Fluorescent dye

In Vitro Stability Study [1]
The fluorescence stability of the Cy-PP microplastics dispersed in corn oil was evaluated for 10 min by measuring the change in signal intensity to confirm the entrapment of Cy-PP microplastics (Figure S4). The fluorescence intensities of all three samples (Cy5.5-COOH, Cy-PP (approximately 5 µm), and Cy-PP (10–50 µm)) were maintained over 95% for 10 min of exposure. Furthermore, in vitro digestion process simulation was performed before in vivo biodistribution study to prove the stability of Cy-PP microplastics in the body. The simulated digestion consisted of three solutions: salivary, gastric, and intestinal fluids (SF, GF, and IF, respectively). Cy-PP microplastics underwent the digestive processes with three phases of simulated digestive system by following a known method (Figure 5a). No significant release of Cy5.5-COOH was observed with little fluorescence signal from each digestive fluid according to serial digestive steps (Figure 5b).

Currently, polypropylene (PP) is used in various products, thus leading to high daily exposure in humans. Thus, it is necessary to evaluate the toxicological effects, biodistribution, and accumulation of PP microplastics in the human body. In this study, administration of two particle sizes of PP microplastics (approximately 5 and 10-50 µm) did not lead to any significant changes in several toxicological evaluation parameters, including body weight and pathological examination, compared with the control group in ICR mice. Therefore, the approximate lethal dose and no-observed-adverse-effect level of PP microplastics in ICR mice were established as ≥2000 mg/kg. Furthermore, we manufactured cyanine 5.5 carboxylic acid (Cy5.5-COOH)-labeled fragmented PP microplastics to monitor real-time in vivo biodistribution. After oral administration of the Cy5.5-COOH-labeled microplastics to the mice, most of the PP microplastics were detected in the gastrointestinal tract and observed to be out of the body after 24 h in IVIS Spectrum CT. Therefore, this study provides a new insight into the short-term toxicity, distribution, and accumulation of PP microplastics in mammals[1].

Fluorescence Labeling of PP Microplastics [1]
PP microplastics were fluorescently labeled using the CSD method for biodistribution analysis. First, 15 g of PP microplastics were added to 150 mL of distilled water and 150 mL of THF and stirred for 10 min. A Cy5.5-COOH solution (50 mg mL−1) in DMSO of 0.5 mL was added to the PP microplastic suspension followed by stirring for 4 days. After the reaction, the Cy-PP microplastics were separated by vacuum filtration using a qualitative filter paper (2 to 3 µm) to remove unlabeled Cy5.5-COOH in the reaction suspension and then washed with distilled water and ethyl alcohol. The Cy-PP microplastics were dried in a dark place at 40 °C. The morphology and chemical structure of the Cy5.5-labeled PP were analyzed using SEM and FT-IR spectroscopy, respectively.

Single Toxicity Test [1]
A single oral administration toxicity test was performed to observe the toxic reactions of two particle sizes of PP microplastics with a single oral administration and to confirm the ALD. Twelve male and female 6-week-old ICR mice were separated into the control, low-dose (500 mg/kg), medium-dose (1000 mg/kg), and high-dose (2000 mg/kg) groups for two different particle sizes of PP microplastics. Corn oil was administered to the control group, and PP microplastic suspended in corn oil at a dosage of 10 mL/kg liquid amount were administered to the other groups as a single oral administration. During the 2-week period, clinical sign observation (once a day), morbidity or dead animal observation (twice a day), and body weight measurement (once a week) were performed. After the end of the observation period, all animals were euthanized using carbon dioxide anesthesia and exsanguinated via the abdominal aorta, followed by necropsy and gross postmortem examination. All conditions for the experiment were set with reference to the OECD Test Guidelines 423 [60].

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Four-Week Repeated Toxicity Test [1]
A 4-week repeated oral administration toxicity test was conducted to evaluate the toxicity response and safety of PP microplastics. Forty male and female 6-week-old ICR mice were separated into the control, low-dose (500 mg/kg), medium-dose (1000 mg/kg), and high-dose (2000 mg/kg) groups. PP microplastic suspension in corn oil at a dosage of 10 mL/kg was orally administered to all the groups except the control group once a day for 4 weeks. During the 4-week observation period, clinical sign and morbidity or dead animal observations were performed once and twice a day, respectively. Body weight and food and water consumption were measured once a week. After the end of the observation period, blood was collected via the abdominal aorta under isoflurane anesthesia. A blood cell analyzer and a serum biochemistry analyzer were used to perform hematological and hematochemical analyses, respectively. Complete gross postmortem examinations were performed on all animals and tissues. The adrenal gland, brain, cecum, colon, duodenum, epididymis, esophagus, heart, ileum, jejunum, kidney, liver, lungs, ovary, pancreas, parathyroid gland, pituitary gland, rectum, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus were harvested. Among the extracted organs, the brain, spleen, heart, kidney, liver, testis, epididymis, and ovary were weighed. All extracted organs were fixed to 10% neutral-buffered formalin. For histopathological evaluation, a tissue processor was used for procedure of the tissues from the formalin-fixed samples. The paraffin-embedded tissue blocks were cut to a 4 µm thickness and mounted onto glass slides. Staining was performed with hematoxylin and eosin using an autostainer. The histopathological evaluation of all the slides was performed in a blind manner.

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In Vivo Biodistribution Study of Cy-PP Microplastics in Mice [1]
Fifteen male and female 6-week-old ICR mice were separated into the control, Cy-PP (approximately 5 µm), and Cy-PP (10–50 µm) groups. ICR mice had their hair removed to minimize autofluorescence generated from the hair and were fasted 8 h before fluorescence imaging. Two particle sizes of Cy-PPs were orally administered to mice as a solution dispersed in corn oil at a concentration of 2000 mg/kg. Fluorescent images were acquired by the IVIS Spectrum CT at excitation and emission wavelength of 675 and 720 nm, respectively. In vivo images were acquired under 2.5% isoflurane anesthesia, and imaging process was performed at 0.2, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. Twenty-four hours after oral administration of corn oil for control, Cy-PP (approximately 5 µm), and Cy-PP (10–50 µm), feces were collected from mice in group cages, and in vitro evaluation was performed. At 24 h after administration, in vivo imaging was the end point, saline perfusion was performed via the left ventricle of the mouse before sacrifice, and ex vivo imaging was performed.

[1]. Toxicity and Biodistribution of Fragmented Polypropylene Microplastics in ICR Mice. Int J Mol Sci. 2023 May 9;24(10):8463.

We labeled the two particle sizes of PP microplastics with fluorescent dye Cy5.5-COOH. After oral administration of the labeled microplastics to the mice, most of labeled microplastics were observed in the gastrointestinal tract. Moreover, most of labeled microplastics were observed to be out of the body after 24 h in IVIS Spectrum CT. We observed that in the extracted organs, some microplastics partially remained in the gastrointestinal tract and were not observed in other organs. From the aforementioned results, it provides a new insight on the toxicological significance of two particle sizes of PP microplastics, along with the distribution and accumulation levels of PP microplastics in mammals. For future studies, long-term administration of microplastics with higher doses than the doses in this study and analysis of additional organs should be considered. [1]

C40H42N2O2

582.773690700531

582.324

1449612-07-0

CY5.5-COOH chloride;2410537-32-3

131637033

Brown to reddish brown solid powder

10

0

3

8

44

1170

0

CC1(C(N(CCCCCC(=O)[O-])C2C=CC3=CC=CC=C3C1=2)=CC=CC=CC1=[N+](C2C=CC3=CC=CC=C3C=2C1(C)C)C)C

MQFHWNVLHOOSOL-UHFFFAOYSA-N

InChI=1S/C40H42N2O2/c1-39(2)34(41(5)32-25-23-28-16-11-13-18-30(28)37(32)39)20-8-6-9-21-35-40(3,4)38-31-19-14-12-17-29(31)24-26-33(38)42(35)27-15-7-10-22-36(43)44/h6,8-9,11-14,16-21,23-26H,7,10,15,22,27H2,1-5H3

6-[(2Z)-1,1-dimethyl-2-[(2E,4E)-5-(1,1,3-trimethylbenzo[e]indol-3-ium-2-yl)penta-2,4-dienylidene]benzo[e]indol-3-yl]hexanoate

Cy5.5-cooh; 1449612-07-0; 6-(1,1-Dimethyl-2-(5-(1,1,3-trimethyl-1H-benzo[e]indol-3-ium-2-yl)penta-2,4-dien-1-ylidene)-1,2-dihydro-3H-benzo[e]indol-3-yl)hexanoate; 1144107-80-1; 6-[(2Z)-1,1-dimethyl-2-[(2E,4E)-5-(1,1,3-trimethylbenzo[e]indol-3-ium-2-yl)penta-2,4-dienylidene]benzo[e]indol-3-yl]hexanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (85.80 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)