Ecole Polytechnique Fédéral Lausanne, EPFL Powder Technology Laboratory

1. Combination of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) and Dynamic Magnetic Fields for Enhanced Transfection Ecole Polytechnique Fédéral Lausanne, EPFL Powder Technology Laboratory H. Hofmann, B. Steitz, A. Fink-Petri, University of Zürich Institute of Veterinary Biochemistry and Molecular Biology, S. Kamau; P.O. Hassa; M. Hottiger MatSearch M. Hofmann University of Zurich Vetsuisse Faculty Zurich, Musculoskeletal Research Unit, Equine Hospital, K. Schulze, L. Galuppo, B. von Rechenberg

2. Aim of study • Explore the use of dynamic magnetic fields to enhance non-viral transfection with superparamagnetic iron oxide (SPION) beads • Uptake of SPION as non-viral vectors into cells (293T, COS and HeLa cell lines& synoviocytes) • Obtain preliminary results with intraarticular application of non-viral vector in vivo • Take advantage of • the smaller sized SPION-beads (70 nm) for intraarticular application • Lower cytotoxicity of PVA coated compared to PEI coated SPION

3. Important steps in transfection 1, 2 extracellular 3, 4 H+ 4 Early Endosome pH 6.5 – 7.5 Late Endosome pH ~ 5.0 Endocytosis intracellular GFP Endosomal Escape Nucleus Gene Products Transcription Nuclear Targeting

4. 1 2 dB/dz Static field gradient Y Y Y Y Cell membrane Force per particle acting on the membrane : Static: 1 10-13 – 1 10-14 N Dynamic: 1 10-15 – 1 10-16 N 3 Y Y 4 Dynamic magnetic Field gradient

5. Materials & methods Transfection with SPIONs SPIONS:PVA-SPIONs, PEI-SPIONs (50 – 70 nm) ; Control: Larger SPIONs (200-250 nm) Cells: HeLa (human cervix carcinoma cells),293T, Cos7 (fibroblast , America Type Culture Collection (ATCC)), synoviocytes, Plasmid and PCR-Products: • pEGFP-C2 plasmid (Clontech) • PCR products from pEGFP-C2 plasmid (Clontech) were amplified using primers designed to include the 5’human cytomegalovirus (CMV) immediate early promoter and a 3’SV40 early mRNA polyadenylation signal. The PCR products (1.6 kilobase pairs) were purified using PCR purification Kit (Qiagen). Experimental procedure:  SPIONs complexed with DNA for 30 min at room temperature  Added to cells, exposure to static magnet for 5 or 20 min, 4 hrs incubation Medium replacement incubation for minimum 24 hrs  FACS and microscopy analysis - 24 hrs (48, 72 and 96 hrs) Transfection controls: Lipofectamine, PEI and Ca2PO4 Detail see : Nucleic Acids Research2006 , accepted,

6. Magnetic flux density distribution (component Mz and Mx) z 30 y Mz M’z x Mag flux density (mT) M’x Mx 0 0 3 6 Distance (cm) Well plate Dynamic magnetic field generator Dynamic magnetic field (Equipment :Matsearch, Pully CH and Stetter Elektronik, D) mZ X

7. 20nm PEI coated SPIONs R = PEI:Fe mass ratios

8. M12: amino-PVA Changes of the Zeta-potential with DNA adsorption

9. Uptake of amino-PVA-Cy3.5 in synoviocytes 300 24 h 48 h 72 h 24 hrs 82.1% 48 hrs 82.9% 72 hrs 85.8% 225 % of positive cells Cell count 150 75 0 10 0 10 1 10 2 10 3 10 4 Fluorescence intensity (Cy 3.5) Time (hours)  Efficient uptake of PVA-coated SPIONs by synoviocytes

10. 293T cells 293T cells Synovial cells 3.8% 96.2% 3.8% 96.2% Counts GFP GFP GFP Expression of GFP in cells transfected with PVA- and PEI-coated SPIONs PEI-SPIONs (24 hrs) PVA-SPIONs (96 hrs) • Efficient gene delivery by the SPIONs in different cells lines • PEI-SPIONs – high gene delivery after 24 hrs • PVA-SPIONs – clear gene expression after 72 hrs

11. 293T cells were transfected with either DNA/polyMAG (A) or DNA/SPIONs (B),

12. Different cells were transfected with DNA/SPIONs (B), static magnetic field 5 or 20 min.

13. Enhancement of transfection rates by the application of dynamic magnetic fields 293T cells SPIONS + magnet • Application of dynamic magnetic field for 5 min significantly increased the transfection efficiency over that with the conventional transfection methods

14. Static and dynamic magnetic field bioweb.wku.edu/.../stokes/131f98chap8.html.

15. Behaviour of SPIONs in vivo after intraarticular application PEI-SPIONs 200-250 nm PEI-SPIONs 50 nm PVA-SPIONs 50 nm PEI-SPIONs 200-250 nm • Very good biocompatibility with PVA-SPIONs • Swelling, reddening, oedema with PEI-SPIONs especially with the larger particles • Immunohistochemistry for GFP identified positive samples in the inguinal lymphnode but was inconclusive for synovial membrane samples.

16. Summary •  The uptake PVA-coated SPIONs is very efficient and they have better biocompatibility than PEI-SPIONs. • Significantly higher transfection rates were achieved with PEI-SPIONs after 5 or 20 min exposure to magnet and additionally 5 min to dynamic magnetic fields (than conventional transfection methods) • PEI-coated SPIONs are very efficient for non-viral gene delivery, resulting in high transfection rates especially with synoviocytes (96.2%) • Smaller PEI-SPIONs are more biocompatible than larger particles • In-vivo transfection with small PEI-SPION and dynamic magnetic fields seems to be possible.

17. Acknowledgement • Swiss National Science Foundation • VETSuisse Research Fund

18. Materials & methods Transfection with SPIONs SPIONS:PVA-SPIONs, PEI-SPIONs (50 – 70 nm) ; Control: Larger SPIONs (200-250 nm) Cells: 293T, HeLa, Cos7, synoviocytes, Plasmid and PCR-Products: • The pEGFP-C2 plasmid (Clontech) was propagated in Escherichia coli and purified using an Endotoxin-free Maxiprep plasmid Kit (Qiagen). • PCR products from pEGFP-C2 plasmid (Clontech) were amplified using primers designed to include the 5’human cytomegalovirus (CMV) immediate early promoter and a 3’SV40 early mRNA polyadenylation signal, (forward primer: 5’-CCG TAT TAC CGC CAT GCA T-3’; reverse primer: 5’-GCC GAT TTC GGC CTA TTG GT-3’). The PCR products (1.6 kilobase pairs) were purified using PCR purification Kit (Qiagen). Experimental procedure:  SPIONs complexed with DNA for 30 min at room temperature  Added to cells, exposure to static magnet for 5 or 20 min, 4 hrs incubation Medium replacement incubation for minimum 24 hrs  FACS and microscopy analysis - 24 hrs (48, 72 and 96 hrs) Optimization:  Aim: Efficient gene delivery (high proportion of green GFP expressing cells) Minimum toxicity (low proportion of red PI stained non-viable cells Transfection controls: Lipofectamine, PEI and Ca2PO4

19. Plasmidic DNA (I)

20. PCR product promotor gene poly A side

21. Transfection with Plasmidic DNA vs. PCR product Plasmid Advantage • Large amounts are cheap • Stable • Large choice of transfection methods, promoters • Protocols optimized Disadvantage: • In vivo transfection regarded as non-safe PCR product Advantage: • In-vivo safe; without viral parts • No insertion into plasmid needed Disadvantage: • Expensive • Fast degredation Expression of unmodified PCR products never shown