HYDROGEL Enzyme Mediated Bioresponsive Hydrogels PEPTIDE ENZYME DRUG

HYDROGEL Enzyme Mediated Bioresponsive Hydrogels PEPTIDE ENZYME DRUG Masters Thesis Defense July 6 th, 2012 A. Nolan Wilson Committee Prof. Anthony Guiseppi-Elie (Chair) Dr. Roberts and Dr. Kitchens TARGET

Enzymatically Sensitive Hydrogel (ESH) Responsive Polymers Bioresponsive Environment Responsive “Sense” Input Response Type Antibodies Degrade Cells Gelation Enzymes Release Metabolites Swelling Self Assembly Ulijn, R. V. , N. Bibi, et al. (2007). J. J. Moon, et al. , Biomaterials, 31, 3840 (2010) R. Jin, C. Hiemstra, Z. Zhong and J. Feijen, A. Guiseppi-Elie, S. I. Brahim and D. M. Hughes, et. al. Soft Matter, 8, 5595 (2012). "Bioresponsive hydrogels. " Materials Today Narinesingh, Advanced Materials, 14, 743 Biomaterials, 28, 2791 (2007) 10(4): 40 -48 (2002). 2

Mechanism and Fundamentals Sense Biological Event • Catalysis • Binding Transduction and Signal Creation • Bio-transducing perturbs system equilibrium (signal) in response to sensing Response • Bio-transducing perturbs system equilibrium (signal) in response to actuation 3

Motivation of Work: MMPs as Bad Actors Cancer “The matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis. ” 1 Chronic Wounds “It is believed that in chronic wounds the activity of MMPs is up-regulated while the expression of TIMPs is decreased, thus affecting ECM turnover. ” 2 Hemorrhagic Shock “MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. ” 3 1. 2. 3. Kessenbrock, K. , V. Plaks, et al. (2010). "Matrix Metalloproteinases: Regulators of the Tumor Microenvironment. " Cell 141(1): 52 -67. Martins, V. , M. Caley, et al. "Matrix metalloproteinases and epidermal wound repair. " Cell and Tissue Research: 1 -14. Altshuler, A. E. , A. H. Penn, et al. (2012). "Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats. " PLo. S ONE 7(3): e 32672. 4

Motivation of Work Research Area: Investigating controlled release mechanisms from hydrogels by using environmental and biological stimuli as release triggers. Inhibitor MAG-283 YLL-224 MMP-1 480 180 IC 50 Value (n. M) MMP-2 MMP-3 MMP-7 3 280 14 63 4500 210 MMP-8 1. 1 5. 9 MMP-9 2. 3 44 Application: Use controlled release of MMPI (inhibitors) in chronic wound environment to restore normal metabolic healing processes. Overactive ECM Proteases Chronic Wound Environment Modulated Release of MMP-Inhibitors Acute Wound Healing 5 Q. -X. A. Sang, M. -C. Jia, M. A. Schwartz, M. C. Jaye, H. K. Kleinman, M. A. Ghaffari and Y. -L. Luo, Biochemical and Biophysical Research Communications, 274, 780 (2000).

Control Loops: A Unique Opportunity Using ESH Metabolic Process … i 1 2 Actuation Transduction Response β α Bio-responsive Hydrogel Metabolic Process … i 1 2 Hydrogel ± ± α' Feedforward Control Loop Bio-responsive α Feedback Control Loop β' ± ± β Actuation Transduction Response 6

Actuated Release by Chymotrypsin: Mechanism Concentration HIGH LOW Diffusion-Reaction Transport Goal 1. Create Chymotrypsin-Hydrogel Model System where: • Control Release Rate • Control Dose Amount Process 1. Diffusion of Enzyme in 2. Cleavage of Peptide-Chromogen Bond 3. Diffusion of Chromophore out D Enzyme Peptide SOLUTION DISK D Chromophore SOLUTION DISK 7

Actuated Release by Chymotrypsin: Hydrogel HEMA TEGDA HMMA AEMA N-Succ. AAPF p. Na Biocompatible Non-degradable Control release rate Enzyme Access Control Release Amount Cleavable Pendant Group Measurable, neutral feedback Design Criteria: • Biocompatible • Non-degradable • Accessible by enzyme • Pendant group cleaved by enzymatic cleavage • Measurable, neutral feedback loop • Control release rate • Control released amount AEMA + N-Succ-AAPF-p. Na EDC (1 m. M, MES, p. H 5. 0, 25 o. C) Sulfo-NHS (5 m. M, PBS, p. H 7. 0, 25 o. C) AEMA-Succ. AAPF-p. Na 8

Actuated Release by Chymotrypsin: Methodology Experimental Variables • Mole% AEMA (0, 5, 10, 20, 30%) • Mole %TEGDA (1, 5, 7, 9, 12%) A. N. Wilson, R. Salas and A. Guiseppi-Elie, J Control Release, 160, 41 (2012). 99

Actuated Release by Chymotrypsin: Results Controlling Release Rate (TEGDA) Controlling Release Amount (AEMA) 10

• Thiele Modulus Approach • Measure rapp and rintrinsic • Calculate η • Goal seek to determine φ • φ< 1 : Kinetically rate limited • φ> 1 : Transport rate limited • Measure KM and Vmax • Calculate Deff (μm 2 / s) Actuated Release by Chymotrypsin: Analysis % TEGDA

Actuated Release by Chymotrypsin: Optimization A. N. Wilson, R. Salas and A. Guiseppi-Elie, J Control Release, 160, 41 (2012). 12

Conclusions and Future Work § Can predictably control release rates and release amount from enzymatically actuated hydrogel § Have proposed a predictive methodology for optimization § To create hydrogel with releasable inhibitor creating integrated feedback loop § To apply inhibitory hydrogel to MMP/chronic wound application in vitro and in vivo 13

Acknowledgements § Clemson Ch. BE Department § Professor Guiseppi § Committee: Dr. Roberts and Dr. Kitchens § C 3 B Research Group § Ruth Salas (UG who also worked on project) § Do. D –Congressionally Directed Medical Research Program (CDMRP) and the Joint Warfighter Medical Research Program (JWMRP) 14

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Water Fickia n Transport Bindin g Response Thermodynamics Kinetics Cataly sis Degrade Reacti on Equili brium