Fluorescein Sodium Loaded By Polyethyleneimine for Fundus Fluorescein Angiography Improves Adhesion

Figures & data

Figure 1. Schematic illustration of the preparation of the PEI–NHAc–FS NPs.

FS: Fluorescein sodium; NP: Nanoparticle; PEI: Polyethyleneimine.

Figure 2. Different characterization methods including ¹H NMR spectrum, Fourier transform infrared spectrum spectrum and differential thermal/thermogravimetric analysis.

(A) ¹H NMR spectrum, (B) Fourier transform infrared spectrum and (C) differential thermal/thermogravimetric analysis of fluorescein sodium (FS) (1), polyethyleneimine (PEI) (2), PEI–NH₂–FS nanoparticles (3) and PEI–NHAc–FS nanoparticles (4), respectively.

Table 1. Mean particle size and zeta potential of PEI–NHAc–FS.

Formulation	Zeta potential (mV)	Mean diameter (nm)	PDI
FS	0.90 ± 0.39	2060.33 ± 477.53	0.733
PEI	0.16 ± 0.11	16.14 ± 2.16	0.403
PEI–NH2–FS	2.32 ± 2.10	42.26 ± 15.25	0.244
PEI–NHAc–FS	0.05 ± 0.02	181.33 ± 88.02	0.302

FS: Fluorescein sodium; PDI: Polymer dispersity index; PEI: Polyethyleneimine.

Figure 3. Different characterization methods including UV-Vis spectrum and EEM fluorescence spectrum.

UV-Vis spectrum (A) and excitation-emission matrix fluorescence spectrum (B) of fluorescein sodium (FS) (1), polyethyleneimine (PEI) (2), PEI–NH₂–FS NPs (3) and PEI–NHAc–FS NPs (4), respectively.

Figure 4. Analysis of cell viability and apoptosis by CCK-8 assay and Annexin V/PI staining.

(A) CCK-8 assay of ARPE-19 cells treated with FS and (B) PEI–NHAc–FS nanoparticles at different FS concentrations for 12 and 24 h. (C) Morphological images of ARPE-19 cells treated with 0, 2.5, 5, 7.5, 10 μM FS and PEI–NHAc–FS nanoparticles for 24 h. (Scale bar: 200 μm). (D) Cell apoptosis was detected by Annexin V/PI staining. (E) Statistical analysis of cells rate (%).

FITC: Fluorescein isothiocyanate; FS: Fluorescein sodium; PEI: Polyethyleneimine; PI: Propidium Iodide.

Figure 5. Celluar uptake was recorded by fluorescence intensity via flow cytometry and fluorescent microscope.

Fluorescence intensity was detected by flow cytometry with a 488-nm Argon-ion laser for the incubation of ARPE-19 cells with free FS and polyethyleneimine–NHAc–FS nanoparticles for (A) 12 h or (B) 24 h at different FS concentration. Statistical analysis of FITC-A mean in varying concentrations of FS and PEI–NHAc–FS in ARPE-19 cells for (C) 12 h and (D) 24 h. Fluorescence images were recorded for the incubation of ARPE-19 cells with free FS and polyethyleneimine–NHAc–FS nanoparticles at different FS concentration for (E) 12 h and (F) 24 h. Scale bar: 200 μm.

FITC: Fluorescein isothiocyanate; FS: Fluorescein sodium.

Figure 6. Fluorescence intensity was detected in ARPE-19 cells for 24 h via confocal microscopy.

Scale bar: 10 μm.

FS: Fluorescein sodium.

Figure 7. In vivo fluorescence imaging of different agents was detected by fundus angiography.

Fundus angiography images by at autofluorescence mode and different time points postintravenous injection of free FS or PEI–NHAc–FS nanoparticles at different FS concentration ([A] 0.5%, [B] 1% and [C] 5%).

FS: Fluorescein sodium; PEI: Polyethyleneimine.

Figure 8. Laser-induced choroidal neovascularization in Brown Norway rats for 28 days, and fundus angiography images at autofluorescence mode and different time points postintravenous injection of free fluorescein sodium or PEI–NHAc–FS nanoparticles at 1% fluorescein sodium concentration.

FS: Fluorescein sodium; PEI: Polyethyleneimine.

Figure 9. In vivo analysis of biosecurity was measured by hematoxylin and eosin staining and electroretinogram.

(A) Histomorphological images of heart, liver, spleen, kidney, brain and muscle after intravenous injection of polyethyleneimine–NHAc–fluorescein sodium nanoparticles (1% fluorescein sodium concentration) after 28 days. Scale bar: 50 μm. (B) The retinal function was measured postintravenous injection of polyethyleneimine–NHAc–fluorescein sodium nanoparticles (1% fluorescein sodium concentration) via electroretinogram. (C) Statistical analysis of the mean amplitudes of the 1 (a-wave) and 2 (b-wave).