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1.
J Nanosci Nanotechnol ; 14(7): 5355-62, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24758031

ABSTRACT

Diagnosis of glioblastoma multiform (GBM) with MRI lacks molecular information and requires a biopsy for pathologic confirmation. The EGFRvIII, is a constitutively active mutant of the EGF receptor, identified in a high percentage of brain cancers and associated with increased invasiveness and resistance, making it a good target to improve imaging and diagnosis. The present study shows that conjugation of near-infrared quantum dot (Qd800) to an anti-EGFRvIII single domain antibody, made of the variable region with an extra cysteine for site-specific conjugation (EG2-Cys), increased its internalization in U87MG-EGFRvIII cells in vitro compared to Qd800 conjugated with the Fc region of the antibody (EG2-hFc) or unconjugated. EG2-Cys also improved the contrast in Near-Infrared Imaging of mice bearing orthotopic glioblastoma. The increased accumulation was confirmed by fluorescence microscopy of brain sections. The specificity of EG2-Cys in brain tumor expressing the EGFRvIII mutant receptor may provide an accurate less invasive diagnosis and determine the level of tumor aggressiveness and resistance.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , ErbB Receptors/metabolism , Glioblastoma/pathology , Molecular Imaging/methods , Quantum Dots , Animals , Cell Line, Tumor , Humans , Infrared Rays , Mice , Mice, Nude , Microscopy, Fluorescence/methods , Reproducibility of Results , Sensitivity and Specificity
2.
Br J Cancer ; 105(11): 1697-707, 2011 Nov 22.
Article in English | MEDLINE | ID: mdl-22027709

ABSTRACT

BACKGROUND: ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood-brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells. METHODS: Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines. RESULTS: Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions. CONCLUSION: ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , Paclitaxel/pharmacokinetics , Receptors, LDL/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Line, Tumor , Endocytosis , Glioma/drug therapy , Glioma/pathology , Hep G2 Cells , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Male , Mice , Mice, Nude , Paclitaxel/pharmacology , Peptides/pharmacokinetics , Peptides/pharmacology , Phenotype , RNA Interference , Receptors, LDL/genetics , Tumor Microenvironment , Tumor Suppressor Proteins/genetics
3.
Br J Cancer ; 103(10): 1606-16, 2010 Nov 09.
Article in English | MEDLINE | ID: mdl-20959824

ABSTRACT

BACKGROUND: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications. METHODS: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe3O4 nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy. RESULTS: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe3O4 NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe3O4 NPs selectively in GBM vessels. CONCLUSIONS: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.


Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Insulin-Like Growth Factor Binding Proteins/immunology , Animals , Antibodies , Brain/immunology , Brain/pathology , Brain Neoplasms/immunology , Brain Neoplasms/ultrastructure , DNA Primers , DNA, Complementary/genetics , DNA, Complementary/immunology , Gene Amplification , Glioblastoma/immunology , Glioblastoma/ultrastructure , Humans , Immunoglobulin G/immunology , Immunohistochemistry , Insulin-Like Growth Factor Binding Proteins/metabolism , Mice , Microscopy, Fluorescence , Polymerase Chain Reaction , Tissue Distribution
4.
Br J Pharmacol ; 160(4): 1016-28, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20590596

ABSTRACT

BACKGROUND AND PURPOSE: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications. EXPERIMENTAL APPROACH: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies. KEY RESULTS: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions. CONCLUSIONS: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.


Subject(s)
Brain Neoplasms/diagnosis , ErbB Receptors/metabolism , Glioblastoma/diagnosis , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/metabolism , Animals , Antibodies/metabolism , Antibody Affinity , Brain Neoplasms/blood supply , Brain Neoplasms/metabolism , Brain Neoplasms/ultrastructure , Cell Line, Tumor , ErbB Receptors/immunology , Glioblastoma/blood supply , Glioblastoma/metabolism , Glioblastoma/ultrastructure , Half-Life , Humans , Immobilized Proteins/metabolism , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Kinetics , Male , Mice , Mice, Nude , Molecular Imaging/methods , Molecular Weight , Tomography, Optical/methods , Whole Body Imaging/methods , X-Ray Microtomography/methods
5.
J Microsc ; 232(2): 225-34, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19017221

ABSTRACT

The localization of asialo-GM1 in ordered membrane raft domains in HeLa cells has been examined using a combination of membrane fractionation and fluorescence imaging. The glycolipid is enriched in Triton X-100 insoluble membrane fractions that contain high concentrations of cholesterol and caveolin-1 but is also found in detergent soluble membrane fractions. Near-field fluorescence microscopy shows that a fraction of the asialo-GM1 is localized in small nanoscale clusters that have an upper limit for the average diameter of approximately 90 nm and are partially colocalized with caveolae membrane domains. In addition to clusters, a diffuse, non-clustered population of asialo-GM1 is observed and is hypothesized to correspond to glycolipid isolated in detergent soluble membrane fractions.


Subject(s)
Cell Membrane/chemistry , G(M1) Ganglioside/analysis , Microscopy, Fluorescence/methods
6.
Pharm Res ; 17(9): 1035-48, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11087034

ABSTRACT

Caveolae are omega-shaped invaginations of the plasma membrane with a diameter of 50-100 nm. Caveolae invaginations can detach from the plasma membrane to form discrete functional caveolae vesicles within the cell cytoplasm. Caveolae are most prominent in adipocytes, fibroblasts, muscle cells (skeletal, smooth and cardiac), capillary endothelium and type I pneumocytes, although other cell types also display these structures but at a lower numerical density. The key structural and functional protein for caveolae is caveolin. At the plasma membrane caveolae serve to compartmentalize and integrate a wide range of signal transduction processes. Caveolae also serve transport functions including that of the vesicular internalisation of small molecules by the process of potocytosis, and the endocytic and transcytotic movements of macromolecules. Opportunities exist for basic and applied investigators working within the pharmaceutical sciences to exploit caveolae membrane interactions with the aim to develop novel cellular or transcellular drug delivery strategies.


Subject(s)
Caveolae/physiology , Caveolins/metabolism , Drug Resistance, Multiple/physiology , Genetic Therapy/methods , Signal Transduction/physiology , Vesicular Transport Proteins , Animals , Biological Transport/physiology , Caveolin 1 , Cholesterol/metabolism , Endothelium, Vascular/metabolism , Folic Acid/metabolism , Humans , Membrane Proteins/metabolism , Pulmonary Alveoli/metabolism , SNARE Proteins
7.
J Drug Target ; 8(4): 247-56, 2000.
Article in English | MEDLINE | ID: mdl-11144235

ABSTRACT

Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer. The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity. Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation [A] to 137% and retention [R] to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and [A] to 147% and [R] to 163% in vinblastine-resistant KBVI nasopharyngeal carcinoma cells. Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 microg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 microg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity. The combination of EPADI (10 microg/ml) with verapamil (1 microM) resulted in a considerable increase in the [A] and [R] of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone. KBV1 cells treated with combination of EPADI (10 microg/ml) and verapamil (1 microM) achieved 160% and 1120% greater [A] and [R] of rhodamine-123, respectively, compared to untreated cells. The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Antibiotics, Antineoplastic/pharmacokinetics , Doxorubicin/pharmacokinetics , Drug Resistance, Multiple , Fatty Acids, Unsaturated/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antibiotics, Antineoplastic/toxicity , Biological Transport, Active/drug effects , Doxorubicin/toxicity , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Drug Synergism , Eicosapentaenoic Acid/analogs & derivatives , Eicosapentaenoic Acid/pharmacology , Humans , Rhodamine 123/pharmacokinetics , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Verapamil/pharmacology
8.
Biochem Biophys Res Commun ; 262(1): 121-6, 1999 Aug 19.
Article in English | MEDLINE | ID: mdl-10448079

ABSTRACT

Human MDR1 P-glycoprotein (P-gp) is a membrane efflux pump for cytotoxics, whereas MDR3 P-gp is a phosphatidylcholine transporter. We have examined a role for MDR1 P-gp in phosphatidylcholine transport in MDR3-negative epithelial cells that have been induced to express the MDR1 P-gp by exposure to cytotoxics. The accumulation and retention of the fluorescently labelled phosphatidylcholine analogue, C12-NBD-PC, was studied in resistant, KBV1 and MCFadr, and sensitive, KB3-1 and MCF7, cells. Lower accumulation and decreased retention of C12-NBD-PC was evident in resistant cells, e.g., KBV1 accumulated 56%, and MCFadr accumulated 60%, of C12-NBD-PC levels in KB3-1 and MCF7, respectively. Treatment with the MDR1 P-gp inhibitor, verapamil, altered the kinetics of C12-NBD-PC in the resistant cells to more closely follow the pattern of C12-NBD-PC handling by sensitive cells. Comparison of C12-NBD-PC to that of the model MDR1 P-gp substrate, rhodamine-123, indicated phosphatidylcholine turnover kinetics by MDR1 P-gp to be relatively low. The transport by MDR1 P-gp of phosphatidylcholine from inner to outer membrane leaflet may regulate P-gp function and fulfill a role in the MDR1 multidrug-resistant phenotype.


Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/metabolism , Epithelial Cells/metabolism , Phosphatidylcholines/metabolism , Verapamil/pharmacology , 4-Chloro-7-nitrobenzofurazan/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/genetics , Biological Transport/drug effects , Doxorubicin/metabolism , Drug Resistance, Neoplasm , Epithelial Cells/drug effects , Flow Cytometry , Fluorescent Dyes/metabolism , Humans , Kinetics , Phenotype , RNA, Messenger/analysis , RNA, Messenger/genetics , Rhodamine 123/metabolism , Tumor Cells, Cultured
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