RESUMO
Functionalization of nanoparticles with cationic moieties, such as polyethyleneimine (PEI), enhances binding to the cell membrane; however, it also disrupts the integrity of the cell's plasma and vesicular membranes, leading to cell death. Primary fibroblasts were found to display high surface affinity for cationic iron oxide nanoparticles and greater sensitivity than their immortalized counterparts. Treatment of cells with cationic nanoparticles in the presence of incremental increases in serum led to a corresponding linear decrease in cell death. The surface potential of the nanoparticles also decreased linearly as serum increased and this was strongly and inversely correlated with cell death. While low doses of nanoparticles were rendered non-toxic in 25% serum, large doses overcame the toxic threshold. Serum did not reduce nanoparticle association with primary fibroblasts, indicating that the decrease in nanoparticle cytotoxicity was based on serum masking of the PEI surface, rather than decreased exposure. Primary endothelial cells were likewise more sensitive to the cytotoxic effects of cationic nanoparticles than their immortalized counterparts, and this held true for cellular responses to cationic microparticles despite the much lower toxicity of microparticles compared to nanoparticles.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Nanocápsulas/química , Nanocápsulas/toxicidade , Polietilenoimina/toxicidade , Soro/química , Animais , Apoptose/fisiologia , Cátions , Linhagem Celular , Materiais Revestidos Biocompatíveis/síntese química , Materiais Revestidos Biocompatíveis/toxicidade , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Camundongos , Polietilenoimina/química , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Acute lung injury (ALI) and its most severe manifestation, acute respiratory distress syndrome (ARDS), is a clinical syndrome defined by acute hypoxemic respiratory failure and bilateral pulmonary infiltrates consistent with edema. In-hospital mortality is 38.5% for AL, and 41.1% for ARDS. Activation of alveolar macrophages in the donor lung causes the release of pro-inflammatory chemokines and cytokines, such as TNF-α. To determine the relevance of TNF-α in disrupting bronchial endothelial cell function, we stimulated human THP-1 macrophages with lipopolysaccharide (LPS) and used the resulting cytokine-supplemented media to disrupt normal endothelial cell functions. Endothelial tube formation was disrupted in the presence of LPS-activated THP- 1 conditioned media, with reversal of the effect occurring in the presence of 0.1µg/ml Enbrel, indicating that TNF-α was the major serum component inhibiting endothelial tube formation. To facilitate lung conditioning, we tested liposomal and porous silicon (pSi) delivery systems for their ability to selectively silence TNFR1 using siRNA technology. Of the three types of liposomes tested, only cationic liposomes had substantial endothelial uptake, with human cells taking up 10-fold more liposomes than their pig counterparts; however, non-specific cellular activation prohibited their use as immunosuppressive agents. On the other hand, pSi microparticles enabled the accumulation of large amounts of siRNA in endothelial cells compared to standard transfection with Lipofectamine(®) LTX, in the absence of non-specific activation of endothelia. Silencing of TNFR1 decreased TNF-α mediated inhibition of endothelial tube formation, as well as TNF-α-induced upregulation of ICAM-1, VCAM, and E-selection in human lung microvascular endothelial cells.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , RNA Interferente Pequeno/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Cátions/metabolismo , Citocinas/metabolismo , Selectina E/genética , Células Endoteliais/metabolismo , Inativação Gênica , Humanos , Molécula 1 de Adesão Intercelular/genética , Lipopolissacarídeos/farmacologia , Lipossomos , Macrófagos/metabolismo , Microvasos/citologia , Microvasos/metabolismo , Especificidade da Espécie , Suínos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.
Assuntos
Vasos Sanguíneos/metabolismo , Dependovirus/imunologia , Técnicas de Transferência de Genes , Nanopartículas/química , Silício/química , Animais , Vasos Sanguíneos/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , Tamanho da Partícula , Suínos , Veias/imunologia , Veias/virologiaRESUMO
AIMS: Endothelial cells are dynamic cells tasked with selective transport of cargo from blood vessels to tissues. Here we demonstrate the potential for nanoparticle transport across endothelial cells in membrane-bound vesicles. MATERIALS & METHODS: Cell-free endothelial-derived biovesicles were characterized for cellular and nanoparticle content by electron microscopy. Confocal microscopy was used to evaluate biovesicles for organelle-specific proteins, and to monitor biovesicle engulfment by naive cells. RESULTS: Nanoparticle-laden biovesicles containing low-density polyethyleneimine nanoparticles appear to be predominately of endosomal origin, combining features of multivesicular bodies, lysosomes and autophagosomes. Conversely, high-density polyethyleneimine nanoparticles stimulate the formation of biovesicles associated with cellular apoptotic breakdown. Secreted LAMP-1-positive biovesicles are internalized by recipient cells, either of the same origin or of novel phenotype. CONCLUSION: Cellular biovesicles, rich in cellular signals, present an important mode of cell-to-cell communication either locally or through broadcasting of biological messages.
Assuntos
Comunicação Celular/fisiologia , Endossomos/química , Células Endoteliais/química , Células Endoteliais/fisiologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Vesículas Transportadoras/química , Materiais Biomiméticos/química , Sistema Livre de Células/química , Células Cultivadas , Humanos , Tamanho da PartículaRESUMO
Delivering genes from surfaces, called substrate-mediated gene delivery or reverse transduction, is a useful method to achieve spatial localization of gene delivery. We tested the compatibility of adeno-associated virus (AAV) vectors with various cell adhesive proteins to mediate gene delivery from surfaces. Our studies demonstrate that AAV vectors can be successfully adsorbed on collagen I, elastin, and laminin substrates leading to robust gene delivery to overlying cells. Notably, AAV immobilization on laminin yields the highest efficiency of gene expression. This increased gene expression cannot be explained by increases in the levels of virus deposition, transcriptional activity of cells, or virus vector uptake into cells. Further refinement of our knowledge of AAV interactions with extracellular matrix proteins may have important implications in a variety of applications ranging from tissue engineering to in vivo gene therapy.
Assuntos
Moléculas de Adesão Celular/farmacologia , Dependovirus/efeitos dos fármacos , Dependovirus/metabolismo , Transdução Genética , Adsorção/efeitos dos fármacos , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Elastina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Laminina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
We have investigated the use of adeno-associated virus (AAV) nanoparticles as platforms for the co-delivery of genes and drugs to cancer cells. With its regular geometry, nanoscale dimensions, lack of pathogenicity, and high infection efficiency in a wide range of human cells and tissues, AAV is a promising vector for such applications. We tested the covalent conjugation of paclitaxel onto surface-exposed lysine residues present on the virus capsid. Immunoblotting results suggest successful attachment of drug molecules to the virus nanoparticles. Favorably, the reaction conditions did not reduce the gene delivery efficiency of the AAV vectors. Unfortunately, decrease in cancer cell viability was not observed with our AAV-taxol conjugates. For future attempts at conjugating drugs to the AAV nanoparticle, we have identified several improvements than can be considered to achieve the desired cytotoxicity in target cells.
Assuntos
Dependovirus/química , Nanopartículas/administração & dosagem , Nanopartículas/química , Paclitaxel/administração & dosagem , Paclitaxel/química , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Dependovirus/genética , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vetores Genéticos/química , Vetores Genéticos/genética , Células HeLa , Humanos , Lisina/metabolismo , Nanopartículas/efeitos adversos , Paclitaxel/efeitos adversosRESUMO
High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.
Assuntos
Dependovirus/genética , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Transdução Genética/métodos , Adesão Celular , Linhagem Celular , Materiais Revestidos Biocompatíveis , Colódio/química , Expressão Gênica/genética , Técnicas de Transferência de Genes/instrumentação , Vetores Genéticos/genética , Humanos , Interferência de RNARESUMO
We have reprogrammed the stimulus-responsive conformational change property of a virus nanoparticle (VNP) to enable the surface exposure of metal binding motifs upon activation with heat. The VNP is based on the widely investigated adeno-associated virus (AAV). An intrinsic bioactive functionality of AAV was genetically replaced with a hexahistidine (His) tag. The peptide domain with the inserted His tag is normally inaccessible. Upon external stimulation with heat, the VNP undergoes a conformational change, resulting in externalization of His tag-containing domains and the conferred ability to bind metal. We show that beyond this newfound functionality of the capsid, the VNPs maintain many of the wild-type capsid properties. Our work lays the groundwork for developing stimulus-responsive VNPs that can be used as "smart" building blocks for the creation of higher order structures.