Dual‐Functioning Metal‐Organic Frameworks: Methotrexate‐Loaded Gadolinium MOFs as Drug Carriers and Radiosensitizers

Abstract

Cancer remains a critical global health challenge, necessitating advanced drug delivery systems through innovations in materials science and nanotechnology. This study evaluates gadolinium metal‐organic frameworks (Gd‐MOFs) as potential drug delivery systems for anticancer therapy, particularly when combined with radiotherapy. Gd‐MOFs were synthesized using terephthalic acid and gadolinium (III) chloride hexahydrate and then loaded with methotrexate (MTX). Characterization via fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), magnetic resonance imaging (MRI), and X‐ray diffraction (XRD) confirmed their correct structure and stability. Effective MTX loading and controlled release were demonstrated. Anticancer effects were assessed on human healthy bronchial epithelial cells (BEAS‐2B) and human lung cancer cells (A549) using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay under in vitro radiation therapy. MTX/Gd‐MOF combined with radiotherapy showed a greater reduction in cancer cell viability (41.89% ± 2.75 for A549) compared to healthy cells (56.80% ± 1.97 for BEAS‐2B), indicating selective cytotoxicity. These findings highlight the potential of Gd‐MOFs not only as drug delivery vehicles but also as radiosensitizers, enhancing radiotherapy efficacy and offering promising evidence for their use in combinatory cancer therapies to improve treatment outcomes.

This study explores gadolinium metal‐organic frameworks (Gd‐MOFs) as dual‐functional platforms for anticancer drug delivery and radiosensitization. Synthesized using terephthalic acid and gadolinium (III) chloride hexahydrate, Gd‐MOFs were loaded with methotrexate (MTX) and characterized via fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), magnetic resonance imaging (MRI), and X‐ray diffraction (XRD). In vitro studies on A549 and BEAS‐2B cells under radiotherapy demonstrated enhanced cytotoxicity in cancer cells, highlighting their potential for selective therapy. The findings support Gd‐MOFs as promising candidates for combinatory cancer treatments, improving therapeutic efficacy. The graphical summary illustrates their role in drug delivery, MRI imaging, and radiosensitization.

1. Introduction

According to the 2020 data from the Global Cancer Observatory, there were approximately 19.3 million new cancer cases and nearly 10 million deaths reported worldwide. Notably, the incidence of lung cancer among males in Turkey was found to be nearly twice the global average, underscoring its significant prevalence.^{[ 1 ]} Lung cancer is primarily attributed to prolonged tobacco use; however, genetic predispositions and environmental exposures also play a significant role in influencing how individuals metabolize carcinogens and repair DNA damage.^{[ 2} , ^{3 ]} The complex interaction of these factors makes the prevention and treatment of lung cancer particularly challenging. Numerous studies in the literature have explored nanoparticle‐based drug delivery systems for lung cancer treatment.^{[ 4 ]}

While traditional chemotherapy is effective in killing cancer cells, it often suffers from significant drawbacks, including non‐specific distribution throughout the body, systemic toxicity, and severe side effects. These limitations not only reduce the quality of life for patients but also restrict the therapeutic efficacy of anticancer agents.^{[ 5 ]} As a result, there has been significant interest in the pharmaceutical and biomedical research communities in the development of advanced drug delivery systems.^{[ 6 ]} Effective drug delivery systems can enhance the therapeutic efficacy of pharmaceutical compounds, provide targeted delivery to specific tissues or cells, minimize adverse side effects, and potentially be compatible with imaging systems for theranostic applications.^{[ 7 ]} Traditional drug delivery methods often face challenges such as poor solubility, low bioavailability, rapid clearance from the body, and lack of specificity, making them less suitable for theranostic treatments. These limitations can lead to suboptimal therapeutic outcomes. Consequently, there is a growing demand for innovative drug delivery platforms that can overcome these challenges, offering controlled, sustained, and targeted release of theranostic agents.^{[ 8 ]}

In this regard, metal‐organic frameworks (MOFs), a family of crystalline materials distinguished by the systematic arrangement of metal ions and organic ligands, may present a novel approach to the therapy of cancer. MOFs are made up of porous hybrid (organic–inorganic) components that can carry out a variety of chemical reactions. They have a high surface area, tunable porous architectures, and are created by organic ligands coupled to metal‐oxo clusters.^{[ 9 ]} These characteristics make MOFs perfect for a wide range of biological applications, including the explanation of gas absorption mechanisms, the discovery of catalytic features, and most notably, the use as drug delivery systems. When compared to organic carriers (liposomes, biopolymers, micelles, and dendrimers) and inorganic carriers (zeolites, silica, and carbon allotropes), MOFs stand out as potential drug carriers due to their configurable pore topologies, high drug‐loading capacities, and adjustable multifunctionality.^{[ 10} , ^{11 ]}

Raju et al. (2022)^{[ 12 ]} investigated the development of pH‐sensitive FU@Eu‐MOF nanoscale MOFs for lung cancer therapy, demonstrating that these structures were synthesized to enhance biocompatibility and ensure targeted drug delivery. Park et al. (2016)^{[ 13 ]} modulated the particle size of the porphyrinic metal‐organic framework (MOF) PCN‐224 to enhance uptake by HeLa human cervical cancer cells, resulting in increased internalization and cytotoxicity through passive targeting and photodynamic therapy. Moreover, MOFs have shown potential for enhancing photodynamic performance. The use of MOF combined hyaluronate modified CaO₂ nanoparticles (PCN‐224‐CaO₂‐HA) in photodynamic therapy was more effective than alone application.^{[ 14 ]} Safinejad et al. (2022)^{[ 9 ]} focused their research on drug delivery systems developed using a lanthanum‐based metal‐organic framework (La‐MOF). In the study, La‐MOF synthesized with 3,4‐dihydroxycinnamic acid was effectively tested on the human breast cancer cell line MDA‐MB‐468.

Radiotherapy has been a widely utilized and effective method for cancer treatment for many years. This therapeutic approach is particularly preferred in cases involving localized tumors, where surgical intervention is either not feasible or inappropriate. Radiotherapy works by damaging the DNA of cancer cells, thereby inhibiting their ability to divide and proliferate.^{[ 15 ]} Radiotherapy employs high‐energy radiation to specifically target cancer cells. The types of radiation used include X‐rays, gamma rays, and protons. During treatment, radiation is precisely directed at the cancerous area with the aim of minimizing damage to surrounding healthy tissues.^{[ 16 ]} Radiotherapy can be administered through two main approaches: external (from outside the body) or internal (from within the body).^{[ 17 ]} Radiotherapy can be effective in the local control of lung cancer and in alleviating symptoms. It is commonly used in the treatment of both small cell lung cancer (SCLC) and non‐small cell lung cancer (NSCLC).^{[ 18 ]} MOFs can enhance the efficacy of radiotherapy due to their heavy metal content, high loading capacity, and controlled release properties.^{[ 19 ]} Additionally, MOFs prepared with metal ions and ligands that are endogenous to the human body or previously known to be non‐toxic, and with particle sizes not exceeding 200 nm, exhibit enhanced biocompatibility and biodegradability. These characteristics are critical for minimizing potential side effects and ensuring safer biomedical applications.^{[ 20 ]}

In this study, Gd‐MOFs were first synthesized, followed by characterization studies to evaluate their structure. Their interactions with methotrexate were examined using in silico methods (Supporting Information). Subsequently, drug loading studies were conducted, and the results were characterized by fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X‐ray diffraction (XRD), MR, and scanning electron microscopy (SEM) devices, followed by in vitro drug release studies. The biological activities of the Gd‐MOFs were then tested on the cancerous lung cell line A549 and the healthy lung cell line BEAS‐2B. Additionally, their radiotherapy‐enhancing effects were investigated using the linear accelerator (LINAK) device. This research is significant in demonstrating the potential of MOFs as drug delivery systems, their compatibility with imaging systems, and their effectiveness in radiotherapy and combination therapy applications, providing valuable insights for future studies.

2. Materials and Methods

2.1. Materials

Acetic acid, gadolinium (III) chloride hexahydrate (GdCl₃·6H₂O), terephthalic acid, sodium dodecyl sulfate (SDS), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), and phosphate‐buffered saline (PBS) were purchased from Sigma Aldrich. N,N‐Dimethylformamide was purchased from Isolab. Distilled water was obtained using the Millipore system.

2.2. Synthesis of Gadolinium Metal‐Organic Frameworks (Gd‐MOFs)

Terephtalic acid and gadolinium (III) chloride hexahydrate (GdCl₃·6H₂O) were used in the production of Gd‐MOFs (CCDC number: 949 513). Quantities of 2 mmol each of terephthalic acid and GdCl₃·6H₂O were employed. First, the designated reagents were dissolved in 50 mL of DMF. To ensure homogeneity, this liquid was agitated using a magnetic stirrer at a low speed. Then the mixture was poured into a hydrothermal jar made of stainless steel and sealed tightly.^{[ 21 ]} The hydrothermal vessel was placed in an oven and heated to 120°C for 48 h in order to perform the synthesis. For the MOF structure to develop and crystallize, this step is essential. The hydrothermal vessel was opened and allowed to cool after the synthesis was finished. A Gooch funnel was then used to filter the solid material. To eliminate any remaining DMF after filtering, ethanol was progressively poured over the solid material. The solid substance was subsequently allowed to dry for approximately 2 h at room temperature with natural airflow. The dried material was then dried again under vacuum for 12 h at 80°C and then for 2 h at 120°C in the last step. The purpose of this process was to guarantee that the MOF structure was completely dried and stabilized. As a result, the resultant powder was fit for characterization and additional investigation.

2.3. Characterization Studies of MTX/Gd‐MOFs

2.3.1. Drug Loading and Release Studies in MOFs

The study investigated the encapsulation efficiency of incorporating methotrexate, a chemical with anticancer properties, into MOFs. Methotrexate was dissolved in purified water with the aid of ultrasonication. The concentration of methotrexate was adjusted to 0.01 mg/mL (w/v) during the preparation of the solution. Ultrasonication was performed for 30 min at 25°C and 40 kHz. The drug loading experiment was conducted using three different dosages: 0.05, 0.1, and 0.2 mg. The loading procedure was carried out at 37°C with constant stirring for 48 h. The quantities of drug loaded were determined using a UV–vis spectrophotometer at a wavelength of 276 nm (Equation 1).

$$\mathrm{Encapsulation}\mathrm{efficiency}\left(EE\%\right)=\frac{W_t}{W_i}\times 100\%$$ (1)

Equation (1) was used to calculate the methotrexate encapsulation efficiency (%), where W_i is the total amount of drug in the MOFs and W_i is the total quantity of drug added initially during preparation.

In the release study, the drug release amount was measured at intervals of 15, 30, 60, 90, 120, 150, 180, 240, and 300 min using UV–vis spectroscopy. At each predetermined time point, 1 mL of the release medium was withdrawn and replaced with fresh buffer solution to maintain environmental conditions. The amount of drug in the collected samples from the release medium was determined spectrophotometrically at 276 nm. Experimental drug release data were analyzed to obtain the regression coefficient (r ²) corresponding to the kinetic model.^{[ 22 ]} The mechanism of drug release was investigated by nonlinear regression. Zero order (Equation 2), first order (Equation 3), Higuchi (Equation 4), and Korsmeyer–Peppas (Equation 5) kinetic models were fitted to the data, and the related kinetic parameters were calculated. Kinetic model fitting and related calculations were conducted by using OriginLab OriginPro 8.5 data analysis and graphing software. The most preferred kinetic models are given below.

$$\mathrm{Zero}\mathrm{order}:Q=Q_0+K_0\times t$$ (2)

$$\mathrm{First}\mathrm{order}:Q/Q_0=1-e^{\left(-K_1\times t\right)}$$ (3)

$$\mathrm{Higuchi}:Q=K_{\mathrm{h}}\times t^{0.5}$$ (4)

$$\mathrm{Korsmeyer}-\mathrm{Peppas}:Q/Q_0=K_{\mathrm{kp}}\times t^n$$ (5)

These models assume that drug release occurs at a constant rate, independent of the drug concentration. Q ₀ is the initial amount of drug, K ₀ is the zero‐order release constant, and t is time, K₁ is the first‐order release constant, K _h is the Higuchi release constant, e is the Euler's number, K _kp is the Korsmeyer–Peppas constant, and n is the release exponent that indicates the mechanism of drug release.

2.3.2. MR Imaging

Magnetic resonance (MR) imaging was performed in the T1‐weighted state using a Philips MR 7700 3T scanner to evaluate the visibility of Gd‐MOF and MTX/Gd‐MOF under MR conditions.

2.3.3. SEM Imaging and EDS Analyses

Scanning electron microscope (SEM) analyses were carried out using a Thermo Fisher Quattro S to assess the size of the Gd‐MOF and MTX/Gd‐MOF. The Thermo Fisher Quattro S EDS detector was employed in EDS analyses to elucidate the molecular composition.

2.3.4. X‐Ray Diffraction (XRD) Analysis

This analysis is crucial for determining the structural properties and crystal arrangements of the samples. X‐ray diffraction (XRD) analyses were conducted using a Rigaku SmartLab X‐ray diffractometer. The crystalline structures and phases of Gd‐MOF were examined. The XRD results were simulated in VESTA 3.5.8 for comparative purposes under the following conditions: Number of wavelengths (no. of lambda): 2, Wavelength range (λ, Å): 1.54059–1.54432, relative intensity: 1–0.5.

2.3.5. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR analysis was employed to identify the chemical compositions and functional groups of Gd‐MOFs and MTX/Gd‐MOF. Analyses were conducted using a Bruker Alpha FTIR Spectrometer. The spectra were recorded in the range of 400–4000 cm⁻¹ (2 cm⁻¹ resolution).

2.3.6. Thermal Gravimetric Analysis (TGA)

Thermogravimetric analyses (TGA) were conducted using a Hitachi STA 7200 Thermogravimetric analyzer. This analysis was performed to determine the thermal stability and weight loss characteristics of Gd‐MOF. The samples were heated in a nitrogen atmosphere over a temperature range from 25 to 1000°C.

2.3.7. Differential Scanning Calorimetry (DSC) Analysis

DSC analysis is crucial for evaluating the thermal behavior of Gd‐MOF. Differential scanning calorimetry (DSC) analyses were conducted using a Perkin Elmer DSC 6002 instrument. The phase transitions and other thermal properties of the samples were examined within the temperature range of −30 to 300°C, with the temperature increasing at a rate of 10°C per minute.

2.4. Cell Culture

For in vitro cell culture experiments, the human lung adenocarcinoma epithelial cell line (A549 cell line, RRID: CVCL_0023) and the human bronchial epithelial cell line immortalized by the SV40 virus (BEAS‐2B cell line, RRID: CVCL_0168) were obtained from the American Type Culture Collection (ATCC). BEAS‐2B plated and maintained in Minimum Essential Medium Eagle (MEM) supplemented with 10% fetal calf serum and 1.0% penicillin/streptomycin (100 Units/mL). A549 were grown in Dulbecco's Modified Eagle Medium (DMEM) enriched with L‐glutamine, 10% fetal calf serum, and a penicillin/streptomycin mixture (10,000 Units/mL). Both cell types were cultured in 75 cm² tissue culture flasks under a humidified environment with 5.0% CO₂ at 37°C. The cultures were passaged twice a week.

2.4.1. Cell Viability

Cell viability was evaluated using the MTT assay,^{[ 23 ]} which involves a water‐soluble tetrazolium salt. The MTT molecule, upon entering viable cells, undergoes enzymatic reduction by the mitochondrial enzyme succinate dehydrogenase, leading to the formation of insoluble purple formazan crystals. The intensity of the color produced is directly proportional to the number of viable cells. The cytotoxicity of Gd‐MOFs, MTX, and MTX/Gd‐MOF were assessed at three different concentrations over a 72‐h period. Cells were seeded at a density of 4 × 10³ cells per well in 96‐well plates and allowed to grow to approximately 80% confluence over three days. Following this, the cells were treated with various concentrations of samples. After treatment, cells were exposed to a 10% MTT solution and incubated for 4 h. Subsequently, the cells were lysed with a 0.1 mg/mL sodium dodecyl sulfate (SDS) solution (0.01 M HCl). Absorbance was measured at 570 nm using a Multiscan Go spectrophotometer (Thermo Fisher, Waltham, MA, USA).

2.4.2. Radiotherapy

In this study, MOF, MTX, and MOF‐MTX were used as radiosensitizing agents to enhance the effectiveness of radiotherapy, applying the method used by Tornaci et al.^{[ 24 ]} on A549 cancerous and BEAS‐2B healthy lung cells. A total of 4 × 10³ cells were seeded into 96‐well plates the day prior to the experiment. The following day, the cells were treated with a medium containing a non‐toxic dose of MOF, MTX, and MOF‐MTX and incubated for an additional 2 h. For the radiotherapy experiment, both cell lines were exposed to X‐rays using a linear accelerator (LINAK) with aid at a dose of 4 Gray at 6 MeV (Electa Versa HD). Wells containing only medium served as the negative control group, while wells with only cells were irradiated with 4 Gray as the positive control group. After radiation exposure, the cells were incubated under standard cell culture conditions for 72 h. At the end of this period, cell viability was assessed using the MTT assay and compared to the control groups

2.5. Statistic Analyses

The data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using one‐way ANOVA, followed by Tukey's post‐hoc test for multiple comparisons, utilizing GraphPad Prism 9 software. A p‐value of <0.05 was considered statistically significant. The sample size is denoted by n.

3. Result and Discussion

3.1. X‐Ray Diffraction (XRD) Analysis

As illustrated in Figure 1, the 2θ values exhibit a pronounced peak at 9.52°, indicative of the high crystallinity of the Gd‐MOFs. The sharp and intense peak at 9.52° suggests that the MOF structure was synthesized with a well‐ordered crystal lattice, reflecting precise and successful fabrication. Similar findings have been previously reported, with PXRD patterns of various MOF crystals exhibiting diffraction peaks at low angles (ca. 2θ = 10°),^{[ 25} , ²⁶ , ²⁷ , ²⁸ , ^{29 ]} which are consistent with the simulated XRD pattern shown in Figure 1. The absence of certain peaks in the simulated pattern (Figure 1B) appears to be due to the exceptionally high normalized intensity of the synthesized material, with slight shifts also observed. However, the major peak is clearly visible in both figures (Figure 1A,B). The high‐intensity and narrow diffraction peaks are indicative of superior crystal quality and minimal microstructural strain. Additionally, the presence of lower intensity peaks may correspond to the phase purity of the material and the potential formation of secondary phases during the crystal growth process.^{[ 30 ]} These observations confirm that the Gd‐MOFs were successfully synthesized, exhibiting a highly ordered crystalline structure. Overall, the XRD data provide a comprehensive evaluation of the crystalline structure and phase purity of the Gd‐MOFs.

Figure 1.

A) X‐ray diffraction (XRD) analysis of the MOF crystal. B) Simulated XRD pattern of the MOF crystal using the VESTA software.

3.2. Drug Loading and Release Studies in MOFs

The encapsulation efficiency of methotrexate‐loaded MOFs (MTX/Gd‐MOFs) was calculated using the calibration curve formula (y = 29.9x – 0.004) (R ² = 0.999) derived from our measurements. A high encapsulation efficiency of 82% was obtained, indicating strong interactions between Gd‐MOF and MTX (Figures S1 and S2). Studies showed that the metal content of MOFs interacting with drug molecules yields noticeably high encapsulation efficiencies.^{[ 31 ]} Additionally, the interaction between methotrexate and Gd‐MOFs was demonstrated in silico using molecular dynamics (MD) simulation methods (Figures S1 and S2). A recent study has reported successful loading of MTX on an iron‐based MOF with 84% encapsulation efficiency.^{[ 32 ]} Hydroxyl groups of terephthalic acid in the Gd‐MOF structure and carboxyl groups of MTX form ester bonds, which contribute to the high encapsulation efficiencies obtained in MTX/Gd‐MOF samples.^{[ 33 ]} Varying initial drug content, however, did not significantly affect the encapsulation.

In this study, the drug release kinetics of methotrexate (MTX)‐loaded MOFs, synthesized from Gadolinium (III) chloride hexahydrate and terephthalic acid, were investigated in PBS (pH 7.4) and acetate buffer (pH 5) environments (Figure 2). The solubility and diffusion properties of methotrexate are directly influenced by pH. At neutral pH (PBS, pH 7.4), the solubility of methotrexate may be lower, resulting in a slower release rate. In contrast, at acidic pH (acetate buffer, pH 5.5), the solubility of methotrexate increases, leading to a more rapid release rate.^{[ 34 ]} However, in their study, Howarth et al. (2016)^{[ 35 ]} demonstrated that various types of MOFs maintain structural stability across nearly all pH levels, whether in acidic or basic environments.

Figure 2.

Cumulative drug release in different release media pH 7.4 in PBS buffer (red line) and pH 5.5 in acetate buffer (black line). (0.1 mg drug loaded Gd‐MOF).

Moreover, the kinetic parameters for several models were calculated and presented in Table 1. Comparing the R‐square values of the drug release in pH 5.5 medium, the highest value is obtained for the Korsmeyer‐Peppas model (R ² = 0.984), showing the best‐fitted model to the experimental data is Korsmeyer–Peppas. Drug release in pH 7.4 medium showed lower fit to the Korsmeyer–Peppas model (R ² = 0.885). However, in studies, similar values were considered acceptable for a meaningful and good fit for other kinetic models, namely first‐order, Higuchi, and Weibull.^{[ 36} , ^{37 ]} The pH of the release medium influenced especially the K value of the Korsmeyer–Peppas model. The K‐value calculated in this model is also named as drug release rate constant and is related to the release rate of the drug molecules in the studied release medium.^{[ 38 ]} Comparing the K‐values of MTX release in pH 5.5 and pH 7.4 media, we observed that MTX release is more rapid in pH 5.5 medium. This result is also predominant in the cumulative release profiles presented earlier. Although the n‐values, which explain the dominant release mechanism, showed a minor change, the dominant release mechanism in both media was Fickian diffusion since the calculated n‐values were lower than 0.45. This finding suggests that during drug release, no additional MOF fragments are transferred to living tissue. Moreover, preserving this stability is crucial for subsequent radiotherapies, as it ensures the MOFs remain intact throughout the treatment process.

Table 1.

Calculated kinetic parameters with different methods.

	Korsmeyer–Peppas			Higuchi		Zero order		First order
	K KP (min− n )	n	R 2	K H (min−0.5)	R 2	K 0 (min−1)	R 2	K 1 (min−1)	R 2
pH 5.5	0.646	0.31	0.984	1.061	0.933	0.015	0.739	0.0003	0.736
pH 7.4	0.117	0.27	0.885	0.352	0.810	0.004	0.536	0.0002	0.449

SEM and EDS analyses were conducted to determine the size and molecular composition of Gd‐MOF and MTX/Gd‐MOF samples (Figure 3). The MOF structures exhibited rod‐like shapes with sizes ranging from 150 to 800 nm. In contrast, Hu et al. (2021)^{[ 30 ]} reported that Gd‐MOFs had needle‐like shapes with average sizes around 100 nm. The differences in size and shape observed in this study may be attributed to the use of terephthalic acid as the organic phase. Additionally, the presence of nitrogen atoms indicated under Figure 3B in the EDS analyses suggests the incorporation of methotrexate. To observe the behavior of the complex structures formed with Gd‐MOF and MTX/Gd‐MOF under magnetic resonance imaging (MRI), a T1‐weighted state using a Philips MR 7700 3T scanner was employed. As shown in Figure 4, both the drug‐loaded and plain Gd‐MOFs retained their properties as contrast agents in the 6‐well plate.

Figure 3.

SEM image and EDS results of A) Gd‐MOF and B) MTX/Gd‐MOF.

Figure 4.

MR Imaging of A) Gd‐MOF; B) MTX/Gd‐MOF; and C,D) pure water.

3.3. Fourier Transform Infrared Spectroscopy (FTIR)

FT‐IR spectra of Gd‐MOF, MTX, and MTX/Gd‐MOF were presented in Figure 5. The spectrum belonging to the Gd‐MOF structure revealed a number of major peaks, which showed the successful synthesis of the MOF structure. The peak observed at 3455 cm⁻¹ was attributed to the O─H stretching vibration of water molecules and hydroxyl groups. The stretching of C─H bonds of terephthalic acid (TPA) was detected at 3059 and 2928 cm⁻¹.^{[ 39 ]} The peak at 1654 cm⁻¹ indicated the coordinate covalent bonding between Gd metal and oxygen of TPA, which caused the elongation of C═O bonds.^{[ 32 ]} Additionally, the nonexistence of a peak at around 1675–1680 cm⁻¹ showed that free TPA was absent in the structure.^{[ 40 ]} The spectrum also showed peaks at 1546, 1404, and 747 cm⁻¹, which were attributed to the asymmetric and symmetric carboxylate stretching and aromatic C─H stretching, respectively.^{[ 39 ]} These findings, in total, confirm the Gd‐MOF structure. The water molecules adsorbed on the MOF structure show a peak at ≈1660 cm⁻¹ belonging to the bending mode.^{[ 41 ]} The characteristic vibrational modes of MOF structure can create an overlap with the vibrations of water molecules, especially if the frequencies are in a similar region. Therefore, we suggest that the disappearance of the frequency belonging to the bending mode of water was caused by the overlapping with the frequency of metal and organic ligand interactions.

Figure 5.

FT‐IR spectra of pure MTX, Gd‐MTX, and MTX/Gd‐MOF.

MTX incorporation within the Gd‐MOF structure caused several shifts in some peaks, which may show the involvement of the related functional groups in the drug‐MOF interactions. First, the peak observed at 1654 cm⁻¹ in the spectrum of Gd‐MOF disappeared in the spectrum of MTX/Gd‐MOF. Additionally, the fingerprint peaks of MTX at 1688–1608 cm⁻¹ belonging to the C═O bonds^{[ 42 ]} were not observed in the MTX/Gd‐MOF structure. The O─H stretching in MTX at 3348 cm⁻¹ was absent in the spectrum of MTX/Gd‐MOF. N─H stretching was observed at 1563–1518 cm⁻¹ in the pure MTX spectrum. In the spectrum of MTX/Gd‐MOF, these peaks shifted to 1506 and 1546 cm⁻¹. Moreover, the N─H stretching of primary amine groups of MTX at 816 cm⁻¹ has shifted to 827 cm⁻¹.^{[ 43 ]} These alterations in the IR spectrum of MTX/Gd‐MOF may be an indication of MTX and Gd‐MOF interactions. Recent studies showed that hydrogen bonds forming between COOH and OH groups^{[ 44 ]} and ionic bonding between primary amines (NH₂) and O─H groups^{[ 42 ]} can cause minor shifts in the frequencies of the related groups. Thus, considering the high drug encapsulation efficiency, we suggest that the mentioned interactions may be present in the MTX/Gd‐MOF complex. Some characteristic peaks of MTX were visible in the MTX/Gd‐MOF spectrum, which verified the existence of MTX in the MOF structure. These peaks are listed as 2949 cm⁻¹, belonging to C─H stretching of CH₃ groups,^{[ 45 ]} 1597 cm⁻¹ belonging to aromatic C─C,^{[ 46 ]} 1099 cm⁻¹ belonging to C─N stretching of tertiary amine groups.^{[ 42} , ^{47 ]}

3.4. Thermal Gravimetric Analysis (TGA)

Thermogravimetric analysis was used to investigate the thermal stability and the thermal degradation patterns of Gd‐MOF and MTX/Gd‐MOF. The thermograms given in Figure 6 showed a similar two‐step decomposition pattern for both Gd‐MOF and MTX/Gd‐MOF. The reason for the absence of the MTX decomposition step was thought to be the dilution effect, meaning the trace amount of MTX in the structure does not create a visible weight loss on the thermogram.^{[ 40 ]} The thermal decomposition peak observed at 149 and 187°C was due to the adsorbed water and residual solvent.^{[ 29} , ^{48 ]} A conjugated decomposition peak at 161°C was detected in MTX/Gd‐MOF, which can be similarly attributed to water decomposition. These decomposition steps corresponded to 13.2% and 11.6% weight loss, respectively. The second weight loss pattern observed at approximately 600°C was the result of the thermal decomposition of the Gd‐MOF crystal structure.^{[ 48 ]} However, it is worth noting that thermal stability was increased with the incorporation of MTX, increasing thermal decomposition temperature from 608 to 614°C. The corresponding weight loss percentages of Gd‐MOF decomposition were calculated as 30% and 34% for Gd‐MOF and MTX/Gd‐MOF, respectively.

Figure 6.

TGA and DTG curves of A) Gd‐MOF and B) MTX/Gd‐MOF.

3.5. Differential Scanning Calorimetry (DSC) Analysis

As shown in Figure 7, there were two consecutive endothermic peaks at 167 and 195°C in the DSC curve of Gd‐MOF. Such consecutive endothermic peaks are typical in the case of polymorphic transition in which the melting process is followed by recrystallization.^{[ 49 ]} The melting peak of MTX/Gd‐MOF did not show any sign of MTX melting, which was expected to occur at around 120–135°C.^{[ 50} , ^{51 ]} It can be inferred from the absence of the melting peak of MTX in the MTX/Gd‐MOF curve that MTX was not in its crystalline state within the Gd‐MOF structure.^{[ 52 ]} A single endothermic peak at 176°C was detected in MTX/Gd‐MOF thermogram. The observed conjugation of two melting peaks into a single one could be attributed to the drug‐MOF interactions interfering with the melting behavior of Gd‐MOF.

Figure 7.

DSC thermograms of Gd‐MOF and MTX/Gd‐MOF.

3.6. Cell Culture

3.6.1. Viability Assay

In recent years, there has been intense interest in drug delivery systems, particularly MOFs, and their effects on biological systems. One of the most commonly used methods in conducting these studies is the in vitro MTT assay. In this study, different formulations and concentrations of experimental groups—methotrexate (MTX), gadolinium‐based MOF (Gd‐MOF), and their combination (MTX/Gd‐MOF)—were tested on two different cell lines over a period of 72 h to investigate their effects on cell viability (Figure 8). According to the results, after 72 h, the MTX group (drug alone) yielded similar outcomes in both cell lines. However, the MTX/Gd‐MOF group exhibited a greater decrease in viability at doses of 25 and 50 ng/mL in the A549 cell line compared to the BEAS‐2B cell line. Specifically, for 25 ng/mL, cell viability in A549 cells was found to be 51.073 ± 2.492 (p < 0.001), and for 50 ng/mL, it was 51.05 ± 3.77 (p < 0.001). In contrast, the viability data for the BEAS‐2B cell line at the same concentrations were 60.189 ± 1.228 and 58.274 ± 2.348, respectively. This is thought to result from the higher metabolic rates of cancerous cell lines and their increased uptake of carrier molecules. In the Gd‐MOF group alone, after 72 h, viability losses at a dose of 20 µg/mL were measured as 70.618 ± 3.72 in the A549 cell line and 72.001 ± 2.085 in the BEAS‐2B cell line, indicating that no significant toxic effect was observed when used alone. Gadolinium nanoparticles are generally used in MRI imaging (T1‐weighted) in a chelated form and have been found to be rapidly excreted directly via the kidneys.^{[ 53 ]} Moreover, although no comprehensive research has been conducted on Gd‐MOFs, studies similar to this one have reported that cytotoxic effects were not observed.^{[ 54 ]} A dose of 2.5 ng/mL was found to be the concentration that did not reduce viability in any of the groups. Therefore, to more clearly observe the combined effects of radiotherapy, a working dose of 2.5 ng/mL was selected for further experiments.

Figure 8.

The cytotoxic effect of MTX, Gd‐MOF, and MTX/Gd‐MOF on A549 and BEAS‐2B cell lines at 72 h. The mean is accompanied by the standard deviation (± SD); (n = 4).

3.6.2. Radiotherapy

In recent years, non‐invasive radiotherapy studies have been at the forefront of modern treatment approaches frequently used in cancer therapy. The ability to control cell proliferation and reach distant tissues without surgical intervention has led healthcare professionals to prefer this treatment method in nearly every other patient.^{[ 55 ]} Radiotherapy exerts its effects on cells by directly inducing DNA strand breaks, generating reactive oxygen species (ROS) in the treated environment leading to apoptosis, or by disrupting the cell cycle and inhibiting proliferation.^{[ 56 ]} Consequently, research into radiosensitizer agents to minimize the side effects of this treatment has become a popular area. In this study, methotrexate (MTX) was encapsulated into Gd‐MOF nanoparticles to utilize radiotherapy in combination with chemotherapy. Changes in cell viability under 4 Gray radiation were examined using the MTT assay. As shown in Figure 9, the application of the MTX/Gd‐MOF group in combination with radiotherapy resulted in a significantly greater reduction in cell viability in both cell lines compared to other groups. The viability of the cancerous A549 cell line was observed to be 41.899 ± 2.751 (p < 0.01), while that of the healthy BEAS‐2B cell line was 56.804 ± 1.971 (p < 0.05). This may be attributed to the better repair mechanisms of healthy cell lines compared to cancerous ones. Lower viability measurements in groups containing Gd‐MOF are expected, as metals with high atomic numbers (Z) are known to be usable as radiosensitizers.^{[ 57 ]} The initial applications of gadolinium‐based particles began with their use as MRI contrast agents and guides; subsequent studies have demonstrated that these nanoparticles and MOF derivatives can enhance the effectiveness of radiotherapy.^{[ 58} , ^{59 ]} This suggests that Gd‐MOF is not only a drug delivery system but also a valuable theranostic agent.^{[ 60} , ^{61 ]} have shown that MTX applications also increase the sensitivity of cells to radiotherapy; the cell losses observed in the groups treated with MTX alone in this study support this finding. Additionally, many studies have demonstrated that radiotherapy has a synergistic effect with chemotherapy.^{[ 62 ]} Therefore, it is thought that another reason for the viability losses in the MTX/Gd‐MOF group is this synergistic effect.

Figure 9.

MTT results for combined effects of MTX, Gd‐MOF, MTX/Gd‐MOF, and radiotherapy. (R−) mean is no radiation and (R+) mean is 4 Gy radiation. The mean is accompanied by the standard deviation (± SD), (n = 4).

4. Conclusion

This study explored the potential of MOFs as dual‐functioning systems, serving both as drug delivery vehicles and radiosensitizers for cancer therapy, with a particular focus on methotrexate (MTX)‐loaded gadolinium‐based MOFs. The synthesized MOF‐MTX structures underwent comprehensive characterization using XRD, DSC, TGA, SEM, MR, and FTIR analyses to confirm their stability, composition, size, and visuality under MR and drug interaction. These findings were further supported by in silico analyses. The FTIR results exhibited characteristic absorption peaks, indicating successful MTX loading into the MOF, while thermal analyses revealed that the incorporation of MTX influenced the thermal stability of the MOF structure. Drug release studies demonstrated the controlled and sustained release of MTX from the MOF, a critical factor in enhancing therapeutic efficacy and minimizing side effects. In vitro cytotoxicity studies conducted on the A549 lung cancer cell line and the BEAS‐2B healthy lung cell line indicated that the MOF‐MTX complex exerted a significantly stronger antiproliferative effect compared to MOF or MTX alone. Additionally, the combination of radiotherapy with MTX/Gd‐MOF further amplified the therapeutic effects, particularly at a radiation dose of 4 gray on A549 cell line (41.89 ± 2.75) and BEAS‐2B cell line (56.80 ± 1.97). These results underscore the potential of MOFs not only as effective drug delivery systems but also as radiosensitizers that enhance the efficacy of radiotherapy treatments. In conclusion, this study presents valuable insights into the application of Gd‐MOFs in drug delivery, MR imaging, and cancer therapy, showcasing their capability to achieve controlled drug release and improve radiotherapy outcomes. Emphasizing the necessity of testing MTX/Gd‐MOF structures in a broader range of cancer cell lines and in vivo models to further elucidate their potential, future studies should focus on evaluating their safety, efficacy, and biodegradability in in vivo systems, as well as assessing their long‐term effects within biological environments to confirm their clinical viability.

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

Supporting Information

Data Availability Statement

The data supporting the findings of this study are available within the article and Supplementary data. Additional data that support the findings of this study are available from the corresponding author upon reasonable request.