The combination of extracellular vesicles in a cardiac extracellular matrix hydrogel with polyethylene glycol presents promising physico-chemical properties and bioactivity for cardiac applications
Authors: Gomez-Cid L , Lopez-Donaire ML , Velasco D , Fernandez-Santos ME , Grigorian-Shamagian L , Fernandez-Aviles F 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain
CIBERCV

Background
Extracellular vesicles derived from cardiosphere-derived cells (EVs) have shown multiple beneficial effects in preclinical models of cardiac diseases. However, common administration routes present poor EV retention within the heart tissue, which can limit the therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) provide structural support and seem promising as drug-delivery materials, but may be limited by their long gelation time and soft mechanical properties.

Purpose
To develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs for its potential use in the treatment of cardiac pathologies.

Methods
Extracellular vesicles were purified from cardiosphere-derived cells previously obtained from human cardiac biopsies (who underwent cardiac surgery for other reasons). For cECMH preparation, decellularized porcine cardiac tissue was lyophilized and milled. After 72 hour-digestion with pepsin in HCl solution, pH and salt concentration were adjusted to physiological values and the solution kept cold until used (final ECM concentration 8 mg/ml). cECMH alone, with PEG at 0, 3, 6, 12 mg/ml, or with PEG at 8 mg/ml + EVs (5 mg/ml) were used. Gels were formed after incubation at 37ºC. Gelation kinetics (half-gelation time, lag phase initiation, and slope of gelation) were obtained through turbidimetry measurements. Rheology measurements (viscosity, storage and loss modulus) were obtained with a TA Instruments AR-G2 rheometer, and injectability confirmed using a Myostar injection catheter. Rate of degradation was determined with collagenase and the Ninhydrin assay and product bioactivity (anti-senescent effect) evaluated coculturing the individual and the combined products with human cardiac stromal cells from two different donors.

Results
The new combined product (cECMH + PEG + EVs) presented improved physicochemical properties with respect to cECMH alone: 13 ± 3 min vs. 42 ± 2 min half gelation time (p &lt; 0.001, Fig. 1A), an increase in storage modulus, (15 ± 5 vs. 4.5 ± 0.9 Pa, p &lt; 0.01, Fig. 1B), preserved injectability through a catheter, and biodegradability. It also maintained in vitro bioactivity of its individual components, as it reduced cardiac stromal cellular senescence by ~55% vs. serum-free medium (p &lt; 0.001, Fig. 1 C-D.), similar to EVs and cECMH alone.

Conclusions
The combination of cECMH, PEG and EVs is a potential advanced therapy product with improved gelation time and mechanical properties capable of maintaining bioactivity. This combination solves some of the product’s individual limitations, which may translate into boosted therapeutic efficacy.