RESUMO
PURPOSE: Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. METHODS: The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. RESULTS: Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. CONCLUSIONS: Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.
Assuntos
Portadores de Fármacos/química , Nanopartículas/química , RNA Interferente Pequeno/química , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Cátions/química , Química Farmacêutica/métodos , Humanos , Lipídeos/química , Metacrilatos/química , Nanopartículas/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Polímeros/química , RNA Interferente Pequeno/administração & dosagemRESUMO
Angiogenesis is one of the hallmarks of cancer which renders it an attractive target for therapy of malignancies. Tumor growth suppression can be achieved by inhibiting angiogenesis since it would deprive tumor cells of oxygen and vital nutrients. Activation of endothelial cells of tumor vasculature is the first step in angiogenesis which is mediated by various factors. One of the major triggers in this process is vascular endothelial growth factor (VEGF) which binds to VEGF receptors on endothelial cells of tumor vessels. This induces a series of signaling cascades leading to activation of cellular processes involved in angiogenesis, and therefore down-regulation of VEGF receptor-2 (VEGFR-2) expression seems a viable option to inhibit angiogenesis. In our investigations, this aim has been pursued by using siRNA interfering with the expression of VEGFR-2. Since the discovery of RNA interference (RNAi) as a gene regulation process, successful delivery of small non-coding RNA has presented itself as a major challenge. In the current study, we have characterized a galectin-1 targeted anginex-coupled lipoplex (Angiplex) containing siRNA against the gene of VEGFR-2 as an angiostatic therapeutic. Angiplex particles had a size of approximately 120 nm with a net negative charge and were stable in vitro. These particles were internalized in a specific manner by HUVECs compared to a non-targeted lipoplex system, and their uptake was higher than Lipofectamine 2000. Gene silencing efficiency of Angiplex was shown to be 61%.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Peptídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Inibidores da Angiogênese/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipídeos/química , Peptídeos/química , RNA Interferente Pequeno/químicaRESUMO
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.
Assuntos
Glioblastoma/terapia , MicroRNAs/administração & dosagem , Animais , Proliferação de Células/genética , Embrião de Galinha , Feminino , Terapia Genética/métodos , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Nus , MicroRNAs/genética , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Distribuição Aleatória , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Delivery of nucleic acids to tumors has received extensive attention in the past few decades since these molecules are capable of treating disease by modulating the source of abnormalities. Although high efficiency and low toxicity of numerous delivery systems for nucleic acids have been approved frequently with in vitro assays, contradictions have been observed in many cases between these results and what has occurred in the dynamic in vivo situation. Filling this gap seems to be crucial for further preclinical development of such systems. In this paper, we discuss various barriers which polymeric DNA or siRNA nanoparticles encounter upon systemic administration with an aim to assist in designing more relevant in vitro assays. Furthermore, individual considerations concerning delivery of DNA and siRNA have been addressed.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Neoplasias/terapia , Polímeros/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , DNA/química , Humanos , Polímeros/química , RNA Interferente Pequeno/químicaRESUMO
In this study, the application of thermotropic liquid crystals embedded in cellulose nitrate membranes as on-off drug permeation control in response to temperature changes is described. Two low-molecular-weight liquid crystals, n-pentyl-cyanobiphenyl (K15) and n-heptyl-cyanobiphenyl (K21), with nematic-to-isotropic phase transition temperatures (T(n-i)) of 36.3 °C and 43.3 °C, respectively, were used to modulate drug permeation through the membrane. Liquid crystal-embedded membranes composed of appropriate blends of K15 and K21 were prepared by vacuum filtration. The permeation of pyrazinamide and metronidazole as drug models with different hydrophilicity and molecular weights through the liquid crystal-embedded membrane was examined. It was found that the drug permeation through the membrane could be modulated by changing the temperature below and above the T(n-i) of liquid crystals. The permeation of pyrazinamide, the hydrophilic drug with smaller molecular weight, was more temperature-dependent than metronidazole, the hydrophobic drug with higher molecular weight. These experiments were also repeated with thermal cycling between 25 °C and 45 °C. The permeation profiles were reversible and followed zero-order kinetics.