RESUMEN

Anti-angiogenic therapies are promising options for diseases with enhanced vessel formation such as tumors or retinopathies. In most cases, a site-specific local effect on vessel growth is required, while the current focus on systemic distribution of angiogenesis inhibitors may cause severe unwanted side-effects. Therefore, in the current study we have developed an approach for the local inhibition of vascularization, using complexes of lentivirus and magnetic nanoparticles in combination with magnetic fields. Using this strategy in the murine embryonic stem cell (ESC) system, we were able to site-specifically downregulate the protein tyrosine phosphatase SHP2 by RNAi technology in areas with active vessel formation. This resulted in a reduction of vessel development, as shown by reduced vascular tube length, branching points and vascular loops. The anti-angiogenic effect could also be recapitulated in the dorsal skinfold chamber of mice in vivo. Here, site-specific downregulation of SHP2 reduced re-vascularization after wound induction. Thus, we have developed a magnet-assisted, RNAi-based strategy for the efficient local inhibition of angiogenesis in ESCs in vitro and also in vivo.

Asunto(s)

Regulación hacia Abajo , Vectores Genéticos/genética , Lentivirus/genética , Células Madre Embrionarias de Ratones/metabolismo , Neovascularización Fisiológica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Animales , Línea Celular , Vectores Genéticos/administración & dosificación , Imanes/química , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/terapia , Interferencia de ARN , Transducción Genética/métodos

RESUMEN

Targeted gene or drug delivery aims to locally accumulate the active agent and achieve the maximum local therapeutic effect at the target site while reducing unwanted effects at nontarget sites. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected magnetic nanoparticles (MNPs), phospholipids, and nucleic acid are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres or microbubbles. This chapter describes the protocols for preparation of magnetic lipospheres and microbubbles for nucleic acid delivery, and it also describes the procedures for labeling the components of the bubbles (lipids, MNPs, and nucleic acids) for the visualization of the vectors and their characterization, such as magnetic responsiveness and ultrasound contrast effects. Protocols are given for the transfection procedure in adherent cells, evaluation of the association of the magnetic vectors with the cells, reporter gene expression analysis, and cell viability assessment.

Asunto(s)

Nanopartículas de Magnetita/química , Nanosferas/química , Transfección/métodos , Animales , Línea Celular , Supervivencia Celular/genética , Genes Reporteros/genética , Humanos , Campos Magnéticos , Ratones , Microburbujas , Ácidos Nucleicos/genética , Fosfolípidos/química , Ondas Ultrasónicas

RESUMEN

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

Asunto(s)

Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Animales , Nanopartículas de Magnetita/efectos adversos , Trasplante de Células Madre

RESUMEN

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

Asunto(s)

Portadores de Fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Nanopartículas de Magnetita/administración & dosificación , Neovascularización Fisiológica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Cicatrización de Heridas/genética , Animales , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Nanopartículas de Magnetita/química , Ratones , Terapia Molecular Dirigida , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteolisis , Piel/lesiones , Piel/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo

RESUMEN

In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

Asunto(s)

Vasos Sanguíneos/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Nanopartículas de Magnetita/administración & dosificación , Microburbujas , Animales , Marcación de Gen/métodos , Ratones

RESUMEN

Targeted delivery systems for anticancer drugs are urgently needed to achieve maximum therapeutic efficacy by site-specific accumulation and thereby minimizing adverse effects resulting from systemic distribution of many potent anticancer drugs. We have prepared folate receptor-targeted magnetic liposomes loaded with doxorubicin, which are designed for tumor targeting through a combination of magnetic and biological targeting. Furthermore, these liposomes are designed for hyperthermia-induced drug release to be mediated by an alternating magnetic field and to be traceable by magnetic resonance imaging (MRI). Here, detailed preparation and relevant characterization techniques of targeted magnetic liposomes encapsulating doxorubicin are described.

Asunto(s)

Doxorrubicina/farmacología , Liposomas/química , Magnetismo , Muerte Celular , Citometría de Flujo , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/química , Células HeLa , Humanos , Microscopía Fluorescente , Nanopartículas/química , Fosfatidiletanolaminas/química , Fosfolípidos/química , Polietilenglicoles/química

RESUMEN

Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.

Asunto(s)

Lípidos/química , Magnetismo/métodos , Nanopartículas de Magnetita/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Transfección/métodos , Tejido Adiposo/metabolismo , Animales , Células Endoteliales/metabolismo , Liposomas/química , Ratones , Músculos/metabolismo , Células 3T3 NIH , ARN Mensajero/genética , Ovinos , Porcinos

RESUMEN

Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

Asunto(s)

NADPH Oxidasas/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Células A549 , Tejido Adiposo/citología , Animales , Área Bajo la Curva , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Genes Reporteros , Semivida , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , NADPH Oxidasas/metabolismo , Células 3T3 NIH , Osteogénesis , Biosíntesis de Proteínas , Estabilidad del ARN , Curva ROC , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Transfección

RESUMEN

Gene therapy is a promising approach for chronic disorders that require continuous treatment such as cardiovascular disease. Overexpression of vasoprotective genes has generated encouraging results in animal models, but not in clinical trials. One major problem in humans is the delivery of sufficient amounts of genetic vectors to the endothelium which is impeded by blood flow, whereas prolonged stop-flow conditions impose the risk of ischemia. In the current study we have therefore developed a strategy for the efficient circumferential lentiviral gene transfer in the native endothelium under constant flow conditions. For that purpose we perfused vessels that were exposed to specially designed magnetic fields with complexes of lentivirus and magnetic nanoparticles thereby enabling overexpression of therapeutic genes such as endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). This treatment enhanced NO and VEGF production in the transduced endothelium and resulted in a reduction of vascular tone and increased angiogenesis. Thus, the combination of MNPs with magnetic fields is an innovative strategy for site-specific and efficient vascular gene therapy.

Asunto(s)

Endotelio Vascular/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Lentivirus/genética , Nanopartículas de Magnetita/química , Animales , Aorta/metabolismo , Aorta/fisiología , Endotelio Vascular/enzimología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Tono Muscular/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Neovascularización Fisiológica/genética , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.

Asunto(s)

Aminas/química , Lípidos/química , Polímeros/química , ARN Mensajero/genética , Animales , Cationes/química , Ratones , Células 3T3 NIH , Relación Estructura-Actividad , Porcinos

RESUMEN

AIM: To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). MATERIALS & METHODS: Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. RESULTS: Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. CONCLUSION: Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.

Asunto(s)

Endotelio Corneal/metabolismo , Magnetismo/métodos , Nanopartículas de Magnetita/química , Dióxido de Silicio/química , Transfección/métodos , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , ADN/administración & dosificación , ADN/genética , Humanos , Subunidad alfa del Receptor de Interleucina-2/análisis , Lectinas Tipo C/análisis , Leucocitos Mononucleares/citología , Campos Magnéticos , Plásmidos/administración & dosificación , Plásmidos/genética , Proteínas Virales/genética

RESUMEN

The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

RESUMEN

The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.

Asunto(s)

Fibroblastos/citología , Magnetismo , Nanotecnología , Animales , Animales Modificados Genéticamente , Línea Celular , Microscopía Electrónica de Transmisión , ARN Mensajero/genética , Porcinos

RESUMEN

Limitations associated to the use of growth factors represent a major hurdle to musculoskeletal regeneration. On the one hand, they are needed to induce neo-tissue formation for the substitution of a necrotic or missing tissue. On the other hand, these factors are used in supraphysiological concentrations, are short lived and expensive and result in many side effects. Here we develop a gene transfer strategy based on the use of chemically modified mRNA (cmRNA) coding for human bone morphogenetic protein 2 (hBMP-2) that is non-immunogenic and highly stable when compared to unmodified mRNA. Transfected stem cells secrete hBMP-2, show elevated alkaline phosphatase levels and upregulated expression of RunX2, ALP, Osterix, Osteocalcin, Osteopontin and Collagen Type I genes. Mineralization was induced as seen by positive Alizarin red staining. hBMP-2 cmRNA transfected human fat tissue also yielded an osteogenic response in vitro as indicated by expression of hBMP-2, RunX2, ALP and Collagen Type I. Delivering hBMP-2 cmRNA to a femur defect in a rat model results in new bone tissue formation as early as 2 weeks after application of very low doses. Overall, our studies demonstrate the feasibility and therapeutic potential of a new cmRNA-based gene therapy strategy that is safe and efficient. When applied clinically, this approach could overcome BMP-2 growth factor associated limitations in bone regeneration.

Asunto(s)

Proteína Morfogenética Ósea 2/genética , Regeneración Ósea , Fémur/lesiones , Osteogénesis , ARN Mensajero/uso terapéutico , Células Madre/citología , Transfección , Animales , Proteína Morfogenética Ósea 2/metabolismo , Células Cultivadas , Fémur/metabolismo , Fémur/patología , Fémur/fisiología , Terapia Genética/métodos , Humanos , Masculino , ARN Mensajero/química , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Células Madre/metabolismo

RESUMEN

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Asunto(s)

Arteria Carótida Común/cirugía , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Endoteliales/trasplante , Endotelio Vascular/cirugía , Nanopartículas de Magnetita/química , Óxido Nítrico Sintasa de Tipo III/genética , Animales , Arteria Carótida Común/citología , Arteria Carótida Común/metabolismo , Células Endoteliales/citología , Células Endoteliales/enzimología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Imanes , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Polietileneimina/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ingeniería de Tejidos , Transducción Genética , Transgenes

RESUMEN

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2(*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2(*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

Asunto(s)

Adenoviridae/metabolismo , Nanopartículas de Magnetita , Viroterapia Oncolítica/métodos , Vesiculovirus/metabolismo , Adenoviridae/genética , Animales , Línea Celular Tumoral , Cricetinae , Cricetulus , Humanos , Masculino , Ratas , Vesiculovirus/genética

RESUMEN

This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type.

Asunto(s)

Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Interferencia de ARN , ARN Interferente Pequeño/química , Transfección/métodos , Células CACO-2 , Línea Celular Tumoral , Respiración de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fluorocarburos/química , Vectores Genéticos , Células HeLa , Humanos , Iminas/química , Radioisótopos de Yodo , Campos Magnéticos , Microburbujas , Fosfolípidos/química , Plásmidos/química , Plásmidos/metabolismo , Polietilenos/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ultrasonido

RESUMEN

PURPOSE: To explore the potential of magnetofection in delivering pDNA to primary mouse embryonic fibroblasts (PMEFs) and porcine fetal fibroblasts (PFFs) and investigate an effect of magnetic cell labeling on transfection efficacy. METHODS: The formulation and a dose of the magnetic vector were optimized. The efficacy of the procedure was quantified by vector internalization, transgene expression and cell iron loading upon specific labeling with Ab-conjugated magnetic beads or non-specific labeling with MNPs. RESULTS: Up to sixty percent of PMEF and PFF cells were transfected at low pDNA doses of 4-16 pg pDNA/cell. Specific labeling of the PMEFs with MNPs, resulted in a 3- and 2-fold increase in pDNA internalization upon magnetofection and lipofection, respectively, that yielded a 2-4-fold increase in percent of transgene-expressing cells. Non-specific cell labeling had no effect on the efficacy of the reporter expression, despite the acquisition of similar magnetic moments per cell. In PFFs, specific magnetic labeling of the cell surface receptors inhibited internalization and transfection efficacy. CONCLUSIONS: Magnetic labeling of cell-surface receptors combined with the application of an inhomogenous magnetic field (nanomagnetic activation) can affect the receptor-mediated internalization of delivery vectors and be used to control nucleic acid delivery to cells.

Asunto(s)

ADN/administración & dosificación , Portadores de Fármacos/administración & dosificación , Fibroblastos/metabolismo , Campos Magnéticos , Nanopartículas de Magnetita/administración & dosificación , Transfección/métodos , Animales , Células Cultivadas , ADN/genética , Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Ratones , Microscopía Electrónica de Transmisión , Plásmidos , Cultivo Primario de Células , Coloración y Etiquetado , Propiedades de Superficie , Porcinos

RESUMEN

We present a low-cost, portable microfluidic platform that uses laminated polymethylmethacrylate chips, peristaltic micropumps and LEGO® Mindstorms components for the generation of magnetoliposomes that does not require extrusion steps. Mixtures of lipids reconstituted in ethanol and an aqueous phase were injected independently in order to generate a combination of laminar flows in such a way that we could effectively achieve four hydrodynamic focused nanovesicle generation streams. Monodisperse magnetoliposomes with characteristics comparable to those obtained by traditional methods have been obtained. The magnetoliposomes are responsive to external magnetic field gradients, a result that suggests that the nanovesicles can be used in research and applications in nanomedicine.

Asunto(s)

Liposomas/química , Imanes/química , Técnicas Analíticas Microfluídicas/métodos , Simulación por Computador , Diseño de Equipo , Etanol , Hidrodinámica , Lípidos/química , Técnicas Analíticas Microfluídicas/instrumentación , Nanopartículas/química , Nefelometría y Turbidimetría

RESUMEN

Targeted gene or drug delivery aims to locally accumulate the active agent and achieve the maximum local therapeutic effect at the target-site while reducing unwanted effects at nontarget sites. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected magnetic nanoparticles (MNPs), phospholipids, and nucleic acid are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres or microbubbles. This manuscript describes the protocols for preparation of magnetic lipospheres and microbubbles for nucleic acid delivery, and it also describes the procedures for labeling the components of the bubbles (lipids, MNPs, and nucleic acids) for the visualization of the vectors and their characterization, such as magnetic responsiveness and ultrasound contrast effects. Protocols are given for the transfection procedure in adherent cells, evaluation of the association of the magnetic vectors with the cells, reporter gene expression analysis, and cell viability assessment.

Asunto(s)

ADN/metabolismo , Fenómenos Magnéticos , Microburbujas , ARN Interferente Pequeño/genética , Transfección/instrumentación , Ultrasonido/instrumentación , Animales , Supervivencia Celular/efectos de los fármacos , ADN/genética , Colorantes Fluorescentes/química , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Nanopartículas/química , Nanopartículas/toxicidad , Plásmidos/genética , Polietileneimina/química , ARN Interferente Pequeño/metabolismo