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Nanoparticle−Hydrogel System for Postmyocardial Infarction Delivery of MicroRNA Sruti Bheri and Michael E. Davis*
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Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322, United States ABSTRACT: Effective therapies for cardiac repair and regeneration after myocardial infarction (MI) are rather limited. Although microRNAs (miRs) are known to play an important role in improving cardiac function after MI at a cellular level, delivering and retaining miRs at the target site has been challenging. To address this dilemma, several miR carriers have been developed, but these face their own limitations such as immunogenicity and poor targeting to the infarct site. In this Perspective, we summarize different mechanisms for miR administration and localization to cardiac tissue, with a specific focus on the clinically relevant injectable hydrogel and nanoparticle system developed by Yang et al. and reported in this issue of ACS Nano. We also highlight future directions for this field and outline the remaining unanswered questions.
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ability of miRs makes them powerful tools for effectively modifying cell function. After an MI, increases in cardiomyocyte (CM) survival and vessel formation are favorable for improved recovery of the damaged tissue. Studies have shown that miRs can play critical roles in both cell survival and cell apoptosis; therefore, increasing the presence of cell-survival-associated miRs could improve cardiac tissue repair in the infarcted site.9 Researchers have identified several relevant miRs and miR families, including antiapoptotic miRs, such as miR-24 and -214;10,11 pro-proliferation miRs, such as miR-19, -199a, and -590;12,13 and angiogenic miRs, such as miR-210.14 Moreover, delivery of paracrine signals containing relevant miRs to an infarct site induces cardiac repair, further highlighting the promise of miRbased therapeutics in the cardiac sphere.15 Despite the potential of miR therapies, there are several challenges associated with miR delivery to and retention at the infarct site. The delivery of free RNA is associated with poor cell entry and off-target effects. Further, RNAs have a short half-life in vivo for reasons including degradation by endogenous ribonucleases, making the direct delivery of naked miRs impractical for therapy.16 To address this challenge, researchers have studied various chemical modifications of miRs, including the use of phosphorothioate bonds or adding fluoro-RNA.17,18 Despite prolonging miR stability in vivo, these modifications were still associated with off-target effects and the formation of toxic byproducts after the chemicals degraded.17 To overcome these limitations, researchers have developed miR carriers to protect the naked
yocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide, with an estimated 805,000 events annually in the United States alone.1 Myocardial infarction occurs due to coronary artery occlusion, which results in local ischemia, tissue damage, and, eventually, heart failure. MicroRNA (miR)-based therapy is one avenue being explored to improve cardiac function at a cellular level after MI.2 However, delivery and retention of these miRs at the infarct site has proven to be a challenge.3 To assist with delivery, several miR carriers have been engineered, including modified, naturally derived vesicles and synthetic nanoparticles.4 Further, different in vivo biomaterial approaches have been developed, which improve localized administration of the miRs and minimize washout.5 Despite significant developments in these areas, few studies have holistically developed a miR carrier with a suitable in vivo administration strategy to facilitate cardiac repair. In this Perspective, we outline the importance of miR therapies for cardiac repair and relevant in vivo delivery and localization mechanisms for cardiac miRs. We focus, in particular, on the research work by Yang et al., published in this issue of ACS Nano, which describes an injectable hydrogel and nanoparticle system for in vivo miR administration.6 We also discuss future directions for miR therapeutics and miR delivery, highlighting prospective research questions that arise from the findings of this group. Significance of MicroRNAs in Myocardial Infarction. MicroRNAs are single-stranded, short noncoding RNA (approximately 20−25 nucleotides) that downregulate complementary messenger RNA (mRNA) through RNA interference.7 A single miR can target several mRNA and, therefore, can regulate many downstream pathways.8 This inherent © XXXX American Chemical Society
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miRs, improve cell uptake, and enable more targeted delivery.19−21 MicroRNA Carriers. Our cells have a built-in mechanism for transporting signaling molecules like miRs, using lipidbilayer vesicles such as exosomes and other larger extracellular vesicles (EVs).22 These EVs are nano- to microscaled vesicles containing protein and/or nucleic acid cargo (including miRs) that are released by cells and taken up by neighboring recipient cells. This EV-mediated cargo transport enables alterations in the recipient cells’ function and is, therefore, a useful vehicle for miR-induced cell-response modifications. However, from a therapeutic standpoint, we have little control over EV production, miR encapsulation, and recipient cells’ EV uptake, which limits our abilities to develop controlled, targeted miR therapies using this naturally present mechanism. Thus, there has been value in developing synthetic mimics to serve as vehicles for miR transport and delivery to cardiac tissue. Several researchers have attempted to develop different carriers to emulate EV function for successful miR transport and delivery to local cells.23,24 These techniques include both viral and nonviral vectors. Viral vectors, such as adenoassociated virus and lentivirus, have demonstrated successful and stable transfection with high levels of gene expression but are expensive and have potential immunogenic and mutagenic responses.23 More recently, with the advancement of nanotechnology, nonviral vectors have been developed to address some of the above concerns. These vectors include liposomes, inorganic nanoparticles, and polymer-based carriers.24,25 These nonviral vectors have limited mutagenic risk and relatively lower immunogenicity and enable greater customization. Moreover, controlling their composition enables modifications to improve cell targeting, although efficient homing of these systems to specific tissues still remains an important challenge to address. Targeting MicroRNAs to Infarct Sites. Delivery of miRs to the target site is critical for directed and controlled miR administration. One method to overcome the nonspecific distribution of miRs is using targeting agents, specifically homing peptides. These peptides have a simple structure with a high level of specificity for target cell receptors and limited off-target effects, making them a suitable candidate for cardiac tissue homing. Several classes of these targeted peptides have been developed, including cell-penetrating peptides (CPPs), which are ∼9−35mer peptides that can traverse the cell membrane and transfer any vesicles or vectors to which they are bound.26 One of the most common CPPs is TAT protein from HIV-1, which is known to have efficient cellular uptake by host cells, making it a suitable peptide for delivery of miR vectors to the infarct site.
Previous studies by the Heilshorn laboratory and others show that such hydrogels with embedded progenitor cells stabilize the infarct border zone size and reduce the myocardial remodeling.27,28 These findings indicate that shear-thinning hydrogels could be a favorable mechanism for miR delivery and retention at the infarct site. Nanoparticles for MicroRNA Delivery within ShearThinning Hydrogels. Based on recent developments in the field of miR delivery for MI, there is a need to develop a therapeutic that could be targeted to the infarct site with a minimally invasive delivery mechanism and limited washout. As outlined above, the field has grown in relevant aspects miR carriers, targeting techniques, and delivery methodsbut research into clinically relevant therapies combining all of these aspects is still in its infancy. In this section, we explore the work by Yang et al., who used nanoparticle-based miR delivery with a shear-thinning hydrogel as a bridge to developing more translatable systems for miR-based MI therapeutics.6
Based on recent developments in the field of microRNA delivery for myocardial infarction, there is a need to develop a therapeutic that could be targeted to the infarct site with a minimally invasive delivery mechanism and limited washout. For in vivo delivery of miR therapies, it is important to have standardized manufacturing of the nanoparticles with stable shelf life and physiologically relevant size. The authors synthesized miR nanoparticles (miNPs) with a DSPE−PEG shell with cardioprotective miR-199a-3p and a CPP conjugated to the surface. The miNP also contained a poly(9,9dioctylfluorene-co-benzothiadiazole) (PFBT) fluorescent core for ease of particle detection and tracking. The miNPs were ∼110 nm in diameter with low variation and similar in size to naturally derived exosomes (30−120 nm), another potentially important parameter for uptake.29 The miNPs were stable over repeated freeze−thaw cycles and maintained their size, surface charge, and distribution, all of which are critical for developing a translatable therapy. Further, the authors demonstrated that the miNPs had low cell toxicity, enabling higher levels of miRs to be safely delivered to the cardiac cells using these nanoparticles. Nondividing cells such as CMs have poor transfection efficiency with nonviral vectors.30 To address these uptake issues, Yang et al. incorporated the TAT CPP onto the miNP shell.6 TAT is a HIV-1 protein with a cationic moiety known to increase its cellular uptake.31 The addition of the TAT CPP increased miNP uptake by human embryonic stem cell (hESC)-derived CMs and hESC-derived endothelial cells (ECs). These preliminary findings make in vivo translation of the therapy highly promising and show that, although a challenge in the past, nonviral miR administration to cardiac tissues can be achieved.30 The authors also tested the miNPs in vivo in an ischemia/ reperfusion rat model through injections into the infarct border zone. For ease of administration, the miNPs were encapsulated in an elastin-like protein−hyaluronic acid (ELP−HA) hydrogel. This shear-thinning hydrogel enabled a smooth and fast injection into the target site, with efficient release of the miNPs
Delivery of microRNAs (miRs) to the target site is critical for directed and controlled miR administration. Another aspect of miR-based therapy is localized retention of the miR vectors in vivo using a minimally invasive approach for a translatable therapy. Injectable hydrogels are a promising option as they are biodegradable, minimize surgical intervention, and assist with prolonged drug retention and controlled drug release. Specifically, shear-thinning hydrogels have clean in vivo delivery with reduced premature gelation and fracture due to their self-healing mechanical properties.5 B
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Elsewhere, the authors investigated the combined role of the cargo, carrier, and administration method on cardiac repair, outlining the clinical relevance of focusing on all aspects of the cardiac therapeutics. Studies like this encourage the field to address in vivo parameters such as homing the miR to the target site and minimizing invasiveness with an injectable hydrogel system. The researchers also focused on the effect of the therapy on several different cardiac subpopulations, including CMs, ECs, and CFs, and specific targeting approaches to differentiate these cells may enhance therapy. Further, although the authors mention observing increases in EC proliferation and angiogenesis, further work is needed to determine if the vessels are poorly interconnected and/or friable, as such structures would not be suitable for cardiac repair in the long term. In addition, Yang et al. did not yet report off-target effects of the miNP on other organ systems, which are important for long-term in vivo particle administration. On an intracellular level, the exact uptake mechanism of the miNP and the miR trafficking needs further study. The authors show that miR-199a-3p increases CM and EC proliferation (Ki67+) with prolonged improvements in cardiac function with just a single injection. Although this result is promising from a tissue repair perspective, this finding suggests limited control over the miR once administered. Previous studies show that miR-199a-3p expression is also associated with breast carcinoma cell proliferation, so methods to silence miR function controllably once the desired cardiac repair is achieved would be valuable and limit risk of oncogenisis.35 Further, as endosomal escape of oligonucleotides can reduce their efficiency, understanding the intracellular trafficking of miNPs and miRs could enable more regulated drug administration. Another aspect that can be further developed is the variety of miR cargo encapsulated. Here, the authors provide proof of concept by focusing on miR-199a-3p delivery, but multi-miR combination therapies can be designed to target several aspects of cardiac regeneration. With the advent of sequencing and computational analysis of cardiac miRs relevant to repair, the roles of miR families and miR combinations on different cardiac gene functions are rapidly being explored.7 Therefore, miNPs can be further optimized for specific reparative outcomes, such as by developing particles with a mixture of proliferation and antifibrotic miRs. Finally, immune response after MI is a complex and inherent part of the disease manifestation, involved in both disease progression and repair, and is a growing focus of research. Macrophages and regulatory T-cells can play key roles in limiting pro-inflammatory responses and increasing regenerative cytokines in the infarct region.36,37 This study briefly looks at in vivo immune activity in the presence of miNPs through hematoxylin and eosin staining, but further temporal and spatial studies are required to understand the long-term response of the immune system to the miNPs, making this a valuable future direction.
and localized retention. Moreover, the presence of miNPs in the hydrogel improved cardiac function through increased ejection fraction, fractional shortening, and end diastolic volume, and preserved end systolic volume. These improvements were sustained over a 3 month period, indicating less need for repeated miNP dosages and minimizing the need for prolonged clinical intervention. Further, ex vivo analysis detected increases in CM proliferation and EC-based angiogenesis. These findings highlight the ease of delivery and retention with the shear-thinning hydrogel−nanoparticle system and the reparative effects observed with successful miR-199a-3p uptake.
Yang et al., as reported in this issue of ACS Nano, used nanoparticle-based microRNA (miR) delivery with a shearthinning hydrogel as a bridge to developing more translatable systems for miR-based myocardial infarction therapeutics. Because MI conditions are typically a result of coronary artery occlusion, the local cardiac tissue environment is hypoxic. Yang et al. demonstrated that proliferation of CMs and ECs is observed when miNPs are delivered in hypoxic conditions, although it is reduced compared to normoxic conditions. Cell cycle re-entry is also observed in CMs and ECs, even under hypoxia, and is not observed with cardiac fibroblasts (CFs). This distinction reflects a cell-specific miR response with proliferation of the reparative CMs and ECs and limited cell cycle re-entry of fibrosis and scar-formationassociated CFs. The group also observed an increase in vascular function and limited fibrotic contraction, again highlighting the reparative nature of the miNP therapy. All in all, the authors conclude that their clinically relevant miR delivery nanoparticle−hydrogel system has significant potential to develop miR therapies further for improving cardiac repair after MI.
FUTURE PERSPECTIVES AND DIRECTIONS The work by Yang et al. advances the field of cardiac regeneration and repair in several ways, justifying future work in cardiac miNPs. Their system adds to the growing list of miR carriers designed to emulate the advantages of naturally derived vesicles while maintaining control over the production and cargo. Although more established in cancer and hepatitis therapeutics, with some early phase clinical trials (Cobomarsen and Miravirsen),32,33 miR therapies for cardiac repair are still emerging. Here, the authors show that miR delivery with miNPs can result in functional cardiac responses, highlighting the relevance and scope of such therapies for cardiac diseases. This work could be further developed by also investigating the effects of miNP size on transport and miR uptake. Studies have shown that exosome size plays a role in vesicle internalization, with smaller exosomes having higher uptake and more effective cell responses than larger ones.34 As miNPs are targeting different cardiac cell populations and could be applicable for other cardiac diseases, as well, understanding the effects of particle size on function could provide further control during delivery.
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Michael E. Davis: 0000-0002-9239-2886 C
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS We thank H.J. Park for her comments during the creation of this perspective. ABBREVIATIONS MI, myocardial infarction; miR, microRNA; mRNA, messenger RNA; CM, cardiomyocyte; EV, extracellular vesicle; CPP, cell-penetrating peptide; miNP, microRNA nanoparticle; EC, endothelial cell; CF, cardiac fibroblast REFERENCES (1) Benjamin, E. J.; Muntner, P.; Alonso, A.; Bittencourt, M. S.; Callaway, C. W.; Carson, A. P.; Chamberlain, A. M.; Chang, A. R.; Cheng, S.; Das, S. R.; Delling, F. N.; Djousse, L.; Elkind, M. S. V.; Ferguson, J. F.; Fornage, M.; Jordan, L. C.; Khan, S. S.; Kissela, B. M.; Knutson, K. L.; Kwan, T. W.; et al. Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation 2019, 139, e56−e528. (2) Fasanaro, P.; Greco, S.; Ivan, M.; Capogrossi, M. C.; Martelli, F. MicroRNA: Emerging Therapeutic Targets in Acute Ischemic Diseases. Pharmacol. Ther. 2010, 125, 92−104. (3) Rupaimoole, R.; Han, H.-D.; Lopez-Berestein, G.; Sood, A. K. MicroRNA Therapeutics: Principles, Expectations, and Challenges. Aizheng 2011, 30, 368−370. (4) Johnsen, K. B.; Gudbergsson, J. M.; Skov, M. N.; Christiansen, G.; Gurevich, L.; Moos, T.; Duroux, M. Evaluation of Electroporation-Induced Adverse Effects on Adipose-Derived Stem Cell Exosomes. Cytotechnology 2016, 68, 2125−2138. (5) Wang, L. L.; Burdick, J. A. Engineered Hydrogels for Local and Sustained Delivery of RNA-Interference Therapies. Adv. Healthcare Mater. 2017, 6, 1601041. (6) Yang, H.; Qin, X.; Wang, H.; Zhao, X.; Liu, Y.; Wo, H. T.; Liu, C.; Nishiga, M.; Chen, H.; Ge, J.; Sayed, N.; Abilez, O. J.; Ding, D.; Heilshorn, S. C.; Li, K. An in Vivo MiRNA Delivery System for Restoring Infarcted Myocardium. ACS Nano 2019, DOI: 10.1021/ acsnano.9b03343. (7) He, L.; Hannon, G. J. MicroRNAs: Small RNAs with a Big Role in Gene Regulation. Nat. Rev. Genet. 2004, 5, 522−531. (8) Li, Y.; He, X. N.; Li, C.; Gong, L.; Liu, M. Identification of Candidate Genes and MicroRNAs for Acute Myocardial Infarction by Weighted Gene Coexpression Network Analysis. BioMed Res. Int. 2019, 2019, 5742608. (9) Boon, R. A.; Dimmeler, S. MicroRNAs in Myocardial Infarction. Nat. Rev. Cardiol. 2015, 12, 135−142. (10) Li, R.-C.; Tao, J.; Guo, Y. B.; Wu, H. D.; Liu, R. F.; Bai, Y.; Lv, Z. Z.; Luo, G. Z.; Li, L. L.; Wang, M.; Yang, H. Q.; Gao, W.; Han, Q. D.; Zhang, Y. Y.; Wang, X. J.; Xu, M.; Wang, S. Q. Vivo Suppression of MicroRNA-24 Prevents the Transition toward Decompensated Hypertrophy in Aortic-Constricted Mice. Circ. Res. 2013, 112, 601− 605. (11) Aurora, A. B.; Mahmoud, A. I.; Luo, X.; Johnson, B. A.; van Rooij, E.; Matsuzaki, S.; Humphries, K. M.; Hill, J. A.; Bassel-Duby, R.; Sadek, H. A.; Olson, E. N. MicroRNA-214 Protects the Mouse Heart from Ischemic Injury by Controlling Ca2+ Overload and Cell Death. J. Clin. Invest. 2012, 122, 1222−1232. (12) Chen, J.; Huang, Z. P.; Seok, H. Y.; Ding, J.; Kataoka, M.; Zhang, Z.; Hu, X.; Wang, G.; Lin, Z.; Wang, S.; Pu, W. T.; Liao, R.; Wang, D. Z. Mir-17−92 Cluster Is Required for and Sufficient to Induce Cardiomyocyte Proliferation in Postnatal and Adult Hearts. Circ. Res. 2013, 112, 1557−1566. (13) Eulalio, A.; Mano, M.; Dal Ferro, M.; Zentilin, L.; Sinagra, G.; Zacchigna, S.; Giacca, M. Functional Screening Identifies MiRNAs Inducing Cardiac Regeneration. Nature 2012, 492, 376−381. D
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