PEG-functionalized UiO-66 MOFs for targeted vincristine delivery: enhanced cytotoxicity in breast and ovarian cancer cell lines

Abstract

Vincristine (VIN) inhibits microtubule formation in the mitotic spindle, effectively arresting cells in mitosis. This study aimed to develop a polyethylene glycol (PEG)-functionalized UiO-66 metal–organic framework (MOF) as a targeted drug delivery system for VIN in MDA-MB-231 (breast cancer) and A2780 (ovarian cancer) cell lines. The synthesized UiO-66–VIN–PEG nanoparticles exhibited a mean diameter of 223.5 ± 7.45 nm with a spherical morphology. Fourier transform infrared spectroscopy (FT-IR) confirmed successful VIN loading onto the UiO-66 structure. Drug release studies demonstrated a gradual, pH-dependent release profile, with VIN release reaching 55% at pH 7.4 and 75% at pH 5.4 over 72 h, highlighting the system's responsiveness to the acidic tumor microenvironment. Stability assessments indicated that size, polydispersity index (PDI), and entrapment efficiency (EE%) remained more stable at 4 °C compared to 25 °C. In vitro experiments demonstrated significant cytotoxicity and apoptosis induction in MDA-MB-231 and A2780 cells. This was evidenced by increased expression of pro-apoptotic genes (BAX, P53) and suppression of anti-apoptotic and cell cycle-regulatory genes (BCL-2, CCND1, and CDK4). In addition, a notable elevation in DCF fluorescence was observed in UiO-66–VIN-treated and UiO-66–VIN–PEG-treated cells compared to controls. These findings underscore the potential of UiO-66–VIN–PEG as a pH-responsive, targeted drug delivery platform for enhancing VIN's anti-cancer efficacy in breast and ovarian cancer.

Graphical Abstract

Introduction

Vincristine (VIN), an alkaloid derived from the periwinkle plant, is commonly administered alongside other chemotherapy agents to treat various cancers (Lena et al. 1975). VIN inhibits microtubule assembly within the mitotic spindle, disrupting cell division and arresting cancer cells in mitosis. However, resistance to VIN frequently arises during breast and ovarian cancer therapy (Bates and Eastman 2017). Chemoresistance can develop through multiple mechanisms, including increased drug metabolism, reduced intracellular drug uptake, alterations in drug target expression, intracellular drug sequestration, and dysregulation of genes involved in apoptosis, cell cycle control, and DNA repair (Mansoori et al. 2017). In addition, resistance is often associated with autophagy modulation and inflammatory cascades (Novototskaya-Vlasova et al. 2022; Chen et al. 2023a).

Targeted drug delivery systems (DDS) can enhance the selective accumulation of therapeutic agents at tumor sites, thereby improving treatment efficacy while minimizing systemic toxicity (Li et al. 2015). Nanocarrier-based platforms address the limitations of conventional drug delivery methods, which often suffer from high toxicity, poor targeting specificity, and the risk of drug resistance (Wong et al. 2007). Among these, metal–organic frameworks (MOFs) have emerged as versatile, porous materials with strong bonding interactions between inorganic metal centers and polydentate organic linkers. Compared to traditional porous materials, MOFs offer superior surface area, tunable porosity, adjustable pore sizes, and enhanced biocompatibility (Pascanu et al. 2019; Parsaei and Akhbari 2022).

MOF-based composites are recognized as the most promising next-generation materials for removing organic dyes. In recent years, extensive research has been conducted to develop advanced adsorbents capable of effectively eliminating a wide range of pollutants (Hegde et al. 2023).

Beyond pollutant removal, these materials have demonstrated broad utility in applications, such as catalysis, sensing, gas storage, electronics, optics/luminescence, magnetism, and drug delivery. MOFs possess several key attributes, including exceptional stability, a large surface area, tunable functionalization, and the ability to integrate specific functional groups, making them highly attractive as drug delivery carriers (Alavijeh and Akhbari 2022; Jarai et al. 2020; Uthappa et al. 2023). Zirconium-based structures have gained increasing attention among MOFs due to their non-toxic nature, exceptional stability, and biocompatibility with living systems (Vermoortele et al. 2013). The outstanding biocompatibility of zirconium has driven extensive research on its MOFs, particularly the well-characterized Zr terephthalate UiO-66, which is widely employed in drug delivery applications. UiO-66 is a MOF composed of octahedral [Zr₆O₄(OH)₄] clusters coordinated with benzene-1,4-dicarboxylic acid (BDC) ligands (Jerozal et al. 2023).

Due to their exceptional chemical and physical properties, various UiO-66-derived MOFs have been utilized for anti-cancer drug packaging. These materials exhibit excellent water resistance, enhanced chemical, thermal, and mechanical stability, ideal biodegradability, and a high specific surface area, making them highly effective for controlled drug release (Bazzazan et al. 2023; Parsaei and Akhbari 2023; Molavi et al. 2018).

Surface modification of nanoparticles has proven to be an effective strategy for regulating drug release, enhancing chemical and colloidal stability, and controlling the in vivo behavior of nano-MOFs in biological environments (Barjasteh et al. 2022). In biomedical applications, there is growing interest in integrating MOF nanoparticles with biocompatible polymers, such as chitosan, polyethylene glycol (PEG), and cyclodextrin (Karimi and Namazi 2023).

PEG is a widely used, biocompatible, non-immunogenic, hydrophilic, and chemically inert polymer. It has become one of the most extensively utilized compounds for nanoparticle surface engineering, allowing evasion of innate immune detection. PEG coating (PEGylation) forms a hydrophilic barrier around nanoparticles, shielding them from environmental degradation, preventing aggregation, and ensuring colloidal stability (Ahmadijokani et al. 2022). Due to its advantageous properties—including improved bioavailability, controlled drug release, excellent biocompatibility and biodegradability, and physicochemical characteristics that extend plasma half-life—PEG is frequently chosen for drug delivery applications (Chen et al. 2023b; Grossen et al. 2017). Incorporating PEG onto the external surfaces of MOFs has been proposed as a strategy to improve stability and efficacy in DDSs (Abánades Lázaro et al. 2017).

The interaction between UiO-66 and PEG chains can occur through physical or chemical bonding, depending on how the material is synthesized and functionalized. In some cases, PEG binds to UiO-66 via hydrogen bonding or van der Waals forces, allowing it to influence the structure of UiO-66 without forming permanent chemical bonds. For instance, PEG has been used as a surfactant in UiO-66 synthesis, helping to shape its structural properties while maintaining its inherent composition (Jia et al. 2022).

On the other hand, covalent bonding can be achieved when PEG is modified with reactive functional groups, such as alkyne or azide, enabling its attachment to UiO-66 through click chemistry. This method significantly improves the stability and functionality of UiO-66–PEG composites, making them more effective in drug delivery and imaging applications (Zeng et al. 2021).

The hydrophilic nature of PEG allows for its easy attachment to the external surfaces of UiO-66, primarily through strong chemical hydrogen bonding (Hu et al. 2020). This property makes PEGylation a widely used strategy in modern cancer therapy to enhance DDSs. PEGylation involves grafting PEG chains onto the surface of drug carriers, forming a protective hydrophilic layer. This coating helps extend the circulation time and half-life of nanoparticles in the bloodstream by preventing rapid clearance through spatial repulsion effects. Given these advantages, PEGylated MOFs offer greater stability, biocompatibility, drug-loading efficiency, and prolonged systemic circulation, making them promising candidates for drug delivery applications (Zeyni et al. 2023).

Breast and ovarian cancers are among the most aggressive malignancies affecting women, necessitating urgent therapeutic and diagnostic interventions (Łukasiewicz et al. 2021; Grabska et al. 2021). To our knowledge, no prior study has explored the encapsulation of VIN within a UiO-66–PEG system for enhanced delivery to breast and ovarian cancer cells. This study introduces a novel smart DDS for VIN, integrating the unique properties of PEGylated MOFs with active targeting mechanisms to advance therapeutic approaches for high-grade breast and ovarian cancers.

Materials and methods

Synthesis of MOF

UiO-66 was synthesized using a modified version of a previously reported method. In a standard procedure, 0.39 g of terephthalic acid (BDC, benzene-1,4-dicarboxylic acid) was employed as the organic linker and dissolved with 0.54 g of ZrCl₄ in 31 mL of N,N-dimethylformamide (DMF) at 25  °C under vigorous stirring until a clear solution formed. The mixture was then placed in a Teflon-lined hydrothermal autoclave and heated at 120  °C for 24 h. After cooling to room temperature, the precipitate was collected via centrifugation and underwent a solvent exchange process to eliminate unreacted components. This purification step included three rounds of ultrasonication and centrifugation in 20 mL of DMF, followed by three rounds in 20 mL of chloroform (15 min per cycle). Furthermore, the synthesized UiO-66 was soaked in 15 mL of chloroform for 5 days, with daily sonication (15 min in fresh chloroform) to further remove residual DMF. Finally, the purified nanoparticles were vacuum-dried at 120 °C to ensure complete solvent removal (Ahmadijokani et al. 2020).

For drug loading, VIN (15 mg) was first dissolved in 15 mL of DMF inside a glass tube sealed with a phenolic cap. To activate the drug for conjugation, CDI (20 mg) was added to the solution, which was then heated at 45 °C under vacuum for 1 h. Once the reaction was complete, the mixture was cooled to room temperature, and UiO-66 (200 mg) was introduced. To ensure uniform dispersion, the solution underwent ultrasonication (50 kHz, 100 W/L) for 5 min, followed by continuous stirring at room temperature for 48 h. After stirring, the drug-loaded particles were isolated using centrifugation (4,000 rpm, 10 min) and washed with ethanol to remove any unbound compounds. The final product was left to dry at room temperature.

To functionalize the MOF, 75 mg of UiO-66–VIN particles was mixed with 0.6 g of PEG 2000 (purity > 99.9%, Merck, Germany) in 100 mL of deionized water. The mixture was stirred for 3 h at room temperature, after which the particles were separated by centrifugation (4000 rpm, 10 min), washed three times with deionized water to remove excess reagents, and dried at room temperature (Gupta et al. 2019). The synthesis process of UiO-66–VIN is illustrated schematically in Fig. 1.

Fig. 1

Synthesis process of the UiO-66–VIN–PEG MOF nanoparticles is depicted in the schematic representation

Characterization tests

The functional groups of VIN, UiO-66, UiO-66–VIN, and UiO-66–VIN–PEG were analyzed using FT-IR (Spectrum Two, PerkinElmer, USA) in the range of 4000–400 cm⁻¹. The morphology of the synthesized MOFs was examined via SEM (TESCAN VEGA 3SB) and TEM (Philips CM30, Netherlands).

The hydrodynamic diameter and size distribution of UiO-66–VIN–PEG were determined using DLS (Malvern Zetasizer Nano, Malvern Instruments, UK). The crystallographic structure was characterized by XRD (Bruker AXS D8 Advance) with Cu Kα radiation, operating within the 2θ range of 10°–90°.

Entrapment efficiency (EE%)

EE% was determined by measuring the concentration of unbound VIN. The free drug was separated from the UiO-66–PEG mixture using Ultracel-30 K Millipore filters (MWCO: 30,000 Da, Merck, Germany). The inner compartment of the filtration device contained 500 μL of the formulation and was centrifuged at 4000 rpm for 20 min at 4 °C (Eppendorf® 580R, Germany). The concentration of unencapsulated drug in the outer compartment was quantified via UV–visible spectrophotometry at 297 nm (JASCO, V-530, Japan). EE% was calculated using the following Eq. (1):

$$\text{EE }(\text{\%}) =\frac{\text{Initial \,vincristine \,added}-\text{Free\, vincristine }}{\text{Initial \,vincristine \,added}} \times 100$$

(1)

In vitro release study and kinetic model

The dialysis diffusion technique was employed to evaluate the in vitro release profile of VIN from UiO-66–VIN and UiO-66–VIN–PEG. A dialysis membrane with a molecular weight cutoff (MWCO) of 12 kDa (Merck, Germany) was used. For this experiment, 2 mL of the MOF suspension was placed into a dialysis bag, which was then sealed and submerged in 50 mL of PBS–SDS (pH 7.4, 37 °C). This solution functioned as the release medium, and the setup was maintained on a magnetic stirrer at 300 rpm. At predefined time intervals, aliquots were withdrawn from the buffer compartment and replaced with fresh PBS–SDS to maintain sink conditions. The optical density of each sample was determined using UV–visible spectrophotometry at 297 nm, and the cumulative amount of released drug was quantified using the standard curve equation. A release profile graph was generated to illustrate drug release over time.

The release kinetics were analyzed using Higuchi, Korsmeyer–Peppas, zero-order, and first-order models. The Higuchi model represents the relationship between the cumulative percentage of drug release and the square root of time, describing a diffusion-controlled release mechanism. The Korsmeyer–Peppas model examines drug release from polymeric nanoparticle formulations and is particularly useful for systems exhibiting a combination of Fickian and non-Fickian diffusion. The zero-order model describes a constant drug release rate, independent of concentration, whereas the first-order model assumes that drug release is concentration-dependent. The correlation coefficient (r) for each model was calculated by linear regression analysis to determine the best-fitting release mechanism (Safari Sharafshadeh et al. 2024).

Physical stability

The physical stability of UiO-66–VIN–PEG was evaluated over 1 month under two distinct storage conditions (25 °C and 4 °C). Changes in particle size, polydispersity index (PDI), and EE% were monitored at 14-day and 30-day intervals to assess formulation stability.

Cytotoxicity assay

The MTT colorimetric assay (3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyltetrazolium bromide; Gibco) was used to assess the cytotoxic effects of UiO-66, free VIN, and UiO-66–VIN–PEG on MDA-MB-231, A2780, and HFF cells. All cell lines were obtained from Iran's National Center for Genetic and Biological Resources. Cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and incubated at 37 °C for 24 h. Subsequently, cells were exposed to increasing concentrations (12.5–200 μg/mL) of the formulations for 48 and 72 h.

Cell viability was measured using an MTT reagent (Sigma-Aldrich, Germany) at 570 nm with a Synergy 2 Multi-Detection Microplate Reader (BioTek Instruments, Inc.). The half-maximal inhibitory concentration (IC₅₀) was determined based on data from six independent experiments. Results were expressed as mean ± standard deviation, normalized to untreated control cells (100% proliferation) (Chamani et al. 2020).

Apoptotic and cell cycle arrest-associated gene expression profile

To investigate the molecular mechanisms underlying apoptosis and cell cycle regulation, MDA-MB-231 and A2780 cells were exposed to UiO-66, free VIN, UiO-66–VIN, and UiO-66–VIN–PEG at their respective IC₅₀ concentrations for 48 h. Following treatment, total RNA was extracted using an RNA isolation kit (Cinnagen, Iran), and complementary DNA (cDNA) was synthesized with the RevertAid™ cDNA Synthesis Kit (Cinnagen, Iran).

Gene expression analysis was conducted using real-time PCR to quantify BCL2, BAX, P53, CCND1, and CDK4 expression levels, with β-actin as an internal control (Dolati et al. 2021). The amplification was done with SYBR^® Green Master Mix (Bio-Rad, USA) in a Rotor-Gene Q Detection System (Qiagen, USA). The thermal cycling protocol included an initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 15 s and an extension phase at 72 °C for 1 min. Gene expression levels were calculated using the 2⁻^ΔΔCt method, ensuring precise quantification based on PCR efficiency (Mahdizadeh et al. 2024).

By integrating these molecular analyses, this study provides insights into the apoptotic pathways and cell cycle regulatory mechanisms influenced by UiO-66-based formulations, highlighting their potential impact on cancer cell viability and therapeutic efficacy.

Flow cytometry for apoptosis/necrosis detection

The Annexin V-FITC/propidium iodide (PI) apoptosis kit (MabTag GmbH, Germany) assessed apoptosis and necrosis in pretreated cells. MDA-MB-231 and A2780 cells were seeded in six-well plates at a density of 5 × 10⁵ cells per plate and maintained in a standard culture medium. After 24 h, the medium was removed and replaced with a formulation-containing medium, exposing the cells to UiO-66, free VIN, UiO-66–VIN, and UiO-66–VIN–PEG for 48 h.

Following treatment, cells were trypsinized and harvested by centrifugation at 1000 rpm for 5 min, then washed with 500 μL of PBS. Subsequently, 100 μL of diluted Annexin V, binding buffer, and PI were added to the cell suspension, and samples were incubated in a light-protected environment for 20 min. After incubation, the samples underwent a second centrifugation at 1,000 rpm for 5 min, the supernatant was discarded, and 100 μL of fresh binding buffer was added. The prepared samples were then analyzed using the FITC-Annexin V Apoptosis Detection Kit in a BD FACS Calibur flow cytometer (BD Bioscience, CA) to differentiate apoptotic and necrotic cell populations (Safari Sharafshadeh et al. 2024).

By incorporating this analysis, the study comprehensively evaluates the apoptotic effects induced by the UiO-66-based formulations, offering mechanistic insights into their therapeutic potential in cancer treatment.

Cell cycle analysis

PI staining was employed to evaluate the cell cycle distribution, a method in which PI binds to genomic DNA, enabling the identification of different cell cycle phases. MDA-MB-231 and A2780 cells were seeded in six-well plates at a density of 1 × 10⁶ cells per well and incubated overnight in a fully supplemented growth medium. Following this, cells were treated with UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG for 48 h. After treatment, cells were washed three times with PBS, transferred to a fresh, complete medium, and incubated for another 48 h. Subsequently, cells from each experimental group were fixed in 70% cold ethanol overnight. The fixed cells were resuspended in 500 μL of PI solution (containing RNase) and incubated at room temperature for 20 min in darkness. Finally, flow cytometry analysis was performed to determine the cell cycle distribution. The experiments were conducted in triplicate, focusing on the IC₅₀ concentration (Shahbazi et al. 2024).

This analysis provides insights into the impact of UiO-66-based formulations on cell cycle regulation, further elucidating their potential role in cancer treatment.

Intracellular reactive oxygen species generation

The levels of ROS generated in response to different treatments were quantified using the H₂DCFDA assay kit (Kiazist, Iran, KCAS37). A2780 and MDA-MB-231 cells were treated with UiO-66, free VIN, UiO-66–VIN, and UiO-66–VIN–PEG at their respective IC₅₀ concentrations for 48 h. Following treatment, cells were washed with PBS and incubated with 80 μL of H₂DCFDA at 37 °C for 30 min. Fluorescence intensity was then measured using a microplate reader to assess intracellular ROS production (Asghari Lalami et al. 2023).

This analysis provides insights into the oxidative stress response induced by UiO-66-based formulations, highlighting their potential role in mediating cytotoxic effects in cancer cells.

Statistical analysis

Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test for multiple comparisons among experimental groups. All analyses were conducted in triplicate and evaluated using GraphPad Prism (version 9). A P value < 0.05 was considered statistically significant.

Results and discussion

Characterization of UiO-66–VIN–PEG

DLS analysis revealed that the average sizes of UiO-66, UiO-66–VIN, and UiO-66–VIN–PEG were 178.3 ± 5.32 nm, 223.5 ± 7.45 nm, and 241.8 ± 5.21 nm, respectively, indicating an optimal size for targeted drug delivery (Fig. 2A). The PDI values of these formulations were 0.109 ± 0.012, 0.137 ± 0.015, and 0.183 ± 0.013, respectively, confirming uniform dispersion (Table 1).

Fig. 2

Physicochemical characterization of UiO-66–VIN–PEG following VIN encapsulation. A DLS analysis of particle size distribution. B SEM image of UiO-66. C SEM image of UiO-66–VIN–PEG, D TEM image of UiO-66–VIN–PEG, showcasing the morphological features of synthesized MOF. E FT-IR spectroscopy confirms the structural composition. F XRD analysis comparing the peaks of (a) UiO-66, (b) UiO-66–VIN, (c) UiO-66–VIN–PEG, and (d) VIN

Table 1 Main physicochemical parameters (size, PDI, and EE%) were measured immediately after the preparation of UiO-66–VIN–PEG

A comparison of the hydrodynamic diameters of UiO-66–VIN and UiO-66–VIN–PEG indicates that PEGylation resulted in a slight size increase, confirming the successful incorporation of PEG into the UiO-66 framework. This expansion can be attributed to PEG’s hydrophilic nature, which attracts water molecules, increasing particle hydration. Furthermore, PEG may interact with VIN, influencing drug distribution within the MOF and its overall interactions Zeng et al. 2021. Since nanoparticles between 100 and 200 nm exhibit enhanced tumor penetration, the formulation falls within the suitable range for passive targeting Longmire et al. 2008.

The morphology of unloaded UiO-66 and UiO-66–VIN–PEG was examined using SEM and TEM. The SEM images of unloaded UiO-66 and UiO-66–VIN–PEG confirmed that the nanoparticles retained their spherical shape and uniform size distribution without visible degradation (Fig. 2B, C, respectively). The TEM image further verified the consistent dispersion of the UiO-66–VIN–PEG particles (Fig. 2D).

FT-IR spectroscopy confirmed structural characteristics and chemical interactions (Fig. 2E). Characteristic peaks for VIN appeared at 655 cm⁻¹ (out-of-plane bending), 1083 cm⁻¹ (C–O ester), 1407 cm⁻¹ (C–N stretching), 1640 cm⁻¹ (N–H deformation), 2084 cm⁻¹ (aromatic C=C), and 3437 cm⁻¹ (N–H stretching). Peaks for UiO-66 were observed at 498 cm⁻¹ and 553 cm⁻¹ (Zr–(OC) asymmetric and symmetric stretching), 1401 cm⁻¹ (C–O in carboxyl), 1505 cm⁻¹ (carboxylate stretching), 670 cm⁻¹ and 749 cm⁻¹ (O–H bending and Zr–O modes), and 1583 cm⁻¹ (C–C aromatic stretching) (39, 40). Upon VIN loading, a new C–O stretching band appeared at 1059 cm⁻¹, confirming drug incorporation. Following PEGylation, a C–H bond peak characteristic of PEG emerged at 1298 cm⁻¹ Liu et al. 2019. A shift in the VIN-related peak to 1056 cm⁻¹ further supports PEG conjugation and drug encapsulation (Fig. 2E).

To examine crystallinity, XRD analysis was performed (Fig. 2F). The UiO-66 diffraction pattern exhibited peaks at 7.56°, 8.68°, 12.15°, 14.90°, 17.30°, 19.17°, 22.63°, 25.95°, 27.58°, 30.17°, 31.08°, 33.46°, 35.87°, 37.71°, 39.72°, 40.91°, 43.77°, 44.77°, 50.57°, 51.94°, and 57.01°, with index peaks at 7.56°, 8.68°, 25.95°, and 31.08°. The presence of distinct peaks confirmed the crystalline nature of UiO-66–VIN, and VIN loading did not alter nanoparticle symmetry. In addition, a PEG-related peak at 27.2°, marked with an asterisk, confirmed PEG's presence in the MOF structure.

In addition, EE% was 65.24% ± 1.72% for UiO-66–VIN and 75.31% ± 1.52% for UiO-66–VIN–PEG, highlighting improved drug loading efficiency after PEGylation. The higher EE% of UiO-66–VIN–PEG compared to UiO-66–VIN suggests that PEGylation enhances drug entrapment, likely due to an increase in molecular weight and a more porous matrix, which facilitates deeper drug incorporation within the MOF Zaman et al. 2022.

These findings demonstrate that UiO-66–VIN–PEG exhibits high structural stability, efficient VIN encapsulation, and preserved crystallinity, making it a promising nano-carrier for targeted cancer therapy.

Drug release and kinetic study

Considering the acidic microenvironment of cancer cells (pH ~ 5.5) compared to physiological conditions (pH ~ 7.4), it is crucial to assess drug release at different pH levels. The release profile of VIN from MOFs was evaluated using UV–visible spectroscopy, monitoring absorbance changes in the release medium while incubating UiO-66–VIN and UiO-66–VIN–PEG. Another key objective was to optimize the release mechanism using kinetic models (Fig. 3A, B, and Table 2).

Fig. 3

In vitro release profile and stability of UiO-66–VIN–PEG. A, B Cumulative release of VIN from UiO-66–VIN and UiO-66–VIN–PEG at pH 5.4 and 7.4. C–E Physical stability evaluation based on (C) particle size, D PDI, and E EE% at 4 °C and 25 °C over 30 days. Data are presented as means ± standard deviations (n = 3). Statistical significance: *P < 0.05,**P < 0.01

Table 2 Kinetic release models and the parameters obtained for UiO-66–VIN–PEG

The release pattern was biphasic, characterized by an initial burst release within the first few hours, followed by sustained release over 72 h. At pH 5.4, approximately 66.10% ± 2.33% of VIN was released in the first 24 h, compared to 50.31% ± 1.53% at pH 7.4 for UiO-66 formulations. This was followed by a prolonged release phase, with cumulative drug release reaching 64.53% ± 1.86% at pH 5.4 and 49.32% ± 2.12% at pH 7.4 for UiO-66–VIN–PEG. Figure 3A, B shows that the release rate increased significantly at pH 5.4, demonstrating a pH-sensitive release behavior.

Free VIN (in aqueous solution) exhibited rapid release, with over 90% released within 12 h. In contrast, UiO-66–VIN showed moderate release, reaching ~ 70% at pH 5.4 and 50% at pH 7.4 after 72 h. The UiO-66–VIN–PEG formulation demonstrated a more controlled release, reaching ~ 80% cumulative release at pH 5.4 within 72 h, indicating a prolonged drug release profile. Notably, PEGylation reduced the initial burst release while maintaining sustained release, which can prolong therapeutic effects while minimizing systemic toxicity.

These findings suggest that the DDS is more effective in acidic conditions, a key characteristic for targeting cancer cells that often exhibit a lower extracellular pH. The presence of PEG in UiO-66–VIN–PEG appears to enhance release control, offering extended drug availability compared to UiO-66–VIN.

MOF-based carriers are expected to retain their drug payload under physiological conditions while selectively releasing the drug at the tumor site, preventing non-specific drug dispersion. The observed pH-responsive release in UiO-66–VIN–PEG can be attributed to the lack of strong interactions between Zr–O clusters and VIN molecules, facilitating acid-triggered drug release (Naderinezhad et al. 2017). In addition, the disintegration of UiO-66 and PEG-modified structures at acidic pH accelerates drug release (Davarpanah et al. 2018). These results suggest that PEGylation enhances VIN retention, allowing for a sustained and tumor-targeted release, ultimately improving drug bioavailability and therapeutic efficacy (Cortesi et al. 2013).

Previous studies support this finding. Nasrabadi et al. reported that 80% of ciprofloxacin was released from UiO-66 at pH 7.4 after 72 h, whereas at pH 5, release increased to 87% (Nasrabadi et al. 2019). Similarly, Rakhshani et al. found that the lack of strong UiO-66–NH₂/PNVCL and Zr–O/DOX interactions resulted in faster drug release at pH 5.5 compared to pH 7.4 (Rakhshani et al. 2021a).

Drug release from MOFs occurs in two phases: an initial rapid release from surface pores and a gradual diffusion of drug molecules from internal pores (Rakhshani et al. 2021b). PEGylation further modifies drug encapsulation efficiency and enhances targeted delivery. Increasing PEG molecular weight can improve drug loading capacity, likely due to the hydrophilic shell formation, which prevents premature drug desorption. In addition, PEGylation delays drug excretion, enabling controlled drug diffusion and sustained therapeutic action. PEGylated MOFs offer enhanced stability, biocompatibility, encapsulation efficiency, and prolonged blood circulation (Zeyni et al. 2023).

To characterize the kinetic release mechanisms, multiple mathematical models were applied to describe the release behavior of VIN from UiO-66–VIN–PEG at pH 5.4 and 7.4 (Table 2). Model selection was based on the highest R² value, indicating the best fit. According to Table 2, the first-order model best describes the release kinetics of free VIN, whereas the Korsmeyer–Peppas model provides the best fit for UiO-66–VIN–PEG at both pH 7.4 and 5.4.

VIN release from MOFs followed a non-Fickian diffusion mechanism, as indicated by the n values from the Korsmeyer–Peppas equation, which were n = 0.5344; R² = 0.9211 (UiO-66–VIN, pH 5.4), n = 0.5857; R² = 0.9380 (UiO-66–VIN–PEG, pH 5.4), n = 0.5515; R² = 0.9374 (UiO-66–VIN, pH 7.4), and n = 0.6440; R² = 0.9537 (UiO-66–VIN–PEG, pH 7.4). These results indicate that PEGylation enhances controlled diffusion, providing a sustained-release system for cancer-targeted therapy.

Stability of MOF

To evaluate the physical stability of UiO-66–VIN–PEG, changes in vesicle size, PDI, and EE% were monitored over 30 days at 4 °C and 25 °C (Fig. 3C–D). During storage, drug leakage from the MOF led to an increase in particle size and PDI, accompanied by a decrease in EE%. After 30 days, a significant rise in particle size (P < 0.05) and PDI (P < 0.05) was observed, while EE% decreased significantly (P < 0.01) at 25 °C compared to 4 °C, indicating greater drug loss at the higher temperature.

The structural rigidity of UiO-66 appears to be temperature-dependent, affecting its stability as a nano-carrier. UiO-66 was selected due to its exceptional chemical resistance to polar solvents (water, alcohol, and organic) and high thermal stability Hasan et al. (2014). Consistent with previous studies, lower temperatures are recommended for the long-term storage of UiO-66–PEG, as they help preserve its structural integrity and prevent premature drug release (Duvnjak Romić et al. 2019; Dastneshan et al. 2021).

Cytotoxicity of UiO-66–VIN–PEG formulation

The cytotoxicity of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG was assessed via MTT assay in MDA-MB-231 (Fig. 4A, C), A2780 (Fig. 4B, D), and HFF cells (Fig. 4E, F) after 48 h and 72 h. Untreated cells served as controls. Among all tested formulations, UiO-66–VIN–PEG exhibited the highest anti-cancer activity against MDA-MB-231 and A2780 cells, with its cytotoxic effect being dose- and time-dependent. Across all concentrations (12.5–200 μg/mL), significant cytotoxicity was observed after 48 h and 72 h in both cancer cell lines (P < 0.0001). At 200 μg/mL, UiO-66–VIN–PEG induced a significant reduction in cell viability (P < 0.0001), though no substantial difference in cell viability reduction was noted between UiO-66–VIN and UiO-66–VIN–PEG at higher concentrations. Interestingly, A2780 cells exhibited a stronger cytotoxic response than MDA-MB-231 cells, suggesting higher susceptibility to the treatment.

Fig. 4

Cytotoxicity assessment of UiO-66–VIN–PEG in breast (MDA-MB-231) and ovarian (A2780) cancer cells, as well as normal (HFF) cells. A, B Viability of MDA-MB-231 and A2780 cells after 48 h of treatment with UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG at varying concentrations. C, D Cell viability after 72 h of exposure. E, F Viability of HFF cells following 48 and 72 h of treatment. Data are expressed as means ± standard deviations (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant

These findings highlight the ability of MOFs to inhibit tumor growth by inducing apoptosis-associated gene expression, leading to cellular disintegration (Nel et al. 2006). In addition, PEGylation enhances drug stability by forming a protective layer, minimizing premature degradation and macrophage uptake, thereby facilitating targeted drug accumulation in malignant cells (Elmehrath et al. 2023). The lower sensitivity of MDA-MB-231 cells may stem from the lack of HER2 amplification, absence of ER/PR expression, and altered multidrug resistance (MDR) gene expression. Consequently, MOF-based DDSs have been extensively explored for treating breast and ovarian cancers (Li et al. 2021; Alves et al. 2021).

The MTT assay results for HFF cells revealed that UiO-66–VIN–PEG exhibited lower toxicity toward normal cells than free VIN, demonstrating selective cytotoxicity (Fig. 4E, F). At 200 μg/mL, UiO-66–VIN–PEG induced 20% cell death after 48 h and 26% after 72 h, whereas cancer cells were significantly more vulnerable to treatment. These findings align with studies showing that encapsulating chemotherapeutics within MOFs reduces toxicity to normal cells while enhancing anti-cancer efficacy.

The IC₅₀ values for UiO-66–VIN–PEG were significantly lower (P < 0.001) than those of free VIN and UiO-66–VIN in MDA-MB-231 and A2780 after 48 h and 72 h. Specifically, the IC₅₀ values for MDA-MB-231 were 273 μg/mL (48 h) and 212 μg/mL (72 h), while for A2780 cells, the values were 188 μg/mL (48 h) and 139 μg/mL (72 h). Notably, no significant difference in IC₅₀ was observed between UiO-66–VIN and UiO-66–VIN–PEG across the 48 h and 72 h timepoints.

Consistent with these results, Hashemzadeh et al. demonstrated that their MOF-based oxaliplatin delivery system significantly reduced IC₅₀ values in CT-26 colorectal cancer cells, where IC₅₀ values for oxaliplatin, UiO-66–oxaliplatin, and UiO-66–oxaliplatin@FA were 21.38, 95.50, and 18.20 μg/mL, respectively Hashemzadeh et al. 2021. Similarly, Bazzazan et al. reported that UiO-66–Curcumin exhibited high biocompatibility with healthy MCF10A breast cells, maintaining 95% viability at 200 μg/mL while significantly reducing IC₅₀ in SKBR3 and MDA-MB-231 cells (72.2 vs. 159.3 μg/mL and 109.3 vs. 269.1 μg/mL, respectively, after 72 h) (Bazzazan et al. 2023).

These results demonstrate that PEGylated MOF-based DDSs enhance anti-cancer efficacy, improve selectivity toward malignant cells, and reduce toxicity to normal tissues, making them promising candidates for targeted cancer therapy.

Real-time PCR findings

The effect of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG on gene expression was evaluated in MDA-MB-231 and A2780 cells. The expression levels of BAX, BCL2, CCND1, P53, and CDK4 were analyzed to determine the impact of IC₅₀ concentrations of each formulation on cancer treatment. The results revealed a significant upregulation of BAX and P53 in all treatment groups compared to the control (P < 0.0001), with UiO-66–VIN–PEG showing the most substantial increase (P < 0.0001). Conversely, BCL2, CCND1, and CDK4 were significantly downregulated in the VIN, UiO-66–VIN, and UiO-66–VIN–PEG groups (P < 0.0001), with the strongest suppression observed in UiO-66–VIN–PEG (Fig. 5A, B). These findings align with the MTT assay results, suggesting that cell cycle regulation and apoptotic gene modulation contribute to the cytotoxic effects of these formulations.

Fig. 5

Gene expression analysis of apoptosis- and cell cycle-related markers. Expression levels of BAX, BCL2, CCND1, P53, and CDK4 in A MDA-MB-231 and B A2780 cells treated with IC₅₀ concentrations of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG for 48 h. Data are presented as means ± standard deviations (n = 3). ***P < 0.001, ****P < 0.0001 for all comparisons

Quantifying apoptotic mechanisms is complex due to multiple interconnected biological pathways. The BCL-2 protein family consists of pro-apoptotic (e.g., BAX) and anti-apoptotic (e.g., BCL-2) regulators that govern mitochondrial outer membrane permeabilization, a key step in programmed cell death (Martinou and Youle 2011). BAX is a pro-apoptotic factor regulated by P53, contributing to mitochondrial dysfunction by activating the voltage-dependent anion channel, leading to cytochrome c release and subsequent caspase activation (Gogvadze et al. 2006). P53 plays a central role in tumor suppression, responding to DNA damage by initiating repair or triggering apoptosis by regulating BCL-2 family proteins (Ramadan et al. 2019).

The cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs), which control G1/S phase transition. The activation of CDK4/6 is critical for cell cycle progression, and it is facilitated by the MAPK signaling pathway, which promotes cyclin D expression. The cyclin D–CDK4/6 complex phosphorylates retinoblastoma protein (Rb), allowing cell cycle progression (Hu and Huang 2023). The observed downregulation of CDK4 and CCND1 in response to UiO-66–VIN–PEG suggests inhibition of G1/S phase transition, contributing to cell cycle arrest and cancer cell suppression.

These findings are consistent with Ashrafi et al., who demonstrated that UiO-66–Ginsenoside (UiO-66–Gin) upregulated BAX, CASP3, and CASP9 while downregulating BCL2 in AGS gastric cancer cells (Kazazi et al. 2023). Similarly, Bazzazan et al. reported that UiO-66–Curcumin significantly inhibited Matrix metalloproteinase 2, Matrix metalloproteinase 9, and cyclin E/D while activating caspase 3 and caspase 9, improving anti-cancer efficacy (Bazzazan et al. 2023). Furthermore, an in vitro study on MCF-7 cells demonstrated significant alterations in the expression of caspase 7, 8, and 9, indicating the pro-apoptotic effects of MOF-based therapies (Haddad et al. 2020).

These findings highlight that MOF-based DDSs effectively modulate apoptosis and cell cycle progression, reinforcing their therapeutic potential in targeted cancer treatment.

Apoptotic/necrotic detection

Apoptosis in MDA-MB-231 and A2780 cells was assessed using flow cytometry, with cells stained using annexin V-FITC and PI for precise quantification. Treatment with VIN, UiO-66–VIN, and UiO-66–VIN–PEG significantly increased apoptosis in both cancer cell lines compared to the control (P < 0.0001) (Fig. 6A, B). In contrast, UiO-66 alone did not significantly increase apoptotic cells relative to the control (P > 0.05).

Fig. 6

Apoptosis analysis using flow cytometry. A, B Flow cytometry diagrams of control and treated (A) MDA-MB-231 and (B) A2780 cells. Q1: necrotic cells, Q2: late apoptotic cells, Q3: early apoptotic cells, Q4: live cells. C, D Apoptosis rates (%) of MDA-MB-231 and A2780 cells treated with UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG. Cells were exposed to half of their IC₅₀ concentration. Data are presented as means ± standard deviations (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Exposure to UiO-66–VIN–PEG resulted in a marked increase in early apoptosis in MDA-MB-231 cells compared to the control (P < 0.0001). In addition, early apoptosis was significantly elevated in A2780 cells treated with VIN, UiO-66–VIN, and UiO-66–VIN–PEG compared to the untreated group and those treated with UiO-66 alone (P < 0.0001). Late apoptosis was also significantly higher in both cancer cell lines following treatment with UiO-66–VIN and UiO-66–VIN–PEG compared to untreated and UiO-66-treated cells (P < 0.0001), with UiO-66–VIN–PEG demonstrating the most pronounced effect. In addition, VIN significantly increased necrotic cell percentages in both cancer cell lines compared to the control group (P < 0.0001) (Fig. 6C, D). These results indicate that loading VIN into UiO-66 and UiO-66–PEG enhances its ability to induce apoptosis while minimizing systemic toxicity, thereby improving selective cytotoxicity against cancer cells (P < 0.0001).

Flow cytometry enables a rapid and precise assessment of apoptosis, making it an essential technique for evaluating pharmaceutical and nanodrug formulations (Khatibi et al. 2022; Pan et al. 2014). Consistent with these findings, UiO-66–VIN–PEG exhibited more significant cytotoxicity against A2780 cells than MDA-MB-231, suggesting its enhanced therapeutic potential for ovarian cancer treatment.

Similar results have been reported in previous studies. Ashrafi et al. demonstrated that UiO-66–Gin significantly increased apoptosis in AGS gastric cancer cells compared to ginsenoside alone (Kazazi et al. 2023). In addition, studies on breast cancer models have shown that Quercetin@Fe₃O₄–COOH@UiO-66 was efficiently internalized by tumor cells, leading to apoptosis. Research by Bazzazan et al. further confirmed that UiO-66–Curcumin induced significantly higher apoptosis in MDA-MB-231 and SKBR3 cells at IC₅₀ concentrations compared to curcumin alone, reinforcing the potential of MOF-based DDSs for improving anti-cancer efficacy (Bazzazan et al. 2023).

Cell cycle arrest

The impact of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG on cell cycle progression in MDA-MB-231 and A2780 cells was assessed using flow cytometry (Figs. 7A and 8B). The results (Fig. 7C, D) indicate that treatment with VIN, UiO-66–VIN, and UiO-66–VIN–PEG significantly inhibited cell proliferation by increasing the sub-G1 phase population in both cancer cell lines compared to the control (P < 0.01, P < 0.001, respectively).

Fig. 7

Cell cycle analysis using flow cytometry. A, B Cell cycle distribution in MDA-MB-231 and A2780 cells treated with UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG. C, D Quantification of cell cycle phases (%) following treatment. Cells were exposed to half of their IC₅₀ concentration. Data are presented as means ± standard deviations (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

ROS generation in A MDA-MB-231 and B A2780 cells treated with IC₅₀ concentrations of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG for 48 h. Data represent means ± standard deviations (n = 3). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

In MDA-MB-231 cells, the percentage of sub-G1 phase cells after treatment with VIN, UiO-66–VIN, and UiO-66–VIN–PEG was approximately 20.65%, 33.17%, and 29.86%, respectively. In A2780 cells, the proportions were 24.13%, 37.43%, and 37.39%, respectively. Moreover, treatment with VIN and UiO-66–VIN–PEG significantly reduced the G1 phase population in MDA-MB-231 cells, whereas UiO-66–VIN and UiO-66–VIN–PEG notably decreased the G1 phase population in A2780 cells. UiO-66–VIN treatment significantly reduced the G2 phase population in both cell lines (P < 0.05).

These findings were further supported by apoptosis analyses, which demonstrated that A2780 cells treated with UiO-66–VIN and UiO-66–VIN–PEG exhibited elevated apoptosis rates and increased cell cycle arrest in the sub-G1 phase. Cell cycle arrest was linked to the downregulation of CCND1 and CDK4 gene expression, consistent with RT-qPCR results. The increase in the sub-G1 phase reflects the proportion of cancer cells undergoing apoptosis (Fotouhi et al. 2021).

These results suggest that the high drug-loading capacity and controlled release properties of MOF-based nanocomposites make them promising candidates for targeted drug delivery. Studies evaluating MOF compounds have also emphasized their role in inducing cell cycle arrest (Wu and Yang 2017). Lin et al. reported that ZJU-64 and ZJU-64-CH3 exhibited minimal cytotoxicity in PC12 cells, whereas ZIF-8 released Zn²⁺, leading to cell cycle arrest and oxidative stress (Lin et al. 2016). Similarly, Bazzazan et al. demonstrated that treatment with IC₅₀ concentrations of Curcumin and UiO-66–Curcumin significantly increased the sub-G1 phase population in breast cancer cells. This effect was more pronounced than in the groups treated with UiO-66 alone or the untreated control (Bazzazan et al. 2023).

ROS generation in treated cells

Elevated levels of ROS have been shown to induce cell death by continuously damaging lipids, proteins, and DNA. Excessive ROS disrupt normal protein function and interfere with redox-modifying enzymes, gene expression, redox receptor-binding proteins, and protein turnover regulation (Joo and Jetten 2010; Sadri et al. 2020). Cancer cells generate higher levels of ROS than normal cells due to factors, such as hypoxia, mutations in nuclear and mitochondrial genes, oncogene activation, and loss of tumor suppressor genes. While moderate ROS levels are essential for cancer cell proliferation, differentiation, and survival, excessive accumulation can trigger apoptosis. In addition, recent studies indicate that ROS play a role in tumor invasion, angiogenesis, and metastasis (Zeng et al. 2016; Tomar et al. 2019).

In this study, IC₅₀ concentrations of UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG were used to treat MDA-MB-231 and A2780 cells, after which intracellular ROS levels were measured (Fig. 8A, B). Compared to the control group, both cell lines treated with UiO-66, VIN, UiO-66–VIN, and UiO-66–VIN–PEG exhibited significantly higher 2′,7′-dichlorodihydrofluorescein (DCF) fluorescence (P < 0.05, P < 0.01, P < 0.001, P < 0.0001, respectively). Notably, MDA-MB-231 and A2780 cells treated with UiO-66–VIN–PEG at IC₅₀ concentrations showed intracellular ROS production increases of 2199.5% and 2505%, respectively. The highest ROS levels were observed in cells treated with UiO-66–VIN–PEG compared to all other groups. These findings align with previous studies demonstrating the potential of nanoscale MOFs to induce ROS production for effective cancer therapy (Zhang et al. 2021; Sk et al. 2018; Ni et al. 2020).

Limitations of the study

A comprehensive evaluation of the anti-cancer properties of UiO-66–VIN–PEG requires further in vivo and clinical investigations. The most effective approach to assessing DDSs involves in vitro, in vivo, and human trials. The process typically begins with preliminary screening in cell cultures, followed by optimization in animal models, and ultimately progresses to clinical evaluation for therapeutic validation.

Conclusion

This study, UiO-66–VIN-PEG was successfully synthesized and demonstrated superior anti-cancer efficacy compared to UiO-66–VIN and free VIN. In addition, UiO-66–VIN–PEG exhibited a protective effect on normal cells, mitigating the adverse effects of the drug. The formulation significantly reduced the viability of breast (MDA-MB-231) and ovarian (A2780) cancer cells by inducing apoptosis and cell cycle arrest. Molecular analyses revealed that UiO-66–VIN–PEG altered the expression of key regulatory genes (BAX, BCL2, P53, CCND1, and CDK4) and promoted ROS generation, contributing to its cytotoxic effects.

The findings suggest that UiO-66–VIN–PEG holds considerable promise as an anti-cancer therapy, primarily due to its pH-responsive drug release, enhanced apoptosis induction, and potent cytotoxic activity against cancer cells. However, further research is necessary to elucidate the mechanisms underlying its therapeutic effects and validate its efficacy in in vivo and clinical settings.