36 research outputs found
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Gold Nanoparticles as a Delivery System of Oligonucleotides into the Brain
The treatment of brain disease is challenging due to the blood-brain barrier, a physiological structure that prevents the majority of potential therapeutic agents from entering the brain. One approach to overcome this problem is the use of nanoparticles as a delivery system. Several types of nanoparticle have shown promise as drug carriers, including gold nanoparticles. They exhibit relatively low cytotoxicity and can enter cells by both active and passive uptake mechanisms and can cross the blood-brain barrier in vivo and in vitro. The aim of this study was to investigate the potential of 5nm gold glyconanoparticles as a delivery system for oligonucleotides into the brain.
Three ligand formulations of gold glyconanoparticles were investigated, covalently coated with glucose, OEG-amine/galactose or OEG-amine/galactose/insulin. The two formulations with OEG-amine showed higher uptake efficiency into both human brain endothelial cells (hCMEC/D3) and primary astrocytes, as determined by electron microscopy. Nanoparticles located in subcellular compartments of endothelium were quantitated. Inhibition studies demonstrated that both active and passive transport mechanisms were involved in the uptake of these nanoparticles. However, knockdown of the insulin-receptor on the endothelium did not reduce transport of insulin-coated nanoparticles. It appeared that the OEG-amine coating alone induced much higher levels of vesicular transport, relative to cytosolic uptake.
The uptake efficiency of OEG-amine/galactose nanoparticles into different endothelial cells (kidney (ciGENC), bone marrow (BMEC) and lung primary (HMVEC-L)) was compared. Kidney endothelium had higher nanoparticle uptake than brain endothelium. Cellular properties that might influence this cell-selective uptake were investigated; the high level of nanoparticle uptake by kidney endothelium was correlated with a higher level of endocytosis and a different glycocalyx composition on these cells.
The transport characteristics of the three formulations of glyconanoparticles were investigated in vivo. The nanoparticles were injected intracarotid into rats and left to circulate for 10 min, in order to capture the first contact of glyconanoparticles with the brain, as detected by electron microscopy. The nanoparticles were observed in brain parenchyma of the cortex, striatum, hippocampus, median eminence and choroid plexus. However, a biodistribution study of the gold, by ICP-mass spectrometry showed that the great majority of the injected nanoparticles were present in the kidney.
Finally, a cargo molecule of DNA oligonucleotide was attached to the gold glyconanoparticles (galactose-coated) by the place-exchange reaction, forming ssDNA/galactose nanoparticles. Nanoparticles with different amounts of bound DNA were fractionated by FPLC and analysed by a novel electrophoretic mobility shift assay (EMSA). When comparing the uptake efficiency of dsDNA/galactose nanoparticles to galactose nanoparticles there was no reduction in uptake efficiency, despite addition of the highly negatively charged cargo.
To conclude, gold glyconanoparticles can cross the blood-brain barrier and enter cells of the brain in vivo and in vitro. Addition of a DNA oligonucleotide cargo does not alter their ability to cross endothelium and hence <5 nm gold glyconanoparticles may be a useful carrier of oligonucleotides into the brain
Nanocarriers for Delivery of Oligonucleotides to the CNS
Nanoparticles with oligonucleotides bound to the outside or incorporated into the matrix can be used for gene editing or to modulate gene expression in the CNS. These nanocarriers are usually optimised for transfection of neurons or glia. They can also facilitate transcytosis across the brain endothelium to circumvent the blood-brain barrier. This review examines the different formulations of nanocarriers and their oligonucleotide cargoes, in relation to their ability to enter the brain and modulate gene expression or disease. The size of the nanocarrier is critical in determining the rate of clearance from the plasma as well as the intracellular routes of endothelial transcytosis. The surface charge is important in determining how it interacts with the endothelium and the target cell. The structure of the oligonucleotide affects its stability and rate of degradation, while the chemical formulation of the nanocarrier primarily controls the location and rate of cargo release. Due to the major anatomical differences between humans and animal models of disease, successful gene therapy with oligonucleotides in humans has required intrathecal injection. In animal models, some progress has been made with intraventricular or intravenous injection of oligonucleotides on nanocarriers. However, getting significant amounts of nanocarriers across the blood-brain barrier in humans will likely require targeting endothelial solute carriers or vesicular transport systems
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Gold nanocarriers for transport of oligonucleotides across brain endothelial cells
Treatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium.
DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with single- or double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA.
Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts.
Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS
Transport of Gold Nanoparticles by Vascular Endothelium from Different Human Tissues
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 transpor
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PPAR<i>γ</i> agonist-loaded PLGA -PEG nanocarriers as a potential treatment for Alzheimer’s disease: in vitro and in vivo studies
Objective: The first aim of this study was to develop a nanocarrier that could transport the peroxisome proliferator-activated receptor agonist, pioglitazone (PGZ) across brain endothelium and examine the mechanism of nanoparticle transcytosis. The second aim was to determine whether these nanocarriers could successfully treat a mouse model of Alzheimer’s disease (AD).
Methods: PGZ-loaded nanoparticles (PGZ-NPs) were synthesized by the solvent displacement technique, following a factorial design using poly (lactic-co-glycolic acid) polyethylene glycol (PLGA-PEG). The transport of the carriers was assessed in vitro, using a human brain endothelial cell line, cytotoxicity assays, fluorescence-tagged nanocarriers, fluorescence-activated cell sorting, confocal and transmission electron microscopy. The effectiveness of the treatment was assessed in APP/PS1 mice in a behavioral assay and by measuring the cortical deposition of β-amyloid.
Results: Incorporation of PGZ into the carriers promoted a 50x greater uptake into brain endothelium compared with the free drug and the carriers showed a delayed release profile of PGZ in vitro. In the doses used, the nanocarriers were not toxic for the endothelial cells, nor did they alter the permeability of the blood–brain barrier model. Electron microscopy indicated that the nanocarriers were transported from the apical to the basal surface of the endothelium by vesicular transcytosis. An efficacy test carried out in APP/PS1 transgenic mice showed a reduction of memory deficit in mice chronically treated with PGZ-NPs. Deposition of β-amyloid in the cerebral cortex, measured by immunohistochemistry and image analysis, was correspondingly reduced.
Conclusion: PLGA-PEG nanocarriers cross brain endothelium by transcytosis and can be loaded with a pharmaceutical agent to effectively treat a mouse model of AD
Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro
The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier
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A hydrogel model of the human blood-brain barrier using differentiated stem cells
An in vitro model of the human blood-brain barrier was developed, based on a collagen hydrogel containing astrocytes, overlaid with a monolayer of endothelium, differentiated from human induced pluripotent stem cells (hiPSCs). The model was set up in transwell filters allowing sampling from apical and basal compartments. The endothelial monolayer had transendothelial electrical resistance (TEER) values >700Ω.cm2 and expressed tight-junction markers, including claudin-5. After differentiation of hiPSCs the endothelial-like cells expressed VE-cadherin (CDH5) and von-Willebrand factor (VWF) as determined by immunofluorescence. However, electron microscopy indicated that at set-up (day 8 of differentiation), the endothelial-like cells still retained some features of the stem cells, and appeared immature, in comparison with primary brain endothelium or brain endothelium in vivo. Monitoring showed that the TEER declined gradually over 10 days, and transport studies were best carried out in a time window 24-72hrs after establishment of the model. Transport studies indicated low permeability to paracellular tracers and functional activity of P-glycoprotein (ABCB1) and active transcytosis of polypeptides via the transferrin receptor (TFR1)
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Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus
Blood-Brain Barrier (BBB) breakdown occurs in ageing and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the ageing female BBB using transmission electron microscopy (TEM) and 3D reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, aged cortex showed thicker basement membrane (BM), higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte – endothelial cell contact and increased tight junction tortuosity compared to young animals. Only increased BM thickness and pericyte mitochondrial volume were observed in aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between cortex and hippocampus of 24-month-old mice. Therefore, the ageing female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in post-menopausal women
Transcriptional control of the multi-drug transporter ABCB1 by transcription factor Sp3 in different human tissues
The ATP-binding cassette (ABC) transporter ABCB1, encoded by the multidrug resistance gene MDR1, is expressed on brain microvascular endothelium and several types of epithelium, but not on endothelia outside the CNS. It is an essential component of the blood-brain barrier. The aim of this study was to identify cell-specific controls on the transcription of MDR1 in human brain endothelium. Reporter assays identified a region of 500bp around the transcription start site that was optimally active in brain endothelium. Chromatin immunoprecipitation identified Sp3 and TFIID associated with this region and EMSA (electrophoretic mobility shift assays) confirmed that Sp3 binds preferentially to an Sp-target site (GC-box) on the MDR1 promoter in brain endothelium. This result contrasts with findings in other cell types and with the colon carcinoma line Caco-2, in which Sp1 preferentially associates with the MDR1 promoter. Differences in MDR1 transcriptional control between brain endothelium and Caco-2 could not be explained by the relative abundance of Sp1:Sp3 nor by the ratio of Sp3 variants, because activating variants of Sp3 were present in both cell types. However differential binding of other transcription factors was also detected in two additional upstream regions of the MDR1 promoter. Identification of cell-specific controls on the transcription of MDR1 indicates that it may be possible to modulate multi-drug resistance on tumours, while leaving the blood brain barrier intact
Gold nanoparticles for imaging and drug transport to the CNS
Gold nanoparticles with a core size of 2 nm covalently coated with glycans to maintain solubility, targeting molecules for brain endothelium, and cargo molecules hold great potential for delivery of therapies into the CNS. They have low toxicity, pass through brain endothelium in vitro and in vivo, and move rapidly through the brain parenchyma. Within minutes of infusion the nanoparticles can be detected in neurons and glia. These nanoparticles are relatively easy to synthesize in association with their surface ligands. They can be detected by electron microscopy, ICP-mass spectrometry, and spectroscopy.
However, modification of the basic gold nanoparticle is required for in vivo imaging by MR or radioactive methods. Depending on their surface coat, the nanoparticles
cross the brain endothelium by the plasma membrane/cytosolic route (passive transport) or by vesicular transcytosis (active transport). A primary aim of current research is to improve the biodistribution of the nanoparticles for CNS drug delivery. Smaller gold
nanoparticles are removed rapidly via the kidney, while larger nanoparticles are taken up by mononuclear phagocytes in various tissues. Receptors selectively located on brain endothelium can act as targets for the nanoparticles, to increase their delivery to the brain