478044 Optimizing Oligonucleotide-Peptide Conjugate Formation for Photothermally-Responsive Fibrin Hydrogels

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Cara Abecunas1,2, Chase Linsley3 and Benjamin Wu3, (1)Chemical and Biomolecular Engineering, Lafayette College, Easton, PA, (2)Amgen Scholars, Los Angeles, CA, (3)Bioengineering, UCLA, Los Angeles, CA

Nearly seven million people suffer from chronic wounds in the United States, and that number is expected to rise as cases of obesity and diabetes increase. Currently, therapies are limited for the treatment of chronic wounds. Regranex®, approved in 1997, is the only growth factor-based therapy for chronic wounds, but long-term use can increase the risk of cancer mortality. To eliminate potential harmful side effects, new strategies for drug delivery have been proposed. On-demand delivery technologies can improve the efficacy and reduce the potential for unwanted side effects of therapeutic molecules. Here, the design and fabrication of light-responsive fibrin hydrogels for the release of growth factors into the wound microenvironment is reported. dsDNA are (photo)thermally responsive and must first be conjugated to Factor XIIIa substrate peptide in order to be connected to fibrin. Oligonucleotide-peptide conjugates were enzymatically incorporated into fibrin hydrogels via Factor XIIIa (FXIIIa) enzyme to make the scaffolds stimuli-responsive. To fabricate the oligonucleotide-peptide conjugate, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) crosslinker was used. Current results show that ethanol precipitation can efficiently separate the unreacted SMCC and SMCC-oligonucleotide conjugates with 99.9% recovery of the oligonucleotide. SMCC-oligonucleotide complexes were then reacted with FXIIIa substrate peptide in varied reaction conditions. Reactions were performed in deoxygenated and oxygenated conditions with molar ratios of 1:12 and 1:1 (SMCC-oligonucleotide: peptide). Conjugation of the oligonucleotide-peptide complexes into fibrin hydrogels was then measured by fluorescent intensity of the scaffolds. Results showed that reacting the oligonucleotide with deoxygenated peptide in a 1:1 mole ratio and conjugating the complex into fibrin with new, more bioactive FXIIIa enzyme led to 14.60% incorporation efficiency. To further improve conjugation efficiency, six spacers composed of glycine and serine amino acids were added between the reactive cysteine group of FXIIIa peptide substrate and the rest of the substrate peptide. The addition of amino acid spacers in the peptide resulted in a significant decrease in conjugation of the complex into the fibrin hydrogel (0.93%). Results of this study determined that using a deoxygenated peptide and 1:1 molar ratio in the formation of oligonucleotide-peptide complexes led to the highest incorporation into the fibrin hydrogel thus far. Ongoing work is aiming to confirm the formation of oligonucleotide-peptide conjugates and quantify the reaction efficiency using mass spectrometry.

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