A study developed a delivery system for the anticancer drug daunorubicin using a dendrimer-drug conjugate. A poly(amidoamine) dendrimer was synthesized and conjugated to daunorubicin through a folic acid linker. Characterization with NMR, mass spectrometry, and HPLC confirmed successful synthesis. In vitro cytotoxicity tests on KB cells showed the conjugate was more effective against folate receptor-positive cancer cells than free daunorubicin. Further studies are needed to evaluate anticancer activity in enzyme-containing systems and in vivo, as well as study the kinetics of daunorubicin release from the dendrimer.

1. Development of delivery system for anticancer agent using dendrimer-drug conjugate 원월(Yuan Yue) 우석대학교 제약공학과

2. Scope of the study Synthesis of dendrimer conjugate of daunorubicin For targeting to cancer cells For reducing the side effects of daunorubicin

3. Nano-sized carriers for anticancer agents Ex.: dendrimers, polymeric nanoparticles, liposomes, etc. Major advantage EPR effect V. P. TorchilinAAPS J 2007, 9(2) E128-147

4. Generation 5 poly(amidoamine) dendrimer- G5 PAMAM dendrimer

5. -NR2 0

6. Advantage of dendrimer Nanometer-size Low polydispersity Nontoxicity of PAMAM dendrimers to biological systems Biocompatible Amphiphilic molecules

7. Designed Dendrimer-drug conjugate daunorubicin folate

8. Selected anti-cancer agent Daunorubicin

9. Targeting molecule Folate (folic acid)

10. Synthesis

12. Potentiometric Titration of Free amino groups Dendrimer was dissolved in NaCl solution Neutralization with excess HCl Back titration of remaining HCl with NaOH Number of primary amino groups in G5 dendrimer Theoretical calculation from the structure = 128 Measured = 99

13. 1H-NMR assign of G5-Ac Acetyl group=e e :면적:0.31 d :면적:0.62, 388 Acetyl group=65 195 1H NMR spectra of acetylated dendrimer(after dialysis)

14. h i h i g

15. 1H-NMR assign of G5-Ac-FA 195 1H NMR spectra of G5-Ac-FA(after dialysis)

16. 1H-NMR assign of G5-Ac-FA-OH-DRC 1H NMR spectra of G5-Ac-FA-OH-DRC(after dialysis)

17. Dendrimer중 daunorubicin농도 측정 DRC dendrimer conjugate (1mg) was dissolved in distilled water (200 microlitre) 0.2 ml of the solution was mixed with 0.8 ml of 2.5 M HCl and 1 ml of methanol The mixture was incubated at 50℃ for 1.5 h.

18. Dendrimer중 daunorubicin농도 측정 Standards Dissolve 5mg of daunorubicin in 5mL methanol 1 mL standard + 0.8mL of 2.5mL HCL+0.2 mL distilled water The mixture was incubated at 50℃ for 1.5 h

19. HPLC 조건 Column: Cadenza CD-C18 (ImtaktCorp , Japan, ODS, 0.003mm, pore size:12nm, 150 x 4.6mm) 0.01 M KH2PO4: acetonitrile: acetic acid 45: 55: 0.27 (v/v/v) 0.5 mL/min 30℃ 490 nm

20. Result of HPLC daunorubicin 0.5mg/mL Dendrimer conjugate 9 daunorubicins for one dendrimer

21. MALDI TOF mass spectrum 27009.36 29921.54 32851.05 G5-Ac G5 35148.14 33289.75 G5-Ac-FA G5-Ac-FA-OH-DRC G5-Ac-FA-OH

23. Cytotoxicity assay(MTT) KB cell lines (Folate 부족한 환경중에서 배양하면 folate receptor를 overexpression) Cell was seeded in a 96 well plate 1×104 cells/ml complete medium or folic acid-deficient medium G5-Ac-FA-OH, G5-Ac-FA-OH-DRC, or free daunorubicin 0.1,0.2,1,2,5,10,20uM 24 h, 48h, 72h and 96h 180 microlitre fresh media 20 microlitre of MTT dye (5 mg/ml) 3h of incubation at 37 ℃. Read OD at 560nm and subtract background at 670nm

24. Result of cytotoxicity assay %survival concentration Folate receptor+,24h Folate receptor+,48h Folae receptor+,72h Folate receptor+,96h

25. Result of cytotoxicity assay Folate receptor-, 24h Folate receptor-, 48h Folate receptor-,72h Folate receptor-,96h

26. Conclusion Folate-modified dendrimer conjugate of daunorubicin was successfully synthesized as proposed The dendrimer conjugates was also well characterized by NMR, MALDI/MS, potentiometry and HPLC. In the future In vitro anticancer activity should be evaluated using enzyme-containing system and also in vivo. A kinetic study for daunorubicin release from dendrimer need to be performed.

27. THANK YOU 감사합니다