Synthesis, radiolabeling, and biodistribution of 99 m-technetium-labeled zif-8 nanoparticles for targeted imaging applications

Abstract

This study investigates the synthesis and radiolabeling of zeolitic imidazolate frameworks (ZIF-8) with the radioisotope technetium-99 m (^99mTc) using a solvothermal method in methanol. The methanolic medium facilitated the formation of nanoparticles with favorable characteristics, including a smaller particle size (198 ± 9.8 nm) and a low polydispersity index (PDI = 0.219 ± 0.011). Radiolabeling efficiency (RE%) and radiochemical purity (RCP%) were optimized by employing SnCl₂ as a reducing agent, resulting in an RE% of 95.2 ± 1.9% and an RCP% of 96.1 ± 1.7% in triplicate (n = 3) at 65 °C. The nanoparticles exhibited high serum stability, retaining 99.05% of RCP% after 24 h, and demonstrated hemocompatibility, with hemolysis rates below 5% across all tested concentrations. In vitro biocompatibility assessments using NIH-3T3 cells indicated cell viability above 70% at concentrations up to 40 μg/mL. Biodistribution studies in rabbits (n = 6) revealed predominant accumulation in the bladder, with radiotracer uptake in the bladder being 6.3, 7.2, and 36.2 times higher than in the liver, kidneys, and heart (p < 0.0001), respectively, suggesting renal clearance. These results underscore the potential of ^99mTc-(ZIF-8) nanoparticles for biomedical applications, particularly in targeted imaging and drug delivery. Future research will focus on improving targeting specificity and enhancing therapeutic efficacy in disease models.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-04145-w.

Introduction

Noninvasive image-guided disease monitoring and therapy represent cutting-edge treatment approaches. This technique involves tagging nanoprobes, radionuclides, or fluorescent dyes to drugs or targeting agents, enabling the tracking of their movement and enhancing the visualization of anatomical and physiological changes within the region of interest (ROI) for a prolonged period, compared to surrounding tissues (Gupta et al. 2021; Zia et al. 2022). Currently, approximately 80% of clinical radiodiagnostic scans are conducted using single-photon emission computed tomography (SPECT), while around 20% utilize positron emission tomography (Zia et al. 2022). To improve contrast in ROIs, strategies such as antibody conjugation and the use of nanocarriers, including liposomes and polymeric micelles for high-payload delivery, are commonly employed (Zia et al. 2022).

Technetium-99 m (^99mTc) is a preferred gamma-emitting radionuclide in this context due to its favorable characteristics, such as an ideal gamma-ray energy of 140 keV, a 6-h half-life, and its availability from a generator. Developing new ^99mTc radiotracers has become a significant focus in radiopharmacy (Gharepapagh et al. 2021). Numerous ^99mTc-labeled nanoplatforms are being designed as SPECT-based contrast agents for molecular imaging of various organs (El-Ghareb et al. 2020; Sakr et al. 2020; Dubey et al. 2023). For example, Zhang et al. (Zhang et al. 2016) engineered a polymeric nanoparticle-based SPECT imaging probe targeting the asialoglycoprotein receptor for detecting liver fibrosis (Zhang et al. 2016). In a separate study, the same group developed a molecular probe targeting vimentin and desmin for SPECT imaging of liver fibrosis (Zhang et al. 2018). These studies underscore the potential of leveraging specific cell surface receptors or molecular pathways to achieve targeted biodistribution within the liver. For instance, palmitic acid accumulates in hepatocytes and plays a critical role in synthesizing and maintaining cell membrane phospholipids and adipose triacylglycerols. The liver is central to the metabolism of saturated fatty acids like palmitic acid (Annevelink et al. 2023). Recent research has shown significant interest in developing smart nanocarriers capable of efficiently loading therapeutic molecules, releasing drugs precisely at target sites, and serving as diagnostic agents (Alavi et al. 2019, 2020, 2021; Foroushani et al. 2021). Among the various materials used for drug delivery (Babaei et al. 2017; Ghaferi et al. 2020, 2024; Koohi Moftakhari Esfahani et al. 2022; Alavi et al. 2023a, b, 2024b, c; Alharthi et al. 2024a; Alrashidi et al. 2024), metal–organic frameworks (MOFs) have garnered attention for their unique advantages as drug carriers (Pan et al. 2020; Tan et al. 2020; Foroushani et al. 2021). Specifically, zeolitic imidazolate framework-8 (ZIF-8), a type of MOF, has shown significant potential for drug delivery applications due to its interesting properties (Zhong et al. 2020; Feng et al. 2021; Ahmad et al. 2023; Reshmi et al. 2023). ZIF-8 is stable in neutral aqueous solutions but disintegrates in acidic environments, making it a promising candidate for pH-targeted cancer therapies (Hou et al. 2022; Zhao et al. 2022).

However, current imaging and therapeutic agents face challenges, such as non-specific distribution, inefficient targeting, and prolonged retention in non-target tissues, leading to off-target toxicity and reduced imaging accuracy (Rangasamy et al. 2019; Cheng et al. 2023; Winuprasith et al. 2023). Traditional SPECT imaging agents often suffer from rapid clearance or poor biodistribution, limiting their ability to provide detailed and specific images for disease diagnosis (Luo et al. 2016; Wang et al. 2024). The use of MOFs, particularly ^99mTc-labeled ZIF-8 nanoparticles, offers a potential solution to these limitations by combining high radiolabeling efficiency (RE%) with improved biodistribution profiles and renal clearance (Ahmadi et al. 2024; Alavi et al. 2024a). These nanoparticles are engineered to minimize off-target effects, reduce long-term tissue accumulation, and enhance imaging precision. Additionally, their modular structure offers opportunities for future functionalization, enabling more targeted therapeutic applications (Ahmadi et al. 2024; Alavi et al. 2024a).

This study aims to address these limitations by providing a novel platform for both imaging and drug delivery with greater accuracy and safety. Specifically, we focus on developing ^99mTc-radiolabeled ZIF-8 nanoparticles and evaluating their preparation methods, RE%, radiochemical purity (RCP%), size, polydispersity index (PDI), zeta potential, morphology, in vitro stability, and biocompatibility. We further investigate their biodistribution, imaging capabilities, and in vivo toxicity. The methodologies employed include solvothermal synthesis, thin-layer chromatography (TLC), dynamic light scattering (DLS), atomic force microscopy (AFM), scanning electron microscopy (SEM), red blood cell (RBC) hemolysis tests, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, SPECT/computed tomography (CT) imaging, spectrophotometry, and histopathological analyses.

Materials and methods

Materials

Methanol, zinc nitrate hexahydrate (Zn(NO₂)0.6H₂O), 2-methylimidazole, tin(II) chloride dihydrate (SnCl₂0.2H₂O), and hydrochloric acid (HCl) were sourced from Sigma-Aldrich. TLC silica gel (SG) 60 F254 aluminum sheets, acetone, ethanol (EtOH), phosphate-buffered saline (PBS) tablets, Roswell Park Memorial Institute 1640 (RPMI-1640) medium, fetal bovine serum (FBS), trypsin, MTT reagent, penicillin/streptomycin antibiotics, dimethyl sulfoxide (DMSO), sodium chloride (NaCl), sodium dodecyl sulfate (SDS), ketamine, xylazine, formalin, hematoxylin and eosin (H&E), triton X-100, and sodium hydroxide (NaOH) were obtained from Merck (Germany). The [⁹⁹Mo]/[^99mTc] generator was acquired from the Pars-Isotope Center (Tehran, Iran). Mouse embryonic fibroblast NIH-3T3 cells, male Wistar rats (200–250 g), and male New Zealand rabbits (2.5–3.0 kg) were purchased from the Pasteur Institute of Iran.

Synthesis of zeolitic imidazolate frameworks nanoparticles using a solvothermal method

ZIF-8 nanoparticles were synthesized via a solvothermal method. The formulation was optimized for particle size and PDI by adjusting the concentrations of reagents and other conditions (Table S1). A series of experiments were conducted to determine the optimal incubation time and reagent concentrations for Zn(NO₂)₂ and 2-methylimidazole. The aim was to produce the smallest possible nanoparticle size with the lowest PDI and highest RE%. Results showed that a 1-h incubation at room temperature (RT) in methanol as the solvent produced nanoparticles with the most desirable properties (198 ± 9.8 nm, PDI = 0.219 ± 0.011).

For the synthesis, 1 g of 2-methylimidazole was dissolved in 75 mL of methanol and mixed with a separate solution containing 1 g of Zn(NO₂)₂0.6H₂O dissolved in 75 mL of methanol. The mixture was incubated at RT for 1 h. After incubation, the nanoparticles were stored at 4 °C for further use (Mendes et al. 2014).

Radiolabeling of zeolitic imidazolate frameworks with ⁹⁹mTc

Radiolabeling of the ZIF-8 nanoparticles was optimized using four different conditions (Table S2). The best radiolabeling results were obtained by incubating the nanoparticles with SnCl₂ and sodium pertechnetate (Na⁹⁹mTcO₄) for 40 min at 65 °C, yielding a high RE% of 95.2 ± 1.9% and a RCP% of 96.1 ± 1.7%. To prepare the radiolabeled ZIF-8 nanoparticles, a 500 µL suspension of the nanoparticles (2 mg/mL) in deionized water (DW) was prepared. Then, 100 µL of a SnCl₂ solution (15 mg/mL) in 0.01 M HCl was added. Before use, the HCl solution was degassed with nitrogen gas. Na⁹⁹mTcO₄ (6 mCi) was added as the technetium source. After incubating the mixture at 65 °C for 40 min, the radiolabeled samples were centrifuged at 12,000 RPM for 20 min. The sediment was washed twice with DW and centrifuged again to remove any unreacted pertechnetate.

RE% was calculated by dividing the radioactivity of the sediment (labeled nanoparticle activity) by the total radioactivity initially added to the sample, and multiplying by 100.

RCP% was determined using instant TLC (iTLC) with SG as the stationary phase and acetone as the mobile phase. After applying the radiolabeled samples onto the TLC strip, the acetone traveled up the strip by capillary action, separating the labeled nanoparticles from the free pertechnetate (⁹⁹mTcO₄). Radioactivity at the top (A₁) and bottom (A₀) of the TLC strip was measured using a gamma counter. The RCP% was calculated as follows:

$$\text{RCP\%}=\frac{{\text{A}}_0}{{\text{A}}_0+{\text{A}}_1}\times 100.$$

All measurements were performed in triplicate (n = 3) to ensure reproducibility.

Nanoparticles characterization

The ⁹⁹mTc-labeled ZIF-8 nanoparticles were characterized for their size, PDI, and zeta potential using a Zetasizer instrument (Nano ZS3600; Malvern Instruments, UK) through DLS analysis. A suspension of nanoparticles at a concentration of 100 μg/mL was prepared in PBS (pH 7.4). Additionally, three-dimensional topographical images of the ⁹⁹mTc-labeled ZIF-8 nanoparticles were obtained using AFM (model 0101/A, Iran). For AFM imaging, 20 µL of the nanoparticle suspension was deposited onto a mica surface and air-dried at RT. Imaging was performed in tapping mode using a silicon cantilever with a spring constant of 40 Nm¹, and the resulting images were analyzed using JPK software (Germany). Furthermore, the morphology of the nanoparticles was examined using SEM (FEIESEM QUANTA 200, USA) under vacuum conditions after lyophilization (Alharthi et al. 2024b).

Serum stability of ^99mTc-(ZIF-8)

Serum stability of the ⁹⁹mTc-labeled nanoparticles was evaluated by mixing 250 µL of the radiolabeled nanoparticles (0.00233 MBq) with 500 µL of freshly prepared human serum, followed by incubation at 37 °C. A control sample containing free Na⁹⁹mTcO₄ was prepared under the same conditions. The release of free ⁹⁹mTcO₄ from the nanoparticles was monitored at different time intervals over a 24-h period using TLC and a gamma counter. The stability of the radiolabeled nanoparticles was assessed by comparing their RCP% to that of the control sample (free Na⁹⁹mTcO₄).

Red blood cell (RBC) hemolysis

Blood was collected from healthy volunteers and treated with sodium citrate to prevent clotting. After centrifugation at 1000 × g for 5 min, the plasma layer was removed, and the remaining RBC pellets were washed with a sterile isotonic 0.9% NaCl solution. The RBCs were then suspended in the same NaCl solution at a 1:20 ratio. ⁹⁹mTc-(ZIF-8) nanoparticles were prepared in isotonic 0.9% NaCl solution at concentrations of 10, 20, 100, 200, and 300 μg/mL. These concentrations were selected based on preliminary biocompatibility studies and MTT assay results, as well as hemocompatibility data from the previous literature (Dashti et al. 2024). This range ensured coverage of both low and high nanoparticle exposure levels, representing a broad spectrum of potential clinical doses. The RBC hemolysis test was performed by mixing 1 mL of the nanoparticle suspension with 1 mL of the RBC suspension, halving the nanoparticle concentrations. The mixture was incubated for 12 h at 37 °C in a water bath with constant shaking. Control samples for 100% hemolysis (positive control) and 0% hemolysis (negative control) were prepared by mixing 1 mL of RBC suspension with 1 mL of 1% triton X-100 and isotonic 0.9% NaCl solution, respectively. After incubation, the samples were centrifuged at 4000 RPM for 5 min, and the absorbance of the supernatant was measured at 540 nm using UV–Vis spectroscopy (Gao et al. 2023). The percentage of hemolysis was calculated using the following formula:

$$\text{Hemolysis}\left(\text{\%}\right)=\frac{A_S-A_N}{A_P^{}-A_N}\times 100,$$

where A_S is the absorbance of the sample, A_N is the absorbance of the negative control, and A_P is the absorbance of the positive control. All measurements were conducted in triplicate.

In vitro biocompatibility assessment

NIH-3T3 cells, a widely used fibroblast model, were chosen for the initial biocompatibility assessment of ⁹⁹Tc-(ZIF-8) nanoparticles due to their relevance in cytotoxicity studies and their robustness in modeling nanoparticle interactions with connective tissue cells (Fu et al. 2022; Al-Musawi et al. 2023). While NIH-3T3 cells were used for this initial study, future experiments will expand to other cell lines, including cancerous or organ-specific cells, to further evaluate therapeutic potential and specificity (Salles et al. 2020).

NIH-3T3 cells were cultured in RPMI-1640 medium supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v) FBS. Cultures were maintained at 37 °C in a humidified incubator with 5% CO₂. NIH-3T3 cells (1 × 10⁵ cells/well) were seeded into 96-well plates and incubated for 48 h to allow for growth. After the media was removed, fresh complete media containing ⁹⁹mTc-(ZIF-8) nanoparticles at concentrations of 5, 10, 20, 40, 80, 160, and 320 μg/mL were added. Following 48 h of incubation, the media was aspirated, and 100 μL/well of MTT solution (0.5 mg/mL in PBS) was added. The cells were incubated for an additional 4 h at 37 °C. The formazan crystals formed were dissolved by adding 100 μL of DMSO to each well. The absorbance was measured at 570 nm using a microplate reader (Bio-Rad 550, USA).

Negative controls consisted of cells with only the medium, while positive controls consisted of cells treated with a solubilizing buffer (10% SDS in 0.1 N HCl). Each treatment concentration was tested in triplicate, and the experiments were repeated three times for reproducibility.

Biodistribution and imaging studies

Six rabbits were used for biodistribution and imaging studies, conducted in accordance with the Ethics Committee of Rafsanjan University of Medical Sciences, Rafsanjan, Iran (IR.RUMS.REC.1402.87). During the acclimatization period, the rabbits were housed in wire cages (0.8 × 0.6 × 0.6 m) at room temperature (22 ± 4 °C), with a 12-h light/dark cycle and a relative humidity of 68 ± 5% in the animal facility. They had unrestricted access to food and water throughout the study.

After a 2-week acclimatization period, all rabbits were anesthetized with a ketamine/xylazine solution (25/5 mg/kg) and placed in a specialized chamber. This anesthesia protocol provided adequate sedation and analgesia for the entire imaging session, which lasted approximately 120 min. Vital signs, including heart rate, respiratory rate, and body temperature, were monitored continuously to ensure the animals’ stability. The rabbits were then injected with 0.4 mL of ^99mTc-labeled ZIF-8 nanoparticles (equivalent to 2.4 mCi) via the marginal vein of the ear. SPECT/CT imaging was performed using a Siemens SymbiaT2 system.

Images were taken at 30, 45, 60, 75, 90, 105, and 120 min post-injection. SPECT images were acquired with a matrix size of 256 * 256 and were analyzed by delineating an iso-contour ROI around the target organs. The percentage dose uptake in each organ was calculated by dividing the counts per second per pixel in each organ by the total radioactivity counts.

In vivo toxicity studies

The in vivo toxicity of the nanoparticles was evaluated using Wistar rats, which were divided into two groups of six. The control group received PBS, while the test group received ^99mTc-labeled ZIF-8 nanoparticles. Each rat in the test group received 0.2 mL of labeled nanoparticles (0.75 mCi), administered intravenously via the tail vein, while the control group received 0.2 mL of PBS. Injections were given at 48-h intervals, for a total of five injections per rat.

Ten days after the final injection, the rats were euthanized, and blood samples were collected from the heart. Kidneys and livers were harvested for histopathological examination. The potential side effects of the treatment were evaluated by monitoring changes in the rats’ body weight and by measuring serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine. Histopathological analysis was conducted on the harvested organs to assess any tissue damage or abnormalities.

Statistical analysis

The experimental data were analyzed using GraphPad Prism software version 8.00 (San Diego, CA, USA). The results, including measurements of nanoparticle size, PDI, zeta potential, serum stability, biocompatibility, and biodistribution, were reported as mean ± standard deviation (SD, n = 3). Statistical significance among groups was determined using the ANOVA test. Nonlinear regression analysis was also performed to further interpret the data. A significance level of P < 0.05 was considered statistically significant, ensuring the accuracy and reliability of the results.

Results and discussion

Synthesis and radiolabeling of zeolitic imidazolate frameworks with ^99mTc

In this study, the solvothermal method was employed due to its established use in synthesizing MOFs, allowing for precise control over particle properties by adjusting reaction conditions (Santoso et al. 2021; Ahmadi et al. 2022). The choice of solvent plays a critical role in determining the characteristics of ZIF-8 nanoparticles (Santoso et al. 2021), with methanol emerging as the preferred solvent. Methanol offers several advantages over other solvents, including lower risks and proven effectiveness in producing nanoparticles with desirable sizes and properties (García-Palacín et al. 2020).

In this work, ZIF-8 nanoparticles were synthesized using methanol, which was found to be more suitable than DW for generating nanoparticles with smaller sizes and lower PDI. Methanol’s ability to promote hydrogen-bond donation enhanced molecular interactions between the reagents and solvent, facilitating nanoparticle formation at RT (Santoso et al. 2021). Additionally, the use of methanol resulted in ZIF-8 nanoparticles with a higher surface area, a key factor for drug delivery applications (Malekmohammadi et al. 2019).

Following synthesis, ZIF-8 nanoparticles were radiolabeled with ^99mTc under four different conditions. The use of SnCl₂ as a reducing agent for Na^99mmTcO₄ was critical in achieving high RE% and RCP%. ^99mTc radioisotope was selected due to its accessibility, cost-effectiveness, and favorable gamma emission properties, making it an ideal candidate for efficient nanoparticle labeling (Alberto et al. 2021).

Nanoparticles characterization

DLS analysis provided valuable insights into the size, PDI, and zeta potential of the radiolabeled nanoparticles (^99mTc-ZIF-8). The average size of the nanoparticles was 198 nm (198 ± 9.8 nm), an ideal size for cellular uptake and systemic distribution (Zhang et al. 2023). Moreover, the low PDI value (0.219 ± 0.011) indicated a narrow size distribution, which is essential for consistent therapeutic performance and biodistribution (Kumar 2021).

The zeta potential of 33 ± 1.8 mV suggested good nanoparticle stability due to electrostatic repulsion, preventing particle aggregation and promoting prolonged circulation time in biological systems (Alavi et al. 2022).

AFM and SEM analyses confirmed the spherical morphology and monodispersity of the ^99mTc-ZIF-8 nanoparticles (Fig. 1a and b), consistent with previous studies (Falsafi et al. 2020). The monodispersity observed in AFM images aligned with the DLS results, further confirming the uniform size distribution of the nanoparticles. The nanoscale dimensions observed through AFM and SEM are advantageous for cellular uptake, supporting efficient intracellular drug delivery.

Fig. 1.

a Atomic force microscopy (AFM) and b Scanning electron microscopy (SEM) of ZIF-8 nanoparticles. The figures (a and b) illustrate that monodisperse spherical nanoparticles with smooth surfaces were formed. c Serum stability of ^99mTc-(ZIF-98). The results are expressed as mean ± SD from three independent experiments

Moreover, the smooth surface morphology detected in both AFM and SEM images suggests favorable interactions with biological membranes, facilitating cellular uptake and the potential for intracellular drug release (Xue et al. 2018). These characteristics position ^99mTc-ZIF-8 nanoparticles as promising candidates for drug delivery and imaging applications.

Serum stability of.^99mTc-(ZIF-8)

The stability of ^99mTc-labeled ZIF-8 nanoparticles was evaluated in freshly prepared human serum over a 24-h period to confirm the retention of the ^99mTc label without significant loss. The findings, which are consistent with the imaging data, indicate that ^99mTc-(ZIF-8) nanoparticles maintain their stability, making them suitable for scanning applications. The results of this investigation are shown in Fig. 1b, further demonstrating the nanoparticles’ potential for biomedical imaging.

Red blood cell (RBC) hemolysis

RBCs, comprising 40–50% of blood volume, are vital for physiological functions. Biomedical materials, particularly those used intravenously, interact with RBCs, making hemocompatibility an essential safety measure (Avsievich et al. 2022). Ensuring hemocompatibility is a prerequisite for advancing any drug candidate to further studies, including biodistribution and pharmacokinetics evaluation (Phillips et al. 2019).

The RBC hemolysis test, a standard method for assessing the safety of nanoparticles in proximity to RBCs, evaluates the potential damage to RBC membranes and the release of hemoglobin (Dashti et al. 2024). The hemocompatibility of ^99mTc-(ZIF-8) nanoparticles was assessed using UV–Vis spectroscopy to determine hemoglobin release after incubation with nanoparticles (Gao et al. 2023). As shown in Fig. 2a, the nanoparticles demonstrated complete safety at all tested concentrations during the 12-h incubation, with a hemolysis rate of less than 5%. However, a slight increase in hemolysis was observed at higher concentrations, warranting careful consideration of nanoparticle dosing in future studies.

Fig. 2.

a Hemolysis rate (%) for ^99mTc-(ZIF-8) nanoparticles compared to the control group at concentrations of 10, 20, 100, 200, and 300 µg/mL; b Effect of ^99mTc-(ZIF-8) nanoparticles on NIH-3T3 cell viability, compared to the control group, measured by MTT assay after 48 h of incubation. Data are presented as mean ± SD (n = 3)

In vitro biocompatibility assessment

Nanoparticles intended for biomedical applications must exhibit good biocompatibility, meaning that they can perform their intended functions without causing adverse biological reactions. The cytotoxicity of nanoparticles is influenced by factors, such as size, concentration, incubation time, and cell type (Zafar et al. 2019; He et al. 2024). In this study, the biocompatibility of ^99mTc-(ZIF-8) nanoparticles was evaluated using NIH-3T3 cells and the MTT assay, a widely accepted method for assessing cytotoxicity and cell viability (Table 1).

Table 1.

Radiolabeling efficiency (RE%) and radiochemical purity (RCP%) of ^99mTc-(ZIF-8) nanoparticles using four different conditions

Condition	T (°C)	SnCl2 concentration (mg/mL)	RE (%)	RCP (%)
1	RT	15	42.3 ± 2.8	57.6 ± 3.9
2	65	15	95.2 ± 1.9%	96.1 ± 1.7%
3	RT	–	8.2 ± 0.4%	14.2 ± 0.7%
4	65	–	-	-

RT room temperature

The results, depicted in Fig. 2b, showed that at concentrations up to 40 μg/mL, the nanoparticles maintained cell viability above 70% compared to the control group, confirming their safety and minimal toxicity at these concentrations. However, as the nanoparticle concentration increased, a dose-dependent decrease in cell viability was observed, highlighting the importance of cautious dosing to mitigate potential cytotoxic effects on normal cells (González-Vega et al. 2022).

Biodistribution and imaging studies

The biodistribution of ^99mTc-(ZIF-8) nanoparticles was evaluated using SPECT imaging in rabbits following intravenous administration (Fig. 3, Table 2). The results indicated that the highest radiotracer accumulation occurred in the bladder, while the heart tissues exhibited the lowest accumulation over the time points observed (bladder > liver > kidney > heart). On average, radiotracer accumulation in the bladder was 6.3, 7.2, and 36.2 times higher than in the liver (p < 0.0001), kidney (p < 0.0001), and heart (p < 0.0001), respectively. The increasing nanoparticle accumulation in the bladder over time can be attributed to renal clearance.

Fig. 3.

SPECT images of ^99mTc-(ZIF-8) nanoparticles were captured at various intervals (30, 45, 60, 75, 90, 105, and 120 min) following intravenous administration in rabbits, using a gamma camera. The results are presented as the mean ± 3SD

Table 2.

Biodistribution percentage of ^99mTc-(ZIF-8) nanoparticles in different organs of rabbits at different times post-injection of the nanoparticles (30, 45, 60, 75, 90, 105, and 120 min)

Organ/time (min)	heart	liver	kidney	Bladder
30	1.4	7.3	6.3	39.6
45	1.3	7.2	6.2	45.3
60	1.2	7.1	6.1	48.3
75	1.2	6.9	6	51.1
90	1.2	6.7	5.9	44.1
105	1.1	6.5	5.8	39.1
120	1	6.4	5.7	36.1

The small size of the nanoparticles (198 ± 9.8 nm) and their surface properties allow them to be efficiently filtered by the kidneys and excreted through the urine, minimizing their retention in other organs such as the liver and heart. The rapid renal excretion of the nanoparticles suggests that they may be favorable for applications where quick clearance is desired to reduce long-term toxicity and off-target effects. These biodistribution patterns highlight the potential of ^99mTc-(ZIF-8) nanoparticles for targeted imaging applications.

Initially, the radiotracer showed high activity in the kidneys, which gradually decreased as bladder activity increased. The observed decrease in bladder radioactivity at the 90-min mark suggests that the rabbit had voided, confirming renal excretion as the primary clearance route. The significant accumulation of ^99mTc-(ZIF-8) nanoparticles in the bladder demonstrates their high clearance rate, making them suitable for applications that require rapid elimination from the body.

In vivo toxicity studies

To comprehensively evaluate the safety profile and potential side effects of ^99mTc-(ZIF-8) nanoparticles, several parameters were assessed, including changes in body weight, serum markers for liver and kidney function (ALT, AST, BUN, and creatinine), and histopathological examinations of tissues.

Monitoring the body weight of the animals throughout the study revealed no significant deviations from the control group, indicating that the ^99mTc-(ZIF-8) nanoparticles did not adversely affect weight gain or cause notable toxicity (Fig. 4). This suggests that the nanoparticles are well tolerated and non-toxic at the administered doses.

Fig. 4.

Weight changes of Wistar rats treated with ^99mTc-(ZIF-8) nanoparticles compared to the PBS (control) group. The results are expressed as mean ± 3SD

Serum markers were analyzed to evaluate hepatic function (ALT and AST) and renal function (BUN and creatinine). The results showed no significant increases in these markers in the ^99mTc-(ZIF-8) nanoparticle-treated group compared to the control group, indicating that the nanoparticles did not induce liver or kidney toxicity in the treated rats (Fig. 5). These findings further support the safety of the nanoparticles for in vivo applications.

Fig. 5.

Serum levels of (i) ALT, (ii) AST, (iii) BUN, and (iv) creatinine in Wistar rats treated with ^99mTc-(ZIF-8) nanoparticles compared to the PBS (control) group. The data in the figure show that the concentrations of these parameters in the ^99mTc-(ZIF-8) nanoparticle group were comparable to those in the control group and fell within the normal range, suggesting that the nanoparticles did not induce any noticeable toxicity to the kidneys and liver of Wistar rats. The results are expressed as mean ± 3SD

Histopathological examinations were performed on the liver and kidney tissues of treated rats to identify any potential adverse effects caused by the nanoparticles. No histopathological lesions or abnormalities were observed in the nanoparticle-treated group compared to the control group (Fig. 6). The absence of tissue damage or inflammation further confirms the biocompatibility and safety of the ^99mTc-(ZIF-8) formulations.

Fig. 6.

Histopathological impacts of ^99mTc-(ZIF-8) nanoparticles were examined in comparison to PBS (control) in the rats. Consistent with the serum marker analysis (ALT, AST, BUN, and creatinine), the liver and kidney tissues of rats treated with ^99mTc-(ZIF-8) nanoparticles displayed normal histopathology (Magnification × 10, scale bar 100 µm)

These findings collectively suggest that ^99mTc-(ZIF-8) nanoparticles exhibit a favorable safety profile, with no observable toxicity in key organs or serum markers. This supports their potential for future biomedical applications, particularly in imaging and drug delivery.

Conclusion

In this study, we successfully synthesized and radiolabeled ZIF-8 nanoparticles with the ^99mTc radioisotope using a solvothermal method in a methanolic medium. The use of methanol as the solvent was pivotal in achieving nanoparticles with desirable characteristics, such as smaller size, lower PDI, and a higher surface area. This method, combined with the use of SnCl₂ as an effective reducing agent, resulted in high RE% and RCP%, making the ^99mTc-(ZIF-8) nanoparticles suitable for biomedical applications.

Characterization of the nanoparticles using DLS, AFM, and SEM confirmed their nano-sized dimensions, uniform particle distribution, and spherical morphology with smooth surfaces. These properties are particularly advantageous for drug delivery applications, promoting efficient cellular uptake and intracellular drug release. Additionally, the serum stability of ^99mTc-(ZIF-8) and their low hemolysis rates further highlight their potential as safe and effective drug delivery systems.

In vitro biocompatibility assessments using NIH-3T3 cells demonstrated minimal toxicity at concentrations up to 40 μg/mL, with cell viabilities remaining above 70%. This indicates that ^99mTc-(ZIF-8) nanoparticles are biocompatible and safe for use in biological systems, provided that dosing is carefully managed. Biodistribution studies in rabbits revealed that the nanoparticles primarily accumulated in the bladder, indicating efficient renal clearance, which is beneficial in minimizing long-term tissue accumulation and reducing potential toxicity.

Future research should focus on further exploring the in vivo therapeutic potential of ^99mTc-(ZIF-8) nanoparticles in disease models, particularly their use as drug delivery vectors for targeted therapies. Optimization of the synthesis and radiolabeling processes could further enhance their properties, such as improving targeting efficiency and enabling controlled release mechanisms. The addition of targeting ligands or functional moieties on the nanoparticle surface could be investigated to improve specificity and reduce off-target effects. Overall, the positive results from this study provide a solid foundation for the development and application of radiolabeled MOFs in targeted imaging and therapeutic interventions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

BA: conceptualization, investigation, formal analysis, and writing—original draft. SA: methodology, and writing—review and editing. ZMZ: conceptualization, resources, and writing—review and editing. HES: supervision, resources, and writing—review and editing. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

The original data are available upon reasonable request to the corresponding author.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Informed consent

All authors contributed to the discussing the results and commenting on the manuscript.