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. 2016 Aug 25;11(8):e0161610.
doi: 10.1371/journal.pone.0161610. eCollection 2016.

Transport of Gold Nanoparticles by Vascular Endothelium From Different Human Tissues

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Free PMC article

Transport of Gold Nanoparticles by Vascular Endothelium From Different Human Tissues

Radka Gromnicova et al. PLoS One. .
Free PMC article

Abstract

The selective entry of nanoparticles into target tissues is the key factor which determines their tissue distribution. Entry is primarily controlled by microvascular endothelial cells, which have tissue-specific properties. This study investigated the cellular properties involved in selective transport of gold nanoparticles (<5 nm) coated with PEG-amine/galactose in two different human vascular endothelia. Kidney endothelium (ciGENC) showed higher uptake of these nanoparticles than brain endothelium (hCMEC/D3), reflecting their biodistribution in vivo. Nanoparticle uptake and subcellular localisation was quantified by transmission electron microscopy. The rate of internalisation was approximately 4x higher in kidney endothelium than brain endothelium. Vesicular endocytosis was approximately 4x greater than cytosolic uptake in both cell types, and endocytosis was blocked by metabolic inhibition, whereas cytosolic uptake was energy-independent. The cellular basis for the different rates of internalisation was investigated. Morphologically, both endothelia had similar profiles of vesicles and cell volumes. However, the rate of endocytosis was higher in kidney endothelium. Moreover, the glycocalyces of the endothelia differed, as determined by lectin-binding, and partial removal of the glycocalyx reduced nanoparticle uptake by kidney endothelium, but not brain endothelium. This study identifies tissue-specific properties of vascular endothelium that affects their interaction with nanoparticles and rate of transport.

Conflict of interest statement

PW holds share options in Midatech Pharma plc. DM was previously on the Scientific advisory board of this company (until May 2016). This association does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
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.
Fig 2
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
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
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
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.
Fig 6
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
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).

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Grant support

This work was supported by Sheffield Teaching Hospitals Trust and the Biotechnology and Biological Sciences Research Council (BB/K009184/1). The funders provided support in the form of salaries for authors (RG, IAR) and consumables, but did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Midatech Pharma plc provided nanoparticles and facilities for ICP mass spectrometry. The specific roles of the authors are articulated in the ‘author contributions’ section.
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