Scott Gleason, Mathew Kalapurayil, Deepa A. Rao (Mentor)Drake University College of Pharmacy and Health Sciences

INTRODUCTIONResveratrol (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.

Fig. 1 Schematic of a micelle3

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. 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 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 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.

METHODOLOGYMicelle 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) DiI

Dio DiI

IFRET Ratio

I I

Statistical Analysis One-way ANOVA with Dunnett’s post test using GraphPad Prism version 5.00 for Windows

RESULTS & DISCUSSION

500 550 600 650 7000

1.0105

2.0105

3.0105

4.0105

IDiO IDiI

Strong FRET

Flu

ore

scen

ce In

ten

sity

Fig 3a

REFERENCES1. 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.html4. 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

ACKNOWLEDGEMENTSDrake 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 testedDilution (1:18)

Dilution (1:18) + AlbuminDilution (1:18) + α-β- globulins

Dilution (1:18) + γ-globulins

Table 2: Protein conc (mg/ml) post-dilutionAlbumin 40

α-β-globulin 14.8γ-globulin 10

Table 3: Average ± SD FRET ratios with & without RES (n = 3)

Experimental ConditionsF127 F127 + RES

0 min 120 min 0 min 120 minDilution (1:18) 0.95 ± 0.267 0.93 ± 0.14 0.43 ± 0.004 0.42 ± 0.004†Dilution (1:18) + Albumin 0.74 ± 0.077 0.63 ± 0.036* 0.50 ± 0.018 0.50 ± 0.032Dilution (1:18) + αβ- globulins 0.76 ± 0.044 0.70 ± 0.035* 0.46 ± 0.013 0.44 ± 0,014 Dilution (1:18) + γ globulins 0.82 ± 0.038 0.92 ± 0.007 0.45 ± 0.018 0.43 ± 0.015

500 550 600 650 7000

2.0105

4.0105

6.0105

IDiO

IDiI

Weak FRET

Flu

ore

scen

ce In

ten

sity

Fig 3b

Dilution

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

0 min15 min30 min45 min60 min75 min90 min105 min120 min

Wavelength

Fluo

resc

ence

Inte

nsity

Dilution + Albumin

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

0 min15 min30 min45 min60 min75 min90 min105 min120 min

Wavelength

Fluo

resc

ence

Inte

nsity

Dilution +--globulins

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

1.0106

0 min

15 min

30 min

45 min

60 min

75 min

90 min

105 min

120 min

wavelength

Fluo

resc

ence

Inte

nsity

F127+ -globulins

500 550 600 650 7000

5.0104

1.0105

1.5105

0 min15 min30 min45 min60 min75 min90 min105 min120 min

wavelength

Fluo

resc

ence

Inte

nsity

Dilution

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

1.0106

0 min15 min30 min45 min60 min75 min90 min105 min120 min

wavelength

Fluo

resc

ence

Inte

nsity

Dilution + Albumin

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

0 min15 min30 min45 min60 min75 min90 min105 min120 min

wavelength

Fluo

resc

ence

Inte

nsity

Dilution +--globulins

500 550 600 650 7000

2.0105

4.0105

6.0105

8.0105

1.0106

0 min

15 min

30 min

45 min

60 min

75 min

90 min

105 min

120 min

wavelength

Fluo

resc

ence

Inte

nsity

Dilution + -globulins

500 550 600 650 7000

5.0105

1.0106

1.5106

2.0106

0 min15 min30 min45 min60 min90 min105 min120 minLegend

wavelength

Fluo

resc

ence

Inte

nsity

Fluorescence data (Fig. 5)

Fig. 5 Representative FRET signals for all formulations Top Panel F127 micelles, bottom panel F127+RES micelles

F127 micelles

0 15 30 45 60 75 90 105 1200.0

0.2

0.4

0.6

0.8

1.0

1.2

Time (min)

I DiI/(

I Dio

+ID

iI)

F127+RES micelles

0 15 30 45 60 75 90 105 1200.0

0.2

0.4

0.6

0.8

1.0

1.2

Dilution

Dilution + Albumin

Dilution + --globulins

Dilution+ -globulins

Time (min)

I DiI/(

I Dio

+ID

iI)

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