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® 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) 4.010 5 2.010 5 0 500 550 600 650 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 6.010 5 4.010 5 2.010 5 0 700 500 550 I DiI 1.010 5 0 500 550 600 650 700 2.010 5 I DiO 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 6.010 5 5 2.010 5 500 550 600 wavelength 500 550 600 650 100 mg F127 0.0 0.0 +/- 10 mg RES Final volume 2 ml 0.4 0 15 30 45 60 75 90 Time (min) 650 700 105 120 0.6 0.4 0 15 30 45 60 75 90 105 120 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. 650 8.010 5 6.010 5 4.010 5 2.010 5 500 5 2.010 5 0 600 wavelength 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 550 600 650 1.010 5 5.010 4 0 500 700 550 650 700 8.010 5 6.010 5 4.010 5 2.010 5 0 600 700 2.010 6 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 550 650 Dilution + -globulins 1.010 6 500 600 wavelength Dilution + --globulins 6.010 5 550 5 wavelength 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 500 1.510 0 700 8.010 5 4.010 F127+ -globulins 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min Dilution + Albumin 0 0 0.2 6 Wavelength 8.010 5 4.010 600 1.010 Fluorescence Intensity I DiO 0.2 Fluorescence Intensity 6.010 5 1.010 6 I DiI 0.05mg DiO 650 700 wavelength 700 Fig. 5 Representative FRET signals for all formulations Top Panel F127 micelles, bottom panel F127+RES micelles Fluorescence Intensity 8.010 5 Fluorescence Intensity 0 min 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min Fluorescence Intensity 8.010 Fluorescence Intensity 2.010 5 4.010 5 Dilution + --globulins Dilution + Albumin Dilution Weak FRET 0.05 mg DiI 0.6 CONCLUSIONS Dilution 5 0.8 Fluorescence data (Fig. 5) Fluorescence Intensity Strong FRET 3.010 5 0.8 RESULTS & DISCUSSION Fig 3b 6.010 5 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 4.010 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 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 /(I Dio +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 0 min 15 min 30 min 45 min 60 min 90 min 105 min 120 min Legend 1.510 6 1.010 6 5.010 5 0 500 550 600 wavelength 650 700 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