Design, fabrication, and characterization of nanoplastics and microplastics

Abstract

The present invention provides nanoplastic particles and / or microplastic particles, reference standards comprising nanoplastic particles and / or microplastic particles, methods of use thereof, and methods of preparation thereof. Uses of the nanoplastic particles and / or microplastic particles of the present invention include tracking the dispersion / distribution of nanoplastic particles and / or microplastic particles in environmental and / or biological systems and organisms present in the environment.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 62 / 978,499, filed on 19 February 2020, and U.S. Provisional Application No. 63 / 089,210, filed on 8 October 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Assessing the presence and downstream impacts of nanoplastics and microplastics in the environment and biological systems is a critical need. Despite the worsening problem, commercially available and well-characterized nanoplastics and microplastics are extremely limited (e.g., primarily polystyrene), which hinders significant progress in understanding their impacts on human health and the environment. For example, the importance of well-characterized standards has been emphasized in the literature for over a decade in fields such as nanotechnology, medicine, and toxicology. 1-5 .

[0003] Society's dependence on plastic is evident from global production, which reached over 330 million tons in 2016. While the usefulness of plastic cannot be denied, its widespread use has resulted in an unforeseen problem: a massive amount of unintended plastic fragments, including nanoplastics and microplastics, are present in the environment. It is estimated that between 4.8 million and 12.7 million tons of plastic fragments flowed into the world's oceans in 2010. In September 2017, microplastics were reported in 94% of tap water samples tested in the United States, and in March 2018, they were detected in 93% of bottled water samples tested.

[0004] Nanoplastics and microplastics can penetrate the environment into ecosystems and products, often undetectable. Microplastics have been found in shellfish, mussels, fish, and products containing honey and sea salt, as well as in drinking water and beverages. Furthermore, these nanoplastics and microplastics may leach foreign chemicals, such as pharmaceutical additives and unreacted monomers. While many plastic-related chemicals found in drinking water and food are known to be toxic to human health, the risks to human health from unintentional exposure to nanoplastics, microplastics, and related chemicals are unknown. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, there is a need to develop compositions / materials for tracking nanoplastics and microplastics in living organisms and the environment, as well as methods for using such compositions / materials. [Means for solving the problem]

[0006] According to one aspect of the present invention, nanoplastic particles or microplastic particles are provided, comprising a nanoplastic polymer or microplastic polymer, a polymer composite or polymer matrix, and a fluorescent tag or radioactive tag.

[0007] According to another aspect of the present invention, a reference standard material comprising nanoplastic particles or microplastic particles is provided, wherein the nanoplastic particles or microplastic particles comprise a nanoplastic polymer or microplastic polymer, a polymer composite or polymer matrix, and a fluorescent tag or radioactive tag.

[0008] According to yet another aspect of the concept of the present invention, a method is provided for monitoring the environmental dispersion of nanoplastic particles or microplastic particles, the method comprising the steps of providing a reference standard material of the concept of the present invention to the environment, and monitoring the dispersion of the standard material in the environment, wherein the step of monitoring the dispersion of the standard material includes detecting the presence of the standard material in at least one sample from the environment.

[0009] According to yet another aspect of the concept of the present invention, a method is provided for monitoring the dispersion of nanoplastic particles or microplastic particles in an object, the method comprising the steps of exposing the object to a reference standard of the concept of the present invention, and monitoring the dispersion of the standard in the object, wherein the step of monitoring the dispersion of the standard includes detecting the presence of the standard in at least one sample from the object.

[0010] According to yet another aspect of the concept of the present invention, a method is provided for monitoring the presence of nanoplastic particles or microplastic particles in a sample, the method comprising the steps of: providing a standard substance containing the nanoplastic particles or microplastic particles, which includes a polymer, a polymer composite or polymer matrix and a fluorescent tag or a radioactive tag, to an environment; and determining whether the standard substance is present in a sample obtained from the environment.

[0011] According to yet another aspect of the concept of the present invention, a method for preparing nanoplastic particles or microplastic particles is provided, the method comprising the steps of: dissolving a plastic in a first solvent to provide a plastic solution; precipitating the plastic solution in a second solvent; and evaporating the first solvent to provide a dispersion of the nanoplastic particles or microplastic particles in the second solvent. [Brief explanation of the drawing]

[0012] [Figure 1]The structure of nanoplastic particles or microplastic particles shows (A) solid, (B) matrix, (C-D) core-shell (C) functionalized with tracer, or chemical group (D). [Figure 2] SEM of polyethylene terephthalate (PET) nanoplastic particles (148 nm) according to an embodiment of the present invention concept is shown. [Figure 3] Fluorescence images visualizing PET nanoplastics containing rhodamine B (RB) (panel A) and PET nanoplastics containing fluorescein (panel B) on BeWo trophoblast cells b30 cells (nuclei are stained blue) are shown. [Figure 4] Fluorescence images visualizing PET-RB and polystyrene (PS) Alexa Fluorophore (AF) 488 nanoparticles on BeWo trophoblast cells b30 cells (nuclei are stained blue) are shown. [Figure 5] An MTS assay examining the cytotoxicity of PET nanoplastic particles and PS nanoplastic particles is shown. PET nanoplastics were determined to exhibit cytotoxicity by the MTS assay measuring metabolic activity. [Figure 6] Exemplary PET nanoplastic particles prepared as described in Example 3 are shown. [Figure 7] SEM image (panel A), TEM image (panel B), and DLS curve (panel C) of PET-RB NPs are shown. [Figure 8] FT-IR spectra of PET NPs (top) and PET-RB NPs (bottom) are shown. [Figure 9] Cytotoxicity of PET-NPs (black) and PET-RB NPs (gray) tested by membrane integrity (LHD release) (panel A) and metabolic activity (MTS assay) (panel B) is shown. The graph shows mean ± standard deviation. One asterisk indicates P value < 0.05 and two asterisks indicate P value < 0.001. [Figure 10]Bright-field (Panel AD) and fluorescence microscopy (Panel EH) images of RAW264.7 cells exposed to control (Panel A+E), 0.005 mg / mL (Panel B+F), 0.05 mg / mL (Panel C+G), and 0.5 mg / mL of PET-RB NP (Panel D+H) are shown. Images from individual fluorescence channels are shown in Figure 14. The cell nucleus is shown in the blue channel, the cell cytoplasm in the green channel, and the PET-RB NP in the red channel. [Figure 11] The FT-IR spectra of the PET starting materials are shown. [Figure 12] The Raman spectra of PET NP and PET-RB NP in BSA 0.5 mg / mL are shown. [Figure 13] The pyrolysis GC / MS chromatograms of the PET fibers (top) used to prepare PET-NP (middle) and PET-RB NP (bottom) are shown. Four of the characteristic peaks were identified as (1) vinyl benzoate, (2) benzoic acid, (3) divinyl terephthalate, and (4) 4-(vinyl benzoate carbonyl). [Figure 14] These are fluorescence micrographs of RAW264.7 cells exposed to PET-RB NP, showing superimposed images of three fluorescence channels (panels A-D), PET-RB NP (panels E-H), cell cytoplasm (panels I-L), and nucleus (panels M-P) for control (panel A+E+I+M), 0.005 mg / mL PET-RB NP (panel B+F+J+N), 0.05 mg / mL PET-RB NP (panel C+G+KO), and 0.5 mg / mL PET-RB NP (panel D+H+L+P). [Modes for carrying out the invention]

[0013] Next, the aforementioned and other aspects of the present invention will be described in more detail with respect to other embodiments described herein. It should be understood that the present invention can be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to make this disclosure thorough and complete and to fully convey the scope of the invention to those skilled in the art.

[0014] The terms used in this description of the invention are used solely for the purpose of describing specific embodiments and are not intended to limit the invention. As used in this description and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural form unless the context clearly indicates otherwise. Furthermore, as used herein, the term "and / or" includes any and all combinations of one or more of the related enumerated items and may be abbreviated as " / ".

[0015] As used herein, the term “comprise” may, in addition to its usual meaning, also include the expressions “consist essentially of” and / or “consist of,” and in some embodiments, may be specifically referred to in this way. Thus, in some embodiments, the expression “comprise” may also refer to a specifically enumerated element included in a claim that does not include any further elements, and embodiments in which the specifically enumerated element included in a claim may include and / or include further elements, or embodiments in which the elements described in the claims may include further elements that do not materially affect the basic and novel characteristics described in the claims. For example, the statement that an element described in a claim, such as a composition, formulation, method, or system, "comprising" the listed elements also includes the meaning that the element described in the claim, such as a composition, formulation, method, or kit, does not contain any further elements, and the meaning that the element described in the claim, such as a composition, formulation, method, or kit, "consisting essentially of," can contain further elements that do not substantially affect the basic and novel characteristics of the element described in the claim.

[0016] The term "about" generally refers to a range of numbers that a person skilled in the art would consider to be equivalent to, or to have the same function or result as, the stated number. For example, the term "about" may refer to a range of ±1%, ±2%, ±5%, ±10%, ±15%, or ±20% of the stated number, depending on the number that a person skilled in the art would consider to be equivalent to, or to have the same function or result as, the stated number. Furthermore, in some embodiments, the number modified by the term "about" may also be the number that is "exactly" the stated number. It will also be understood that any number presented without modification includes both the number that is "about" the stated number and the number that is "exactly" the stated number. Similarly, the term "substantially" means that the form, method, or degree is largely the same, but not entirely, and that certain elements will have a range of configurations that a person skilled in the art would consider to have the same function or result. When a particular element is expressed approximately by the use of the term "substantially," it will be understood that the particular element forms another embodiment.

[0017] All technical and scientific terms used herein have the same meanings as those generally understood by a person of ordinary skill in the art to which this invention pertains, unless otherwise defined.

[0018] (composition) Embodiments of the present invention include artificial nanoplastic particles and / or microplastic particles that are chemically designed and processed into a shape that can be used as a reference standard material. We have demonstrated that these materials can be used in vivo.

[0019] The material of the nanoplastic particles and microplastic particles may be a polymer, a polymer composite, or a polymer matrix. In some embodiments, the nanoplastic particles and / or microplastic particles include polyethylene terephthalate (PET), polyethylene (PE), high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), polylactic acid (PLA), polycarbonate (PC), polymethyl methacrylate (PMMA), polyamide (PA), polyacrylic acid (PAA), polyacrylonitrile (PAN), polyoxymethylene (POM), polyurethane (PUR), silicone, nylon, or acrylonitrile butadiene styrene (ABS). In some embodiments, the polymer, polymer composite, or polymer matrix includes PET. In some embodiments, the polymer, polymer composite, or polymer matrix includes PS.

[0020] In some embodiments, the nanoplastic particles and microplastic particles are prepared by a bottom-up approach. In some embodiments, the nanoplastic particles and microplastic particles are prepared by a top-down approach. Methods for preparing nanoplastic particles and microplastic particles include, but are not limited to, self-assembly, condensation, nucleation, colloidal methods, sol-gel treatment, oil-water micromulsion, hydrothermal synthesis, polyol methods, sonochemical methods, emulsion polymerization, dispersion polymerization, and microemulsion polymers. In certain embodiments, the particles are prepared by chain growth polymerization. Non-limiting examples of chain growth polymerization for preparing particles include radical chain polymerization, anionic chain polymerization, and cationic chain polymerization. In one non-limiting example, the material for the particles is prepared using radical chain polymerization of monomers containing one or more acrylate or vinyl functional groups.

[0021] The particles can be prepared using chemical processes, physicochemical processes, physicomechanical processes, or a combination thereof. Non-limiting examples of chemical processes for preparing particles include suspension polymerization, emulsion polymerization, dispersion polymerization, polycondensation polymerization, and combinations thereof. Non-limiting examples of physicochemical processes for preparing particles include coacervation, layer-by-layer assembly, sol-gel encapsulation, supercritical CO2 encapsulation, and combinations thereof. Non-limiting examples of physicomechanical processes for preparing particles include spray drying, multi-nozzle drying, fluidized bed coating, centrifugation techniques, vacuum encapsulation, electrostatic encapsulation, and combinations thereof. In some embodiments, core-shell particles are formed by an interfacial reaction between two immiscible monomers at the interface between the core and the surrounding solution.

[0022] A method for preparing nanoplastic particles and / or microplastic particles according to the concept of the present invention may include the steps of: dissolving a plastic in a first solvent to provide a plastic solution; precipitating the plastic solution in a second solvent; and evaporating the first solvent to provide a dispersion of nanoplastic particles or microplastic particles in the second solvent. The methods / techniques for dissolution, precipitation, and / or evaporation are not particularly limited and may be carried out using any methods / techniques that can be understood by those skilled in the art.

[0023] In some embodiments, the plastic may be, but is not limited to, polyethylene terephthalate (PET), polyethylene (PE), high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), polylactic acid (PLA), polycarbonate (PC), polymethyl methacrylate (PMMA), polyamide (PA), polyacrylic acid (PAA), polyacrylonitrile (PAN), polyoxymethylene (POM), polyurethane (PUR), silicone, nylon, acrylonitrile butadiene styrene (ABS), or any combination thereof. In some embodiments, the plastic is PET. In some embodiments, the first solvent may be, but is not limited to, phenol, DMSO, nitrobenzene, o-chlorophenol, o-cresol, diphenylamine, dichloromethane, HFIP, or any combination thereof. In some embodiments, the solvent is HFIP. In some embodiments, the plastic solution may contain plastic in a concentration / amount between about 0.1% by weight and about 0.5% by weight. In some embodiments, the second solvent may be water, but is not limited thereto.

[0024] The precipitation of the plastic solution may be carried out, for example, by adding the plastic solution to the second solvent at rates of approximately 0.1 mL / min and approximately 5 mL / min, thereby precipitating the plastic solution in the second solvent, but is not limited to these rates. In some embodiments, the plastic solution is added to the second solvent at a rate of approximately 1 mL / min.

[0025] The amount and temperature of the solution / solvent used in dissolving and / or precipitating nanoplastic particles and / or microplastic particles according to the method of preparing nanoplastic particles and / or microplastic particles of the present invention may be any amount and / or temperature that a person skilled in the art would assume for carrying out the method of the present invention. For example, the amount of the plastic solution may be about 10 mL, the amount of the second solvent may be about 50 mL to about 5000 mL, and the temperature of the second solvent may be about 0°C to about 20°C.

[0026] The particles of the present invention can be modified to allow monitoring of the particles through a biological material. In some embodiments, the plastic particles include fluorescent tags dispersed through a polymer matrix. Non-limiting examples of fluorescent tags include rhodamine such as rhodamine B (RB), fluorescein, Alexa fluorescent compounds, Nile Red, R-phycoerythrin, Pacific Blue, Cascade Blue, Texas Red, Cy5, Cy3, Cy7, hydroxycoumarin, aminocoumarin, methoxycoumarin, and the like. In one non-limiting example, the fluorescent compound is a bioconjugate. In other embodiments, the particles are, for example, 14 C or 3 The particles may have, but are not limited to, radioactive tags or labels such as H. Particles of the concept of the present invention, modified as described herein, can be prepared, for example, by dissolving a plastic in a first solvent having a fluorescent tag as described herein.

[0027] The structure of the labeled particle system includes solid, matrix, or surface functionalization (Figure 1). In one non-limiting example, the nanoplastic particles are matrix-type, having fluorescent tracers dispersed throughout the polymer matrix. In another non-limiting example, the nanoplastic particles are surface-functionalized, having fluorescent tracers associated with the surface of the particles. Surface functionalization can also include chemical groups. Non-limiting examples of such chemical groups include -COOH, -COO - , -NH3 +Examples include -NH2, -OH, -PEG, streptavidin, streptavidin-biotin complex, and antibodies. Non-limiting examples of the form of nanoplastic particles or microplastic particles according to embodiments of the concept of the present invention include spheres, fibers, rods, and dendrimers.

[0028] In some embodiments, the particle size or average particle size is less than approximately 1 micron, less than approximately 0.9 microns, less than approximately 0.8 microns, less than approximately 0.7 microns, less than approximately 0.6 microns, less than approximately 0.5 microns, less than approximately 0.4 microns, less than approximately 0.3 microns, less than approximately 0.2 microns, or less than approximately 0.1 microns. In some embodiments, the particle size or average particle size is less than 500 nm. In some embodiments, the particle size or average particle size is less than 200 nm. In some embodiments, the particle size or average particle size is less than 150 nm. In some embodiments, the particle size or average particle size is less than 100 nm. In some embodiments, the particles of the present invention are sized to represent the particle size distribution of nanoplastic particles and / or microplastic particles found in the environment.

[0029] In some embodiments, fabricated nanoplastic particles and / or microplastic particles, such as PET nanoplastic particles, are provided. The PET particle system of the present invention can remain in an aqueous suspension, enabling use in biological systems.

[0030] (method) In other embodiments of the present invention, for example, a method is provided for monitoring the presence and / or dispersion of nanoplastics or microplastics, such as nanoplastic particles or microplastic particles dispersed in the environment, ecosystems and / or living organisms. The nature of the method is not particularly limited and may be any monitoring method that can be understood by those skilled in the art. For example, a method for monitoring nanoplastics or microplastics may be in vitro, in situ, in vivo, or ex vivo without departing from the spirit of the disclosure. Monitoring the presence and / or dispersion of nanoplastics or microplastics may include providing a sample or obtaining a sample from the environment or ecosystem, and qualitatively or quantitatively determining / detecting whether nanoplastics or microplastics are present in the sample.

[0031] The nature of the environmental system or biological system is not particularly limited. For example, the environmental system or biological system may be a marine, freshwater, or terrestrial environment, or a marine, freshwater, or terrestrial biological system. The biological system may include biological organisms, for example, marine, freshwater, or terrestrial organisms. The organisms may be unicellular or multicellular, and may be plant organisms or animal organisms, without departing from the scope of the concept of the present invention. In some embodiments, the animal organisms may be mammalian organisms, for example, rodents, primates, or human organisms, but are not limited thereto. The presence and / or dispersion of nanoplastics or microplastics may be monitored by any method that will be understood by those skilled in the art, for example, in vitro, in situ, in vivo, or ex vivo, or any combination thereof. In some embodiments, samples taken from organisms may include, but are not limited to, fecal or waste samples, organ or tissue samples, and / or placental samples that can be analyzed for the presence and / or dispersion of nanoplastics and / or microplastics. In some embodiments, the environment or biosystem may include soil, sediment, or water from which samples can be taken and analyzed for the presence and / or dispersion of nanoplastics and / or microplastics. In some embodiments, samples may be taken from food and / or consumer products and analyzed for the presence and / or dispersion of nanoplastics and / or microplastics.

[0032] Methods for monitoring the presence and / or dispersion of nanoplastics and / or microplastics may include, for example, analytical methods such as high-resolution pyrolysis GC-MS. In some embodiments, monitoring the presence and / or dispersion of nanoplastics and / or microplastics may include tracking the fluorescence emitted by a fluorescently labeled nanoplastic and / or microplastic reference standard, as described herein. In other embodiments, monitoring the presence and / or dispersion of nanoplastics and / or microplastics may include tracking the radioactivity emitted by a radioactively labeled nanoplastic and / or microplastic reference standard, as described herein.

[0033] Various aspects of the present invention have been described, and these will be described in more detail in the following examples. These examples are included herein for illustrative purposes only and are not intended to limit the present invention.

[0034] (Example 1) (Fabrication of PET nanoplastic particles) A PET solution was prepared from PET fibers and hexafluoroisopropanol (HFIP). Next, the solution was precipitated in pure water cooled to 0°C in a beaker (i.e., a non-solvent to solvent ratio of 7:1). Then, the entire contents of the precipitation container were rotated and evaporated under vacuum at 37°C, and all remaining HFIP was removed by distillation. The water-dispersed PET nanoplastic particles were recovered by centrifugation. The nanoplastic particles or microplastic particles were imaged using a scanning electron microscope (SEM) or bright-field microscope. The hydrodynamic diameter was characterized by dynamic light scattering (DLS, Malvern Zetasizer Nano-ZS, Malvern Panalytical). The diameter of the microplastic particles was measured using a Mastersizer 2000 (Malvern Zetasizer Nano-ZS, Malvern Panalytical). Figure 2 shows an example of a scanning electron microscope (SEM) image of 148 nm PET nanoplastic particles prepared by the method described herein.

[0035] (Example 2) (Preparation of PET nanoplastic particles containing fluorescent tracers) A PET solution was prepared from PET fibers and hexafluoroisopropanol (HFIP). The formulation contained trace amounts of fluorescein or rhodamine B. The solution was precipitated in pure water cooled to 0°C in a beaker (i.e., a non-solvent to solvent ratio of 7:1). The entire contents of the precipitation container were then rotated and evaporated under vacuum at 37°C to remove all remaining HFIP by distillation. The water-dispersed PET nanoplastic particles were recovered by centrifugation. The PET nanoplastic particles were imaged using a fluorescence microscope (Figure 3).

[0036] (Example 3) (Biological effects of nanoplastics and microplastics on human health) Microplastics have been found in shellfish, mussels, fish, and products containing honey and sea salt, as well as in drinking water and beverages. The health effects of microplastics present in the environment and consumer products are unknown.

[0037] (the purpose) The objective of this project is to investigate how ingested nanoplastic and microplastic particles (NMPs), as well as the accompanying plastic-related exogenous chemicals (e.g., plasticizers and contaminants) released from these particles, interact with biological systems in vitro and in vivo. The goal is to investigate the risks to human health associated with exposure to these composite materials. We hypothesize that both NMPs and released plastic-related chemicals affect biological systems after ingestion. Therefore, exposure studies of NMPs differ from exposure studies of other nanomaterials and micromaterials because the outcomes of the particles and the outcomes of the associated chemicals must be considered equally.

[0038] (method) (Fabrication of PET nanoplastic particles) A 1.67% (v:v) PET solution was prepared by mixing 0.25 g of PET fiber and 15 mL of hexafluoroisopropanol (HFIP, CAS #920-66-1) in a scintillation vial with a 0.5-inch stirring bar. Preparations containing fluorescein or rhodamine B were prepared in the same manner, and a dye was further added at a concentration of 0.0001% by weight. The preparations were then stirred at 600 rpm for 10 minutes to obtain a clear solution, or a colored solution if a dye was present. Each solution was then precipitated in 105 mL of DI water cooled to 0°C in a 500 mL beaker (i.e., a non-solvent to solvent ratio of 7:1). The cooled DI water was rapidly stirred with a 2-inch magnetic stirring bar, and the HFIP solution was added dropwise to produce a turbid dispersion of particles. The entire contents of the precipitation container were then rotated and evaporated under vacuum at 37°C to remove all remaining HFIP by distillation. The water-dispersed PET nanoparticles were centrifuged at 4000g for 10 minutes to obtain a pellet of fine particles densely packed at the bottom of a 50ml centrifuge tube. Then, most of the water was removed by decantation, and the slurry was analyzed using a scanning electron microscope and DLS analysis to determine the particle size and polydispersity. The PET nanoplastic particles prepared as described above are shown in Figure 6.

[0039] (Characterization of nanoplastic and microplastic particles) Nanoplastic and microplastic particles were imaged using SEM or fluorescence microscopy. Their hydrodynamic diameters were characterized by dynamic light scattering (DLS, Malvern Zetasizer Nano-ZS, Malvern Panalytical). The diameters of microplastic particles were measured using Mastersizer 2000 (Malvern Zetasizer Nano-ZS, Malvern Panalytical).

[0040] (result) The creation of libraries of nanoplastic and microplastic particles was initiated by the manufacturing and procurement of materials. Each material was characterized and formulated into vehicles suitable for oral administration to experimental animals. Figure 4 shows images of PET-RB NPs on BeWo b30 cells. Figure 5 shows the MTS assay measuring metabolic activity in trophoblast cells exposed to PET nanoplastic and PS nanoplastic particles. PET nanoplastic particles were observed to induce a cytotoxic response, while PS nanoplastic particles did not.

[0041] (Conclusion) (PET fabrication and plastic particle library) • We successfully fabricated PET nanoplastic particles and nanoplastic fibers with and without contrast agents.

[0042] • We have launched a plastic particle library to capture the vitality of benchmark standard materials.

[0043] (Biological effects of nanoplastic particles and related chemicals) • Fluorescence microscopy images show that PET nanoplastic particles and PS nanoplastic particles are taken up by trophoblast cells (Figure 4).

[0044] PET nanoplastic particles induced cytotoxic reactions in trophoblast cells, but PS nanoplastic particles did not (Figure 5).

[0045] (significance) Federal and public concern about nanoplastics and microplastics, and their potential health effects, is rapidly increasing. Federal agencies are emphasizing the need for effective detection methods, characterization methods, and standards for nanoplastics and microplastics.

[0046] · Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP), 2010: Knowledge on the distribution and fate of microplastics has only just begun to emerge. 1

[0047] · European Food Safety Authority (EFSA), 2016: Published the report "Presence of microplastics and nanoplastics in food, with particular focus on seafood", and concluded that research on toxicokinetics and toxicity, including research on the degradation of microplastics and the potential for nanoplastics formation in the human gastrointestinal (GI) tract, as well as research on local effects in the GI tract, is needed. 2

[0048] · US Environmental Protection Agency (EPA), 2017: In June 2017, held a microplastics expert workshop focusing on four areas: 1) method needs, 2) source, transport, and fate needs of microplastics, 3) ecological assessment needs, and 4) human health assessment needs. 3

[0049] · World Health Organization (WHO), 2019: "The World Health Organization (WHO) today called for further assessment of microplastics in the environment and their potential impact on human health, following the release of an analysis of current research on microplastics in drinking water." 4

[0050] • The National Science Foundation (NSF) - Topics for FY 2020 Emerging Frontiers in Research and Innovation (NSF 19-599), 2019: “Engineering the Elimination of End-of-Life Plastics (E3P): …Their inherent durability leads to ever-increasing accumulation in landfills and the environment, where they eventually fragment into microplastics that contaminate waterways, wildlife, and human bodies.”

[0051] • National Toxicology Program (NTP, workshop presentation), October 2019: What are nanoplastics and microplastics present in the environment?

[0052] • Food and Drug Administration (FDA, "What are we exposed to? If we can detect and analyze nanoplastics, we can detect and analyze them," presentation at NSF workshop, December 2019): There is a need for validated methods and standards in the detection and characterization of microplastics.

[0053] Currently, there is a shortage of benchmark nanoplastics and microplastics, which raises the challenge of developing effective detection and characterization methods. This limitation is addressed by fabricating the nanoplastic and microplastic particles described herein.

[0054] References 1.GESAMP, Proceedings of the GESAMP International Workshop on Microplastic particles as a vector in transporting persistent, bioaccumulating and toxic substances in the ocean. 2010, The Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection 2.EFSA, Presence of microplastics and nanoplastics in food, with particular focus on seafood. EFSA Journal 2016. 14(6): p. 4501. 3.EPA, Microplastics Expert Workshop Report - Trash Free Waters Dialogue Meeting. 2018. 4.WHO, Microplastics in drinking-water. 2019.

[0055] (Example 4) (Fabrication of fluorescent tracer-attached polyethylene terephthalate (PET) nanoparticles for research in mammalian cells) This report describes the synthesis of PET NPs with a precise size distribution using a simple bottom-up approach. Furthermore, it demonstrates that incorporating fluorescent tracers into the NPs enables visualization and characterization of these PET NPs within mammalian cells.

[0056] (Materials and Methods) (Preparation of PET NPs) A PET solution was prepared by mixing 0.58 g of PET fiber (IZO Home Goods) and 35 mL of hexafluoroisopropanol (HFIP) (Sigma-Aldrich, St. Louis, MO, USA) in a 40 mL scintillation vial equipped with a magnetic stirrer. The PET solution (10 mL) was added dropwise at a rate of 1 mL / min to 75 mL of ultrapure water (18.2 MΩ-cm resistance) at room temperature using a syringe pump with a Fortuna® Optima® 10-mL glass syringe (Model # NE-300, New Era Pump Systems, Inc., Farmingdale, NY, USA) to precipitate PET NPs. The entire contents of the precipitation tank were transferred to a 250 mL round-bottom flask and removed by rotary evaporation under vacuum at 55°C to remove any remaining HFIP. The volume in the round-bottom flask was reduced (~30 mL), and ultrapure water (~75 mL) was added, followed by a second rotational evaporation of the flask. The concentrated suspension of particles was pipetted into a 20 mL scintillation vial. Particles containing rhodamine B (Sigma-Aldrich, St. Louis, MO, USA) were prepared using the same approach as described above. A tracer solution (0.05 mg / mL) in HFIP was prepared from a 1 mg / mL stock solution. Next, an aliquot (1 mL) of the 0.05 mg / mL tracer solution was added to the PET solution before precipitation in ultrapure deionized water.

[0057] To remove residual HFIP, the particle suspension was centrifuged and resuspended. Each washing step included centrifuging the suspension at 13.1 rpm for 5 minutes at room temperature, removing the supernatant, and resuspending in an equal volume of 0.5 mg / mL of Bovine Serum Albumin (BSA) to maintain the particle concentration in the suspension. The particles were resuspended after a 30-second vortex step, followed by discrete sonication at a total of 840 J / mL using a cup-horn sonicator (Ultrasonic Liquid Processor S-400, Misonic Inc., Farmingdale, NY). In the first washing step, BSA was added to the initial particle suspension before the first centrifugation step to achieve a final concentration of 0.5 mg / mL. The particles were washed three times. After the final resuspendion, the hydrodynamic diameter of the particles was measured by dynamic light scattering (DLS) (Malvern Zetasizer Nano-ZS, Malvern Panalytical, Westborough, MA). Zeta potential (Malvern Zetasizer Nano-ZS, Malvern Panalytical, Westborough, MA) was measured using disposable Folded Capillary Zeta Cells (Malvern Panalytical, Westborough, MA). The suspension of particles used for FT-IR and pyrolysis gas chromatography / mass spectrometry (Pyro-GC / MS) was washed with water instead of 0.5 mg / mL BSA. To determine the particle concentration, aliquots (1 mL) of PET particles were transferred to 2 mL Eppendorf tapered tubes and left overnight in a vacuum oven under ambient conditions. The next day, the weight of the tubes was measured to determine the dry particle weight. To determine the concentration of rhodamine B in the particles, the dry particles were then dissolved in HFIP (1 mL), and their fluorescence was determined using a Synergy MX multimode plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Calibration curves for rhodamine B in HFIP were obtained by serial dilution of the phosphor (1.25 μg / mL stock solution, λ ex= 550nm, λ em (= 580nm).

[0058] (Characterization of PET NPs) Fourier transform infrared spectroscopy (FT-IR): Dried samples were analyzed using a Nicolet 6700 FTIR with a Smart Orbit® single-bounce diamond crystal ATR accessory. This instrument is equipped with a DTGS detector and a KBr beam splitter. Method parameters were set to resolutions of 4 and 32 scans, at 4000–400 cm⁻¹. -1 The region was scanned. A background was performed on a cleaned crystal before each sample. After the background acquisition was complete, a small amount of sample was added to the diamond crystal, pressure was applied, and data was acquired.

[0059] 19 F nuclear magnetic resonance spectroscopy ( 19 F-NMR): The presence of residual hexafluoro-2-propanol in PET NPs was detected. 19 The determination was made by 1H-NMR. Fluorine NMR experiments were performed using a Varian Unity Inova 500 mHz NMR (Palo Alto, CA) equipped with a Nalorac Cryogenics Corporation-specific HF observation probe (Martinez, CA). 19 The 1F-NMR sample was mixed with 10% D2O. The total recycling time was 8 seconds. Residual fluorine was calibrated and quantified using Agilent VnmrJ ver. 4.2 software (Santa Clara, CA) with a detection limit of 0.02 mM, using an external reference standard.

[0060] Transmission electron microscopy (TEM): PET NPs were prepared using a drop-mount method for liquid deposition. The PET NPs were pipetteed onto a 200-mesh carbon-coated copper transmission electron microscope (TEM) grid. The liquid suspension was air-dried on the copper grid in a fume hood with a HEPA filter. Two TEM grids were prepared for each sample. These grids were analyzed using a Hitachi H-7000 transmission electron microscope. Multiple images of each sample were taken using an AMT digital camera. The analysis magnification ranged from 40,000x to 300,000x.

[0061] Scanning electron microscopy (SEM): SEM analysis was performed using a Zeiss Auriga field emission scanning electron microscope (FESEM) (Carl Zeiss Microscopy, White Plains, NY) with an acceleration voltage of 5 kV and a beam current of 10 μA. Before SEM analysis, all samples were coated with gold / palladium by sputtering. Particle size was measured using ImageJ (NIH).

[0062] X-ray photoelectron spectroscopy (XPS) was performed using an Escalab Xi+ XPS (Thermo Fisher Scientific, Waltham, MA). All scans were charge-compensated. Survey scans were performed with a pass energy of 200 eV, a step size of 1.0 eV, and a residence time of 10 milliseconds. Single-element scans were performed with a pass energy of 50 eV, a step size of 0.1 eV, and a residence time of 50 milliseconds.

[0063] Raman spectroscopy: The spectra of all samples were measured at room temperature using a Horiba XploRA Raman Confocal Microscope (Horiba Scientific, Piscataway, NJ) with a wavelength excitation of 532 nm and a 1200 L mm-1 grating.

[0064] Ultraviolet-Visible Spectrophotometer (UV-VIS): Samples were analyzed in the 200–800 nm wavelength range using a Shimadzu UV-2600 UV-Visible Spectrophotometer (Columbia, MD) with LabSolutions software version 1.03 (Atlanta, GA). Samples were diluted 1:10 and 1:100 with BSA, and the BSA was used as a blank. A slit width of 2 nm and a data interval of 0.5 nm were used.

[0065] Pyrolysis gas chromatography / mass spectrometry (Pyro-GC / MS): Pyrolysis was performed using a CDS Analytical 5250-T Trapping Pyrolysis Autosampler (Oxford, PA) connected to a Thermo Scientific Trace 1310 gas chromatograph coupled to a Q-Exactive mass spectrometer (Waltham, MA). Sample vials consisted of quartz rods in quartz tubes with the upper headspace filled with quartz wool. Samples were prepared by transferring them to vials in microgram units. The initial thermal desorption step was performed at 50°C for 60 seconds, and the samples were sent to the GC-MS. Subsequently, a washing step was performed at 350°C for 20 seconds to expel all sufficiently volatile sample contents to the exhaust port to prevent unwanted substances from reaching the column. The final step involved heating at 50°C for 3 seconds, then increasing the temperature to 700°C at 10°C / millisecond, and holding for 60 seconds while all samples were sent to the column for analysis. Data analysis was performed using Xcalibur software version 4.1.31.9 (Thermo) and the National Institute of Standards and Technology version 17 (Gaithersburg, MD) library to help identify the target spectral peaks.

[0066] (Research using mammalian cells) Endotoxin assay: Endotoxin detection and quantification were performed according to the manufacturer's protocol using a Pyrochrome test kit (Associates of Cape Cod Inc., East Falmouth, MA) containing GlucoShield reconstitution buffer and control standard endotoxin. The supernatants of PET-NP and PET-RB NP were tested with Limulus amoeba cell lysate (LAL) reagent water (Associates of Cape Cod Inc., East Falmouth, MA). The BSA solution used for particle washing and suspension was also tested. To ensure that PET NPs did not interfere with the assay, a positive product control (PPC) containing a final concentration of 0.5 EU / mL was tested in parallel at the same concentration. No interference was detected between the two PET NPs and the assay.

[0067] Cell Culture: The toxicity of PET NP was tested using mouse alveolar macrophage cells RAW264.7 (ATCC® TIB-71®, ATCC, Manassas, VA). RAW264.7 cells were cultured in Dulbecco's modified Eagle medium (Gibco, Life Technologies, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY) and 100U penicillin / streptomycin (P / S) (Gibco, Life Technologies, Grand Island, NY). The cells were cultured at 37°C in 5% humidified CO2 for 1 × 10⁶ cells. 4 The cells were maintained at a concentration of cells / mL, washed with pre-warmed phosphate-buffered saline (PBS) (Gibco, Life Technologies, Grand Island, NY), and passaged twice a week. RAW264.7 cells were used between passage numbers 41 and 45.

[0068] Cytotoxicity assay: RAW264.7 1 x 10 5Cells were seeded in 96-well plates at a concentration of cells / mL and incubated for 24 hours. PET NPs suspended in fresh medium were added to the cells at a 2-fold dilution at a concentration of 0.0005–0.5 mg / mL. After 24 hours of exposure to the NPs, the medium was collected for lactate dehydrogenase (LDH) release measurement. An LDH assay (TOX7, Sigma-Aldrich, St. Louis, MO) was performed according to the manufacturer's protocol to measure the level of LDH released into the medium. Briefly, 75 μL of medium was analyzed to assess cell viability as a function of cell membrane integrity. After collecting the medium for LDH measurement, the monolayer was washed with PBS, and cell viability and metabolic activity were measured using an MTS assay. The MTS [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI) was performed according to the manufacturer's protocol. Briefly, metabolic activity was measured by adding the cell reagent solution to the cells and colorimetrically measuring the reduction of MTS to colored formazan by the metabolically active cells. Data were expressed as a percentage relative to a representative control. All studies were performed using biological duplication and at least experimental triple replication.

[0069] Fluorescence microscopy observation: Cells are placed in a glass-bottomed Petri dish (MatTek, Ashland, MA) at 1x10 5Cells were seeded at a concentration of cells / mL and exposed to PET-RB NP at concentrations of 0.005 mg / mL, 0.05 mg / mL, and 0.5 mg / mL for 16 hours after 24 hours. Simultaneously with PET-RB NP exposure, CellLight Lysosomes-GFP *BacMam 2.0* (Life Technologies, Grand Island, NY) was added to the cells, and lysosomes were stained with 25 particles per cell. The cells were then fixed at room temperature with 3% paraformaldehyde and 0.1% glutaraldehyde for 30 minutes. After washing the cells three times with PBS, they were stained with 1:200 DAPI (Life Technologies, Grand Island, NY) at room temperature for 15 minutes. After washing the cells three times with PBS, bright-field imaging and fluorescence imaging were performed with a 40x objective lens. The aforementioned imaging was performed using an Olympus IX71 inverted microscope equipped with a CCD microscope camera (INFINITY3-3URF, 3.0 Megapixel, manufactured by CoolLED). Image processing was performed using ImageJ (NIH).

[0070] Data Analysis: Data was expressed as mean ± standard deviation using the software Prism (GraphPad 7.4, GraphPad Software, San Diego, CA). Student's t-test was used for statistical analysis, and the statistical significance was P < 0.05.

[0071] (Results and Discussion) (Preparation and characterization of PET NPs) PET NPs were prepared by a precipitation method in which PET and HFIP solutions were slowly added to ultrapure water to form NPs. After multiple washes to remove residual HFIP solvent from the NP formulation, 19The fluorine signal was no longer detected by F-NMR. During washing of the PET NPs with ultrapure water, the particles aggregated; therefore, a 0.5 mg / mL BSA protein solution was used instead to maintain the dispersion of the particles. Here, the use of BSA was also suitable for subsequent cell culture studies, as described in the next section. However, the use of species-specific proteins or alternative surfactants as stabilizers for these NPs may be required to be consistent with the biological system under investigation. To enable the detection of PET NPs within cells, the particles were labeled with rhodamine B (PET-RB) by incorporating a tracer into the NPs during preparation. The round morphology of the PET-RB NPs was evident by SEM (Figure 7, panel A) and TEM (Figure 7, panel B), while no morphological difference was evident in PET NPs without the tracer (Figure 11). After washing and resuspending the particles with BSA solution, the hydrodynamic diameters were 170 nm ± 3 nm for PET-NPs and 158 nm ± 2 nm for PET-RB NPs (Figure 7, Panel C, Figure 11). The washing step with BSA solution slightly increased the hydrodynamic diameter compared to the unwashed sample, but the average size distribution remained below 200 nm, and the polydispersity index was 0.2 for PET and 0.1 for PET-RB. Furthermore, the average diameters of the NPs calculated from SEM images were 95 nm ± 14 nm for PET NPs and 88 nm ± 14 nm for PET-RB NPs. The difference between the hydrodynamic diameter and the diameter calculated from SEM images is expected and may be due to the presence of BSA corona in the particle suspension. 43 The zeta potentials of NPs suspended in BSA solution were -37mV for PET NPs and -38mV for PET-RB NPs, supporting the high dispersibility and stability of the particles. For example, PET NPs were measured at 164±4 nm (PDI 0.2) after being stored at room temperature for one month.

[0072] FT-IR analysis was performed to investigate the composition of NP (Figure 8). The FT-IR profile of NP showed characteristic absorption bands of PET bulk polymer (Figure 11), and has been reported previously. 44-46 As stated, 1715cm -1(C = O telescopic), 1578cm -1 (C=C expansion and contraction within the ring), 1505cm -1 (In-plane bending of CH within the ring; expansion and contraction of C=C within the ring), 1240cm -1 (Bending within plane C=O, expansion / contraction of CC, expansion / contraction of C(=O)-O) 46 and 724cm -1 (Interaction between ester group and benzene ring) 44 ) was included. As shown in Figure 8, the prominent IR absorption bands were similar between PET and PET-RB NP. Interestingly, despite the validation of the fluorescence tracer by fluorescence microscopy, 1690 cm⁻¹ -1 Typical bands associated with rhodamine B, such as (CC stretching), were not present in PET-RB NP. The absence of rhodamine B absorption bands in FT-IR is thought to be due to the low concentration of the tracer, which prevented detection in the IR spectrum. Additional tests using Raman spectroscopy also identified various locations within PET and PET-RB NP (Figure 12). 1612.92 cm -1 The main peak corresponds to Raman scattering caused by the benzene rings in the PET structure. Other secondary peaks are at 1725.16 cm⁻¹. -1 (Carbonyl stretching), 1446.24 and 1287.60 cm -1 (Weak CC bond), as well as 1177 and 1116.98 cm -1 It was located in a weak COC asymmetric stretching vibration. Further analysis of PET and PET-RB NPs was performed by pyro-GC-MS (Figure 13).

[0073] The surface chemical state of PET NPs in BSA containing and without rhodamine B was investigated by XPS analysis. Table 1 shows the bond energies of all elements present in the samples. The shifts in bond energies of the C 1s, N 1s, O 1s, Zn 2p, and S 2p spectra correspond to differences in the interaction between these elements and the PET structure. The C 1s peak centered at 284.4 eV is present in both samples and is associated with the phenyl carbon in the PET structure. The satellite peak centered around 291 eV is due to the π-π* fluctuation process of the aromatic ring in the structure. The O 1s spectrum centered around 530.5 eV corresponds to the C=O bond. The N 1s peak centered around 399.5 eV is due to the CN bond between nitrogen and the aromatic PET ring. Furthermore, a Zn 2p peak with two spin-orbit divisions, 2p3 / 2 and 2p1 / 2, was observed, with a bond energy difference of ~23 eV. 2p3 / 2, centered at 1021.3 eV, is Zn +2 The presence of zinc in the chemical environment was confirmed. No significant shifts in binding energy were observed in either sample. Finally, in the S 2p spectra, both samples showed an S 2p3 / 2 peak around 163 eV.

[0074] [Table 1]

[0075] (Evaluation of PET NPs in mammalian cells) Prior to evaluation in mammalian cells, a kinematic viscosity turbidity LAL assay was used to confirm the possibility of endotoxin contamination of PET NPs. Endotoxin levels were detectable, but the values ​​were low, showing 0.1 EU / mL for PET-NPs and 0.064 EU / mL for PET-RB NPs. Cytotoxicity and uptake of PET NPs were evaluated dose-response using mouse alveolar macrophages RAW264.7. Cytotoxicity was assessed by determining cell membrane integrity (LHD release) and metabolic activity (MTS) (Figure 9). Significant increases in LHD release were observed: 0.0625 mg / mL (P-value = 0.0016) for PET-NPs and 0.0010 mg / mL (P-value = 0.0034) for PET-RB NPs. At a concentration of 0.125 mg / mL for both PET NPs (160±27.5% of control for PET NPs and 178±18.3% of control for PET-RB NPs), LDH release continued to increase with increasing concentration, reaching 506±85% of control for PET-NPs and 447±46.1% of control for PET-RB NPs at 0.5 mg / mL. On the other hand, the MTS assay showed a slight increase at the lowest concentration of PET NPs. Only at the highest concentration of PET NPs tested, 0.5 mg / mL, did the MTS assay show a decrease in mitochondrial activity (82.9±8.77% of control for PET-NPs and 71.3±29.4% of control for PET-RB). Taken together, these findings suggest that cell membrane integrity is affected at lower NP concentrations before mitochondrial activity changes.

[0076] The uptake of PET-RB NPs into cells and the resulting morphological changes in RAW264.7 cells were revealed by bright-field and fluorescence microscopy. After exposure to a low concentration of 0.005 mg / mL of PET-RB NPs, individual particles were visible in the cytoplasm (Figure 10, panels B and F), but at concentrations of 0.05 mg / mL and 0.5 mg / mL of PET-RB NPs, large clusters of NPs within cells were observed in both bright-field (Figure 10, panels A-D) and fluorescence microscopy (Figure 10, panels E-H). Individual fluorescence channels from fluorescence microscopy (Figure 10, panels E-H) are shown in Figure 14. Cell nuclei (blue channels) are shown in panels M-P of Figure 14, cell cytoplasm (green channels) are shown in panels I-L of Figure 14, and PET-RB NPs (red channels) are shown in panels E-H of Figure 14. The fluorescence intensity of larger NP aggregates became supersaturated during the exposure time required to visualize individual PET-RB NP particles, making the aggregates appear larger in fluorescence microscopy images compared to bright-field images. Because PET-RB NPs exhibited low levels of autofluorescence at green wavelengths, it was not possible to determine whether PET-RB NPs were associated with lysosomes. At 0.05 mg / mL of PET-RB NPs, particles were observed within phagocytose cells, with some cells forming rigid phagosomes around the NPs, while higher concentrations resulted in vacuoles with large gaps around the NPs. At the highest concentration, phagosomes expanded, and elongated crescent-shaped nuclei formed around the cell periphery. At 0.005 mg / mL, bubble-like morphological changes were observed, indicating detachment of the cell membrane from the cortical cytoskeleton. 47 These bubbles increased at 0.05 mg / mL but not at 0.5 mg / mL. At 0.5 mg / mL, nuclear condensation and increased fluorescence intensity were observed, supporting cytotoxicity data indicating that many cells were killed at this concentration.

[0077] (Conclusion) The presence of fragmented plastics derived from high-value polymers in the environment is a new concern due to the unknown impact on biosystems and human health. As a significant source of high-value polymers and plastic waste, PET infiltrates drinking water, food, and beverages in the form of tiny fragments (i.e., microplastics), as indicated in various reports. While current reports focus on micron-sized plastics, nano-sized PET also poses a potential environmental pollution risk.

[0078] PET nanoparticles (PET NPs) with a hydrodynamic diameter of less than 200 nm were synthesized. To support studies in a cell model, rhodamine B fluorescence tracer was incorporated into the PET NPs, and their uptake in RAW264.7 macrophages was measured. The results confirmed that PET-RB NPs were taken up by macrophages in a dose-response manner. This finding indicated that the concentration of PET NPs required to affect the integrity of the macrophage cell membrane (0.0010 mg / mL) was lower than the concentration required to alter mitochondrial activity (0.5 mg / mL). Higher concentrations of PET NPs (0.5 mg / mL) resulted in clear morphological changes, with enlarged phagosomes causing nuclear elongation and likely leading to cell death. This study demonstrates that mammalian macrophage cells are affected by PET nanoplastics.

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[0080] Various features of the present invention may be described in the context of a single embodiment, but such features can also be provided separately or in any suitable combination. Conversely, for clarity, the present invention may be described in the context of separate embodiments in this specification, but the present invention can also be implemented in a single embodiment.

[0081] The above is illustrative of the concept of the present invention and should not be construed as limiting it. Further embodiments of the concept of the present invention are illustrated in the following claims, and their equivalents are included within the claims.

Claims

1. A method for preparing nanoplastic particles and / or microplastic particles, the following: A step of dissolving the plastic in a first solvent to provide a plastic liquid; The step of precipitating the plastic liquid in a second solvent; and The step of evaporating the first solvent to provide a dispersion of the nanoplastic particles or microplastic particles in the second solvent. Includes, The aforementioned plastic is selected from the group consisting of polyethylene terephthalate (PET), polyethylene (PE), low-density polyethylene (LDPE), and polyamide (PA). The first solvent is HFIP, and The second solvent is water. The above method.

2. The method according to claim 1, wherein the plastic is PET.

3. The method according to claim 1 or 2, wherein the plastic liquid contains 0.1 to 5% by weight of plastic.

4. The method according to any one of claims 1 to 3, wherein the plastic liquid is added to the second solvent at a rate of 0.1 to 5 mL / min to precipitate the plastic liquid in the second solvent.

5. The method according to claim 4, wherein the plastic liquid is added to the second solvent at a rate of 1 mL / min.

6. The method according to any one of claims 1 to 5, wherein the plastic liquid is in an amount of 10 mL, and the second solvent is in an amount between 50 mL and 5000 mL.

7. The method according to any one of claims 1 to 6, wherein the second solvent has a temperature between 0°C and 20°C.

8. The method according to any one of claims 1 to 7, wherein the plastic is dissolved in the first solvent having a fluorescent tag.

9. The method according to claim 8, wherein the fluorescent tag is selected from the group consisting of rhodamine, fluorescein, Alexa fluorescent compound, Nile Red, R-phycoerythrin, Pacific Blue, Cascade Blue, Texas Red, Cy5, Cy3, Cy7, hydroxycoumarin, aminocoumarin, and methoxycoumarin.

10. The method according to any one of claims 1 to 9, wherein the prepared nanoplastic particles or microplastic particles have an average size of less than 1 micron.

11. The method according to any one of claims 1 to 10, wherein the prepared nanoplastic particles or microplastic particles have an average size of less than 500 nm.

12. The method according to any one of claims 1 to 11, wherein the prepared nanoplastic particles or microplastic particles have an average size of less than 150 nm.

13. The method according to any one of claims 1 to 12, wherein the prepared nanoplastic particles or microplastic particles have an average size of less than 100 nm.

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