60. utilizing injectable decellularized extracellular matrix hydrogels for the slow release of micrornas

Department: Bioengineering
Faculty Advisor(s): Karen L. Christman

Primary Student
Name: Melissa Jenee Hernandez
Email: mjh003@ucsd.edu
Phone: 858-246-1593
Grad Year: 2019

Abstract
Introduction: One main challenge for utilizing microRNAs (miRNAs) as therapeutics has been ineffective delivery. Upon injection, miRNAs rapidly diffuse from the injection site, and circulating RNases also quickly degrade these small oligonucleotides. Our objective was to evaluate the capacity of decellularized extracellular matrix (ECM) hydrogels for delivering miRNAs. We hypothesized that encapsulation of miRNAs within ECM hydrogels would prolong the release profile, allowing for greater therapeutic potential with later in vivo applications. Methods: Fabrication of porcine-derived ECM hydrogels included decellularization with detergent, lyophilization, milling, and pepsin digestion. Antimir and antagomir, miRNA inhibitors, were synthesized with the same 22-bp complimentary sequence, but the antagomir had a cholesterol group conjugated to the 3? end. Four μg of Cy3-labeled antimir (n=3) or antagomir (n=3) were loaded in 200 μl of decellularized myocardial ECM hydrogels. After the initial rinse, 250 μl of PBS was added, and 200 μl of supernatant was collected daily. Collagenase and 1.5M NaCl were added in place of PBS at the end of the study to dissociate residual miRNA. Fluorescence measurements to quantify miRNA release were recorded with a microplate reader. Values are reported as mean SD. Results: Although the antimir and antagomir shared the same oligonucleotide sequence, the presence of a cholesterol group on the antagomir largely affected the release profile. At day 3, the cumulative amount of miRNA released was 61.53.1% for the antimir and 29.03.6% for the antagomir. By day 15, the amount of miRNA released was increased to 85.13.7% for the antimir and 68.22.6% for the antagomir. The remaining miRNA was dissociated upon addition of collagenase and NaCl. Conclusions: ECM hydrogels resulted in a steady release of the miRNAs over a two-week period in vitro. Slower release of the antagomir was likely due to electrostatic interactions between the attached cholesterol group and ECM hydrogel, as indicated by further release of the antagomir following incubation with 1.5M NaCl. This study establishes the ability of decellularized hydrogels to modulate the release of miRNAs, which could allow for increased efficacy by localizing the therapeutic benefits in the region of interest.

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