Journal: bioRxiv

Article Title: Cell-nanoplastics association impacts cell proliferation and motility

doi: 10.64898/2026.04.03.716369

Figure Lengend Snippet: (A) Representative confocal images of MDCK II monolayers expressing CaaX-GFP to label cell membrane (green), stained with Hoechst 33342 to label cell nucleus (cyan), and pre-incubated with 20 µg/mL red fluorescent NPs (red) for 24 hours. Panel on the right is a z -stack image. (B) Cell-associated NP fluorescence of different cell lines quantified using flow cytometry. The median fluorescence intensity (MFI) of N = 3 biological replicates is plotted in the log scale. (C) A method to quantify the mass of NPs using epifluorescence microscopy and microfluidic devices. 20 to 1600 µg/mL of NPs were injected into a microfluidic channel with known height similar to tissue cells, and imaged using an epifluorescence microscope. The total fluorescence per unit area is a linear function of the NP density. n = 3 technical repeats per condition. (D) Quantification of cell-associated NP density at different NP loading times in the MDCK II monolayer. 20 µg/mL NPs were loaded for 1, 6, and 24 hours, followed by washing and epifluorescence imaging. The mass density was then calculated based on the cell height measured in (A) and the calibration curve in (C). N = 4. (E) The relative NP release at different times after 24 hours of NP loading in the MDCK II monolayer. The release fraction is the loss of fluorescence intensity compared to the intensity measured immediately after NP loading by tracking the same region of cells. N = 4. (F) Schematic of the in vivo model. C57BL/6 mice were administered 100 µL of NPs (2 mg/mL) via intravenous (i.v.) injection, then sacrificed at 1, 7, and 30 days post-injection. NP retention was assessed by IVIS imaging. (G) NP distribution in major organs and brain 30 days post-injection. (H) Representative images and total radiant efficiency of NPs in liver at 1, 7, and 30 days post-injection. (B, C, D, E, H) Error bars represent standard deviation. Student’s t -test or one-way ANOVA was used. Representative statistical significance is shown: ns for p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (A) Scale bar = 10 µm.

Article Snippet: Microfluidic channel masks were designed using AutoCAD and fabricated by FineLineImaging.

Techniques: Expressing, Membrane, Staining, Incubation, Fluorescence, Flow Cytometry, Epifluorescence Microscopy, Injection, Microscopy, Imaging, In Vivo, Standard Deviation

Journal: bioRxiv

Article Title: Cell-nanoplastics association impacts cell proliferation and motility

doi: 10.64898/2026.04.03.716369

Figure Lengend Snippet: (A) Relative cell-associated NP fluorescence in MDCK II monolayers under treatment with different inhibitors. Cells were co-incubated with 20 µg/mL NPs and inhibitors for 6 hours. NP fluorescence intensity was normalized to vehicle control (DMSO). N = 3-6. (B) Dosedependent response of NP association under EIPA (NHE1 inhibitor) treatment. N = 6. (C) Relative NP release from MDCK II monolayers under various drug treatments. All samples were first incubated with 20 µg/mL NPs for 6 hours without drug treatment, followed by extensive washing and imaging to quantify the initial level of cell-associated NPs. Cells were then incubated in NP-free media containing inhibitors for 24 hours, after which NP fluorescence was measured again. The release fraction was calculated as the relative decrease in fluorescence intensity over this period. Conditions that compromised monolayer integrity due to cytotoxicity were excluded. N = 3-6. (D and E) Dose-dependent NP release fractions under treatment with CID-1067700 (Rab7 inhibitor; D) and LY294002 (PI3K inhibitor; E). N = 3-6. (F) Quantification of trans-epithelial NP transport sing a transwell system. MDCK II monolayers grown on 1 µm pore transwells were treated with 50 µg/mL NPs and inhibitors for 24 hours. NPs in the lower compartment were quantified using microfluidic devices. Empty transwells were used as baseline controls. N = 5. (G) Quantification of trans-epithelial NP transport in the presence of Dynasore or LY294002 using the assay in (F). N = 4-5. (H) Schematic illustration of pathways affecting NP-cell association. (A-G) Error bars represent standard deviation. (A and B) Paired t -test or RM one-way ANOVA was used. (C-G) Student’s t -test or one-way ANOVA was used.

Article Snippet: Microfluidic channel masks were designed using AutoCAD and fabricated by FineLineImaging.

Techniques: Fluorescence, Incubation, Control, Imaging, Standard Deviation