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An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium.

ABSTRACT: A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

SUBMITTER: Yang H

PROVIDER: S-EPMC7930012 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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An <i>in Vivo</i> miRNA Delivery System for Restoring Infarcted Myocardium.

Yang Huaxiao H Qin Xulei X Wang Huiyuan H Zhao Xin X Liu Yonggang Y Wo Hung-Ta HT Liu Chun C Nishiga Masataka M Chen Haodong H Ge Jing J Sayed Nazish N Abilez Oscar J OJ Ding Dan D Heilshorn Sarah C SC Li Kai K

ACS nano 20190607 9

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an <i>in vivo</i> delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cel ...[more]

PMID: 31149806

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Project description:Myocardial infarction (MI) results in debilitating remodeling of the myocardial extracellular matrix (ECM). In this proof-of-principle study it was sought to modulate this aggressive remodeling by injecting a hyaluronic acid-based reservoir delivering exogenous microRNA-29B (miR-29B). This proof-of-principal study was executed whereby myocardial ischemia/reperfusion was performed on C57BL/6 mice for 45 min after which five 10 μL boluses of a hydrogel composed of thiolated hyaluronic acid cross-linked with poly (ethylene glycol) diacrylate, containing exogenous miR-29B as an active therapy, were injected into the border zone of the infarcted myocardium. Following surgery, the myocardial function of the animals was monitored up to 5 weeks. Delivering miR-29B locally using an injectable hyaluronan-based hydrogel resulted in the maintenance of myocardial function at 2 and 5 weeks following MI in this proof-of-principle study. In addition, while animals treated with the control of a nontargeting miR delivered using the hyaluronan-based hydrogel had a significant deterioration of myocardial function, those treated with miR-29B did not. Histological analysis revealed a significantly decreased presence of elastin and significantly less immature/newly deposited collagen fibers at the border zone of the infarct. Increased vascularity of the myocardial scar was also detected and Raman microspectroscopy discovered significantly altered ECM-specific biochemical signals at the border zone of the infarct. This preclinical proof-of-principle study demonstrates that an injectable hyaluronic acid hydrogel system could be capable of delivering miR-29B toward maintaining cardiac function following MI. In addition, Raman microspectroscopy revealed subtle, yet significant changes in ECM organization and maturity. These findings have great potential with regard to using injectable biomaterials as a local treatment for ischemic tissue and exogenous miRs to modulate tissue remodeling.

Project description:Cardiac tissue engineering is a promising approach to provide large-scale tissues for transplantation to regenerate the heart after ischemic injury, however, integration with the host myocardium will be required to achieve electromechanical benefits. To test the ability of engineered heart tissues to electrically integrate with the host, 10 million human embryonic stem cell (hESC)-derived cardiomyocytes were used to form either scaffold-free tissue patches implanted on the epicardium or micro-tissue particles (~1000 cells/particle) delivered by intramyocardial injection into the left ventricular wall of the ischemia/reperfusion injured athymic rat heart. Results were compared to intramyocardial injection of 10 million dispersed hESC-cardiomyocytes. Graft size was not significantly different between treatment groups and correlated inversely with infarct size. After implantation on the epicardial surface, hESC-cardiac tissue patches were electromechanically active, but they beat slowly and were not electrically coupled to the host at 4 weeks based on ex vivo fluorescent imaging of their graft-autonomous GCaMP3 calcium reporter. Histologically, scar tissue physically separated the patch graft and host myocardium. In contrast, following intramyocardial injection of micro-tissue particles and suspended cardiomyocytes, 100% of the grafts detected by fluorescent GCaMP3 imaging were electrically coupled to the host heart at spontaneous rate and could follow host pacing up to a maximum of 300-390 beats per minute (5-6.5 Hz). Gap junctions between intramyocardial graft and host tissue were identified histologically. The extensive coupling and rapid response rate of the human myocardial grafts after intramyocardial delivery suggest electrophysiological adaptation of hESC-derived cardiomyocytes to the rat heart's pacemaking activity. These data support the use of the rat model for studying electromechanical integration of human cardiomyocytes, and they identify lack of electrical integration as a challenge to overcome in tissue engineered patches.

Project description:Injectable, acellular biomaterials hold promise to limit left ventricular remodeling and heart failure precipitated by infarction through bulking or stiffening the infarct region. A material with tunable properties (eg, mechanics, degradation) that can be delivered percutaneously has not yet been demonstrated. Catheter-deliverable soft hydrogels with in vivo stiffening to enhance therapeutic efficacy achieve these requirements. We developed a hyaluronic acid hydrogel that uses a tandem crosslinking approach, where the first crosslinking (guest-host) enabled injection and localized retention of a soft (<1 kPa) hydrogel. A second crosslinking reaction (dual-crosslinking) stiffened the hydrogel (41.4±4.3 kPa) after injection. Posterolateral infarcts were investigated in an ovine model (n≥6 per group), with injection of saline (myocardial infarction control), guest-host hydrogels, or dual-crosslinking hydrogels. Computational (day 1), histological (1 day, 8 weeks), morphological, and functional (0, 2, and 8 weeks) outcomes were evaluated. Finite-element modeling projected myofiber stress reduction (>50%; P<0.001) with dual-crosslinking but not guest-host injection. Remodeling, assessed by infarct thickness and left ventricular volume, was mitigated by hydrogel treatment. Ejection fraction was improved, relative to myocardial infarction at 8 weeks, with dual-crosslinking (37% improvement; P=0.014) and guest-host (15% improvement; P=0.058) treatments. Percutaneous delivery via endocardial injection was investigated with fluoroscopic and echocardiographic guidance, with delivery visualized by magnetic resonance imaging. A percutaneous delivered hydrogel system was developed, and hydrogels with increased stiffness were found to be most effective in ameliorating left ventricular remodeling and preserving function. Ultimately, engineered systems such as these have the potential to provide effective clinical options to limit remodeling in patients after infarction.

Project description:Coronary artery disease is a severe ischemic condition characterized by the reduction of blood flow in the arteries of the heart that results in the dysfunction and death of cardiac tissue. Despite research over several decades on how to reduce long-term complications and promote angiogenesis in the infarct, the medical field has yet to define effective treatments for inducing revascularization in the ischemic tissue. With this work, we have developed functional biomaterials for the controlled release of immunomodulatory cytokines to direct immune cell fate for controlling wound healing in the ischemic myocardium. The reparative effects of colony-stimulating factor (CSF-1), and anti-inflammatory interleukins 4/6/13 (IL4/6/13) have been evaluated in vitro and in a predictive in vivo model of ischemia (the skin flap model) to optimize a new immunomodulatory biomaterial that we use for treating infarcted rat hearts. Alginate hydrogels have been produced by internal gelation with calcium carbonate (CaCO3) as carriers for the immunomodulatory cues, and their stability, degradation, rheological properties and release kinetics have been evaluated in vitro. CD14 positive human peripheral blood monocytes treated with the immunomodulatory biomaterials show polarization into pro-healing macrophage phenotypes. Unloaded and CSF-1/IL4 loaded alginate gel formulations have been implanted in skin flap ischemic wounds to test the safety and efficacy of the delivery system in vivo. Faster wound healing is observed with the new therapeutic treatment, compared to the wounds treated with the unloaded controls at day 14. The optimized therapy has been evaluated in a rat model of myocardial infarct (ischemia/reperfusion). Macrophage polarization toward healing phenotypes and global cardiac function measured with echocardiography and immunohistochemistry at 4 and 15 days demonstrate the therapeutic potential of the proposed immunomodulatory treatment in a clinically relevant infarct model.

Project description:Rationale: Exosomes are emerging as a promising drug delivery carrier. However, rapid uptake of exosomes by the mononuclear phagocyte system (MPS) remains an obstacle for drug delivery into other targeted organs, including the heart. We hypothesized that prior blocking of uptake of exosomes by the MPS would improve their delivery to the targeted organs. Methods: Exosomes were isolated from the cell culture medium. Fluorescence-labeled exosomes were tracked in vitro and in vivo by fluorescence imaging. The expression of clathrin heavy chain (Cltc), cavolin1, Pak1 and Rhoa, known genes for endocytosis, were profiled in various cell lines and organs by qPCR. The knockdown efficiency of siRNA against Cltc was analyzed by Western blotting. Exosomecontrol and exosomeblocking were constructed by encapsulating isolated exosomes with siControl or siClathrin via electroporation, while exosometherapeutic was constructed by encapsulating isolated exosomes with miR-21a. Doxorubicin-induced cardiotoxicity model was used to verify the therapeutic efficiency of the exosome-based miR-21a delivery by echocardiography. Results: Exosomes were preferentially accumulated in the liver and spleen, mainly due to the presence of abundant macrophages. Besides the well-known phagocytic effect, efficient endocytosis also contributes to the uptake of exosomes by macrophages. Cltc was found to be highly expressed in the macrophages compared with other endocytosis-associated genes. Accordingly, knockdown of Cltc significantly decreased the uptake of exosomes by macrophages in vitro and in vivo. Moreover, prior injection of exosomeblocking strikingly improved the delivery efficiency of exosomes to organs other than spleen and liver. Consistently, compared with the direct injection of exosometherapeutic, prior injection of exosomeblocking produced a much better therapeutic effect on cardiac function in the doxorubicin-induced cardiotoxicity mouse model. Conclusions: Prior blocking of endocytosis of exosomes by macrophages with exosomeblocking successfully and efficiently improves the distribution of following exosometherapeutic in targeted organs, like the heart. The established two-step exosome delivery strategy (blocking the uptake of exosomes first followed by delivery of therapeutic exosomes) would be a promising method for gene therapy.

Dataset Information

An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium.

Publications

An <i>in Vivo</i> miRNA Delivery System for Restoring Infarcted Myocardium.

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