Poster U3 - Engineered Lipid Nanoparticles as a Vehicle of Novel Endogenous Therapeutics for Treatment of Chronic Cardiovascular Disease

Introduction: Cardiovascular disease (CVD) is the leading cause of death in the United States and worldwide, and chief among these is myocardial infarction (MI). While initial incidence of MI is typically non-fatal, the accumulation of irreversible damage to the tissue significantly increases the mortality and risk of future MI. This damage most commonly presents in the form of cardiac fibrosis (CFB), stiffening of the myocardium co-occurring with pro-inflammatory changes to the microenvironment, for which there is currently no established direct pharmaceutical treatment. Interestingly, the likelihood and severity of CFB post-MI is highly dependent on the age and sex of the subject. While the reasons for this are largely unknown in literature, we have previously described age- and sex-dependent changes in the myocardium resulting from differences in the cargo and morphology of tissue embedded small extracellular vesicles (EVs) in human hearts. We utilized some select compounds identified from these sEVs which demonstrated synergistic therapeutic effects to develop a preliminary treatment. Herein, we evaluate the therapeutic potential of additional cargo in these sEVs from various human hearts compared to clinical plasma samples in order to develop a highly targeted therapeutic cocktail consisting of endogenous agents for anti-fibrotic effects. We then engineer a lipid nanoparticle (LNP) carrier with cardiac-targeting capabilities and miRNA-friendly composition and evaluate the efficacy and selectivity of our system using our previously established human heart anoxia and reperfusion (HEART) heart-on-a-chip model.

Materials and

Methods: Tissue embedded sEVs were isolated from donor human hearts or from clinical plasma samples. Subjects were separated based on age and sex, being age-matched when possible. After sEV characterization, both cohorts were lysed and profiled for total miRNA content via Nanostring or total protein content via mass spectrometry. Targets were pulled from both of these populations via unsupervised analysis, and evaluated for downstream function via MetaCore analysis and KEGG proteomapping. Cardiac-targeting LNPs were constructed with lipid ratios which enhance uptake of LNPs in non-liver organs in vivo and conjugation of tetraspanins to take advantage of endogenous tetraspanin web targeting. To enhance miRNA encapsulation and stability in LNPs, miRNA agents were first conjugated to smaller, ionized LNPs then encapsulated in larger, targeting LNPs. Population characteristics, drug loading efficiency, and stability of engineered LNPs was evaluated. Selectivity, uptake rate, and cargo delivery of the LNPs were evaluated in 2D culture for cardiomyocytes (CMs) and cardiac fibroblasts (CFs), and in the HEART model. The HEART model was fabricated by standard polydimethylsiloxane (PDMS) soft-lithography and bonded to a glass slide. The HEART model consists of terminal (cardiac) and non-terminal (flow) channels separated by micropillars. Cardiac channels were loaded with a matrix-mimetic hydrogel with encapsulated CMs and CFs to mimic the myocardium, while the flow channel was seeded with endothelial cells. LNPs were introduced through the flow channel and HEARTs were kept subjected to normal of MI-like conditions to induce CFB. Cell survival, reperfusion damage, and fibrotic signaling were quantified.

Results, Conclusions, and Discussions: Tissue and plasma sEVs were successfully isolated and quantified, with tissue sEVs showing age-dependent morphological changes that were not seen in the plasma sEVs. Full miRNA profiling revealed 798 miRNAs present in any of the three biological replicates for any cohort. Unsupervised analysis revealed 125 targets of interest, and 37 potential therapeutic agents were identified in addition to our previously established 4 agents. These 45 agents were evaluated via MetaCore analysis and some were revealed to be involved in pathways relating to regulated exosome trafficking and inflammation inhibition, apoptotic signaling, inhibition of Ang2-mediated CFB cascade, CVD-associated inflammation cascades, the promotion of stem cell recruitment for cardiac healing, and pro-regenerative recovery from MI. Full protein profiling revealed 2547 proteins present in any of the three biological replicates for any cohort. Of these, 274 were found to be unique to sEVs from aged donor hearts, 202 were unique to sEVs from aged donor plasma, and these proteins were mapped using the KEGG proteomapping. This revealed unique regulation of cell metabolism in aged cardiac sEVs and direct implication in the development of CVDs. Additionally, these proteins were assessed via MetaCore pathway analysis. Aged donor heart sEVs were involved in pathways which disrupt cardiac immunoregulation and matrix homeostasis, and promotion of pathways which have been implicated in CFB and other pathological cardiac remodeling. LNPs were successfully formed with our formulation strategy, with larger LNPs forming a mostly monodisperse (PDI < 0.3) spherical population with diameter of 200 nm, which is the maximum size for efficient delivery of LNP cargos. LNPs were successfully taken up by cardiac cells, as was previously shown using sEVs, and could be loaded with the desired compounds. The HEART model was successfully constructed integrating CMs and CFs in the device and was capable of modeling cardiac fibrosis after MI-like conditions. The LNPs successfully integrated into the HEART system and provided therapeutic effects in the post-MI model. In conclusion, we profiled the miRNA and protein content of sEVs isolated from donor hearts and age-matched plasma. These compounds were successfully integrated into LNPs and used to provide anti-fibrotic effects in a microfluidic model.

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