Stress-induced Gene Expression Sensing Intracellular Heating Triggered by Magnetic Hyperthermia

It is known that alternating magnetic field applications on eukaryotic cells loaded with single domain iron oxide nanoparticles result in high hyperthermic citotoxicity leading to cell dead. Although magnetic hyperthermia therapy for cancer tumours is being developed under this idea, some in vitro assays have shown controversial results indicating that alternating magnetic field triggers large apoptotic effect without significant culture-temperature increase. In agreement with these observations a huge lowering in nanoparticle specific heating rates, when going from the colloidal suspension to cell endosomes, together with cell death, has been reported. Here, we propose a new methodology to determine the occurrence of local heating in cells when alternating magnetic fields in the radiofrequency field range are applied to cell cultures holding very low iron oxide concentrations, being these concentrations insufficient to produce a global cell-culture temperature increase up to therapeutic values. To this end, human lung adenocarcinoma cells (A549 cell line) were transduced with a lentiviral vector encoding the expression of the enhanced green fluorescence protein, EGFP, under the action of the inducible human heat shock protein 70B promoter. This modified A549 cell line was incubated with aqueous suspensions of magnetite core nanoparticles (uncoated or covered with coating agents like citric acid or silicon oxide), and exposed to radiofrequency fields. The application of an alternating magnetic field to cell cultures loaded with nanoparticles resulted in no global temperature increase but EGFP expression. Stress-inducible gene expression scales with uptake and nanoparticle properties like saturation magnetization and heat dissipation efficiency. Our analysis demonstrates that EGFP expression is linked to a localized intracellular temperature increase.Fil: de Sousa, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Carrea, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Mendoza Zélis, Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Muraca, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidade Estadual de Campinas; BrasilFil: Mykhaylyk, Olga. Technische Universitat Munchen; AlemaniaFil: Sosa, Yolanda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Sánchez, Francisco Homero. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Dewey, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Fernández van Raap, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; Argentin

Transferencia génica asistida por magnetovectores en músculo esquelético

El objetivo del presente trabajo es determinar si la magnetofección puede constituir una alternativa eficiente para la transferencia génica in vitro e in vivo en células maduras del musculo esquelético.Facultad de Ciencias Médica

Transferencia génica asistida por magnetovectores en músculo esquelético

El objetivo del presente trabajo es determinar si la magnetofección puede constituir una alternativa eficiente para la transferencia génica in vitro e in vivo en células maduras del musculo esquelético.Facultad de Ciencias Médica

Transferencia génica asistida por magnetovectores en músculo esquelético

El objetivo del presente trabajo es determinar si la magnetofección puede constituir una alternativa eficiente para la transferencia génica in vitro e in vivo en células maduras del musculo esquelético.Facultad de Ciencias Médica

Stress-induced Gene Expression Sensing Intracellular Heating Triggered by Magnetic Hyperthermia

It is known that alternating magnetic field applications on eukaryotic cells loaded with single domain iron oxide nanoparticles result in high hyperthermic citotoxicity leading to cell dead. Although magnetic hyperthermia therapy for cancer tumours is being developed under this idea, some in vitro assays have shown controversial results indicating that alternating magnetic field triggers large apoptotic effect without significant culture-temperature increase. In agreement with these observations a huge lowering in nanoparticle specific heating rates, when going from the colloidal suspension to cell endosomes, together with cell death, has been reported. Here, we propose a new methodology to determine the occurrence of local heating in cells when alternating magnetic fields in the radiofrequency field range are applied to cell cultures holding very low iron oxide concentrations, being these concentrations insufficient to produce a global cell-culture temperature increase up to therapeutic values. To this end, human lung adenocarcinoma cells (A549 cell line) were transduced with a lentiviral vector encoding the expression of the enhanced green fluorescence protein, EGFP, under the action of the inducible human heat shock protein 70B promoter. This modified A549 cell line was incubated with aqueous suspensions of magnetite core nanoparticles (uncoated or covered with coating agents like citric acid or silicon oxide), and exposed to radiofrequency fields. The application of an alternating magnetic field to cell cultures loaded with nanoparticles resulted in no global temperature increase but EGFP expression. Stress-inducible gene expression scales with uptake and nanoparticle properties like saturation magnetization and heat dissipation efficiency. Our analysis demonstrates that EGFP expression is linked to a localized intracellular temperature increase.Facultad de Ciencias ExactasInstituto de Física La PlataFacultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Magnetofection enhances adenoviral vector-based gene delivery in skeletal muscle cells

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.Instituto de Investigaciones Bioquímicas de La Plat

Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo

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

Nucleic acid delivery to magnetically-labeled cells in a 2D array and at the luminal surface of cell culture tube and their detection by MRI

The magnetic labeling of living cells has become of major interest in the areas of cell therapy and tissue engineering. Magnetically labeled cells have been reported to allow increased and controlled seeding, tracking, and targeting of cells. In this work, we comprehensively characterize magnetic nanoparticles (MNPs) possessing a magnetite core of about 11 nm, and which are coated with the fluorinated surfactant F(CF2)nCH2CH2SCH2CH2C(O)OLi and 1,9-nonandithiol (NDT) for the nonspecific labeling of human pulmonary epithelial (H441) cells. We achieved a non-specific cell loading of 38 pg Fe/cell. In this work we combine magnetic cell labeling with subsequent genetic modification of the cells with non-viral transfection complexes associated with PEI-Mag2 magnetic nanoparticles upon gradient magnetic field application called magnetofection. The magnetic responsiveness and magnetic moment of the MNP-labeled cells and the magnetic transfection complexes were evaluated by measuring changes in the turbidity of prepared cells suspensions and complexes in a defined magnetic gradient field. The magnetic responsiveness of cells that were loaded with NDT-Mag1 MNPs (20-38 pg Fe/cell) was sufficient to engraft these labeled cells magnetically onto the luminal surface of a culture tube. This was achieved using a solenoid electromagnet that produced a radial magnetic field of 20-30 mT at the seeding area and an axial gradient field of approx. 4 T/m. The MNP-labeled cells were magnetofected in 2D arrays (well plates) and at the luminal surface of cell culture tube. The optimized magnetic pre-labeling of cells did not interfere with, or even increased, the efficiency of magnetofection in both culture systems without causing cell toxicity. Cell loading of 38 pg Fe/cell of NDT-Mag1 MNPs resulted in high transverse relaxivities r2*, thus allowing the MRI detection of cell concentrations that were equivalent to (or higher than) 1.2 microg Fe/ml. Multi-echo gradient echo imaging and R2* mapping detected as few as 1533 MNP-labeled H441 cells localized within a 50 microl fibrin clot and MNP-labeled cell monolayers that were engrafted on the luminal surface of a cell culture tube. Further loading of cells with MNPs did not increase either the magnetic responsiveness of the cells or the sensitivity of MR imaging. In summary, the NDT-Mag1 magnetic nanoparticles provided a high cell-loading efficiency, resulting in strong cell magnetic moments and a high sensitivity to MRI detection. The transfection ability of the labeled cells was also maintained, thereby increasing the magnetofection efficiency