Therapeutic Efficacy of Cryopreserved, Allogeneic Extracellular Vesicles for Treatment of Acute Myocardial Infarction.

Extracellular vesicles (EV) that are derived from endothelial progenitor cells (EPC) have been determined to be a novel therapy for acute myocardial infarction, with a promise for immediate "off-the-shelf" delivery. Early experience suggests delivery of EVs from allogeneic sources is safe. Yet, clinical translation of this therapy requires assurances of both EV stability following cryopreservation and absence of an adverse immunologic response to EVs from allogeneic donors. Thus, more bioactivity studies on allogeneic EVs after cold storage are necessary to establish quality standards for its widespread clinical use. Thus, in this study, we aimed to demonstrate the safety and efficacy in delivering cryopreserved EVs in allogeneic recipients as a therapy for acute myocardial infarction.In this present study, we have analyzed the cardioprotective effects of allogeneic EPC-derived EVs after storage at -80°C for 2 months, using a shear-thinning gel (STG) as an in vivo delivery vehicle. EV size, proteome, and nucleic acid cargo were observed to remain steady through extended cryopreservation via nanoparticle tracking analysis, mass spectrometry, and nanodrop analysis, respectively. Fresh and previously frozen EVs in STG were delivered intramyocardially in a rat model of myocardial infarction (MI), with both showing improvements in contractility, angiogenesis, and scar thickness in comparison to phosphate-buffered saline (PBS) and STG controls at 4 weeks post-MI. Pathologic analyses and flow cytometry revealed minimal inflammatory and immune upregulation upon exposure of tissue to EVs pooled from allogeneic donor cells.Allogeneic EPC-EVs have been known to elicit minimal immune activity and retain therapeutic efficacy after at least 2 months of cryopreservation in a post-MI model.

Injectable Supramolecular Hydrogel/Microgel Composites for Therapeutic Delivery.

Shear-thinning hydrogels are useful for biomedical applications, from 3D bioprinting to injectable biomaterials. Although they have the appropriate properties for injection, it may be advantageous to decouple injectability from the controlled release of encapsulated therapeutics. Toward this, composites of hydrogels and encapsulated microgels are introduced with microgels that are fabricated via microfluidics. The microgel cross-linker controls degradation and entrapped molecule release, and the concentration of microgels alters composite hydrogel rheological properties. For the treatment of myocardial infarction (MI), interleukin-10 (IL-10) is encapsulated in microgels and released from composites. In a rat model of MI, composites with IL-10 reduce macrophage density after 1 week and improve scar thickness, ejection fraction, cardiac output, and the size of vascular structures after 4 weeks when compared to saline injection. Improvements are also observed with the composite without IL-10 over saline, emphasizing the role of injectable hydrogels alone on tissue repair.

Injectable and protease-degradable hydrogel for siRNA sequestration and triggered delivery to the heart.

Injectable hydrogels have significant therapeutic potential for treatment of myocardial infarction (MI) through tissue bulking and local drug delivery, including the delivery of small interfering RNAs (siRNAs). As siRNA targets are identified as potential treatments for MI, hydrogels may bolster efficacy through local and sustained release. Here, we designed an injectable hydrogel to respond to local upregulation in proteolytic activity after MI to erode and release siRNA against MMP2 (siMMP2), a target implicated in deleterious remodeling. Specifically, hyaluronic acid (HA) was modified with hydrazides or aldehydes and mixed to form shear-thinning and self-healing hydrogels through dynamic hydrazone bonds and with peptide crosslinkers that degrade in response to protease activity. HA was further modified with ß-cyclodextrin to sequester cholesterol-modified siRNA, limiting passive diffusion. Hydrogels eroded in response to proteases and released active siRNA that knocked down MMP2 in primary cardiac fibroblasts. In a rat model of MI, hydrogels delivering siMMP2 attenuated hydrogel erosion by ~46% at 4 weeks when compared to hydrogels delivering control siRNA, ultimately improving myocardial thickness in the infarct. Delivery of the siMMP2 hydrogel led to significant functional improvements, including increased ejection fraction (27%, 66%), stroke volume (32%, 120%), and cardiac output (20%, 128%) when compared to controls (% increase versus hydrogels with control siRNA, % increase versus saline injection alone). This report demonstrates the utility of biomaterial-based RNA delivery systems for cardiac applications.

Effects of a novel microtubule-depolymerizer on pro-inflammatory signaling in RAW264.7 macrophages.

The Nuclear Factor-kappa B (NF-κB) pathway is vital for immune system regulation and pro-inflammatory signaling. Many inflammatory disorders and diseases, including cancer, are linked to dysregulation of NF-κB signaling. When macrophages recognize the presence of a pathogen, the signaling pathway is activated, resulting in the nuclear translocation of the transcription factor, NF-κB, to turn on pro-inflammatory genes. Here, we demonstrate the effects of a novel microtubule depolymerizer, NT-07-16, a polysubstituted pyrrole compound, on this process. Treatment with NT-07-16 decreased the production of pro-inflammatory cytokines in RAW264.7 mouse macrophages. It appears that the reduction in pro-inflammatory mediators produced by the macrophages after exposure to NT-07-16 may be due to activities upstream of the translocation of NF-κB into the nucleus. NF-κB translocation occurs after its inhibitory protein, IκB-α is phosphorylated which signals for its degradation releasing NF-κB so it is free to move into the nucleus. Previous studies from other laboratories indicate that these processes are associated with the microtubule network. Our results show that exposure to the microtubule-depolymerizer, NT-07-16 reduces the phosphorylation of IκB-α and also decreases the association of NF-κB with tubulin which may affect the ability of NF-κB to translocate into the nucleus. Therefore, the anti-inflammatory activity of NT-07-16 may be explained, at least in part, by alterations in these steps in the NF-κB signaling pathway leading to less NF-κB entering the nucleus and reducing the production of pro-inflammatory mediators by the activated macrophages.