Kerrie Venner

Carbon-nanotube shape and individualization critical for renal excretion

In the past few years there has been an accumulating amount of evidence to suggest that the structural features of nanoparticles are responsible for dramatically different pharmacokinetic and body-excretion profiles. For example, a recent study convincingly illustrated that the mean hydrodynamic diameter of quantum dots (spherically shaped nanocrystals) is a determinant factor in achieving effective urinary excretion through the renal filtration barrier. In that study, however, the effect of nanoparticle shape on renal filtration was not examined at all. Our group has investigated the pharmacokinetic and excretion profiles of nonspherical, fibrilous carbon nanotubes (CNTs) and, in the present study, we have attempted to elucidate the mechanism by which such cylindrical nanoparticles can be excreted through the renal route. We have previously reported that surface-functionalized, water-dispersible, single-walled carbon nanotubes (SWNTs; average diameter 1 nm; average length 300–1000 nm) were capable of rapid and effective renal clearance and urinary excretion with a blood-circulation half-life of a few hours. These observations were in agreement with other studies

Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress

Following our identification of PTEN‐induced putative kinase 1 (PINK1) gene mutations in PARK6‐linked Parkinsons disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG‐132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

The interaction of carbon nanotubes with an in vitro blood-brain barrier model and mouse brain in vivo

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.

Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Autophagic vacuolar myopathy in twin girls

Hereditary autophagic vacuolar myopathy (AVM) may occur in several diseases including the rimmed vacuolar myopathies, acid maltase deficiency, Danon disease, infantile autophagic vacuolar myopathy and X‐linked myopathy with excessive autophagy (XMEA). In the latter three conditions the vacuoles are lined by membranes with sarcolemmal features. We present two unusual cases of autophagic vacuolar myopathy in twin girls born at term with no family history of neurological disease. After initial normal developmental milestones they developed progressive leg weakness and wasting with contractures from the age of 12 years. Investigations showed raised CK, normal female karyotype, normal acid maltase activity, normal nerve conduction and myopathic EMG features. Frozen sections of skeletal muscle were stained using routine tinctorial and histochemical methods. Immunohistochemical staining for spectrin, merosin, dystrophin, complement membrane attack complex and sarcoglycans was performed and ultrastructural examination undertaken. Direct sequence analysis of the lamp‐2 gene using genomic DNA extracted from lymphocytes was performed. Histological analysis of the muscle biopsies demonstrated myofibres with vacuoles lacking glycogen and lipid many of which were delineated using immunohistochemistry for merosin, dystrophin and sarcoglycans. Ultrastructural examination showed duplication of the myofibre basal lamina with associated autophagic material. Vacuoles within myofibres were either membrane bound containing autophagic material or lined by plasma membrane and basal lamina. Intermyofibrillar glycogen was increased. Sequence analysis of the coding region and intron/exon boundaries of the lamp‐2 gene was normal. This is the first report of female cases of AVM with sarcolemmal features. We suggest that these patients may represent manifesting carriers of XMEA, or alternatively, a new form of disease with a similar phenotype having autosomal recessive inheritance.