Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo.
ABSTRACT: Brain glioblastoma and neurodegenerative diseases are still largely untreated due to the inability of most drugs to cross the blood-brain barrier (BBB). Nanoparticles have emerged as promising tools for drug delivery applications to the brain; in particular carbon nanotubes (CNTs) that have shown an intrinsic ability to cross the BBB in vitro and in vivo. Angiopep-2 (ANG), a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), has also shown promising results as a targeting ligand for brain delivery using nanoparticles (NPs). Here, we investigate the ability of ANG-targeted chemically-functionalised multi-walled carbon nanotubes (f-MWNTs) to cross the BBB in vitro and in vivo. ANG was conjugated to wide and thin f-MWNTs creating w-MWNT-ANG and t-MWNT-ANG, respectively. All f-MWNTs were radiolabelled to facilitate quantitative analyses by ?-scintigraphy. ANG conjugation to f-MWNTs enhanced BBB transport of w- and t-MWNTs-ANG compared to their non-targeted equivalents using an in vitro co-cultured BBB model consisting of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Additionally, following intravenous administration w-MWNTs-ANG showed significantly higher whole brain uptake than the non-targeted w-MWNT in vivo reaching ~2% injected dose per g of brain (%ID/g) within the first hour post-injection. Furthermore, using a syngeneic glioma model, w-MWNT-ANG showed enhanced uptake in glioma brain compared to normal brain at 24h post-injection. t-MWNTs-ANG, on the other hand, showed higher brain accumulation than w-MWNTs. However, no significant differences were observed between t-MWNT and t-MWNT-ANG indicating the importance of f-MWNTs diameter towards their brain accumulation. The inherent brain accumulation ability of f-MWNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-MWNT-ANG to deliver active therapeutics for brain glioma therapy.
Project description:Carbon nanotubes (CNTs) are a novel nanocarriers with interesting physical and chemical properties. Here we investigate the ability of amino-functionalized multi-walled carbon nanotubes (MWNTs-NH3(+)) to cross the Blood-Brain Barrier (BBB) in vitro using a co-culture BBB model comprising primary porcine brain endothelial cells (PBEC) and primary rat astrocytes, and in vivo following a systemic administration of radiolabelled f-MWNTs. Transmission Electron microscopy (TEM) confirmed that MWNTs-NH3(+) crossed the PBEC monolayer via energy-dependent transcytosis. MWNTs-NH3(+) were observed within endocytic vesicles and multi-vesicular bodies after 4 and 24 h. A complete crossing of the in vitro BBB model was observed after 48 h, which was further confirmed by the presence of MWNTs-NH3(+) within the astrocytes. MWNT-NH3(+) that crossed the PBEC layer was quantitatively assessed using radioactive tracers. A maximum transport of 13.0 ± 1.1% after 72 h was achieved using the co-culture model. f-MWNT exhibited significant brain uptake (1.1 ± 0.3% injected dose/g) at 5 min after intravenous injection in mice, after whole body perfusion with heparinized saline. Capillary depletion confirmed presence of f-MWNT in both brain capillaries and parenchyma fractions. These results could pave the way for use of CNTs as nanocarriers for delivery of drugs and biologics to the brain, after systemic administration.
Project description:The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3(+)) and amino-functionalized MWNT (MWNT-NH3(+)). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS applications.
Project description:Herein, we reported for the first time that RGD-conjugated silica-coated gold nanorods on the surface of multiwalled carbon nanotubes were successfully used for targeted photoacoustic imaging of in vivo gastric cancer cells. A simple strategy was used to attach covalently silica-coated gold nanorods (sGNRs) onto the surface of multiwalled carbon nanotubes (MWNTs) to fabricate a hybrid nanostructure. The cross-linked reaction occurred through the combination of carboxyl groups on the MWNTs and the amino group on the surface of sGNRs modified with a silane coupling agent. RGD peptides were conjugated with the sGNR/MWNT nanostructure; resultant RGD-conjugated sGNR/MWNT probes were investigated for their influences on viability of MGC803 and GES-1 cells. The nude mice models loaded with gastric cancer cells were prepared, the RGD-conjugated sGNR/MWNT probes were injected into gastric cancer-bearing nude mice models via the tail vein, and the nude mice were observed by an optoacoustic imaging system. Results showed that RGD-conjugated sGNR/MWNT probes showed good water solubility and low cellular toxicity, could target in vivo gastric cancer cells, and obtained strong photoacoustic imaging in the nude model. RGD-conjugated sGNR/MWNT probes will own great potential in applications such as targeted photoacoustic imaging and photothermal therapy in the near future.
Project description:Earlier studies proved the success of using chemically functionalised multi-walled carbon nanotubes (f-MWNTs) as nanocarriers to the brain. Little insight into the kinetics of brain distribution of f-MWNTs in vivo has been reported. This study employed a wide range of qualitative and quantitative techniques with the aim of shedding the light on f-MWNT's brain distribution following intravenous injection. ?-Scintigraphy quantified the uptake of studied radiolabelled f-MWNT in the whole brain parenchyma and capillaries while 3D-single photon emission computed tomography/computed tomography imaging and autoradiography illustrated spatial distribution within various brain regions. Raman and multiphoton luminescence together with transmission electron microscopy confirmed the presence of intact f-MWNT in mouse brain, in a label-free manner. The results evidenced the presence of f-MWNT in mice brain parenchyma, in addition to brain endothelium. Such information on the rate and extent of regional and cellular brain distribution is needed before further implementation into neurological therapeutics can be made.
Project description:Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~386nm), bearing net positive charge (5.8mV), or short MWNTs-OVA (~122nm) of increasing negative charges (-23.4, -35.8 or -39mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system.
Project description:MnO2-MWNT-Ni foam supercapacitor electrodes were developed based on directly grown multiwalled carbon nanotubes (MWNTs) and hydrothermal MnO2 nanostructures on Ni foam substrates. The electrodes demonstrated excellent electrochemical and battery properties. The charge transfer resistance dropped 88.8% compared with the electrode without MWNTs. A high specific capacitance of 1350.42 F·g-1 was reached at the current density of 6.5 A·g-1. The electrode exhibited a superior rate capability with 92.5% retention in 25,000 cycles. Direct MWNT growth benefits the supercapacitor application for low charge transfer resistance and strong MWNT-current collector binding.
Project description:Potential applications of carbon nanotubes have attracted many researchers in the field of drug delivery systems. In this study, multiwalled carbon nanotubes (MWNTs) were first functionalized using hyperbranched poly citric acid (PCA) to improve their hydrophilicity and functionality. Then, paclitaxel (PTX), a potent anticancer agent, was conjugated to the carboxyl functional groups of poly citric acid via a cleavable ester bond to obtain a MWNT-g-PCA-PTX conjugate. Drug content of the conjugate was about 38% (w/w). The particle size of MWNT-g-PCA and MWNT-g-PCA-PTX was approximately 125 and 200 nm, respectively. Atomic force microscopy and transmission electron microscopy images showed a curved shape for MWNT-g-PCA and MWNT-g-PCA-PTX, which was in contrast with the straight or linear conformation expected from carbon nanotubes. It seems that the high hydrophilicity of poly citric acid and high hydrophobicity of MWNTs led to conformational changes from a linear state to a curved state. Paclitaxel can be released from the MWNT-g-PCA-PTX conjugates faster at pH 6.8 and 5.0 than at pH 7.4, which was suitable for the release of the drug in tumor tissues and tumor cells. In vitro cytotoxicity studies were evaluated in the A549 and SKOV3 cell lines. MWNT-g-PCA had an insignificant cytotoxic effect on both cell lines. MWNT-g-PCA-PTX had more of a cytotoxic effect than the free drug over a shorter incubation time (eg, 24 hours versus 48 hours), which suggests improved cell penetration of MWNT-g-PCA-PTX. Therefore, paclitaxel conjugated to MWNT-g-PCA is promising for cancer therapeutics.
Project description:Alzheimer's disease (AD) is a neurodegenerative disorder characterised by brain accumulation of toxic protein aggregates, including extracellular amyloid beta (A?) plaques, inflammation, neuronal death and progressive cognitive dysfunction. Current diagnostic modalities, based on cognitive tests, fail to detect early AD onset, thus emphasising the need to develop improved methods for pre-symptomatic disease detection. Building on the properties of the Pittsburgh Compound B (PiB), an A?-binding molecule suitable to use as positron emission tomography (PET) imaging agent, and aiming at using a more clinically available modality (like magnetic ressonance imaging, MRI), PiB derivatives have been conjugated to the macrocyclic chelator 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A) monoamide. However, these derivatives do not readily cross the highly selective blood-brain barrier (BBB). Taking advantage of the capacity of functionalised carbon nanotubes (f-CNTs) to cross biological barriers, including the BBB, this manuscript reports on the conjugation of two PiB derivative Gd3+ complexes - Gd(L2) and Gd(L3) - to multi-walled f-CNTs (f-MWNTs) and assessment of their in vivo biodistribution and brain uptake. It is shown that Gd(L2) and Gd(L3) can be efficiently loaded onto different f-MWNTs, with significant improvement in brain accumulation of the conjugates compared to the free metal complexes. Overall, this study demonstrates that f-MWNTs have potential to be used as carriers in theranostic applications involving brain delivery of BBB impermeable compounds.
Project description:Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic, quantitative and multi-scale techniques in understanding the different cellular processing routes of functionalised nanomaterials. This correlative approach has wide implications for assessing the biopersistence of MWNT aggregates elsewhere in the body, in particular their interaction with macrophages in the lung.
Project description:Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection reagent for siRNAs. Our results indicate that the PEI-NH-SWNTs and PEI-NH-MWNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression and may therefore be considered as novel nonviral gene delivery reagents.