Histopathological and biochemical profiling of Carfilzomib-loaded Fe–Co MOFs

Abstract

In recent years, new medications like proteasome inhibitors (PIs) have significantly improved cancer patients’ response rate and overall survival. Carfilzomib (CFZ), a second-generation proteasome inhibitor, has shown promising results in clinical trials for treating multiple myeloma patients. In the current study, a Fe–Co metal-organic framework (MOF) was developed as a drug delivery system for targeted therapy of cancer cells. CFZ-loaded Fe–Co MOFs were synthesized and characterized using DLS, VSM, SEM-EDS, and BET analyses. The in vivo effects of CFZ-loaded Fe–Co MOFs were compared with standard drugs using a male Wistar rat model. Based on the results, DLS revealed a polydisperse size distribution, while VSM showed strong magnetic properties with 20 emu/g saturation magnetization. SEM-EDS confirmed a well-defined crystalline structure with uniform elemental distribution, and BET analysis indicated a mesoporous structure with a surface area of 84.984 m²/g. The MOFs demonstrated a high drug loading efficiency of 74.86% and a controlled release profile, with an initial burst followed by sustained release. When administered intravenously to rats, free CFZ at doses of 0.4 mg/kg and 0.8 mg/kg led to significant increases in serum liver enzymes, kidney function markers, and liver malondialdehyde content. Furthermore, high doses of CFZ-loaded Fe–Co MOFs caused significant histopathological changes in the rats. These findings provide a basis for further research on using Fe–Co MOFs as carriers of proteasome inhibitors like CFZ for targeted drug delivery.

Introduction

Cancer remains a significant global health challenge, affecting millions of individuals across all age groups and organs. Under normal conditions, human cells follow a regulated division, repair, and death cycle, which maintains healthy tissue function [1]. When the process of cell regulation becomes disrupted, abnormal cells can multiply uncontrollably, leading to the development of tumors. These tumors can be either benign or malignant, with the latter having the ability to spread to other parts of the body, posing serious health risks. With the increasing incidence of cancer, there is a pressing need for more effective treatment options. Chemotherapy is a commonly used method for treating cancer, involving the use of potent drugs to kill rapidly dividing cells. However, traditional chemotherapy has its limitations, including issues like uneven drug distribution, lack of precise targeting of cancer cells, and severe side effects. To address these challenges, improved drug delivery systems are needed to enhance the effectiveness of chemotherapy while minimizing harm to healthy tissues. Recent advancements in nanotechnology have provided a promising solution by enabling the targeted delivery of chemotherapy drugs directly to cancerous tissues. Nanotechnology has transformed cancer treatment by introducing innovative approaches to drug delivery. Nanocarriers have emerged as a cutting-edge method for delivering chemotherapy agents. These nanoparticles can specifically target cancer cells, sparing healthy tissues and reducing the side effects associated with traditional chemotherapy. For instance, nanoparticles can release drugs in response to specific triggers, such as X-ray radiation, allowing localized treatment at the tumor site [2]. Another paper developed carboxymethyl cellulose/hydroxyapatite/Fe₃O₄ hydrogel beads with 28.73 emu/g saturation magnetization; they achieved pH-responsive DOX release—minimal at pH 1.2 and enhanced at physiological pH—and demonstrated potent cytotoxicity against SW480 colon cancer cells [3]. Also, researchers synthesized an NH₂-MIL-101(Fe)/graphene oxide composite for co-delivery of luteolin and matrine, which improved drug stability and exhibited significant in vitro anti-colorectal cancer activity [4].

MOFs have shown great potential in improving drug delivery by providing controlled release, targeted delivery, and enhanced drug stability. The tunable pore sizes of MOFs allow for the encapsulation of various drugs, protecting them from degradation and improving their bioavailability. Additionally, the high surface area of MOFs enables efficient drug loading and release, leading to improved drug delivery to cancer cells while minimizing off-target effects [5]. These unique properties make them promising candidates for advanced drug delivery systems in cancer treatment [6, 7]. For example, the NH₂-MIL‑101(Fe)@GO nanocomposite co‑delivered luteolin and matrine with loading efficiencies of approximately 9.8% and 14.1%, respectively, exhibited pH-responsive release (significantly higher at pH 5 vs. 7.4), and enhanced anti‑colorectal cancer activity—including inhibited tumor cell migration, increased ROS generation, and upregulated caspases‑3/9—compared to MOF or GO alone [8]. Separately, the NH₂-MIL‑101(Fe)/DOX@DAS core‑shell system loaded doxorubicin at 92 ± 0.5% efficiency and achieved highly selective pH-triggered release (~ 70% at pH 5 vs. < 8% at pH 7.4 over 10 days), resulting in significant cytotoxicity against HeLa cells (IC₅₀ ≈ 100 µg/mL) [9].

Carfilzomib (CFZ), as a second-generation covalent inhibitor of the proteasome, has high potency and selectivity. It irreversibly targets the chymotrypsin-like β5 subunit, leading to robust induction of apoptosis, with superior efficacy compared to the first-generation inhibitor bortezomib and reduced off‑target toxicity (such as neurotoxicity) [10]. Furthermore, CFZ has demonstrated activity in bortezomib-resistant and refractory multiple myeloma cell lines, underscoring its ability to overcome drug resistance mechanisms. Its favorable pharmacokinetics—rapid extrahepatic metabolism, short half-life (~ 30 min), minimal CYP450 involvement, and wide tissue distribution—facilitate controlled and safe therapeutic use, including both once- and twice-weekly dosing. Finally, while highly effective in hematological malignancies, CFZ’s limited efficacy in solid tumors—due to poor stability and bioavailability—makes it an ideal candidate for nanoformulation strategies aimed at enhancing tumor targeting and drug delivery [11]. To enhance CFZ’s effectiveness and reduce toxicity, various nanocarrier systems, including metal-organic frameworks (MOFs) [12], daratumumab-decorated polypeptide micelles (Dar-PMs) [13], oil-in-water-based microemulsions [14], and ternary polypeptide NPs [15] have been developed. Fe–Co metal–organic frameworks (MOFs) have demonstrated significant advantages over traditional nanocarriers like liposomes and dendrimers in delivering chlorpromazine (CPZ), a neuroleptic agent used in treating schizophrenia. Fe–Co MOFs possess a high surface area and tunable porosity, allowing for substantial drug encapsulation. For instance, iron-based MOFs can achieve a drug loading of up to 25 wt%, significantly higher than liposomes, which have a drug loading capacity of only 0.4 wt%. The porous nature of Fe–Co MOFs enables controlled release of encapsulated drugs. By modifying the framework’s structure, the release rate can be tailored to match the therapeutic needs, providing sustained drug delivery and minimizing side effects. Fe–Co MOFs are composed of biocompatible metals and organic linkers, which reduce the risk of toxicity. Additionally, their inherent biodegradability ensures that they do not accumulate in the body, mitigating long-term adverse effects. The incorporation of iron and cobalt imparts magnetic properties to the MOFs, facilitating external magnetic field-guided delivery. This feature enhances the precision of drug targeting, particularly in complex anatomical regions. Fe–Co MOFs can be functionalized with targeting ligands, such as peptides or antibodies, to improve specificity towards particular cell types or tissues. This functionalization enhances the therapeutic index by concentrating the drug at the desired site and reducing off-target effects. Compared to other porous materials, Fe–Co MOFs demonstrated several outstanding advantages, such as high surface area and porosity for high loading of therapeutic agents and facile modification of physical (e.g., pore size and shape) and chemical properties of MOFs through inorganic clusters and/or organic ligands. These features made them more versatile and efficient as drug delivery systems compared to traditional vectors [5, 16, 17].

This study investigates the potential of iron-cobalt (Fe–Co) MOFs for delivering carfilzomib (CFZ) as a potential cancer treatment. We synthesized and analyzed CFZ-loaded Fe–Co MOF nanoparticles to evaluate their properties, drug-loading efficiency, and release behavior. Additionally, we assessed the safety of these nanoparticles in a rat model compared to conventional CFZ treatment. This research aims to explore the use of MOFs as a targeted and controlled delivery system for proteasome inhibitors, offering a promising approach to cancer therapy. A schematic overview of the Fe–Co MOF–based delivery system for CFZ is shown in Scheme 1. This visual summary clarifies the workflow, from synthesis and characterization to in vivo evaluation.

Scheme 1.

Schematic overview of Fe–Co MOF–based delivery system for Carfilzomib (CFZ)

Materials & methods

Chemicals, reagents, and assay kits

Dimethyl sulfoxide (DMSO), 3-(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT), FeCl₂·4H₂O, FeCl₃·6H₂O, ammonia solution (25%), terephthalic acid, Co(NO₃)₂·6H₂O, DMF, and ethanol (96%) and Trypan blue were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). CFZ with Catalog No. A11278 was purchased from AdooQ, BioScience. The antimycotic-antibiotic solution (0.5 µg/mL amphotericin, 100 u/mL penicillin, and 100 mg/mL streptomycin), Phosphate-buffered saline (PBS), and trypsin-ethylenediaminetetraacetic acid (EDTA) solution were procured from INOCOLON Company (Karaj, Iran).

Synthesis of CFZ-loaded Fe–Co MOF NPs

Fe₃O₄ NPs were synthesized using a co-precipitation method, where FeCl₂·4H₂O and FeCl₃·6H₂O, in a 2:1 molar ratio, were dissolved in 30 ml of distilled water under mechanical stirring at room temperature. Ammonia solution (25%, 1.5 ml) was then added dropwise under a nitrogen atmosphere over 20 min while the mixture was heated to 80 °C. The resulting black solution was stirred at 80 °C under nitrogen for one hour. The precipitate was magnetically separated, washed with deionized water, and dried at 70 °C under vacuum.

To prepare Fe–Co MOFs, the Fe₃O₄ NPs were dispersed in 15 mL of DMF. Terephthalic acid (0.453 g) was added, and the mixture was ultrasonicated for 30 min. Co(NO₃)₂·6H₂O solution was added dropwise to this mixture, followed by another 30 min of ultrasonication. The solution was transferred to a Teflon-lined stainless-steel autoclave and heated at 80 °C for 24 h. The magnetic catalyst was then separated using an external magnet, washed with DMF and ethanol (96%), and dried in a vacuum oven at 60 °C for 12 h.

Finally, 5 mL of Fe–Co MOF was dispersed in PBS (pH = 7.4) and mixed with 1 mL of CFZ solution in DMSO. The mixture was ultrasonicated for 3 min and stirred for 24 h at 25 °C, followed by centrifugation to yield the CFZ-loaded Fe–Co MOFs. The supernatant was collected to evaluate the CFZ loading, and UV–vis spectroscopy (Cary 60, Agilent, USA) was used to measure the optical density of any remaining CFZ. The experiments were conducted in triplicate, and the drug loading percentage was calculated using the total CFZ concentration.

Characterization of CFZ-loaded Fe–Co MOF NPs

The Fe–Co MOF was characterized using advanced instruments to assess its physical, morphological, magnetic, and surface properties. Surface morphology and elemental analysis were analyzed using Field Emission Scanning Electron Microscopy (FE-SEM-EDS) on a ZEISS Sigma 300 from Carl Zeiss AG, Germany. Samples were prepared by depositing the MOF onto conductive carbon tape and sputter-coating with gold for enhanced imaging. The SEM operated at 5–15 kV to capture high-resolution images for particle size and distribution analysis. Dynamic Light Scattering (DLS) measured particle size distribution using a Nano DS instrument (CILAS, France). Samples were dispersed in water, sonicated, and measured at room temperature to assess particle stability in suspension. Magnetic properties were examined using a Vibrating Sample Magnetometer (VSM1100) from WEISTRON, Taiwan. Hysteresis loops were recorded at room temperature under a magnetic field of − 10,000 to + 10,000 Oe, revealing key magnetic characteristics such as coercivity and saturation magnetization. The specific surface area and porosity were determined using the Brunauer–Emmett–Teller (BET) method on a BELSORP Mini II, also from Microtrac B.E.L. Corp, Japan. The samples were degassed at 150 °C, and nitrogen adsorption-desorption isotherms at − 196 °C provided data on surface area, pore size distribution, and total pore volume.

The in vitro drug release assay was conducted by gently stirring 0.01 g of CFZ-loaded Fe–Co MOFs in 50 mL of PBS (pH 7.4) at room temperature. Additionally, CFZ-loaded Fe–Co MOFs were dispersed in 50 mL of acetate buffer (pH 5.2) to simulate the acidic tumor environment. At specific time intervals, 1 mL of the medium was withdrawn to measure CFZ release and replaced with an equal volume of fresh buffer to maintain a constant volume. The amount of released CFZ was quantified at 245 nm using UV–Vis spectroscopy on a spectrophotometer (Cary 60, Agilent, USA). Each experiment was performed in triplicate, and the total drug concentration was used to calculate the percentage of CFZ released from the Fe–Co MOFs.

Animal treatments and experimental design

In this experimental study, we utilized fifty male adult Wistar rats obtained from the animal breeding colony of the Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran. All animals were healthy and free of any specific pathogens at the time of acquisition.

These rats were kept in standard laboratory conditions with a stable temperature range of 23–24 °C and a light schedule of 12 h light and 12 h dark, and they had continuous access to regular food and tap water. For the experimental procedures, the rats were subjected to intravenous injections of free CFZ at doses of 0.4 mg/kg and 0.8 mg/kg, as well as CFZ-loaded Fe–Co MOF at doses of 0.4 mg/kg and 0.8 mg/kg. Each rat received a total of ten intravenous injections at 72-hour intervals. It’s important to note that all injection protocols and handling were carried out strictly to the guidelines of care outlined in the NIH publication. 85−23, ensuring the ethical treatment of laboratory animals. The specific doses for the intravenous injections were determined following preliminary experimental assessments.

Liver histopathology

The fixed liver samples were then dehydrated in a series of ethanol solutions of increasing concentrations to remove water and prepare them for embedding in paraffin wax. After dehydration, the samples were cleared in xylene and infiltrated with molten paraffin wax. Finally, the samples were embedded in paraffin blocks, which were then sectioned into thin slices using a microtome for further histological analysis. The stained sections were observed at various magnifications to assess the overall tissue architecture and the presence of any pathological features by using an Olympus Tokyo light microscope (Tokyo, Japan). An overall score of liver damage severity was semi-quantitatively assessed as follows: pyknotic nuclei, inflammatory cell infiltration, bleeding, and necrosis. Scores were given as 0, none; 1, mild; 2, moderate; 3, severe for each lesion based on the references. Histopathological investigation and scoring were carried out by two specialists blindly. The liver, kidney, heart, and testis sections were examined for semi-quantitative analysis. Histological changes were assessed on a scale of one to four, with one representing normal histology and four indicating severe pathological lesions, following established references [18].

Table 1 shows the classification of haematoxylin and eosin-stained sections injuries - hepatic damage score (HDS), Renal damage score (RDS).

Table 1.

The classification of haematoxylin and Eosin stained sections includes injuries—hepatic damage score (HDS), renal damage score (RDS)

Description	Degree
	1	2	3	4
Liver	Normal	Individual hepatocytes with mild fatty change. Slight sinusoidal disarrangement	Severe fatty change. Severe sinusoidal disarrangement.	Hepatocyte necrosis and iron granules
Kidney	Normal	Narrowing of the proximal and distal tubules. Expansion of Bowman’s capsule	Vacuolation of proximal and distal tubules.	Iron granules and glomerulosclerosis
Heart	Normal	Mild inflammatory cell infiltration,	Eosinophilic stained pyknotic nuclei cells, Congestion of blood vessels	Severe necrosis, Bleeding.
Testis	Normal	Hypo-spermatogenesis: Disorganization of Sertoli cell pattern	Multinucleated giant Sertoli cell	Spermatocyte arrest, Germ cell aplasia

Serum biochemical parameters

Blood samples were collected from the retro-orbital blood plexus of rats and immediately centrifuged at 5,000 rpm for ten minutes. This processing allowed for the evaluation of various biochemical parameters in the rats. The analysis was carried out using a laboratory autoanalyzer (Selectra Pro M) along with commercially available chemical kits from Pars Azmoon, Tehran, Iran. At the end of the study, the animals were euthanized using pentobarbital sodium at a dose of 200 mg/kg. Liver MDA levels were measured according to the method described by Ohkawa et al., with minor changes [19].

Statistical analysis

The results were analyzed for significant relationships or patterns using SPSS software (v.23). Differences between data sets were tested using analysis of variance (ANOVA) followed by post hoc comparison. The results are expressed as mean ± SD. All assays were repeated three times. Data set differences were tested using variance analysis (ANOVA) followed by post hoc comparison.

Results and discussions

Characterization of CFZ-loaded Fe–Co MOFs

Size distribution analysis by DLS

Figure 1 shows a Dynamic Light Scattering (DLS) analysis of a CFZ-loaded Fe–Co MOF DLS was used to measure the size distribution of particles in a suspension, providing information on the hydrodynamic diameter of the particles. The x-axis represented the hydrodynamic diameter of the particles in nanometers (nm), which referred to the diameter of a sphere that diffused at the same rate as the particle being measured. This effective size included the core particle and any associated solvent molecules. The y-axis on the right showed the intensity distribution (qi %) in blue, while the y-axis on the left displayed the cumulative distribution (Qi %) in red. Two distinct peaks were observed in the analysis. The first peak was sharp and prominent at around 10 nm, indicating the presence of smaller particles. The second peak was broader and appeared at around 200 nm. The DLS data showed populations of two distinct sizes, indicating that the sample had a polydisperse nature. This meant the sample consisted of particles of different sizes. The red curve representing the cumulative distribution (Qi %) showed the percentage of the total particle population below a certain size. For instance, at around 100 nm on the x-axis, about 90% of the particles were smaller in size. Other researchers reported the same results for inorganic NPs. For example, the DLS analysis of AgNPs-RR showed a Z-average size of 48.11 nm and a polydispersity index (PDI) of 0.472, indicating a bimodal distribution and potential agglomeration in the solution [20]. Also, the DLS analysis indicated the polydispersity of the Fe, Ag/Fe, APTES–Ag/Fe, and PEI–Ag/Fe NPs [21].

Fig. 1.

Size distribution of Fe–Co MOFs measured by DLS at 25 °C

Magnetic properties of CFZ-loaded Fe–Co MOFs by VSM analysis

The VSM analysis for the CFZ-loaded Fe–Co MOFs is shown in Fig. 2. The graph depicted a magnetic hysteresis loop, where the y-axis represented the magnetic moment in emu/gram, and the x-axis showed the applied magnetic field in Oersted. The material had reached a saturation magnetization of around 20 emu/g at high applied fields, indicating a good magnetic response. Additionally, the narrow hysteresis loop reflected low coercivity, meaning the material could easily reverse its magnetization direction. The presence of a small but non-zero remanence showed that the material retained some magnetization even after the external magnetic field was removed. In the context of drug delivery, these magnetic properties were particularly relevant. The magnetic nature of the CFZ-loaded Fe–Co MOFs suggested that it could have been guided to specific locations within the body using an external magnetic field. The high saturation magnetization indicated that the particles would have been strongly attracted to such a field, enhancing their potential for targeted delivery. For example, the saturation magnetization (MS) decreases significantly from 79.4 emu/g in CoFe₂O₄ to 19.2 emu/g in CoFe₂O₄/Zn-MOF and 18.9 emu/g in CoFe₂O₄/[Cu/Zn-MOF] due to the growth of MOFs on magnetic particles. Still, the MS remains unchanged during transmetalation, indicating that the CoFe2O4 cores are preserved within the framework [22].

Fig. 2.

Magnetic properties of CFZ-loaded Fe–Co MOFs by VSM analysis

Morphology and elemental composition by SEM-EDS

The SEM image of CFZ-loaded Fe–Co MOFs likely reveals a well-defined crystalline structure characteristic of MOFs (Fig. 3). The particles appear uniformly distributed with distinct boundaries, suggesting successful loading of CFZ. Into the Fe–Co MOF matrix. The size of these particles might range from 100 to 140 nm, depending on the magnification, showing a porous structure typical of MOFs. This porosity is essential for encapsulating drugs like CFZ and providing a controlled release mechanism. The observed morphology and size of the CFZ-loaded Fe–Co MOFs make it an excellent candidate for drug delivery applications. The uniform particle size ensures consistent drug release rates, while the porous structure allows for high drug loading capacity and sustained release [12]. The stability and integrity of the MOF framework ensure that the drug is protected from degradation until it reaches the target site, enhancing the efficacy and safety of the drug delivery system [23]. In a similar report, FESEM and TEM analysis reveal that the as-prepared magnetic Fe₃O₄@Co-MOF exhibits an irregular crystal structure, with spherical Fe₃O₄ NPs (~ 35 nm in diameter) attached directly to the MOF surface without compromising its integrity [24].

Fig. 3.

Morphology image of CFZ-loaded Fe–Co MOFs by FE-SEM analysis

The SEM-EDS analysis of the CFZ-loaded Fe–Co Metal-Organic Framework (MOF) provided comprehensive insights into its elemental composition and distribution (Fig. 4A and B). The analysis revealed that oxygen was the most abundant element, constituting 50.74 wt% with an atomic percentage of 75.51%. Iron was also significantly present at 29.08 wt%, corresponding to an atomic percentage of 12.40%, while cobalt accounted for 17.69 wt% and 7.15% in atomic percentage. Carbon, the least abundant element, comprised 2.49 wt% with an atomic percentage of 4.94%. The elemental mapping confirmed the uniform distribution of oxygen, iron, cobalt, and carbon throughout the MOF structure, indicating the successful integration of these elements within the framework. The high oxygen content likely originated from the MOF’s organic ligands and the possible oxidation of the metal components. At the same time, the significant presence of iron and cobalt confirmed their incorporation, which was crucial for achieving properties such as magnetic responsiveness or catalytic activity. In the context of drug delivery, the data suggested that the CFZ-loaded Fe–Co MOFs, with their considerable metal content and uniform elemental distribution, could be advantageous for controlled drug release. The structure’s composition, particularly the metal centers, likely played a key role in facilitating responsive or targeted drug delivery, potentially through magnetic guidance. Additionally, though minimal, the presence of carbon might have indicated the retention of drug molecules within the MOF structure, which was essential for sustained release profiles [25, 26]. For example, the EDX spectroscopy analysis confirms the presence and uniform distribution of C, N, O, Fe, Cl, and Co in the NPs, with carbon comprising 62.3% of the catalyst, alongside TEM images indicating agglomerated Fe₃O₄ NPs within the core-shell structure of the Fe₃O₄@Co-MOF composite [24].

Fig. 4.

SEM-EDS mapping A and elemental composition B of CFZ-loaded Fe–Co MOF

Porosity assessment of CFZ-Loaded Fe–Co MOF by BET analysis

The nitrogen adsorption-desorption isotherm, depicted in Fig. 5a, exhibited a type (III) isotherm according to the IUPAC classification, indicative of materials with mesoporous structures. The isotherm displayed a hysteresis loop of type H3, which is typically associated with materials that possess slit-shaped pores. This behavior suggested that synthesized CFZ-loaded Fe–Co MOFs had a mesoporous framework with distinctive pore structures. The pore size distribution shown in Fig. 5b further supported this observation, highlighting a range of pore sizes within the material. The BET surface area of synthesized CFZ-loaded Fe–Co MOFs was measured at 84.984 m²/g, indicating a high surface area that is advantageous for applications in drug delivery. However, the total pore volume was 0.1 cm³/g. The average pore diameter, calculated to be 4.7073 nm, corresponded with the mesoporous nature of the material. This was consistent with the observed isotherm and hysteresis loop types, which both pointed to a predominance of smaller mesopores. These results indicated that CFZ-Loaded Fe–Co MOFs possessed a high surface area with well-defined mesopores, making them suitable for applications where controlled pore characteristics are crucial, such as drug delivery. For example, the N₂ adsorption-desorption isotherms revealed that MIL-88, Fe₃O₄@C@MIL-88, and Fe₃O₄@C@MIL-88-DOX-FC exhibited microporous and macroporous structures, with surface area and pore volume decreasing sequentially due to the filling of pores and channel structures by Fe₃O₄, DOX, and FC [27].

Fig. 5.

Nitrogen adsorption-desorption isotherm a and Pore size distribution profile b of CFZ-Loaded Fe–Co MOFs

Loading efficiency and drug release of CFZ-Loaded Fe–Co MOFs

The loading efficiency and release behavior of a CFZ-loaded Fe–Co MOF were critical aspects in evaluating its performance as a drug delivery system. The initial loading efficiency was 74.86%, indicating that 74.86% of the drug had been successfully incorporated into the Fe–Co MOF’s structure, which suggested a high capacity for drug encapsulation. The release behavior data illustrated the drug release profile from CFZ-loaded Fe–Co MOFs over 24 h at two different pH levels (Fig. 6). At pH 5.2, a significant initial burst release was observed, with the drug release reaching approximately 40% within the first hour. This rapid release suggests that the acidic environment facilitated a faster release of the drug from the MOFs. The release then continued to increase more gradually, reaching around 58% at 8 h and ultimately exceeding 60% by 24 h, indicating a sustained and controlled release phase. In contrast, at pH 7.4, the release was considerably slower. An initial burst release of about 25% occurred in the first hour, followed by a more gradual increase, reaching approximately 34% by 8 h. After this point, the release plateaued, with minimal further increase, reaching only about 35% at 24 h. This pH-dependent release behavior can be attributed to the structural sensitivity of Fe–Co MOFs to acidic conditions. At lower pH, protonation of coordination bonds within the MOF framework may occur, leading to partial degradation or increased porosity, thereby facilitating faster drug diffusion. Additionally, the solubility of CFZ might be enhanced in acidic environments, further contributing to the higher release rate. In contrast, the stability of the MOF structure under neutral pH conditions restricts drug diffusion, resulting in slower and more limited release. These findings support the potential use of CFZ-loaded Fe–Co MOFs for targeted drug delivery to acidic tumor microenvironments, where such pH-responsive behavior could enhance therapeutic efficacy while minimizing off-target effects. In a similar study, the CS/DOX@5-Fu@Al-MOF/GO microspheres demonstrated a more sustained and pH-sensitive drug release profile compared to DOX@5-Fu@Al-MOF/GO, with 63.1% of 5-Fu and 54.47% of DOX released at pH 5.0 over 72 h, and 27.69% of 5-Fu and 25.10% of DOX released at pH 7.4 [28]. To minimize the initial burst release of the drug, several strategies had been employed to reduce the undesired burst release in MOF-based drug delivery systems akin to the CFZ–Fe–Co MOF. First, surface coatings—particularly PEGylation—had been applied to the MOF exterior to block outer pores and slow early drug leakage. For example, PEG-modified UiO‑66 exhibited a delayed release profile during the initial hours, effectively suppressing the burst effect [29]. Second, bilayer coatings had been introduced: zirconium-based MOFs had been coated with a secondary layer that significantly slowed the diffusion of surface-adsorbed drug and prevented rapid release, with only about 8% released in the first 2 h compared to ~ 40% from uncoated MOF [30]. Third, in situ (one-pot) encapsulation had been used, which embedded drug molecules more deeply within the porous structure, as demonstrated in studies on MOF-801 loaded with 5‑FU [31]. Finally, stimuli-responsive frameworks had been designed: pH-sensitive zirconium-based MOFs had remained stable at physiological pH and only released their cargo under acidic conditions similar to tumor environments, avoiding burst release at neutral pH [32]. Together, these modifications had significantly mitigated initial burst release in MOF carriers and provided a strong rationale for applying similar approaches—PEGylation, bilayer coatings, in situ loading, and stimuli-responsive linkers.

Fig. 6.

Release behavior of CFZ-loaded Fe–Co MOFs at pH 7.4 and 5.2

Biochemical findings

In this study, a total of 50 male Wistar rats were randomly assigned to five different groups. Two groups received plain CFZ, while the other two groups received CFZ encapsulated in Fe–Co MOF through intravenous administration. Figure 7A shows the serum level of aspartate aminotransferase. Significant elevation in serum AST levels was observed following intravenous injection of free CFZ at the dose of 0.8 mg/kg (P < 0.05). Treatment with CFZ-loaded Fe–Co MOFs (0.8 mg/kg) also increased serum AST levels (P < 0.05). Intravenous therapy with free CFZ and also with CFZ-loaded Fe–Co MOFs (0.8 mg/kg) significantly increased ALT levels (P < 0.001) (Fig. 7B). In rats receiving CFZ-loaded Fe–Co MOFs (0.8 mg/kg), serum BUN levels were significantly higher than control rats (P < 0.001) (Fig. 7C). On the other hand, intravenous administration of free CFZ (0.8 mg/kg) had no significant effect on serum BUN levels (Fig. 7C) (P > 0.05). Intravenous injections of free CFZ and CFZ-loaded Fe–Co MOFs (0.8 mg/kg) resulted in higher serum creatinine levels in rats (P < 0.001). This elevation was not observed in rats treated with a 0.4 mg/kg dose of CFZ. (Fig. 7D). Treatment with CFZ-loaded Fe–Co MOFs (0.4 mg/kg) did not affect serum creatinine (P > 0.01). Furthermore, treatment with CFZ-loaded Fe–Co MOFs at a concentration of 0.8 mg/kg dose of free CFZ significantly increased liver MDA level (P < 0.05) (Fig. 7E).

Fig. 7.

Effects of free CFZ and CFZ-loaded Fe–Co MOFs on serum biochemical parameters of different experimental groups.* Significant compared with the control group (P < 0.05)

Histopathological findings

The histopathological structure of liver tissue is illustrated in Fig. 8A. In our histopathological examinations, rats administered CFZ-loaded Fe–Co MOFs (0.4 mg/kg) exhibited a consistent liver structure (Fig. 8B). However, rats treated with a higher dose of 0.8 mg/kg of CFZ-loaded Fe–Co MOFs displayed morphological damage to the liver tissue, including the accumulation of inflammatory cells within the liver parenchyma (Fig. 8C). The group injected with 0.4 mg/kg of free CFZ showed a normal liver appearance. In contrast, rats injected with 0.8 mg/kg of free CFZ exhibited necrosis, inflammatory cell accumulation, and disarray of hepatic cords (Fig. 8E).

Fig. 8.

Liver sections of both the control and treated groups. A Photo of a healthy liver in a control rat. B Liver of a rat treated with CFZ-loaded Fe–Co MOFs (0.4 mg/kg), demonstrating normal liver histopathology. C Liver necrosis in rats treated with CFZ-loaded Fe–Co MOFs (0.8 mg/kg) showing accumulation of inflammatory cells in liver parenchyma (Arrow). D Liver of a rat from the group treated with free CFZ (0.4 mg/kg), showing well-arranged hepatic cords and liver sinusoids. E Accumulation of inflammatory cells and disarrangement of hepatic cords in rats treated with free CFZ (0.8 mg/kg) (arrowhead)

Our histopathological findings indicated that rats injected with CFZ-loaded Fe–Co MOFs at 0.8 mg/kg (Fig. 9B) and free CFZ (Fig. 9D) maintained well-organized tubular and glomerular structures. In contrast, rats treated with free CFZ at 0.8 mg/kg (Fig. 9E) and CFZ-loaded Fe–Co MOFs (Fig. 9C) exhibited cloudy swelling and glomerulosclerosis, the lining epithelia were swollen (Fig. 9E), and the proximal tubule lumen was narrow and star-shaped (Fig. 9C).

Fig. 9.

Microscopic examination of kidney sections from different groups of rats using Masson trichrome staining at 40× original magnification. A Kidney of a healthy rat from the control group showing a normal glomerulus. B Kidney of a rat from the group treated with CFZ-loaded Fe–Co MOFs (0.4 mg/kg), displaying an intact glomerulus. C Kidney section of a rat treated with CFZ-loaded Fe–Co MOFs (0.8 mg/kg), presenting signs of cloudy swelling mainly in the proximal and distal renal tubules. D Kidney section of rats treated with free CFZ (0.4 mg/kg), demonstrating normal proximal and distal tubules with normal-sized nuclei. E Kidney micrograph of a rat from the group treated with free CFZ (0.8 mg/kg), showing glomerulosclerosis characterized by swollen lining epithelium and narrowing of the proximal tubule lumen

Figure 10A depicts the intact appearance of heart cells. Rats receiving Carfilzomib-loaded Fe–Co MOFs at 0.4 mg/kg showed healthy heart tissue (Fig. 10B). Microscopic examination of the heart from the group injected with Carfilzomib-loaded Fe–Co MOFs at 0.8 mg/kg revealed necrosis of heart cells (Fig. 10C). The heart section from rats treated with free Carfilzomib at 0.4 mg/kg exhibited mild inflammatory cell infiltration (Fig. 10D). Our histopathological findings indicated that rats receiving the higher dose of free Carfilzomib displayed signs of heart damage, including inflammatory cell infiltration, bleeding, and necrosis (Fig. 10E). Furthermore, heart sections of rats treated with 0.8 mg/kg of free Carfilzomib showed severe necrosis (Fig. 10F)

Fig. 10.

Heart sections of rats from control and treatment groups, Hematoxylin and eosin; ×40 original magnification. A The normal appearance of cardiac cells of a control rat. The nuclei appeared normal in size and situation; B heart section of a rat receiving CFZ-loaded Fe–Co MOFs at 0.4 mg/kg dose showing a healthy heart; C microscopic examination of a heart of a rat receiving CFZ-loaded Fe–Co MOFs at 0.8 mg/kg showing necrosis of heart cells (arrow); D heart section of rats treated with free CFZ (0.4 mg/kg) showing mild inflammatory cell infiltration; E inflammatory cell infiltration (arrowhead) and eosinophilic stained pyknotic nuclei (arrow) in rats treated with 0.8 mg/kg of free CFZ. (arrow); F heart section of a rat treated with 0.8 mg/kg of free CFZ, necrosis (arrow) and hemorrhage are visible (star).

Figure 11 presents photomicrographs of the testes from both control and experimental groups. Control rats displayed well-organized testicular structures (Fig. 11A). In testicular photomicrographs from the experimental groups, all stages of spermatogenesis were present in rats subjected to various doses of CFZ-loaded Fe–Co MOFs. Specifically, rats treated with 0.4 mg/kg and 0.8 mg/kg of CFZ-loaded Fe–Co MOFs exhibited uniformly sized seminiferous tubules (Fig. 11B and C, respectively). Rats receiving the 0.4 mg/kg dose of free CFZ also showed no evidence of inflammation, necrosis, or cellular infiltrates (Fig. 11D). However, signs of injury, such as multinucleated giant Sertoli cells, were observed in the testes of rats treated with the 0.8 mg/kg dose of free CFZ (Fig. 11F).

Fig. 11.

Histopathological examination of the testis of control and experimental groups. A normal testis histopathology of control rats; B histopathological pattern of a testis section of a rat treated with CFZ-loaded Fe–Co MOFs (0.4 mg/kg dose); C testis of rats injected with CFZ-loaded Fe–Co MOFs (0.8 mg/kg dose) showing normal histopathology; D normal feature of seminiferous tubules in the testis section of a rat from the group treated with free CFZ (0.4 mg/kg dose); E testis of a rat treated with free CFZ (0.8 mg/kg) (arrowhead). F Testis of a rat treated with free CFZ (0.8 mg/kg dose), the arrow shows a multinuclear giant Sertoli cell

The histopathological scores are illustrated in Table 2. A semi-quantitative analysis revealed that rats treated with higher doses of free CFZ exhibited significantly higher degrees of hepatic damage (P < 0.05 and P < 0.01, respectively).

Table 2.

The liver damage score (LDS), renal damage score (RDS), heart damage score (HDS), and testis damage score (TDS)

	Liver	Heart	Testis	Kidney
Control	1.12  ±  0.3	1.14 ± 0.37	1.0 ± 0.0	1.25 ± 0.46
CFZ 0.4 mg/kg	1.6  ±  0.51	1.25 ± 0.46	1.2 ± 0.4	1.5 ± 0.75
CFZ 0.8 mg/kg	2.5***±0.53	2.37***±0.5	1.42 ± 0.53	2.5*±0.92
CFZ-loaded Fe–Co MOFs 0.4 mg/kg	1.37 ± 0.51	1.3 ± 0.57	1.28 ± 0.48	1.62 ± 0.51
CFZ-loaded Fe–Co MOFs 0.8 mg/kg	2.5***±0.53	2.42***±0.53	1.87***±0.35	3.12***±0.83

*Significant compared with the control group (P < 0.05)

**Significant compared with the control group (P < 0.01)

***Significant compared with the control group (P < 0.001)

Our semi-quantitative histopathological findings were in line with the microscopic investigation results. The interpretation of biochemical data showed that both free CFZ and CFZ-loaded Fe–Co MOFs have nephrotoxic and hepatotoxic potential at higher doses; however, the low dose of CFZ-loaded Fe–Co MOFs showed no toxic effects. It seems that CFZ-loaded Fe–Co MOFs have dose-dependent toxicity. Previous studies have demonstrated the dose-dependent toxicity of NPs [33].

‏On the other hand, the histopathological data showed that CFZ-loaded Fe–Co MOFs are less hepatotoxic than the free CFZ. In this case, we observed that signs of liver injury, such as necrosis and sinusoidal disarrangement, were less prominent in rats treated with CFZ-loaded Fe–Co MOFs than in the group treated with free CFZ. The current study compared the in vivo toxicity of free CFZ and CFZ-loaded Fe–Co MOFs. Biochemical analysis revealed that high doses of free CFZ and CFZ-loaded Fe–Co MOFs can be toxic to the kidneys and liver. However, the low dose of CFZ-loaded Fe–Co MOFs showed no adverse effects. The toxicity of CFZ-loaded Fe–Co MOFs increased with higher doses, consistent with previous studies on nanoparticle toxicity [34]. Carfilzomib has a low risk of elevated serum enzyme levels but may rarely cause significant acute liver injury. The exact mechanisms behind the liver injury, which leads to elevated serum enzymes and hepatic toxicity during carfilzomib therapy, are not fully understood. Carfilzomib is primarily metabolized in the body by plasma peptidases, with only a partial metabolism occurring in the liver via the CYP 3A4 pathway [35]. Histopathological analysis indicates that the administration of CFZ-loaded Fe–Co metal-organic frameworks (MOFs) results in reduced nephrotoxicity compared to the use of free CFZ. These findings suggest that CFZ-loaded Fe–Co MOFs may provide a promising therapeutic strategy for minimizing renal structural damage associated with CFZ treatment. Additionally, our histopathological data revealed severe cardiotoxicity linked to free CFZ. One of the most significant adverse effects of carfilzomib is its cardiovascular complications, which can include heart failure, arrhythmias, hypertension, and ischemic heart disease [36].

In recent years, various studies have investigated the application of different nanocarriers to enhance the anticancer effectiveness of CFZ [24], self-assembled nanostructures [25], albumin-coated nanocarriers [26], lipid nanodisks [27], zinc ferrite magnetic nanoparticles [28], cobalt oxide nanostructures [29], and nanoparticles designed to mimic neutrophil behavior [30]. Notably, recent findings indicated that Pluronic F127/CFZ nanomicelles displayed greater cytotoxicity compared to free CFZ, leading to significant morphological changes in cancer cells [31]. The administration of a low dose of the nano-drug does not produce nephrotoxic effects in male Wistar rats. Metal-organic frameworks are emerging as a promising alternative to conventional drug delivery systems, attributed to their capacity to hold substantial drug loads, ease of functionalization, efficient clearance from biological systems, and low toxicity profiles. Despite some advancements, there are still relatively few reports on the therapeutic uses of Fe–Co-based MOFs. In contrast, Ni-based MOFs have found a range of applications in the biomedical field. For instance, nickel-based frameworks are known for their unique structure, which offers high selectivity, sensitivity, and detection limits, making them particularly effective for glucose detection in human serum [32].

In the current study, the administration of free carfilzomib and carfilzomib-loaded metal-organic frameworks led to a significant increase in liver lipid peroxidation. Lipid peroxidation occurs when there are elevated levels of reactive oxygen species (ROS), which can damage proteins, lipids, cell membranes, and DNA. This process is associated with various diseases, including diabetic complications, neurological disorders, and cancer. The reduced hepatotoxicity and nephrotoxicity observed with CFZ-loaded Fe–Co MOFs could allow for safer administration of CFZ, particularly in patients with pre-existing liver or kidney conditions. The dose-dependent toxicity of CFZ-loaded Fe–Co MOFs highlights the importance of careful dose optimization to balance therapeutic benefits and adverse effects. Our study aligns with previous research indicating that free CFZ or nanoparticle forms of CFZ can elevate lipid peroxidation and oxidative stress [37].

Also, this study assesses the biocompatibility and toxicity of Metal-Organic Frameworks (MOFs) on key organs such as the liver, kidneys, testes, and heart. Exposure to MOFs caused significant liver damage with necrosis and inflammation, potentially leading to fibrosis. The kidneys showed signs of tubular degeneration and inflammation, indicating possible renal toxicity. Testes had lower levels of reproductive toxicity, while the heart exhibited mild inflammation and congestion. Variability in organ responses to MOFs suggests varying distribution and susceptibility. Factors like particle size, surface area, and dosage play a key role in organ-specific toxicity. Systemic exposure to MOFs leads to widespread effects. Previous studies have shown that metal-organic framework nanoparticles (MOF NPs) have therapeutic potential and toxicological risks. While MOF-74(Co) NPs were relatively safe in mice, other MOF NPs [38], such as Cu-MOF, ZIF-8, and ZIF-90, showed significant toxicity in zebrafish, causing liver damage and mitochondrial disruption [39]. The composition and structure of MOF NPs play a crucial role in determining their toxicity profiles. Species-specific toxicity assessments are necessary to understand the environmental and biomedical implications of MOF NPs. In terms of therapeutic potential, DIBc NMOF showed promising results in diabetic nephropathy models, improving kidney function and reducing blood glucose levels. However, the potential toxicity of NMOFs, as seen in the zebrafish study, highlights the need for careful consideration of their safety [40].

MOFs can cause histopathological changes by releasing reactive oxygen species from metal ions, leading to oxidative stress and inflammation. MOF degradation in the body may also release toxic metal ions or ligands. This study’s limitation is the use of an animal model that may not fully represent human responses, and a short exposure duration. Future research should explore chronic exposure, utilize in vitro models, and investigate strategies to mitigate the toxicity of MOFs.

Conclusion

In this study, a novel Fe–Co metal-organic framework (MOF) was developed and evaluated as a carrier for the anticancer drug carfilzomib (CFZ). The CFZ-loaded Fe–Co MOFs exhibited a high drug loading efficiency of 74.86%, strong magnetic responsiveness (saturation magnetization of 20 emu/g), and a mesoporous structure with a surface area of 84.98 m²/g. Drug release studies showed an initial burst followed by sustained release, indicating controlled delivery. In vivo results demonstrated that the low dose (0.4 mg/kg) of CFZ-loaded MOFs caused minimal toxicity, while the higher dose (0.8 mg/kg) led to moderate liver and kidney damage, although less severe than free CFZ. The novelty of this work lay in the use of a bimetallic Fe–Co MOF system that combined high drug loading, controlled release, and reduced systemic toxicity—features not previously reported for CFZ delivery. These findings highlighted the potential of Fe–Co MOFs as a safer and more efficient platform for targeted cancer therapy.

Author contributions

Mohammad Reza Hajinezhad, Mahmood Barani, Saman Sargazi: Conceptualization. Mahmood Barani, Mohammad Reza Hajinezhad: Methodology, Investigation. Mohammad Reza Hajinezhad, Saman Sargazi, Mahmood Barani: writing-original draft preparation. Mohammad Reza Hajinezhad, Saman Sargazi, Mahmood Barani: writing-review and editing. Mohammad Reza Hajinezhad, Mahmood Barani: Supervision. All authors have read and agreed to the published version of the manuscript.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT in order to proofread some parts of the text . After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Funding

The current research was funded by a research project from the University of Zabol, Zabol, Iran. Authors thank funding from the University of Zabol (Project number: PR-UOZ1402-4).

Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval and consent to participate

The protocol of this study was approved by the ethics committee of the University of Zabol (Ethical code: IR.UOZ.REC.1403.003). All injection protocols and handling were carried out strictly to the guidelines of care outlined in the NIH publication no. 85–23, ensuring the ethical treatment of laboratory animals.

Consent for publications

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.