RESUMO
Superparamagnetic iron-oxide based contrast agents can provide important diagnostic information regarding the assessment of cardiac inflammatory diseases. The aim of the study was to analyze whether nanoparticles conjugated to recombinant 70-kDa heat shock protein (Hsp70-SPION) can be applied for the detection of acute myocardium infarct by MRI. Cellular experiments demonstrated increased CD40-mediated uptake of Hsp70-SPIONs in comparison to non-conjugated SPIONs. Following induction of an acute infarct in rats by ligation of the left anterior descending artery SPIONs and Hsp70-SPION conjugates were injected intravenously on day 4. The animals underwent sequential MRI that showed the presence of the particles in the infarcted zone. Subsequent biodistribution analyses with the help of method on non-linear magnetic response indicated the preferential accumulation of the Hsp70-SPIONs in the heart tissue that was further confirmed with histological analyses. The study demonstrated that an acute infarct can be visualized by MRI using Hsp70-functionalized SPION conjugates. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxides nanoparticles (SPIONs) have been studied extensively as a contrast agent for MRI. Their tissue specificity can be further enhanced by conjugation with various ligands. In this study, the authors conjugated superparamagnetic nanoparticles to 70-kDa heat shock protein (Hsp70-SPION) to investigate the feasibility for the detection of acute myocardium infarct. The positive findings would suggest that this approach might be used clinically in the future.
Assuntos
Meios de Contraste/química , Compostos Férricos/química , Proteínas de Choque Térmico HSP70/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Infarto do Miocárdio/diagnóstico por imagem , Animais , Meios de Contraste/farmacocinética , Compostos Férricos/farmacocinética , Proteínas de Choque Térmico HSP70/farmacocinética , Nanopartículas de Magnetita/análise , Masculino , Miocárdio/patologia , Ratos Wistar , Distribuição TecidualRESUMO
The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T(2)-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of T*(2) values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M(2) measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.
Assuntos
Anticorpos Monoclonais Murinos , Neoplasias Encefálicas/terapia , Quimiorradioterapia/métodos , Sistemas de Liberação de Medicamentos/métodos , Raios gama/uso terapêutico , Glioma/terapia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Nanopartículas de Magnetita/química , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/farmacologia , Proteínas de Choque Térmico HSP70/química , Células HeLa , Humanos , Células K562 , Masculino , Ratos , Ratos WistarRESUMO
Nanovaccines based on superparamagnetic iron oxide nanoparticles (SPIONs) provide a novel approach to induce the humoral and cell-based immune system to fight cancer. Herein, we increased the immunostimulatory capacity of SPIONs by coating them with recombinant heat shock protein 70 (Hsp70) which is known to chaperone antigenic peptides. After binding, Hsp70-SPIONs deliver immunogenic peptides from tumor lysates to dendritiÑ cells (DCs) and thus stimulate a tumor-specific, CD8+ cytotoxic T cell response. We could show that binding activity of Hsp70-SPIONs to the substrate-binding domain (SBD) is highly dependent on the ATPase activity of its nucleotide-binding domain NBD), as shown by (31)P NMR spectroscopy. Immunization of C6 glioma-bearing rats with DCs pulsed with Hsp70-SPIONs and tumor lysates resulted in a delayed tumor progression (as measured by MRI) and an increased overall survival. In parallel an increased IFNγ secretion were detected in the serum of these animals and immunohistological analysis of subsequent cryosections of the glioma revealed an enhanced infiltration of memory CD45RO+ and cytotoxic CD8+ T cells. Taken together the study demonstrates that magnetic nanocarriers such as SPIONs coated with Hsp70 can be applied as a platform for boosting anti-cancer immune responses.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Vacinas Anticâncer/administração & dosagem , Dextranos/administração & dosagem , Portadores de Fármacos , Glioma/tratamento farmacológico , Proteínas de Choque Térmico HSP70/administração & dosagem , Nanopartículas de Magnetita/administração & dosagem , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dextranos/química , Dextranos/imunologia , Dextranos/metabolismo , Composição de Medicamentos , Glioma/sangue , Glioma/imunologia , Glioma/metabolismo , Glioma/patologia , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização , Interferon gama/sangue , Células K562 , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Masculino , Melanoma Experimental , Camundongos , Nanomedicina , Domínios e Motivos de Interação entre Proteínas , Espectroscopia de Prótons por Ressonância Magnética , Ratos Wistar , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacosRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION-EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION-EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION-EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION-EGF conjugates in animals provided receptor-mediated targeted delivery across the blood-brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION-EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Dextranos/administração & dosagem , Dextranos/química , Fator de Crescimento Epidérmico/farmacocinética , Glioma/tratamento farmacológico , Glioma/metabolismo , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/química , Animais , Apoptose , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Dextranos/ultraestrutura , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Glioma/patologia , Nanopartículas de Magnetita/ultraestrutura , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Resultado do TratamentoRESUMO
BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma. METHODS: The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner. RESULTS: Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. CONCLUSION: The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.