Fig 1.
Characteristics of PEG-amine/galactose gold nanoparticles.
(A) Schematic of nanoparticle organization. (B) Size distribution of nanoparticles (mean size ± s.d.). (C) High resolution electron microscopy of <4 nm nanoparticles (scale bar = 5 nm). (D) Absorbance spectrum of the nanoparticles.
Table 1.
Internalisation of gold nanoparticles by brain endothelial cells.
Fig 2.
Uptake of PEG-amine/galactose gold nanoparticles by brain endothelial cells.
(A) Localisation of nanoparticles at 3hrs (t-test, ** P<0.01). (B) Effect of temperature on nanoparticle uptake into vesicles at 3hrs (t-test, ** P<0.01). (C). Effect of inhibitors of active transport sodium azide/2-deoxy glucose on uptake of nanoparticles at 2 hrs (one-way ANOVA, Tukey’s post test ** P<0.001). (D) Cell membrane integrity/viability test after treatment with sodium azide/2-deoxy glucose, for 2 or 4hrs. Digitonin is a positive control for cell death. All bars show mean ± SEM and are all based on 3-independent experiments.
Fig 3.
Location of PEG-amine/galactose nanoparticles in a brain vessel in vivo.
Silver-enhanced electron micrograph of a microvessel of rat cerebral cortex, 10 minutes after intracarotid infusion of 50 μg (Au) of nanoparticles. Arrows indicate nanoparticles in the endothelium. Scale bar = 500 nm.
Fig 4.
Uptake of nanoparticles by kidney or brain endothelial cells.
Internalisation of PEG-amine/galactose gold nanoparticles into vesicles or cytosol of brain (hCMEC/D3) or kidney (ciGENC) endothelial cells at 3 hrs. Data are mean ± SEM of 3 independent experiments (ANOVA followed by Tukey’s multiple comparison test, P<0.01).
Fig 5.
TEM of PEG-amine/galactose nanoparticles in endothelial cells.
Silver-enhanced nanoparticles in endothelial cells, 3 hrs after their application to the apical (upper) cell surface. (A) Brain endothelial cells, hCMEC/D3. (B) Kidney endothelial cells, ciGENC. Scale bar = 0.5 μm.
Table 2.
Glycan-binding properties of lectins on endothelium.
Fig 6.
Effect of the endothelial glycocalyx on nanoparticle internalisation.
(A) Binding profile of lectins WGA, WFL and PNA on brain and kidney endothelium. Binding was standardised as a percentage of UEA (standard endothelial marker), showing the mean ± SEM from 3 independent experiments (ANOVA and Tukey’s multiple comparison test, *** P<0.001). Effect of partial removal of glycocalyx (with neuraminidase or endopeptidase) on nanoparticle uptake into cytosol or vesicles, of brain endothelium (B) or kidney endothelium (C), compared with untreated cells (= 100%). (Data shown are mean ± SEM from 3 independent experiments, paired t-test, * P<0.05).
Fig 7.
Comparison of the rate of endocytosis and vesicular size in endothelial cells.
(A) Endocytosis of dextran measured by FACS, after 1hr incubation, comparing brain and kidney endothelial cells (mean ± SEM, 3 independent experiments, repeated-measures ANOVA with Bonferroni multiple comparison post test, * P< 0.05). (B) Diameter of vesicles in brain and kidney endothelial cells (mean ± SEM of 3 independent experiments, unpaired t-test, no-significant difference).