Multilayer polyion complex nanoformulations of superoxide dismutase 1 for acute spinal cord injury.

As one of the most devastating forms of trauma, spinal cord injury (SCI) remains a challenging clinical problem. The secondary processes associated with the primary injury, such as overproduction of reactive oxygen species (ROS) and inflammation, lead to concomitant compression of the injured spinal cord and neuronal death. Delivery of copper-zinc superoxide dismutase (SOD1), an efficient ROS scavenger, to the site of injury can mitigate SCI-induced oxidative stress and tissue damage. Towards this goal catalytically active nanoformulations of SOD1 ("nanozymes") are developed as a modality for treatment of SCI. Along with the cross-linked polyion complex of SOD1 with polycation poly(ethylene glycol) (PEG)-polylysine (single-coat (SC) nanozyme), we introduce for the first time the chemically cross-linked multilayer polyion complex in which SOD1 is first incorporated into a polyion complex with polycation, then coated by anionic block copolymer, PEG-polyglutamic acid (double-coat (DC) nanozyme). We developed DC nanozymes with high enzymatic activity and ability to retain and protect SOD1 under physiological conditions. Pharmacokinetic study revealed that DC nanozymes significantly prolonged circulation of active SOD1 in the blood stream compared to free SOD1 or SC nanozymes (half-life was 60 vs 6min). Single intravenous injection of DC nanozymes (5kU of SOD1/kg) improved the recovery of locomotor functions in rats with moderate SCI, along with reduction of swelling, concomitant compression of the spinal cord and formation of post-traumatic cysts. Thus, based on the testing in a rodent model the SOD1 DC nanozymes are promising modality for scavenging ROS, decreasing inflammation and edema, and improving recovery after SCI.

Internalization of Vectorized Liposomes Loaded with Plasmid DNA in C6 Glioma Cells.

We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.

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.

Internalization of Vectored Liposomes in a Culture of Poorly Differentiated Tumor Cells.

Internalization of liposomal nanocontainers conjugated with monoclonal antibodies to VEGF, VEGFR2 (KDR), and proteins overproduced in the tumor tissue was studied in vitro on cultures of poorly differentiated tumor cells. Comparative analysis of accumulation of vectored liposomes in the tumor cells was performed by evaluating co-localization of labeled containers and cell organelles by laser scanning confocal microscopy. We observed nearly 2 times more active penetration and accumulation of liposomes vectored with antibodies in the tumor cells in comparison with non-vectored liposomes. Selective clathrin-dependent penetration of vectored liposomes into tumor cells was demonstrated by using pharmacological agents inhibiting endocytosis.

Relationship between the Size of Magnetic Nanoparticles and Efficiency of MRT Imaging of Cerebral Glioma in Rats.

BSA-coated Fe3O4 nanoparticles with different hydrodynamic diameters (36±4 and 85±10 nm) were synthesized, zeta potential and T2 relaxivity were determined, and their morphology was studied by transmission electron microscopy. Studies on rats with experimental glioma C6 showed that smaller nanoparticles more effectively accumulated in the tumor and circulated longer in brain vessels. Optimization of the hydrodynamic diameter improves the efficiency of MRT contrast agent.

Cuprizone Model as a Tool for Preclinical Studies of the Efficacy of Multiple Sclerosis Diagnosis and Therapy.

To study demyelination and remyelination processes and their response to different drugs, a protocol for modeling multiple sclerosis using the copper chelator cuprizone was developed. Magnetic resonance imaging confirmed the presence of demyelination lesions on week 4 of 0.6% cuprizone-containing diet. Immunohistochemical staining with polyclonal antibodies to glial fibrillary acidic protein (pAb GFAP) confirmed the increase in the number of reactive astrocytes on week 4 of diet and during remyelination (week 2 after diet). Analysis of neurophysiological functions in mice with cuprizone-induced demyelination revealed motor and behavioral deficits. This model can be used as a tool for preclinical studies of the efficiency of multiple sclerosis diagnostic and therapy.

[Contrast agents in MRI-diagnosis of multiple sclerosis].

Magnetic resonance imaging using contrast agents plays an important role in diagnosis and assessment of treatment efficacy in multiple sclerosis. The development of contrast agents on the basis of gadolinium or iron oxide nanoparticles has potential for diagnosis of pathological foci (tumors, amyloid plaques, inflammation and foci of demyelination or necrosis) in nervous system diseases. Newly developing types of diagnostic substances for visualization of pathological foci in multiple sclerosis are presented in this review.

Site-directed delivery of VEGF-targeted liposomes into intracranial C6 glioma.

The efficiency of conventional chemotherapy for aggressive tumors in the CNS remains low and new strategies for the targeted delivery of anti-tumor substances are now actively developed. Pegylated liposomes covalently conjugated with monoclonal antibodies to VEGF synthesized by us are nanoparticle characterized by narrow size distribution and high dispersion stability. Immunochemical activity of antibodies after conjugation was 70% of initial level. The anti-VEGF liposomes developed by us were highly specific for VEGF(+) tumor cells (in vitro and in vivo). Intravenous injection of VEGF-liposomes to rats with intracranial C6 glioma was followed by their specific accumulation in the malignant tissues and engulfment by glioma cells, which attested to target delivery and selective accumulation of anti-VEGF-liposomes in the brain tumor. Thus, the use of targeting molecules can significantly increase the distribution and efficiency of delivery of nanocontainers to a tumor characterized by hyperexpression of the target proteins.

Targeted delivery of cisplatin by сonnexin 43 vector nanogels to the focus of experimental glioma C6.

The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors.

Tumor-specific contrast agent based on ferric oxide superparamagnetic nanoparticles for visualization of gliomas by magnetic resonance tomography.

The aim of this study was to create vector superparamagnetic nanoparticles for tumor cell visualization in vivo by magnetic resonance tomography. A method for obtaining superparamagnetic nanoparticles based on ferric oxide with the magnetic nucleus diameter of 12 ± 3 nm coated with BSA and forming stable water dispersions was developed. The structure and size of the nanoparticles were studied by transmissive electron microscopy, dynamic light scattering, and x-ray phase analysis. Their T2 relaxivity was comparable with that of the available commercial analog. Low cytotoxicity of these nanoparticles was demonstrated by MTT test on primary and immortalized cell cultures. The nanoparticles were vectorized by monoclonal antibodies to connexin 43 (Cx43). Specific binding of vectorized nanoparticles to C6 glioma Cx43-positive cell membranes was demonstrated. Hence, vector biocompatible nanoparticles with high relaxivity, fit for use as MRT contrast for the diagnosis of poorly differentiated gliomas, were created.

Visualization of experimental glioma C6 by MRI with magnetic nanoparticles conjugated with monoclonal antibodies to vascular endothelial growth factor.

We developed a method for obtaining iron oxide nanoparticles and their conjugation with monoclonal antibodies to vascular endothelial growth factor. The resultant vector nanoparticles were low-toxic and the antibodies retained their immunochemical activity after conjugation. The study was carried out on rats with intracranial glioma C6 on day 14 after its implantation. The intravenously injected nanoparticles visualized the brain tumor in contrast to nanoparticles conjugated with nonspecific immunoglobulins that did not accumulate in the tumor.