1. Developing Thyroid Cancer Targeting Liposomes for Therapeutic and Diagnostic Agents Research Presentation 3-31-09 Presented by Fai

2. Research Objective Conjugation of the superagonist (TR1401) to the liposomes encapsulating drug in order to improve delivery of drug to the thyroid tumor cells → specific targeting and high doses. TR1401 bound liposome encapsulating drug TSH receptor Thyroid tumor cell

3. Methodology 1)Conjugate TR1401 to liposome with EDC 1.1) TR1401 -- liposome* (* = Alaxa488) 1.2) TR1401* -- liposome 2) Conjugate TR1401to His tag with SMCC → NTA liposome TR1401-- His tag -- NTA liposome

4. Methodology 1)Conjugate TR1401 to liposome with EDC 1.1) TR1401 -- liposome* (* = Alaxa488) 1.2) TR1401* -- liposome 2) Conjugate TR1401to His tag with SMCC → NTA liposome TR1401-- His tag -- NTA liposome

5. Flotation Assay Red = 50 K RPM Blue =35 K RPM Conclusions: 1)Protein is completely on the bottom 2)Liposomes are not completely on the surface 3)50K = 30K (in consideration of liposome)

6. Flotation Assay Red = 50 K RPM Blue =35 K RPM Conclusion: 1)After conjugating protein to liposome, the complex acts like liposome

7. Flotation Assay Red = 50 K RPM Blue =35 K RPM 8.77% 7.28% 15.83%14.93%

8. Flotation Assay Red = 50 K RPM Blue =35 K RPM 8.77% 7.28% 15.83%14.93% 7.06% 7.66%

9. Flotation Assay Red = 50 K RPM Blue =35 K RPM Conclusions: 1)Amount of protein >> liposome 2)Maximum capacity of liposome 3)Liposome 25 ul: TR1401 2.4 ug 8.77% 7.28% 15.83%14.93% 7.06% 7.66%

10. Flotation Assay Red = 50 K RPM Blue =35 K RPM

11. FACS Lipo*--TR Lipo*--BSA Liposome*

12. FACS JPO9 TR* 1 ul TR* 5 ul TR-lipo* (Max) TR-lipo* (Max-1) TR-lipo* (Max+1) TR-lipo* (Middle) TR-lipo* (Bottom)

13. FACS JPO9 BSA-lipo* (Max) BSA-lipo* (Max-1) BSA-lipo* (Max-1)(x2) BSA-lipo* (Max+1) BSA-lipo* (Middle) BSA-lipo* (Bottom) TR* 1 ul TR* 5 ul TR-lipo* (Max-1) TR-lipo* (Max+1)

14. Lipo*--TR1401, Max, 7hr

16. Lipo*-- BSA, Max, 7hr

18. Liposome*, Max+1, 46hr

20. Lipo*-- BSA, Max, 57 hr

22. Methodology 1)Conjugate TR1401 to liposome with EDC 1.1) TR1401 -- liposome* (* = Alaxa488) 1.2) TR1401* -- liposome 2) Conjugate TR1401to His tag with SMCC → NTA liposome TR1401-- His tag -- NTA liposome

23. SDS page after conjugation TR+SMCC TR His TR+SMCC TR His TR+SMCC TR His TR+SMCC+His TR+SMCC+His TR+SMCC+His Non-reducedReduced Reduced with boiling

24. Conjugation & Purification FPLC TR-His NTA column TR--His (Imidazole) Dialysis TR--His, free TR Conjugation TR--His (SMCC) TR--His, free TR, free His

25. Dialysis and NTA column

27. TR TR before TR after FT Wash Elute Elute Dialysis Dialysis #1 #1 #2 Non-reducedReduced TR TR before TR after FT Wash Elute Elute Dialysis Dialysis #1 #1 #2

28. Conjugation & Purification FPLC TR-His NTA column TR--His (Imidazole) Dialysis TR--His, free TR Conjugation TR--His (SMCC) TR--His, free TR, free His TR : His 969 ug : 2907 ug 3,876 ug

29. Dialysis and NTA column 1425 ug

30. Conjugation & Purification FPLC TR-His NTA column TR--His (Imidazole) Dialysis TR--His, free TR Conjugation TR--His (SMCC) TR--His, free TR, free His TR : His 969 ug : 2907 ug 1,425 ug 3,876 ug

31. Dialysis and NTA column 1425 ug 755 ug

32. Conjugation & Purification FPLC TR-His NTA column TR--His (Imidazole) Dialysis TR--His, free TR Conjugation TR--His (SMCC) TR--His, free TR, free His TR : His 969 ug : 2907 ug 1,425 ug 3,876 ug 755 ug

33. Dialysis and NTA column

34. FPLC – Elution #2 320 ug

35. Conjugation & Purification FPLC TR-His NTA column TR--His (Imidazole) Dialysis TR--His, free TR Conjugation TR--His (SMCC) TR--His, free TR, free His TR : His 969 ug : 2907 ug 1,425 ug 3,876 ug 755 ug 320 ug (415 ug ) (340 ug)

36. FPLC – Elution #2

37. A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 TR Elute#2 Reduced with boiling Reduced

38. FPLC – Elution #2

39. FACS (JPO9 & TR-His) JPO9 (non-washed) JPO9 (washed) TR* TR-His (A5) TR-His (A6) TR-His (A10) TR SA-9R-His Sup. Hybridoma TSHR