N. F. Grinenko

VEGF-targeted magnetic nanoparticles for MRI visualization of brain tumor.

UNLABELLED This work is focused on synthesis and characterization of targeted magnetic nanoparticles as magnetic resonance imaging (МRI) agents for in vivo visualization of gliomas. Ferric oxide (Fe3O4) cores were synthesized by thermal decomposition and coated with bovine serum albumin (BSA) to form nanoparticles with Deff of 53±9nm. The BSA was further cross-linked to improve colloidal stability. Monoclonal antibodies against vascular endothelial growth factor (mAbVEGF) were covalently conjugated to BSA through a polyethyleneglycol linker. Here we demonstrate that 1) BSA coated nanoparticles are stable and non-toxic to different cells at concentration up to 2.5mg/mL; 2) conjugation of monoclonal antibodies to nanoparticles promotes their binding to VEGF-positive glioma С6 cells in vitro; 3) targeted nanoparticles are effective in MRI visualization of the intracranial glioma. Thus, mAbVEGF-targeted BSA-coated magnetic nanoparticles are promising MRI contrast agents for glioma visualization. FROM THE CLINICAL EDITOR This work focuses on synthesis and characterization of targeted magnetic nanoparticles as magnetic resonance imaging (МRI) agents for in vivo visualization of gliomas. The authors utilize the fact that high-grade gliomas have extensive areas of necrosis and hypoxia, which results in increased secretion of angiogenesis vascular endothelial growth factor (VEGF). Monoclonal antibodies against vascular endothelial growth factor (mAbVEGF) were covalently conjugated to crosslinked BSA coated ferric oxide (Fe3O4) nanoparticles. The results show that these targeted nanoparticles are effective in MRI visualization of the intracranial glioma and may provide a new and promising contrast agent.

Targeted delivery of liposomal nanocontainers to the peritumoral zone of glioma by means of monoclonal antibodies against GFAP and the extracellular loop of Cx43.

UNLABELLED The selectivity of PEGylated immunoliposomes based on monoclonal antibodies against GFAP and the E2 extracellular loop of connexin 43 (MAbE2Cx43) with respect to the focus of a glioma was estimated in experiments on animals with intracranial C6 glioma. Stealth immunoliposomes were labeled with 2 alternative labels, a fluorescent (Dil C18) and a paramagnetic (Gd-DTPA) one. Fluorescent-labeled liposomal nanocontainers were detected at the periphery of the glioma, where the target antigens were overexpressed, 48 hours after injection. Dynamic T1 MRI of rats injected with paramagnetic immunoliposomes carrying MAbE2Cx43 showed distinct accumulation of the paramagnetic contrast agent at the periphery of the glioma, which began 6 hours after administration. These data suggest that immunoliposomal nanocontainers based on antibodies against GFAP and the E2 extracellular fragment of connexin 43 are suitable for targeted delivery of diagnostic and therapeutic drugs to the peritumoral invasion zone of high-grade gliomas. FROM THE CLINICAL EDITOR PEGylated immunoliposomes based on monoclonal antibodies against GFAP and the E2 extracellular loop of connexin 43 were investigated in animals with intracranial C6 glioma. These immunoliposomal nanocontainers were found suitable for targeted delivery of diagnostic and therapeutic drugs to the peritumoral invasion zone of high-grade gliomas.

Core–shell–corona doxorubicin-loaded superparamagnetic Fe3O4 nanoparticles for cancer theranostics

Superparamagnetic iron oxide magnetic nanoparticles (MNPs) are successfully used as contrast agents in magnetic-resonance imaging. They can be easily functionalized for drug delivery functions, demonstrating great potential for both imaging and therapeutic applications. Here we developed new pH-responsive theranostic core-shell-corona nanoparticles consisting of superparamagentic Fe3O4 core that displays high T2 relaxivity, bovine serum albumin (BSA) shell that binds anticancer drug, doxorubicin (Dox) and poly(ethylene glycol) (PEG) corona that increases stability and biocompatibility. The nanoparticles were produced by adsorption of the BSA shell onto the Fe3O4 core followed by crosslinking of the protein layer and subsequent grafting of the PEG corona using monoamino-terminated PEG via carbodiimide chemistry. The hydrodynamic diameter, zeta-potential, composition and T2 relaxivity of the resulting nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, thermogravimetric analysis and T2-relaxometry. Nanoparticles were shown to absorb Dox molecules, possibly through a combination of electrostatic and hydrophobic interactions. The loading capacity (LC) of the nanoparticles was 8 wt.%. The Dox loaded nanoparticles release the drug at a higher rate at pH 5.5 compared to pH 7.4 and display similar cytotoxicity against C6 and HEK293 cells as the free Dox.

Immunofluorescent Analysis of Connexin-43 Using Monoclonal Antibodies to Its Extracellular Domain

Immunofluorescent analysis of connexin-43 was carried out on preparations of fixed and living cultures of rat and human glioma cells, HEK293 cells, and frozen sections of the rat brain with experimental glioma using monoclonal antibodies to recombinant extracellular fragment of connexin-43 (E2 second extracellular loop). These monoclonal antibodies visualized membrane and cytoplasmic pools of connexin-43 in preparations fixed with paraformaldehyde. Incubation of monoclonal antibodies to E2 extracellular loop with living cells led to visualization of only connexin hemichannels on cell membranes. No immunofluorescence characteristic of dimer connexons, organizing the gap junction, was detected. This fact indicates that antibodies to connexin-43 extracellular loop E2, obtained in our study, specifically react with target antigen solely at the stage of connexon presentation on the membrane in the form of hemichannels. These monoclonal antibodies can be used for immunophenotyping and sorting of connexin-43-positive cells in vitro and as the guide molecules in addressed delivery of diagnostic preparations and drugs to glioma cells in vivo.

Generation of Monoclonal Antibodies to Recombinant Vascular Endothelial Growth Factor

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Expression of Tight Junction Proteins by Umbilical Vein Epithelial Cells Co-Cultured with Allogenic Astrocytes

We studied expression of tight junction proteins and formation of the barrier properties in the culture of umbilical vein endothelial cells under conditions of co-culturing with allogenic GFAP-positive astrocytes. This culturing signifi cantly increases of expression of tight junction proteins (claudin-5, occludin, and ZO-1). The formation of tight junctions signifi cantly increased transendothelial resistance and reduced permeability for sodium fl uorescein. Thus, reproducible in vitro model for the study of endothelial barrier properties was created based on co-culturing of umbilical endothelial cells and human astrocytes. This model can be used for evaluation of the permeability of the tissue-blood barriers for substances of different chemical structure and for studies of factors modulating the state of cell-cell contacts.

Connexin 43-targeted T1 contrast agent for MRI diagnosis of glioma

Glioblastoma multiforme is the most aggressive form of brain tumor. Early and accurate diagnosis of glioma and its borders is an important step for its successful treatment. One of the promising targets for selective visualization of glioma and its margins is connexin 43 (Cx43), which is highly expressed in reactive astrocytes and migrating glioma cells. The purpose of this study was to synthesize a Gd-based contrast agent conjugated with specific antibodies to Cx43 for efficient visualization of glioma C6 in vivo. We have prepared stable nontoxic conjugates of monoclonal antibody to Cx43 and polylysine-DTPA ligands complexed with Gd(III), which are characterized by higher T1 relaxivity (6.5 mM(-1) s(-1) at 7 T) than the commercial agent Magnevist® (3.4 mM(-1) s(-1)). Cellular uptake of Cx43-specific T1 contrast agent in glioma C6 cells was more than four times higher than the nonspecific IgG-contrast agent, as detected by flow cytometry and confocal analysis. MRI experiments showed that the obtained agents could markedly enhance visualization of glioma C6 in vivo after their intravenous administration. Significant accumulation of Cx43-targeted contrast agents in glioma and the peritumoral zone led not only to enhanced contrast but also to improved detection of the tumor periphery. Fluorescence imaging confirmed notable accumulation of Cx43-specific conjugates in the peritumoral zone compared with nonspecific IgG conjugates at 24 h after intravenous injection. All these features of Cx43-targeted contrast agents might be useful for more precise diagnosis of glioma and its borders by MRI.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-12-4) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purifi ed (metal affi nity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-12-4 showed that biotinylated VEGF specifi cally binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fl uorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specifi c receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confi rmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Magnetic Resonance Imaging of Tumors with the Use of Iron Oxide Magnetic Nanoparticles as a Contrast Agent

We studied the possibility of using BSA-coated magnetic iron oxide nanoparticles for magnetic resonance imaging diagnosis of C6 glioblastoma, 4T1 mammary adenocarcinoma, and RS-1 hepatic mucous carcinoma. In all three cases, magnetic nanoparticles accumulated in the tumor and its large vessels. Magnetic resonance imaging with contrast agent allows visualization of the tumor tissue and its vascularization.

Glioma Cell and Astrocyte Co-cultures As a Model to Study Tumor–Tissue Interactions: A Review of Methods

Astrocytes are a dominant cell type that envelopes the glioma bed. Typically, that is followed by formation of contacts between astrocytes and glioma cells and accompanied by change in astrocyte phenotype, a phenomenon known as a ‘reactive astrogliosis.’ Generally considered glioma-promoting, astrocytes have many controversial peculiarities in communication with tumor cells, which need thorough examination in vitro. This review is devoted to in vitro co-culture studies of glioma cells and astrocytes. Firstly, we list several fundamental works which allow understanding the modalities of co-culturing. Cell-to-cell interactions between astrocytes and glioma cells, the roles of astrocytes in tumor metabolism, and glioma-related angiogenesis are reviewed. In the review, we also discuss communications between glioma stem cells and astrocytes. Co-cultures of glioma cells and astrocytes are used for studying anti-glioma treatment approaches. We also enumerate surgical, chemotherapeutic, and radiotherapeutic methods assessed in co-culture experiments. In conclusion, we underline collisions in the field and point out the role of the co-cultures for neurobiological studies.