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

Abstract

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.

Keywords: COVID-19; Organ on-a-chip; microfluidics; mutations, variants of concerns; nano-liposomes; viral genomics.

Figure 1

Organ-on-a-chip for viral infection. Application of OoC affords an opportunity to uncover virus mutation and transmission, as well as patient-specific analyses and therapeutic targets. This illustration highlights the capacity of the integrated microfluidic platform to simulate organ-specific tissues for (1) screening the SARS-CoV-2 heterogeneity and variants, (2) identifying the population heterogeneity with different susceptibilities to SARS-CoV-2 variants, (3) detecting animal-to-human transmission (zoonosis), (4) analyzing the host immune responses, including the antibody titers against SARS-CoV-2 variants, and (5) performing bioinformatics for drug development and repurposing Adapted from , with permission from Trends Microbiol, copyright 2020. Created using BioRender.com.

Figure 2

Structural proteins of SARS-CoV-2 in microfluidic platforms. (A) Schematic structure of the Spike (S) protein in relation to envelope (E), membrane (M), and nucleocapsid (N) proteins. (B) Spike protein is composed of S₁ and S₂ subunits. The RBD of S₁ protein binds to the hACE2 receptor. (C) In response to viral infection, inflammatory cells are recruited to activate the coagulation pathways, leading to blood clot formation or known as thrombosis. The multi-array microfluidic platform enables high-throughput screening of patient-specific COVID-19-associated coagulation in response to pseudo-SARS-CoV-2 inoculation, obviating the need to perform the studies in the viral containment Biosafety Laboratory 3 (BSL3). Created using BioRender.com.

Figure 3

Lung-on-a-chip. (A) Schematic diagram of human airway chips. (B) Immunofluorescence micrographs of cells: ZO-1 shows tight junctions, cilia in the epithelium, and VE-cadherin in the junctions of the endothelium in the airway-on-a-chip without (Control) and without virus (+ Virus) for 48 h (Blue, DAPI: stained nuclei, Scale bar is 50 µm). Reproduced from , with permission from Cold Spring Harbor Laboratory. (C) Schematic diagram represents configuration of human alveolus-on-a-chip infected by SARS-CoV-2. The chip is divided into two chambers by a PDMS membrane: upper alveolar epithelial chamber (blue) and lower pulmonary microvascular endothelial chamber (red). (D) The alveolar-capillary boundary is formed by co-culture with alveolar epithelial cells (HPAEpiC) and pulmonary microvascular endothelial cells (HULEC-5a) under flow condition. The constructed alveolus chip is exposed to SARS-CoV-2 via the epithelial layer. After virus infection, human immune cells are infused into the vascular chamber. Image of the chip is shown. Reproduced from , with permission from Wiley, copyright 2020.

Figure 4

Gut-on-a-chip. The chip has two chambers separated by an ECM coated with the PDMS membrane. The upper chamber is seeded with the intestinal epithelial cells (Caco-2 and mucin-secreting HT-29 cells) and the lower chamber with human umbilical vein endothelial cells (HUVECs). SARS-CoV-2 is inoculated into the upper chamber. Reproduced from , with permission from Am J Gastroenterol, copyright 2020. Created using BioRender.com.

Figure 5

Engineering Lipo-Spike and Lipo-hACE2. (A) Fluorochromes such as coumarin-6 or rhodamine are encapsulated in the liposomes during self-assembly. Biotinylated liposomes are conjugated non-covalently (d = 100 nm) with Neutravidin protein to form Liposomes-biotinyl Cap-Neutravidin (88). (B) Next, biotinylated SARS-CoV-2 S-protein, His, Avitag (Acrobiosystem), are bound to the Liposomes-biotinyl Cap-Neutravidin, forming Liposome-Spike conjugation. hACE2 (Acrobiosystem: Biotinylated Human ACE2/ACEH Protein, Fc, Avitag AC2-H82F9-25ug) is biotinylated with Liposomes-biotinyl Cap-Neutravidin, forming liposome-hACE2 conjugation. Lipo-Spike competes with SARS-CoV-2 for the host ACE2 receptors. (C) Recombinant-Glycoprotein-S-Spikes are biotinylated to the liposomes, promoting binding to the ACE2 receptors expressed on the host cell membrane; thus, competing with SARS-CoV-2 for internalization into the host cells. (D) Lipo-ACE2 decoy competes with SARS-CoV-2 for the host ACE2 receptors. Recombinant-ACE2 proteins are biotinylated to the liposomes, promoting SARS-CoV-2 binding to the liposomes; thereby, preventing SARS-CoV-2 from binding to the host ACE2 receptors. (E) Patient-specific blood is used to predict COVID-19-Associated Coagulation. A schematic of the human artery endothelial cells (HAEC)-seeded microfluidic chip mimics liposome Spike entry into the endothelial cells. Reproduced from , , with permission from Wiley and Theranostics, copyright 2021-2022. Created using BioRender.com.

Figure 6

Engineering SARS-CoV-2 genomes to screen COVID-19-associated coagulation. (A) Sub-genomic and SARS-CoV-2 Spike sequences are cloned and inserted into the nano-liposomes. Specific regions of NSP 1-16) for 5' untranslated region (UTR) and open reading frame (ORF 3, 6, 7, 8, & 9) for 3' UTR or the CoV-2-Spike can be incorporated into the nano-liposomes that are conjugated with Spike proteins. (B) A schematic of SARS-CoV-2 viral nanoparticles highlight the incorporation of genomic sequence-specific into the nano-liposomes. (C) Nanoparticles can be infused into a multi-array microfluidic platform for screening a host of genomic sequences, including Spike mutations and variants. Shear stress activated ACE2 receptors facilitate the binding of the nanoparticles to the endothelial cells. Microfluidic channel (400 µm x 100 µm x 2 cm) is seeded with tissue-specific cell types to simulate viral interaction in an organ-specific chip. Created using BioRender.com.

Figure 7

Mutations arising from SARS-CoV-2 Variants of Concern: Omicron vs. Delta. (A) Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute to the high binding affinity to hACE2. In comparison to the Delta variant, both the Spike protein and RBD in Omicron are comprised of a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein's core to provide structural stability of the Spike protein. It is postulated that a disorder-order transition in the Omicron variant between Spike protein RBD regions 468-473 may influence the disordered residues/regions on Spike protein stability and binding to ACE2. (B) Nab targeting VoC in the microfluidic platforms. Schematic representation of VoC. Spike proteins derived from the specific VoC can be conjugated with C-Lipo encapsulated with fluorochrome . Abs from the vaccinated or recovered COVID-19 blood can be assessed for their efficiency against the specific VoC. (C) A mix of c-Lipo and Abs from blood sample can be infused into a multi-array microfluidic platform for ELISA-like detection of VoC. Fluorescent signals can be used to quantify the optimal combination of Ab candidates targeting the VoC. Abs: antibodies; VoC: Variants of Concern. Created using BioRender.com.