Magnetofection

1. In the name of God

2. Magnetofection
Using Magnetic Particles and Magnetic Force to
Enhance and Target Nucleic Acid Delivery
Presented by: Elaheh Emadi-Andani

3. 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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4. 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

5. 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: Endocytosis
185 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 nanoparticle
2.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/chitosan
FSA/polyethylene imine
Dextran
starch
chitosan carboxymethyl cellulose
Agar
Poly l-lysine
polyvinyl alcohol
:their material properties
:the characteristics of the magnetic field
magnetic susceptibility
saturation magnetization
size and shape
magnetic momen
magnetic force
magnetic field gradient
magnetic 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 particles
3.2) 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 particles
3.2) 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 particles
3.3) 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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Cont.
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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Magnets
Permanent 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 targeting
To 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 magnetofection
Usage 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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Cont.
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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25. • 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 equipment
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Magnetofection Benefits

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Transfection efficiency & toxicity & quantity

27. 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.

30. All the best
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