(250b) DNA-Directed Patterning to Validate a Liposome Model of Sars-Cov-2
AIChE Annual Meeting
Tuesday, November 9, 2021 - 8:18am to 8:36am
Lipid-based nanoparticles, or liposomes, have found wide applications in combatting the COVID-19 pandemic. Most notably, lipid-based nano-formulations encapsulate mRNA in the recently-approved COVID-19 vaccines from Moderna and Pfizer-BioNTech . As enveloped viruses have been shown to fuse with liposomes , lipid-based nanoparticles have been suggested as models of enveloped viruses themselves [3, 4]. Based on their ability to display surface antigens, liposomes have been used to model the immune response to antigens  and to trigger a T-cell response to SARS-CoV . These lipid-based nanoparticle applications arose from the demands of an urgent health crisis and necessitated high-throughput, highly flexible methods for nanoparticle characterization.
We have developed a novel, high-throughput method, DNA-directed patterning , to validate lipid-based nanoparticles. This method relies on lithographically patterning single-stranded oligonucleotides on a glass substrate and subsequently hybridizing the complementary oligonucleotides to which liposomes have been tagged, ultimately leading to the controlled and precise patterning of liposomes. Through the development of S-liposomesâan ideal, safe model of SARS-CoV-2 that preserves its most basic features and function as a lipid bilayer nanoparticle that displays the SARS-CoV-2 spike (S) proteinâwe demonstrate the rapid characterization that DNA-directed patterning provides .
Liposomes were fabricated by rehydrating lipid thin films (dioleoyl phosphatidyl choline, dioleoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl glycerol acid, and cholesterol in a 4:3:2:7; 1,2-dioleoyl-sn-glycero-3-((N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl) (nickel salt) at 5 mol%.; DiO, DiI, or DiD at 1 mol%) followed by sequential extrusion and labeling with either a His-tagged SARS-CoV-2 spike protein or a His-tagged CD63.
Amine-terminated single-stranded 20 nt DNA oligos were immobilized on glass substrates by selectively exposing regions of a Shipley 1813 photoresist-coated slide to UV light using a photomask and dropcasting the appropriate oligonucleotide solution. Following reductive amination with 0.25% sodium borohydride, photoresist could be stripped and reapplied to iteratively pattern subsequent oligonucleotide sequences for multi-component patterns.
Liposomes were labeled with an anchor DNA oligonucleotide containing a cholesterol molecule that intercalated in the membrane as well as an adaptor sequence that facilitated hybridization with the surface patterned oligo. This anchor was stabilized using a complementary co-anchor oligo-cholesterol molecule.
Liposome-associated cholesterol-oligonucleotide tags were hybridized to complementary oligonucleotides patterned on a glass substrate surface. To verify the presence of spike protein on the liposomes, biotinylated ACE2 (ACROBiosystems) in 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS was introduced and incubated for 1 hr at room temperature followed by Cy5-labeled streptavidin (Invitrogen) in 2% BSA (incubated for 45 minutes at room temperature). For neutralization assays, prior to ACE2 incubation, different concentrations of anti-SARS-CoV-2 receptor binding domain neutralizing antibody (range: 0-100 µg mL-1; Human IgG1, ACROBiosystems) in 2% BSA were incubated with the patterned liposomes for 1 hr. Internalization assays were performed by incubating liposomes with 293T cells engineered to express ACE2 followed by time-lapse microscopy.
RESULTS AND DISCUSSION
Following the fabrication of S-liposomes, we utilized DNA-directed patterning to validate our ability to fluorescently label and visualize these lipid nanoparticles. We mixed three labeled lipid-based nanoparticles and flowed the mixture across the pre-patterned substrate. The complementary oligonucleotide tags hybridized to the patterned oligonucleotides, leading to distinctive patterns of labeled liposomes.
To verify that the S trimer was oriented correctly using DNA-directed patterning, we patterned fluorescent S-liposomes onto a glass substrate in a 50 x 50 array of squares, each 141 Âµm2 in size. To verify binding specificity, we measured fluorescence for conditions with intermediate components removed (i.e. no oligonucleotide tag, liposomes without the S protein, no addition of biotinylated ACE2). The condition which included all components showed significantly higher fluorescence (p<0.0001, one-way ANOVA with Tukeyâs multiple comparison test) than those of the controls in which each component of the binding chemistry was systematically removed.
Using S-liposomes and CD63-liposomes labeled with different oligo sequences and dyes, we showed that specificity of Watson-Crick base pairing allowed for quick, specific, and simultaneous separation of the tagged liposomes to distinct patterns. After patterning, the introduction of biotinylated ACE2 followed by Cy5-streptavidin allowed us to measure relative ACE2 binding, which occurred only in the presence of S-liposomes and not in the presence of CD63-liposomes (p < 0.0001).
We then employed DNA-directed patterning to assess the ability of a commercially available neutralizing antibody (clone: HTS0483) to interfere with ACE2 binding. We patterned fluorescent S-liposomes and incubated them with different concentrations (0-100 µg mL-1) of neutralizing antibody. As expected, the concentration of neutralizing antibody inversely correlated with Cy5 intensity.
To confirm that S-liposomes, as validated by DNA-directed patterning, exhibit biological function, we investigated the ability of ACE2-expressing cells to internalize these lipid nanoparticles. We seeded cells transduced to express ACE2 (293T+ACE2) and visualized internalization of fluorescent liposomes without surface protein (plain) or with S or CD63. To compare the effects of the neutralizing antibody in this system against our DNA-directed patterning assay, we next added a range (0-10 µg mL-1) of neutralizing antibody concentrations with fluorescent S-liposomes to 293T+ACE2 cells. As expected, we found that increasing concentrations of antibody led to decreasing fluorescence, indicating successful measurement of antibody interference with the interaction between the S-liposomes and ACE2.
In conclusion, we have demonstrated a flexible, easy-to-use, high-throughput methodâDNA-directed patterningâto validate and characterize lipid-based nanoparticles, in this case, a model of SARS-CoV-2. The flexibility of DNA-directed patterning of these lipid bilayer nanoparticles enables high-throughput studies and/or screening capabilities against liposomes displaying different surface proteins or variants of a surface protein, all simultaneously on a single substrate. An attractive feature of our method is that it uses entirely off-the-shelf componentsâoligonucleotides of any 20-nucleotide sequence can be readily purchased. Moreover, multiple surface proteins that could be used to target liposomes containing therapeutics or the release of such therapeutics could be characterized or tested in a multiplexed way. Taken together, S-liposomes and DNA-directed patterning can readily be adapted and customized to facilitate the development of new diagnostics and therapeutics for SARS-CoV-2. Looking beyond the current pandemic, DNA-directed patterning of liposomes can be applied in the rapid formulation and implementation of a wide variety of custom assays for rapid implementation and read-out, including competitive binding, drug targeting, and particle-cell interactions.
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