Engineering organ-on-a-chip systems to model viral infections.

Infectious diseases remain a public healthcare concern worldwide. Amidst the pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, increasing resources have been diverted to investigate the therapeutics targeting COVID-19 Spike glycoprotein and to develop various classes of vaccines. Most of the current investigations employ two-dimensional (2D) cell culture and animal models. However, 2D culture negates the multicellular interactions and 3D microenvironment, and animal models cannot mimic human physiology because of interspecies differences. On the other hand, organ-on-a-chip (OoC) research devices introduce a game-changer to model viral infections in human tissues, facilitating high-throughput screening of antiviral therapeutics. In this context, this review provides an overview of the in vitro OoC-based modeling of viral infection, highlighting the strengths and challenges for the future directions.

Engineering viral genomics and nano-liposomes in microfluidic platforms for patient-specific analysis of SARS-CoV-2 variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuing to spread globally, contributing to the persistence of the COVID-19 pandemic. Increasing resources have been focused on developing vaccines and therapeutics that target the Spike glycoprotein of SARS-CoV-2. Recent advances in microfluidics have the potential to recapitulate viral infection in the organ-specific platforms, known as organ-on-a-chip (OoC), in which binding of SARS-CoV-2 Spike protein to the angiotensin-converting enzyme 2 (ACE2) of the host cells occurs. As the COVID-19 pandemic lingers, there remains an unmet need to screen emerging mutations, to predict viral transmissibility and pathogenicity, and to assess the strength of neutralizing antibodies following vaccination or reinfection. Conventional detection of SARS-CoV-2 variants relies on two-dimensional (2-D) cell culture methods, whereas simulating the micro-environment requires three-dimensional (3-D) systems. To this end, analyzing SARS-CoV-2-mediated pathogenicity via microfluidic platforms minimizes the experimental cost, duration, and optimization needed for animal studies, and obviates the ethical concerns associated with the use of primates. In this context, this review highlights the state-of-the-art strategy to engineer the nano-liposomes that can be conjugated with SARS-CoV-2 Spike mutations or genomic sequences in the microfluidic platforms; thereby, allowing for screening the rising SARS-CoV-2 variants and predicting COVID-19-associated coagulation. Furthermore, introducing viral genomics to the patient-specific blood accelerates the discovery of therapeutic targets in the face of evolving viral variants, including B1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), c.37 (Lambda), and B.1.1.529 (Omicron). Thus, engineering nano-liposomes to encapsulate SARS-CoV-2 viral genomic sequences enables rapid detection of SARS-CoV-2 variants in the long COVID-19 era.

An engineered nano-liposome-human ACE2 decoy neutralizes SARS-CoV-2 Spike protein-induced inflammation in both murine and human macrophages.

Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation. Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo. Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1ß, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro, whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase ß (IKKß), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses. Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKß/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses.

Development of targeted nanoparticles loaded with antiviral drugs for SARS-CoV-2 inhibition.

Recently, a novel coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised global concerns, being the etiological agent of the current pandemic infectious coronavirus disease 2019 (COVID-19). Specific prophylactic treatments like vaccines, have been authorized for use by regulatory bodies in multiple countries, however there is an urgent need to identify new, safe, and targeted therapeutics as post-exposure therapy for COVID-19. Among a plethora of potential pharmacological targets, the angiotensin-converting enzyme 2 (ACE2) membrane receptor, which plays a crucial role in viral entry, is representing an attractive intervention opportunity for SARS-CoV-2 antiviral discovery process. In this scenario, we envisioned that binding to ACE2 by multivalent attachment of ligands to nanocarriers incorporating antiviral therapeutics, it would increase receptor avidity and impart specificity to these nanovectors for host cells, particularly in the pulmonary tract, which is the primary entry route for SARS-CoV-2. Herein, we report the design and development of novel polymeric nanoparticles (NP), densely grafted with various ligands to selectively bind to ACE2, as innovative nanovectors for targeted drug delivery. We first evaluated the impact of these biocompatible targeted NP (TNP) on ligand binding toward ACE2 and measured their competition ability vs a model of spike protein (Lipo-S1). Next, we tested the effectiveness of the most performing nanoprotopype, TNP-1, loaded with a model anti-SARS-CoV-2 drug such as remdesivir (RDV), on antiviral activity against SARS-CoV-2 infected Vero E6 cells. The RDV-TNP-1 exhibited a significantly improved antiviral effect compared to RDV at the same concentration. Interestingly, unloaded TNP (TNP-1E) also exhibited a basal antiviral activity, potentially due to a direct competitive mechanism with viral particles for the ACE2 binding site. We also measured the anti-exopeptidase activity of TNP-1E against ACE2 protein. Collectively, these insights warrant in-depth preclinical development for our nanoprototypes, for example as potential inhalable drug carriers, with the perspective of a clinical translation.

Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.

Association of COVID-19 transmission with high levels of ambient pollutants: Initiation and impact of the inflammatory response on cardiopulmonary disease.

Ambient air pollution contributes to 7 million premature deaths annually. Concurrently, the ongoing coronavirus disease 2019 (COVID-19) pandemic, complicated with S-protein mutations and other variants, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in over 2.5 million deaths globally. Chronic air pollution-mediated cardiopulmonary diseases have been associated with an increased incidence of hospitalization and mechanical ventilation following COVID-19 transmission. While the underlying mechanisms responsible for this association remain elusive, air pollutant-induced vascular oxidative stress and inflammatory responses have been implicated in amplifying COVID-19-mediated cytokine release and vascular thrombosis. In addition, prolonged exposure to certain types of particulate matter (PM2.5, d < 2.5 µm) has also been correlated with increased lung epithelial and vascular endothelial expression of the angiotensin-converting enzyme-2 (ACE2) receptors to which the SARS-CoV-2 spike glycoproteins (S) bind for fusion and internalization into host cells. Emerging literature has linked high rates of SARS-CoV-2 infection to regions with elevated levels of PM2.5, suggesting that COVID-19 lockdowns have been implicated in regional reductions in air pollutant-mediated cardiopulmonary effects. Taken together, an increased incidence of SARS-CoV-2-mediated cardiopulmonary diseases seems to overlap with highly polluted regions. To this end, we will review the redox-active components of air pollutants, the pathophysiology of SARS-CoV-2 transmission, and the key oxidative mechanisms and ACE2 overexpression underlying air pollution-exacerbated SARS-CoV-2 transmission.

Flow-Mediated Susceptibility and Molecular Response of Cerebral Endothelia to SARS-CoV-2 Infection.

BACKGROUND AND PURPOSE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with an increased rate of cerebrovascular events including ischemic stroke and intracerebral hemorrhage. The mechanisms underlying cerebral endothelial susceptibility and response to SARS-CoV-2 are unknown yet critical to understanding the association of SARS-CoV-2 infection with cerebrovascular events. METHODS: Endothelial cells were isolated from human brain and analyzed by RNA sequencing. Human umbilical vein and human brain microvascular cells were used in both monolayer culture and endothelialized within a 3-dimensional printed vascular model of the middle cerebral artery. Gene expression levels were measured by quantitative polymerase chain reaction and direct RNA hybridization. Recombinant SARS-CoV-2 S protein and S protein-containing liposomes were used to measure endothelial binding by immunocytochemistry. RESULTS: ACE2 (angiotensin-converting enzyme-2) mRNA levels were low in human brain and monolayer endothelial cell culture. Within the 3-dimensional printed vascular model, ACE2 gene expression and protein levels were progressively increased by vessel size and flow rates. SARS-CoV-2 S protein-containing liposomes were detected in human umbilical vein endothelial cells and human brain microvascular endothelial cells in 3-dimensional middle cerebral artery models but not in monolayer culture consistent with flow dependency of ACE2 expression. Binding of SARS-CoV-2 S protein triggered 83 unique genes in human brain endothelial cells including upregulation of complement component C3. CONCLUSIONS: Brain endothelial cells are susceptible to direct SARS-CoV-2 infection through flow-dependent expression of ACE2. Viral S protein binding triggers a unique gene expression profile in brain endothelia that may explain the association of SARS-CoV-2 infection with cerebrovascular events.