Transcript DUCURS post 27 - eScholarShare

®
PLURONIC
ASSESSING RESVERATROL
F127 MICELLE STABILITY IN THE PRESENCE OF
PLASMA PROTEINS USING FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
Scott Gleason, Mathew Kalapurayil, Deepa A. Rao (Mentor)
Drake University College of Pharmacy and Health Sciences
METHODOLOGY
Micelle stability assessed over 2 hours at 15 min
intervals using FRET (n = 3 ± SD)

DiO excitation λ = 484 nm
DiI excitation λ = 568 nm

Emission detected from 495 – 695 nm

Experimental conditions tested (Table 1)

Protein concentrations post dilution (Table 2)

FRET ratios were used to normalize data (equation)
4.010 5
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100 mg F127
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+/- 10 mg RES
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Final volume 2 ml
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Time (min)
Fig. 6 FRET ratios for F127 micelles with & without RES
Table 3: Average ± SD FRET ratios with & without RES (n = 3)
F127
F127
Experimental Conditions
0 min
120 min
0 min
Dilution (1:18)
0.95 ± 0.267 0.93 ± 0.14
0.43 ± 0.004
Dilution (1:18) + Albumin
0.74 ± 0.077 0.63 ± 0.036* 0.50 ± 0.018
Dilution (1:18) + αβ- globulins 0.76 ± 0.044 0.70 ± 0.035* 0.46 ± 0.013
Dilution (1:18) + γ globulins
0.82 ± 0.038 0.92 ± 0.007 0.45 ± 0.018
*Represents statistical significance as compared to F127 dilution micelles (p <0.05)
† represents statistical significance using F127 micelles as control
Further exploration of F
formulations in an in vi
needed to validate the
findings. Additionally dete
life of RES can be extended
this formulation.
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Dilution + -globulins
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Dilution +
--globulins
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Dilution + Albumin
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Fluorescence Intensity
I DiI
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Fluorescence Intensity
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1.010 6
I DiI
0.05mg DiO
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wavelength
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Fig. 5 Representative FRET signals for all formulations
Top Panel F127 micelles, bottom panel F127+RES micelles
Fluorescence Intensity
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Fluorescence Intensity
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Fluorescence Intensity
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Fluorescence Intensity
I DiO
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Dilution +
--globulins
Dilution + Albumin
Dilution
Weak FRET
0.05 mg DiI
0.6
CONCLUSIONS
Dilution
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Fluorescence data (Fig. 5)
Fig 3b
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0.8
RESULTS & DISCUSSION
Fluorescence Intensity
Strong FRET
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Fluorescence Intensity
Fluorescence Intensity
Fig 3a
1.0
One-way ANOVA with Dunnett’s post test using GraphPad Prism version 5.00 for Windows
Wavelength
4.010 5
1.0
I DiI
FRET Ratio 
I Dio  I DiI
Statistical Analysis

1.2
Solvent casting method used (Fig. 4)
Table 2: Protein conc (mg/ml) post-dilution
Albumin
40
α-β-globulin
14.8
γ-globulin
10

1.2
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Table 1: Experimental conditions tested
Dilution (1:18)
Dilution (1:18) + Albumin
Dilution (1:18) + α-β- globulins
Dilution (1:18) + γ-globulins
Evaluation of Micelle Stability

IDiI /(IDio +I DiI )
Micelle preparation:
Fluorescence Intensity
Resveratrol (RES) is a polyphenolic compound possessing chemopreventative &
chemotherapeutic potential1. However, the pharmaceutical effects of RES have not
been fully explored due to its short biologic half-life of ~6 minutes2.
Polymeric micelles are dynamic core-shell structures
(Fig.1) which self assemble at critical micelle
concentrations from amphiphilic polymers in solution.
Micelles can alter the pharmacokinetics of hydrophobic
compounds, such as RES, by harboring them in their
core. This can be advantageous in cancer treatment as
the nanoscopic size of micelles allows them to
selectively permeate leaky vasculature associated with
affected tissues4.
Fig. 1 Schematic of a micelle3
Pluronics® are triblock copolymers with both hydrophobic(poly(propylene oxide))
and hydrophilic (poly(ethylene oxide)) blocks. These polymers can form micelles
capable of increasing a drug’s residence time in the body. The Pluronic® used in
this study, F127, has shown to increase solubility and metabolic stability of various
compounds5.
The objective of the study is to assess timebased stability of F127 micelles with &
without RES in the presence of plasma
proteins using Fluorescence Resonance
Energy Transfer (FRET). FRET utilizes the
coinciding
excitation
and
emission
wavelengths of a two dye pair: DiO (the
donor) and DiI (the acceptor) (Fig. 2). When
DiO is excited in close proximity to DiI, two
emission peaks are detected, one for DiO & a
second for DiI. When the dye pair is loaded in
a micelle and the micelle remains stable, a strong FRET signal is observed (Fig. 3a).
In theory, the presence of this signal indicates a stable system with fluorophores
loaded into a micelle. Whereas, reduction of the signal (Fig. 3b) implies micelles in
solution have been destabilized6. Using this technique, it is possible to distinguish
whether or not Pluronic® F 127 micelles are able to serve as potential drug carriers
for RES.
F127+RES micelles
IDiI /(I Dio +I DiI )
INTRODUCTION
F127 micelles
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Legend
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6
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 F127 micelles without resveratrol
 stable to dilution & addition of γ-globulins
 de-stabilized in a statistically significant manner in the presenc
αβ- globulins
 F127 micelles with resveratrol
 de-stabilized predominantly by dilution in a statistically signific
 re-stabilized in the presence of plasma proteins but not compl
compared to F127 micelles without resveratrol
REFERENCES
1. Heynekamp JJ, Weber WM, Hunsaker LA, Gonzales AM, Orlando RA, Deck LM, Vander Jagt DL. Substituted trans-Stilbenes, Including Analogues of
Inhibit the Human Tumor Necrosis Factor Alpha-Induced Activation of Transcription Factor Nuclear Factor KappaB. J Med Chem. 2006, 49: 7182-71
2. Walle T, Hsieh F, DeLegge MH, Oatis JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metabolism a
1382.
3. http://atrp.gatech.edu/pt18-3/18-3_p3.html
4. Chowdhary RK, Chansarkar N, Sharif I, Hioka N, Dolphin D. Formulation of Benzoporphyrin Derivatives in Pluronics. Photochem Photobiol 2003,77:
5. Heynekamp JJ, Weber et.al. Substituted trans-Stilbenes, Including Analogues of the Natural Product Resveratrol, Inhibit the Human Tumor Necrosi
of Transcription Factor Nuclear Factor KappaB. J Med Chem 2006, 49:7182-7189.
6. Hongtao Chen et. al. Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer
6597-6601
ACKNOWLEDGEMENTS
Drake University for funding