RESUMEN
The present work aims at determining the most effective dose (number) of mesenchymal stem cells (MSC) for its transplantation in order to treat chronic spinal cord injury (SCI) in mature Sprague-Dawley rats (n=24). MSC were obtained from bone marrow of 4-6-month-old Sprague-Dawley rats. Four weeks after SCI, MSC suspension (4 µl) was injected to experimental animals into the injured area in doses of 4×105, 8×105, or 106. Using MRI, diffusion tensor imaging (DTI), diffusion tensor tractography (DTT), immunohistochemistry, histological staining, and behavioral tests, we studied the effect of transplantation of MSC in different doses on the following parameters in rats with SCI: the size of lesion cavity and post-traumatic syrinx (PTS), glial scar formation, neuronal fibers remodeling, axonal regeneration and sprouting, vascularization, expression of neuronal factors, and motor functions. MSC administration improved motor function in rats after SCI due to stimulation of regeneration and sprouting of the axons, enhanced recovery of locomotor functions, reduction of PTS and the glial scar, and stimulation of vascularization and expression of the neurotrophic factors. The effects of MSC were dose-dependent; the most effective dose was 106 cells.
RESUMEN
Despite the progress of modern medicine, oncological diseases are still among the most common causes of death of adult populations in developed countries. The current therapeutic approaches are imperfect, and the high mortality of oncological patients under treatment, the lack of personalized strategies, and severe side effects arising as a result of treatment force seeking new approaches to therapy of malignant tumors. During the last decade, cancer immunotherapy, an approach that relies on activation of the host antitumor immune response, has been actively developing. Cancer immunotherapy is the most promising trend in contemporary fundamental and practical oncology, and restoration of the pathologically altered tumor microenvironment is one of its key tasks, in particular, the reprogramming of tumor macrophages from the immunosuppressive M2-phenotype into the proinflammatory M1-phenotype is pivotal for eliciting antitumor response. This review describes the current knowledge about macrophage classification, mechanisms of their polarization, their role in formation of the tumor microenvironment, and strategies for changing the functional activity of M2-macrophages, as well as problems of targeted delivery of immunostimulatory signals to tumor macrophages using nanoparticles.
Asunto(s)
Inmunoterapia , Macrófagos/metabolismo , Nanopartículas/metabolismo , Neoplasias/terapia , Animales , Polaridad Celular/fisiología , Humanos , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Microscopía Intravital , Activación de Macrófagos/efectos de los fármacos , Macrófagos/química , Macrófagos/clasificación , Ratones , Nanopartículas/química , Fenotipo , Corona de Proteínas/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Olfactory ensheathing cells showed significant effects on the regeneration of the spinal cord in experimental models and in clinical trials. However, the use of these cells in the therapy of posttraumatic cysts of the spinal cord has not been studied. Cultures of human and rat olfactory mucosa were obtained according to the protocols developed by us. Passage 3-4 cultures are most enriched with olfactory ensheathing cells and are preferable for transplantation. We performed transplantation of 750,000 olfactory ensheathing cells into the region of modeled cysts. The therapeutic effect of human cells was more pronounced. The positive dynamics of recovery of motor activity in the hind limbs of rats can reflect regenerative processes in the spinal cord after transplantation of olfactory ensheathing cells into the region of posttraumatic cysts.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Mucosa Olfatoria/citología , Traumatismos de la Médula Espinal/terapia , Animales , Células Cultivadas , Femenino , Humanos , Regeneración Nerviosa/fisiología , Ratas , Ratas Wistar , Recuperación de la Función/fisiologíaRESUMEN
Magnetic resonance imaging (MRI) is one of the most perspective methods of noninvasive visualization in medicine, and use of contrast agents significantly its potentialities extends. Iron oxide nanoparticles are promising contrast agents, but in fact all the data on their efficiency were obtained in high-field tomographs for experimental animals. We studied the possibility of using magnetic nanoparticles for MRI visualization of rat brain glioblastoma at the most common clinical field 1.5 T The data indicate the efficiency of iron oxide magnetic nanoparticles as contrast agents for 1.5 T MR tomographs.
Asunto(s)
Medios de Contraste/química , Compuestos Férricos/química , Glioblastoma/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Animales , Encéfalo/diagnóstico por imagen , Línea Celular Tumoral , RatasRESUMEN
We studied dependences of T2 relaxation time on magnetic field and concentration of nanoparticles. It was found that nanocontrast media are effective under the influence of the magnetic fields in the range 0.3-7 T. Data of electron paramagnetic resonance confirm the assumption on aggregation of nanoparticles not coated with proteins in high magnetic fields.
Asunto(s)
Medios de Contraste/química , Compuestos Férricos/química , Campos Magnéticos , Imagen por Resonancia Magnética/métodos , Nanopartículas/químicaRESUMEN
Intravital microscopy is widely used for in vivo studies of the mechanisms of carcinogenesis and response to antitumor therapy. For visualization of tumor cells in vivo, cell lines expressing fluorescent proteins are needed. Expression of exogenous proteins can affect cell growth rate and their tumorigenic potential. Therefore, comprehensive analysis of the morphofunctional properties of transduced cells is required for creating appropriate models of tumor microenvironment. In the present study, six lines of mouse tumor cells expressing green and red fluorescent proteins were derived. Analysis of cells morphology, growth kinetics, and response to chemotherapy in vitro revealed no significant differences between wild-type and transduced cell lines. Introduction of fluorescent proteins into the genome of 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells did not affect tumor growth rate after subcutaneous implantation to mice, while both CT26-GFP and CT26-RFP cells (murine colon cancer) were rejected starting from day 8 after implantation. Elucidation of the mechanisms underlying CT26-GFP/RFP rejection is required to modify transduction technique for creating the models of tumor microenvironment accessible for in vivo visualization. Transduced 4T1 and B16-F10 cell lines can be used for intravital microscopic imaging of tumor cells, neoplastic vasculature, and leukocyte subpopulations.
Asunto(s)
Microscopía Intravital/métodos , Proteínas Luminiscentes/análisis , Microambiente Tumoral/fisiología , Animales , Línea Celular Tumoral , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Proteínas Fluorescentes Verdes/análisis , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microambiente Tumoral/genética , Proteína Fluorescente RojaRESUMEN
We evaluated the efficacy of rat olfactory ensheathing cells in the therapy of experimental cysts of the spinal cord. Improvement of the motor function of the hind limbs after transplantation of the olfactory ensheathing cells into the posttraumatic spinal cord cysts rats was found. We also determined the required number of cells for transplantation and demonstrated a neuroprotective effect of this dosage. For further clinical studies, autologous tissue-specific cell preparation of olfactory ensheathing cells has to be created. Cell therapy in combination surgical and pharmacological treatment will substantially improve the quality of life of patients with posttraumatic spinal cord cysts.
Asunto(s)
Quistes/terapia , Bulbo Olfatorio/citología , Traumatismos de la Médula Espinal/terapia , Animales , Femenino , Regeneración Nerviosa/fisiología , Ratas , Ratas Wistar , Recuperación de la Función/fisiología , Médula Espinal/fisiologíaRESUMEN
We studied survival of rat ensheathing cells after transplantation into experimental posttraumatic cysts. These cells were prepared according to our original protocol, labeled with intravital membrane dye PKH26, and transplanted into posttraumatic cysts of the spinal cord. The presence of cysts was verified by magnetic resonance imaging. Olfactory ensheathing cells were detected in the spinal cord by the immunofluorescence method. It was shown that rat olfactory ensheathing cells survived in the spinal cord over 4 weeks and their migration was observed. High survival rate and the possibility of obtaining olfactory ensheathing cells from the olfactory mucosa of patients for creation of an autologous preparation allow considering them as very promising material for the treatment of patients with posttraumatic cysts of the spinal cord.
Asunto(s)
Quistes/terapia , Mucosa Olfatoria/citología , Traumatismos de la Médula Espinal/terapia , Animales , Supervivencia Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos , Regeneración Nerviosa/fisiología , RatasRESUMEN
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.
Asunto(s)
Adenocarcinoma Mucinoso/diagnóstico por imagen , Adenocarcinoma/diagnóstico por imagen , Neoplasias Encefálicas/diagnóstico por imagen , Medios de Contraste/farmacocinética , Glioma/diagnóstico por imagen , Neoplasias Hepáticas/diagnóstico por imagen , Nanopartículas de Magnetita/administración & dosificación , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Adenocarcinoma/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Medios de Contraste/química , Femenino , Compuestos Férricos/administración & dosificación , Compuestos Férricos/química , Glioma/metabolismo , Inyecciones Subcutáneas , Neoplasias Hepáticas/metabolismo , Imagen por Resonancia Magnética/métodos , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/diagnóstico por imagen , Medios de Contraste/química , Glioma/diagnóstico por imagen , Nanopartículas de Magnetita/química , Animales , Animales no Consanguíneos , Línea Celular Tumoral , Medios de Contraste/farmacocinética , Femenino , Imagen por Resonancia Magnética , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Tamaño de la Partícula , Albúmina Sérica Bovina/químicaRESUMEN
We obtained the morphologically, cytofluorometrically, and functionally mature dendritic cells from rats that were pulsed with antigens of the C6 glioma tissue extract. The concentrations of angiogenesis antigens (VEGF, VEGFR-1, and VEGFR-2) and periglioma zone proteins (GFAP, connexin 43, and BSAT1) in the pulsing extract were measured by ELISA. Our results drove us to a conclusion that despite mature phenotype of pulsed dendritic cell, the antigenic composition of glioma tissue extracts should be modified.
Asunto(s)
Antígenos de Neoplasias/genética , Neoplasias Encefálicas/química , Mezclas Complejas/farmacología , Células Dendríticas/efectos de los fármacos , Glioma/química , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Química Encefálica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mezclas Complejas/química , Conexina 43/genética , Conexina 43/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/inmunología , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Inmunofenotipificación , Trasplante de Neoplasias , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/inmunología , Cultivo Primario de Células , Ratas , Técnicas Estereotáxicas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
We report the synthesis and characterization of a new sulfur-containing derivative of bacteriochlorophyll a. The latter was isolated from biomass of the nonsulfur purple bacterium Rhodobacter capsulatus strain B10. The developed photosensitizer is N-aminobacteriopurpurinimide with an exocyclic amino group acylated with a lipoic acid moiety, which is a biogenic substance that acts as a cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes in the body. The disulfide moiety of lipoic acid confers the compound aurophilicity, thus allowing its conjugation with gold nanoparticles (NP-Au) via S-Au bonds. The shape and the size of the resulting nanoconjugate with immobilized photosensitizer (PS-Au) were assessed by dynamic light scattering and transmission electron microscopy. The conjugated nanoparticles are spherical with hydrodynamic diameter of 100-110 nm. The PS-Au conjugate absorbs light at 824 nm and emits strong fluorescence at 830 nm, which allowed in vivo study of its dynamic biodistribution in rats bearing sarcoma M-1. Compared to the free photosensitizer, PS loaded on the gold nanoparticles (PS-Au) showed extended circulation time in the blood and enhanced tumor uptake due to nonspecific passive targeting when the drug accumulates in tumor sites through the leaky tumor neovasculature and does not return to the circulation.
Asunto(s)
Bacterioclorofila A/farmacología , Oro/farmacología , Nanopartículas del Metal/administración & dosificación , Neoplasias/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Animales , Bacterioclorofila A/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Oro/química , Membrana Dobles de Lípidos/química , Nanopartículas del Metal/química , Fármacos Fotosensibilizantes/síntesis química , Ratas , Distribución TisularRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Liposomas/administración & dosificación , Liposomas/química , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Línea Celular Tumoral , Femenino , Ratas , Ratas Wistar , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.
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Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Modelos Biológicos , Metástasis de la Neoplasia/fisiopatología , Animales , Femenino , Luciferasas/farmacología , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/farmacología , Imagen por Resonancia Magnética/métodos , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/diagnóstico por imagen , Metástasis de la Neoplasia/ultraestructura , Análisis de Supervivencia , Tomografía Óptica , Microtomografía por Rayos X , Proteína Fluorescente RojaRESUMEN
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.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias Encefálicas/terapia , Cisplatino/farmacología , Conexina 43/inmunología , Glioblastoma/terapia , Inmunoconjugados/farmacología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Cisplatino/química , Conexina 43/química , Portadores de Fármacos , Femenino , Geles , Glioblastoma/inmunología , Glioblastoma/mortalidad , Glioblastoma/patología , Inmunoconjugados/química , Ratones , Ratones Endogámicos BALB C , Nanoestructuras/química , Trasplante de Neoplasias , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Técnicas Estereotáxicas , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Glioma/patología , Imagen por Resonancia Magnética/métodos , Magnetismo , Nanopartículas/química , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Medios de Contraste/química , Femenino , RatasRESUMEN
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.
Asunto(s)
Medios de Contraste/química , Compuestos Férricos/química , Glioma/patología , Espectroscopía de Resonancia Magnética/métodos , Nanopartículas/química , Anticuerpos Monoclonales/química , Línea Celular Tumoral , HumanosRESUMEN
We propose a method for obtaining superparamagnetic nanoparticles based on iron oxide and their water suspensions. The structure and size of nanoparticles were confirmed by transmission electron microscopy, dynamic light scattering, and X-ray diffraction analysis. The nanoparticles also contained a fluorescent dye Dil C18. Cytotoxicity of obtained aqueous suspension was studied by MTT assay; low toxicity of nanoparticles was demonstrated. High T2-relaxivity of nanoparticles allows using them as a contrast agent for MRI. After incubation of cerebellar sections with nanoparticles vectorized with antibodies to antigen AMVB1, specific visualization of blood vessels was detected.
Asunto(s)
Compuestos Férricos/química , Imagen por Resonancia Magnética/métodos , Nanopartículas/química , Medios de Contraste/química , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Difracción de Rayos XRESUMEN
Gd-based complexes are widely used as magnetic resonance imaging (MRI) contrast agents. The safety of previously approved contrast agents is questionable and is being re-assessed. The main causes of concern are possible gadolinium deposition in the brain and the development of systemic nephrogenic fibrosis after repeated use of MRI contrasts. Thus, there is an urgent need to develop a new generation of MRI contrasts that are safe and that have high selectivity in tissue accumulation with improved local contrast. Here, we report on a new type of theranostic MRI contrast, namely dextran stabilised, gadolinium doped cerium dioxide nanoparticles. These ultra-small (4-6 nm) Ce0.9Gd0.1O1.95 nanoparticles have been shown to possess excellent colloidal stability and high r1-relaxivity (3.6 mM-1 s-1). They are effectively internalised by human normal and cancer cells and demonstrate dose-dependent selective cytotoxicity to cancer cells.