NanoCell Therapy Evaluation for Liver Cancer Treatment: In Vitro & In Vivo Studies

1. Evaluation of the novel nanocell(invitro/in vivo) Dr.Ramraj Panthee Chulabhorn Research Institute

2. Outline • Introduction • In vitro studies • In vivo studies • Summary

3. Introduction • Novel nanocell • Confocal microscope • In vitro • Cell culture studies • Co- culture studies • In vivo • Nude mice tumor studies

4. 200 nm 100 nm The novel nanocellstructure FTY720 (F) Doxorubisin+5FU(D+5F) PLGAconjugation(Both) Nanocell

5. 200 nm 100 nm The novel nanocell Function 2 4 1 Nc into tumor cell Tumor cell 3 Nanocell BVs shutdown

6. Confocal microscopeStructure and function • Laser ray • 3 dimensional picture • Computerized device

7. In vitro studiescontrols v • 5FU • FTY720 Doxo +5FU Dox Doxorubicin Dox+FTY Vehicle is phosphate buffer solution(PBS)

8. Cell culture studies Drugs action on liver cancer cells Survival Cells + Drugs Viable rate Liver cancer cells culturemedium + Calf serum 5% 24 hours

9. Cont.Cell culture studies • Plated for 24 hrs, • Doxorubicin +5FU Conjugated mixed for 48hrs. • Evaluation: Trypan blue exclusion method. • Ec 50 calculation curve fitting Cell culture Trypan blue – cell viability Ec50 - 50% test cells effective by drugs

10. Tumor-endothelium co-culture studies • Liver cancer cells with GFP Expre.. • Endothelial cells • Matrigelmatrix • Paraformaldehyde(4%) • Incubation- propidium Iodide • Exposed to different treatment Liver cancer cell Endothelial cell

11. Microscopic studies of cell Control 30 h FTY720 DOXO+5FU Control 12 h Nanocell 12h Nanocell 30h Nanocell in 12h action ,BV Nanocell in 30h tumor Modified picture from: Nature vol. 436 July 2005 S.sengupta et.al..

12. Microscopic studies (cont.) • Yellow stained – Liver cancer cells • Red stained - endothelial cells • Doxorubicin +5FU –Tumor reduced • FTY720 – Vascular shutdown • Nanocell in 12hours little effect • Nanocell in 30hours complete tumor reduced

13. Stereological quantificationof co-culture • Vascular component: VEHICLE 12hr-no change In 30hrs. Vascular changed 5Fluouracil +Doxo Vehicle FTY720 Vehicle Nanocell ------ 12 h--- -------30 h--------- Model picture from Nature vol.436 2005

14. Stereological quantification of co-culture • Tumor component: Vehicle 12hrs no change Doxo+5FU 30hrs FTY720 Doxo+5F Nanocell Vehicle vehicle Tumor component Model picture from Naturevol.436 2005

15. Physicochemical release kinetic of drugs • Doxorubicin + 5FU– slow release • FTY720 —rapid release FTY720 Doxorubicin+5FU Model picture from Nature vol. 4362005

16. In vivo nanocell studiesNude mouse • High sensitive to antigen • Well manifestation of tumor Nude mouse

17. In vivo nanocells (cont.) in vivo tumor studies • GFP expression • Liver cancer cells • Implanted male nude mice • Tumor vol. 50mm3 • Treatment started with 100µ l iv • FTY720 and • doxorbicin+5FU • Animals were killed in periods • Necropsies tumor - histopathology

18. In vivo NanocellNude mouse tumor Tumor 50mm3 in size Liver cancer cells injected into sub -cutaneous tissue

19. Tumor studies Size of tumor with different controls D+F V F 5F D+5F+F N Modified picture from Nature vol.436,july 2005 S. sengupta et.al.

20. Effects of treatment • Survival • Toxicity Traditional chemotherapy 30days survive Nanocell combined 65 days survive Less toxic WBC count show - normal Without any treatment20days survive Source-Nature:436:2005 S.Sengupta et.al

21. Tissue distribution studies • NC + Fluorescence dye • Injected – mice • Mice were killed – 5, 10, 24 hrs. after injection • Organs collection –liver, lungs, spleen, tumor and serum after necropsed • Wt. – organs and fluorescence extracted Purpose - Distribution of drugs into the tissues Result – Nanocells were detected within 5 hours

22. SummaryA novel nanocell therapy Slow release Target specific Less toxic Cytotoxic Lifespan