1. Introduction
Air pollution has aroused widespread public health concerns in China with the rapid growth of the economy and industry. According to the Global Health Observatory data from the WHO, almost seven million people are killed by air pollution every year[1,2]. Among them, particulates with aerodynamic diameters of less than 2.5 μm (PM 2.5), which comprise an air-suspended mixture of solid and liquid particles, have become the most criticized pollutant, attracting considerable attention. These particulates can penetrate human lungs and cause many adverse health effects, such as asthma, pneumonia, stroke, chronic bronchitis, and arrhythmia[3-5]. In addition, exposure to a polluted atmosphere for a prolonged period of time is associated with a high incidence of stroke and impaired cognitive function[6]. When the level of PM 2.5 increases by 10 μg/m3 in one day from standard levels, hospital admissions for respiratory disease increase by 4% to 14%[7-9]. It is estimated that the contribution of air pollution to premature mortality might double by 2050[10].
To date, various studies concerning the effects of PM 2.5 on health have been conducted. For example, PM 2.5 may contribute to systemic oxidative stress and damage mammalian cells, which is considered an essential molecular mechanism of PM 2.5-mediated toxicity[11,12]. Numerous organic chemicals coated on PM 2.5 can produce or increase levels of intracellular reactive oxygen species, which have been identified as signaling molecules in various pathways regulating cell survival and death [13,14]. In addition, PM 2.5 alters the expression of antioxidant enzymes, including superoxide dismutase and catalase, and decreases their activities [15,16]. PM 2.5 may also lead to global DNA hypomethylation, P16 gene promoter hypermethylation, and decreased DNA methyltransferase activity in normal subjects as well as notably chronic obstructive pulmonary diseased cells [17-20]. The presence of PM 2.5 can also cause local and systemic inflammation [21-23]. Although these findings from in vitro cell and ex vivo animal experiments are encouraging, there remains a lack of a strategy for systematically and comprehensively evaluating the in vivo behaviors of the particles. To better protect ourselves from PM 2.5, its characteristics, such as biodistribution, metabolic activation, excretion, toxicity, and cellular responses, need to be better understood.
Molecular imaging technology is a growing biomedical research discipline that enables the visualization of physiological or pathological processes in living subjects as well as the quantification of biological processes at the cellular level [24,25]. Compared with traditional ex vivo studies, molecular imaging allows numerous in vivo experiments to occur repeatedly in the same animal, which markedly reduces costs and shortens the length of time of the experiments. Due to low instrumentation and radionuclide costs, single photon emission tomography (SPECT) is widely used clinically for noninvasive particle detection [26,27].
Radio-halogen 131I is a theranostic radioactive isotope used in nuclear medicine [28,29]. For example, 131I-labeled metaiodobenzylguanidine has been applied in the treatment of neuroendocrine tumors. As well as therapy, radioiodine can be used for SPECT imaging by emitting high-energy gamma radiation with more extended half-life periods (T1/2 = 8.3 days). Radioiodine is commercially available in China [15]. Due to its complex composition, it is difficult to label neutral PM 2.5 particles with 131I. However, the particles are easy to assemble because of their low surface hydrophobicity. SiO2 particles are a reasonable substitute because their diameter is similar to that of PM 2.5 [30]. In addition, the interior hollow-porous (approximately 5 nm aperture) section is convenient for the adsorption of nuclides. To avoid accumulation, the SiO2 particles are modified with polyvinyl pyrrolidone (PVP) or ethyl cellulose (EC). PVP is a water-soluble macromolecule commonly used in pharmaceutics. Coating PVP on the surface of SiO2 particles is beneficial for increasing the hydrophilicity and dispersion of the particles. EC is another water-insoluble but ethanol-soluble polymer commonly used in pharmaceutical preparations. To better evaluate the in vivo properties, the modified SiO2 nanoparticles were labeled with 131I. After administration of radiolabeled particles to rodents via inhalation, the performance of the radiolabeled compounds, including biodistribution, retention, and metabolism, was determined using SPECT imaging. A schematic illustration of the process is provided in Fig. 1.
2. Methods and Materials
2.1 Materials
All commercial reagents were of analytical grade. Sprague Dawley (SD) rats were purchased from the Shanghai Laboratory Animal Co. (SLAC), Ltd., China. The 131I sodium iodide solution was obtained from the Chengdu Gaotong Isotope Corporation (China Nuclear Group). Dialysis bags (Amicon centrifugal filter device, MWCO = 30 kDa) were purchased from Merck Millipore, Germany. Phosphate-buffered saline (PBS) was obtained from Sangon Biotechnology (Shanghai, China). PVP and EC were purchased from Sigma-Aldrich. SiO2 nanoparticles were gifted from China Pharmaceutical University. All reagents were used without further purification.
2.2 Modified SiO2 nanoparticles with polyvinyl pyrrolidone (PVP/SiO2)
The SiO2 nanoparticles modified with PVP (PVP/SiO2) were prepared according to a previously reported procedure [31]. In brief, 100 mg PVP in 2.5 mL ethanol was mixed with SiO2 nanoparticles (200 mg) at 25 ℃ for 2 h. Then, the mixtures were dried at 140°C to remove residual ethanol. After milling with a pneumatic cracker, PVP/SiO2 nanoparticles were obtained. The morphological characteristics of the products were determined by scanning electron microscopy (SEM) and dynamic light scattering (DLS).
2.3 Modified SiO2 nanoparticles with ethyl cellulose (EC/SiO2)
To prepare EC/SiO2, ethylene carbonate (500 mg) and SiO2 nanoparticles (200 mg) were mixed at room temperature for 2 h in the presence of 2 mL ethanol. Then, the mixtures were dried at 140°C to remove residual ethanol. After milling with a pneumatic cracker, EC/SiO2 nanoparticles were obtained. The morphological characteristics of the products were determined by SEM and DLS.
2.4 Preparation of [131I]-EC/SiO2 nanoparticles
A 2 mL Na131I (3700 MBq) solution was mixed with 500 mg of EC/SiO2 nanoparticles in 1 mL of ethanol and 1 mL water at room temperature for 240 h. The resulting complexes were then placed in dialysis tubing and dialyzed in deionized water, which was replaced with fresh water every 24 h. The radioactivity in the dialysis bag was monitored using a radioactivity meter (CAPINTEC. INC CRC-25). After removing free 131I, [131I]-EC/SiO2 nanoparticles were obtained by evaporating the solvents in a vacuum drying oven (DZF-6053 YiHeng, Shanghai) at 50°C.
2.5 In vitro stability of [131I]-EC/SiO2 nanoparticles
The in vitro stability of 131I-labeled EC/SiO2 particles was determined by incubating the radiolabeled compound in PBS and human plasma at 37°C. The purified [131I]-EC/SiO2 (200 μL, 3.7 MBq, 0.5 mg, respectively) was placed in a dialysis bag (MWCO = 10 K) then suspended in 20 mL of plasma or PBS with magnetic stirring. At the selected time intervals (2, 4, 8, 12, and 24 h), 0.5 mL dialyzate was removed to calculate the radioactivity using a PerkinElmer 1470 γ-counter.
2.6 Inhalation administration
All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China’s requirements. The Animal Study Committee of the Jiangsu Institute of Nuclear Medicine approved the experiments. The rats were routinely screened for common rat pathogens and housed in specific pathogen-free facilities under standard conditions (24 ± 2°C, 50 ± 5% humidity) with a 12 h light/dark cycle and food and water available ad libitum.
To ensure the best image quality from SPECT, the [131I]-EC/SiO2 particles were administrated using a non-surgical intratracheal installation method.[32] Briefly, five SD rats (SLAC Laboratory Animal Co., Ltd, China; 6-8 weeks old, 200-300 g) were anesthetized via an intraperitoneal injection of pentobarbital. A small animal laryngoscope was used throughout the process. A ball tipped needle was maneuvered through the epiglottis, after which contact with the tracheal rings provided confirmation that the needle was inside the trachea. Then, an injector containing 0.2 mL of [131I]-EC/SiO2 (111 ± 18.5 MBq) was inserted into the ball tripped needle. After gently instilling the substance into the trachea, the animal was maintained in an upright position for 2 min to allow the fluid to drain into the respiratory tree.
2.7 SPECT imaging
After administration via inhalation, the rats were placed in a prone position using a handmade holding device under anesthetization. SPECT imaging was performed at 6 and 24 h post-administration using a Philips Skylight SPECT fitted with a high-energy pinhole collimator[33]. Static images (10 min) of the animals were obtained with a 256 × 256 matrix and 16 bits pixel depth. After imaging, the SPECT data were reconstructed using an ordered-subset expectation maximization algorithm.
Semi-quantitative assessment was performed by calculating the target-to-background ratio (TBR). The lungs, liver, and kidneys were considered the target areas, and the blood pool was considered the background area.
2.8 Ex vivo autoradiograph
After SPECT imaging, rats were euthanized with excessive amounts of isoflurane and dissected. The lungs were sectioned, and the slices were laid on X-film in a darkroom and imaged on a Cyclone Plus Storage Phosphor system (PerkinElmer, USA). Region of interest analysis of ex vivo images was carried out to compare uptake in the lobus, lobus medius, and lobus inferior. Quantitative [131I]-EC/SiO2 uptake was determined by gamma counting of two serially sectioned lung tissue specimens.
2.9. Postmortem examinations
For hematoxylin and eosin (HE) staining, the rats were euthanized after SPECT imaging and their tissues, including heart, liver, spleen, kidneys, and lungs, were harvested and fixed in 4% formalin solution at room temperature for 48 h. The tissues were then embedded in paraffin blocks and sectioned into slices with a thickness of 4 µm. The tissue sections were then mounted onto glass slides and stained with HE. The stained sections were examined using an optical microscope.
2.10 Statistical analysis
Statistical analyses were performed using GraphPad Prism software. Data were analyzed using the unpaired, 2-tailed Student’s t-test. Differences at the 95% confidence level (p < 0.05) were considered statistically significant.
3. Results and Discussion
3.1 Characterization of modified SiO2 nanoparticles
As shown in Fig. 2, the average size of the unmodified SiO2 particles was approximately 3.5 μm. The PVP/SiO2 and EC/SiO2 nanoparticles were both well-dispersed in water. DLS showed that the uniform sizes of PVP/SiO2 and EC/SiO2 nanoparticles were approximately 3 and 1.2 μm, respectively (Fig. 2). The findings of DLS and SEM revealed that both PVP/SiO2 and EC/SiO2 nanoparticles possessed spherical shapes with diameters of nearly 3 and 1.2 μm, respectively (Fig. 2). Since the diameter was larger than 2.5 μm, PVP/SiO2 particles may not be applicable for the following studies.
In addition, considering the water solubility of PVP, PVP/SiO2 nanoparticles may be unstable in vivo. On the contrary, EC/SiO2 nanoparticles can be applied to mimic PM 2.5 particles because EC has been widely used in biomedicine and life sciences.
3.2 Preparation of 131I-labeled EC/SiO2 nanoparticles
The EC/SiO2 nanoparticles were further labeled with 131I to investigate the characteristics of PM 2.5 particles. To determine whether the diameter of the [131I]-EC/SiO2 nanoparticles met the requirements to mimic PM 2.5 for the in vivo study, non-radioactive iodine was first adsorbed on EC/SiO2 nanoparticles. The particle size was found to be smaller than 2.5 μm (Fig. 3a). After radiolabeling with 131I, the radiolabeling yield and stability were measured. The labeling yield gradually decreased because of the dissociation of free 131I from the 131I-EC/SiO2 nanoparticles. After dialyzing for 168 h, the labeling yields of [131I]-EC/SiO2 nanoparticles remained constant (~30%), and no further free 131I was found outside the dialysis bag (Fig. 3b). This indicated that 131I was successfully labeled in the EC/SiO2 nanoparticles. Less than 3% 131I was released from the [131I]-EC/SiO2 after 24 h of incubation in PBS or serum (Fig. 3c). This implied that 131I can be easily labeled on the EC/SiO2 nanoparticles, and [131I]-EC/SiO2 nanoparticles exhibit high radiostability in vitro, indicating that the particles could be further applied to tracing PM 2.5 in vivo.
3.3 Inhalation and in vivo SPECT imaging
We analyzed the characteristics of PM 2.5 via molecular imaging. The in vivo biodistribution of [131I]-EC/SiO2 nanoparticles, the "mimic PM 2.5," was further investigated. The [131I]-EC/SiO2 nanoparticles were administered to the rats (n = 5) by endotracheal intubation. The inhaled radio dose of [131I]-EC/SiO2 was 111 ± 18.5 MBq, with a total mass of 300 mg.
SPECT imaging was performed at 6 and 24 h post-administration. After 6 h of administration, SPECT images showed that the TBR of the lungs was 11.85 ± 3.90 (Fig. 4). At the same time, the TBRs of the liver, kidneys, and bladder were 7.27 ± 0.72, 2.56 ± 0.24, and 0.76 ± 0.05, respectively. After 24 h of administration, the TBRs of the lungs and bladder were 2.33 ± 0.86 and 5.33 ± 1.63, respectively.
In vivo SPECT imaging showed that the radiotracer was mainly located in the lungs, which was in accordance with the finding that the lungs are the target organ of PM 2.5 [21,30,18]. Radioactivity was also identified in other healthy organs, such as the heart, kidneys, and liver. It appears that PM 2.5 could penetrate human body tissue. Delayed images at 24 h post-administration showed that the particles were mostly eliminated from the urinary passage. Moreover, moderate radio signals appeared in the abdomen, especially in the intestine. This implies that particles might also be excreted via feces.
Molecular imaging technology, including PET and SPECT, has been widely used clinically for tumor diagnosis, cancer staging, and therapeutic response monitoring. This is the first time, to our knowledge, that molecular imaging technology has been used for the visualization of PM 2.5 particles in vivo. Image-based quantitation of PM 2.5 generated by SPECT also enabled real-time noninvasive data analysis. This study provides accurate measurements of PM 2.5.
However, limitations of this study still exist. The association between the adverse effects of air pollution and cardiovascular and respiratory health as well as cognitive functioning has been well-documented [7,34,35], but this study did not investigate these features. In addition, air pollution is multifaceted, as it comprises numerous environmental toxins [31]. However, in the present study, the mimic PM 2.5 particles were composed of only one component. Meanwhile, the long-term effects were not evaluated. In addition, the thyroid of the SD rats was not blocked, which resulted in mild uptakes into the thyroid. All these challenges will be solved in future with further research.
3.4 In vivo autoradiograph and HE staining
To verify the SPECT results, ex vivo autoradiography and HE staining were conducted. After SPECT imaging, rats were euthanized and autoradiographs were performed. Exposure to the phosphor imaging plates for 12 h provided a favorable autoradiographic signal-to-noise ratio. It was found that a higher density of radioactivity was distributed in the inside fields of the lungs (red areas)(Fig. 4e) than in the outside fields of the lungs(black areas)(Figs. 4c,d). Particles were primarily localized in the trachea (red areas; Figs. 4c,d,e), which was consistent with the results of SPECT imaging.
Briefly, for HE staining, the lung tissues of [131I]-EC/SiO2-inhaled rats and normal rats were collected, sliced, and stained with HE to assess the PM 2.5-induced injury. The lung tissues of the normal rats exhibited an intact structure (Fig. 4f). The alveolar space was bright, and there was no edema in the alveolar septum. However, increased exudates, congestion in the pulmonary alveolus, and collapse, rupture, and fusion of the pulmonary alveoli were observed in the lung tissues of the [131I]-EC/SiO2-inhaled rats. Compared with the normal group, the thickness of the alveolar septum significantly increased. PM 2.5 particles may cause serious lung injury.
4. Conclusion
In summary, we successfully developed a novel molecular imaging measurement method for PM 2.5 distribution and metabolism detection in SD rats. EC and PVP were used to screen for the most favorable substitute. EC/SiO2 exhibited a smaller size and a uniform spherical shape. EC/SiO2 not only possesses the appropriate particle size but can also be successfully labeled on 131I with high stability. SPECT imaging showed the biodistribution of the "mimic PM 2.5" after administration via inhalation. Subsequently, the autoradiograph and HE-stained images were consistent with SPECT imaging results. Therefore, we conclude that this molecular imaging method could be a novel and effective technique for the detection and visualization of PM 2.5 metabolism. The preclinical studies were satisfactory, and further studies are currently being undertaken.
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