Yuanyue
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Development of delivery system for anticancer agent using dendrimer-drug conjugate 원원 (Yuan Yue) 원원원원원 원원원원원
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Development of delivery system for anticancer agent using dendrimer-drug conjugate
원월 (Yuan Yue)우석대학교 제약공학과
Scope of the study Synthesis of dendrimer conjugate of
daunorubicinFor targeting to cancer cells For reducing the side effects of daunorubicin
Nano-sized carriers for anticancer agentsEx.: dendrimers, polymeric nanoparticles,
liposomes, etc. Major advantage
EPR effect
V. P. Torchilin AAPS J 2007, 9(2) E128-147
Generation 5 poly(amidoamine) dendrimer
- G5 PAMAM dendrimer
-NR2
0
Advantage of dendrimerNanometer-sizeLow polydispersityNontoxicity of PAMAM dendrimers to
biological systemsBiocompatibleAmphiphilic molecules
daunorubicin
folate
Designed Dendrimer-drug conjugate
Selected anti-cancer agentDaunorubicin
Targeting moleculeFolate (folic acid)
Synthesis
Potentiometric Titration of Free amino groups Dendrimer was dissolved in NaCl solutionNeutralization with excess HClBack titration of remaining HCl with NaOH
Number of primary amino groups in G5 dendrimer
Theoretical calculation from the structure = 128
Measured = 99
1H-NMR assign of G5-Ac
1H NMR spectra of acetylated dendrimer(after dialysis)
e : 면적 :0.31 d : 면적 :0.62, 388
Acetyl group=e
Acetyl group=65
195
h
h i
i
g
Proton Integral ratio
g 1
i 2
h 2
1H NMR spectra of G5-Ac-FA(after dialysis)
195
1H-NMR assign of G5-Ac-FA
1H NMR spectra of G5-Ac-FA-OH-DRC(after dialysis)
1H-NMR assign of G5-Ac-FA-OH-DRC
Dendrimer 중 daunorubicin 농도 측정DRC 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 methanolThe mixture was incubated at 50℃ for 1.5 h.
Dendrimer 중 daunorubicin 농도 측정StandardsDissolve 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
0.5mg/mL
0.2mg/mL
0.1mg/mL
0.05mg/mL
0.02mg/mL
0.01mg/mL
HPLC 조건Column: 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/min30℃ 490 nm
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mAU
-100
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
mAU
-100
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mAU
-100
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
mAU
-100
-75
-50
-25
0
25
50
75
100
125
150
175
200
225
250
daunorubicin0.5mg/mL
Dendrimer conjugate
Result of HPLC
9 daunorubicins for one dendrimer
MALDI TOF mass spectrum27009.36
G5 G5-Ac
G5-Ac-FA
G5-Ac-FA-OH G5-Ac-FA-OH-DRC
29921.54
32851.05
33289.75
35148.14
NMR calculatio
n
Titration
HPLC(after
cleavage)
Mass
Amino (G5) - 99 - -
Acetyl 65 74 - 69
Folate 3 - - 7
Daunorubicin - - 9 3
Compounds Mass
G5 27009.36
G5-Ac 29921.54
G5-Ac-FA 32851.05
G5-Ac-FA-OH 33289.75
G5-Ac-FA-OH-D 35148.14
Cytotoxicity assay(MTT)KB cell lines (Folate 부족한 환경중에서 배양하면 folate
receptor 를 overexpression)Cell was seeded in a 96 well plate1×104 cells/mlcomplete medium or folic acid-deficient mediumG5-Ac-FA-OH, G5-Ac-FA-OH-DRC, or free daunorubicin 0.1,0.2,1,2,5,10,20uM24 h, 48h, 72h and 96h180 microlitre fresh media20 microlitre of MTT dye (5 mg/ml)3h of incubation at 37 ℃.Read OD at 560nm and subtract background at 670nm
Result of cytotoxicity assay
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
140
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
Folate receptor+,24h
Folate receptor+,48h
Folae receptor+,72h
Folate receptor+,96h
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
daunorubicinG5-Ac-FA-OHG5-Ac-FA-OH-D
%su
rvival
concentration
Result of cytotoxicity assay
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
Folate receptor-, 24h0 0.1 0.2 1 2 5 10 20
0
20
40
60
80
100
120
Folate receptor-, 48h
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
Folate receptor-,72h
0 0.1 0.2 1 2 5 10 200
20
40
60
80
100
120
daunorubicinG5-Ac-FA-OHG5-Ac-FA-OH-D
Folate receptor-,96h
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 futureIn 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.
THANK YOU감사합니다
