1. Presented By O.A.NAZEER HUSSAIN Roll No: 43 S7 AE&I Guided By: Mr. Suresh Babu V

2. Overview  Introduction  Experiments  Results  Discussion  Conclusion

3. Introduction  Gene Transfection Deliberately introducing nucleic acids into cells. “Infection by transformation”  Viral methods Carrier Viruses. ‘Virus Transduction’.

4.  Non-Viral methods Chemical based transfection Non chemical methods Paticle based methods  Merits  Demerits

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

7. Schematic diagram of forming magnetic gene complexes via electrostatic interaction

8. Transfection agents Highly charged macromolecules Formation of Magnetic Gene vectors Composition. Toxicity. PEI(Poly ethyleneimine)

9. 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.

10. 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

11. 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

12. Structural formulas for coating materials used in this method

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

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

15. Results A. Materials

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

18. 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

19. TEM images of prepared magnetic nanoparticles with different modifications

20. Hydrodynamic diameters(Dh) of prepared magnetic nanoparticles with different modification

22. 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)

23. 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

24. 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

25. 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

26. 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.

27. THANK YOU

28. QUESTIONS???????