Labeling of PET and PP nanoplastic test materials with non-leachable π-conjugated fluorescent polymers

Labeling of PET and PP nanoplastic test materials with non-leachable π-conjugated fluorescent polymers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Labeling of PET and PP nanoplastic test materials with non-leachable π-conjugated fluorescent polymers My Vanessa Nguyen Hoang, Maria Anzengruber, Yoel Negrin Montecelo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8842721/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Micro- and nanoplastic (MNP) particles are widely present in nature, mainly due to the extensive overuse of single-use plastics combined with poor waste management. Despite the diversity in the environment, many experimental studies still rely almost exclusively on polystyrene as a model plastic test material, while other environmentally relevant polymers remain underrepresented. In addition, labeled MNP test materials suitable for biological studies are still limited. In this study, nanosized polyethylene terephthalate (PET) and polypropylene (PP) particles were produced using a co-precipitation approach with the fluorescent π-conjugated polymer, poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) at low (0.8 % w/w) and high (3–5 % w/w) F8BT content. Fluorescently labeled MNPs (75–85 % dye internalization) could be produced with > 85 % of the particles in the submicron size range. Co-precipitation of F8BT with PET produced a subset of spindle-shaped particles, while F8BT-labeled PP particles were primarily spherical. The fluorescence limit of detection of the F8BT labeled PET and PP was ~0.2 µg/mL for both systems. The strong fluorescence enabled measurements of cell uptake using an innovative exposure system, the FlowCube, which overcomes dosimetry issues with buoyant particles. This work provides an innovative approach to producing fluorescently labeled PET and PP nanoplastic test materials for environmental and biological studies. microplastics nanoplastics fluorescently labeled nanoplastics polyethylene terephthalate polypropylene F8BT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Micro- and nanoplastics (MNPs) are increasingly recognized as widespread environmental contaminants, having been detected in, e.g., marine water [ 1 ], [ 2 ], [ 3 ], air [ 4 ], [ 5 ], and food [ 6 ], [ 7 ]. These materials originate from the fragmentation of larger plastic debris through mechanical, thermal, and chemical degradation processes. With only about 9 % of global plastic waste beng recycled, plastic pollution continues to rise. In 2019, global plastic production reached 368 million metric tons, with an expected annual growth rate of approximately 5 % [ 8 ]. This trend contributes t the increasing environmental accumulation of plastic debris and highlights the urgent need for further investigation, particularly given the clear evidence of human exposure to MNPs. Concerns have been raised regarding the potential of MNPs to interact with biological systems, cross cellular barriers, and accumulate in organisms [ 9 ], [ 10 ], [ 11 ], [ 12 ]. While numerous studies have explored cellular uptake and the effects of MNPs in various in vitro and in vivo systems, the findings remain inconclusive [ 13 ]. Many MNP exposure studies rely primarily on commercially available polystyrene (PS) beads, even though PS accounts for less than 10% of environmental plastic waste [ 14 ]. While these particles are easy to handle and available in well-defined sizes, their smooth, spherical, and monodisperse nature does not reflect the diversity of plastic debris found in the environment. As a result, the environmental relevance of PS-based model systems remains limited. More representative test materials made from polymers such as polyethylene terephthalate (PET) and polypropylene (PP), both extensively used in packaging, are underrepresented in MNP research. These materials differ in density, crystallinity, and weathering behavior, making them valuable for comparative studies on cellular transportation and toxicological effects. In recent years, efforts have expanded the range of available model MNPs through both top-down methods (milling, ultrasonication) and bottom-up methods (nanoprecipitation) [ 15 ]. Nanoprecipitation has been employed to produce a variety of plastic types including PET, PP [ 16 ], low-density polyethylene (LDPE), high-density polyethylene (HDPE), and polyvinyl chloride (PVC) particles [ 17 ]. Nevertheless, there remains a pressing need to further develop and diversify nanosized plastic test materials. The desired attributes for such materials include environmental relevance, microbiological stability, well-defined physicochemical properties, suitability for biological assays, and analytical detectability. Besides the lack of well-characterized test materials, another major challenge lies in the identification and quantification of MNPs, especially in complex biological matrices. [ 18 ] Thermal analytical techniques combined with chromatographic separation and mass spectrometric detection, such as pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) [ 19 ] and thermal extraction desorption-gas chromatography-mass spectrometry (TED-GC-MS) [ 20 ] are currently among the most sensitive approaches, enabling polymer-specific quantification of unlabeled materials at low limits of detection (LOD) with the highest sensitivity observed for water and air samples (LOD: < 1 ng/mL; see Table S1 ). It is important to note that method sensitivity greatly depends on polymer type, matrix complexity and content of organic matter, with reported LOD values ranging from 0.02–10.8 µg/mL (or µg/g), depending on the sample matrix (see Table S1 ). Mass-based quantification methods are destructive, require complex sample preparation, pose a risk of MNP loss or sample contamination, and are susceptible to matrix effects which can strongly influence detection limits [ 21 ]. Different polymers can also produce identical pyrolysis products, resulting in false positive polymer identifications and concentrations [ 22 ]. In contrast, labeling strategies focus on the detection of an incorporated tracer rather than the polymer itself and are therefore primarily used for particle tracking, visualization, and uptake studies [ 18 ]. Approaches include the incorporation of radioactive or stable isotopes into the polymer. Despite their excellent detectability, disadvantages include safety concerns, strict regulatory requirements, and limited half-lives [ 23 ], [ 24 ]. Stable isotopes like ¹³C or ²H are safer alternatives but often lack spatial resolution and require specialized instruments like isotope ratio mass spectrometers (IRMS), which are not widely available [ 25 ]. Metal tracers embedded in the polymer matrix enable highly sensitive detection through inductively coupled plasma mass spectrometry (ICP-MS), while avoiding issues such as photobleaching and dye leaching. However, their production may require toxic metals and specialized equipment [ 26 ]. A third and widely used strategy is fluorescence labeling. This technique is popular due to its ease of use and compatibility with microscopy, flow cytometry, and spectroscopy methods (see supplementary Table S2) [ 27 ], [ 28 ]. Fluorescent labeling can be achieved either by surface staining or by incorporating a dye into the polymer matrix [ 29 ]. While surface staining is simple, it often suffers from poor dye retention, leaching in protein rich media and interference from autofluorescence. In the current study, the high molecular weight, π-conjugated polymer, poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT), was explored as a fluorescent polymeric label for MNPs. Due to the presence of π-conjugated double bonds in the F8BT backbone (Fig. 1 ), the polymer exhibits semiconducting properties and a bright yellow-green fluorescence, which is characterized by its thermal stability, strong emission and resistance to photobleaching. F8BT has been primarily used in organic light-emitting diode and photovoltaic technologies but has also been explored as an optical contrast agent in bioimaging applications [ 30 ], [ 31 ], [ 32 ], [ 33 ]. When using a polymeric dye to label nanoplastics, it is important to recognize that most polymer blends are immiscible due to their relatively low entropy of mixing and high interfacial tension [ 41 ]. Co-precipitation of two immiscible polymers can produce nanoparticles with different structural morphologies depending on the interfacial tension between the two polymers. Such diverse morphologies include dimer particles, occluded particles, core-shell capsules and hetero-aggregate systems (Fig. 1 ). The type of solvents/anti-solvent systems, the polymer structure and molecular weight, ratio of the polymers in the blend, as well as the mixing temperature and speed determine the final equilibrium particle structure [ 40 ]. Despite this complexity, Hildebrand and Flory-Huggins solubility parameters have been used to estimate how well a given polymer will interact with other polymers [ 42 ], whereby similar values indicate a higher potential for miscibility. The reported Hildebrand solubility parameters (HSP) [ 43 ], [ 44 ] for F8BT, PET and PP, the nanoplastics chosen for this study, indicate that F8BT may exhibit a higher miscibility with PET compared to PP. For the purposes of nanoplastic labeling, particle morphologies where the F8BT label is miscible with the plastic or embedded in the PET/PP core (internalized F8BT) are more desirable than morphologies where F8BT is located externally, since external F8BT could leach or change the particle surface properties compared to unlabeled plastics (Fig. 1 ). Building on a previously established preparation method for PET and PP nanoplastics intended for use in biological assays [ 16 ], F8BT-labeling of these two materials was explored. First, F8BT incorporation into PET/PP nanoplastics was characterized and the physicochemical properties of systems with optimal loading was evaluated. Key criteria included morphology, size reproducibility, good dispersibility, optical properties, and low, reproducible LOD/LOQ values. Cellular uptake was then investigated in Calu-3 epithelial cells using both a conventional static well plate setup and an innovative dynamic exposure system known as the FlowCube. The FlowCube is designed to expose cells to low-density particles, like PP, that would otherwise float to the surface of the culture medium in standard submerged cell culture formats. The FlowCube simulates a 3D exposure scenario by vertically inserting cell-seeded slides into a cube structure, ensuring continuous interaction between particles and cells. 2. Materials and methods Materials The starting material polyethylene terephthalate (CAS:25038-59-9) and polypropylene (CAS: 9003-07-0) granulates were kindly provided by Plastics Europe (Brussels, Belgium). The fluorescent dye F8BT (average MN ≤ 25000) was purchased from Sigma-Aldrich (St. Louis, USA). As well as benzyl alcohol (BA), xylene (isomeric mixture), glycerol, Fluorsave mounting medium and the non-ionic surfactant Tween 80. Bovine serum albumin (BSA, fraction V) was supplied by Carl Roth (Karlsruhe, Germany). Tetrahydrofuran (THF) was obtained from Fluka (Buchs, Switzerland) whereas ethanol (≥ 99.8%) was purchased from Brenntag (Guntramsdorf, Austria). Polycarbonate (PC) membrane filters (2.0 µm) were bought from Merck Millipore (Billerica, USA) and nylon membrane filters (0.8 µm) were obtained from Cytiva (Marlborough, USA). Experiments involving the human bronchial epithelial cell line Calu-3 (ATCC HTB-55), which was obtained from LGC Standards Ltd. (Teddington, UK), were performed using following products provided from different suppliers. Dulbecco’s phosphate buffered saline (PBS) and fetal bovine serum (FBS) superior from Sigma-Aldrich, cell culture media Dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM/F-12) + GlutaMAX, trypsin-EDTA (0.25%) and penicillin streptomycin from Gibco (Thermo Fisher Scientific, Waltham, USA). Poly-L-lysine hydrobromide, paraformaldehyde (PFA) and ammonium chloride were also obtained from Sigma-Aldrich. Hoechst 33342 was purchased from Invitrogen. Thermanox plastic coverslips and square glass microscope slides were purchased from Nunc (Thermo Fisher Scientific, Waltham, USA). Fluorescent polystyrene (PS) particles labeled with Nile Red were purchased from Kisker Biotech (Steinfurt, Germany) and used as a commercial reference material. All reagents were of analytical grade or higher and used without further purification. Ultrapure water (18.2 MΩ·cm) was used in all aqueous dilution preparations. Test material production The procedure for producing unlabeled submicron-range PET and PP nano-sized test materials developed by Wimmer et al . [ 16 ] was used with modifications. An updated detailed standard operating procedure is available in the electronic supplementary information. PET granules (260 mg; m granules ) were dissolved in benzyl alcohol (10 mL) without (0% w/w) and with F8BT (0.8% and 5% w/w). The mixture was heated to 215 ± 0.2°C, stirred at 250 rpm for 45 min and was rapidly transferred into chilled ethanol (125 mL) under more stirring (400 rpm) to induce precipitation. The precipitate was washed five times with ethanol (100 mL each) by filtration using a polycarbonate membrane (pore size: 2 µm). Nanoparticles were retained, presumably due to rapid agglomeration during this step. The particles were resuspended in ethanol (26 mL) and sonicated in a sonication bath (Sonocool SC 255, Bandelin electronic, Berlin, Germany) for 2 h (referred to as intermediate product; 10 mg/mL). All sonication steps were performed with the same instrument under the following settings: 21 ± 1°C, 100% intensity, 35 kHz frequency, and 180 W nominal power. To prepare PP nanoplastics, a similar approach was followed with minor modifications: PP granules (165 mg) were dissolved in xylene (80 mL) at 185 ± 0.2°C, without (0% w/w) and with F8BT (0.8 and 3% w/w). The hot solution was added to cooled ethanol (240 mL) and the suspension was filtered through a 0.8 µm nylon membrane, undergoing the same rigorous ethanol washing steps to remove residual solvent. The material retained on the filter membrane was resuspended in ethanol (16 mL) and subsequently sonicated for 45 min prior to weighing (m susp ). After sonication, the concentration and product yield of the intermediate product were determined gravimetrically by weighing a microcentrifuge tube before ( m empty ) and after addition of 1 mL of ethanolic suspension ( m filled ). The sample was then dried (55°C, 20 mbar), and the dry mass was recorded ( m dry ). When the gravimetrically determined mass loss between measurements was below 1%, concentration and yield were calculated using equations ( 1 ) and ( 2 ), respectively. Eq. ( 1 ): The concentration (c) of nanosuspension calculated as mg/g. $$\:c\:\left[\frac{mg}{g}\right]=\:\frac{\left({m}_{dry}-{m}_{empty}\right)\:x\:1000}{({m}_{filled}-{m}_{empty})}$$ 1 Eq. ( 2 ): Yield (Y) of particle production calculated in %. $$\:Y\:\left[\%\right]=\:\frac{c\:x\:{m}_{susp}}{{m}_{granules}}$$ 2 To prepare for exchanging the storage medium from ethanol to glycerol (final product), glycerol was heated to 60°C and the required amount to achieve a final plastic concentration of 40 mg nanoplastics per 1 g glycerol was calculated. The heated glycerol was added to the ethanolic suspension, followed by vortexing and sonication (5 min) to ensure even distribution. Ethanol was evaporated (55°C, 20 mbar), and the process was considered complete once the mass loss between measurements was below 1%. The final product was stored in 1.5 mL glass vials under light exclusion. Encapsulation efficiency Two calibration curves for F8BT quantification were prepared by dissolving the dye in either pure tetrahydrofuran or a tetrahydrofuran/benzyl alcohol mixture (1:1; v/v) to create a stock solution (500 µg/mL). This stock was further diluted with the respective solvents to obtain calibration standards ranging from 1 to 12.5 µg/mL. Absorption spectra (300–700 nm) were recorded using a quartz cuvette and a UV/Vis spectrophotometer (Epoch, BioTek Instruments, Winooski, USA). The absorption maximum of F8BT in THF (λ max = 454 nm) was determined and used for subsequent quantification. Measurements were repeated three times for each solvent and two linear calibration curves were generated (in THF: R² = 0.9766; in THF/BA: R² = 0.9993). To calculate the encapsulation efficiency, three potential sources of unencapsulated dye were considered: (1) the filtrate (dye particles washed away during filtration), (2) the wash fractions (additional particles removed during washing), and (3) the retentate (particles retained on the filter and resuspended in ethanol). PET particles (0; 0.8; 5% F8BT) were produced as previously described. During filtration and washing, the filtrate (W1) and all subsequent ethanol wash fractions (W2–W6) were collected to recover unencapsulated or non-adsorbed dye. 1) The filtrate was collected, and ethanol was evaporated under vacuum (55°C; 20 mbar; 30 min). Benzyl alcohol could not be removed under these conditions; therefore, the remaining residue was weighed and mixed with an equal volume of THF (1:1) to fully dissolve the dye. Absorbance was recorded at the previously determined maximum. 2) Each wash fraction, consisting mainly of ethanol, was evaporated under the same conditions. The dry residues were redissolved in 2 mL THF, and absorbance was measured. F8BT content in each wash step was quantified using the corresponding calibration curve. 3) Unencapsulated and or externally present dye in the retentate was determined using the intermediate product in ethanol (10 mg/mL) by taking 5 mL, evaporating the ethanol, redissolving the residue in THF (5 mL), centrifuging the particles (15,000 rpm; 5 min), and recording the absorbance of the supernatant. For PP particles (0; 0.8; 3%), the same procedure was applied, except that the filtrate was dissolved directly in 2 mL pure THF. The undesired morphology, in which F8BT remained external to the particle, was defined as the retentate, whereas the desired morphology corresponded to the internalized fraction of F8BT. Dye leaching This experiment for PET and PP was performed using the intermediate product in ethanol. Falcon tubes were prepared with 5 mL of the sample. The ethanol was evaporated (55°C; 20 mbar; 30 min), and the tubes were refilled with either THF (extreme condition) or PBS (physiological condition). Samples were centrifuged (15000 rpm; 5 min), and the absorbance of the supernatant was recorded. Afterwards, the supernatant was replaced with fresh medium, the pellet was resuspended, and the tubes were stored under light exclusion until the next measurement. Measurements were taken at the time points 0, 1, and 7 days. Dispersion and size characterization Transmission electron microscopy (TEM) TEM images were obtained from the ethanolic intermediate product using a JEOL JEM-1400 Flash transmission electron microscope operated at an accelerating voltage of 80–120 kV. Quality control (QC) dispersion protocol (intermediate product) To assess the particle size distribution of the test material a quality control dispersion protocol using the non-ionic surfactant Tween 80 was developed. The goal was to optimize the deagglomeration process to obtain an accurate understanding of the primary particle size. The MNP ethanol suspension (10 mg/mL; 200 µL) was mixed with 800 µL Tween 80 (5% w/w in ultrapure water) and was subsequently sonicated for 15 (PET) or 40 minutes (PP), resulting in a first dilution (1 mg/mL). This suspension was then diluted further by adding it directly into the laser diffraction measuring chamber until a laser obscuration of 3–5% was achieved (second dilution; ~0.3–0.4% Tween 80; ~0.2–0.4 mg/mL nanoplastic content). Quality control (QC) dispersion protocol (final product) When dispersing the final glycerol-dispersed product (40 mg/g), the test material was heated to 60°C and vortexed to ensure a homogeneous suspension and reduce viscosity. One drop (~ 25 mg suspension containing 1 mg nanoplastic) was weighed and dispersed in 1 mL of Tween 80 (5% w/w in ultrapure water) making the first dilution (1 mg/mL). This suspension was further diluted by filling up the laser diffraction measuring chamber with water and Tween 80 (400 µL; 5% w/w in ultrapure water) and adding the suspension dropwise into the device until a final Tween 80 concentration of 0.35–0.39% was reached and depending on when a laser obscuration of 3–5% was achieved (second dilution; ~0.3–0.4% Tween 80; ~ 0.02–0.04 mg/mL nanoplastic content). Biorelevant dispersion (BR) protocol (final product) When investigating aspects such as cell uptake it might be important to omit Tween 80 as a dispersing agent and use a biorelevant option instead. Therefore, BSA and serum-supplemented cell culture medium (10% FBS, 1% Penicillin/Streptomycin) were explored as BR dispersing agents. Briefly, the final glycerol-based product was heated to 60°C and sonicated as described above. MNP samples (25 ± 0.25 mg) were dispensed into 1 mL BSA solution (10% m/v in ultrapure water, filtered through a 0.45 µm PVDF membrane) to obtain a nanoplastic concentration of 1 mg/mL (first dilution). The mixture was sonicated for 30 minutes, then serum-supplemented cell culture medium was added to reach the desired concentration for further studies. This second dilution was followed by a second sonication step of 15 min (PET) and 40 min (PP due to its higher hydrophobicity) before further use. The final theoretical BSA concentration (above what is present in the serum) was 1.5% for a plastic concentration of 150 µg/mL. Particle size measurements were carried out using laser diffraction (LD) with the Mastersizer 3000 instrument (Malvern Panalytical, Malvern, UK). For each sample, 10 individual, consecutive measurements were performed, and the average particle size distribution was calculated. The measuring chamber was filled with 5–6 mL of distilled water or serum-supplemented cell culture medium (for biorelevant dispersion protocol), and the sample dispersed as described above was added until a laser obscuration of 3–5% was reached. The Mie scattering theory was applied using the following parameters: for PET, a refractive index (RI) of 1.636 and absorption of 0.01 and for PP, an RI of 1.490 and absorption of 0.01. Since the RI of the PET-F8BT and PP-F8BT mixtures was unknown, the Fraunhofer approximation setting were also used. Measurements were conducted at room temperature. Contact angle measurements Ethanolic nanoplastic suspensions (intermediate product) were dried via vacuum filtration using a 0.8 µm nylon membrane. The dried powder was transferred onto a glass slide, which had been prepared with double-sided adhesive tape, by pressing it firmly onto the tape. Full surface coverage had to be ensured to prevent gaps between particles and a leveled surface. This compression also minimized adsorption of the water droplet onto the adhesive surface. Contact angle measurements were conducted for PET nanoplastics containing 0, 0.8 and 5% w/w F8BT and PP nanoplastics containing 0, 0.8, and 3% F8BT. A drop shape analyzer (DSA30S, Krüss, Hamburg, Germany) was used applying the sessile drop technique with deionized water as the test liquid. The baseline was manually adjusted for each measurement and droplets were dispensed onto a fresh, dry plastic surface each time. The droplet volume was set to 2 µL with a dispense rate of 0.16 µL/min. Following a stabilization period of 5 sec, measurements were recorded at one frame per second, and each contact angle value was determined as the mean of six frames. For each sample, 30 individual measurements were performed and the mean value was calculated. Optical properties To evaluate the optical characteristics of F8BT, the glycerol stock dispersion was prepared according to the biorelevant dispersion protocol, resulting in a final sample concentration of 50 µg/mL. Spectral scans were performed by fixing the excitation wavelength at 460 nm and recording the corresponding emission spectrum from 300–650 nm or fixing the emission maximum at 540 nm and scanning the excitation from 300–650 nm. The background was subtracted and the final spectra were calculated and plotted. This experiment was performed three individual times. Fluorescence quantification studies The fluorescence properties of PET (0.8% and 5% F8BT) and PP (0.8% and 3% F8BT) suspensions were characterized to determine the limit of detection (LOD) and limit of quantification (LOQ) of measurements using the Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland) and furthermore the variability of sample handling was also investigated. As a comparator material, commercially available PS particles fluorescently labeled with Nile Red were used. Test materials were dispersed using the two-step biorelevant dispersion protocol. In the second dilution step, serum-supplemented cell culture medium was replaced with PBS or cell lysate to mimic procedures typically used for quantifying uptake of fluorescently labeled nanomaterials in cells [ 30 ]. Final concentrations ranging from 0.008–500 µg/mL were tested by using serial dilution steps. Briefly, cell lysate was obtained by culturing Calu-3 cells, a human bronchial epithelial cell line, at a density of 4 × 10⁵ cells/mL in 24-well plates. After 48 h incubation, the medium was changed to a fresh one, followed by another 24 h incubation at 37°C in 5 % C₂. For lysis, cells were washed three times with PBS, and 1 mL of a Triton X-100 solution (0.1 % i PBS) was added per well. After 10 min incubation to ensure cell membrane disruption, the cell layer was mechanically detached by scraping the surface with a pipette tip. Cell lysate from all wells were pooled and used as a diluent for the preparation of the dilution series. Following dispersion, diluted samples were transferred to black 96-well plates, and fluorescence was measured using the Tecan plate reader. Each dilution series was prepared as three independent experiments with three technical replicates per concentration, resulting in a total of nine data points per concentration. The plate reader settings were as follows: orbital shaking for 60 seconds at an amplitude of 1 mm prior to measurement; excitation was fixed at 460 nm and emission wavelength was set to 544 nm for F8BT-labeled particles whereas for Nile Red-labeled PS particles wavelengths at 552/636 nm were used. The LOD and LOQ were calculated using Equations 3 and 4 , which take the residual standard deviation of the linear regression into account: Eq. ( 3 ): LOD in µg/mL. $$\:LOD\left[\frac{{\mu\:}g}{mL}\right]=\:\frac{{\sigma\:}\:x\:3.3}{slope}$$ 3 Eq. ( 4 ): LOQ in µg/mL. $$\:LOQ\left[\frac{\mu\:g}{mL}\right]=\:\frac{{\sigma\:}\:x\:10}{slope}\:$$ 4 Two commonly used approaches for the determination of the standard deviation (σ) were compared: the residual standard deviation method and the intercept-based method [ 45 ], [ 46 ]. Even though LOD and LOQ are widely applied, the literature is often unclear about which approach is used, and even the Q2R1 of International Conference on harmonization (ICH) guidelines state only that standard deviation must be considered, without specifying which one [ 47 ]. This lack of clarity leads to inconsistencies in the literature and values reported there should therefore be interpreted with caution. The residual method estimates the standard deviation from the residuals (σ residuals ) of the calibration regression. This approach accounts for the spread of the actual data points and is therefore often seen as more robust. An advantage of this method is that it reflects the real scatter of the measurements and is less sensitive to outliers in the lower concentration range. However, it tends to overestimate LOD and LOQ, especially when the calibration dataset has limited points or higher noise. In contrast to that, the intercept method uses the standard deviation of the y-intercept (σ intercept ) of the calibration curve. This method usually yields lower LOD and LOQ values because it focuses on the precision of the baseline signal rather than the residual variance. It is simple to implement and widely used, but a major drawback is that it can lead to overly optimistic detection limits that might not reflect realistic conditions when the calibration curve is not perfectly linear or when there is significant heteroscedasticity (non-uniform variance of the data). Furthermore, the coefficient of variation (CV%) of the individual fluorescence values (n = 9) was calculated for each concentration to provide information on the variability of fluorescence signal associated with the dispersion and dilution process, as well as the magnitude of dilution. The following equation was used: Eq. ( 5 ): Coefficient variation in %. $$\:CV\left[\%\right]=\:\frac{{\sigma\:}\:x\:100}{mean}$$ 5 Size stability of dispersion in different media Using the biorelevant dispersion protocol, the size stability of both the first (1 mg/mL) and second dilution (100 µg/mL) was assessed over time. The particle size distribution of the first dilution was tested after storage at 4°C over a period of 7 days, to assess whether the more concentrated dispersion could be prepared in advance without losing the particle characteristics. Before each measurement, the samples were vortexed and sonicated for 30 minutes to ensure uniform dispersion. The second dilution was tested under conditions simulating in vitro exposure. i.e., the diluted suspension was incubated for 48 h at 37°C under 5% CO₂, without agitation. This setup was intended to mimic static conditions in a microtiter plate and assess potential particle agglomeration. Prior to measurement, the vial was not disturbed, replicating exposure conditions. Both stability tests were performed using the same batch of particles. Particle size measurements were performed as described above with the exception that the measuring chamber was filled with 6 mL of serum supplemented cell culture medium instead of water. Cell uptake All cell uptake experiments were performed using Calu-3 with passage numbers from 24 to 28. Conventional 2D monolayer studies The standard exposure setup was performed using a 24-well plate, in which Calu-3 cells were seeded at a density of 4 × 10⁵ cells/mL. Preliminary data (see supplementary Figure S1 ) showed that incubation of F8BT-labeled PET and PP directly in the well plates resulted in substantial adsorption of the nanomaterials to the exposed surfaces, increasing the fluorescence background to unrealistic levels (Figure S1 , electronic supplementary information). To circumvent this issue, removable Thermanox plastic coverslips were placed into the wells prior to seeding. Cells were seeded onto the coverslips and incubated for 48 h at 37°C and 5% CO₂ until approximately 80% confluency was reached. PET (5% F8BT) and PP (3% F8BT) with high dye loadings were dispersed using the biorelevant dispersion protocol (final concentration: 100 µg/mL) and 500 µL was added to each well. A vehicle control containing the equivalent amount of glycerol in BSA (10%) and cell culture medium was also included. Incubation was performed for 24 h after which the supernatant was aspirated and the wells were washed three times with PBS (500 µL) to remove particles non-internalized by cells. The coverslips were then transferred into a fresh 24-well plate and 1 mL of Triton X-100 (0.1% in PBS) was added to lyse the cells. After 10 min incubation, the cell layer was gently scraped using a pipette tip to ensure complete detachment. The resulting lysate, including cellular debris, was transferred directly to a black 96-well plate for fluorescence measurement utilizing the Tecan plate reader. The same measurement settings, as previously described in the section about fluorescence quantification studies were used. The experiment was repeated independently three times, each with technical triplicates. FlowCube: monolayers in vertical placement Due to the low density of PP test materials, the classic static exposure setup in a well plate is not suited to achieve measurable cellular uptake, as the particles tend to float and do not interact with the cell monolayer on the bottom. To address this, the FlowCube system was developed to ensure consistent particle-cell contact. In this setup, cells were grown on square glass slides that formed the four vertical walls of a cube-like chamber. In addition to that, a magnetic stirrer was positioned at the bottom of the chamber to maintain homogenous particle suspension and further promote continuous contact with the cell monolayer. Prior to cell seeding, glass slides were functionalized with poly-L-lysine (0.05% w/v in water) by placing them in a shaker for 20 min with the solution fully covering them, followed by three washing steps with ultrapure water (30 min each). The slides were then air-dried and sterilized under UV light for 30 min. Treated slides were placed into a 24-well plate designed for suspension cells to prevent cell attachment to the well surface. Calu-3 cells were seeded onto the slides at a density of 4 × 10⁵ cells/mL and incubated for 48 h. After medium change, cells were incubated for another 24 h to reach ~ 80% confluency. Once confluent, the slides were transferred into the cube chamber, where they formed the inner walls or were placed into a 24-well plate as a control. PET (5% F8BT) and PP (3% F8BT) were dispersed using the biorelevant dispersion protocol at a concentration of 100 µg/mL and added to the FlowCube chamber (1.2 mL per cube) or to the well plate (500 µL per well). The following exposure conditions were tested: 1) cubes without magnetic stirring (non-stir), 2) cubes with magnetic stirring (stir) to simulate shear stress, and 3) conventional well plate exposure (well). After 24 hours of incubation, cellular uptake was evaluated via fluorescence microscopy and flow cytometry. Fluorescence microscopy Following exposure, cells were fixed and stained directly on the slides. The slides were placed into fresh well plates and washed with PBS. Slides were fixed with 4% paraformaldehyde (100 µL, 30 min) and washed twice with PBS (1 mL each). For staining, 100 µL of Hoechst 33342 (1 mg/mL) was added per well and incubated in the dark for 30 min. The staining solution was then removed, and slides were washed again twice with PBS (1 mL each). One drop of mounting medium was placed on a microscope slide and the stained coverslip was placed cell-side down onto it to preserve the monolayer for imaging. Fluorescence microscopy was performed using a Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with a 63× oil immersion objective. Filter sets used were: blue (excitation 335–383 nm, emission 420–470 nm), green (excitation 460–488 nm, emission 500–557 nm) and red (excitation 567–602 nm, emission 615–4095 nm). Exposure time was kept constant at 360 ms for all samples, except for slides that were exposed to PET under well conditions (12 ms). Images were post-processed using the ZEN software (version 3.1.1, Carl Zeiss Microscopy, Jena, Germany) for brightness and contrast adjustment. Flow cytometry For flow cytometry the exposed slides were washed with PBS (1.2 mL for cube samples; 500 µL for well samples) and transferred into new well plates. Trypsin (150 µL) was added to each well and incubated for 5 minutes at 37°C and 5% CO₂. To stop enzymatic activity, complete cell culture medium (400 µL of) was added. The resulting cell suspension was transferred to fluorescence activated cell sorting (FACS) tubes and analyzed immediately using a Gallios flow cytometer (Beckman Coulter, Brea, USA). Fluorescence of internalized F8BT-labeled particles was detected in the FL1 channel (525/40 nm, FITC settings). Data acquisition was performed using the Kaluza software (Beckman Coulter, Brea, USA). All experiments were conducted independently three times, each with four technical replicates per treatment condition. Statistical analysis All statistical analyses were performed with Prism Graphpad (version 10.3.1, GraphPad Software, San Diego, USA). 3. Results and discussion Impact of F8BT co-precipitation on PET and PP nanoparticle properties The production of PET and PP nanoplastics was previously optimized [ 16 ] and used as a starting point for this labeling study. Pilot studies demonstrated that colloidally stable nanoparticles could be produced with up to 5 % ww F8BT per mass of PET and up to 3 % ww F8BT per mass PP. Increasing F8BT content above these values led to irreversible particle aggregation. To investigate the impact of F8BT incorporation on particle characteristics, all further studies were conducted with a low (0.8 % ww) and a higher F8BT content (5 % ad 3 % ww F8BT per mass PET / PP, respectively). Particle production resulted in yields of 90.4 ± 3.9 % fr PET across eight batches and 90.6 ± 6.1 % fr PP across 17 produced batches, with no effect of F8BT labeling on the production yield observed. TEM was used to investigate particle morphology, while laser diffraction was used as a complementary method to characterize the particle size distribution, including the presence of aggregates[ 16 ] (Fig. 2 ). When preparing the samples for TEM, it should be noted that the intermediate product (ethanolic PET/PP suspension) was applied directly to the TEM grid without use of Tween 80 as a dispersion agent, since Tween 80 can reduce image quality. With this preparation technique aggregation could not be prevented. In contrast, Tween 80 was used as a dispersant for particle size measurements using the quality control (QC) dispersion protocol which was previously validated and results in only minor aggregate formation [ 16 ]. During TEM analysis, PET nanoparticles were more stable than PP nanoparticles, which is attributed to the higher melting point of PET and its increased thermal stability under the electron beam. TEM images of PET samples unexpectedly revealed the presence of spindle-shaped particles next to smaller, more spherical particle aggregates for both 0.8% and 5% F8BT content (Fig. 2 A-B). Previous scanning electron microscopy studies of unlabeled PET test materials did not show spindle-shaped particles, indicating that this unexpected morphology is caused by the co-precipitation with F8BT. This hypothesis is further supported by a comparison of the particle size distribution curves of F8BT-labeled PET versus non-labeled PET nanomaterials (Fig. 2 and Table 1 ). Labeled PET nanoparticles exhibit an unusual shift towards smaller particles sizes (Fig. 2 A-B; right panel), which is likely the result of altered diffraction patterns caused by the anisotropic, spindle-shaped particles in the suspension. This phenomenon is more pronounced with a higher F8BT content (5%), indicating that increases in F8BT are likely to increase the number of spindle-shaped particles in the suspension. It is postulated that these structures may form during precipitation due to co-localization and enhanced miscibility of F8BT and PET at the interface between solvent and non-solvent, thus influencing precipitation kinetics and particle morphology. A positive aspect of this observation is that a mixture of spherical and spindle-shaped particles can better reflect the more heterogeneous morphology of environmental nanoplastics, while still maintaining a similar size distribution and surface properties characteristic of test materials. While most fluorescently labeled MNPs reported in the literature exhibit a spherical morphology, this study expands the diversity of existing test material (see supplementary Table S2). As mentioned, TEM images of PP nanomaterials were difficult to produce due to beam-induced melting of the materials during the imaging procedure. Images of the deformed materials are nonetheless depicted (Fig. 2 C-D). The laser diffraction data (Fig. 2 C-D; right panel) is more informative, showing that co-precipitation of F8BT and PP has only a minor impact on the particle size distribution, indicating a lack of particles with a spindle shape. This may be a result of the relatively large difference in solubility parameters between F8BT and PP, indicating a reduced miscibility between the two polymers and therefore a different co-precipitation behavior. Interestingly, a statistical comparison of the D10 and D50 values from n = 5 batches of the labeled vs non-labeled PP nanomaterials (Table 1 ) does reveal a small but significant decrease in particles sizes for batches produced with 3% F8BT content. This could indicate that a small fraction of elongated particles could also exist within the labeled PP test materials. Table 1 also demonstrates that the batch-to-batch variability in particle size distribution is suitably low and the fraction of particles in the submicron size range generally meets previously defined targets (i.e. 85% of the test materials should be < 1 µm [ 16 ]). Table 1 Batch-to-batch reproducibility of particle size distributions of the intermediate product. Student T-tests were used to compare differences between D10 and D50 values in the labeled vs their corresponding non-labeled group. * Denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001. Polymer % F8BT content (# batches) D10 [µm] Mean ± SD (CV%) D50 [µm] Mean ± SD (CV%) D90 [µm] Mean ± SD (CV%) % < 1 µm Mean ± SD (CV%) PET 0 (n = 5) 0.075 ± 0.009 (11.8%) 0.173 ± 0.023 (13.6%) 0.569 ± 0.198 (34.7%) 92.4 ± 1.3 (1.4%) 0.8 (n = 3) 0.079 ± 0.010 (12.7%) 0.225 ± 0.040 (17.8%) 3.043 ± 0.511 (16.8%) 81.5 ± 3.5 (4.3%) 5 (n = 5) 0.039 ± 0.010*** (25.4%) 0.108 ± 0.019** (17.6%) 1.168 ± 0.737 (63.2%) 89.2 ± 5.3 (5.9%) PP 0 (n = 5) 0.130 ± 0.014 (10.5%) 0.256 ± 0.025 (9.6%) 2.900 ± 3.270 (112.8%) 87.3 ± 6.4 (7.3%) 0.8 (n = 5) 0.132 ± 0.010 (7.4%) 0.257 ± 0.022 (8.7%) 3.365 ± 2.671 (79.4%) 87.5 ± 4.6 (5.2%) 3 (n = 7) 0.109 ± 0.012* (11.3%) 0.221 ± 0.025* (11.3%) 2.913 ± 3.416 (117.3%) 89.2 ± 5.0 (5.5%) To examine whether the medium exchange between ethanol (intermediate product) and glycerol (final product) influenced the particle size distribution, particle size distributions of test materials with the higher F8BT content were compared before and after medium exchange. For both PET and PP, no significant differences were observed across four batches (PET: p = 0.41; PP: p = 0.30) in the particle size distribution between the intermediate and final product. (Fig. 3 A-B). The influence of the type of dispersing agent on the particle size distribution was also compared (Fig. 3 C-D). As discussed previously, the QC dispersion protocol uses an initial high concentration of the synthetic surfactant, Tween 80, to achieve complete dispersion with the aim of characterizing the “true” particle size distribution of the materials. In contrast, the presence of Tween 80 may negatively impact many biological assays devised for testing the effects of MNPs. To avoid this, biorelevant (BR) dispersion protocols have been developed and tested [ 16 ]. Here we report an optimized BR dispersion method, which uses a concentrated aqueous BSA solution (10 % wv) to dilute the highly concentrated glycerol suspension to a 1 mg/mL stock suspension (first dilution), followed by a second dilution in cell culture medium to the desired working concentration. BSA was chosen as a dispersing aid based on pilot studies showing that it achieves dispersion more effectively than complex protein mixtures, such as FBS which led to major aggregation in comparison (ESI Figure S2). Since BSA is a pure protein, it likely interacts more consistently with the particle surface, resulting in improved colloidal stability. Typically, dispersions in cell culture media are more challenging than in water due to their high salt concentrations, which promote aggregation of hydrophobic particles [ 48 ], [ 49 ], [ 50 ], [ 51 ], [ 52 ]. Similar to observations by Wimmer et al. [ 16 ], labeled PET showed excellent dispersion using the BR protocol, while labeled PP agglomerated forming 1–10 µm sized aggregates. Despite extensive optimization of the BR dispersion protocol, the extremely hydrophobic surface of the pristine PP nanoplastics (with or without labels) promotes aggregation, unless more aggressive dispersion protocols using synthetic dispersants, such as Tween 80, are used. Encapsulation efficiency and leaching studies As summarized in Fig. 1 , the two particle production methods differ considerably in the choice of solvents, concentration of polymer in the solvent, temperature used, and the volume ratio of anti-solvent to solvent. All these factors are presumed to influence the incorporation of F8BT within the nanoplastics as well as the morphology of the nanoparticles. Given the complexity, it is not possible to accurately predict F8BT location in the particle systems, although some hypotheses may be generated. Given the similar solubility parameter values of F8BT and PET, we hypothesized that these two polymers would show a better miscibility, resulting in a higher amount of F8BT within the particle interior (either miscible, core-shell or occluded). In contrast, the larger differences in F8BT and PP solubility parameters were hypothesized to result in a lower miscibility between the two particles and possibly a higher amount of non-encapsulated F8BT or F8BT on the particle exterior. Due to the very low F8BT concentrations present in the systems, it is challenging to characterize encapsulation efficiency and particle morphology using commonly reported methods such as nuclear magnetic resonance (NMR)[ 53 ], [ 54 ] or TEM [ 55 ]. Instead, we exploited the excellent solubility of F8BT in THF (PET and PP are insoluble in THF) to separate internal F8BT from the external fraction. First, the fraction of free F8BT recovered in the ethanol washing liquid was determined. Secondly, particles captured on the filter following washing were subjected to incubation in THF to isolate and quantify non-encapsulated or externally bound F8BT. Finally, the theoretical amount of internal F8BT was calculated by subtracting the sum of the two fractions (wash loss and external F8BT) from the original mass. For both PET and PP test materials (Fig. 4 A-B), the amount of F8BT present in the ethanol wash was < 1.5 %. As hypotheized, the fraction of non-encapsulated or externally bound F8BT was lower for PET test materials (~ 15 %) compared t PP test materials (~ 25 %). No signifcant differences were observed between the lower and higher F8BT contents in both test material types. A fundamental challenge in fluorescently labeling MNPs is dye leaching [ 25 ]. It has been reported that even with commercially available products, the fluorophore can leach out over time [ 56 ], [ 57 ]. To evaluate F8BT leaching from the labeled PET/PP nanoplastics, samples were incubated for seven days at room temperature in either 1) THF, to simulate aggressive conditions most favorable for F8BT extraction, or 2) PBS, mimicking an aqueous biological medium (Fig. 4 C-D). No F8BT release into the PBS was measurable for any of the samples at any timepoint, indicating that even externally bound F8BT remained particle bound. When incubated in THF, non-encapsulated and externally bound F8BT was dissolved immediately (day 0 value) followed by a marginal further leaching of F8BT into the solvent over the next 24 hours, after which no further leaching was observed. A pilot study explored washing the ethanolic suspension with THF to remove external F8BT. Unfortunately, this resulted in irreversible aggregation and must be further optimized to be useful (supplementary Figure S3). Surface hydrophobicity To further assess whether F8BT alters the surface hydrophobicity of the labeled test materials, contact angle measurements were conducted on dried films of compacted PET and PP nanoparticles with varying F8BT content. It was hypothesized that increasing the amount of F8BT could lead to altered surface hydrophobicity, which could be reflected in the water contact angle. This would have implications for dispersion behavior, biological interactions and wettability. The contact angles of unlabeled PET (108.8 ± 16.8°) and PP (135.1 ± 10.2°) (Fig. 5 ) are very similar to previous investigations [ 16 ], showing that unlabeled PP nanoplastics are significantly more hydrophobic than PET nanoplastics (i.e. larger contact angle). Co-precipitation of PP with increasing amounts of F8BT showed a trend towards very minor decreases in median contact angle, which were not significant. In contrast, addition of either 0.8 % o 5 % ww F8BT to PET increased the median contact angle significantly, indicating an increase in hydrophobicity and thus the presence of F8BT at the particle surface. It should be noted that the contact angle measurements show a high level of variability, likely due to the more complex sample preparation, where surface irregularities or incomplete coverage of the measuring area could have affected the outcome. While the F8BT label does not appear to affect surface hydrophobicity by much under these preparation conditions, further evaluation using complementary techniques like zeta potential or other surface characterization methods such as X-ray photoelectron spectroscopy (XPS) [ 58 ], [ 59 ] or time-of-flight secondary ion mass spectrometry (ToF-SIMS) [ 60 ] could provide more detail about surface composition and dye localization. Optical properties Particle morphology impacts the optical properties of π-conjugated polymers, such as F8BT [ 31 ], [ 61 ]. For example, nanoparticles made of F8BT alone (stabilized with surfactants) exhibit very low limits of detection (LOD) / quantification (LOQ), whereas co-precipitation of F8BT with a second polymer decreases the sensitivity ~ 10-fold (Table 2 ) [ 30 ], [ 32 ]. Fluorescence quenching of F8BT following encapsulation is a commonly observed phenomenon associated with low amounts of the conjugated polymer in the system, aggregation-induced quenching and a coiled conformation of the embedded fluorescent polymer in the matrix [ 31 ]. Despite quenching, these detection levels are still comparable to current methods for nanoplastic quantification, such as pyrolysis-GC/MS and TED-GC-MS (supplementary Table S1 ). Table 2 Limits of detection/quantification for different F8BT nanoparticle systems compared to fluorescence-labeled polystyrene beads. System LOD [µg/mL] LOQ [µg/mL] F8BT 100% [ 30 ] ~ 0.03 ~ 0.09 5% w/w F8BT / 95% w/w poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) [ 31 ], [ 32 ] ~ 0.3 ~ 0.9 Commercially available fluorescence labeled yellow-green polystyrene beads [ 30 ] 1.2 3.6 4-[1-Cyano- 2-[4-(Diethylamino) − 2-hydroxyphenyl]ethenyl] − 1-ethylpyridinium (PCP) / PS [ 62 ] 0.53 1.59 (E)-2-(2-(4-(dimethylamino)nanphthalen-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (HCY) / PS [ 63 ] 0.15 0.45 9-(2,2-dicyanovinyl)julolidine (DCVJ) / PS [ 64 ] 0.56 1.68 Fluorescence excitation and emission scans were performed (Fig. 6 A-B), showing no significant changes to the excitation and emission spectra between fully soluble F8BT measured in THF (λ Ex = 480 nm; λ Em = 545 nm) and encapsulated F8BT in PET or PP nanoparticles (PET: λ Ex = 480 nm and λ Em = 542 nm; PP: λ Ex = 480 nm and λ Em = 541 nm). The spectra for F8BT encapsulated in PET and PP nanoplastics are very similar to those reported for F8BT embedded within a PEG-PLGA polymer matrix, which ranged from 537–541 nm [ 31 ], depending on manufacturing conditions. Detection limits (LOD and LOQ) and experimental variability were also determined using different dispersion protocols. The method (residual vs intercept) used for LOD and LOQ determination strongly influences reported values. Since guidelines often remain vague, both calculation methods were compared (LOD: Fig. 6 C-D ) . LOD values for F8BT-labeled PET ranged between 0.2–0.7 µg/mL (intercept method) with a higher sensitivity when 5% F8BT was used. These values are comparable to previously reported PEG-PLGA systems also containing 5% F8BT [ 30 ], [ 31 ], [ 32 ] as well as to other systems using various fluorophores and MNPs reported in the literature (Table 2 ). LOD values of PP (intercept method) also varied between 0.2–0.9 µg/mL but showed no influence of F8BT content or dispersion protocol on the LOD. Since the QC dispersion protocol resulted in a much lower LOD, it is hypothesized that aggregation of PP test materials during the BR dispersion process may cause aggregation-induced quenching and increased variability in the LOD/LOQ. In all cases, F8BT-labeled PET and PP nanoplastics had significantly lower LOD values (p < 0.001) compared to commercially available Nile Red-labeled polystyrene beads, which were diluted using the BR dispersion protocol into PBS (LOD residual : 32.1 ± 8.3 µg/mL; LOD intercept : 11.4 ± 2.9 µg/mL). Handling of suspensions, especially performing dilutions, is known to be associated with greater error than handling solutions. When diluting into complex media, the variability increases further [ 65 ]. In serial dilutions, generally the lower concentrations near the LOD/LOQ are associated with the greatest error, which is why it can be informative to routinely assess the CV% of measured fluorescence values of each concentration in the serial dilution across multiple experimental replicates. In an early pilot study testing the variability of serial dilutions prepared using non-optimized BR dispersion protocols with FBS (supplementary Figure S4) was investigated. Concentrations > 15 µg/mL were associated with low variability (CV < 10 %),while concentrations < 15 µg/mL showed test material-specific differences: PET CV: 30–80 %; P CV: 50–130 %. ollowing optimization of the BR dispersion protocol, the variability in the critical concentration range < 15 µg/mL could be reduced to < 10 % fr nearly all media (Fig. 6 E-F), with the exception PP diluted in cell lysate (CV: 20–50 %).This experiment highlights the importance of optimized dispersion protocols and well-trained operators when handling fluorescently labeled nanoplastic suspensions, whereby a CV < 10 % btween replicate dilutions is ideal. Application of F8BT-labeled PET and PP test materials in cell uptake studies using the FlowCube The next step was to characterize the in vitro performance of the F8BT-labeled test systems in cell culture studies. To evaluate how well the F8BT-labeled nanoplastics remain in dispersion over time, two complementary experiments were conducted. The first focused on the size stability of the stock dispersions (i.e. first dilution of the glycerol suspension into an aqueous medium at 1 mg/mL) when stored at 4°C. This is useful for users who would like to store stock dispersions for multiple use over a given period. The second experiment aimed to simulate the particle size stability in cell culture medium under incubation conditions (37°C, 5% CO₂) over 48 h, representing a typical cell exposure duration. Stability studies over one week storage at 4°C showed that PET materials diluted from glycerol into 10% BSA (1 mg/mL) maintained dispersion stability for at least three days, while from day 5 onwards, a shift towards larger particles became more pronounced (Fig. 7 A). Dispersion stability was defined as the percentage of particles < 1 µm. With PP test materials, two distinct size populations were present at the first timepoint. However, a slight trend toward deagglomeration, possibly due to improved surface coating of the PP sample with BSA over time, was observed but requires further investigation (Fig. 7 B), indicating that storage of the first aqueous dilution (1 mg/mL) at 4°C is feasible. The colloidal stability of test materials diluted into cell culture medium and incubated at 37°C was also tested. Both PET and PP test materials were colloidally stable up to 24 h but showed evidence of aggregation after 48 h (Fig. 7 C, D). This suggests that particles incubated in cell culture medium up to 24 h will show diffusion and sedimentation/flotation behavior characteristic of single particles, while beyond 48 h, the diffusion and sedimentation/flotation behavior of agglomerate structures will need to be considered for dosimetry purposes, as well as mechanisms of particle cell interactions [ 30 ], [ 66 ], [ 67 ]. To assess whether F8BT-labeled PET and PP particles are suitable for quantifying cellular uptake, Calu-3 cells, a commonly used model for evaluating effects on lung epithelial tissues, were employed as a model. Two complementary experimental setups were compared, both with a 24 h exposure duration and an administered dose of 100 µg/mL. The first set up was comprised of a conventional well plate-format, where cells were grown under submerged conditions on round glass cover slips positioned at the bottom of the well. The well-known limitation of this set up is that it is unable to accommodate low-density test materials like PP, which will float to the surface of the medium during incubation thereby reducing particle-cell interactions [ 67 ], [ 68 ], [ 69 ]. This behavior was verified using fluorescence spectroscopy to quantify nanoparticle uptake in the Calu-3 cells. Only the fluorescence associated with F8BT-labeled PET was quantifiable (Fig. 8 A) and the amount detected (0.22 ± 0.11 µg/mL) was only marginally above the LOD (~ 0.2 µg/mL). This equated to ~ 0.2 % of the adminitered dose. As a comparison, the uptake of F8BT-labeled PEG-PLGA nanoparticles by a fibroblast cell line was 0.5–0.8 % [ 32 ] . To overcome the limitations associated with particle buoyancy, the dynamic flow-based exposure system, FlowCube (Fig. 8 B), was used. Calu-3 cell monolayers grown on square cover slips were placed vertically into the walls of the chamber and test materials are added to the medium in the center of the chamber. Particle-cell interactions occur either by diffusion only (cube static; non-stirred conditions) or diffusion combined with stirring-induced advection and hydrodynamic shear forces (cube dynamic; stirred conditions). As a control, slides can also be placed at the bottom of the cube to simulate conventional well-plate conditions (well static). In the FlowCube experiment, fluorescence spectroscopy could not be used for quantification, since the amount of particle uptake was below the F8BT detection limits. Instead, semi-quantification of the F8BT-fluorescence signal was measured by flow cytometry. The results show that the FlowCube is indeed able to overcome density-related issues associated with cell exposure to buoyant nanomaterials. Flow cytometry showed that similar amounts of F8BT-labeled PET and PP test materials were detected above the background fluorescence in the cube set up (Fig. 8 C-D). Since no significant differences were observed between the static (non-stirred) and dynamic (stirred) conditions, it may be assumed that uptake was primarily caused by consistent diffusion-driven particle-cell contact rather than by centrifugal force. As expected, when cells were positioned horizontally at the bottom of the cube (well static conditions), PET-associated fluorescence was approximately 6-fold higher than that of the vertical cell placement, while PP-associated fluorescence was not detectable. The flow cytometry data was supported by fluorescence microscopy of stained Calu-3 monolayers, which shows the same trend (Fig. 8 E-H; images from the static condition are included in the supplementary; Figure S5). These results confirm that co-precipitated F8BT-labeled nanoplastics are suitable for tracking and quantifying cellular uptake via fluorescence spectroscopy, flow cytometry, and fluorescence confocal imaging. 4. Conclusions This study provides an innovative approach to fluorescence labeling of submicron sized PET and PP test materials using the bright and stable polymer dye, F8BT. Using a combination of complementary methodologies, we showed that the co-precipitation of the fluorescent polymer, F8BT, with PET leads to a high encapsulation efficiency of the dye (~ 85%) with very low leaching potential but also promotes the formation of a subset of spindle-shaped particles within the suspension. This mixture of particle morphologies is interesting from both a polymer chemistry perspective and as an example of an MNP test material that shows greater morphological diversity and therefore better mimics environmentally relevant nanoplastics. F8BT was also suitable for labeling PP test materials, with an encapsulation efficiency of ~ 75% and little impact on particle morphology. The fluorescence label provided a detection limit as low at ~ 0.2 µg/mL using fluorescence spectroscopy, which is comparable to other current MNP quantification methods. Leaching experiments confirmed that F8BT was retained within the plastic particles even after prolonged exposure to PBS, an aqueous biological medium. Additionally, fluorescence labeling of the materials provided insights into the reproducibility of handling suspensions, which was useful in validating dispersion protocols within different bulk media. In vitro experiments were used to investigate particle-cell interactions and uptake using a novel cell exposure model, the FlowCube. The vertical exposure system of the FlowCube overcame dosimetry issues associated with buoyant particles. Cellular uptake of both low and high-density nanoplastics could be measured using the F8BT label, indicating sufficient label sensitivity for future biological applications. Overall, this work contributes valuable information on the development of nanoplastic test materials for environmental and biological studies. Declarations Funding This work was supported by funding from the EU's H2020 framework program for research and innovation under grant agreement no. 965173 (Imptox). Author Contribution M.V.N.H. and L.A.D. wrote the main manuscript text. M.V.N.H., M.A., Y.N.M. and L.W. generated experimental data. M.V.N.H. prepared all figures with contributions from Y.N.H. (Figure 2) and M.A. (Figure 8). D.J.S.V. compiled the literature overview in Tables 2, S1 and S2. T.C.V., M.A.G., F.K., R.D.H. and L.A.D. contributed to the study design, supervision and funding acquisition. All authors reviewed the manuscript. Acknowledgement This work was supported by funding from the EU H2020 framework program for research and innovation under grant agreement no. 965173 (Imptox). The authors would like to warmly thank Ing. Claudia Mitterer for granting access to the drop shape analyser and Patrick Treacy and Johannes Szilvassy for participating in the pilot dilution studies. We also want to thank Markus Kirchner for providing the fluorescently labeled polystyrene test particles. Data Availability The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. References Amelia TSM, Khalik WMAWM, Ong MC, Shao YT, Pan HJ, Bhubalan K. Marine microplastics as vectors of major ocean pollutants and its hazards to the marine ecosystem and humans, Progress in Earth and Planetary Science 2021 8:1 , vol. 8, no. 1, pp. 1–26, Jan. 2021, 10.1186/S40645-020-00405-4 Yang H, Chen G, Wang J. Microplastics in the Marine Environment: Sources, Fates, Impacts and Microbial Degradation. Toxics. Feb. 2021;9(2). 10.3390/TOXICS9020041 . Marcharla E, et al. Microplastics in marine ecosystems: A comprehensive review of biological and ecological implications and its mitigation approach using nanotechnology for the sustainable environment. Environ Res. Sep. 2024;256:119181. 10.1016/J.ENVRES.2024.119181 . O’Brien S, et al. There’s something in the air: A review of sources, prevalence and behaviour of microplastics in the atmosphere. Sci Total Environ. May 2023;874:162193. 10.1016/J.SCITOTENV.2023.162193 . Torres-Agullo A, Karanasiou A, Moreno T, Lacorte S. Overview on the occurrence of microplastics in air and implications from the use of face masks during the COVID-19 pandemic. Sci Total Environ. Dec. 2021;800:149555. 10.1016/J.SCITOTENV.2021.149555 . Rainieri S, Barranco A. Microplastics, a food safety issue? Trends Food Sci Technol. Feb. 2019;84:55–7. 10.1016/J.TIFS.2018.12.009 . Mir MA et al. Jun., Microplastics in food products: Prevalence, artificial intelligence based detection, and potential health impacts on humans, Emerg. Contam. , vol. 11, no. 2, p. 100477, 2025, 10.1016/J.EMCON.2025.100477 Rajvanshi J, et al. An analytical review on revamping plastic waste management: exploring recycling, biodegradation, and the growing role of biobased plastics. Environ Sci Pollut Res. Apr. 2024;1–19. 10.1007/S11356-024-33333-7/FIGURES/5 . Zhao H, Mu S, Wang W, Li X. Potential threats of environmental microplastics to the skeletal system: current insights and future directions. Front Endocrinol (Lausanne). 2025;16:1658056. 10.3389/FENDO.2025.1658056 . Nozari asl R, Jaafarzadeh Haghighi Fard N, Jahedi F, Khaksar MA, Shenavar B. Systematic review of pulmonary toxicity induced by microplastics and nanoplastics: Insights from in vivo and in vitro studies, Toxicologie Analytique et Clinique , vol. 37, no. 2, pp. 223–241, Jun. 2025, 10.1016/J.TOXAC.2024.12.002 Marycleopha M, Balarabe BY, Kumar S, Adjama I. Exploring the Impact of Microplastics and Nanoplastics on Macromolecular Structure and Functions. J Appl Toxicol. Jan. 2026;46(1):22–41. 10.1002/JAT.4915 . Al-Sid-Cheikh M, Rowland SJ, Stevenson K, Rouleau C, Henry TB, Thompson RC. Uptake, Whole-Body Distribution, and Depuration of Nanoplastics by the Scallop Pecten maximus at Environmentally Realistic Concentrations, 2018, 10.1021/acs.est.8b05266 Lopez GL, Lamarre A. The impact of micro- and nanoplastics on immune system development and functions: Current knowledge and future directions. Reprod Toxicol. Aug. 2025;135:108951. 10.1016/J.REPROTOX.2025.108951 . van den Akker K, Mandemaker LDB, Dorresteijn JM, Amaral-Zettler LA, Weckhuysen BM, Meirer F. Fluorescent nanoplastics: What steps are needed towards a representative toolkit? Microplastics and Nanoplastics 2025 6:1 , vol. 6, no. 1, p. 4-, Dec. 2025, 10.1186/s43591-025-00159-0 Crosset-Perrotin G et al. Jun., Production, labeling, and applications of micro- and nanoplastic reference and test materials, Environ. Sci. Nano , vol. 12, no. 6, pp. 2911–2964, 2025, 10.1039/D4EN00767K Wimmer L, et al. A quality-by-design inspired approach to develop PET and PP nanoplastic test materials for use in in vitro and in vivo biological assays. Environ Sci Nano. 2025. 10.1039/d4en01186d . Merdy P et al. Aug., Nanoplastic production procedure for scientific purposes: PP, PVC, PE-LD, PE-HD, and PS, Heliyon , vol. 9, no. 8, p. e18387, 2023, 10.1016/J.HELIYON.2023.E18387 Chen H, Hu Q, Li W, Cai X, Mao L, Li R. Approaches to Nanoparticle Labeling: A Review of Fluorescent, Radiological, and Metallic Techniques. Environ Health. Aug. 2023;1(2):75–89. 10.1021/ENVHEALTH.3C00034 . Sullivan GL, Gallardo JD, Jones EW, Hollliman PJ, Watson TM, Sarp S. Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF). Chemosphere. Jun. 2020;249:126179. 10.1016/J.CHEMOSPHERE.2020.126179 . Duemichen E, Eisentraut P, Celina M, Braun U. Automated thermal extraction-desorption gas chromatography mass spectrometry: A multifunctional tool for comprehensive characterization of polymers and their degradation products. J Chromatogr A. May 2019;1592:133–42. 10.1016/J.CHROMA.2019.01.033 . Zytowski E, Baldermann S. Thermal Desorption and Extraction Coupled With Gas Chromatography and Mass Spectrometry for the Quantification of Polystyrene Nanoplastic in Pak Choi, Rapid Communications in Mass Spectrometry , vol. 39, no. 14, p. e10046, Jul. 2025, 10.1002/RCM.10046 Rødland ES, et al. A novel method for the quantification of tire and polymer-modified bitumen particles in environmental samples by pyrolysis gas chromatography mass spectroscopy. J Hazard Mater. Feb. 2022;423(10):127092. 10.1016/j.jhazmat.2021.127092 . Al-Sid-Cheikh M, Rowland SJ, Kaegi R, Henry TB, Cormier MA, Thompson RC. Synthesis of 14C-labelled polystyrene nanoplastics for environmental studies, Communications Materials 2021 1:1 , vol. 1, no. 1, p. 97-, Dec. 2020, 10.1038/s43246-020-00097-9 Clark NJ, et al. Determining the accumulation potential of nanoplastics in crops: An investigation of 14C-labelled polystyrene nanoplastic into radishes. Environ Res. Nov. 2025;284:122687. 10.1016/J.ENVRES.2025.122687 . Liu Y, Li J, Parakhonskiy BV, Hoogenboom R, Skirtach A, De Neve S. Labelling of micro- and nanoplastics for environmental studies: state-of-the-art and future challenges. J Hazard Mater. Jan. 2024;462:132785. 10.1016/J.JHAZMAT.2023.132785 . Staufer T, et al. Biodistribution of nanoplastics in mice: advancing analytical techniques using metal-doped plastics. Commun Biol. Dec. 2025;8(1):1247. 10.1038/S42003-025-08709-1 . Villacorta A, et al. Fluorescent labeling of micro/nanoplastics for biological applications with a focus on true-to-life tracking. J Hazard Mater. Sep. 2024;476:135134. 10.1016/J.JHAZMAT.2024.135134 . Merdy P, Bonneau A, Delpy F, Lucas Y. Fluorescent labelling as a tool for identifying and quantifying nanoplastics, RSC Adv. , vol. 14, no. 50, pp. 37610–37617, Nov. 2024, 10.1039/D4RA04526B Behnke T, Würth C, Laux EM, Hoffmann K, Resch-Genger U. Simple strategies towards bright polymer particles via one-step staining procedures. Dyes Pigm. Aug. 2012;94(2):247–57. 10.1016/J.DYEPIG.2012.01.021 . Ahmad Khanbeigi R et al. Mar., Surface Chemistry of Photoluminescent F8BT Conjugated Polymer Nanoparticles Determines Protein Corona Formation and Internalization by Phagocytic Cells, Biomacromolecules , vol. 16, no. 3, pp. 733–742, 2015, 10.1021/bm501649y Abelha TF, et al. Bright conjugated polymer nanoparticles containing a biodegradable shell produced at high yields and with tuneable optical properties by a scalable microfluidic device. Nanoscale. Feb. 2017;9(5):2009–19. 10.1039/C6NR09162H . Modicano P, et al. Does Encapsulation of π-Conjugated Polymer Nanoparticles within Biodegradable PEG–PLGA Matrices Mitigate Photoinduced Free Radical Production and Phototoxicity? Adv Ther (Weinh). Jan. 2025;8(1). 10.1002/ADTP.202400190 . de Brito EB, et al. Synthesis and characterization of novel fluorene–based green copolymers and their potential application in organic light-emitting diodes. J Mater Res Technol. Jan. 2024;28:4317–33. 10.1016/J.JMRT.2023.12.249 . Vandenburg HJ, Clifford AA, Bartle KD, Carlson RE, Carroll J, Newton ID. A simple solvent selection method for accelerated solvent extraction of additives from polymers, Analyst , vol. 124, no. 11, pp. 1707–1710, Jan. 1999, 10.1039/A904631C Hanschmann B. Precipitation of Polypropylene and Polyethylene Terephthalate Powders Using Green Solvents via Temperature and Antisolvent-Induced Phase Separation, Advances in Polymer Technology , vol. 2023, no. 1, p. 7651796, Jan. 2023. 10.1155/2023/7651796 Wu Y, et al. Molecular behavior of silicone adhesive at buried polymer interface studied by molecular dynamics simulation and sum frequency generation vibrational spectroscopy †. Royal Soc Chem Soft Matter. 2024. 10.1039/d4sm00407h . Urbano L, et al. Influence of Surfactant Structure on Photoluminescent π-Conjugated Polymer Nanoparticles: Interfacial Properties and Protein Binding. Langmuir. 2018;34:6125. 10.1021/acs.langmuir.8b00561 . Dovgolevsky E, Kirmayer S, Lakin E, Yang Y, Brinker CJ, Frey GL. Self-assembled conjugated polymer–surfactant–silica mesostructures and their integration into light-emitting diodes. J Mater Chem. Jan. 2008;18(4):423–36. 10.1039/B713170D . Costa GP, Choi P, Stoyanov SR, Liu Q. The temperature dependence of the Hildebrand solubility parameters of selected hydrocarbon polymers and hydrocarbon solvents: a molecular dynamics investigation. J Mol Model. Jul. 2024;30(7):196. 10.1007/S00894-024-05929-W . Sun Z, et al. Diverse Particle Carriers Prepared by Co-Precipitation and Phase Separation: Formation and Applications. ChemPlusChem. Jan. 2021;86(1):49–58. 10.1002/CPLU.202000497 . Neuman A, Zhang S, Lee D, Riggleman RA. Increases in Miscibility of a Binary Polymer Blend Confined within a Nanoparticle Packing. Macromolecules. Feb. 2023;56(3):954–63. 10.1021/ACS.MACROMOL.2C01918 . Lindvig T, Michelsen ML, Kontogeorgis GM. A Flory–Huggins model based on the Hansen solubility parameters. Fluid Phase Equilib. Dec. 2002;203:1–2. 10.1016/S0378-3812(02)00184-X . Miranda-Quintana RA, Chen L, Smiatek J. Insights into Hildebrand Solubility Parameters – Contributions from Cohesive Energies or Electrophilicity Densities?**. ChemPhysChem. Jan. 2024;25(1). 10.1002/CPHC.202300566 . Venkatram S, Kim C, Chandrasekaran A, Ramprasad R. Critical Assessment of the Hildebrand and Hansen Solubility Parameters for Polymers, J. Chem. Inf. Model. , vol. 59, no. 10, pp. 4188–4194, Oct. 2019, 10.1021/ACS.JCIM.9B00656 Shrivastava A, Gupta VB. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles Young Scientists. 2011. 10.4103/2229-5186.79345 . Zaari Lambarki L, et al. Comparison of approaches for assessing detection and quantitation limits in bioanalytical methods using HPLC for sotalol in plasma OPEN. Nature. 2025. 10.1038/s41598-024-83474-5 . International Conference on Harmonisation of Technical Requirements for Registration. of Pharmaceuticals for Human Use (ICH), Validation of Analytical Procedures: Text and Methodology Q2(R1), 2005. Caputo F, et al. Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range. J Colloid Interface Sci. Apr. 2021;588:401–17. 10.1016/J.JCIS.2020.12.039 . Kato H, et al. Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol In Vitro. Aug. 2009;23(5):927–34. 10.1016/J.TIV.2009.04.006 . Marucco A, et al. Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS). Mater 2019. Nov. 2019;12(23):3833. 10.3390/MA12233833 . Ompala C, Renault JP, Taché O, Cournède É, Devineau S, Chivas-Joly C. Stability and dispersibility of microplastics in experimental exposure medium and their dimensional characterization by SMLS, SAXS, Raman microscopy, and SEM. J Hazard Mater. May 2024;469:134083. 10.1016/J.JHAZMAT.2024.134083 . Zanoni I et al. Feb., Characterization of polyethylene and polyurethane microplastics and their adsorption behavior on Cu2 + and Fe3 + in environmental matrices, Environmental Sciences Europe 2025 37:1 , vol. 37, no. 1, p. 21-, 2025, 10.1186/S12302-025-01061-5 Zhang XM, Patel AB, De Graaf RA, Behar KL. Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem Phys Lipids. Jan. 2004;127(1):113–20. 10.1016/J.CHEMPHYSLIP.2003.09.013 . Williams WA, Aravamudhan S. Micro-Nanoparticle Characterization: Establishing Underpinnings for Proper Identification and Nanotechnology-Enabled Remediation, Polymers (Basel). , vol. 16, no. 19, p. 2837, Oct. 2024, 10.3390/POLYM16192837 Fang C, Luo Y, Naidu R. Microplastics and nanoplastics analysis: Options, imaging, advancements and challenges. TRAC Trends Anal Chem. Sep. 2023;166:117158. 10.1016/J.TRAC.2023.117158 . Catarino AI, Frutos A, Henry TB. Use of fluorescent-labelled nanoplastics (NPs) to demonstrate NP absorption is inconclusive without adequate controls, Science of The Total Environment , vol. 670, pp. 915–920, Jun. 2019, 10.1016/j.scitotenv.2019.03.194 Schür C, Rist S, Baun A, Mayer P, Hartmann NB, Wagner M. When Fluorescence Is not a Particle: The Tissue Translocation of Microplastics in Daphnia magna Seems an Artifact, Environ. Toxicol. Chem. , vol. 38, no. 7, pp. 1495–1503, Jul. 2019, 10.1002/etc.4436 Melo-Agustín P, Kozak ER, M. de Jesús Perea-Flores, and, Mendoza-Pérez JA. Identification of microplastics and associated contaminants using ultra high resolution microscopic and spectroscopic techniques, Science of The Total Environment , vol. 828, p. 154434, Jul. 2022, 10.1016/J.SCITOTENV.2022.154434 Sobhani Z, Zhang X, Gibson C, Naidu R, Megharaj M, Fang C. Identification and visualisation of microplastics/nanoplastics by Raman imaging (i): Down to 100 nm. Water Res. May 2020;174:115658. 10.1016/J.WATRES.2020.115658 . Jungnickel H, et al. Time-of-flight secondary ion mass spectrometry (ToF-SIMS)-based analysis and imaging of polyethylene microplastics formation during sea surf simulation. Sci Total Environ. Sep. 2016;563–4. 10.1016/J.SCITOTENV.2016.04.025 . Modicano P, et al. Enhanced optical imaging properties of lipid nanocapsules as vehicles for fluorescent conjugated polymers. Eur J Pharm Biopharm. Sep. 2020;154:297–308. 10.1016/J.EJPB.2020.07.017 . Wu T, Hu G, Ning J, Yang J, Zhou Y. A photoluminescence strategy for detection nanoplastics in water and biological imaging in cells and plants. J Hazard Mater. Jan. 2024;461:132695. 10.1016/j.jhazmat.2023.132695 . Li L, et al. Identification of Nanoplastics by Probing the Viscous Nanoenvironment. Small Sci. Dec. 2025;5(12). 10.1002/smsc.202500430 . Moraz A, Breider F. Detection and Quantification of Nonlabeled Polystyrene Nanoparticles Using a Fluorescent Molecular Rotor. Anal Chem. Nov. 2021;93(45):14976–84. 10.1021/acs.analchem.1c02055 . Altmann K et al. Dec., Characterizing nanoplastic suspensions of increasing complexity: inter-laboratory comparison of size measurements using dynamic light scattering, Environ. Sci. Nano , vol. 12, no. 12, pp. 5242–5256, 2025, 10.1039/D5EN00645G Sharma G, et al. Iron oxide nanoparticle agglomeration influences dose rates and modulates oxidative stress-mediated dose–response profiles in vitro. Nanotoxicology. Sep. 2013;8(6):663. 10.3109/17435390.2013.822115 . Watson CY, DeLoid GM, Pal A, Demokritou P. Buoyant Nanoparticles: Implications for Nano-Biointeractions in Cellular Studies. Small. Jun. 2016;12(23):3172–80. 10.1002/SMLL.201600314 . Moore TL, et al. Nanoparticle administration method in cell culture alters particle-cell interaction. Nature. 2019. 10.1038/s41598-018-36954-4 . Schröter L, Ventura N. Nanoplastic Toxicity: Insights and Challenges from Experimental Model Systems. Small. Aug. 2022;18(31). 10.1002/SMLL.202201680 . Additional Declarations No competing interests reported. Supplementary Files Nguyenetal2026Supplementary.docx floatimage1.png Graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 29 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 11 Mar, 2026 Editor assigned by journal 17 Feb, 2026 Submission checks completed at journal 17 Feb, 2026 First submitted to journal 10 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8842721","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605743387,"identity":"644645d6-8713-4155-9ae3-c33374399ee7","order_by":0,"name":"My Vanessa Nguyen Hoang","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"My","middleName":"Vanessa Nguyen","lastName":"Hoang","suffix":""},{"id":605743388,"identity":"5b66c9f2-5809-4ee4-ba07-a3bf10141f26","order_by":1,"name":"Maria Anzengruber","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Anzengruber","suffix":""},{"id":605743389,"identity":"9dcebc9e-7c3f-4de4-96f0-d86fc2f87cee","order_by":2,"name":"Yoel Negrin Montecelo","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Yoel","middleName":"Negrin","lastName":"Montecelo","suffix":""},{"id":605743390,"identity":"ee7ed8a7-364f-4c3b-9c4c-535e309c270c","order_by":3,"name":"Lukas Wimmer","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Lukas","middleName":"","lastName":"Wimmer","suffix":""},{"id":605743391,"identity":"539580da-5c3b-441f-a8c9-f59c106039bd","order_by":4,"name":"Dragana J. Stanić-Vučinić","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Dragana","middleName":"J.","lastName":"Stanić-Vučinić","suffix":""},{"id":605743392,"identity":"6d21ce99-a7b6-4d4c-ab85-4cab4394d6f6","order_by":5,"name":"Tanja Ćirković Veličković","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"Ćirković","lastName":"Veličković","suffix":""},{"id":605743393,"identity":"5c9feec8-dec8-4d10-9cd0-dc44a7cbfe9e","order_by":6,"name":"Frank von der Kammer","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"von der","lastName":"Kammer","suffix":""},{"id":605743394,"identity":"429de5df-b35d-4b25-8ffe-26ae796a3145","order_by":7,"name":"Mark A. Green","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"A.","lastName":"Green","suffix":""},{"id":605743395,"identity":"cbda61cc-0c2a-43cc-a7c9-a5cce3e99464","order_by":8,"name":"Richard D. Harvey","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"D.","lastName":"Harvey","suffix":""},{"id":605743396,"identity":"ce5b2e08-2ef4-49e6-af7e-acca5e5fbb00","order_by":9,"name":"Lea Ann Dailey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHElEQVRIiWNgGAWjYBACPijN2MDM3MDAUMHAwAZk49XChtACUnkGWQsbIS1g1IZNDl0Le/PDz4U7GGT72xkbH/POO5zHJ5Hc+IChxo6Bfz52B7LxHDOWnnmGwXjGYcZmY95th4vZJBKbDRiOJTNIHMNhi0SCgTRvG0Niw2HGNmmglsQ2noNtEgxsBxgYcGmRf/75N0jL/MOM7b9554C1tP9g+HeAQR6nLTxmYFs2AG1h5m0AamFvbAOGwwEGA1xaeHLKrHnbJIw3Av0iOedYOkhLs0RiXzKP4bEErFr42Y9vvs3bZiM77/zhgx/e1Fgnzm9mf/jhwzc7ObnDB7BbAwESYJKJB8YHms+DSy0KYPxBlLJRMApGwSgYaQAABORYNhCMLI8AAAAASUVORK5CYII=","orcid":"","institution":"University of Vienna","correspondingAuthor":true,"prefix":"","firstName":"Lea","middleName":"Ann","lastName":"Dailey","suffix":""}],"badges":[],"createdAt":"2026-02-10 15:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8842721/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8842721/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104867507,"identity":"12a90d67-2318-4b34-8968-417ceb4237b0","added_by":"auto","created_at":"2026-03-18 07:12:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e(A) Chemical structures and HSP values of PET [34], [35], [36], F8BT [37], [38]and PP [34], [39]. (B) Conditions used to prepare F8BT-labeled PET and PP nanoparticles. (C) An overview of different particle morphologies possible when co-precipitating two polymers (adapted from Sun et al.) [40].\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/313cd0000d154cb95330d916.png"},{"id":104867513,"identity":"ad1c152d-f143-47bb-a641-3feb812805e5","added_by":"auto","created_at":"2026-03-18 07:13:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":471605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTEM images of PET nanoparticles with 0.8 % (A) and 5 % (B) F8BT content. In the right panel, the corresponding particle size distribution curves of labeled vs. non-labeled PET materials are shown. TEM images of PP with 0.8 % (C) and 3 % (D) F8BT content are also depicted. Note: Due to the low melting temperature, PP particles exhibited deformation during TEM imaging, which could not be improved by optimization of the imaging conditions. In the right panel, the corresponding particle size distribution curves of labeled vs. non-labeled PP materials are shown.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/50fca32a95b91b895eb5fe4b.png"},{"id":104867528,"identity":"d80ef03c-bca1-498c-b7dd-7956b4312a44","added_by":"auto","created_at":"2026-03-18 07:13:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":284848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentative size distribution comparisons between the intermediate product in ethanol and the final product in glycerol for PET (A) and PP (B) systems with their respective F8BT concentrations. Effects of dispersion protocol (QC vs BR) on the particle size distribution of PET with 5 % F8BT (C) and PP with 3 % F8BT (D).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/af5c1abb517f102945b5c9c6.png"},{"id":104867520,"identity":"0b6c6a16-5a5d-4c24-98f0-d433e325ab9e","added_by":"auto","created_at":"2026-03-18 07:13:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eF8BT present in wash fluid and externally bound to particles was measured directly and internal F8BT content extrapolated for PET (A) and PP (B) test materials. F8BT leaching into PBS or THF from labeled PET (C) or PP (D) test materials. Values represent the mean ± standard deviation of n=3 experiments.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/bd326ea522a6a5fd70954c98.png"},{"id":104867416,"identity":"7429b593-8850-4d5a-9cd7-5b31c77e7403","added_by":"auto","created_at":"2026-03-18 07:12:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eContact angle measurements of labeled vs unlabeled PET and PP test materials (n=30 measurements per sample). Statistical analysis was performed by using one-way ANOVA applying Dunnett's multiple comparisons test. ** Denotes p \u0026lt; 0.01; *** denotes p \u0026lt; 0.001; ns denotes not significant.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/b91089927f29eba33fd5eb3f.png"},{"id":104867379,"identity":"2b579bdd-98f2-4226-a94f-913892e20139","added_by":"auto","created_at":"2026-03-18 07:12:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":491258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eExcitation spectra with fixed emission at 540 nm (A) and emission scan with fixed excitation at 460 nm (B) of F8BT-labeled PET and PP nanoplastics compared to F8BT dissolved in THF. LOD values for PET (C) and PP (D) with different F8BT content following different dispersion protocols: QC = quality control dispersion SOP; BR PBS= biorelevant dispersion SOP with the second dilution step in PBS; BR CL = biorelevant dispersion SOP with the second dilution step in cell lysate. The CV% of selected concentrations from the dilution series are depicted for PET (E) and PP (F). CV% values were calculated from n = 9 replicates per concentration from a single test material batch.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/a7556ebcb51bce5db6cf65b9.png"},{"id":104867522,"identity":"feb1e15f-f539-4de7-a3d2-c24f463ab31c","added_by":"auto","created_at":"2026-03-18 07:13:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":425741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eParticle size distribution of labeled PET (A) and PP (B) over seven days storage at 4 °C following initial dilution from the glycerol storage medium (final product) to a 10 % BSA solution (in water; 1 mg/mL). The colloidal stability of working dilutions (100 µg/mL) of labeled PET (C) and PP (D) following from the BSA stock suspension into cell culture medium and incubation for 48 h at 37 °C.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/1936513fd985f07c359d93ab.png"},{"id":104867539,"identity":"f69f4210-2be7-4812-9081-cfd9855d6b28","added_by":"auto","created_at":"2026-03-18 07:13:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":587851,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence quantification of cell-associated F8BT-labeled PET test materials following 24 h incubation in conventional well plates (n=3). (B) The FlowCube system with vertical and horizontal placements for monolayers grown on coverslips and stirring capabilities. Semi-quantitative analysis of F8BT-labeled PP (C) and PET (D) following 24 h incubation in the FlowCube under different conditions (n=3). Representative fluorescence microscope images of Calu-3 cells treated with PP (E-F) and PET (G-H) in the FlowCube under different conditions.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/e5331a8bc3eb59b181592993.png"},{"id":104867659,"identity":"5a4305fe-0fb8-429b-8699-8662e2029e33","added_by":"auto","created_at":"2026-03-18 07:13:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4070960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/bd316d4b-74fb-46fb-a9c3-f651c0445979.pdf"},{"id":104867440,"identity":"63073ce0-da0e-47fd-a148-f66eb7bc5e8f","added_by":"auto","created_at":"2026-03-18 07:12:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1848977,"visible":true,"origin":"","legend":"","description":"","filename":"Nguyenetal2026Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/745ebe765af54be5e5e817d6.docx"},{"id":104867460,"identity":"84bf3b33-8e60-4ac1-89de-4cea09a26e4e","added_by":"auto","created_at":"2026-03-18 07:12:56","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":66652,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8842721/v1/ca157664fff0da2de7cbf72f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Labeling of PET and PP nanoplastic test materials with non-leachable π-conjugated fluorescent polymers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicro- and nanoplastics (MNPs) are increasingly recognized as widespread environmental contaminants, having been detected in, e.g., marine water [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], air [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and food [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These materials originate from the fragmentation of larger plastic debris through mechanical, thermal, and chemical degradation processes. With only about 9 % of global plastic waste beng recycled, plastic pollution continues to rise. In 2019, global plastic production reached 368\u0026nbsp;million metric tons, with an expected annual growth rate of approximately 5 % [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This trend contributes t the increasing environmental accumulation of plastic debris and highlights the urgent need for further investigation, particularly given the clear evidence of human exposure to MNPs. Concerns have been raised regarding the potential of MNPs to interact with biological systems, cross cellular barriers, and accumulate in organisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While numerous studies have explored cellular uptake and the effects of MNPs in various \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e systems, the findings remain inconclusive [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany MNP exposure studies rely primarily on commercially available polystyrene (PS) beads, even though PS accounts for less than 10% of environmental plastic waste [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. While these particles are easy to handle and available in well-defined sizes, their smooth, spherical, and monodisperse nature does not reflect the diversity of plastic debris found in the environment. As a result, the environmental relevance of PS-based model systems remains limited. More representative test materials made from polymers such as polyethylene terephthalate (PET) and polypropylene (PP), both extensively used in packaging, are underrepresented in MNP research. These materials differ in density, crystallinity, and weathering behavior, making them valuable for comparative studies on cellular transportation and toxicological effects. In recent years, efforts have expanded the range of available model MNPs through both top-down methods (milling, ultrasonication) and bottom-up methods (nanoprecipitation) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nanoprecipitation has been employed to produce a variety of plastic types including PET, PP [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], low-density polyethylene (LDPE), high-density polyethylene (HDPE), and polyvinyl chloride (PVC) particles [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nevertheless, there remains a pressing need to further develop and diversify nanosized plastic test materials. The desired attributes for such materials include environmental relevance, microbiological stability, well-defined physicochemical properties, suitability for biological assays, and analytical detectability.\u003c/p\u003e \u003cp\u003eBesides the lack of well-characterized test materials, another major challenge lies in the identification and quantification of MNPs, especially in complex biological matrices. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Thermal analytical techniques combined with chromatographic separation and mass spectrometric detection, such as pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and thermal extraction desorption-gas chromatography-mass spectrometry (TED-GC-MS) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] are currently among the most sensitive approaches, enabling polymer-specific quantification of unlabeled materials at low limits of detection (LOD) with the highest sensitivity observed for water and air samples (LOD: \u0026lt; 1 ng/mL; see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). It is important to note that method sensitivity greatly depends on polymer type, matrix complexity and content of organic matter, with reported LOD values ranging from 0.02\u0026ndash;10.8 \u0026micro;g/mL (or \u0026micro;g/g), depending on the sample matrix (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Mass-based quantification methods are destructive, require complex sample preparation, pose a risk of MNP loss or sample contamination, and are susceptible to matrix effects which can strongly influence detection limits [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Different polymers can also produce identical pyrolysis products, resulting in false positive polymer identifications and concentrations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, labeling strategies focus on the detection of an incorporated tracer rather than the polymer itself and are therefore primarily used for particle tracking, visualization, and uptake studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Approaches include the incorporation of radioactive or stable isotopes into the polymer. Despite their excellent detectability, disadvantages include safety concerns, strict regulatory requirements, and limited half-lives [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Stable isotopes like \u0026sup1;\u0026sup3;C or \u0026sup2;H are safer alternatives but often lack spatial resolution and require specialized instruments like isotope ratio mass spectrometers (IRMS), which are not widely available [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Metal tracers embedded in the polymer matrix enable highly sensitive detection through inductively coupled plasma mass spectrometry (ICP-MS), while avoiding issues such as photobleaching and dye leaching. However, their production may require toxic metals and specialized equipment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A third and widely used strategy is fluorescence labeling. This technique is popular due to its ease of use and compatibility with microscopy, flow cytometry, and spectroscopy methods (see supplementary Table S2) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Fluorescent labeling can be achieved either by surface staining or by incorporating a dye into the polymer matrix [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While surface staining is simple, it often suffers from poor dye retention, leaching in protein rich media and interference from autofluorescence.\u003c/p\u003e \u003cp\u003eIn the current study, the high molecular weight, π-conjugated polymer, poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT), was explored as a fluorescent polymeric label for MNPs. Due to the presence of π-conjugated double bonds in the F8BT backbone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the polymer exhibits semiconducting properties and a bright yellow-green fluorescence, which is characterized by its thermal stability, strong emission and resistance to photobleaching. F8BT has been primarily used in organic light-emitting diode and photovoltaic technologies but has also been explored as an optical contrast agent in bioimaging applications [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen using a polymeric dye to label nanoplastics, it is important to recognize that most polymer blends are immiscible due to their relatively low entropy of mixing and high interfacial tension [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Co-precipitation of two immiscible polymers can produce nanoparticles with different structural morphologies depending on the interfacial tension between the two polymers. Such diverse morphologies include dimer particles, occluded particles, core-shell capsules and hetero-aggregate systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The type of solvents/anti-solvent systems, the polymer structure and molecular weight, ratio of the polymers in the blend, as well as the mixing temperature and speed determine the final equilibrium particle structure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Despite this complexity, Hildebrand and Flory-Huggins solubility parameters have been used to estimate how well a given polymer will interact with other polymers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], whereby similar values indicate a higher potential for miscibility. The reported Hildebrand solubility parameters (HSP) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] for F8BT, PET and PP, the nanoplastics chosen for this study, indicate that F8BT may exhibit a higher miscibility with PET compared to PP. For the purposes of nanoplastic labeling, particle morphologies where the F8BT label is miscible with the plastic or embedded in the PET/PP core (internalized F8BT) are more desirable than morphologies where F8BT is located externally, since external F8BT could leach or change the particle surface properties compared to unlabeled plastics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBuilding on a previously established preparation method for PET and PP nanoplastics intended for use in biological assays [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], F8BT-labeling of these two materials was explored. First, F8BT incorporation into PET/PP nanoplastics was characterized and the physicochemical properties of systems with optimal loading was evaluated. Key criteria included morphology, size reproducibility, good dispersibility, optical properties, and low, reproducible LOD/LOQ values. Cellular uptake was then investigated in Calu-3 epithelial cells using both a conventional static well plate setup and an innovative dynamic exposure system known as the FlowCube. The FlowCube is designed to expose cells to low-density particles, like PP, that would otherwise float to the surface of the culture medium in standard submerged cell culture formats. The FlowCube simulates a 3D exposure scenario by vertically inserting cell-seeded slides into a cube structure, ensuring continuous interaction between particles and cells.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e \u003cb\u003eMaterials\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe starting material polyethylene terephthalate (CAS:25038-59-9) and polypropylene (CAS: 9003-07-0) granulates were kindly provided by Plastics Europe (Brussels, Belgium). The fluorescent dye F8BT (average MN \u0026le; 25000) was purchased from Sigma-Aldrich (St. Louis, USA). As well as benzyl alcohol (BA), xylene (isomeric mixture), glycerol, Fluorsave mounting medium and the non-ionic surfactant Tween 80. Bovine serum albumin (BSA, fraction V) was supplied by Carl Roth (Karlsruhe, Germany). Tetrahydrofuran (THF) was obtained from Fluka (Buchs, Switzerland) whereas ethanol (\u0026ge;\u0026thinsp;99.8%) was purchased from Brenntag (Guntramsdorf, Austria). Polycarbonate (PC) membrane filters (2.0 \u0026micro;m) were bought from Merck Millipore (Billerica, USA) and nylon membrane filters (0.8 \u0026micro;m) were obtained from Cytiva (Marlborough, USA). Experiments involving the human bronchial epithelial cell line Calu-3 (ATCC HTB-55), which was obtained from LGC Standards Ltd. (Teddington, UK), were performed using following products provided from different suppliers. Dulbecco\u0026rsquo;s phosphate buffered saline (PBS) and fetal bovine serum (FBS) superior from Sigma-Aldrich, cell culture media Dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM/F-12) + GlutaMAX, trypsin-EDTA (0.25%) and penicillin streptomycin from Gibco (Thermo Fisher Scientific, Waltham, USA). Poly-L-lysine hydrobromide, paraformaldehyde (PFA) and ammonium chloride were also obtained from Sigma-Aldrich. Hoechst 33342 was purchased from Invitrogen. Thermanox plastic coverslips and square glass microscope slides were purchased from Nunc (Thermo Fisher Scientific, Waltham, USA). Fluorescent polystyrene (PS) particles labeled with Nile Red were purchased from Kisker Biotech (Steinfurt, Germany) and used as a commercial reference material. All reagents were of analytical grade or higher and used without further purification. Ultrapure water (18.2 MΩ\u0026middot;cm) was used in all aqueous dilution preparations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTest material production\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe procedure for producing unlabeled submicron-range PET and PP nano-sized test materials developed by Wimmer \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] was used with modifications. An updated detailed standard operating procedure is available in the electronic supplementary information.\u003c/p\u003e \u003cp\u003ePET granules (260 mg; \u003cem\u003em\u003c/em\u003e\u003csub\u003egranules\u003c/sub\u003e) were dissolved in benzyl alcohol (10 mL) without (0% w/w) and with F8BT (0.8% and 5% w/w). The mixture was heated to 215\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C, stirred at 250 rpm for 45 min and was rapidly transferred into chilled ethanol (125 mL) under more stirring (400 rpm) to induce precipitation. The precipitate was washed five times with ethanol (100 mL each) by filtration using a polycarbonate membrane (pore size: 2 \u0026micro;m). Nanoparticles were retained, presumably due to rapid agglomeration during this step. The particles were resuspended in ethanol (26 mL) and sonicated in a sonication bath (Sonocool SC 255, Bandelin electronic, Berlin, Germany) for 2 h (referred to as intermediate product; 10 mg/mL). All sonication steps were performed with the same instrument under the following settings: 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 100% intensity, 35 kHz frequency, and 180 W nominal power.\u003c/p\u003e \u003cp\u003eTo prepare PP nanoplastics, a similar approach was followed with minor modifications: PP granules (165 mg) were dissolved in xylene (80 mL) at 185\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C, without (0% w/w) and with F8BT (0.8 and 3% w/w). The hot solution was added to cooled ethanol (240 mL) and the suspension was filtered through a 0.8 \u0026micro;m nylon membrane, undergoing the same rigorous ethanol washing steps to remove residual solvent. The material retained on the filter membrane was resuspended in ethanol (16 mL) and subsequently sonicated for 45 min prior to weighing (m\u003csub\u003esusp\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eAfter sonication, the concentration and product yield of the intermediate product were determined gravimetrically by weighing a microcentrifuge tube before (\u003cem\u003em\u003c/em\u003e\u003csub\u003eempty\u003c/sub\u003e) and after addition of 1 mL of ethanolic suspension (\u003cem\u003em\u003c/em\u003e\u003csub\u003efilled\u003c/sub\u003e). The sample was then dried (55\u0026deg;C, 20 mbar), and the dry mass was recorded (\u003cem\u003em\u003c/em\u003e\u003csub\u003edry\u003c/sub\u003e). When the gravimetrically determined mass loss between measurements was below 1%, concentration and yield were calculated using equations (\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): The concentration (c) of nanosuspension calculated as mg/g.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:c\\:\\left[\\frac{mg}{g}\\right]=\\:\\frac{\\left({m}_{dry}-{m}_{empty}\\right)\\:x\\:1000}{({m}_{filled}-{m}_{empty})}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e): Yield (Y) of particle production calculated in %.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Y\\:\\left[\\%\\right]=\\:\\frac{c\\:x\\:{m}_{susp}}{{m}_{granules}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTo prepare for exchanging the storage medium from ethanol to glycerol (final product), glycerol was heated to 60\u0026deg;C and the required amount to achieve a final plastic concentration of 40 mg nanoplastics per 1 g glycerol was calculated. The heated glycerol was added to the ethanolic suspension, followed by vortexing and sonication (5 min) to ensure even distribution. Ethanol was evaporated (55\u0026deg;C, 20 mbar), and the process was considered complete once the mass loss between measurements was below 1%. The final product was stored in 1.5 mL glass vials under light exclusion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEncapsulation efficiency\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo calibration curves for F8BT quantification were prepared by dissolving the dye in either pure tetrahydrofuran or a tetrahydrofuran/benzyl alcohol mixture (1:1; v/v) to create a stock solution (500 \u0026micro;g/mL). This stock was further diluted with the respective solvents to obtain calibration standards ranging from 1 to 12.5 \u0026micro;g/mL. Absorption spectra (300\u0026ndash;700 nm) were recorded using a quartz cuvette and a UV/Vis spectrophotometer (Epoch, BioTek Instruments, Winooski, USA). The absorption maximum of F8BT in THF (λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;454 nm) was determined and used for subsequent quantification. Measurements were repeated three times for each solvent and two linear calibration curves were generated (in THF: R\u0026sup2; = 0.9766; in THF/BA: R\u0026sup2; = 0.9993).\u003c/p\u003e \u003cp\u003eTo calculate the encapsulation efficiency, three potential sources of unencapsulated dye were considered: (1) the filtrate (dye particles washed away during filtration), (2) the wash fractions (additional particles removed during washing), and (3) the retentate (particles retained on the filter and resuspended in ethanol). PET particles (0; 0.8; 5% F8BT) were produced as previously described. During filtration and washing, the filtrate (W1) and all subsequent ethanol wash fractions (W2\u0026ndash;W6) were collected to recover unencapsulated or non-adsorbed dye. 1) The filtrate was collected, and ethanol was evaporated under vacuum (55\u0026deg;C; 20 mbar; 30 min). Benzyl alcohol could not be removed under these conditions; therefore, the remaining residue was weighed and mixed with an equal volume of THF (1:1) to fully dissolve the dye. Absorbance was recorded at the previously determined maximum. 2) Each wash fraction, consisting mainly of ethanol, was evaporated under the same conditions. The dry residues were redissolved in 2 mL THF, and absorbance was measured. F8BT content in each wash step was quantified using the corresponding calibration curve. 3) Unencapsulated and or externally present dye in the retentate was determined using the intermediate product in ethanol (10 mg/mL) by taking 5 mL, evaporating the ethanol, redissolving the residue in THF (5 mL), centrifuging the particles (15,000 rpm; 5 min), and recording the absorbance of the supernatant. For PP particles (0; 0.8; 3%), the same procedure was applied, except that the filtrate was dissolved directly in 2 mL pure THF. The undesired morphology, in which F8BT remained external to the particle, was defined as the retentate, whereas the desired morphology corresponded to the internalized fraction of F8BT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDye leaching\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis experiment for PET and PP was performed using the intermediate product in ethanol. Falcon tubes were prepared with 5 mL of the sample. The ethanol was evaporated (55\u0026deg;C; 20 mbar; 30 min), and the tubes were refilled with either THF (extreme condition) or PBS (physiological condition). Samples were centrifuged (15000 rpm; 5 min), and the absorbance of the supernatant was recorded. Afterwards, the supernatant was replaced with fresh medium, the pellet was resuspended, and the tubes were stored under light exclusion until the next measurement. Measurements were taken at the time points 0, 1, and 7 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDispersion and size characterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e \u003cp\u003eTEM images were obtained from the ethanolic intermediate product using a JEOL JEM-1400 Flash transmission electron microscope operated at an accelerating voltage of 80\u0026ndash;120 kV.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuality control (QC) dispersion protocol (intermediate product)\u003c/strong\u003e \u003cp\u003eTo assess the particle size distribution of the test material a quality control dispersion protocol using the non-ionic surfactant Tween 80 was developed. The goal was to optimize the deagglomeration process to obtain an accurate understanding of the primary particle size. The MNP ethanol suspension (10 mg/mL; 200 \u0026micro;L) was mixed with 800 \u0026micro;L Tween 80 (5% w/w in ultrapure water) and was subsequently sonicated for 15 (PET) or 40 minutes (PP), resulting in a first dilution (1 mg/mL). This suspension was then diluted further by adding it directly into the laser diffraction measuring chamber until a laser obscuration of 3\u0026ndash;5% was achieved (second dilution; ~0.3\u0026ndash;0.4% Tween 80; ~0.2\u0026ndash;0.4 mg/mL nanoplastic content).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuality control (QC) dispersion protocol (final product)\u003c/strong\u003e \u003cp\u003eWhen dispersing the final glycerol-dispersed product (40 mg/g), the test material was heated to 60\u0026deg;C and vortexed to ensure a homogeneous suspension and reduce viscosity. One drop (~\u0026thinsp;25 mg suspension containing 1 mg nanoplastic) was weighed and dispersed in 1 mL of Tween 80 (5% w/w in ultrapure water) making the first dilution (1 mg/mL). This suspension was further diluted by filling up the laser diffraction measuring chamber with water and Tween 80 (400 \u0026micro;L; 5% w/w in ultrapure water) and adding the suspension dropwise into the device until a final Tween 80 concentration of 0.35\u0026ndash;0.39% was reached and depending on when a laser obscuration of 3\u0026ndash;5% was achieved (second dilution; ~0.3\u0026ndash;0.4% Tween 80; ~ 0.02\u0026ndash;0.04 mg/mL nanoplastic content).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBiorelevant dispersion (BR) protocol (final product)\u003c/strong\u003e \u003cp\u003eWhen investigating aspects such as cell uptake it might be important to omit Tween 80 as a dispersing agent and use a biorelevant option instead. Therefore, BSA and serum-supplemented cell culture medium (10% FBS, 1% Penicillin/Streptomycin) were explored as BR dispersing agents. Briefly, the final glycerol-based product was heated to 60\u0026deg;C and sonicated as described above. MNP samples (25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mg) were dispensed into 1 mL BSA solution (10% m/v in ultrapure water, filtered through a 0.45 \u0026micro;m PVDF membrane) to obtain a nanoplastic concentration of 1 mg/mL (first dilution). The mixture was sonicated for 30 minutes, then serum-supplemented cell culture medium was added to reach the desired concentration for further studies. This second dilution was followed by a second sonication step of 15 min (PET) and 40 min (PP due to its higher hydrophobicity) before further use. The final theoretical BSA concentration (above what is present in the serum) was 1.5% for a plastic concentration of 150 \u0026micro;g/mL.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eParticle size measurements were carried out using laser diffraction (LD) with the Mastersizer 3000 instrument (Malvern Panalytical, Malvern, UK). For each sample, 10 individual, consecutive measurements were performed, and the average particle size distribution was calculated. The measuring chamber was filled with 5\u0026ndash;6 mL of distilled water or serum-supplemented cell culture medium (for biorelevant dispersion protocol), and the sample dispersed as described above was added until a laser obscuration of 3\u0026ndash;5% was reached. The Mie scattering theory was applied using the following parameters: for PET, a refractive index (RI) of 1.636 and absorption of 0.01 and for PP, an RI of 1.490 and absorption of 0.01. Since the RI of the PET-F8BT and PP-F8BT mixtures was unknown, the Fraunhofer approximation setting were also used. Measurements were conducted at room temperature.\u003c/p\u003e \u003cp\u003e \u003cb\u003eContact angle measurements\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEthanolic nanoplastic suspensions (intermediate product) were dried via vacuum filtration using a 0.8 \u0026micro;m nylon membrane. The dried powder was transferred onto a glass slide, which had been prepared with double-sided adhesive tape, by pressing it firmly onto the tape. Full surface coverage had to be ensured to prevent gaps between particles and a leveled surface. This compression also minimized adsorption of the water droplet onto the adhesive surface. Contact angle measurements were conducted for PET nanoplastics containing 0, 0.8 and 5% w/w F8BT and PP nanoplastics containing 0, 0.8, and 3% F8BT. A drop shape analyzer (DSA30S, Kr\u0026uuml;ss, Hamburg, Germany) was used applying the sessile drop technique with deionized water as the test liquid. The baseline was manually adjusted for each measurement and droplets were dispensed onto a fresh, dry plastic surface each time. The droplet volume was set to 2 \u0026micro;L with a dispense rate of 0.16 \u0026micro;L/min. Following a stabilization period of 5 sec, measurements were recorded at one frame per second, and each contact angle value was determined as the mean of six frames. For each sample, 30 individual measurements were performed and the mean value was calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptical properties\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the optical characteristics of F8BT, the glycerol stock dispersion was prepared according to the biorelevant dispersion protocol, resulting in a final sample concentration of 50 \u0026micro;g/mL. Spectral scans were performed by fixing the excitation wavelength at 460 nm and recording the corresponding emission spectrum from 300\u0026ndash;650 nm or fixing the emission maximum at 540 nm and scanning the excitation from 300\u0026ndash;650 nm. The background was subtracted and the final spectra were calculated and plotted. This experiment was performed three individual times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence quantification studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence properties of PET (0.8% and 5% F8BT) and PP (0.8% and 3% F8BT) suspensions were characterized to determine the limit of detection (LOD) and limit of quantification (LOQ) of measurements using the Infinite 200 PRO plate reader (Tecan, M\u0026auml;nnedorf, Switzerland) and furthermore the variability of sample handling was also investigated. As a comparator material, commercially available PS particles fluorescently labeled with Nile Red were used. Test materials were dispersed using the two-step biorelevant dispersion protocol. In the second dilution step, serum-supplemented cell culture medium was replaced with PBS or cell lysate to mimic procedures typically used for quantifying uptake of fluorescently labeled nanomaterials in cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Final concentrations ranging from 0.008\u0026ndash;500 \u0026micro;g/mL were tested by using serial dilution steps. Briefly, cell lysate was obtained by culturing Calu-3 cells, a human bronchial epithelial cell line, at a density of 4 \u0026times; 10⁵ cells/mL in 24-well plates. After 48 h incubation, the medium was changed to a fresh one, followed by another 24 h incubation at 37\u0026deg;C in 5 % C₂. For lysis, cells were washed three times with PBS, and 1 mL of a Triton X-100 solution (0.1 % i PBS) was added per well. After 10 min incubation to ensure cell membrane disruption, the cell layer was mechanically detached by scraping the surface with a pipette tip. Cell lysate from all wells were pooled and used as a diluent for the preparation of the dilution series.\u003c/p\u003e \u003cp\u003eFollowing dispersion, diluted samples were transferred to black 96-well plates, and fluorescence was measured using the Tecan plate reader. Each dilution series was prepared as three independent experiments with three technical replicates per concentration, resulting in a total of nine data points per concentration. The plate reader settings were as follows: orbital shaking for 60 seconds at an amplitude of 1 mm prior to measurement; excitation was fixed at 460 nm and emission wavelength was set to 544 nm for F8BT-labeled particles whereas for Nile Red-labeled PS particles wavelengths at 552/636 nm were used. The LOD and LOQ were calculated using Equations \u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, which take the residual standard deviation of the linear regression into account:\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e): LOD in \u0026micro;g/mL.\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:LOD\\left[\\frac{{\\mu\\:}g}{mL}\\right]=\\:\\frac{{\\sigma\\:}\\:x\\:3.3}{slope}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;(\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e): LOQ in \u0026micro;g/mL.\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:LOQ\\left[\\frac{\\mu\\:g}{mL}\\right]=\\:\\frac{{\\sigma\\:}\\:x\\:10}{slope}\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTwo commonly used approaches for the determination of the standard deviation (σ) were compared: the residual standard deviation method and the intercept-based method [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Even though LOD and LOQ are widely applied, the literature is often unclear about which approach is used, and even the Q2R1 of International Conference on harmonization (ICH) guidelines state only that standard deviation must be considered, without specifying which one [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This lack of clarity leads to inconsistencies in the literature and values reported there should therefore be interpreted with caution. The residual method estimates the standard deviation from the residuals (σ\u003csub\u003eresiduals\u003c/sub\u003e) of the calibration regression. This approach accounts for the spread of the actual data points and is therefore often seen as more robust. An advantage of this method is that it reflects the real scatter of the measurements and is less sensitive to outliers in the lower concentration range. However, it tends to overestimate LOD and LOQ, especially when the calibration dataset has limited points or higher noise. In contrast to that, the intercept method uses the standard deviation of the y-intercept (σ\u003csub\u003eintercept\u003c/sub\u003e) of the calibration curve. This method usually yields lower LOD and LOQ values because it focuses on the precision of the baseline signal rather than the residual variance. It is simple to implement and widely used, but a major drawback is that it can lead to overly optimistic detection limits that might not reflect realistic conditions when the calibration curve is not perfectly linear or when there is significant heteroscedasticity (non-uniform variance of the data).\u003c/p\u003e \u003cp\u003eFurthermore, the coefficient of variation (CV%) of the individual fluorescence values (n\u0026thinsp;=\u0026thinsp;9) was calculated for each concentration to provide information on the variability of fluorescence signal associated with the dispersion and dilution process, as well as the magnitude of dilution. The following equation was used:\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;(\u003cspan refid=\"Equ5\" class=\"InternalRef\"\u003e5\u003c/span\u003e): Coefficient variation in %.\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:CV\\left[\\%\\right]=\\:\\frac{{\\sigma\\:}\\:x\\:100}{mean}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eSize stability of dispersion in different media\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing the biorelevant dispersion protocol, the size stability of both the first (1 mg/mL) and second dilution (100 \u0026micro;g/mL) was assessed over time. The particle size distribution of the first dilution was tested after storage at 4\u0026deg;C over a period of 7 days, to assess whether the more concentrated dispersion could be prepared in advance without losing the particle characteristics. Before each measurement, the samples were vortexed and sonicated for 30 minutes to ensure uniform dispersion. The second dilution was tested under conditions simulating \u003cem\u003ein vitro\u003c/em\u003e exposure. i.e., the diluted suspension was incubated for 48 h at 37\u0026deg;C under 5% CO₂, without agitation. This setup was intended to mimic static conditions in a microtiter plate and assess potential particle agglomeration. Prior to measurement, the vial was not disturbed, replicating exposure conditions. Both stability tests were performed using the same batch of particles. Particle size measurements were performed as described above with the exception that the measuring chamber was filled with 6 mL of serum supplemented cell culture medium instead of water.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell uptake\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll cell uptake experiments were performed using Calu-3 with passage numbers from 24 to 28.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConventional 2D monolayer studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe standard exposure setup was performed using a 24-well plate, in which Calu-3 cells were seeded at a density of 4 \u0026times; 10⁵ cells/mL. Preliminary data (see supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) showed that incubation of F8BT-labeled PET and PP directly in the well plates resulted in substantial adsorption of the nanomaterials to the exposed surfaces, increasing the fluorescence background to unrealistic levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, electronic supplementary information). To circumvent this issue, removable Thermanox plastic coverslips were placed into the wells prior to seeding. Cells were seeded onto the coverslips and incubated for 48 h at 37\u0026deg;C and 5% CO₂ until approximately 80% confluency was reached. PET (5% F8BT) and PP (3% F8BT) with high dye loadings were dispersed using the biorelevant dispersion protocol (final concentration: 100 \u0026micro;g/mL) and 500 \u0026micro;L was added to each well. A vehicle control containing the equivalent amount of glycerol in BSA (10%) and cell culture medium was also included. Incubation was performed for 24 h after which the supernatant was aspirated and the wells were washed three times with PBS (500 \u0026micro;L) to remove particles non-internalized by cells. The coverslips were then transferred into a fresh 24-well plate and 1 mL of Triton X-100 (0.1% in PBS) was added to lyse the cells. After 10 min incubation, the cell layer was gently scraped using a pipette tip to ensure complete detachment. The resulting lysate, including cellular debris, was transferred directly to a black 96-well plate for fluorescence measurement utilizing the Tecan plate reader. The same measurement settings, as previously described in the section about fluorescence quantification studies were used. The experiment was repeated independently three times, each with technical triplicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlowCube: monolayers in vertical placement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDue to the low density of PP test materials, the classic static exposure setup in a well plate is not suited to achieve measurable cellular uptake, as the particles tend to float and do not interact with the cell monolayer on the bottom. To address this, the FlowCube system was developed to ensure consistent particle-cell contact. In this setup, cells were grown on square glass slides that formed the four vertical walls of a cube-like chamber. In addition to that, a magnetic stirrer was positioned at the bottom of the chamber to maintain homogenous particle suspension and further promote continuous contact with the cell monolayer.\u003c/p\u003e \u003cp\u003ePrior to cell seeding, glass slides were functionalized with poly-L-lysine (0.05% w/v in water) by placing them in a shaker for 20 min with the solution fully covering them, followed by three washing steps with ultrapure water (30 min each). The slides were then air-dried and sterilized under UV light for 30 min. Treated slides were placed into a 24-well plate designed for suspension cells to prevent cell attachment to the well surface. Calu-3 cells were seeded onto the slides at a density of 4 \u0026times; 10⁵ cells/mL and incubated for 48 h. After medium change, cells were incubated for another 24 h to reach\u0026thinsp;~\u0026thinsp;80% confluency. Once confluent, the slides were transferred into the cube chamber, where they formed the inner walls or were placed into a 24-well plate as a control. PET (5% F8BT) and PP (3% F8BT) were dispersed using the biorelevant dispersion protocol at a concentration of 100 \u0026micro;g/mL and added to the FlowCube chamber (1.2 mL per cube) or to the well plate (500 \u0026micro;L per well). The following exposure conditions were tested: 1) cubes without magnetic stirring (non-stir), 2) cubes with magnetic stirring (stir) to simulate shear stress, and 3) conventional well plate exposure (well). After 24 hours of incubation, cellular uptake was evaluated via fluorescence microscopy and flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence microscopy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing exposure, cells were fixed and stained directly on the slides. The slides were placed into fresh well plates and washed with PBS. Slides were fixed with 4% paraformaldehyde (100 \u0026micro;L, 30 min) and washed twice with PBS (1 mL each). For staining, 100 \u0026micro;L of Hoechst 33342 (1 mg/mL) was added per well and incubated in the dark for 30 min. The staining solution was then removed, and slides were washed again twice with PBS (1 mL each). One drop of mounting medium was placed on a microscope slide and the stained coverslip was placed cell-side down onto it to preserve the monolayer for imaging.\u003c/p\u003e \u003cp\u003eFluorescence microscopy was performed using a Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with a 63\u0026times; oil immersion objective. Filter sets used were: blue (excitation 335\u0026ndash;383 nm, emission 420\u0026ndash;470 nm), green (excitation 460\u0026ndash;488 nm, emission 500\u0026ndash;557 nm) and red (excitation 567\u0026ndash;602 nm, emission 615\u0026ndash;4095 nm). Exposure time was kept constant at 360 ms for all samples, except for slides that were exposed to PET under well conditions (12 ms). Images were post-processed using the ZEN software (version 3.1.1, Carl Zeiss Microscopy, Jena, Germany) for brightness and contrast adjustment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor flow cytometry the exposed slides were washed with PBS (1.2 mL for cube samples; 500 \u0026micro;L for well samples) and transferred into new well plates. Trypsin (150 \u0026micro;L) was added to each well and incubated for 5 minutes at 37\u0026deg;C and 5% CO₂. To stop enzymatic activity, complete cell culture medium (400 \u0026micro;L of) was added. The resulting cell suspension was transferred to fluorescence activated cell sorting (FACS) tubes and analyzed immediately using a Gallios flow cytometer (Beckman Coulter, Brea, USA). Fluorescence of internalized F8BT-labeled particles was detected in the FL1 channel (525/40 nm, FITC settings). Data acquisition was performed using the Kaluza software (Beckman Coulter, Brea, USA). All experiments were conducted independently three times, each with four technical replicates per treatment condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll statistical analyses were performed with Prism Graphpad (version 10.3.1, GraphPad Software, San Diego, USA).\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e \u003cb\u003eImpact of F8BT co-precipitation on PET and PP nanoparticle properties\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe production of PET and PP nanoplastics was previously optimized [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and used as a starting point for this labeling study. Pilot studies demonstrated that colloidally stable nanoparticles could be produced with up to 5 % ww F8BT per mass of PET and up to 3 % ww F8BT per mass PP. Increasing F8BT content above these values led to irreversible particle aggregation. To investigate the impact of F8BT incorporation on particle characteristics, all further studies were conducted with a low (0.8 % ww) and a higher F8BT content (5 % ad 3 % ww F8BT per mass PET / PP, respectively). Particle production resulted in yields of 90.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 % fr PET across eight batches and 90.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1 % fr PP across 17 produced batches, with no effect of F8BT labeling on the production yield observed.\u003c/p\u003e \u003cp\u003eTEM was used to investigate particle morphology, while laser diffraction was used as a complementary method to characterize the particle size distribution, including the presence of aggregates[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When preparing the samples for TEM, it should be noted that the intermediate product (ethanolic PET/PP suspension) was applied directly to the TEM grid without use of Tween 80 as a dispersion agent, since Tween 80 can reduce image quality. With this preparation technique aggregation could not be prevented. In contrast, Tween 80 was used as a dispersant for particle size measurements using the quality control (QC) dispersion protocol which was previously validated and results in only minor aggregate formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring TEM analysis, PET nanoparticles were more stable than PP nanoparticles, which is attributed to the higher melting point of PET and its increased thermal stability under the electron beam. TEM images of PET samples unexpectedly revealed the presence of spindle-shaped particles next to smaller, more spherical particle aggregates for both 0.8% and 5% F8BT content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Previous scanning electron microscopy studies of unlabeled PET test materials did not show spindle-shaped particles, indicating that this unexpected morphology is caused by the co-precipitation with F8BT. This hypothesis is further supported by a comparison of the particle size distribution curves of F8BT-labeled PET versus non-labeled PET nanomaterials (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Labeled PET nanoparticles exhibit an unusual shift towards smaller particles sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B; right panel), which is likely the result of altered diffraction patterns caused by the anisotropic, spindle-shaped particles in the suspension. This phenomenon is more pronounced with a higher F8BT content (5%), indicating that increases in F8BT are likely to increase the number of spindle-shaped particles in the suspension. It is postulated that these structures may form during precipitation due to co-localization and enhanced miscibility of F8BT and PET at the interface between solvent and non-solvent, thus influencing precipitation kinetics and particle morphology. A positive aspect of this observation is that a mixture of spherical and spindle-shaped particles can better reflect the more heterogeneous morphology of environmental nanoplastics, while still maintaining a similar size distribution and surface properties characteristic of test materials. While most fluorescently labeled MNPs reported in the literature exhibit a spherical morphology, this study expands the diversity of existing test material (see supplementary Table S2).\u003c/p\u003e \u003cp\u003eAs mentioned, TEM images of PP nanomaterials were difficult to produce due to beam-induced melting of the materials during the imaging procedure. Images of the deformed materials are nonetheless depicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). The laser diffraction data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D; right panel) is more informative, showing that co-precipitation of F8BT and PP has only a minor impact on the particle size distribution, indicating a lack of particles with a spindle shape. This may be a result of the relatively large difference in solubility parameters between F8BT and PP, indicating a reduced miscibility between the two polymers and therefore a different co-precipitation behavior. Interestingly, a statistical comparison of the D10 and D50 values from n\u0026thinsp;=\u0026thinsp;5 batches of the labeled vs non-labeled PP nanomaterials (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) does reveal a small but significant decrease in particles sizes for batches produced with 3% F8BT content. This could indicate that a small fraction of elongated particles could also exist within the labeled PP test materials. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also demonstrates that the batch-to-batch variability in particle size distribution is suitably low and the fraction of particles in the submicron size range generally meets previously defined targets (i.e. 85% of the test materials should be \u0026lt;\u0026thinsp;1 \u0026micro;m [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBatch-to-batch reproducibility of particle size distributions of the intermediate product. Student T-tests were used to compare differences between D10 and D50 values in the labeled vs their corresponding non-labeled group. * Denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** denotes p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% F8BT content\u003c/p\u003e \u003cp\u003e(# batches)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD10 [\u0026micro;m]\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003cp\u003e(CV%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD50 [\u0026micro;m]\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003cp\u003e(CV%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD90 [\u0026micro;m] Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003cp\u003e(CV%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e% \u0026lt; 1 \u0026micro;m\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003cp\u003e(CV%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePET\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.075\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003cp\u003e(11.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003cp\u003e(13.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.569\u0026thinsp;\u0026plusmn;\u0026thinsp;0.198\u003c/p\u003e \u003cp\u003e(34.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003cp\u003e(1.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.079\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003cp\u003e(12.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.040\u003c/p\u003e \u003cp\u003e(17.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.043\u0026thinsp;\u0026plusmn;\u0026thinsp;0.511\u003c/p\u003e \u003cp\u003e(16.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e81.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003cp\u003e(4.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010***\u003c/p\u003e \u003cp\u003e(25.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.108\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019**\u003c/p\u003e \u003cp\u003e(17.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.737\u003c/p\u003e \u003cp\u003e(63.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003cp\u003e(5.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003cp\u003e(10.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.256\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003cp\u003e(9.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.900\u0026thinsp;\u0026plusmn;\u0026thinsp;3.270\u003c/p\u003e \u003cp\u003e(112.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003cp\u003e(7.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.132\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003cp\u003e(7.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.257\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003cp\u003e(8.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.365\u0026thinsp;\u0026plusmn;\u0026thinsp;2.671\u003c/p\u003e \u003cp\u003e(79.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e \u003cp\u003e(5.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.109\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012*\u003c/p\u003e \u003cp\u003e(11.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.221\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025*\u003c/p\u003e \u003cp\u003e(11.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.913\u0026thinsp;\u0026plusmn;\u0026thinsp;3.416\u003c/p\u003e \u003cp\u003e(117.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0\u003c/p\u003e \u003cp\u003e(5.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo examine whether the medium exchange between ethanol (intermediate product) and glycerol (final product) influenced the particle size distribution, particle size distributions of test materials with the higher F8BT content were compared before and after medium exchange. For both PET and PP, no significant differences were observed across four batches (PET: p\u0026thinsp;=\u0026thinsp;0.41; PP: p\u0026thinsp;=\u0026thinsp;0.30) in the particle size distribution between the intermediate and final product. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe influence of the type of dispersing agent on the particle size distribution was also compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). As discussed previously, the QC dispersion protocol uses an initial high concentration of the synthetic surfactant, Tween 80, to achieve complete dispersion with the aim of characterizing the \u0026ldquo;true\u0026rdquo; particle size distribution of the materials. In contrast, the presence of Tween 80 may negatively impact many biological assays devised for testing the effects of MNPs. To avoid this, biorelevant (BR) dispersion protocols have been developed and tested [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Here we report an optimized BR dispersion method, which uses a concentrated aqueous BSA solution (10 % wv) to dilute the highly concentrated glycerol suspension to a 1 mg/mL stock suspension (first dilution), followed by a second dilution in cell culture medium to the desired working concentration. BSA was chosen as a dispersing aid based on pilot studies showing that it achieves dispersion more effectively than complex protein mixtures, such as FBS which led to major aggregation in comparison (ESI Figure S2). Since BSA is a pure protein, it likely interacts more consistently with the particle surface, resulting in improved colloidal stability. Typically, dispersions in cell culture media are more challenging than in water due to their high salt concentrations, which promote aggregation of hydrophobic particles [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similar to observations by Wimmer \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], labeled PET showed excellent dispersion using the BR protocol, while labeled PP agglomerated forming 1\u0026ndash;10 \u0026micro;m sized aggregates. Despite extensive optimization of the BR dispersion protocol, the extremely hydrophobic surface of the pristine PP nanoplastics (with or without labels) promotes aggregation, unless more aggressive dispersion protocols using synthetic dispersants, such as Tween 80, are used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEncapsulation efficiency and leaching studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the two particle production methods differ considerably in the choice of solvents, concentration of polymer in the solvent, temperature used, and the volume ratio of anti-solvent to solvent. All these factors are presumed to influence the incorporation of F8BT within the nanoplastics as well as the morphology of the nanoparticles. Given the complexity, it is not possible to accurately predict F8BT location in the particle systems, although some hypotheses may be generated. Given the similar solubility parameter values of F8BT and PET, we hypothesized that these two polymers would show a better miscibility, resulting in a higher amount of F8BT within the particle interior (either miscible, core-shell or occluded). In contrast, the larger differences in F8BT and PP solubility parameters were hypothesized to result in a lower miscibility between the two particles and possibly a higher amount of non-encapsulated F8BT or F8BT on the particle exterior.\u003c/p\u003e \u003cp\u003eDue to the very low F8BT concentrations present in the systems, it is challenging to characterize encapsulation efficiency and particle morphology using commonly reported methods such as nuclear magnetic resonance (NMR)[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] or TEM [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Instead, we exploited the excellent solubility of F8BT in THF (PET and PP are insoluble in THF) to separate internal F8BT from the external fraction. First, the fraction of free F8BT recovered in the ethanol washing liquid was determined. Secondly, particles captured on the filter following washing were subjected to incubation in THF to isolate and quantify non-encapsulated or externally bound F8BT. Finally, the theoretical amount of internal F8BT was calculated by subtracting the sum of the two fractions (wash loss and external F8BT) from the original mass. For both PET and PP test materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B), the amount of F8BT present in the ethanol wash was \u0026lt;\u0026thinsp;1.5 %. As hypotheized, the fraction of non-encapsulated or externally bound F8BT was lower for PET test materials (~\u0026thinsp;15 %) compared t PP test materials (~\u0026thinsp;25 %). No signifcant differences were observed between the lower and higher F8BT contents in both test material types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA fundamental challenge in fluorescently labeling MNPs is dye leaching [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It has been reported that even with commercially available products, the fluorophore can leach out over time [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. To evaluate F8BT leaching from the labeled PET/PP nanoplastics, samples were incubated for seven days at room temperature in either 1) THF, to simulate aggressive conditions most favorable for F8BT extraction, or 2) PBS, mimicking an aqueous biological medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). No F8BT release into the PBS was measurable for any of the samples at any timepoint, indicating that even externally bound F8BT remained particle bound. When incubated in THF, non-encapsulated and externally bound F8BT was dissolved immediately (day 0 value) followed by a marginal further leaching of F8BT into the solvent over the next 24 hours, after which no further leaching was observed. A pilot study explored washing the ethanolic suspension with THF to remove external F8BT. Unfortunately, this resulted in irreversible aggregation and must be further optimized to be useful (supplementary Figure S3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSurface hydrophobicity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further assess whether F8BT alters the surface hydrophobicity of the labeled test materials, contact angle measurements were conducted on dried films of compacted PET and PP nanoparticles with varying F8BT content. It was hypothesized that increasing the amount of F8BT could lead to altered surface hydrophobicity, which could be reflected in the water contact angle. This would have implications for dispersion behavior, biological interactions and wettability. The contact angles of unlabeled PET (108.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8\u0026deg;) and PP (135.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u0026deg;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) are very similar to previous investigations [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], showing that unlabeled PP nanoplastics are significantly more hydrophobic than PET nanoplastics (i.e. larger contact angle). Co-precipitation of PP with increasing amounts of F8BT showed a trend towards very minor decreases in median contact angle, which were not significant. In contrast, addition of either 0.8 % o 5 % ww F8BT to PET increased the median contact angle significantly, indicating an increase in hydrophobicity and thus the presence of F8BT at the particle surface. It should be noted that the contact angle measurements show a high level of variability, likely due to the more complex sample preparation, where surface irregularities or incomplete coverage of the measuring area could have affected the outcome. While the F8BT label does not appear to affect surface hydrophobicity by much under these preparation conditions, further evaluation using complementary techniques like zeta potential or other surface characterization methods such as X-ray photoelectron spectroscopy (XPS) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] or time-of-flight secondary ion mass spectrometry (ToF-SIMS) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] could provide more detail about surface composition and dye localization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOptical properties\u003c/b\u003e \u003c/p\u003e \u003cp\u003eParticle morphology impacts the optical properties of π-conjugated polymers, such as F8BT [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. For example, nanoparticles made of F8BT alone (stabilized with surfactants) exhibit very low limits of detection (LOD) / quantification (LOQ), whereas co-precipitation of F8BT with a second polymer decreases the sensitivity\u0026thinsp;~\u0026thinsp;10-fold (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Fluorescence quenching of F8BT following encapsulation is a commonly observed phenomenon associated with low amounts of the conjugated polymer in the system, aggregation-induced quenching and a coiled conformation of the embedded fluorescent polymer in the matrix [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Despite quenching, these detection levels are still comparable to current methods for nanoplastic quantification, such as pyrolysis-GC/MS and TED-GC-MS (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLimits of detection/quantification for different F8BT nanoparticle systems compared to fluorescence-labeled polystyrene beads.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLOD [\u0026micro;g/mL]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLOQ [\u0026micro;g/mL]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF8BT 100% [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e~\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e~\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5% w/w F8BT / 95% w/w poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e~\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e~\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCommercially available fluorescence labeled yellow-green polystyrene beads [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-[1-Cyano- 2-[4-(Diethylamino)\u0026thinsp;\u0026minus;\u0026thinsp;2-hydroxyphenyl]ethenyl]\u0026thinsp;\u0026minus;\u0026thinsp;1-ethylpyridinium (PCP) / PS [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(E)-2-(2-(4-(dimethylamino)nanphthalen-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium (HCY) / PS [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9-(2,2-dicyanovinyl)julolidine (DCVJ) / PS [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFluorescence excitation and emission scans were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B), showing no significant changes to the excitation and emission spectra between fully soluble F8BT measured in THF (λ\u003csub\u003eEx\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;480 nm; λ\u003csub\u003eEm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;545 nm) and encapsulated F8BT in PET or PP nanoparticles (PET: λ\u003csub\u003eEx\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;480 nm and λ\u003csub\u003eEm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;542 nm; PP: λ\u003csub\u003eEx\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;480 nm and λ\u003csub\u003eEm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;541 nm). The spectra for F8BT encapsulated in PET and PP nanoplastics are very similar to those reported for F8BT embedded within a PEG-PLGA polymer matrix, which ranged from 537\u0026ndash;541 nm [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], depending on manufacturing conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetection limits (LOD and LOQ) and experimental variability were also determined using different dispersion protocols. The method (residual vs intercept) used for LOD and LOQ determination strongly influences reported values. Since guidelines often remain vague, both calculation methods were compared (LOD: Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. LOD values for F8BT-labeled PET ranged between 0.2\u0026ndash;0.7 \u0026micro;g/mL (intercept method) with a higher sensitivity when 5% F8BT was used. These values are comparable to previously reported PEG-PLGA systems also containing 5% F8BT [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] as well as to other systems using various fluorophores and MNPs reported in the literature (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). LOD values of PP (intercept method) also varied between 0.2\u0026ndash;0.9 \u0026micro;g/mL but showed no influence of F8BT content or dispersion protocol on the LOD. Since the QC dispersion protocol resulted in a much lower LOD, it is hypothesized that aggregation of PP test materials during the BR dispersion process may cause aggregation-induced quenching and increased variability in the LOD/LOQ. In all cases, F8BT-labeled PET and PP nanoplastics had significantly lower LOD values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to commercially available Nile Red-labeled polystyrene beads, which were diluted using the BR dispersion protocol into PBS (LOD\u003csub\u003eresidual\u003c/sub\u003e: 32.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 \u0026micro;g/mL; LOD\u003csub\u003eintercept\u003c/sub\u003e: 11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eHandling of suspensions, especially performing dilutions, is known to be associated with greater error than handling solutions. When diluting into complex media, the variability increases further [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In serial dilutions, generally the lower concentrations near the LOD/LOQ are associated with the greatest error, which is why it can be informative to routinely assess the CV% of measured fluorescence values of each concentration in the serial dilution across multiple experimental replicates. In an early pilot study testing the variability of serial dilutions prepared using non-optimized BR dispersion protocols with FBS (supplementary Figure S4) was investigated. Concentrations\u0026thinsp;\u0026gt;\u0026thinsp;15 \u0026micro;g/mL were associated with low variability (CV\u0026thinsp;\u0026lt;\u0026thinsp;10 %),while concentrations\u0026thinsp;\u0026lt;\u0026thinsp;15 \u0026micro;g/mL showed test material-specific differences: PET CV: 30\u0026ndash;80 %; P CV: 50\u0026ndash;130 %. ollowing optimization of the BR dispersion protocol, the variability in the critical concentration range\u0026thinsp;\u0026lt;\u0026thinsp;15 \u0026micro;g/mL could be reduced to \u0026lt;\u0026thinsp;10 % fr nearly all media (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F), with the exception PP diluted in cell lysate (CV: 20\u0026ndash;50 %).This experiment highlights the importance of optimized dispersion protocols and well-trained operators when handling fluorescently labeled nanoplastic suspensions, whereby a CV\u0026thinsp;\u0026lt;\u0026thinsp;10 % btween replicate dilutions is ideal.\u003c/p\u003e \u003cp\u003e \u003cb\u003eApplication of F8BT-labeled PET and PP test materials in cell uptake studies using the FlowCube\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe next step was to characterize the \u003cem\u003ein vitro\u003c/em\u003e performance of the F8BT-labeled test systems in cell culture studies. To evaluate how well the F8BT-labeled nanoplastics remain in dispersion over time, two complementary experiments were conducted. The first focused on the size stability of the stock dispersions (i.e. first dilution of the glycerol suspension into an aqueous medium at 1 mg/mL) when stored at 4\u0026deg;C. This is useful for users who would like to store stock dispersions for multiple use over a given period. The second experiment aimed to simulate the particle size stability in cell culture medium under incubation conditions (37\u0026deg;C, 5% CO₂) over 48 h, representing a typical cell exposure duration.\u003c/p\u003e \u003cp\u003eStability studies over one week storage at 4\u0026deg;C showed that PET materials diluted from glycerol into 10% BSA (1 mg/mL) maintained dispersion stability for at least three days, while from day 5 onwards, a shift towards larger particles became more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Dispersion stability was defined as the percentage of particles\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;m. With PP test materials, two distinct size populations were present at the first timepoint. However, a slight trend toward deagglomeration, possibly due to improved surface coating of the PP sample with BSA over time, was observed but requires further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating that storage of the first aqueous dilution (1 mg/mL) at 4\u0026deg;C is feasible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe colloidal stability of test materials diluted into cell culture medium and incubated at 37\u0026deg;C was also tested. Both PET and PP test materials were colloidally stable up to 24 h but showed evidence of aggregation after 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). This suggests that particles incubated in cell culture medium up to 24 h will show diffusion and sedimentation/flotation behavior characteristic of single particles, while beyond 48 h, the diffusion and sedimentation/flotation behavior of agglomerate structures will need to be considered for dosimetry purposes, as well as mechanisms of particle cell interactions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo assess whether F8BT-labeled PET and PP particles are suitable for quantifying cellular uptake, Calu-3 cells, a commonly used model for evaluating effects on lung epithelial tissues, were employed as a model. Two complementary experimental setups were compared, both with a 24 h exposure duration and an administered dose of 100 \u0026micro;g/mL. The first set up was comprised of a conventional well plate-format, where cells were grown under submerged conditions on round glass cover slips positioned at the bottom of the well. The well-known limitation of this set up is that it is unable to accommodate low-density test materials like PP, which will float to the surface of the medium during incubation thereby reducing particle-cell interactions [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. This behavior was verified using fluorescence spectroscopy to quantify nanoparticle uptake in the Calu-3 cells. Only the fluorescence associated with F8BT-labeled PET was quantifiable (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) and the amount detected (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026micro;g/mL) was only marginally above the LOD (~\u0026thinsp;0.2 \u0026micro;g/mL). This equated to ~\u0026thinsp;0.2 % of the adminitered dose. As a comparison, the uptake of F8BT-labeled PEG-PLGA nanoparticles by a fibroblast cell line was 0.5\u0026ndash;0.8 % [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo overcome the limitations associated with particle buoyancy, the dynamic flow-based exposure system, FlowCube (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), was used. Calu-3 cell monolayers grown on square cover slips were placed vertically into the walls of the chamber and test materials are added to the medium in the center of the chamber. Particle-cell interactions occur either by diffusion only (cube static; non-stirred conditions) or diffusion combined with stirring-induced advection and hydrodynamic shear forces (cube dynamic; stirred conditions). As a control, slides can also be placed at the bottom of the cube to simulate conventional well-plate conditions (well static). In the FlowCube experiment, fluorescence spectroscopy could not be used for quantification, since the amount of particle uptake was below the F8BT detection limits. Instead, semi-quantification of the F8BT-fluorescence signal was measured by flow cytometry.\u003c/p\u003e \u003cp\u003eThe results show that the FlowCube is indeed able to overcome density-related issues associated with cell exposure to buoyant nanomaterials. Flow cytometry showed that similar amounts of F8BT-labeled PET and PP test materials were detected above the background fluorescence in the cube set up (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). Since no significant differences were observed between the static (non-stirred) and dynamic (stirred) conditions, it may be assumed that uptake was primarily caused by consistent diffusion-driven particle-cell contact rather than by centrifugal force. As expected, when cells were positioned horizontally at the bottom of the cube (well static conditions), PET-associated fluorescence was approximately 6-fold higher than that of the vertical cell placement, while PP-associated fluorescence was not detectable. The flow cytometry data was supported by fluorescence microscopy of stained Calu-3 monolayers, which shows the same trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-H; images from the static condition are included in the supplementary; Figure S5). These results confirm that co-precipitated F8BT-labeled nanoplastics are suitable for tracking and quantifying cellular uptake via fluorescence spectroscopy, flow cytometry, and fluorescence confocal imaging.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study provides an innovative approach to fluorescence labeling of submicron sized PET and PP test materials using the bright and stable polymer dye, F8BT. Using a combination of complementary methodologies, we showed that the co-precipitation of the fluorescent polymer, F8BT, with PET leads to a high encapsulation efficiency of the dye (~\u0026thinsp;85%) with very low leaching potential but also promotes the formation of a subset of spindle-shaped particles within the suspension. This mixture of particle morphologies is interesting from both a polymer chemistry perspective and as an example of an MNP test material that shows greater morphological diversity and therefore better mimics environmentally relevant nanoplastics. F8BT was also suitable for labeling PP test materials, with an encapsulation efficiency of ~\u0026thinsp;75% and little impact on particle morphology. The fluorescence label provided a detection limit as low at ~\u0026thinsp;0.2 \u0026micro;g/mL using fluorescence spectroscopy, which is comparable to other current MNP quantification methods. Leaching experiments confirmed that F8BT was retained within the plastic particles even after prolonged exposure to PBS, an aqueous biological medium. Additionally, fluorescence labeling of the materials provided insights into the reproducibility of handling suspensions, which was useful in validating dispersion protocols within different bulk media. \u003cem\u003eIn vitro\u003c/em\u003e experiments were used to investigate particle-cell interactions and uptake using a novel cell exposure model, the FlowCube. The vertical exposure system of the FlowCube overcame dosimetry issues associated with buoyant particles. Cellular uptake of both low and high-density nanoplastics could be measured using the F8BT label, indicating sufficient label sensitivity for future biological applications. Overall, this work contributes valuable information on the development of nanoplastic test materials for environmental and biological studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by funding from the EU's H2020 framework program for research and innovation under grant agreement no. 965173 (Imptox).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.V.N.H. and L.A.D. wrote the main manuscript text. M.V.N.H., M.A., Y.N.M. and L.W. generated experimental data. M.V.N.H. prepared all figures with contributions from Y.N.H. (Figure 2) and M.A. (Figure 8). D.J.S.V. compiled the literature overview in Tables 2, S1 and S2. T.C.V., M.A.G., F.K., R.D.H. and L.A.D. contributed to the study design, supervision and funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by funding from the EU H2020 framework program for research and innovation under grant agreement no. 965173 (Imptox). The authors would like to warmly thank Ing. Claudia Mitterer for granting access to the drop shape analyser and Patrick Treacy and Johannes Szilvassy for participating in the pilot dilution studies. We also want to thank Markus Kirchner for providing the fluorescently labeled polystyrene test particles.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmelia TSM, Khalik WMAWM, Ong MC, Shao YT, Pan HJ, Bhubalan K. Marine microplastics as vectors of major ocean pollutants and its hazards to the marine ecosystem and humans, \u003cem\u003eProgress in Earth and Planetary Science 2021 8:1\u003c/em\u003e, vol. 8, no. 1, pp. 1\u0026ndash;26, Jan. 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/S40645-020-00405-4\u003c/span\u003e\u003cspan address=\"10.1186/S40645-020-00405-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Chen G, Wang J. Microplastics in the Marine Environment: Sources, Fates, Impacts and Microbial Degradation. Toxics. Feb. 2021;9(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/TOXICS9020041\u003c/span\u003e\u003cspan address=\"10.3390/TOXICS9020041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcharla E, et al. Microplastics in marine ecosystems: A comprehensive review of biological and ecological implications and its mitigation approach using nanotechnology for the sustainable environment. Environ Res. Sep. 2024;256:119181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ENVRES.2024.119181\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVRES.2024.119181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Brien S, et al. There\u0026rsquo;s something in the air: A review of sources, prevalence and behaviour of microplastics in the atmosphere. Sci Total Environ. May 2023;874:162193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.SCITOTENV.2023.162193\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2023.162193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorres-Agullo A, Karanasiou A, Moreno T, Lacorte S. Overview on the occurrence of microplastics in air and implications from the use of face masks during the COVID-19 pandemic. Sci Total Environ. Dec. 2021;800:149555. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.SCITOTENV.2021.149555\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2021.149555\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRainieri S, Barranco A. Microplastics, a food safety issue? Trends Food Sci Technol. Feb. 2019;84:55\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.TIFS.2018.12.009\u003c/span\u003e\u003cspan address=\"10.1016/J.TIFS.2018.12.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMir MA et al. Jun., Microplastics in food products: Prevalence, artificial intelligence based detection, and potential health impacts on humans, \u003cem\u003eEmerg. Contam.\u003c/em\u003e, vol. 11, no. 2, p. 100477, 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.EMCON.2025.100477\u003c/span\u003e\u003cspan address=\"10.1016/J.EMCON.2025.100477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajvanshi J, et al. An analytical review on revamping plastic waste management: exploring recycling, biodegradation, and the growing role of biobased plastics. Environ Sci Pollut Res. Apr. 2024;1\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/S11356-024-33333-7/FIGURES/5\u003c/span\u003e\u003cspan address=\"10.1007/S11356-024-33333-7/FIGURES/5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Mu S, Wang W, Li X. Potential threats of environmental microplastics to the skeletal system: current insights and future directions. Front Endocrinol (Lausanne). 2025;16:1658056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/FENDO.2025.1658056\u003c/span\u003e\u003cspan address=\"10.3389/FENDO.2025.1658056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNozari asl R, Jaafarzadeh Haghighi Fard N, Jahedi F, Khaksar MA, Shenavar B. Systematic review of pulmonary toxicity induced by microplastics and nanoplastics: Insights from in vivo and in vitro studies, \u003cem\u003eToxicologie Analytique et Clinique\u003c/em\u003e, vol. 37, no. 2, pp. 223\u0026ndash;241, Jun. 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.TOXAC.2024.12.002\u003c/span\u003e\u003cspan address=\"10.1016/J.TOXAC.2024.12.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarycleopha M, Balarabe BY, Kumar S, Adjama I. Exploring the Impact of Microplastics and Nanoplastics on Macromolecular Structure and Functions. J Appl Toxicol. Jan. 2026;46(1):22\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/JAT.4915\u003c/span\u003e\u003cspan address=\"10.1002/JAT.4915\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sid-Cheikh M, Rowland SJ, Stevenson K, Rouleau C, Henry TB, Thompson RC. Uptake, Whole-Body Distribution, and Depuration of Nanoplastics by the Scallop Pecten maximus at Environmentally Realistic Concentrations, 2018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.est.8b05266\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.8b05266\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez GL, Lamarre A. The impact of micro- and nanoplastics on immune system development and functions: Current knowledge and future directions. Reprod Toxicol. Aug. 2025;135:108951. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.REPROTOX.2025.108951\u003c/span\u003e\u003cspan address=\"10.1016/J.REPROTOX.2025.108951\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Akker K, Mandemaker LDB, Dorresteijn JM, Amaral-Zettler LA, Weckhuysen BM, Meirer F. Fluorescent nanoplastics: What steps are needed towards a representative toolkit? \u003cem\u003eMicroplastics and Nanoplastics 2025 6:1\u003c/em\u003e, vol. 6, no. 1, p. 4-, Dec. 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s43591-025-00159-0\u003c/span\u003e\u003cspan address=\"10.1186/s43591-025-00159-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrosset-Perrotin G et al. Jun., Production, labeling, and applications of micro- and nanoplastic reference and test materials, \u003cem\u003eEnviron. Sci. Nano\u003c/em\u003e, vol. 12, no. 6, pp. 2911\u0026ndash;2964, 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/D4EN00767K\u003c/span\u003e\u003cspan address=\"10.1039/D4EN00767K\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWimmer L, et al. A quality-by-design inspired approach to develop PET and PP nanoplastic test materials for use in in vitro and in vivo biological assays. Environ Sci Nano. 2025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d4en01186d\u003c/span\u003e\u003cspan address=\"10.1039/d4en01186d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerdy P et al. Aug., Nanoplastic production procedure for scientific purposes: PP, PVC, PE-LD, PE-HD, and PS, \u003cem\u003eHeliyon\u003c/em\u003e, vol. 9, no. 8, p. e18387, 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.HELIYON.2023.E18387\u003c/span\u003e\u003cspan address=\"10.1016/J.HELIYON.2023.E18387\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H, Hu Q, Li W, Cai X, Mao L, Li R. Approaches to Nanoparticle Labeling: A Review of Fluorescent, Radiological, and Metallic Techniques. Environ Health. Aug. 2023;1(2):75\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ENVHEALTH.3C00034\u003c/span\u003e\u003cspan address=\"10.1021/ENVHEALTH.3C00034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSullivan GL, Gallardo JD, Jones EW, Hollliman PJ, Watson TM, Sarp S. Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF). Chemosphere. Jun. 2020;249:126179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.CHEMOSPHERE.2020.126179\u003c/span\u003e\u003cspan address=\"10.1016/J.CHEMOSPHERE.2020.126179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuemichen E, Eisentraut P, Celina M, Braun U. Automated thermal extraction-desorption gas chromatography mass spectrometry: A multifunctional tool for comprehensive characterization of polymers and their degradation products. J Chromatogr A. May 2019;1592:133\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.CHROMA.2019.01.033\u003c/span\u003e\u003cspan address=\"10.1016/J.CHROMA.2019.01.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZytowski E, Baldermann S. Thermal Desorption and Extraction Coupled With Gas Chromatography and Mass Spectrometry for the Quantification of Polystyrene Nanoplastic in Pak Choi, \u003cem\u003eRapid Communications in Mass Spectrometry\u003c/em\u003e, vol. 39, no. 14, p. e10046, Jul. 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/RCM.10046\u003c/span\u003e\u003cspan address=\"10.1002/RCM.10046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026oslash;dland ES, et al. A novel method for the quantification of tire and polymer-modified bitumen particles in environmental samples by pyrolysis gas chromatography mass spectroscopy. J Hazard Mater. Feb. 2022;423(10):127092. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jhazmat.2021.127092\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2021.127092\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sid-Cheikh M, Rowland SJ, Kaegi R, Henry TB, Cormier MA, Thompson RC. Synthesis of 14C-labelled polystyrene nanoplastics for environmental studies, \u003cem\u003eCommunications Materials 2021 1:1\u003c/em\u003e, vol. 1, no. 1, p. 97-, Dec. 2020, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s43246-020-00097-9\u003c/span\u003e\u003cspan address=\"10.1038/s43246-020-00097-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark NJ, et al. Determining the accumulation potential of nanoplastics in crops: An investigation of 14C-labelled polystyrene nanoplastic into radishes. Environ Res. Nov. 2025;284:122687. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ENVRES.2025.122687\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVRES.2025.122687\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Li J, Parakhonskiy BV, Hoogenboom R, Skirtach A, De Neve S. Labelling of micro- and nanoplastics for environmental studies: state-of-the-art and future challenges. J Hazard Mater. Jan. 2024;462:132785. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.JHAZMAT.2023.132785\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2023.132785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStaufer T, et al. Biodistribution of nanoplastics in mice: advancing analytical techniques using metal-doped plastics. Commun Biol. Dec. 2025;8(1):1247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/S42003-025-08709-1\u003c/span\u003e\u003cspan address=\"10.1038/S42003-025-08709-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillacorta A, et al. Fluorescent labeling of micro/nanoplastics for biological applications with a focus on true-to-life tracking. J Hazard Mater. Sep. 2024;476:135134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.JHAZMAT.2024.135134\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2024.135134\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerdy P, Bonneau A, Delpy F, Lucas Y. Fluorescent labelling as a tool for identifying and quantifying nanoplastics, \u003cem\u003eRSC Adv.\u003c/em\u003e, vol. 14, no. 50, pp. 37610\u0026ndash;37617, Nov. 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/D4RA04526B\u003c/span\u003e\u003cspan address=\"10.1039/D4RA04526B\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehnke T, W\u0026uuml;rth C, Laux EM, Hoffmann K, Resch-Genger U. Simple strategies towards bright polymer particles via one-step staining procedures. Dyes Pigm. Aug. 2012;94(2):247\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.DYEPIG.2012.01.021\u003c/span\u003e\u003cspan address=\"10.1016/J.DYEPIG.2012.01.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad Khanbeigi R et al. Mar., Surface Chemistry of Photoluminescent F8BT Conjugated Polymer Nanoparticles Determines Protein Corona Formation and Internalization by Phagocytic Cells, \u003cem\u003eBiomacromolecules\u003c/em\u003e, vol. 16, no. 3, pp. 733\u0026ndash;742, 2015, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bm501649y\u003c/span\u003e\u003cspan address=\"10.1021/bm501649y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbelha TF, et al. Bright conjugated polymer nanoparticles containing a biodegradable shell produced at high yields and with tuneable optical properties by a scalable microfluidic device. Nanoscale. Feb. 2017;9(5):2009\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/C6NR09162H\u003c/span\u003e\u003cspan address=\"10.1039/C6NR09162H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eModicano P, et al. Does Encapsulation of π-Conjugated Polymer Nanoparticles within Biodegradable PEG\u0026ndash;PLGA Matrices Mitigate Photoinduced Free Radical Production and Phototoxicity? Adv Ther (Weinh). Jan. 2025;8(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ADTP.202400190\u003c/span\u003e\u003cspan address=\"10.1002/ADTP.202400190\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Brito EB, et al. Synthesis and characterization of novel fluorene\u0026ndash;based green copolymers and their potential application in organic light-emitting diodes. J Mater Res Technol. Jan. 2024;28:4317\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.JMRT.2023.12.249\u003c/span\u003e\u003cspan address=\"10.1016/J.JMRT.2023.12.249\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVandenburg HJ, Clifford AA, Bartle KD, Carlson RE, Carroll J, Newton ID. A simple solvent selection method for accelerated solvent extraction of additives from polymers, \u003cem\u003eAnalyst\u003c/em\u003e, vol. 124, no. 11, pp. 1707\u0026ndash;1710, Jan. 1999, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/A904631C\u003c/span\u003e\u003cspan address=\"10.1039/A904631C\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanschmann B. Precipitation of Polypropylene and Polyethylene Terephthalate Powders Using Green Solvents via Temperature and Antisolvent-Induced Phase Separation, \u003cem\u003eAdvances in Polymer Technology\u003c/em\u003e, vol. 2023, no. 1, p. 7651796, Jan. 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2023/7651796\u003c/span\u003e\u003cspan address=\"10.1155/2023/7651796\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, et al. Molecular behavior of silicone adhesive at buried polymer interface studied by molecular dynamics simulation and sum frequency generation vibrational spectroscopy \u0026dagger;. Royal Soc Chem Soft Matter. 2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d4sm00407h\u003c/span\u003e\u003cspan address=\"10.1039/d4sm00407h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrbano L, et al. Influence of Surfactant Structure on Photoluminescent π-Conjugated Polymer Nanoparticles: Interfacial Properties and Protein Binding. Langmuir. 2018;34:6125. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.langmuir.8b00561\u003c/span\u003e\u003cspan address=\"10.1021/acs.langmuir.8b00561\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDovgolevsky E, Kirmayer S, Lakin E, Yang Y, Brinker CJ, Frey GL. Self-assembled conjugated polymer\u0026ndash;surfactant\u0026ndash;silica mesostructures and their integration into light-emitting diodes. J Mater Chem. Jan. 2008;18(4):423\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/B713170D\u003c/span\u003e\u003cspan address=\"10.1039/B713170D\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta GP, Choi P, Stoyanov SR, Liu Q. The temperature dependence of the Hildebrand solubility parameters of selected hydrocarbon polymers and hydrocarbon solvents: a molecular dynamics investigation. J Mol Model. Jul. 2024;30(7):196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/S00894-024-05929-W\u003c/span\u003e\u003cspan address=\"10.1007/S00894-024-05929-W\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Z, et al. Diverse Particle Carriers Prepared by Co-Precipitation and Phase Separation: Formation and Applications. ChemPlusChem. Jan. 2021;86(1):49\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/CPLU.202000497\u003c/span\u003e\u003cspan address=\"10.1002/CPLU.202000497\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeuman A, Zhang S, Lee D, Riggleman RA. Increases in Miscibility of a Binary Polymer Blend Confined within a Nanoparticle Packing. Macromolecules. Feb. 2023;56(3):954\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACS.MACROMOL.2C01918\u003c/span\u003e\u003cspan address=\"10.1021/ACS.MACROMOL.2C01918\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindvig T, Michelsen ML, Kontogeorgis GM. A Flory\u0026ndash;Huggins model based on the Hansen solubility parameters. Fluid Phase Equilib. Dec. 2002;203:1\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0378-3812(02)00184-X\u003c/span\u003e\u003cspan address=\"10.1016/S0378-3812(02)00184-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiranda-Quintana RA, Chen L, Smiatek J. Insights into Hildebrand Solubility Parameters \u0026ndash; Contributions from Cohesive Energies or Electrophilicity Densities?**. ChemPhysChem. Jan. 2024;25(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/CPHC.202300566\u003c/span\u003e\u003cspan address=\"10.1002/CPHC.202300566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenkatram S, Kim C, Chandrasekaran A, Ramprasad R. Critical Assessment of the Hildebrand and Hansen Solubility Parameters for Polymers, \u003cem\u003eJ. Chem. Inf. Model.\u003c/em\u003e, vol. 59, no. 10, pp. 4188\u0026ndash;4194, Oct. 2019, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACS.JCIM.9B00656\u003c/span\u003e\u003cspan address=\"10.1021/ACS.JCIM.9B00656\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShrivastava A, Gupta VB. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles Young Scientists. 2011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/2229-5186.79345\u003c/span\u003e\u003cspan address=\"10.4103/2229-5186.79345\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaari Lambarki L, et al. Comparison of approaches for assessing detection and quantitation limits in bioanalytical methods using HPLC for sotalol in plasma OPEN. Nature. 2025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-024-83474-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-024-83474-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInternational Conference on Harmonisation of Technical Requirements for Registration. of Pharmaceuticals for Human Use (ICH), Validation of Analytical Procedures: Text and Methodology Q2(R1), 2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaputo F, et al. Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range. J Colloid Interface Sci. Apr. 2021;588:401\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.JCIS.2020.12.039\u003c/span\u003e\u003cspan address=\"10.1016/J.JCIS.2020.12.039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato H, et al. Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol In Vitro. Aug. 2009;23(5):927\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.TIV.2009.04.006\u003c/span\u003e\u003cspan address=\"10.1016/J.TIV.2009.04.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarucco A, et al. Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS). Mater 2019. Nov. 2019;12(23):3833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/MA12233833\u003c/span\u003e\u003cspan address=\"10.3390/MA12233833\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmpala C, Renault JP, Tach\u0026eacute; O, Courn\u0026egrave;de \u0026Eacute;, Devineau S, Chivas-Joly C. Stability and dispersibility of microplastics in experimental exposure medium and their dimensional characterization by SMLS, SAXS, Raman microscopy, and SEM. J Hazard Mater. May 2024;469:134083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.JHAZMAT.2024.134083\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2024.134083\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanoni I et al. Feb., Characterization of polyethylene and polyurethane microplastics and their adsorption behavior on Cu2\u0026thinsp;+\u0026thinsp;and Fe3\u0026thinsp;+\u0026thinsp;in environmental matrices, \u003cem\u003eEnvironmental Sciences Europe 2025 37:1\u003c/em\u003e, vol. 37, no. 1, p. 21-, 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/S12302-025-01061-5\u003c/span\u003e\u003cspan address=\"10.1186/S12302-025-01061-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XM, Patel AB, De Graaf RA, Behar KL. Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem Phys Lipids. Jan. 2004;127(1):113\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.CHEMPHYSLIP.2003.09.013\u003c/span\u003e\u003cspan address=\"10.1016/J.CHEMPHYSLIP.2003.09.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams WA, Aravamudhan S. Micro-Nanoparticle Characterization: Establishing Underpinnings for Proper Identification and Nanotechnology-Enabled Remediation, \u003cem\u003ePolymers (Basel).\u003c/em\u003e, vol. 16, no. 19, p. 2837, Oct. 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/POLYM16192837\u003c/span\u003e\u003cspan address=\"10.3390/POLYM16192837\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang C, Luo Y, Naidu R. Microplastics and nanoplastics analysis: Options, imaging, advancements and challenges. TRAC Trends Anal Chem. Sep. 2023;166:117158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.TRAC.2023.117158\u003c/span\u003e\u003cspan address=\"10.1016/J.TRAC.2023.117158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCatarino AI, Frutos A, Henry TB. Use of fluorescent-labelled nanoplastics (NPs) to demonstrate NP absorption is inconclusive without adequate controls, \u003cem\u003eScience of The Total Environment\u003c/em\u003e, vol. 670, pp. 915\u0026ndash;920, Jun. 2019, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.scitotenv.2019.03.194\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2019.03.194\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026uuml;r C, Rist S, Baun A, Mayer P, Hartmann NB, Wagner M. When Fluorescence Is not a Particle: The Tissue Translocation of Microplastics in Daphnia magna Seems an Artifact, \u003cem\u003eEnviron. Toxicol. Chem.\u003c/em\u003e, vol. 38, no. 7, pp. 1495\u0026ndash;1503, Jul. 2019, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/etc.4436\u003c/span\u003e\u003cspan address=\"10.1002/etc.4436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelo-Agust\u0026iacute;n P, Kozak ER, M. de Jes\u0026uacute;s Perea-Flores, and, Mendoza-P\u0026eacute;rez JA. Identification of microplastics and associated contaminants using ultra high resolution microscopic and spectroscopic techniques, \u003cem\u003eScience of The Total Environment\u003c/em\u003e, vol. 828, p. 154434, Jul. 2022, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.SCITOTENV.2022.154434\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2022.154434\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSobhani Z, Zhang X, Gibson C, Naidu R, Megharaj M, Fang C. Identification and visualisation of microplastics/nanoplastics by Raman imaging (i): Down to 100 nm. Water Res. May 2020;174:115658. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.WATRES.2020.115658\u003c/span\u003e\u003cspan address=\"10.1016/J.WATRES.2020.115658\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJungnickel H, et al. Time-of-flight secondary ion mass spectrometry (ToF-SIMS)-based analysis and imaging of polyethylene microplastics formation during sea surf simulation. Sci Total Environ. Sep. 2016;563\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.SCITOTENV.2016.04.025\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2016.04.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eModicano P, et al. Enhanced optical imaging properties of lipid nanocapsules as vehicles for fluorescent conjugated polymers. Eur J Pharm Biopharm. Sep. 2020;154:297\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.EJPB.2020.07.017\u003c/span\u003e\u003cspan address=\"10.1016/J.EJPB.2020.07.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu T, Hu G, Ning J, Yang J, Zhou Y. A photoluminescence strategy for detection nanoplastics in water and biological imaging in cells and plants. J Hazard Mater. Jan. 2024;461:132695. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jhazmat.2023.132695\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2023.132695\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L, et al. Identification of Nanoplastics by Probing the Viscous Nanoenvironment. Small Sci. Dec. 2025;5(12). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/smsc.202500430\u003c/span\u003e\u003cspan address=\"10.1002/smsc.202500430\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoraz A, Breider F. Detection and Quantification of Nonlabeled Polystyrene Nanoparticles Using a Fluorescent Molecular Rotor. Anal Chem. Nov. 2021;93(45):14976\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.analchem.1c02055\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c02055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltmann K et al. Dec., Characterizing nanoplastic suspensions of increasing complexity: inter-laboratory comparison of size measurements using dynamic light scattering, \u003cem\u003eEnviron. Sci. Nano\u003c/em\u003e, vol. 12, no. 12, pp. 5242\u0026ndash;5256, 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/D5EN00645G\u003c/span\u003e\u003cspan address=\"10.1039/D5EN00645G\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma G, et al. Iron oxide nanoparticle agglomeration influences dose rates and modulates oxidative stress-mediated dose\u0026ndash;response profiles in vitro. Nanotoxicology. Sep. 2013;8(6):663. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/17435390.2013.822115\u003c/span\u003e\u003cspan address=\"10.3109/17435390.2013.822115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatson CY, DeLoid GM, Pal A, Demokritou P. Buoyant Nanoparticles: Implications for Nano-Biointeractions in Cellular Studies. Small. Jun. 2016;12(23):3172\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/SMLL.201600314\u003c/span\u003e\u003cspan address=\"10.1002/SMLL.201600314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoore TL, et al. Nanoparticle administration method in cell culture alters particle-cell interaction. Nature. 2019. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-018-36954-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-36954-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchr\u0026ouml;ter L, Ventura N. Nanoplastic Toxicity: Insights and Challenges from Experimental Model Systems. Small. Aug. 2022;18(31). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/SMLL.202201680\u003c/span\u003e\u003cspan address=\"10.1002/SMLL.202201680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microplastics-and-nanoplastics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mina","sideBox":"Learn more about [Microplastics and Nanoplastics](http://microplastics.springeropen.com)","snPcode":"43591","submissionUrl":"https://submission.nature.com/new-submission/43591/3","title":"Microplastics and Nanoplastics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"microplastics, nanoplastics, fluorescently labeled nanoplastics, polyethylene terephthalate, polypropylene, F8BT","lastPublishedDoi":"10.21203/rs.3.rs-8842721/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8842721/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicro- and nanoplastic (MNP) particles are widely present in nature, mainly due to the extensive overuse of single-use plastics combined with poor waste management. Despite the diversity in the environment, many experimental studies still rely almost exclusively on polystyrene as a model plastic test material, while other environmentally relevant polymers remain underrepresented. In addition, labeled MNP test materials suitable for biological studies are still limited. In this study, nanosized polyethylene terephthalate (PET) and polypropylene (PP) particles were produced using a co-precipitation approach with the fluorescent π-conjugated polymer, poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) at low (0.8 % w/w) and high (3–5 % w/w) F8BT content. Fluorescently labeled MNPs (75–85 % dye internalization) could be produced with \u0026gt; 85 % of the particles in the submicron size range. Co-precipitation of F8BT with PET produced a subset of spindle-shaped particles, while F8BT-labeled PP particles were primarily spherical. The fluorescence limit of detection of the F8BT labeled PET and PP was ~0.2 µg/mL for both systems. The strong fluorescence enabled measurements of cell uptake using an innovative exposure system, the FlowCube, which overcomes dosimetry issues with buoyant particles. This work provides an innovative approach to producing fluorescently labeled PET and PP nanoplastic test materials for environmental and biological studies.\u003c/p\u003e","manuscriptTitle":"Labeling of PET and PP nanoplastic test materials with non-leachable π-conjugated fluorescent polymers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 07:09:58","doi":"10.21203/rs.3.rs-8842721/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-29T17:43:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20832588212492345155539170135207274183","date":"2026-04-09T10:08:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197311591497711147114277584431370929503","date":"2026-04-08T06:43:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-11T15:02:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T10:44:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-17T10:41:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microplastics and Nanoplastics","date":"2026-02-10T14:21:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microplastics-and-nanoplastics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mina","sideBox":"Learn more about [Microplastics and Nanoplastics](http://microplastics.springeropen.com)","snPcode":"43591","submissionUrl":"https://submission.nature.com/new-submission/43591/3","title":"Microplastics and Nanoplastics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4efc74a7-3e3e-44b6-a0b2-aab61ee92e0b","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-04-29T17:43:34+00:00","index":32,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T07:10:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 07:09:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8842721","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8842721","identity":"rs-8842721","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}