PET Tracing of Biodistribution for Orally Administered 64Cu-Labeled Polystyrene in Mice

Visual Abstract

Mi croplastics with diameters of less than 5 mm are recognized as a new environmental threat and human health risk (1). Microplastics have been observed to accumulate in many different marine animals, including fish (2)(3)(4)(5), copepods (6,7), mussels (8)(9)(10), European flat oysters (11), and others (12)(13)(14). Fiber-type microplastics have been found in mussels purchased at markets in Belgium (15). Considering that microplastics are widely detected in food, we can assume that microplastics are ingested along with the contaminated food. Therefore, it is highly likely that human consumption of microplastics is widespread. To understand the full significance of microplastic ingestion, the absorption path for microplastics ingested with foods needs to be visualized.
PET imaging is a powerful tool for observing absorption, distribution, metabolism, and excretion (16). PET can also be used to visualize the in vivo distribution of toxic substances labeled with radioactive isotopes, including diesel exhaust (17), and inhaled aerosols of toxic household disinfectants (18). Figure 1 shows a schematic of the study. We first identified the absorption path and distribution of microplastics using PET. Microplastic polystyrene was labeled with 64 Cu ([ 64 Cu]Cu, to yield [ 64 Cu]Cu-DOTA-polystyrene) and then was orally administered to mice. In a separate experiment, 64 Cu was orally administered as a control to assess the effects of the harsh stomach conditions on dechelated 64 Cu. PET was performed to monitor the absorption and distribution of [ 64 Cu]Cu-DOTA-polystyrene or 64 Cu over 48 h. The ex vivo biodistributions of [ 64 Cu]Cu-DOTA-polystyrene or 64 Cu was measured. Ex vivo tissue radio-thin-layer chromatography (TLC) was performed to identify whether g-rays emitted from the tissue originated from [ 64 Cu]Cu-DOTA-polystyrene or from 64 Cu.

Synthesis and Radiolabeling
To 300 mL of 0.1 M sodium carbonate buffer (pH 9.0), 2.5 mg of amino-polystyrene (0.2-0.3 mm; Spherotech) were added. Then, 260 mg (471.70 nmol) of S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) in 50 mL of deionized water were added, and the mixture (pH 9.0) was shaken at 1,000 rpm and 25 C for 20 h. Unconjugated p-SCN-Bn-DOTA was removed using an Amicon centrifugal filter (30-kDa cutoff; Millipore). DOTA conjugation was confirmed using Fourier-transform infrared spectroscopy (Nicolet iS5; Thermo Fisher Scientific), and the resulting spectra were analyzed using Omnic software from Nicolet Instrument Corp. To determine moles of DOTA per milligram of plastic, 50 mL of filtrate were analyzed by high-performance liquid chromatography (Waters). The quantity of DOTA in the filtrate was calculated from a standard curve (prepared from an analysis of known concentrations of DOTA). The conjugated moles of DOTA to polystyrene were then calculated by subtracting the moles of DOTA in the filtrate from the total moles of DOTA for the reaction. Physicochemical characterization of DOTA-polystyrene was performed using a field-emission scanning electron microscopy and dynamic light scattering. Concentrated DOTA-polystyrene was subsequently buffer-exchanged to isotonic buffered saline for subsequent radiolabeling. The final concentration before radiolabeling was 2.5 mg/100 mL.
Cyclotron-produced [ 64 Cu]CuCl 2 was dried and redissolved in 0.01 N HCl (final concentration, 9.25 MBq/mL). In a 1.5-mL tube, 155.4 MBq of [ 64 Cu]CuCl 2 were added to 80 mL of 0.1 M NaOAc buffer (pH 5). DOTA-polystyrene (2 mg in 80 mL) was added, and the mixture was shaken in a Thermomixer C (Eppendorf AG) at 40 C and 1,000 rpm for 30 min. 64 Cu-labeled DOTA-polystyrene was purified using an Amicon centrifugal filter at 25 C, 3,000 rpm, for 30 min. By repeating this procedure, reaction buffer was exchanged to 1 3 phosphate-buffered saline for further studies.

In Vitro Stability Study
[ 64 Cu]Cu-DOTA-polystyrene in phosphate-buffered saline (1.85 MBq/30 mL) was diluted in 270 mL of phosphate-buffered saline, hydrochloric acid-potassium chloride buffer (pH 2), human serum, or mouse serum. Each sample was incubated at 25 C (buffer) or 37 C (serum) for 48 h. Percentage stability was analyzed using instant TLC (0.1 M citric acid in water as a mobile phase).

PET/CT
All animal experiments were performed under the institutional guidelines of the Korea Institute of Radiological and Medical Sciences. BALB/ c nude mice (n 5 5-7, 5 wk old; Shizuoka Laboratory Center) were used.
PET/CT images were acquired with an Inveon PET scanner (Siemens Medical Solutions). [ 64 Cu]CuCl 2 (4.81 MBq/100 mL) or [ 64 Cu]Cu-DOTA-polystyrene (4.81 MBq/57.8 mg/100 mL) was orally administered to the mice. PET was performed at 1, 6, 12, 24, and 48 h afterward. The PET data were acquired for 15 min within 350-650 keV and were reconstructed using a maximum a priori with shifted Poisson distribution (SP-MAP) algorithm (target resolution 3). The voxel size was 0.776 3 0.776 3 0.796 mm. Regions of interest were drawn in the stomach, liver, and intestine using ASIpro (Siemens Medical Solutions) after coregistration of CT and PET images. SUV max was then calculated.

Biodistribution Study
The accumulated radioactivity concentration (percentage injected dose [%ID]/g) in each organ was measured at corresponding times after administration of [ 64 Cu]Cu-DOTA-polystyrene or 64 Cu.

Ex Vivo Radio-TLC
Ex vivo radio-TLC assays were performed to determine whether the detected g-rays emitted from the tissues were emitted from 64 Cu or from [ 64 Cu]Cu-DOTA-polystyrene at each time point. Homogenized samples were lysed in 10% sodium dodecyl sulfate phosphatebuffered saline (pH 7.4) instead of strong acid because low pH (#1) induces dechelation of 64 Cu from DOTA within 1 min (19). Similarly, a low pH in the stomach can disrupt stable chelation of [ 64 Cu]Cu-DOTA, and this phenomenon was identified from ex vivo radio-TLC of the stomach at later time points.

Statistical Analysis
The data are presented as the mean with SD. The Student t test was performed using Prism (version 5.0; GraphPad).

Ex Vivo Radio-TLC
The ex vivo radio-TLC assay results for other tissues (liver, small, and large intestine) demonstrated that the radiation signal was from [ 64 Cu]Cu-DOTA-polystyrene, not from 64 Cu (Supplemental Fig. 4). DISCUSSION We first identified the in vivo distribution of microplastics in mice by labeling microplastic polystyrene with the radioisotope 64 Cu, orally administering [ 64 Cu]Cu-DOTA-polystyrene (radiolabeled microplastic polystyrene) to mice, and using PET to trace its absorption and distribution. Next, ex vivo biodistribution studies confirmed [ 64 Cu]Cu-DOTA-polystyrene accumulation in specific organs. Ex vivo radio-TLC was used to confirm that the detected g-rays originated from [ 64 Cu]Cu-DOTA-polystyrene. Exposure to microplastics in food and water through oral administration is a significant environmental and health problem (21)(22)(23). However, it is extremely likely that microplastics are widely distributed within the food we eat.
The advantage of PET is that it is possible to observe the in vivo absorption, distribution, metabolism, and excretion of substances labeled with radioactive isotopes without killing the animal (16). Although fluorescence is commonly used for in vivo exposure and biodistribution studies, fluorescence in animal bodies can be absorbed by bone and soft tissues, and prolonged exposure to ultraviolet light can result in bleaching and loss of fluorescence intensity (24). Therefore, quantification of fluorescent images is limited, compared with PET images. In addition, when microplastic-conjugated fluorescent dyes are used, animals must be killed to observe the absorption and accumulation of the microplastics over time. [ 64 Cu]Cu-DOTA-polystyrene transit and absorption were observed within the same animal using PET, without killing the animal.
In this study, we first observed the in vivo pathways (absorption, distribution, metabolism, and excretion) of microplastics labeled with a radioisotope using PET. To trace the polystyrene after oral administration, we selected 64 Cu and p-SCN-Bn-DOTA for the radiolabeling of plastic particles. We subsequently confirmed that the detected radiation was emitted from the [ 64 Cu]Cu-DOTApolystyrene, not from 64 Cu, using ex vivo radio-TLC. DOTA-Nhydroxysuccinimide ester and p-SCN-Bn-DOTA are frequently used chelators (19). DOTA conjugation was confirmed by Fouriertransform infrared spectroscopy, because the functional groups of DOTA show specific bands (Supplemental Fig. 1).
The biodistribution study also demonstrated that the distribution of [ 64 Cu]Cu-DOTA-polystyrene was different from that of 64 Cu. The biodistribution study provided quantification of [ 64 Cu]Cu-DOTA-polystyrene accumulation in each organ, even at low levels of emitted g-rays. Using the biodistribution, we observed the transit and accumulation of [ 64 Cu]Cu-DOTA-polystyrene within the gastrointestinal tract (stomach, intestine, and liver), circulatory organs (heart, lung, and blood), renal system (kidney and bladder), and even brain, at 1 h after oral administration.
In contrast, orally administered 64 Cu was rapidly removed from the stomach, small intestines, and large intestine, before transit to the other organs, including the liver (Fig. 4). We also observed a higher SUV in the liver on PET for the group that was orally administered 64 Cu (Fig. 3). In a previous report, accumulation of 64 Cu in the liver was observed on PET (20). For kidney and spleen, the levels of ID/g (1 , %ID/g , 10) at 1 h were 3.47 and 1.08, respectively. For bladder, testis, heart, lung, and blood, the levels of ID/g (%ID/ g , 1) at 1 h were 0.70, 0.22, 0.55, 0.92, and 0.21, respectively. The rapid distribution of orally administered 64 Cu to the other organs may have occurred because digestive fluid may facilitate solubilization of 64 Cu in the stomach. 64 Cu was partly cleared in feces after transit through the gastrointestinal tract, and the remaining 64 Cu was distributed to other organs, including the liver.
In mice, the normal gastric pH is approximately 3.0 (25). During transit through the stomach, [ 64 Cu]Cu-DOTA-polystyrene may encounter harsh conditions, possibly dechelating 64 Cu. However, our ex vivo radio-TLC assay-through comparison data between 64 Cu and [ 64 Cu]Cu-DOTA-polystyrene-ensured that there was no dechelation of 64 Cu in the stomach or liver at 1 h. According to the data, the detected signal from PET and the biodistribution at 1 h in all other organs, including the liver, was from [ 64 Cu]Cu-DOTA-polystyrene, not from dechelated 64 Cu. Although the acidity of the stomach did affect dechelation at 6 h after administration, the other organs were not influenced by dechelation of 64 Cu from radio-TLC (Supplemental Fig. 4). Therefore, each data point obtained from PET and biodistribution was confirmed with [ 64 Cu]Cu-DOTA-polystyrene. Consequently, the dechelation of 64 Cu could be negligible (Fig. 4). Recently, several animal studies have been published on the effects of microplastics (26)(27)(28)(29). Microplastic ingestion may induce behavioral disorders in mice (30). Therefore, it is important to observe how microplastics are distributed in the body after ingestion. Remarkably, biodistribution demonstrated that [ 64 Cu]Cu-DOTA-polystyrene was distributed to all tested organs, including the testis, even after a one-time single dose. Thus, [ 64 Cu]Cu-DOTA-polystyrene may transit and accumulate in all organs even 1 h after oral administration. According to a recent report, a 4-wk exposure to polystyrene (1.0% w/v, 10 mL) induced male reproductive dysfunction in mice (31). On the basis of that mouse study and our present results, we assumed that at least 4 wk of polystyrene exposure may induce hazardous effects on tract (stomach, intestine, and liver), circulatory organs (heart, lung, and blood), renal system (kidney and bladder), and brain. Overall, accumulation pattern of biodistribution was similar to that of SUV in PET images. %ID/ g of [ 64 Cu]Cu-DOTA-polystyrene in stomach, small intestine, and large intestine was significantly higher than that of 64 Cu. However, in liver, %ID/g of [ 64 Cu]Cu-DOTA-polystyrene was lower than that of 64 Cu. Additionally, [ 64 Cu]Cu-DOTA-polystyrene transited to gastrointestinal tract (liver and spleen), circulatory system (heart, blood, and lung), renal system (kidney and bladder), and even to brain and testis. In contrast, most 64 Cu accumulated in large intestine, stomach, and small intestine at 1 h after administration. Subsequently, 64 Cu transited quickly to other organs, including liver. %ID/g in all other organs tested, including liver, spleen, heart, blood, lung, kidney, bladder, brain, and testis, was greater for 64 Cu than for [ 64 Cu]Cu-DOTA-polystyrene. n 5 5. *P , 0.05, Student t test. **P , 0.005 Student t test. n.s. 5 not statistically significant.
individual organs such as digestive organs, circulatory organs, and excretory organs.
We used BALB/c nude mice because we aimed to assess the tumorigenesis after longitudinal polystyrene exposure for further study. When different strains of mouse were used, possibly different degrees of absorption, distribution, metabolism, and elimination of polystyrene might be observed during PET.
The polystyrene used in these experiments was surface-coated with amines, and it seems likely that this process might affect their biodistribution. Polystyrene is a highly hydrophobic particle, and the addition of multiple primary amines (hydrophilic and positively charged at physiologic pH) and DOTA chelators (hydrophilic and negatively charged at physiologic pH) on the surface may influence biodistribution. Hydrophobic compounds and aggregates tend to show uptake and retention in the liver, and uptake in the liver may therefore be influenced by the surface modifications. Even if radiotracers were prepared from the same material, differences in size, shape, and surface charge can affect biodistribution and clearance. Generally, small nanoparticles penetrate capillary walls more easily than large nanoparticles, and positively charged nanoparticles are cleared more quickly by macrophages (32)(33)(34). Smaller and negatively charged silica nanoparticles have enhanced intestinal permeation by opening tight junctions (35). In this study, we selected a sphere-shaped and 0.2-mm-sized polystyrene and observed no significant differences in size or shape after DOTA conjugation. 64 Cu-labeled DOTA-polystyrene contains uncoordinated carboxylic acids, which have negative charges, and free amines, which have positive charges at physiologic pH. These surface charges may affect the permeability of the gastrointestinal tract and distribution. Recent fluorescence-conjugated microplastic studies indicated that the biodistribution of microplastics was dependent on the size of the particles (26,36). According to the result of Deng et al. (26), the accumulation in the kidney and gut was greater for 5-mm microplastics than for 20-mm microplastics. Therefore, it is possible that a smaller amount of radioisotope-labeled microplastics might accumulate in mouse organs when larger microplastics are used.

CONCLUSION
Our results demonstrate the utility of PET for visualizing the absorption and distribution of polystyrene microplastics radiolabeled with 64 Cu. PET provides information on the accumulation of microplastics in vivo and can provide information on how each organ might be affected after continuous microplastic exposure. The biologic effects of long-term exposure to microplastics in each organ affected in this study will be evaluated in future studies.