DOCSLIB.ORG

Magnetic Nanoparticles Mediated-Gene Delivery for Simpler

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Magnetic Nanoparticles Mediated-Gene Delivery for Simpler Nanoscale Advances View Article Online PAPER View Journal | View Issue Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia Cite this: Nanoscale Adv.,2021,3, 4482 pastoris† Seyda Yildiz, a Kubra Solak, b Melek Acar, a Ahmet Mavi bc and Yagmur Unver *a The introduction of exogenous DNA into a cell can be used to produce large quantities of protein. Here, we describe a novel gene delivery method for Pichia pastoris based on recombinant DNA delivery using magnetic nanoparticles (MNPs) under magnetic forces. For this purpose, a linear plasmid (pGKB-GFP) containing the Green Fluorescent Protein (GFP) gene is loaded on polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs at doses that are non-toxic to the yeast cells. The pGKB-GFP loaded MNPs combined with enhancer PEI (Fe3O4@PEI + pGKB-GFP + PEI) are directly transferred to non-competent cells. An effective GFP expression was observed by the selection of antibiotic-resistant yeast cells and Received 30th January 2021 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. heterologous gene integration into the P. pastoris genome was provided. This method, which is very Accepted 5th June 2021 simple, effective, and advanced equipment-free compared to traditional methods, uses smaller amounts DOI: 10.1039/d1na00079a of DNA and the process can be performed in a shorter time. The suggested method might also be rsc.li/nanoscale-advances adapted for the transformation of other yeast species. Introduction different recombinant proteins have been produced in the P. pastoris expression system, containing either 30% of the total Proteins are used in a number of industrial applications such as cell protein or 80% of the total secreted protein6 [http:// This article is licensed under a medicine, food, detergent, biofuel, textile, paper, etc.1,2 www.pichia.com]. There are over 300 licensed industrial Recombinant protein production, which comprises a consider- processes using this yeast and more than 70 commercial able research eld both in academia and industry, represents products are produced in P. pastoris. This powerful eukaryotic ff Open Access Article. Published on 07 June 2021. Downloaded 10/2/2021 5:31:06 AM. a multibillion-dollar market. The increasing demand for expression system is applied in di erent elds including animal recombinant proteins is leading to substantial growth in these feed additives (e.g. phytase), industrial enzymes (e.g. nitrate markets, which offers a large diversity of applications from reductase), and biopharmaceutical proteins (e.g. human – industrial enzymes to biopharmaceutical protein complexes.3,4 insulin).7 9 Generating a specic protein in an organism is typically ach- Spheroplast generation, electroporation, polyethylene glycol ieved by the manipulation of gene expression in the organism. (PEG), alkali cation (LiCl), gene gun, etc. methods are used to Production of desired proteins can be possible in different hosts introduce DNA into P. pastoris for recombinant protein with recombinant DNA technology which can result in much production. These methods make it possible to integrate the higher expression levels than those produced naturally.5 The vectors into the P. pastoris genome or introduce them as selection of a suitable host is crucial to produce a recombinant autonomously replicating elements. However, challenges such protein. The yeast Pichia pastoris (Komagataella phaffii) is widely as high toxicity, low delivery efficiency, high cost of reagents or accepted as one of the most efficient and versatile expression equipment and the complexity of the application became the platforms among the different host organisms commonly used key drawbacks in the gene delivery to yeast. The rapid devel- for recombinant protein production.3 Up to now thousands of opment of nanobiotechnology suggests innovative solutions to overcome these problems. The process of nucleic acid delivery to target cells by using aDepartment of Molecular Biology and Genetics, Ataturk¨ University, Erzurum 25240, magnetic nanocarriers through a magnet is dened as magne- Turkey. E-mail: [email protected]; [email protected] tofection.10 It is known as a highly efficient and ideal method bDepartment of Nanoscience and Nanoengineering, Ataturk¨ University, Erzurum that uses magnetic force to support the uptake of DNA.11,12 25240, Turkey Furthermore, cell membrane permeability is increased effi- cDepartment of Chemistry Education, Kazım Karabekir Faculty of Education, Ataturk¨ ciently by magnetic force.13 Current applications of magnetic University, Erzurum 25240, Turkey † Electronic supplementary information (ESI) available. See DOI: nanoparticles (MNPs) as gene carriers have rapidly progressed 10.1039/d1na00079a in medical research and animal science, especially in the area of 4482 | Nanoscale Adv.,2021,3, 4482–4491 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Nanoscale Advances washed with deionized water several times by centrifuging (at 9000 rpm for 15 minutes). The Fe3O4@PEI MNPs were stored under inert conditions in 20 mL PBS (Phosphate Buffered Saline; Fluka). Acidication was performed to increase the water dis- persibility and reduce the aggregation of Fe3O4@PEI MNPs. Basically, 100 mg of Fe3O4@PEI MNPs were re-dispersed in water (15 mL). The pH value was adjusted to 2 with 0.5 M HCl (Sigma-Aldrich) and then 7 with 0.5 M NaOH (Sigma-Aldrich). The mixture was stirred for 10 minutes in each step.23 Finally, À1 the Fe3O4@PEI MNPs (4 mg mL ) were stored in an inert atmosphere at +4 C. Fluorescently tagged Fe3O4@PEI MNPs were prepared according to the manufacturer's protocol to investigate cellular Fig. 1 Schematic representation of the method for gene delivery to P. uptake of the MNPs by yeast cells. Brie y, 6 mg Fe3O4@PEI pastoris cells using MNPs. Fe3O4@PEI MNPs together with extra PEI MNPs in 0.5 mL of 2-propanol (Sigma-Aldrich) and 0.2 mg have been used as a carrier platform for linear pGKB-GFP plasmid to TRITC (5/6-tetramethylrhodamine isothiocyanate, Thermo Sci- form magnetofectin (Fe O @PEI + pGKB-GFP + PEI). Magnetofectin 3 4 entic™) in 200 mL of DMSO (Merck) were added in 5 mL of 2- was delivered to cells with the help of a magnet. Antibiotic-resistant transformants were selected on YPD–geneticin agar. P. pastoris propanol. The milieu was kept under nitrogen gas for 10 colonies containing the integrated green fluorescence protein (GFP) minutes and then shaking at room temperature in the dark fi coding sequence in their genome were con rmed by a series of PCR overnight. TRITC-labeled Fe3O4@PEI MNPs were collected by analyses. Then, GFP expression in the cells was confirmed. centrifugation at 10 300g for 10 minutes and washed with 2- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. propanol. The TRITC-labeled MNPs were stored in an inert atmosphere and under dark conditions at À20 C. gene therapy for various diseases.14–16 Currently, magneto- fection has been used for the introduction of DNA into Characterization bacteria,17 mammalian cells,16,18,19 and plant pollens.20 Studies The size and morphology of the Fe3O4@PEI MNPs were ob- showing that high doses of Fe3O4 MNPs signi cantly inhibit the growth of Saccharomyces cerevisiae have been found in the tained via transmission and scanning electron microscopy literature21 However, no study has yet been reported concerning (TEM, Hitachi HighTech HT7700, and SEM, Carl Zeiss), the gene delivery of yeast cells like P. pastoris by MNPs. respectively. A carbon-coated copper grid was used to take TEM images, and SEM images were recorded aer gold coating. The This article is licensed under a Here, a rapid and effective method for DNA delivery into P. pastoris cells was suggested based on using polyethylenimine crystalline phase of the dry Fe3O4@PEI MNPs was examined by X-ray diffraction (XRD, D2 Bruker). The magnetic hysteresis (PEI)-coated iron oxide (Fe3O4@PEI) MNPs. The DNA-loaded loops of the sample were determined at room temperature (T ¼ Open Access Article. Published on 07 June 2021. Downloaded 10/2/2021 5:31:06 AM. Fe3O4@PEI MNPs called magnetofectin could pass through the P. pastoris cell wall under the inuence of a magnetic eld. The 305 K) by using a Vibrating Sample Magnetometer (VSM gene reaching the yeast cell is integrated into the yeast genome Quantum Design PMS9T), with an applied magnetic eld from À ¼ and permanent expression is provided. A schematic represen- 20 kOe to +20 kOe (Oe oersted). Fourier transform infrared tation of the suggested magneto-transformation method for P. spectroscopy (FTIR) was used to determine the surface func- tional groups by scanning dry samples in the range of 400–4000 pastoris is given in Fig. 1. À cm 1 wavelength. Dynamic light scattering (DLS, Malvern ff – Zetasizer Nano Zs) was performed in bu er (Na2HPO4 Experimental section NaH2PO4, 10 mM) at different pH (5.00–8.00) values to deter- z The preparation of magnetic nanoparticles mine hydrodynamic diameter (Rh), surface charge ( -potential) and polydispersity index (PDI) of Fe3O4@PEI MNPs. The Fe3O4@PEI MNPs were obtained using a modied proce- dure from the literature by the co-precipitation method.22 $ Cell strains, media, and growth conditions Brie y, in a three-necked ask, 2.4 g FeCl3 6H2O (Sigma- Aldrich) and 1.0 g FeCl2$4H2O (Sigma-Aldrich) were dissolved in Escherichia coli One Shot TOP10 and Pichia pastoris strain X-33 30 mL deionized water. The solution was stirred vigorously for were purchased from Invitrogen (USA) and incubated on LB 30 minutes under an inert atmosphere, and heated to 80 C. (Luria Bertani) agar (Miller) and Yeast extract Peptone Dextrose Then, 6 mL of ammonium hydroxide (25 wt%, Sigma-Aldrich) (YPD) agar to maintain the culture, respectively. YPD contains was rapidly added and the color of the mixture turned black 1% yeast extract (Merck), 2% peptone (Merck), 2% glucose immediately.
Load more

Recommended publications
  • Magneto-Electroporation: Enhancement and Targeting of Gene Transfection Using Magnetic Nanoparticles and Microchips
    MAGNETO-ELECTROPORATION: ENHANCEMENT AND TARGETING OF GENE TRANSFECTION USING MAGNETIC NANOPARTICLES AND MICROCHIPS Min. Li’, Yu-Cheng Lin’, Kai-Chun Su’, May-I Liao2 and Chen-Sheng Yeh2 ‘Department of Engineering Science, ;bepautment of Chemistry, National Cheng Kung Givers& University Road, Tuinan, Taiwan 101 Tel: +886-6-276-2395, Fax: +8&M-276-2329, E-mail: yucEin~~maiI.ncliu.edu.DN ABSTRACT This study demonstrated that DNA associated with magnetic nanoparticles can be attracted to specific areas of cell surface under magnetic fields, which highly increased the DNA concentration at specific areas and finther enhanced the gene transfection in an electroporation (EP) method. Simulations and experimental verifications were conducted and showed a good correlation. This report focused on enhancement and targeting of gene transfection by using 6 nm dispersed superparamagnetic iron oxide (FezOj> and electroporation microchips. Keywords: Electroporation, Magnetic Nanopartiele, Transfection 1. INTRODUCTION Gene therapy is a potential therapeutic modality, which requires effective gene delivery into cells. Different types of gene delivery systems have been developed. Electroporation has been widely applied on gene delivery. The Microchip used to gene transfection had been reported [l]. For enhancing the transfection rate, nanoparticles have shown their potential on this domain. Using silica nanoparticles (diameter -225nm) together with sedimentation methodology were employed to deliver genes. The calculated sedimentation rate is 8.3 mm/hr [2]. Magnetofection utilizing magnetic nanoparticles, the diameters of 400 nm- 1 l.trn, have significant progress in delivering genes by means of magnetic forces [3]. In this study, DNA plasmids associated with 6 run dispersed superparamagnetic iron oxide (y-Fez03) nanoparticles were attracted to specific cell surface areas using strong magnetic fields, followed by electroporation to deliver genes into cells, further providing enhancement and site-specific gene transfection.
    [Show full text]
  • Materials Chemistry B Accepted Manuscript
    Journal of Materials Chemistry B Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/materialsB Page 1 of 11 Journal of Materials Chemistry B Journal of Materials Chemistry B RSC Publishing ARTICLE Assembly of Polyethylenimine-functionalized Iron Oxide Nanoparticles as Agents for DNA Transfection Cite this: DOI: 10.1039/x0xx00000x with Magnetofection Technique a a a# b b Lei Zhang, Yecheng Li, Jimmy C. Yu, Ying Ying Chen and King Ming Chan Manuscript Received 00th January 2012, Accepted 00th January 2012 Various kinds of inorganic nanoparticles have been used as non-viral gene carriers. Two DOI: 10.1039/x0xx00000x fundamental roles of gene carriers are to bind the DNA molecules and to protect them from enzymatic attacks after internalization into the cells.
    [Show full text]
  • Nonviral Locally Injected Magnetic Vectors for in Vivo Gene Delivery: a Review of Studies on Magnetofection
    nanomaterials Review Nonviral Locally Injected Magnetic Vectors for In Vivo Gene Delivery: A Review of Studies on Magnetofection Artem A. Sizikov 1, Marianna V. Kharlamova 1 , Maxim P. Nikitin 1,2, Petr I. Nikitin 3,* and Eugene L. Kolychev 1,* 1 Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia; [email protected] (A.A.S.); [email protected] (M.V.K.); [email protected] (M.P.N.) 2 Sirius University of Science and Technology, 354340 Sochi, Russia 3 Prokhorov General Physics Institute of the Russian Academy of Sciences, 117942 Moscow, Russia * Correspondence: [email protected] (P.I.N.); [email protected] (E.L.K.) Abstract: Magnetic nanoparticles have been widely used in nanobiomedicine for diagnostics and the treatment of diseases, and as carriers for various drugs. The unique magnetic properties of “magnetic” drugs allow their delivery in a targeted tumor or tissue upon application of a magnetic field. The approach of combining magnetic drug targeting and gene delivery is called magnetofection, and it is very promising. This method is simple and efficient for the delivery of genetic material to cells using magnetic nanoparticles controlled by an external magnetic field. However, magnetofection in vivo has been studied insufficiently both for local and systemic routes of magnetic vector injection, and the relevant data available in the literature are often merely descriptive and contradictory. In this review, we collected and systematized the data on the efficiency of the local injections of magnetic nanoparticles that carry genetic information upon application of external magnetic fields. We also Citation: Sizikov, A.A.; Kharlamova, investigated the efficiency of magnetofection in vivo, depending on the structure and coverage of M.V.; Nikitin, M.P.; Nikitin, P.I.; magnetic vectors.
    [Show full text]
  • Potential of Magnetic Field-Assisted Gene
    Schwerdt et al. 1 MAGNETIC FIELD-ASSISTED GENE DELIVERY: ACHIEVEMENTS AND THERAPEUTIC POTENTIAL José I. Schwerdt1, Gerardo F. Goya2, 3 , Pilar Calatayud2, Claudia B. Hereñú1, Paula C. Reggiani1, Rodolfo G. Goya1 1 Institute for Biochemical Research-Histology B-Pathology B, Faculty of Medicine, National University of La Plata, La Plata, Argentina; 2Instituto de Nanociencia de Aragón (INA), University of Zaragoza, Zaragoza; 3Departamento de Física de la Materia Condensada, Facultad de Ciencias, University of Zaragoza, Zaragoza, Spain. Keywords: gene delivery - magnetic nanoparticles – magnetofection - magnetic gene targeting - minimal invasiveness- nanomedicine ABSTRACT The discovery in the early 2000’s that magnetic nanoparticles (MNPs) complexed to nonviral or viral vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells has raised much interest. This technique, called magnetofection, was initially developed mainly to improve gene transfer in cell cultures, a simpler and more easily controllable scenario than in vivo models. These studies provided evidence for some unique capabilities of magnetofection. Progressively, the interest in magnetofection expanded to its application in animal models and led to the association of this technique with another technology, magnetic drug targeting (MDT). This combination offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies like cancer, neurodegeneration and myocardial infarction. The goal of MDT is to concentrate MNPs functionalized with therapeutic drugs, in target areas of the body by means of properly focused external magnetic fields. The availability of stable, nontoxic MNP-gene vector complexes now offers the opportunity to develop magnetic gene targeting (MGT), a variant of MDT in which the gene coding for a therapeutic molecule, rather than the molecule itself, is delivered to a therapeutic target area in the body.
    [Show full text]
  • Magnetic Hyperthermia in Dendritic Cells and Magnetofection in Brain Cells
    Tesis Doctoral BIOMEDICAL APPLICATIONS OF MAGNETIC NANOPARTICLES: MAGNETIC HYPERTHERMIA IN DENDRITIC CELLS AND MAGNETOFECTION IN BRAIN CELLS Autor Asín Pardo, Laura Director/es Ibarra García, Manuel Ricardo Goya Rossetti, Gerardo Fabián FACULTAD DE CIENCIAS Departamento de Física de la Materia Condensada 2012 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es BIOMEDICAL APPLICATIONS OF MAGNETIC NANOPARTICLES: MAGNETIC HYPERTHERMIA IN DENDRITIC CELLS AND MAGNETOFECTION IN BRAIN CELLS. Memoria presentada para optar al grado de Doctor Europeo por la Universidad de Zaragoza Laura Asín Pardo Directores: M. Ricardo Ibarra Gerardo F. Goya Departamento de Física de la Materia Condensada Zaragoza, Marzo 2012. 1 Index Index .......................................................................................................................... 1 Abbreviations ............................................................................................................ 5 Preface ....................................................................................................................... 8 I. Introduction .................................................................................................... 11 I.I. Nanoscience and nanoparticles ............................................................. 13 I.II. Magnetism basis ................................................................................... 17 I.III. Magnetic hyperthermia ........................................................................
    [Show full text]
  • Robust Uptake of Magnetic Nanoparticles (Mnps) by Central Nervous System (CNS) Microglia: Implications for Particle Uptake in Mixed Neural Cell Populations
    Int. J. Mol. Sci. 2010, 11, 967-981; doi:10.3390/ijms11030967 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Communication Robust Uptake of Magnetic Nanoparticles (MNPs) by Central Nervous System (CNS) Microglia: Implications for Particle Uptake in Mixed Neural Cell Populations Mark R. Pickard and Divya M. Chari * Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Staffordshire, ST5 5BG, UK; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: 00-44-1782-734-673; Fax: 00-44-1782-584-634. Received: 5 January 2010; in revised form: 2 March 2010 / Accepted: 4 March 2010 / Published: 8 March 2010 Abstract: Magnetic nanoparticles (MNPs) are important contrast agents used to monitor a range of neuropathological processes; microglial cells significantly contribute to MNP uptake in sites of pathology. Microglial activation occurs following most CNS pathologies but it is not known if such activation alters MNP uptake, intracellular processing and toxicity. We assessed these parameters in microglial cultures with and without experimental ‘activation’. Microglia showed rapid and extensive MNP uptake under basal conditions with no changes found following activation; significant microglial toxicity was observed at higher particle concentrations. Based on our findings, we suggest that avid MNP uptake by endogenous CNS microglia could significantly limit uptake by other cellular subtypes in mixed neural cell populations. Keywords: magnetic; nanoparticles; microglia; toxicity; uptake 1. Introduction Microglia are a major class of glial cells (the non-neuronal, support cells of the brain) that constitute the macrophages of the central nervous system (CNS); these cells are widely believed to be of haematopoetic origin and populate the CNS during development (reviewed in [1–3]).
    [Show full text]
  • In Vitro Evaluation of Hyperthermia Magnetic Technique Indicating the Best Strategy for Internalization of Magnetic Nanoparticles Applied in Glioblastoma Tumor Cells
    pharmaceutics Article In Vitro Evaluation of Hyperthermia Magnetic Technique Indicating the Best Strategy for Internalization of Magnetic Nanoparticles Applied in Glioblastoma Tumor Cells Javier B. Mamani 1 , Taylla K. F. Souza 1 , Mariana P. Nucci 1,2 , Fernando A. Oliveira 1 , Leopoldo P. Nucci 3, Arielly H. Alves 1, Gabriel N. A. Rego 1, Luciana Marti 1,† and Lionel F. Gamarra 1,*,† 1 Hospital Israelita Albert Einstein, São Paulo 05652-000, SP, Brazil; [email protected] (J.B.M.); [email protected] (T.K.F.S.); [email protected] (M.P.N.); [email protected] (F.A.O.); [email protected] (A.H.A.); [email protected] (G.N.A.R.); [email protected] (L.M.) 2 LIM44-Hospital das Clínicas da Faculdade Medicina da Universidade de São Paulo, São Paulo 05403-000, SP, Brazil 3 Centro Universitário do Planalto Central, Brasília 72445-020, DF, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-11-2151-0243 † These authors contributed equally to this work. Abstract: This in vitro study aims to evaluate the magnetic hyperthermia (MHT) technique and the best strategy for internalization of magnetic nanoparticles coated with aminosilane (SPIONAmine) Citation: Mamani, J.B.; Souza, T.K.F.; in glioblastoma tumor cells. SPIONAmine of 50 and 100 nm were used for specific absorption rate Nucci, M.P.; Oliveira, F.A.; Nucci, L.P.; (SAR) analysis, performing the MHT with intensities of 50, 150, and 300 Gauss and frequencies Alves, A.H.; Rego, G.N.A.; Marti, L.; varying between 305 and 557 kHz.
    [Show full text]
  • Magnetofection Enhances Adenoviral Vector-Based Gene Delivery In
    dicine e & N om a n n a o t N e f c o h l n Journal of a o Pereyra et al., J Nanomed Nanotechnol 2016, 7:2 n l o r g u y o J DOI: 10.4172/2157-7439.1000364 ISSN: 2157-7439 Nanomedicine & Nanotechnology Research Article Open Access Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells Andrea Soledad Pereyra1, Olga Mykhaylyk2, Eugenia Falomir Lockhart1, Jackson Richard Taylor3, Osvaldo Delbono3, Rodolfo Gustavo Goya1, Christian Plank2 and Claudia Beatriz Hereñu1,4* 1Biochemistry Research Institute of La Plata (INIBIOLP)/National Scientific and Technical Research Council (CONICET), School of Medicine, National University of La Plata, La Plata, BA, Argentina (ZC 1900) 2Ismaninger Street 22, Institute of Immunology and Experimental Klinikum rechts der Isar, Technical University of Munich, Munich, Germany (ZC 81675) 3Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA (ZC 27157) 4IFEC-CONICET, Farmacology Department, School of Chemistry, National University of Cordoba, (ZC 5000) Córdoba, Argentina Abstract 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.
    [Show full text]
  • Polymag Polymag Neo OZ Biosciences Protocol V1-2
    Magnetofection™ PolyMag & PolyMag Neo INSTRUCTION MANUAL www.ozbiosciences.com Magnetofection : PolyMag , PolyMag Neo Instruction Manual Magnetofection is a simple and highly efficient in vitro and in vivo transfection method. List of Magnetofection™ Kits Catalog Description Volume (µL) Size (number of Number of Number transfections / µg of transfections / DNA) 96 well plates PN30100 PolyMag reagent 100 100 1000 PN30200 PolyMag reagent 200 200 2000 PN31000 PolyMag reagent 1000 1000 10000 PG60100 PolyMag neo reagent 100 100 1000 PG60200 PolyMag neo reagent 200 200 2000 PG61000 PolyMag neo reagent 1000 1000 10000 KC30200 Magnetofection Starting Kit 1 3 x 100 200 2000 KC30300 SiRNA Starting kit 2 200 +2x100 KC30400 Super Starting Kit 3 200 + 3 x 100 200 400 - 2000 MF10000 Super Magnetic Plate N/A N/A N/A MF14000 Mega Magnetic Plate N/A N/A N/A MF10096 96-Magnets, Magnetic Plate N/A N/A N/A 1 Contains 1 vial of each reagent ( PolyMag, PolyMag Neo and CombiMag ) + one Super Magnetic Plate (MF10000) 2 Contains 1 vial of each reagent ( SilenceMag, PolyMag PolyMag Neo ) + one Super Magnetic Plate (MF10000) 3 Contains 1 vial of each reagent ( PolyMag, PolyMag Neo , CombiMag and SilenceMag ) + one Super Magnetic Plate (MF10000) Use the content of the table above to determine the appropriate catalog number for your needs. You can order these products by contacting us (phone, fax, email, website). Several kits containing different Magnetofection reagents are also available, please contact us ( [email protected] ) for a more detailed offer. For all other supplementary information, do not hesitate to contact our dedicated technical support ([email protected] ) and/or to visit our website: www.ozbiosciences.com .
    [Show full text]
  • Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles
    Pharmaceuticals 2013, 6, 204-222; doi:10.3390/ph6020204 OPEN ACCESS pharmaceuticals ISSN 1424-8247 www.mdpi.com/journal/pharmaceuticals Article Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles Suman Chaudhary 1, Carol Anne Smith 2, Pablo del Pino 3, Jesus M. de la Fuente 3, Margaret Mullin 2, Andrew Hursthouse 4, David Stirling 4 and Catherine C. Berry 1,* 1 Centre for Cell Engineering, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK; E-Mail: [email protected] (C.A.S.) 2 Integrated Microscopy Facility, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK; E-Mail: [email protected] (M.M.) 3 Instituto de Nanociencia de Aragon, University of Zaragoza, Edif I+D, C/ Mariano Esquillor s/n, 50018 Zaragoza, Spain 4. School of Science, University of the West of Scotland, Paisley, PA1 2BE, UK; E-Mail: [email protected] (H.A.); [email protected] (D.S.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +44-141-330-8840; Fax: +44-141-330-3783. Received: 19 November 2012; in revised form: 22 January 2013 / Accepted: 23 January 2013 / Published: 6 February 2013 Abstract: Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues.
    [Show full text]
  • Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles Into Porcine Fibroblast
    Molecular Biotechnology https://doi.org/10.1007/s12033-018-0145-9 ORIGINAL PAPER Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles into Porcine Fibroblast Magdalena Hryhorowicz1 · Bartosz Grześkowiak2 · Natalia Mazurkiewicz1 · Paweł Śledziński1 · Daniel Lipiński1 · Ryszard Słomski1,3 © The Author(s) 2018 Abstract Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantita- tive assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity.
    [Show full text]
  • Magnetofection™ in Vivo INSTRUCTION MANUAL
    Magnetofection™ In vivo INSTRUCTION MANUAL Magnetofection™ In vivo Kits In vivo PolyMag In vivo DogtorMag In vivo ViroMag www.ozbiosciences.com OZ Biosciences – In vivo Magnetofection – Instruction Manual - 1 - OZ Biosciences / Protocol Magnetofection in vivo / www.ozbiosciences.com www.ozbiosciences.com OZ Biosciences / Protocol Magnetofection in vivo / www.ozbiosciences.com In vivo Magnetofection™ Instruction Manual In vivo Magnetofecti on™ is an original, simple and efficient method to transfect target cells/tissue in vivo . List of In vivo Magnetofection™ kits Catalog Number of Product Volume Suitable for Number injections IV-KC30210 In vivo PolyMag Starting kit 1 500 µL 5-50 injections 500 µg 500 µL In vivo Dogtor + IV-KC30220 In vivo DogtorMag Starting kit 2 5-50 injections 500 µg 500 µL In vivo CombiMag IV-KC30230 In vivo ViroMag Starting kit 3 250 µL 10-25 injections 1-2 x 10 7 pfu IV-PN30500 In vivo PolyMag 500 500 µL 5-50 injections 500 µg DNA 10-100 IV-PN31000 In vivo PolyMag 1000 1000 µL 1000 µg DNA injections 500 µL In vivo Dogtor + IV-DM30500 In vivo DogtorMag 500 5-50 injections 500 µg DNA 500 µL In vivo CombiMag 1000 µL In vivo Dogtor + 10-100 IV-DM31000 In vivo DogtorMag 1000 1000 µg DNA 1000 µL In vivo CombiMag injections IV-VM30250 In vivo ViroMag 250 250 µL 10-25 injections 1-2 x 10 7 pfu IV-VM30500 In vivo ViroMag 500 500 µL 20-50 injections 2.5-5 x 10 7 pfu IV-MAG1 Magnets set 4 / / / IV-MAG2 Square magnets set 5 / / / IV-MAG3 Cylinder magnets set 6 / / / 1 contains 1 vial of In vivo PolyMag and a Magnets set 2 contains 1 vial of In vivo Dogtor, In vivo CombiMag and a Magnets set 3 contains 1 vial of In vivo ViroMag and a Magnets set 4 contains 1 extra small cylinder (ø 2 mm), 1 small cylinder (ø 5 mm), 1 cylinder (ø 10 mm) and 1 square (18x18 mm) magnets 5 contains 4 square magnets (18x18 mm) 6 contains 4 extra small cylinder (ø 2 mm), 4 small cylinder (ø 5 mm), 4 cylinder (ø 10 mm) magnets Use the content of the list above to determine the appropriate catalog number for your needs.
    [Show full text]