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Christian Plank - One of the best experts on this subject based on the ideXlab platform.
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Magnetofection enhances adenoviral vector based gene delivery in skeletal muscle cells
Journal of Nanomedicine & Nanotechnology, 2016Co-Authors: Andrea S Pereyra, Christian Plank, Olga Mykhaylyk, Rodolfo G Goya, Eugenia Falomir Lockhart, Jackson Taylor, Osvaldo Delbono, Claudia Beatriz HerenuAbstract: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 difficultto- 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.
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enhancement of nucleic acid delivery to hard to transfect human colorectal cancer cells by Magnetofection at laminin coated substrates and promotion of the endosomal lysosomal escape
RSC Advances, 2015Co-Authors: Christian Plank, Martina Anton, Maria Belen Cerda, Milena Batalla, Eduardo G Cafferata, Osvaldo L Podhajcer, Olga MykhaylykAbstract:Despite a great diversity of nanomaterials, such as cationic lipid, polymers or inorganic nanoparticles, that have been developed in order to carry nucleic acids across plasma membranes, these methodologies have still insufficient efficacy in cells named hard-to-transfect cells, such as the colorectal HT29 and Caco-2 cell lines. This paper describes the improvement of plasmid DNA (pDNA) and small interfering RNA (siRNA) transfer in these cells through the combination of Magnetofection, a simplified extracellular matrix of laminin and endosomal/lysosomal escape promotion using the endosome-disruptive peptide INF-7. Magnetofection of pDNA complexes using selected vector formulations resulted in up to 2-fold enhancement in luciferase expression, as compared to lipofection. Further enhances in pDNA transfer in HT29 cells was obtained when Magnetofection was applied on cells grown on laminin coated substrates, increasing 6-fold the luciferase expression compared to lipofection at uncoated substrates. This technique was also applied to siRNA delivery in cells expressing stably luciferase (Caco-2Luc and HT29Luc) and selected magnetic vector formulations resulted in 61 ± 5% and 50 ± 5% of luciferase silencing in HT29Luc and Caco-2Luc, respectively. Further improvement in reporter gene silencing was obtained when the magnetic complexes were modified with INF-7, reaching more than 95% of luciferase silencing in Caco-2Luc cells, while pre-treatment of HT29Luc cells by chloroquine resulted in 80 ± 4% of down regulation of luciferase expression. Thus, Magnetofection applied on cells grown over laminin coated substrates and the optimization of endosomal escape of magnetic complexes would be a good alternative to enhance nucleic acid transfer in hard-to-transfect colorectal cancer cells.
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360 Magnetofection a versatile approach for messenger rna delivery
Molecular Therapy, 2015Co-Authors: Zohreh Sadat Badieyan, Manish K Aneja, Christian PlankAbstract:Transcript therapy, using stabilized non-immunogenic messenger RNA (SNIM RNA), overcomes the potential risks of mutagenicity and immunogenicity, normally associated with DNA based gene delivery and recombinant protein therapy, respectively.Magnetofection, as a delivery system, has been shown to enhance DNA based viral and non-viral gene delivery. In this method, viral or non-viral gene carriers containing nucleic acids are associated with magnetic nanoparticles and a gradient magnetic field is applied to pull them on to the target cell surface.In this study, we established a Magnetofection based protocol for enhanced mRNA delivery, using non-viral carriers. Firstly, we compared Magnetofection, using different magnetic nanoparticles, to lipofection and polyfection, to find the best protocol for in vitro mRNA delivery. Then, the established protocol was generalized for co-transfection of up to three reporter SNIM RNAs (Luc, eGFP and RFP) in mono-culture of Primary Mouse Embryonic Fibroblasts (PMEF), or co-culture of PMEF and Porcine Fetal Fibroblast (PFF). In parallel, expression kinetics post Magnetofection of d2eGFP SNIM RNA were determined.Magnetofection significantly improved transfection efficiency in mono culture as well as co-culture of applied cells. Using this protocol, co-transfection of different SNIM RNAs did not inhibit the transfection of every single SNIM RNA into PMEF or PFF cells. Moreover, rapid and higher peak of expression was observed with using magnetic nanoparticles.To conclude, Magnetofection of mRNA presents itself as an efficient mRNA delivery system, thereby bringing transcript therapies a step closer to their clinical uses.
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magnetically enhanced nucleic acid delivery ten years of Magnetofection progress and prospects
Advanced Drug Delivery Reviews, 2011Co-Authors: Christian Plank, Olivier Zelphati, Olga MykhaylykAbstract:Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as Magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of Magnetofection, and the application of Magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using Magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.
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recent advances in Magnetofection and its potential to deliver sirnas in vitro
Methods of Molecular Biology, 2009Co-Authors: Olga Mykhaylyk, Martina Anton, Joseph Rosenecker, Olivier Zelphati, Edelburga Hammerschmid, Christian PlankAbstract:This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells 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. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for Magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, Magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by Magnetofection in every cell type.
Olga Mykhaylyk - One of the best experts on this subject based on the ideXlab platform.
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physics of in vitro Magnetofection effect of magnetic transport and redistribution of nanoparticles
Journal of Magnetism and Magnetic Materials, 2020Co-Authors: Olga Mykhaylyk, Magda Lorena Arciniegas Vaca, G A Pasquevich, Nicolas Mele, Rodolfo G Goya, F H SanchezAbstract:Abstract A comprehensive study of relevant aspects of the physics behind in vitro magnetic assisted transfection, or Magnetofection is presented. Magnetofection experiments were performed as a function of culture-magnet distance for a fixed time of 30 min, as a function of time for a fixed separation of about 1 mm, and under special geometries chosen to elucidate the relative effects of gravitational and magnetic forces. It is shown that under appropriate conditions, in vitro Magnetofection can be performed with almost equal easiness in any desired space direction, even against gravity. Redistribution of magnetic nanoparticles in a colloid, with an initial uniform distribution, was studied with the same experimental setup used in Magnetofection experiments, as a function of time, and as a function of culture-magnet relative position. It was found that magnetic nanoparticles tend to arrange into concentrated regions with ring or circle shapes, and that final distribution depends strongly on relative position. Cellular uptake of magnetic nanoparticles was estimated for standard Magnetofection and for no applied magnetic field experiments. A consistent description of results is given by comparing Magnetofection efficiency, nanoparticles uptake and redistribution experiments. The results presented here constitute novel information that will contribute to gaining deeper understanding of how Magnetofection proceeds and how can be improved. They impact directly on in vivo procedures, providing conceptual tools to optimize setup geometry and magnetic field application time.
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Magnetofection enhances adenoviral vector based gene delivery in skeletal muscle cells
Journal of Nanomedicine & Nanotechnology, 2016Co-Authors: Andrea S Pereyra, Christian Plank, Olga Mykhaylyk, Rodolfo G Goya, Eugenia Falomir Lockhart, Jackson Taylor, Osvaldo Delbono, Claudia Beatriz HerenuAbstract: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 difficultto- 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.
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enhancement of nucleic acid delivery to hard to transfect human colorectal cancer cells by Magnetofection at laminin coated substrates and promotion of the endosomal lysosomal escape
RSC Advances, 2015Co-Authors: Christian Plank, Martina Anton, Maria Belen Cerda, Milena Batalla, Eduardo G Cafferata, Osvaldo L Podhajcer, Olga MykhaylykAbstract:Despite a great diversity of nanomaterials, such as cationic lipid, polymers or inorganic nanoparticles, that have been developed in order to carry nucleic acids across plasma membranes, these methodologies have still insufficient efficacy in cells named hard-to-transfect cells, such as the colorectal HT29 and Caco-2 cell lines. This paper describes the improvement of plasmid DNA (pDNA) and small interfering RNA (siRNA) transfer in these cells through the combination of Magnetofection, a simplified extracellular matrix of laminin and endosomal/lysosomal escape promotion using the endosome-disruptive peptide INF-7. Magnetofection of pDNA complexes using selected vector formulations resulted in up to 2-fold enhancement in luciferase expression, as compared to lipofection. Further enhances in pDNA transfer in HT29 cells was obtained when Magnetofection was applied on cells grown on laminin coated substrates, increasing 6-fold the luciferase expression compared to lipofection at uncoated substrates. This technique was also applied to siRNA delivery in cells expressing stably luciferase (Caco-2Luc and HT29Luc) and selected magnetic vector formulations resulted in 61 ± 5% and 50 ± 5% of luciferase silencing in HT29Luc and Caco-2Luc, respectively. Further improvement in reporter gene silencing was obtained when the magnetic complexes were modified with INF-7, reaching more than 95% of luciferase silencing in Caco-2Luc cells, while pre-treatment of HT29Luc cells by chloroquine resulted in 80 ± 4% of down regulation of luciferase expression. Thus, Magnetofection applied on cells grown over laminin coated substrates and the optimization of endosomal escape of magnetic complexes would be a good alternative to enhance nucleic acid transfer in hard-to-transfect colorectal cancer cells.
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rotational magnetic pulses enhance the Magnetofection efficiency in vitro in adherent and suspension cells
Journal of Magnetism and Magnetic Materials, 2013Co-Authors: Ch Dahmani, Olga Mykhaylyk, Fl Helling, St Gotz, Th Weyh, Hansgeorg Herzog, Ch PlankAbstract:Abstract The association of magnetic nanoparticles with gene delivery vectors in combination with the use of gradient magnetic fields (Magnetofection) enables improved and synchronised gene delivery to cells. In this paper, we report a system comprising rotating permanent magnets to generate defined magnetic field pulses with frequencies from 2.66 to 133 Hz and a field amplitude of 190 or 310 mT at the location of the cells. Low-frequency pulses of 2.66–10 Hz with a magnetic flux density of 190 mT were applied to the examined cells for 30–120 s after Magnetofection. These pulses resulted in a 1.5–1.9-fold enhancement in the transfection efficiency compared with Magnetofection with only a static magnetic field in both adherent and suspension cells. The magnetic field amplitudes of 190 and 310 mT had similar effects on the transfection efficacy. No increase in the percentage of transgene-expressing suspension cells and no cytotoxic effects (based on the results of the MTT assay) were observed after applying alternating magnetic fields.
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magnetically enhanced nucleic acid delivery ten years of Magnetofection progress and prospects
Advanced Drug Delivery Reviews, 2011Co-Authors: Christian Plank, Olivier Zelphati, Olga MykhaylykAbstract:Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as Magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of Magnetofection, and the application of Magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using Magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.
Gregor Sersa - One of the best experts on this subject based on the ideXlab platform.
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Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma
Biomaterials, 2017Co-Authors: Mojca Kržan, Jaka Lavrenčak, Vladimir Boštjan Bregar, Rok Romih, Sara Prijic, Andrej Coer, Gregor Sersa, Maja Cemazar, Andrej Žnidaršič, Janez Ščančar, Lara ProsenAbstract:Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; Magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for Magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reportergene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted insignificant antitumor effect and could be further refined for cancer immuno-gene therapy.
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Research Article Magnetofection: A Reproducible Method for
2016Co-Authors: Gene Delivery, Maja Cemazar, Melanoma Cells, Gregor SersaAbstract:Copyright © 2013 Lara Prosen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in someproof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueousmedium,were tested after varying different parameters of the synthesis procedure.The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence Magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting i
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magnetic field contributes to the cellular uptake for effective therapy with Magnetofection using plasmid dna encoding against mcam in b16f10 melanoma in vivo
Nanomedicine: Nanotechnology Biology and Medicine, 2016Co-Authors: Lara Prosen, Rok Romih, Maja Cemazar, Janez Ščančar, Samo Hudoklin, Monika Stimac, Ursa Lampreht Tratar, Maja Ota, Gregor SersaAbstract:Aim: We explored the distribution and cellular uptake of intratumorally injected SPIONs-PAA-PEI-pDNA (Magnetofection complexes), and antitumor effectiveness of Magnetofection with plasmid DNA encoding short hairpin RNA (shRNA) against Mcam (pDNAanti-MCAM). Materials & methods: Analyses were made based on the histology, ultrastructure and quantitative measurements of Magnetofection complexes, and quantification of the antitumor effectiveness in B16F10 melanoma in vivo. Results: Injected Magnetofection complexes were distributed around the injection site. Exposure of tumors to external magnetic field contributed to the uptake of Magnetofection complexes from extracellular matrix into melanoma cells. Three consecutive Magnetofections of tumors with pDNAanti-MCAM resulted in significant reduction of tumor volume. Conclusion: Magnetofection is effective for gene delivery to melanoma tumors, but requires a magnetic field for cellular uptake and antitumor effect.
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Magnetofection an effective selective and feasible non viral gene delivery method
2016Co-Authors: L Prosen, Maja Cemazar, Gregor SersaAbstract:Due to the safety issues of viral vectors, non-viral gene delivery systems, such as chemical carriers, gene electrotransfer, and Magnetofection are developing. Magnetofection is a particle mediated transfection method. Our research group developed unique polyacrilyc acid (PAA) coated and polyethlenimine (PEI) functionalized superparamagnetic iron oxide nanoparticles (SPIONs) for the introduction of reporter and therapeutic plasmid DNA (pDNA) into the cells and tumors.
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mcam silencing with rna interference using Magnetofection has antitumor effect in murine melanoma
Molecular therapy. Nucleic acids, 2014Co-Authors: Lara Prosen, Maja Cemazar, Branka Music, Bostjan Markelc, Tanja Dolinsek, Gregor SersaAbstract:The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by Magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by Magnetofection.
Divya M Chari - One of the best experts on this subject based on the ideXlab platform.
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part ii functional delivery of a neurotherapeutic gene to neural stem cells using minicircle dna and nanoparticles translational advantages for regenerative neurology
Journal of Controlled Release, 2016Co-Authors: Alinda R Fernandes, Divya M ChariAbstract:Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs. Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with Magnetofection technology to safely engineer NSCs to overexpress BDNF. Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/Magnetofection technology for therapeutic delivery of key neurotrophic agents.
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part i minicircle vector technology limits dna size restrictions on ex vivo gene delivery using nanoparticle vectors overcoming a translational barrier in neural stem cell therapy
Journal of Controlled Release, 2016Co-Authors: Alinda R Fernandes, Divya M ChariAbstract:Genetically engineered neural stem cell (NSC) transplant populations offer key benefits in regenerative neurology, for release of therapeutic biomolecules in ex vivo gene therapy. NSCs are 'hard-to-transfect' but amenable to 'Magnetofection'. Despite the high clinical potential of this approach, the low and transient transfection associated with the large size of therapeutic DNA constructs is a critical barrier to translation. We demonstrate for the first time that DNA minicircles (small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids) deployed with Magnetofection achieve the highest, safe non-viral DNA transfection levels (up to 54%) reported so far for primary NSCs. Minicircle-functionalized magnetic nanoparticle (MNP)-mediated gene delivery also resulted in sustained gene expression for up to four weeks. All daughter cell types of engineered NSCs (neurons, astrocytes and oligodendrocytes) were transfected (in contrast to conventional plasmids which usually yield transfected astrocytes only), offering advantages for targeted cell engineering. In addition to enhancing MNP functionality as gene delivery vectors, minicircle technology provides key benefits from safety/scale up perspectives. Therefore, we consider the proof-of-concept of fusion of technologies used here offers high potential as a clinically translatable genetic modification strategy for cell therapy.
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magnetic nanoparticle mediated gene transfer to oligodendrocyte precursor cell transplant populations is enhanced by Magnetofection strategies
ACS Nano, 2011Co-Authors: Stuart I Jenkins, Mark R Pickard, Nicolas Granger, Divya M ChariAbstract:This study has tested the feasibility of using physical delivery methods, employing static and oscillating field "Magnetofection" techniques, to enhance magnetic nanoparticle-mediated gene transfer to rat oligodendrocyte precursor cells derived for transplantation therapies. These cells are a major transplant population to mediate repair of damage as occurs in spinal cord injury and neurological diseases such as multiple sclerosis. We show for the first time that magnetic nanoparticles mediate effective transfer of reporter and therapeutic genes to oligodendrocyte precursors; transfection efficacy was significantly enhanced by applied static or oscillating magnetic fields, the latter using an oscillating array employing high-gradient NdFeB magnets. The effects of oscillating fields were frequency-dependent, with 4 Hz yielding optimal results. Transfection efficacies obtained using Magnetofection methods were highly competitive with or better than current widely used nonviral transfection methods (e.g., electroporation and lipofection) with the additional critical advantage of high cell viability. No adverse effects were found on the cells' ability to divide or give rise to their daughter cells, the oligodendrocytes-key properties that underpin their regeneration-promoting effects. The transplantation potential of transfected cells was tested in three-dimensional tissue engineering models utilizing brain slices as the host tissue; modified transplanted cells were found to migrate, divide, give rise to daughter cells, and integrate within host tissue, further evidencing the safety of the protocols used. Our findings strongly support the concept that magnetic nanoparticle vectors in conjunction with state-of-the-art Magnetofection strategies provide a technically simple and effective alternative to current methods for gene transfer to oligodendrocyte precursor cells.
Maria Belen Cerda - One of the best experts on this subject based on the ideXlab platform.
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enhancement of nucleic acid delivery to hard to transfect human colorectal cancer cells by Magnetofection at laminin coated substrates and promotion of the endosomal lysosomal escape
RSC Advances, 2015Co-Authors: Christian Plank, Martina Anton, Maria Belen Cerda, Milena Batalla, Eduardo G Cafferata, Osvaldo L Podhajcer, Olga MykhaylykAbstract:Despite a great diversity of nanomaterials, such as cationic lipid, polymers or inorganic nanoparticles, that have been developed in order to carry nucleic acids across plasma membranes, these methodologies have still insufficient efficacy in cells named hard-to-transfect cells, such as the colorectal HT29 and Caco-2 cell lines. This paper describes the improvement of plasmid DNA (pDNA) and small interfering RNA (siRNA) transfer in these cells through the combination of Magnetofection, a simplified extracellular matrix of laminin and endosomal/lysosomal escape promotion using the endosome-disruptive peptide INF-7. Magnetofection of pDNA complexes using selected vector formulations resulted in up to 2-fold enhancement in luciferase expression, as compared to lipofection. Further enhances in pDNA transfer in HT29 cells was obtained when Magnetofection was applied on cells grown on laminin coated substrates, increasing 6-fold the luciferase expression compared to lipofection at uncoated substrates. This technique was also applied to siRNA delivery in cells expressing stably luciferase (Caco-2Luc and HT29Luc) and selected magnetic vector formulations resulted in 61 ± 5% and 50 ± 5% of luciferase silencing in HT29Luc and Caco-2Luc, respectively. Further improvement in reporter gene silencing was obtained when the magnetic complexes were modified with INF-7, reaching more than 95% of luciferase silencing in Caco-2Luc cells, while pre-treatment of HT29Luc cells by chloroquine resulted in 80 ± 4% of down regulation of luciferase expression. Thus, Magnetofection applied on cells grown over laminin coated substrates and the optimization of endosomal escape of magnetic complexes would be a good alternative to enhance nucleic acid transfer in hard-to-transfect colorectal cancer cells.