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INTRODUCTION
Myocardial infarction (MI) leads to a significant loss of cardiomyocytes followed by the progressive formation of a non-contractile fibrotic scar. Recently, the use of microRNA (miRNA) has emerged as a promising strategy for cardiac regeneration. Paoletti et al. demonstrated that the transient transfection with four microRNA mimics (termed “miRcombo”) can induce the direct reprogramming of human cardiac fibroblasts (CF) into induced cardiomyocytes (iCMs)[1]. To overcome the limitations of in vivo miRNAs administration, proper nanocarriers are required. Moreover, alginate-based injectable hydrogels can be exploited for controlled in situ release of miRNAs-loaded nanocarriers [2]. However, alginate presents some limitations such as low degradability in vivo and limited cell adhesion.
In this work, new lipoplexes (LPs) loaded with miRcombo were developed to obtain more efficient encapsulation and release of miRcombo to CFs compared to a commercial agent. Then, LPs were combined with an optimized alginate dialdehyde (ADA) hydrogel for potential in situ controlled release of miRNA in the damaged tissue.
METHODS
LPs containing negmiR (negative control) or miRcombo based on a mixture of cationic and helper lipid were prepared at different N/P ratio (N/P 3 - 0.35) and physiochemically characterized [3]. Adult human CFs (AHCFs) were treated with miRcombo-loaded LPs to assess transfection efficiency and ability to promote direct reprogramming of AHCFs into iCMs through gene expression analysis at 15 days culture time. ADA was prepared by ALG oxidation using sodium metaperiodate [2]. ADA-based hydrogels with different compositions were characterised for their physicochemical properties. Preliminarily, siRNA-Cy5-loaded LPs were encapsulated into ADA-based hydrogels with selected composition and Cy5-siRNA release was studied.
RESULTS
MiRcombo-loaded LPs with optimal N/P ratio of 3 were selected based on stability studies (evaluated in different media by DLS analysis), showing high encapsulation efficiency (99%). In vitro cell test of LPs with AHCFs showed high biocompatibility and miRNA cellular uptake. Moreover, treatment of AHCFs with miRcombo-loaded LPs favoured their direct reprogramming into cardiomyocyte-like cells, evaluated through the expression of cardiomyocyte markers such as cardiac troponin C (cTnT).
ADA was prepared with an average yield of 68% and an oxidation degree of 23%. ADA concentration and ionic crosslinking were optimised to develop injectable hydrogels with cardiac-like viscoelastic properties. Model Cy5-siRNA-loaded LPs were physically entrapped within ADA-based hydrogels, completely releasing Cy5-siRNA within 24h.
CONCLUSION
A novel miRNA-delivery system, consisting of miRNA-loaded LPs encapsulated in an ADA-based injectable hydrogel, was developed for cardiac regenerative medicine. Cy5-siRNA release data suggested the need for tailoring the surface charge of miRNA-loaded LPs by proper coating, to minimize the electrostatic interactions between ADA and the positively charged miRNA-loaded LPs. This activity is currently in progress and preliminary data showed an enhancement in the stability of hydrogel-embedded LPs by tailoring their surface charge.
1 Paoletti C, et al. Front Bioeng Biotechnol. ,8,529 (2020). 2 . Sarker, B et al., J. Mater. Chem. B, 2(11), 1470-1482 (2014). 3. Nicoletti et al., Nanomed.: Nanotechnol. Biol. Med., under submission.
BIORECAR is supported from the European Research Council (ERC) under the EU H2020 research and innovation programme (GA N° 772168).
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