1. Scott Gleason, Mathew Kalapurayil, Deepa A. Rao (Mentor) Drake University College of Pharmacy and Health Sciences INTRODUCTION Resveratrol (RES) is a polyphenolic compound possessing chemopreventative & chemotherapeutic potential 1. However, the pharmaceutical effects of RES have not been fully explored due to its short biologic half-life of ~6 minutes 2. Fig. 1 Schematic of a micelle 3 Polymeric micelles are dynamic core-shell structures (Fig.1) which self assemble at critical micelle concentrations from amphiphilic polymers in solution. M icelles 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 tissues 4. 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 compounds 5. The objective of the study is to assess time- based 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 destabilized 6. 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. METHODOLOGY Micelle preparation:  Solvent casting method used (Fig. 4)  0.05 mg DiI  0.05mg DiO  100 mg F127  +/- 10 mg RES  Final volume 2 ml Evaluation of Micelle Stability  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) Statistical Analysis  One-way ANOVA with Dunnett’s post test using GraphPad Prism version 5.00 for Windows RESULTS & DISCUSSION REFERENCES 1.Heynekamp JJ, Weber WM, Hunsaker LA, Gonzales AM, Orlando RA, Deck LM, Vander Jagt DL. Substituted trans-Stilbenes, Including Analogues of the Natural Product Resveratrol, Inhibit the Human Tumor Necrosis Factor Alpha-Induced Activation of Transcription Factor Nuclear Factor KappaB. J Med Chem. 2006, 49: 7182-7189. 2.Walle T, Hsieh F, DeLegge MH, Oatis JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metabolism and Disposition. 2004, (12): 1377- 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 2003,77:299-303. 5.Heynekamp JJ, Weber et.al. Substituted trans-Stilbenes, Including Analogues of the Natural Product Resveratrol, Inhibit the Human Tumor Necrosis Factor Alpha-Induced Activation 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 imaging. PNAS 2008, 105(18): 6597-6601 ACKNOWLEDGEMENTS Drake University for funding CONCLUSIONS  F127 micelles without resveratrol  stable to dilution & addition of γ-globulins  de-stabilized in a statistically significant manner in the presence of albumin & αβ- globulins  F127 micelles with resveratrol  de-stabilized predominantly by dilution in a statistically significant manner  re-stabilized in the presence of plasma proteins but not completely as compared to F127 micelles without resveratrol Further exploration of F127 +/- RES formulations in an in vivo model is needed to validate these in vitro findings. Additionally determine if half- life of RES can be extended in vivo using this formulation. ASSESSING RESVERATROL PLURONIC ® F127 MICELLE STABILITY IN THE PRESENCE OF PLASMA PROTEINS USING FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Table 1: Experimental conditions tested Dilution (1:18) Dilution (1:18) + Albumin Dilution (1:18) + α-β- globulins Dilution (1:18) + γ-globulins Table 2: Protein conc (mg/ml) post-dilution Albumin40 α-β-globulin14.8 γ-globulin10 Table 3: Average ± SD FRET ratios with & without RES (n = 3) Experimental Conditions F127F127 + RES 0 min120 min0 min120 min Dilution (1:18)0.95 ± 0.2670.93 ± 0.140.43 ± 0.0040.42 ± 0.004† Dilution (1:18) + Albumin0.74 ± 0.0770.63 ± 0.036*0.50 ± 0.0180.50 ± 0.032 Dilution (1:18) + αβ- globulins0.76 ± 0.0440.70 ± 0.035*0.46 ± 0.0130.44 ± 0,014 Dilution (1:18) + γ globulins0.82 ± 0.0380.92 ± 0.0070.45 ± 0.0180.43 ± 0.015 Fluorescence data (Fig. 5) Fig. 5 Representative FRET signals for all formulations Top Panel F127 micelles, bottom panel F127+RES micelles Fig. 6 FRET ratios for F127 micelles with & without RES *Represents statistical significance as compared to F127 dilution micelles (p <0.05) † represents statistical significance using F127 micelles as control