In the name of God

Magnetofection Using Magnetic Particles and Magnetic Force to Enhance and Target Nucleic Acid Delivery

Presented by: Elaheh Emadi-Andani

Introduction

• Magnetofection is a novel, simple and highly efficient transfection method.

• In essence, the idea was 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 the unique technology suitable for viral and non viral gene delivery applications.

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PRINCIPLE

• Magnetofection principle is to associate nucleic acids, transfection reagents or viruses with specific cationic magnetic nanoparticles.

• The resulting molecular complexes are then concentrated and transported into cells supported by an appropriate magnetic field and their uptake starts immediately. (vector contact with target cells is a prerequisite for a successful transfection/transduction process).

• Viral and nonviral vectors usually are nanoparticles and as such can reach target cells in culture only by diffusion.

• As diffusion is a slow process and many vector types are subject to time-dependent inactivation under cell culture conditions (as well as being toxic in very high concentrations) measures that accelerate vectors toward target cells and thus increase the vector concentration at the cell surface can improve transfection/transduction efficiency. 4

PRINCIPLE• In magnetofection, the exploitation of a 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, and promotes cellular uptake.

• In the late 1970s, Widder et al. introduced the concept of magnetic drug targeting, designed for accumulating drugs mostly in tumors upon administration into the circulation.

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HOW DOES IT WORK?• The magnetic nanoparticles are made of iron oxide (that can be of natural or synthetic

origin), which are coated with specific proprietary cationic molecules varying upon applications.

• Magnetic nanoparticles are associated with nucleic acids by salt-induced colloidal aggregation (this strategy is based on the natural tendency of polyelectrolyte nanoparticles to aggregate at elevated ionic strength) and electrostatic interaction (polycations ).

• Magnetic force drives these complexes towards the target cells, allowing a rapid concentration of the vector dose onto cells.

• The cellular uptake of the genetic materials is accomplished by endocytosis and pinocytosis.

• The major uptake pathway of the vectors is endocytosis via clathrin-coated pits rather than via a caveolae-dependent pathway or via macropinocytosis.

• Mechanism: depend on nanoparticle size> 375 nm: Endocytosis185 nm – 240 nm: Pore formation

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Cont.• Endocytosis and pinocytosis are two natural biological processes. Consequently,

membrane structure stay intact in contrast to other physical transfection methods.

• The nucleic acids are then released into the cytoplasm by different mechanisms depending upon the formulation used:

1.First is the proton sponge effect caused by cationic polymers coated on the nanoparticle2.Second is the destabilization of the endosome 3.Third one is the usual viral mechanism when virus is used

• After 24, 48 or 72 hours, most of the particles are localized in the cytoplasm, in vacuoles and occasionally in the nucleus.

• Scientists do not fully understand the processes that govern nucleic acid uptake, their intracellular interactions, intracellular trafficking, and the regulation of nucleic acid action inside cells.

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WHAT ARE THE APPLICATIONS?

• It is the only versatile technology adapted to all types of nucleic acids (DNA, siRNA, dsRNA, shRNA, mRNA, antisense oligodesoxynucleotides (ODN) and PCR products ).

• It has been successfully tested on a broad range of cell lines.

• It is perfect for non-dividing or slowly dividing cells and genetic materials can go to the nucleus without cell division.

• 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).

An essential finding using viral vectors is that magnetofection, due to its favorable dose–response relationship, can compensate for low viral titers.

• Magnetofection is both suitable to achieve overexpression of a transfected/transduced gene but also to achieve silencing of gene expression.

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• Several optimized reagents have been designed according to defined applications.

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REQUIREMENTS FOR PRACTICING MAGNETOFECTION

1) Nucleic acids and gene delivery vectors The chemical, physical, and biological characteristics of nucleic acids and vectors are as

essential in magnetofection as they are in conventional nucleic acid delivery.

Magnetofection is dependent on the potency of the parent vector composition, which is associated with magnetic nanoparticles.

Important characteristics of vectors and their components to be assembled with magnetic nanoparticles are predominantly charge, size, and the possibility of chemical coupling reactions.

The surface characteristics of vectors (size, zeta potential) influence the aggregation behavior and the adsorption of serum proteins, the timing and the extent of vector uptake and the intracellular processing.

The overall vector dose is an important parameter and must be optimized. the optimal dose is usually lower in magnetofection than in standard transfections.

Toxicity is a question of vector dose, results have shown that highly efficient and rather nontoxic vectors can be obtained with magnetic lipoplexes

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REQUIREMENTS FOR PRACTICING MAGNETOFECTION

2) Magnetic particles i. They must comprise functionality that allows them to be associated with nucleic acids, gene

vectors, and, if necessary, third components.

ii. The association with nucleic acids, gene vectors, or third components must not impair their functionality in delivery and intracellular processing.

iii. Their magnetic properties must be such that they or at least their formulations with nucleic acids, and vectors can be attracted toward target cells using “reasonable” magnetic force.

iv. They must be physically and chemically stable enough to be stored over several months in a suspension medium.

v. Their magnetic core and their surface coatings must be biocompatible so that they can be applied in living cells and organisms.

vi. Toxicity of the particles and their formulations with delivery vectors must be investigated through a series of in vitro and in vivo experiments

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Cont.

Dendriworms

Encapsulating iron oxide nanoparticles:

The responsiveness of magnetic particles to magnetic fields is dependent on :

Mixed surfactant-polymer layers:

Polymeric coating materials :

Pluronic/chitosanFSA/polyethylene imine

Dextranstarchchitosan carboxymethyl celluloseAgarPoly l-lysine

polyvinyl alcohol

:their material properties

:the characteristics of the magnetic field

magnetic susceptibilitysaturation magnetization size and shape

magnetic moment

magnetic forcemagnetic field gradientmagnetic flux density

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Cont.

3) Associating nucleic acids and/or vectors with magnetic particles

3.1) Assembly of virus and magnetic particles by specific ligand–ligand interactions, , especially using (strept)avidin and biotin technology.

Used three different biological strategies to bind viral vectors to magnetic microparticles:

I. Conjugating streptavidinylated magnetic particles to a biotinylated antibody directed against the viral vectors

II.Conjugating streptavidinylated magnetic particles to biotinylated lectin that binds to retroviral vectors

III.Conjugating streptavidinylated magnetic particles to biotinylated viruses

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Cont.

3) Associating nucleic acids and/or vectors with magnetic particles3.2) Self-assembly of magnetic vectors by electrostatic and hydrophobic interactions

The negatively charged phosphate backbone of nucleic acids as well as the negative zeta potential of all types of viral particles in aqueous dispersions allow their assembly with cationic particles due to electrostatically induced aggregation.

The hydrophobic regions on the surface of the virus particles provide adsorption sites that make association through hydrophobic interactions with magnetic nanoparticles comprising hydrophobic species.

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Cont.

3) Associating nucleic acids and/or vectors with magnetic particles3.2) Self-assembly of magnetic vectors by electrostatic and hydrophobic interactions

Although salt-induced colloid aggregation provides a simple and efficient binding method, it must be considered that aggregates that are too large could lead to embolism when injected into blood vessels.

Appropriate aggregate sizes can be chosen by incubation times in salt-containing medium.

Noncovalently associated magnetic vector preparations may dissociate in the blood stream due to opsonization or mediated by the extracellular matrix in target organs.

A useful test can be performed by incubating the preformed complexes at high protein concentration to identify the formulations and/or the particles ensuring desirable stability/integrity of the magnetic vectors in biological fluids.

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Cont.

3) Associating nucleic acids and/or vectors with magnetic particles3.3) Covalent coupling of vectors and magnetic nanoparticles

Covalent coupling of the magnetic nanoparticles is relatively rarely used.

3.4) Characteristics of self-assembled magnetic vector formulations

Characteristics for magnetic vector formulations:

i. Hydrodynamic diameter

ii. Electrokinetic potential and stability in the presence of proteins

iii. Efficacy in functional nucleic acid delivery for intended applications

iv. Magnetic responsiveness or magnetophoretic mobility

These results indicated that the size and charge of the magnetic vectors didn’t have critical importance for

gene delivery. apparently, fine differences in the composition of the surface layer of the particles

cause more significant differences in the efficiency of the delivery.

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Cont.

The magnetophoretic mobility defined as an average velocity υ under a magnetic

field gradient is evaluated as υ = L/t0.1

L=the average path of the complex movement perpendicular to the measuring light beam

t0.1= the time required for a 10-fold decrease in optical density

The change in the turbidity is immediately recorded

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Cont.

4) Magnets The magnetic force acting on magnetic nanoparticles is described by the following formula:

This means that the essential parameters for magnetic accumulation are the volume (and thus the third power of the radius) of the particle and the field gradient.

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4) Magnets

Magnetic nanoparticles will move in a magnetic field only if it is not homogenous, and if the particles experience a field gradient (measured in T/m).

Magnetic nanoparticles will migrate toward the highest density of magnetic field lines.

In magnetofection, the applied magnet has the task to move the magnetic particle-nucleic acid conjugates to the target site.

For in vivo applications, magnets should be tailor-made according to the anatomy of the target site in order to optimize magnetic retention. This characteristic is all the more important as magnetic flux density and field gradient rapidly decrease with the distance from the source of the magnetic field.

The magnetic force acting on magnetic vectors is proportional to the size of the magnetic particle associated with the vector.

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MagnetsPermanent magnets for in vitro and in vivo magnetofection

Rare-earth permanent magnets are used (e.g., neodymium-iron-boron magnets), which produce magnetic flux densities of approximately 1 T at their surfaces.

The field gradients they produce are dependent on the magnet geometry.

Magnetic retention is feasible even upon first pass at more moderate flow rates and is increased if a magnetic formulation passes the target site several times (this is the so-called avalanche effect).

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Magnets

Implant-assisted magnetic targetingTo increase the magnetic field gradient at the target site and to capture magnetic delivery

vehicles more efficiently, external magnets can provide the magnetizing field and the low-dimensional magnetizable “implants” (wires, needles, stents) are positioned in close proximity to the target site to create high local magnetic field gradients.

This effect can be used advantageously to trap magnetic particles against hydrodynamic force.

Dynamic magnetic fields in magnetofectionUsage of alternating or pulsating fields to further improve the efficiency of magnetofection to

further improve nucleic acid delivery deserves increased interest.

It is speculated that such alternating fields may cause some oscillation of magnetic particles, which may facilitate cellular uptake when the particles are bound to cell surfaces. What really happens is not understood.

The transformation efficiency and cell viability was dependent the number of magnetic field pulses.

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5) The ratio of magnetic particles to nucleic acids/vectors is an essential prerequisite for magnetofection

In nonviral magnetofections, usually magnetic particle:DNA ratios between 0.5 and 1 (Fe w/w) yields optimum efficiencies.

MNPs:virus ratios (2.5–10 fg iron per virus particle further referred to as fg Fe/VP)

iron-to-siRNA ratio of 2.8–1 (w/w)

Using an insufficient amount of magnetic nanoparticles may lead to insufficient vector binding and insufficient magnetic sedimentation.

An excessive amount of magnetic nanoparticles may lead to toxicity and to the decrease in transfection efficacy

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HOW DO YOU USE MAGNETOFECTION REAGENTS?

• The protocol is a easy procedure:

1. Dilute nucleic acids or vectors in serum free medium or buffer and add

Magnetofection reagent

2. Incubate 20-30 minutes

3. Add these complexes directly to cells or injection

4. Apply the magnetic field

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• Concentrate genetic material onto cells (higher number of vector–cell contacts within a few minutes) as:

accelerate transfect kinetics (exceed transgene expression) high transfection efficiency with any nucleic acids - increases efficiency from 30 to 500% with lower toxicity

than other methods high performance even with low dose of nucleic acids (enable to use 10 to 100 times less nucleci acids) (Save

materials) (improvement of the dose–response relationship in nucleic acid delivery) Save time (decrease of incubation times )

• Powerful on hard-to-transfect and primary cells

• Biodegradable iron oxide nanoparticles as are safe and cytotoxicity remained at acceptable levels

• Targeted transfer (the possibility to localize nucleic acid delivery to an area that is under the influence of a gradient magnetic field). magnetic targeting has been demonstrated as an effective strategy to decrease side effects of gene transfer.

• Magnetic nanoparticles do not influence cell function

• It does not need expensive equipment25

Magnetofection Benefits

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Transfection efficiency & toxicity & quantity

The feature of saturation as a result of natural uptake mechanism

Hurdles of the kind of used vectors

Needing for long-circulating magnetic vectors

Fate of nanoparticles

Access to the deeper organs (not any target structure is accessible to the source of a

magnetic field of high enough flux density and field gradient)

Hydrodynamic forces reside in vivo

Extracellular barriers

Tissue penetration and uptake into target cells also remain among the major

limitations when magnetic vectors are administered directly into a target tissue

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Limitations of magnetofection

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In vivo Magnetofection

• In vivo Magnetofection has been designed for in vivo targeted transfection and transduction.

• Access to the target cells in vivo is limited by multiple barriers ( e.g. opsonization , vector inactivation , dissociation) in addition to those that are prevalent on the cellular level (overcoming the plasma membrane, escape from internal vesicles and suitable intracellular localization)

• In this way, systemic distribution is minimized 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).

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Cont.

• In animal models, it has been clearly demonstrated that:magnetic drug targeting is feasible even if the drug administration site is remote

from the target site under magnetic field influence magnetic particles can extravasate under the influence of the magnetic field the magnetic carriers can be well tolerated

It becomes clear that potential advances must come from two sources, namely, from improved magnetic formulations and from the physical forces that are applied.

The objective is optimizing retention, tissue penetration, and cellular uptake and The major goal is to maximize magnetic field gradients at target sites.

It can be expected that progress will come from a combination of physical targeting with biological targeting.

All the best30