Protein-based nanoparticles for therapeutic nucleic acid delivery.

To realize the full potential of emerging nucleic acid therapies, there is a need for effective delivery agents to transport cargo to cells of interest. Protein materials exhibit several unique properties, including biodegradability, biocompatibility, ease of functionalization via recombinant and chemical modifications, among other features, which establish a promising basis for therapeutic nucleic acid delivery systems. In this review, we highlight progress made in the use of non-viral protein-based nanoparticles for nucleic acid delivery in vitro and in vivo, while elaborating on key physicochemical properties that have enabled the use of these materials for nanoparticle formulation and drug delivery. To conclude, we comment on the prospects and unresolved challenges associated with the translation of protein-based nucleic acid delivery systems for therapeutic applications.

Combinatorial development of nebulized mRNA delivery formulations for the lungs.

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

Differential modulation of endothelial cytoplasmic protrusions after exposure to graphene-family nanomaterials.

Engineered nanomaterials offer the benefit of having systematically tunable physicochemical characteristics (e.g., size, dimensionality, and surface chemistry) that highly dictate the biological activity of a material. Among the most promising engineered nanomaterials to date are graphene-family nanomaterials (GFNs), which are 2-D nanomaterials (2DNMs) with unique electrical and mechanical properties. Beyond engineering new nanomaterial properties, employing safety-by-design through considering the consequences of cell-material interactions is essential for exploring their applicability in the biomedical realm. In this study, we asked the effect of GFNs on the endothelial barrier function and cellular architecture of vascular endothelial cells. Using micropatterned cell pairs as a reductionist in vitro model of the endothelium, the progression of cytoskeletal reorganization as a function of GFN surface chemistry and time was quantitatively monitored. Here, we show that the surface oxidation of GFNs (graphene, reduced graphene oxide, partially reduced graphene oxide, and graphene oxide) differentially affect the endothelial barrier at multiple scales; from the biochemical pathways that influence the development of cellular protrusions to endothelial barrier integrity. More oxidized GFNs induce higher endothelial permeability and the increased formation of cytoplasmic protrusions such as filopodia. We found that these changes in cytoskeletal organization, along with barrier function, can be potentiated by the effect of GFNs on the Rho/Rho-associated kinase (ROCK) pathway. Specifically, GFNs with higher surface oxidation elicit stronger ROCK2 inhibitory behavior as compared to pristine graphene sheets. Overall, findings from these studies offer a new perspective towards systematically controlling the surface-dependent effects of GFNs on cytoskeletal organization via ROCK2 inhibition, providing insight for implementing safety-by-design principles in GFN manufacturing towards their targeted biomedical applications.

Endothelial extracellular vesicles contain protective proteins and rescue ischemia-reperfusion injury in a human heart-on-chip.

Extracellular vesicles (EVs) derived from various stem cell sources induce cardioprotective effects during ischemia-reperfusion injury (IRI). These have been attributed mainly to the antiapoptotic, proangiogenic, microRNA (miRNA) cargo within the stem cell-derived EVs. However, the mechanisms of EV-mediated endothelial signaling to cardiomyocytes, as well as their therapeutic potential toward ischemic myocardial injury, are not clear. EV content beyond miRNA that may contribute to cardioprotection has not been fully illuminated. This study characterized the protein cargo of human vascular endothelial EVs (EEVs) to identify lead cardioactive proteins and assessed the effect of EEVs on human laminar cardiac tissues (hlCTs) exposed to IRI. We mapped the protein content of human vascular EEVs and identified proteins that were previously associated with cellular metabolism, redox state, and calcium handling, among other processes. Analysis of the protein landscape of human cardiomyocytes revealed corresponding modifications induced by EEV treatment. To assess their human-specific cardioprotection in vitro, we developed a human heart-on-a-chip IRI assay using human stem cell-derived, engineered cardiac tissues. We found that EEVs alleviated cardiac cell death as well as the loss in contractile capacity during and after simulated IRI in an uptake- and dose-dependent manner. Moreover, we found that EEVs increased the respiratory capacity of normoxic cardiomyocytes. These results suggest that vascular EEVs rescue hlCTs exposed to IRI possibly by supplementing injured myocytes with cargo that supports multiple metabolic and salvage pathways and therefore may serve as a multitargeted therapy for IRI.

Quantifying the effects of engineered nanomaterials on endothelial cell architecture and vascular barrier integrity using a cell pair model.

Engineered nanomaterials (ENMs) are increasingly used in consumer products due to their unique physicochemical properties, but the specific hazards they pose to the structural and functional integrity of endothelial barriers remain elusive. When assessing the effects of ENMs on vascular barrier function, endothelial cell monolayers are commonly used as in vitro models. Monolayer models, however, do not offer a granular understanding of how the structure-function relationships between endothelial cells and tissues are disrupted due to ENM exposure. To address this issue, we developed a micropatterned endothelial cell pair model to quantitatively evaluate the effects of 10 ENMs (8 metal/metal oxides and 2 organic ENMs) on multiple cellular parameters and determine how these parameters correlate to changes in vascular barrier function. This minimalistic approach showed concerted changes in endothelial cell morphology, intercellular junction formation, and cytoskeletal organization due to ENM exposure, which were then quantified and compared to unexposed pairs using a "similarity scoring" method. Using the cell pair model, this study revealed dose-dependent changes in actin organization and adherens junction formation following exposure to representative ENMs (Ag, TiO2 and cellulose nanocrystals), which exhibited trends that correlate with changes in tissue permeability measured using an endothelial monolayer assay. Together, these results demonstrate that we can quantitatively evaluate changes in endothelial architecture emergent from nucleo-cytoskeletal network remodeling using micropatterned cell pairs. The endothelial pair model therefore presents potential applicability as a standardized assay for systematically screening ENMs and other test agents for their cellular-level structural effects on vascular barriers.

Mussel-inspired 3D fiber scaffolds for heart-on-a-chip toxicity studies of engineered nanomaterials.

Due to the unique physicochemical properties exhibited by materials with nanoscale dimensions, there is currently a continuous increase in the number of engineered nanomaterials (ENMs) used in consumer goods. However, several reports associate ENM exposure to negative health outcomes such as cardiovascular diseases. Therefore, understanding the pathological consequences of ENM exposure represents an important challenge, requiring model systems that can provide mechanistic insights across different levels of ENM-based toxicity. To achieve this, we developed a mussel-inspired 3D microphysiological system (MPS) to measure cardiac contractility in the presence of ENMs. While multiple cardiac MPS have been reported as alternatives to in vivo testing, most systems only partially recapitulate the native extracellular matrix (ECM) structure. Here, we show how adhesive and aligned polydopamine (PDA)/polycaprolactone (PCL) nanofiber can be used to emulate the 3D native ECM environment of the myocardium. Such nanofiber scaffolds can support the formation of anisotropic and contractile muscular tissues. By integrating these fibers in a cardiac MPS, we assessed the effects of TiO2 and Ag nanoparticles on the contractile function of cardiac tissues. We found that these ENMs decrease the contractile function of cardiac tissues through structural damage to tissue architecture. Furthermore, the MPS with embedded sensors herein presents a way to non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. These results demonstrate the utility of our MPS as an analytical platform for understanding the functional impacts of ENMs while providing a biomimetic microenvironment to in vitro cardiac tissue samples. Graphical Abstract Heart-on-a-chip integrated with mussel-inspired fiber scaffolds for a high-throughput toxicological assessment of engineered nanomaterials.

Cardiac microphysiological devices with flexible thin-film sensors for higher-throughput drug screening.

Microphysiological systems and organs-on-chips promise to accelerate biomedical and pharmaceutical research by providing accurate in vitro replicas of human tissue. Aside from addressing the physiological accuracy of the model tissues, there is a pressing need for improving the throughput of these platforms. To do so, scalable data acquisition strategies must be introduced. To this end, we here present an instrumented 24-well plate platform for higher-throughput studies of engineered human stem cell-derived cardiac muscle tissues that recapitulate the laminar structure of the native ventricle. In each well of the platform, an embedded flexible strain gauge provides continuous and non-invasive readout of the contractile stress and beat rate of an engineered cardiac tissue. The sensors are based on micro-cracked titanium-gold thin films, which ensure that the sensors are highly compliant and robust. We demonstrate the value of the platform for toxicology and drug-testing purposes by performing 12 complete dose-response studies of cardiac and cardiotoxic drugs. Additionally, we showcase the ability to couple the cardiac tissues with endothelial barriers. In these studies, which mimic the passage of drugs through the blood vessels to the musculature of the heart, we regulate the temporal onset of cardiac drug responses by modulating endothelial barrier permeability in vitro.