent chemical stability, large surface area, high crystallinity, and tunable pores that can trap small molecules such as drugs, dyes, or other nanostructures in the pores make them potential materials for industrial and biomedical applications, especially for drug delivery.⁵⁶

In one research study, six distinct cell lines representing various physiological parts—the kidney, skin, breast, blood, bone, and connective tissue—were used to examine the cellular compatibility of ZIF-8. The results showed that bioactive ZIF-8 did not exhibit significant cytotoxicity up to a threshold level of 30 mg mL⁻¹.⁵⁷ This study investigated the toxicity of bioactive ZIF-67 and ZIF-8 nanocarriers, alone and in combination with saponin and apigenin. The results showed that bioactive ZIF-67 and ZIF-8 samples at 25 μg mL⁻¹ had less than 10% toxicity to PDL cells. The viability rates of Raji cells treated with the above samples at 25 μg mL⁻¹ were 88.41% and 46.82%, respectively. The bioactive ZIF-67-saponin and ZIF-8-saponin compounds also had excellent effects on the studied cancer cells. Hence, the viability rates of Hep-G2 cells treated with the mentioned compounds at 100 μg mL⁻¹ were 61.71% and 67.72%, respectively. The viability rates of cells from PDL treated with these compounds at 100 μg mL⁻¹ were 81.115% and 77.15%, respectively.

Moreover, the toxicity of saponin in PDL cells at a concentration of 100 μg mL⁻¹ was >60%. Among the various biological qualities of apigenin are its anti-inflammatory, anti-cancer, and antioxidant capabilities.⁵⁸ According to the MTT results, the low toxicity of apigenin was evident in all cell lines, but the combination of apigenin with the nanocarrier bioactive ZIF-67 and ZIF-8 increased its anticancer properties. The viability of the PDL cells treated with bioactive ZIF-67 and ZIF-8 apigenin compounds at a concentration of 150 mg mL⁻¹ was 76.57% and 81.9%, respectively. In contrast, the viability of Raji cells treated with the above-mentioned compounds was only 46.57% and 58.67%, respectively. At a concentration of 150 μg mL⁻¹, apigenin exhibited less than 30% toxicity in Raji cells. Fluorescence images of MCF-7 and PDL cells were analyzed to track the internalization of the samples. According to the results, MCF-7 cancer cells showed stable fluorescence emission after 24 h, and the morphology and small number of samples confirmed the internalization of the samples in the cancer cell membrane. Fluorescence images of the ZIF-8-apigenin samples (Z₂ and Z₃) showed the highest emission intensity in fluorescent imaging. ZIF-67 and ZIF-8 nanocarriers have great potential for biomedical applications, including drug delivery, owing to their porous structures, large surface areas, and low toxicity at low concentrations.

5. Conclusion

Evaluation of bioactive ZIF-67 and ZIF-8 nanocarriers with drug loading using new reagents with saponin and apigenin for cancer treatment. One of the significant advantages of this material is that it has few side effects on normal cells. Conventional chemotherapy often damages healthy cells, resulting in several undesirable side effects. Bioactive materials with new drug-loading reagents can target cancer cells more specifically and reduce the side effects on normal cells. In addition, the absence of apoptosis and metastasis confirms the safety and efficacy of this approach. This is an essential breakthrough in cancer treatment because these processes are often responsible for the spread of cancer to other parts of the body. The use of saponins and apigenin as new reagents for drug loading is a promising development. This approach could potentially lead to the discovery of new drugs and treatments for cancer, and provide patients with more treatment options.

Moreover, the results obtained using flow cytometry provide a promising outlook for future cancer treatment. The use of bioactive ZIFs with new drug-loading reagents can transform the field of medicine and lead to safer and more effective patient treatments. This approach has the potential to revolutionize cancer treatment and provide a safer and more effective alternative to traditional chemotherapy. Large surface area, adjustable pore size, rapid drug release under acidic tumor circumstances, biodegradability, biocompatibility, and strong chemical stability are only a few of the benefits and distinctive characteristics of bioactive ZIF MOFs. These features make them excellent candidates for potential use in drug delivery in cancer treatment. Finally, the vital issue confirmed in this study was the reduction of side effects on normal cells. The successful curative combination of bioactive MOFs with natural products in cancer therapy is due to low cross-resistance and drug additivity, which can achieve high fractional cell killing and reduce side effects.

Ethics approval and consent to participate

This study did not involve any animal experiments. No local ethics committee approval was required for any examination or sample collection.

Author contributions

SMM: conceptualization, data curation, formal analysis, methodology, validation, visualization, writing the original draft. AG: data curation, methodology, formal analysis, writing – original draft, writing – review, and editing. WHC: supervision, data curation, formal analysis, investigation, methodology. Project administration and investigation. Funding acquisition.

Data availability

All data generated or analyzed during this study are included in this published article.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the National Science and Technology Council, Taiwan (grant no. NSTC 111-2223-E-011-002-MY3, NSTC 111-2628-E-011-002-MY2, NSTC 113-2927-I-011-504), Ministry of Education, Taiwan (“Sustainable Electrochemical Energy Development Center” (SEED) project), and the National Taiwan University of Science and Technology.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb01303d

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