Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

Figures & data

Figure 1. Schematic representation of the protocol used to prepare cRGD-conjugated PICsomes with tunable cRGD densities (yellow spheres, acetal groups; orange pentagons, cyclic RGD ligands).

Figure 2. Characterization of the cRGD-PICsomes. (A) ¹H NMR spectrum of a cRGD-conjugated PICsome (400 MHz, D₂O, 80 °C), (B) a TEM image of uranyl acetate staining (scale bar, 100 nm).

Table 1. Characteristics of the obtained cRGD-conjugated PICsomes.

Sample	cRGD content^a (mol %)	Diameter^b (nm)	Diameter^c (nm)	PDI^b	Zeta potential^d (mV)
Ctrl-PICsome	0	92 ± 5	90 ± 12	0.085	−28
20%-cRGD-PICsome	23.4	86 ± 10	82 ± 21	0.094	−27
40%-cRGD-PICsome	40.4	94 ± 8	86 ± 21	0.016	−23
100%-cRGD-PICsome	97.5	92 ± 2	85 ± 23	0.072	−20

^a The cRGD content was determined using ¹H NMR.

^b The number average diameter, as determined using DLS (n = 10, mean ± s.d., s.d. = standard deviation).

^c The number average diameter, as determined using TEM (n = 200, mean ± s.d.).

^d The zeta-potentials were determined using a zetasizer.

Figure 3. Uptake of cRGD-PICsomes and Ctrl-PICsomes into HUVECs. (A) High-throughput automated analysis of fluorescence images using an IN Cell Analyzer 1000. Data are presented as the mean ± standard error of the mean (s.e.m.), n = 3. Two-way ANOVA was used to analyze the differences in the fluorescence intensity, and ∗P < 0.005 and ∗∗P < 0.001 were considered significant. (B) Images were captured after a 24 h incubation of HUVECs with PICsomes (red) using confocal laser scanning microscopy (CLSM); nuclei were stained using Hoechst 33342 (blue). Scale bar (white) = 20 μm. CLMS images of 40%-cRGD-PICsomes (C) and 100%-cRGD-PICsomes (D); the late-endosomes and lysosomes were stained using Lyso Tracker Green (green). Scale bar (purple) = 50 μm. (E) Quantitative analysis of fluorescence intensity with quenching method using trypan blue. Data are presented as the mean ± standard deviation, n = 30. The data were analyzed using Student’s t-test, and P∗ < 0.001 was considered significant.

Figure 4. Inhibition of the cellular uptake of cRGD-PICsomes using echistatin in HUVECs. Black line, untreated cells; red line, cells incubated with Cy3-labeled 100%-cRGD-PICsomes; blue line, cells treated with 10 nM echistatin and incubated with Cy3-labeled 100%-cRGD-PICsomes.

Figure 5. Plasma clearance (A), tumor- (B), and liver- accumulation (C) of PICsomes. Blue line, Ctrl-PICsomes; red line, 20%-cRGD-PICsomes, green line, 40%-cRGD-PICsomes; purple line, 100%-cRGD-PICsomes. The data were analyzed using Student’s t-test. Data are presented as the mean ± s.e.m., n = 4; ∗P < 0.05.

Figure 6. Neovascular-targeting of PICsomes following tail-vein injection. IVCLSM images observed in tumor blood vessels (A) 1 h and (B) 6 h after administration of PICsomes. Red, Cy5-labeled Ctrl-PICsomes; green, DyLight488-labeled 40%-cRGD-PICsomes; yellow, co-localization of red and green signals. Scale bar = 100 μm in all images.

Figure 7. IVCLSM analysis of PICsomes in the tumor vasculature. Blood vessels were pre-stained (blue) with anti-CD31 (PECAM-1) antibodies, and tumor vasculature snapshots were then taken (A) 6 h and (B) 24 h after the administration of 40%-cRGD-PICsomes (red). Scale bar = 100 μm in all images. Co-localizations of blood vessels (blue) and 40%-cRGD-PICsomes (red) in the region selected (indicated by a white rectangle in figures 7(A) and (B)). (C) Confocal laser scanning microscopy (CLMS) analysis of a tumor section. The tumor was harvested 3 h after injection of 40%-cRGD-PICsomes (red). The vasculature was stained with PECAM-1 (green), and nuclei were stained using Hoechst 33342 (blue). Scale bar = 20 μm.

Figure 8. Schematic representation of the method used to prepare SPIO-loaded PICsomes and TEM images of the resulting PICsomes. (A) Schematic representation of the protocol used to prepare SPIO-loaded PICsomes. (B) TEM image of SPIO-loaded 40%-cRGD-PICsomes stained with uranyl acetate. (C) TEM image of unstained SPIO-loaded PICsomes. Scale bars = 100 nm in all images.

Figure 9. Changes in the normalized R₂ value over time in orthotopic tumors in the brain. Quantitative R₂ values were normalized to the value obtained before PICsome administration. The data were analyzed using Student’s t-test. Data are presented as the mean ± s.e.m. (n = 4 for 40%-cRGD PICsomes, and n = 3 for Ctrl-PICsomes). ∗P < 0.05.

Figure 10. MRI of U87MG orthotopic glioblastoma with SPIO-loaded PICsomes. T₂∗-weighted MR images of (A) 40%-cRGD-PICsomes (n = 2) and (C) Ctrl-PICsomes (n = 2) before, and 1 and 6 h after administration. (B) ΔR₂ maps in the tumor 1 and 6 h after PICsome administration compared with pre-administration. Tumor lesions were identified using T₂-weighted MRI, and are circled with white dashed lines. Orange arrows represent a reduction of the signal of the tumors. Red arrows represent the sites with significantly increased ΔR₂ values.