Oz Biosciences and Magnofection

Oz Biosciences and Magnofection

TABLE OF CONTENTS

THE PRINCIPLE

HOW DOES IT WORK?

BIODISTRIBUTION OF MAGNETIC NANOPARTICLES

MAGNETOFECTION MINIMIZES TOXICITY

WHAT ARE THE APPLICATIONS?

HOW DO I USE MAGNETOFECTION™ REAGENTS?

DO I NEED SPECIFIC EQUIPMENT?

CONCLUSION

 

Magnetofection™ is a simple and highly effective transfection method to transfect primary cells and hard to transfect cells manufactured by Oz Biosciences and distributed in the UK by Caltag Medsystems.

Inspired by the validated and recognised magnetic drug targeting technology, this original method is a revolution for transfection and infection. In essence, with this technology, Oz Biosciences aimed to unite the advantages of the popular biochemical (cationic lipids or polymers) and physical (electroporation, gene gun) transfection methods in one system while excluding their inconveniences (low efficiency, toxicity, difficulty to handle). It is a unique technology suitable for viral and non-viral gene delivery applications. Magnetofection can be defined as the delivery of nucleic acids, either ‘naked’ or packaged (in complexes with lipids or polymers, and viruses), using magnetic nanoparticles (MNP) under the guidance of an external magnetic field.1,2,3

THE PRINCIPLE

The principle behind Magnetofection™ is to associate nucleic acids, transfection reagents or viruses with specific magnetic nanoparticles. The resulting molecular complexes are then concentrated and transported into cells with the support of an appropriate magnetic field. In this way, the magnetic force exerted upon gene vectors allows a very rapid concentration of the entire applied vector dose on cells, so that 100% of the cells get in contact with a significant vector dose, promoting cellular uptake.2,3

HOW DOES IT WORK?

The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, coated with specific cationic molecules that vary upon application. Their association with the gene vectors (DNA, siRNA, ODN, virus, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated onto cells by the influence of an external magnetic field generated by a specific magnetic plate. The cellular uptake of the genetic material is accomplished by endocytosis and pinocytosis, two natural biological processes.4 Consequently, membrane architecture & structure stay intact, in contrast to other physical transfection methods that damage, create holes, or electroshock the cell membranes. The magnetic force exerted upon the gene vectors allows a very rapid concentration of the entire applied vector dose onto cells/organs. The nucleic acids are then released into the cytoplasm by three different mechanisms depending upon the formulation used:

      1. Nucleic acid delivery can occur due to the “Proton Sponge Effect” caused by the cationic polymers that coat the nanoparticles. This effect promotes endosome osmotic swelling followed by disruption of the endosomal membrane which results in the intracellular release of DNA.
      2. The destabilisation of the endosome can also be caused by the flip-flop of negatively charged lipids located in the endosome’s cytoplasmic-facing monolayer. These negatively charged lipids diffuse into the complex formed between the nucleic acids and the cationic lipids, neutralising the charge of the latter and promoting the release of the nucleic acid.
      3. Delivery of the nucleic acids can also occur by the usual viral mechanism when a virus is used.

BIODISTRIBUTION OF MAGNETIC NANOPARTICLES

The biodegradable cationic magnetic nanoparticles are not toxic at the recommended doses and even higher. The toxicity of these iron-based particles has been extensively studied mainly due to their widespread scientific, diagnosis and medical use (cell separation, MRI, hyperthermia or cancer therapies…).5 Gene vectors / magnetic nanoparticles complexes are internalised into cells after 10-15 minutes i.e., much faster than any other transfection method. After 24, 48 or 72 hours, most of the particles are localised in the cytoplasm in vacuoles and occasionally in the nucleus. In addition, magnetic nanoparticles do not influence cell function. In vivo, without the application of an external magnetic field, MNP accumulates preferentially into the spleen, lung, and liver (although distribution depends on the charge, size, and composition of the particles), and degradation occurs through natural iron metabolism pathways.

MAGNETOFECTION MINIMIZES TOXICITY

Magnetofection™ is effective even with low doses of nucleic acids resulting in minimized cytotoxicity (Fig. 3). As an example, a combination of two technologies, Lipofectamine™ 2000* and CombiMag (Magnetofection™ reagent), enables using smaller amounts of nucleic acids and reagent while increasing the overall efficiency of your transfection

Magnetofection™ has also been compared to electroporation. Electroporation is one of the most efficient transfection tools but it is also known to generate high cytotoxicity. The mechanism of electroporation is the creation of nanometer-scale water-filled holes in the membrane that cause toxicity. It has been shown that Magnetofection can be as efficient as electroporation, but cytotoxicity will be significantly lower (Fig. 4).

WHAT ARE THE APPLICATIONS?

Magnetofection™ is the only versatile and universal technology adapted to all types of nucleic acids (DNA, siRNA, dsRNA, shRNA, mRNA, ODN…), non-viral transfection systems (transfection reagents) and viruses. It has been successfully tested on a broad range of cell lines, hard-to-transfect and primary cells.2,6,7 It is perfect for non-dividing or slowly dividing cells, meaning that the genetic materials can go to the nucleus without cell division. We have shown that combining magnetic nanoparticles to gene vectors of any kind results in a dramatic increase of uptake of these vectors and high transfection efficiency. It is the only technology suitable both for viruses and non-viral nucleic acid delivery applications.

  • For non-viral nucleic acid delivery, it is perfect for primary and hard-to-transfect adherent cells.
  • For viral applications, it is ideal for any cells including primary cells (adherent and suspension).

Using Magnetofection, up to 75 % transfection efficiency can be achieved in many primary cells, such as Chondrocytes, Endothelial cells (HUVEC, HMEC), Epithelial cells, and Fibroblasts. Magnetofection is also highly efficient on classic and hard-to-transfect cell lines, such as SH-SY5Y, PC-12, MEF, C6, etc…

Consequently, several optimized reagents have been designed according to defined applications:

NON VIRAL APPLICATIONS
PolyMag/ PolyMag NeoPolymer complex for all nucleic acids transfection
CombiMagImproves the efficiency of any transfection reagents
Magnetofectamine O2 KitFor all nucleic acids - Association of CombiMag + MTX transfection reagent
NeuroMagFor Neurons transfection
Glial-MagFor Glial cells transfection
SilenceMagFor siRNA applications
FluoMagFluorescent Magnetofection reagents
SelfMagFor creating personalized magnetic delivery system
In vivo APPLICATIONS
In vivo PolyMag & DogtorMagFor all nucleic acids
In vivo ViroMagFor enhancing viral transduction efficiency
VIRAL APPLICATIONS
ViroMagFor enhancing viral transduction efficiency (suitable for all viruses)
ViroMag R/LFor Lentiviral and Retroviral transduction
ViroMag StemLentiviral Transduction Enhancer for stem cells
AdenoMagFor Adenoviral and AAV transduction
Mag4C-LV / Mag4C-ADFor capturing and concentrating Lentiviruses and Adenoviruses

Non-viral applications

PolyMag Neo

PolyMag Neo, a versatile polymer-based transfection reagent, is composed of magnetic nanoparticles coated with specific cationic molecules. It enhances transfection efficiency on primary cells and hard to-transfect cells. Over 120 cells tested!

        • High transgene expression.
  • High transfection efficiency on primary cells.
  • Multipurpose: successfully tested with various cells and nucleic acids.
  • High performance even with low doses of nucleic acids

«Primary human neonatal cardiomyocytes successfully transfected with plasmid DNA using Polymag.» Bittel DC. et al, Cells. 2014.

 

«DNA Transfection, gene silencing & cotransfection (DNA + siRNA) in HUVEC using PolyMag.» Acosta MI. et al, Scientific Rep. 2018.

CombiMag

CombiMag is a magnetic nanoparticle formulation that enables improvement transfection efficiency of any commercial transfection reagent. It can be used with all types of nucleic acids.

  • Improves transfection efficiency without changing your standard protocol.
  • Allows creating your own optimal delivery system with improved efficiency from 30% to 500%.
  • Save materials and time

«Discover how to use CombiMag to efficiently transfect primary cultures of bovine endometrial cells (fibroblasts & epithelial) with DNA.» Lesage-Padilla A. et al, PLoS One. 2017.

Magnetofectamine O2

Magnetofectamine O2, the alliance of MTX transfection reagent and CombiMag reagent, is the perfect one to lead to increased transfection efficiency, minimised toxicity and enhanced gene expression.

  • Boost transfection efficiency.
  • Low amount of nucleic acids – minimized toxicity.
  • No need to change your standard protocol.
  • Serum compatible.

NeuroMag

NeuroMag is the first dedicated transfection reagent for neurons. It is perfect for primary neurons but also for neural cells. Due to its unique properties, NeuroMag allows following the maturation of transfected neurons several days after transfection. It is now routinely used in numerous laboratories to transfect any kind of primary neurons (hippocampal, cortical, motor neurons, dorsal root ganglion, neural stem cells…).8,9

«Transfection of small RNAs (siRNAs, siPOOLs or sgRNAs) in primary Retinal Ganglion Cells using NeuroMag.» Welsbie DS et al, Neuron. 2017.

 

«Transfection efficiency of primary cortical neurons was in the range of 20–30% for overexpression, and 10–15% for TDP-43 knockdown experiments.» Chou C.C. et al, Nature Neuroscience. 2018.

Glial-Mag

Glial-Mag transfection reagent is a new powerful formulation for delivery of nucleic acids into microglial cell lines and primary microglia. This kit is the association of a specific magnetic nanoparticles formulation (Glial-Mag reagent) and a booster (Glial-Boost) designed to enhance transfection efficiency.

  • For transfection of microglial cells line such as BV2, N9, N13, HMO6, MG-5, SIM-A9 and primary microglia.
  • Low nucleic acid amount – minimized toxicity.
  • High level of nucleic acid compaction.

«Magnetofection is superior to other chemical transfection methods in a microglial cell line.» Smolders S. et al, Journal Neuroscience Methods. 2018.

SilenceMag

SilenceMag uses the magnetic force to enhance transfection efficiency on primary and hard-totransfect cells or target silencing into tissues. Based on the Magnetofection technology, SilenceMag reagent gives high protein knockdown at very low doses of siRNA in numerous cell types and tissues.

  • Increased silencing efficiency.
  • Minimized toxicity and off-target effects.
  • Low siRNA/miRNA doses required.
  • Targeted silencing (magnetically-driven).

«90% gene silencing in primary human endothelial colony-forming cells.» Hubert L. et al, J Thromb Haemost. 2014

«Gene Silencing in Endothelial Colony Forming Cells (ECFC) using magnetofection SilenceMag – Approximatively 85- 90% ECFC transfection efficiency was achieved.» Essaadi K. et al, Scientific Reports. 2018

«siRNA transfection on THP-1 cells and RAW 264.7 was performed by using Magnetofection SilenceMag.» Iwata H. et al, Nat. Commun. 2016.

In vivo applications

In vivo Magnetofection has been designed for in vivo targeted transfection and transduction. This original system combines magnetic nanoparticles and nucleic acid vectors that are retained after injection at the magnetically targeted site. in this way, systemic distribution is minimised and toxicity is reduced. DNA complexes can be easily administrated through various injection routes such as systemic administration (intravenous, intra-artery) or local administration (intratumoral, intracerebroventricular).

Magnetofection™ has been tested on several animal models and different organs (such as brain, lung, stomach) and tumours such as fibrosarcoma.2 Researchers showed that injection of magnetofection complexes into mice tumours enables to slow down tumour growth by 50%. “131I-hVEGF siRNA/SilenceMag exhibited an antitumor effect. The synergic therapy of 131IhVEGF siRNA/SilenceMag might be a promising future treatment option against HCC with the dual functional properties of tumour therapy and imaging”.10 Researchers have also transfected DNA plasmid in rat visual cortex neurons using NeuroMag, a Magnetofection reagent dedicated to neural cells. The transfection rates reached values of up to 97% after 30 days, comparable to those achieved by viral vectors.11

Viral applications

Magnetofection™ is also a successful method in enhancing viral transduction efficiency. ViroMag, ViroMag R/L, ViroMag Stem and AdenoMag, the 4 magnetofection reagents dedicated to viruses, enable to increase transduction efficiency up to 10-fold compared to virus alone (Fig. 5). In addition, this technology accelerates and synchronises the transduction process, and enables the concentration of the viral dose onto cells for optimal performance.12,13,14 

HOW DO I USE MAGNETOFECTION™ REAGENTS?

The protocol is a very straightforward and easy procedure (Fig. 1):

  1. Solutions of nucleic acids, viral particles or lipoplexes/polyplexes are prepared in buffer or serum-free culture medium
  2. Vectors are mixed with magnetic nanoparticles formulation composing the Magnetofection™ reagents and incubated 20-30 minutes at room temperature (RT).
  3. Magnetic complexes are added directly onto cells. Then, the cell culture dish is placed on a magnetic plate for 5 to 20 minutes.
  4. Magnetic plate is removed and cells are cultured until experimental assay

DO I NEED SPECIFIC EQUIPMENT?

The only requirement for Magnetofection™ is a magnetic plate specifically designed for this application.The magnetic plate is a one-time buy and completely reusable, so you do not need expensive equipment contrary to approaches such as electroporation or gene gun. Basically, the magnetic field required is produced by specific magnets. Three magnetic plates are available: Super Magnetic Plate, Magnetic Plate with 96 individual magnets and Mega Magnetic Plate. Their design allows producing a heterogeneous magnetic field that magnetises the nanoparticles in solution, forms a very strong gradient to attract the nanoparticles and covers all the surface of the plate. The plate can be washed with ethanol 70% and used within incubators or robots.

CONCLUSION

The different studies carried out demonstrate that Magnetofection™ is a powerful method for introducing nucleic acids into cells. Indeed, magnetic targeting was successful in overcoming the limitations encountered by traditional drug delivery systems: low concentration of vectors on target cells, potentially high toxicity of synthetic vectors, and uncontrolled immune responses and complement activation following systemic diffusion of viral carriers in vivo.

If you have any questions relating to the Magnetofection reagents or would like a quote, please contact us.

BIBLIOGRAPHIC REFERENCES

  1. Scherer F et al, Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 2002 Jan;9(2):102-9.
  2. Plank C et al, Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Adv. Drug Deliv. Rev. 2011 Nov : 1300–1331
  3. Laurent N et al, Nucleic acid delivery using magnetic nanoparticles: the Magnetofection technology. Ther Deliv. 2011 Apr;2(4):471-82.
  4. Khalil IA et al, Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev 2006 ; 58 : 32- 45
  5. Papanikolaou G et al, Toxicol. Appl. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev. 2006 Mar;58(1):32-45
  6. Zhang SQ et al, Exome sequencing identifies MVK mutations in disseminated superficial actinic porokeratosis, Nat Genet. 2012 Oct;44(10):1156-60.
  7. François M et al, Sox18 induces development of the lymphatic vasculature in mice, Nature. 2008 Dec 4;456(7222):643-7
  8. Buerli T et al, Efficient transfection of DNA or shRNA vectors into neurons using magnetofection. Nat Protoc. 2007;2(12):3090-.101
  9. Charrier C et al, Inhibition of SRGAP2 function by its humanspecific paralogs induces neoteny during spine maturation. Cell. 2012 May 11;149(4):923-35.
  10. Chen J at al, Superparamagnetic iron oxide nanoparticles mediated (131)I-hVEGF siRNA inhibits hepatocellular carcinoma tumor growth in nude mice. BMC Cancer. 2014 Feb 21;14:114. doi: 10.1186/1471-2407-14-114.
  11. Soto-Sánchez C et al, Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications. Nanomedicine. 2015 May;11(4):835-43
  12. Sacha JB et al, Synchronous infection of SIV and HIV in vitro for virology, immunology and vaccine-related studies. Nat Protoc. 2010 Feb;5(2):239-46.
  13. Naka K et al, TGF-beta-FOXO signalling maintains leukaemia initiating cells in chronic myeloid leukaemia. Nature. 2010 Feb 4;463(7281):676-80.
  14. Sugimura R et al, Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in the Niche, Cell. 2012 Jul 20;150(2):351-

 

Oz Biosciences and Magnofection

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