Charged Magnetic Nanoparticles for enhancing gene transfection
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01-11-2010, 12:46 AM


Charged Magnetic Nanoparticles for enhancing gene transfection
Presented By
O.A.NAZEER HUSSAIN
S7 AE&I
College Of Engineering, Trivandrum
2007-11 batch


.ppt   Charged Magnetic Nanoparticles for enhancing gene transfection1.ppt (Size: 422.23 KB / Downloads: 70)

Overview
Introduction
Experiments
Results
Discussion
Conclusion
Introduction
Gene Transfection
Deliberately introducing nucleic acids into cells.
“Infection by transformation”

Viral methods
Carrier Viruses.
‘Virus Transduction’.




Non-Viral methods
Chemical based transfection
Non chemical methods
Paticle based methods

Merits
Demerits

Magnetofection

Invented by Christian Plank and Christian Bergemann
Simplicity and efficiency.
Application of magnetic field
Processing time
Principle


Schematic diagram of forming magnetic gene complexes via electrostatic interaction
Transfection agents
Highly charged macromolecules
Formation of Magnetic Gene vectors
Composition.
Toxicity.
PEI(Poly ethyleneimine)
Hypothesis
Any surface charged magnetic nanoparticle is capable of enhancing PEI transfection.
Theory
cationic polymers ,cationic liposomes and genes
Formation of complexes
electrostatic adsorption
Preparation of magnetic nanoparticles independent of genes or vectors used.


Testing
Preparation of Magnetic nanoparticles
Creation of charges
Surface modifications
(MP)-CA, (MP)-CMD, (MP)-APTES, (MP)-betaine etc
25kDA PEI ,green fluroscent protein(GFP) plasmids, NIH-3T3 cells


EXPERIMENTS

A. Preparation of Magnetic nanoparticles with various surface modifications

Different modifications were done.
1. CA modification
2.CMD modification
3.APTES modification
4.Betaine modification
5.PEI modification
Structural formulas for coating materials used in this method

B. Transfection
25kDa PEI ,GFP plasmids, NIH-3T3 cells
mass ratio O.8
Different for each modification
Serum culture medium
Magneto FACTOR

Contd…..
Replaced by medium supplemented with fetal calf serum.
Flow cytometry
All experiments performed in triplicatae
Results A. Materials

In the room temperature magnetization curve we can find that
No coercive force.
Specifice saturation
Attraction towards bottom of the cell plate




The graph shows the zeta potential of magnetic nanoparticles with various surface modification.
Zeta potential
CA or CMD negatively charged
APTES or betaine or PEI positively charged

TEM images of prepared magnetic nanoparticles with different modifications
Hydrodynamic diameters(Dh) of prepared magnetic nanoparticles with different modification


Transfection efficiencies of standard PEI transfection and magnetofection with various surface modifications.
Only 25% cells express GFP after standard PEI transfection
Magnetic field improves the efficiencies for all methods except fot betaine
Magnetofection better than Standard PEI for MP-PEI and MP-CMD
If magnetic nanoparticles are only added and the cell culture plate not placed on a magnetic plate, no enhancing effect is seen(black column)

Fluroscent photos of cell after magnetofection(using MP-PEI) or standard PEI transfection with same cell density
Standard PEI transfection needs 4hr s of cell incubation
Discussions
Results prove that surface-charged magentic nanoparticles enhance gene transfection efficiencies by applying a magnetic field
CA and CMD have great biocompatibility, have equal or even better transfection efficiency than PEI
They can be easily coated on magnetic particles since they have multicarboxyl groups. So they are more preferred than PEI
MP-betaine suppress transfection efficiency. Also transfection efficiency is low if MP-PEI is alone used

Conclusion

If the particle have sufficiently high surface potential, either positive or negative, they enhance gene delivery of PEI
Particles’ surface charge is correlated with the efficiency of magnetofection
Besides PEI, the carboxyl compounds CA and CMD are good coating agents
Refernces
F. Scherer,M. Anton, U. Schillinger, J. Henkel, C. Bergemann, A. Kruger,B. Gansbacher, and C. Plank, “Magnetofection: Enhancing and targeting gene delivery by magnetic force in vitro and in vivo,” Gene Ther.,vol.9,no. 2, pp. 102–109, 2002.
S. C.McBain, H. H. P. Yiu, A. E. Haj, and J. Dobson, “Polyethyleneimine
functionalized iron oxide nanoparticles as agents for DNA delivery and
transfection,” J. Mater. Chem., vol. 17, no. 24, pp. 2561–2565, 2007
X. Pan, J. Guan, J.W. Yoo, A. J. Epstein, L. J. Lee, and R. J. Lee, “Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery,” Int. J. Pharma., vol. 358, no. 1–2, pp. 263–270, 2008.


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