Transcript DUCURS poster 21 - eScholarShare

IN VITRO CHARACTERIZATION OF RESVERATROL-LOADED
®
PLURONIC MICELLES
Michelle M. Stammet, Deepa A. Rao (Mentor)
Drake University College of Pharmacy and Health Sciences
Stability of RES in Micelles Prepared by Solvent Casting Method
Resveratrol (RES), a polyphenol, has shown various biological activities but its use is
hindered due to its hydrophobicity and short biological half-life. Biological studies require
mega or frequent doses of RES to be effective. Pluronics® F88, F98, F108 and F127 form
polymeric micelles & can be used to deliver RES. Micelles were prepared by equilibrium (E)
or solvent casting (SC) with either 10 % or 20 % w/w RES loading. Micelles prepared by E
of 20% w/w RES showed a correlation with the critical micelle concentrations of the
Pluronic® used. Micelles prepared by SC with 10 % w/w RES were stable for 48 hr and
approached equilibrium solubility over 12 days. Micelles prepared by SC with 20 % w/w
RES were able to fully solubilize RES but approached equilibrium solubility rapidly.
Cytotoxicity (IC50) studies in human ovarian cancer cell line, SK-OV3 using RES and all
Pluronic® micellar formulations with 10 % w/w RES were done. RES alone had IC50 values
of 91.99 ± 17.24 μM. RES Pluronic® formulations F88, F98, F108 & F127 had IC50 values of
49.69 ± 9.28 μM, 84.57 ± 7.48 μM, 53.12 ± 11.45 μM and 79.15 ± 21.84 μM respectively,
indicating slightly greater potency than RES alone. In conclusion, RES Pluronic®
formulations prepared by SC form metastable systems of different stabilities based on initial
loading. Cytotoxicity studies show all RES formulations are at least as potent as RES alone.
Preparation of RES-Loaded Micelles
Resveratrol (RES, fig. 1) is a polyphenolic, antioxidative
compound found in grapes, wine and peanuts. It has potent
chemopreventative properties in cardiovascular & cancer
diseases1. The full potential of RES cannot be evaluated due to
its rapid metabolism & short half-life (~ 6 min). To overcome
these issues RES can be delivered using polymeric micelles.
Fig. 1 - Resveratrol (RES)
Res Conc (mg/ml)
Res Conc (mg/ml)
RES-F98
15
10
5
0
5
10
15
Time (days)
RES-F108
15
10
5
0
0
15
10
5
0
0
5
10
15
10
5
0
0
Time (days)
5
10
15
0
Time (days)
Fig. 5 RES - Pluronic® Formulations
TABLE 2 - RES-Pluronic® Formulations
Pluronic® 100 mg F88 F98 F108 F127 F88 F98 F108 F127
RES (mg)
10 10 10 10 20 20 20 20
Assessment of RES–Loaded Micelle Stability
Reverse Phase High Pressure Liquid Chromatography (HPLC) using a C18 column with UV
detection at 306nm. Micelles were stored at 23 ± 2 oC in the dark at for 14 days. N=4 ± SD.
INTRODUCTION
RES-F88
15
Res Conc (mg/ml)
EXPERIMENTAL
Res Conc (mg/ml)
ABSTRACT
Stability of 10 % RES SC formulations:
TABLE 3 – RES Conc. (mg/ml) in Pl
at 14 Days (n= 4 ± SD
F127 ~ F98 > F108 ~F88
All formulations were metastable for Pluronic®
E
10 % RES SC
2 days
F88
2.35 ± 0.051 3.67 ± 0.259
Stability of 20 % RES SC formulations:
F98
3.85
±
0.131
5.64
±
0.756
F127>F88~F98~F108
F108
3.29 ± 0.057 4.27 ± 1.144
All formulations approached equilibrium
solubility
F127
5.39 ± 0.185 5.25 ± 0.679
Cytotoxicity Data For 10 % RES Formulations Prepared By SC
OH
In Vitro Cytotoxicity Studies in SK–OV3
SK-OV3 cells were seeded at a density of 3000 cells/well in 96 well plates & tested with
formulations over 72 hr. Cell viability was assayed using Cell Titer Blue. IC50 concentration &
Hill slope were obtained using Microsoft Excel© Solver Add-in.
HO
OH
Statistical Analysis: One-Way ANOVA with Dunnett’s Post-test (p<0.05).
The goal of this work is to determine if the loading and stability of RES in
various Pluronic® micelles is same/ different using solvent casting (SC) &
equilibrium (E) methods and to evaluate RES potency in these formulations in a
cancer cell line, SK-OV3.
RESULTS
Stability of RES in Micelles Prepared by Equilibrium Method
CONCLUSIONS
10
Res Conc (mg/ml)
Polymeric micelles (fig. 2) are nanoscopic drug
carriers with a core/shell structure that selfassemble in an aqueous environment above a
threshold concentration known as the critical
micelle concentration (CMC)2. The core
provides a suitable encapsulation environment
for a hydrophobic drug. Pluronics® are triblock
copolymers with hydrophobic polypropylene
TABLE 1 – Pluronics®
blocks
(PPO)
flanked
by
hydrophilic Pluronic® PPO MW PEO MW CMC (M)
polyoxyethylene blocks (PEO) blocks. They are
F88
2400
9000
2.5 X10-4
FDA approved excipients for internal use2.
F98
2700
10300
7.7 X10-5
Pluronics® F88, F98, F108 and F127 were chosen
F108
3000
11600
2.2 X10-5
based on their increasing PPO:PEO block ratios
F127
3600
9000
2.8 X10-6
(Table 1). Many different methods are available
to load drugs into micelles. These include simple equilibrium, dialysis, O/W emulsion,
solvent casting and freeze-drying.
TABLE 4 – Cytotoxicity Data for Formulations Tested
F98 & F127
Formulation Conc. Tested (µM) IC50 (µM ±SD) Hill Slope
were as potent
Resveratrol
0.01 – 438
91.99 ± 17.24 0.81 ± 0.16 F88 & F108
F88
0.01 – 219
49.69 ± 9.28* 0.68 ± 0.16
potent than
F98
0.01 – 219
84.57 ± 7.48 0.81 ± 0.11
possibly due
F108
0.01 – 219
53.12 ± 11.45* 0.89 ± 0.15
concentration
in solution
F127
0.01 – 219
79.15 ± 21.83 0.84 ± 0.27
* Denotes statistical significance compared to control (p< 0.05)
F88-RES
F98-RES
F108-RES
F127-RES
8
6
4
2
n=4  SD
0
0
5
10
15
Time (days)
Fig. 4 RES Equilibrium Conc in Pluronics®
All Pluronics® were able to solublize RES according to their respective CMCs
As the CMC decreases RES solubility increases
Order of CMC : F127 < F98 < F108 < F88
RES solubility :F127 > F98 > F108 > F88
 Micelles prepared by equilibrium method solubilized resveratrol based o
 Micelles prepared by solvent casting at 10 % resveratrol loading exhibi
phases with all formulations before approaching equilibrium solubility
 Micelles prepared by solvent casting at 20 % resveratrol loadin
equilibrium solubility faster that the 10 % formulations
 Cytotoxicity studies with Pluronic® 10 % resveratrol formulations exh
similar/greater than resveratrol alone
REFERENCES
1Jannin
B, et. al. 2004. Biochemical Pharmacology 2004, 68, 1113.
2Croy, S. University of Madison-Wisconsin, 2005.
ACKNOWLEDGEMENTS
Drake University for Funding
Drake Undergraduate Science Collaborative Institute for Fellowship Support for M. Stammet
University of Wisconsin-Madison for SK-OV3 Cell Line