www.acsami.org Research Article OligoBinders: Bioengineered Soluble Amyloid-like Nanoparticles to Bind and Neutralize SARS-CoV‑2 Molood Behbahanipour, Roger Benoit, Susanna Navarro,* and Salvador Ventura* Cite This: ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become a primary health concern. Molecules that prevent viral entry into host cells by interfering with the interaction between SARS-CoV-2 spike (S) protein and the human angiotensin-converting enzyme 2 receptor (ACE2r) opened a promising avenue for virus neutralization. Here, we aimed to create a novel kind of nanoparticle that can neutralize SARS-CoV-2. To this purpose, we exploited a modular self-assembly strategy to engineer OligoBinders, soluble oligomeric nanoparticles decorated with two miniproteins previously described to bind to the S protein receptor binding domain (RBD) with high affinity. The multivalent nanostructures compete with the RBD−ACE2r interaction and neutralize SARS-CoV-2 virus-like particles (SC2-VLPs) with IC50 values in the pM range, preventing SC2-VLPs fusion with the membrane of ACE2r-expressing cells. Moreover, OligoBinders are biocompatible and significantly stable in plasma. Overall, we describe a novel protein-based nanotechnology that might find application in SARS-CoV-2 therapeutics and diagnostics. KEYWORDS: SARS-CoV-2, spike protein, coronavirus, nanoparticles, soluble oligomers, protein assemblies, virus inactivation, antiviral agents ■ INTRODUCTION The coronavirus disease 2019 (COVID-19) has become a global muscle, and thyroid gland.4−7 As the first step in the viral replication cycle, developing neutralizing agents that interfere pandemic and one of humanity’s major health challenges. with the S protein−ACE2r interaction has gained significant Although it seems that the most substantial impact of the interest. These reagents include antibodies, peptides, small pandemic has already happened, as of September 30, 2022, there molecules, and DNA aptamers.8,9 However, except for antibod- were still over 600 million confirmed cases worldwide, including ies, these molecules tend to exhibit moderate binding affinity. 6.5 million deaths documented (https://covid19.who.int/). High-affinity monoclonal antibodies (mAbs) developed Moreover, COVID-19 has been reported to be related to an utilizing B cells from COVID-19 patients have been at the increasing prevalence of anxiety and depression,1 and suggested forefront of neutralizing therapies.10−13 However, monomeric to be connected with neurodegenerative disorders.2 antibodies suffer from rapid clearance and short lung retention The rapid development of mRNA vaccines to protect against time,14,15 which may restrict the efficacy of mAb-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) treatments, particularly for local delivery. Furthermore, clinical has rendered the most effective therapy against COVID-19. experiences showed that effective treatments of COVID-19 However, although they elicit a robust antibody response to viral require a high density of inhibitory domains to maximize proteins, after the dose, the antibody levels decline six months blockage efficiency.16 Nanoparticle-based strategies provide postvaccination, indicating a waning of the immune response alternatives to using mAbs alone for virus capture and over time.3 neutralization, because they exhibit longer retention times and The intense research effort during these past two years has allow high-density clustering of binding domains.11,17−19 allowed us to gain comprehensive knowledge of the disease biology and the structural determinants responsible for SARS- CoV-2 internalization in human cells. The virus entry in host Received: October 14, 2022 cells is mediated by the interaction between the receptor binding Accepted: February 9, 2023 domain (RBD) from viral spike (S) protein and the cell surface Published: February 22, 2023 angiotensin-converting enzyme 2 receptor (ACE2r) expressed by nasal ciliated cells, pneumocytes, exocrine pancreas, intestinal tract, seminal vesicle, epididymis, proximal renal tubules, heart © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsami.2c18305 11444 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 1. Schematic representation and sequence of Sup35-DHFR-LCB1/LCB3 fusion proteins. (A, B) Cartoon representation and sequence of Sup35-DHFR-LCB1/LCB3 fusion proteins. The SAC (residues 100−118 of Sup35 protein) fused to DHFR followed by LCB1/LCB3 SARS-CoV-2- RBD minibinders (PDB: 7JZU and 7JZM) are shown in red, gray, and blue, respectively. The three different moieties are linked by S/G linkers shown in green. Amyloid-inspired nanomaterials are gaining momentum in IC50 values ranging from 24 pM16 to 14 nM,31 depending on the nanotechnology because of their modularity, controlled self- assay. assembly, stability, biocompatibility, and high surface/volume The employed modular approach rendered two types of ratios.20−26 A key benefit of these protein-based materials is the spherical particles that share an amyloid-like nature but expose possibility to incorporate the desired functionality through different folded and active minibinder domains, recognizing the straightforward genetic redesign, provided that the integrated S protein with half-maximal inhibitory concentration (IC50) domain remains folded and functionally active upon self- values in the pM range. Accordingly, these multivalent and high- assembly. Despite these advantages, the large majority of density decorated nanostructures neutralized the fusion of developed amyloid-based materials correspond to large, SARS-CoV-2 virus-like particles (SC2-VLPs) to the membrane insoluble, and rigid fibrils of variable length, making them not of ACE2r-expressing human cells with very high potency. Our suitable for biomedical applications that require the action of the OligoBinders are biocompatible, stable, effective, and easily embedded protein activity to occur in human body fluids, such produced, purified, and assembled, representing a conceptually as in circulating SARS-CoV-2 virus capture, an application in novel and convenient alternative to abrogate SARS-CoV-2 which spherical nanoparticles functionalized with either ACE2 internalization into human tissues. recombinant proteins or neutralizing antibodies are becoming an alternative to the use of individual mAbs.17 In this study, we took advantage of our expertise in protein design to develop highly pure and homogeneous spherical ■ RESULTS AND DISCUSSION Design and Conformational Characterization of nanoparticles of an amyloid-like nature able to block the Soluble Sup35-DHFR-LCB Proteins. Soluble oligomeric interaction between the SARS-CoV-2 spike-RBD protein and amyloid-like particles have advantages for in vivo applications the host receptor ACE2, which we named OligoBinders. We relative to the insoluble and infinite assemblies formed by exploited the self-assembly ability of the soft amyloid core amyloid fibrils. Both structures share an intermolecular β-sheet (SAC) of Sup35 protein27−29 and the steric impediment architecture,32 and their formation is governed by the same type imposed by an adjacent globular domain30 to generate of contacts, with hydrophobicity, net charge, and secondary biocompatible soluble and stable oligomers of defined size, structure propensity being the main intrinsic properties which we engineered to display the de novo designed protein determining the amyloid propensity.33 In this scenario, because minibinders LCB1 and LCB3 in a favorable orientation. These fibrils constitute a thermodynamic minimum,34,35 obtaining two domains were developed by David Baker’s lab, inspired by homogeneous and stable oligomers that do not evolve into fibrils the minimal part of the ACE2r, but designed entirely from with time is challenging.36,37 Some natural proteins form fibrils scratch.16 They are small, highly stable, all-α domains consisting in which globular domains hang from the amyloid core,38 but a of 3 α-helices (Figure 1A,B) through broad shape comple- long linker always separates the two regions to avoid steric mentary interfaces mediated by two of the three α-helices. The repulsion, which can prevent the formation of the in-register LCB1 and LCB3 binding sites, like in ACE2, are hidden in the cross-β amyloid fibrils. Recently, a general relationship between closed S conformational state and need at least two RBDs to the size of the globular domains and the length of the linkers that open to allow simultaneous recognition of all three binding sites. would allow infinite fibril growth was established.39 Using this LCB1 and LCB3 form many hydrogen bonds and salt bridges relationship, we proposed that if the linker between the amyloid with the RBD, consistent with their high potency, displaying and the globular sequences is short enough but still flexible, 11445 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 2. Biophysical characterization of soluble Sup35-DHFR-LCB1/LCB3 fusion proteins. Far-UV CD spectra of Sup35-DHFR-LCB1 (A) and Sup35-DHFR-LCB3 (E) were recorded from 200−260 nm at 25 °C in PBS pH 7.4. Normalized thermal denaturation curves of Sup35-DHFR-LCB1 (B) and Sup35-DHFR-LCB3 (F) were monitored by following the CD signal at 222 nm from 20 to 80 °C in PBS pH 7.4. Intrinsic Trp fluorescence emission spectra of Sup35-DHFR-LCB1 (C) and Sup35-DHFR-LCB3 (G) were measured in the range 300−400 nm at 25 °C after excitation at 280 nm. Normalized intrinsic fluorescence of Trp residues for Sup35-DHFR-LCB1 (D) and Sup35-DHFR-LCB3 (H) were recorded at 350 nm in the 25− 90 °C temperature range using 280 nm excitation in PBS pH 7.4. Soluble Sup35-DHFR-LCB1 and Sup35-DHFR-LCB3 proteins were prepared at 10 μM in PBS. 11446 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 3. Biophysical characterization of oligomeric Sup35-DHFR-LCB1/LCB3 fusion proteins. (A, D) Fluorescence emission spectra of Th-T were recorded upon 445 nm excitation in the absence (green line) and the presence of 20 μM monomeric (black) and oligomeric (blue) proteins. (B, E) CR normalized absorbance spectra were recorded in the range 375−700 nm in the absence (red line) and the presence of 20 μM monomeric (black) and oligomeric (blue) proteins. (C, F) The secondary structure of oligomeric particles was derived from the deconvolution of the second-derivative ATR- FTIR absorbance spectra in the amide I region (solid black line); the fitted individual bands are indicated. Amyloid-like intermolecular β-sheet and α- helical components are shown in blue and brown, respectively. these molecules will not assemble into amyloid fibrils; instead, affinity chromatography (Ni-NTA chromatography) with a they would form oligomers with a defined and limited size.40 yield of 83 and 134 mg/L, respectively (Figures S1A,B and Using full-atom and coarse-grained targeted molecular S2A,B). dynamics together with rigid body simulations, we rationalized The secondary structure of soluble recombinant Sup35- that fusing the 19-residue SAC of the Sup35 protein (residues DHFR-LCB1/LCB3 proteins was analyzed by far-UV circular 100−118) to a globular protein of ∼20 kDa through a short 5- dichroism (CD) in the 200−260 nm range (Figure 2A,E). For residue Gly/Ser (SGSGS) flexible linker would sterically hinder both molecules, α-helical signals at 208 and 222 nm were the formation of a fibrillar zipper and result in the assembly of evident. This is consistent with α-helix conformations amyloid-like oligomers of defined dimensions in which the embedded protein can remain in a folded conformation.40 accounting for 35% and 38% of the secondary structure in Subsequently, we demonstrated the accuracy of these LCB1- and LCB3-containing constructs, respectively (Figure 1). predictions using dihydrofolate reductase (DHFR) as a model Next, the tryptophan (Trp) intrinsic fluorescent signal was used protein, with the Sup35-DHFR fusion forming enzymatically as a probe for the Sup35-DHFR-LCB1/LCB3 tertiary structure. active oligomeric structures.27 For both proteins, the intrinsic fluorescence emission spectra The above-described strategy permits the generation of showed a maximum at ∼345 nm, indicative of Trp residues tailored spherical nanoparticles for different purposes. With the being partially hidden from the solvent as expected for the native intention of targeting and neutralizing SARS-CoV-2, we conformations of DHFR and LCB1 (as a note, LCB3 does not incorporated LCB1 or LCB3 minibinders at the C-terminus of have Trp) (Figure 2C,G). Sup35-5aa-DHFR construct. LCB molecules are de novo The stability of soluble Sup35-DHFR-LCB1/LCB3 proteins designed small and stable proteins that can tightly bind SARS- against thermal unfolding was evaluated by monitoring the CoV-2-S-RBD.16 Therefore, the resulting plasmid encoded a changes in CD and fluorescence emission upon heating. The CD tripartite fusion construct consisting of Sup35-SAC, DHFR, and signal was measured at 222 nm, to report on α-helical secondary LCB1/LCB3 domains, separated by Gly/Ser-based linkers. The primary sequence and cartoon representation of Sup35-DHFR- structure (Figure 2B,F) and the fluorescence signal at 350 nm to LCB1 and Sup35-DHFR-LCB3 constructs are detailed in Figure monitor changes in the local environment of Trp residues 1A,B. (Figure 2D,H). Sup35-DHFR-LCB1/LCB3 proteins’ thermal The two Sup35-DHFR-LCB1/LCB3 monomeric proteins denaturation curves evidenced cooperative but multistate were recombinantly expressed and purified from the soluble unfolding profiles, as expected for multidomain fusions, with cellular fraction of Escherichia coli BL21 cells using metal ion overall mid transitions occurring at ∼50 °C. 11447 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Overall, the biophysical analysis of soluble Sup35-DHFR- oligomers, despite their amyloid-like features, the majority of LCB1/LCB3 proteins suggested a folded conformation for the which would necessarily correspond to the LCB domains, purified polypeptides. suggesting that they were folded correctly and potentially active Sup35-DHFR-LCB1/LCB3 Spontaneously Self-Assem- in the oligomeric state. bles into Amyloid-like Oligomers. Monomeric Sup35- Sup35-DHFR-LCB1/LCB3 Oligomers are Spherical DHFR-LCB1/LCB3 proteins were incubated at an initial Nanoparticles. To investigate the macromolecular nature of concentration of 150 μM in 20 mM sodium phosphate buffer Sup35-DHFR-LCB1/LCB3 amyloid-like soluble oligomers, pH 8 for 4 days at 37 °C. To obtain a pure oligomeric fraction, they were imaged by transmission electron microscopy highly aggregated and monomeric species were removed at the (TEM) (Figure 4). Samples were deposited onto copper grids end of the reaction. To this aim, samples were first ultra- and negatively stained with 2% uranyl acetate. Representative centrifuged to remove insoluble aggregates and later filtered TEM images evidenced the presence of abundant isolated through a 100 kDa filter to eliminate nonassembled monomers. spherical nanoparticles of 21/22 nm in diameter (Figure 4A,C, By absorbance measurements, an oligomeric yield of 93% and Table 2). 57% was estimated, relative to the initial protein concentration The homogeneity and size distribution of the nanoparticles in the reaction for LCB1 and LCB3 protein fusions, respectively. was then analyzed by dynamic light scattering (DLS) (Figure The purity of the oligomers was confirmed by native-PAGE 4B,D). In good agreement with TEM images, DLS measure- (Figures S1C and S2C). ments rendered an average size of 20/22 nm, with 99.9% The amyloid-like nature of the purified assemblies was homogeneity and a polydispersity index of 0.3 for both proteins assessed by monitoring the binding to the amyloid-specific dye (Table 2). We named the highly homogeneous spherical thioflavin-T (Th-T) (Figure 3). The oligomeric Sup35-DHFR- nanoparticles displaying the LCB1 and LCB3 domains as LCB1/LCB3 fraction exhibited a significant fluorescence signal, OligoBinder-1 and OligoBinder-3, respectively. whereas their soluble counterparts had negligible Th-T binding The molecular weight (MW) for OligoBinder-1 and Oligo- (Figure 3A,D). The Congo red (CR) dye was used to Binder-3, as derived from the mass distribution DLS data, was corroborate the amyloid nature of the assemblies. In agreement estimated using an empirical mass vs size calibration curve, with Th-T data, the CR absorbance spectrum displayed a red- obtaining average sizes of 731 and 791 kDa, respectively. shift to ∼540 nm typical of amyloid binding for both oligomeric Considering the MW of their monomeric constituents, Oligo- fractions, which was absent in the respective soluble forms Binder-1 and OligoBinder-3 were calculated to consist of 25 and (Figure 3B,E). 27 subunits on average, respectively. Next, we assessed these assemblies’ secondary structure We tested the stability of the two nanoparticles when content, employing attenuated total reflectance Fourier-trans- incubated at different pHs. The size of OligoBinder-1 and form infrared (ATR-FTIR) spectroscopy. FTIR spectra were OligoBinder-3 before and after 24 h of incubation was evaluated recorded in the amide I region (1700−1600 cm−1), correspond- at pH 6.5, 7.0, 7.5, and 8.0 by DLS (Figure S3) and TEM (Figure ing to the absorption of the main chain carbonyl group. The S4). Except for OligoBinder-3 at pH 6.5, for which a discrete maxima of the deconvoluted curves under the absorbance increase in size was observed, no changes in the diameter of the spectra allowed for correlating the wavenumber with defined particles were evidenced under any of the conditions. To further secondary structure elements (Figure 3C,F, Table 1). confirm that oligomeric nanoparticles were not disassembled during incubation, samples incubated for 24 h at the different Table 1. Secondary Structure Assignments of Amide I Band pHs were loaded onto native-PAGE gels (Figure S3). The Components in Purified Assemblies by ATR-FTIR analysis confirmed the absence of monomeric molecules for Spectroscopya both OligoBinders in all assayed conditions. Consistent with the DLS data, a high-molecular-weight band was observed for Sup35-DHFR- Sup35-DHFR- LCB1 LCB3 OligoBinder-3 at pH 6.5, indicating partial association at this wavenumber area wavenumber area pH. This likely responds to the fact that the pI of LCB3 (5.9) is band assignment (cm−1) (%) (cm−1) (%) higher than that of LCB1 (5.3). intermolecular β- 1622.1 13% 1620.6 16% OligoBinders Effectively Compete with the Spike sheet RBD−ACE2 Receptor Interaction. The interaction between intramolecular β- 1637.2 23% 1635.4 25% the RBD from viral S glycoprotein homotrimers and cellular sheet α-helix 1652.4 31% 1650.2 29% ACE2 receptors is indispensable for SARS-CoV-2 to enter the β-turn/loop + 1666.3 19% 1665 19% host cell. Accordingly, it is assumed that molecules able to turn compete with S protein−ACE2r interaction would exert a β-sheet 1680.8 13% 1680.6 10% neutralizing effect. To elucidate the binding capacity of our a The percentage of contribution to the total area of the absorbance multivalent bioengineered particles, OligoBinder-1 and Oligo- spectra is indicated for the different types of secondary structure Binder-3 were immobilized onto a membrane, in the elements, as estimated from the deconvolution of the spectra shown concentration range from 0.04 to 0.4 μg, and incubated with in Figure 3C,F. recombinantly produced S protein RBD, N-terminally tagged with YFP (0.5 ng/μL),41 as a fluorescent reporter (YFP-S- As was expected from the dye binding properties, we could RBD). As shown in Figure 5, OligoBinders can bind S-RBD detect a signal at 1621/1622 cm−1, which can be assigned to the protein in a concentration-dependent manner. In order to intermolecular β-sheet structure characteristic of amyloids. discard potential unspecific binding of YFP-S-RBD to non-LCB Importantly, in addition to the band at 1635/1637 cm−1, domains in the fusion, a construct containing Sup35-SAC fused attributable to the native β-sheet in the DHFR moiety, we to DHFR and the Z-domain (Sup35-DHFR-Z) was produced. identified a band accounting for 29−30% of the spectra at 1650/ The Z-domain is an engineered analogue of the B domain of 1652 cm−1 indicative of an α-helical conformation in the Staphylococcus aureus,42 selected because of its size, about 6.5 11448 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 4. Morphology characterization and size distribution of Sup35-DHFR-LCB1/LCB3 oligomeric assemblies. Transmission electron micrographs of negatively stained Sup35-DHFR-LCB1 (A) and Sup35-DHFR-LCB3 (C) oligomer solutions. The scale bar is 200 nm in size. Size distribution of Sup35-DHFR-LCB1 (B) and Sup35-DHFR-LCB3 (D) oligomeric assemblies characterized by DLS. Table 2. Size of Purified Amyloid Oligomeric Nanoparticles to the target in this qualitative assay. As a trend, OligoBinder-1 Determined by TEM Micrographs and DLSa dots exhibit a higher fluorescent signal than OligoBinder-3. However, the differences in intensities are only statistically nanoparticle TEM (nm) DLS (nm) significant at the highest tested nanoparticle quantities. OligoBinder-1 21.6 ± 4.7 20.3 ± 5.8 Next, for quantifying the interaction between S-RBD and OligoBinder-3 21.0 ± 6.4 21.9 ± 8.0 ACE2r in the presence of OligoBinder-1 and OligoBinder-3, we a The diameter of particles visualized by TEM was determined by used a homogeneous bioluminescent immunoassay based on the ImageJ software [mean ± standard deviation (SD)], and DLS mean interaction between Fc-tagged SARS-CoV-2 spike RBD and size values ± SD were obtained by considering the volume-based human ACE2r, and its detection by secondary antibodies distribution. labeled with NanoLuc luciferase fragments LgBit and SmBit.43 Briefly, the interaction of S-RBD and ACE2r, incubated with kDa, and its fold. The Z-domain adopts a helical bundle-like their corresponding secondary antibodies bearing one subunit of structure formed by 3 α-helices topologically equivalent to those NanoBiT Luciferase, reconstitutes the active enzyme to generate of LCB1 and LCB3 (Figure S5). light (Figure 6A). To guarantee that Sup35-DHFR-Z constructs share a similar In the presence of neutralizing compounds, the luminescent oligomeric disposition, they were biophysically characterized signal decreases inversely proportional to the strength of the S- (Figure S5). The Z-domain is an antibody high-affinity binder, RBD−molecule interaction, since this contact competes with and as such, in contrast to oligomers decorated with LCB1 and the S-RBD−ACE2r one and impedes luciferase reconstitution. LCB3 minibinders, Sup35-DHFR-Z nanoparticles did not retain In this way, serially diluted OligoBinder-1 and OligoBinder-3 any fluorescent signal upon incubation with YFP-S-RBD, were incubated with SARS-CoV-2 RBD-rabbit Fc (rFc-RBD) pointing to the specificity of the LCB1 and LCB3 domains and human ACE2r-mouse Fc (mFc-ACE2), followed by the displayed in OligoBinders as the moieties recognizing the viral S addition of an antibody mix containing anti-mouse Ab-LgBiT protein RBD domain (Figure 5). To validate this extent, we and anti-rabbit Ab-SmBiT and revealed with the Lumit assayed as positive control a rabbit polyclonal antibody raised detection reagent. OligoBinder-1 and OligoBinder-3 showed against SARS-CoV-2 spike (IC50 for RBD binding: 5.4−11.4 IC50 values of 57.2 and 49.4 pM in this assay, respectively nM); as expected, binding of YFP-S-RBD to the antibody in the (Figure 6B). These values are in the same range as or better than membrane was observed. However, when used at the same the ones recorded with the same assay for four different concentration, the fluorescent signal was lower than that for investigational anti-SARS-CoV-2 spike RBD antibodies of OligoBinders, which suggests that the nanoparticles bind better chimeric mouse/human (Sino Biological 40150-D001 and 11449 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 5. Functional assessment of amyloid oligomeric nanoparticles. (A) Dot blot of amyloid OligoBinder-1 and OligoBinder-3 nanoparticles in different amounts (0.40, 0.20, 0.10, and 0.04 μg), loaded onto a membrane. Negative control corresponds to the amyloid-like oligomer of Sup35- DHFR-Z, which shares conformation with OligoBinders. The positive control is a rabbit anti-RBD antibody. The membrane was incubated with the YFP-S-RBD construct (0.5 ng/μL). (B) Bar charts representing the mean of the fluorescence of each dot over the corresponding integrated area. SD was calculated from three independent dot blot assays. * indicates p value <0.05. Figure 6. Neutralization effect of OligoBinder-1/3 nanoparticles against RBD:hACE2r interaction. (A) Schematic representation of the Lumit SARS- CoV-2 spike RBD:hACE2r immunoassay. Created by BioRender.com. (B) Interfering effect of amyloid oligomeric nanoparticles for RBD:hACE2r with the Lumit SARS-CoV-2 spike RBD:hACE2r immunoassay. Results come from the mean calculation of two technical replicates ± SD. IC50 values were calculated using a nonlinear regression (least-squares) fitting method. 40150-D002) and human origin (Active Motif 91361 and SARS-CoV-2 spike RBD with a potency equivalent to the one Biolegend 938502), which exhibited IC50 values of 1870, 250, shown by investigational antibodies. OligoBinder-1 and OligoBinder-3 Block Viral Entry. 430, and 370 pM, respectively. Similar to neutralizing antibodies, molecules able to bind the S Overall, our results indicated that the multivalent Oligo- glycoprotein on the viral surface might prevent the entry of the Binder-1 and OligoBinder-3 efficiently use the previously virus into the host cell, thus offering protection against described high affinity of LCB1 and LCB3 domains16 to bind infections. Therefore, we investigated the ability of Oligo- 11450 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 7. Neutralization effect of OligoBinder-1/3 nanoparticles against SARS-CoV-2 virus. (A) Schematic representation of the SARS-CoV-2 HiBiT- pseudotype VLPs system. Created by BioRender.com. (B) Maximal neutralizing effect of amyloid oligomeric nanoparticles using the HiBiT- pseudotype VLP-based assay. Results come from the mean calculation of two technical replicates ± SD. IC50 values were calculated using a nonlinear regression (least-squares) fitting method. Binder-1 and OligoBinder-3 to block the entry of the virus into with SARS-CoV-2 entry, the entry/fusion processes of SC2- human cells. VLPs are blocked, thereby preventing HiBiT internalization, and Because of safety restrictions to work with SARS-CoV-2 at our no luminescence is produced (Figure 7A). institution, we used HiBiT bioluminescence technology instead, To assess the neutralizing potency of our nanoparticles, which is a well-validated technology for SARS-CoV-2 SARS-CoV-2 S (G614) HiBiT-pseudotype VLPs were investigation.44−46 It employs SARS-CoV-2 pseudotyped virus- incubated with OligoBinder-1 and OligoBinder-3 in the range like particles (SC2-VLPs)47 that resemble the virus structurally 0.2−40000 pM and then added to the hACE2-HEK293T and contain the spike protein at their surface but are (LgBiT) target cells, and the neutralizing IC50 values were noninfectious due to the lack of viral genetic material. Briefly, when HiBiT-tagged VLPs pseudotyped with SARS-CoV-2 S determined (Figure 7B). Consistent with the binding data protein are added to engineered human ACE2-HEK293T cells obtained in the previous assays, the neutralization IC50 values that stably express ACE2r at its surface and LgBiT in their were 120.7 and 127.8 pM for OligoBinder-1 and OligoBinder-3, cytosol, the spike−ACE2r interaction results in membrane respectively. These values are in the same range as or better than fusion, and HiBiT is released into target cells, where it binds to the IC50 values obtained for bamlanivimab,48 imdevimab,49 or LgBiT to generate a luminescent signal in the presence of etesevimab,50 when tested using the same HiBiT assay (Table substrate. However, in the presence of molecules interfering 3). These are mAbs against the spike (S) protein of SARS-CoV- 11451 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article 2 that are being used individually or in combination as treatment a calibrated SEC column to map the fractions in which the and prophylaxis for COVID-19.51 natively assembled particles and potential dissociated mono- mers would elute. Both oligomers and monomers rendered a Table 3. SARS-CoV-2 HiBiT-PsVLP Assay Response to single peak, indicative of their homogeneity (Figure S7A,B). Neutralizing Antibodies and OligoBinders Molecular weights of 813 and 851 kDa were calculated for OligoBinder-1 and OligoBinder-3, in good agreement with the anti-SARS-CoV-2 spike mAb/binder IC50 (ng/mL) IC50 (pM) sizes obtained from DLS analysis. bamlanivimaba 34 232.9 In a next step, we added the oligomers to plasma and, at time 0 imdevimaba 72 499.5 h and after 24 h of incubation, chromatographed the mixture and etesevimaba 230 1629.1 collected the fractions corresponding to native oligomers and OligoBinder-1 88.2 120.7 potential monomers (Figure S7C−F). We then detected Sup35- OligoBinder-3 101.1 127.8 DHFR-LCB1/LCB3 subunits in these fractions by immuno- a Data as provided by Promega Biotech Ibérica, S.L. blotting using an anti-His tag antibody. The experiment confirmed that the integrity of the oligomeric particles did not change upon incubation in plasma, since the signals correspond- Biocompatibility and Stability of OligoBinder-1/3 ing to the oligomers do not vary significantly with time, and no Nanoparticles. Every nanomaterial intended for biomedical monomers were detected for either of the two nanoparticles purposes should be biocompatible. It has been reported that (Figure S7I,J). certain amyloid-based nanomaterials might elicit cytotoxicity, especially in the oligomeric states.52 We wanted to discard this possibility for our nanoparticles. Therefore, we determined the metabolic activity of human cells treated with OligoBinder-1 and ■ CONCLUSION As we enter the third year of the COVID-19 pandemic, we are OligoBinder-3. To this aim, purified oligomers were added to still far from the end of it. An incredible effort has been put into HeLa cultured cells and nontumor MRC-5 cells at different developing therapeutic agents such as new vaccines against concentrations, ranging from 1 to 10 μM. After 72 h of SARS-CoV-2 or antiviral repurposed drugs. There is no doubt incubation, the assemblies’ toxicity was assessed using the that vaccines represent the first barrier of protection against the PrestoBlue fluorescent reagent, a cell-permeable resazurin-based virus. Vaccination conveys less severe illness and is associated solution that is reduced by metabolically active cells, with with lower patient mortality, but older adults and other people fluorescence being a quantitative measure of cell viability. Figure with compromised immune systems might not develop or 8 shows that, compared to the control group, OligoBinders did maintain an adequate immune response to vaccines. In addition, not reduce cell viability at any of the tested concentrations, repurposing antiviral drugs usually takes a long time.53−55 meaning that these spherical nanostructures are biocompatible. Therefore, we will only succeed in defeating the pandemic by The bioavailability of active protein-based materials is critical combining existing treatments with novel approaches. Because for their biomedical use, since polypeptides are intrinsically COVID-19 disease manifests when SARS-CoV-2 enters into vulnerable to plasma proteolytic enzymes. Therefore, we host cells through the interaction between the S protein and investigated the stability of OligoBinder-1 and OligoBinder-3 ACE2r, research is focused on discovering molecules intended in plasma for 48 h at room temperature (RT). Nanoparticles in to target them, thus interfering with their binding.56,57 plasma at time zero were used as a positive control, and plasma Our strategy produced highly pure assemblies of defined size alone was loaded to discard any unspecificity of the used anti-His by incorporating the LCB1 and LCB3 domains through a short tag antibodies. The His tag is at the C-terminus of the two linker at the C-terminus of the Sup35-DHFR construct. This tripartite fusions, and proteolysis should be easily monitored if it strategy has been previously shown to generate spherical occurs. As shown in Figure S6, OligoBinder-1 and OligoBinder- nanoparticles of an amyloid-like nature but devoid of any 3 remained stable for 24 h in plasma. toxicity and having mesoscopic properties of interest.27 We Once confirmed that OligoBinders were not proteolytically exploited the self-assembling properties of this tripartite fusion processed in plasma, we further monitored their stability in this to generate high-surface/volume-ratio functionalized nanostruc- biological fluid by measuring their eventual disassembly into tures that can target and neutralize SARS-CoV-2. their intact individual subunits. To this aim, first, we injected OligoBinders are biocompatible soluble oligomeric particles OligoBinder-1 or OligoBinder-3, as well as their monomers, into exposing functional LCB minibinders, whose properties endorse Figure 8. Cytotoxicity of OligoBinders. Cell viability of HeLa cells (A) and MRC-5 cells (B) after 72 h of incubation in the presence of different concentrations of oligomeric nanoparticles. Results are presented as means ± SD, n = 3. 11452 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article this nanomaterial with the ability to bind to the S protein RBD mM NaCl, EDTA-free protease inhibitor cocktail tablets (Thermo and inhibit the RBD/ACE2-receptor protein−protein inter- Scientific), 1 mM PMSF, and 1 μg/mL DNase]. Cells were then lysed action in the pM range, interfering with the fusion of SC2-VLPs by sonication on ice. Lysates were centrifuged at 27216g for 30 min at 4 to human host cells with higher potency than that observed for °C, and the clarified supernatants were filtered through a 0.45 μm filter and loaded into a Ni-NTA nickel column pre-equilibrated with wash neutralizing mAbs in the same assay. The amyloid-like nature of buffer (30 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole) and the assembly provides these nanoparticles with high stability, as eluted with elution buffer (30 mM Tris-HCl pH 8.0, 300 mM NaCl, 500 shown by their resistance to proteolysis and dissociation in mM imidazole). Eluted samples were loaded into a Superdex 75 plasma. The trick resides in taking advantage of the strength of increase 10/300 GL (GE Healthcare) size exclusion column using PBS the short intermolecular contacts that characterize the amyloid pH 7.4. fold without forming large insoluble fibrillar assemblies. The The protein fraction of cell homogenates and purity of the samples spherical shape and defined size of OligoBinders make them were confirmed by 15% SDS-PAGE. Protein concentration was comparable to other nanostructures of inorganic origin, with the determined by measuring absorbance at 280 nm in a SPECORD 200 unique property that they display functional protein domains in Plus spectrophotometer (Analytik Jena, Jena, Germany). Fractions containing purified proteins were snap-frozen in liquid nitrogen and active conformations ready for binding. In addition, these stored at −80 °C. assemblies have a multimeric nature that might potentially Circular Dichroism Spectroscopy. Sup35-DHFR-LCB1 and enable engagement of all 3 RBDs in a single S trimer; whether Sup35-DHFR-LCB3 proteins were diluted at 10 μM in 1× PBS. Far- this is the reason for the higher apparent neutralizing potency of UV CD spectra were acquired in the range 200−260 nm, at 25 °C, in a OligoBinders relative to therapeutic antibodies in the HiBiT- Jasco J-815 CD spectropolarimeter (Jasco Corporation). The CD PsVLP assay should be further investigated. spectra were obtained from the average of 12 accumulations by LCB1 and LCB3 bind to the viral glycoprotein in different continuous scanning at 0.5 nm intervals, a response time of 1 s at a modes,16 but our assays did not detect significant differences in scanning speed of 100 nm/min, and a bandwidth of 1 nm. their neutralizing activities; in the absence of structural To study the thermal stability by far-UV CD, Sup35-DHFR-LCB1 and Sup35-DHFR-LCB3 proteins were prepared at 10 μM in PBS pH information reporting on the conformation of these domains 7.4. The ellipticity at 222 nm, corresponding to the α-helical in the oligomer and the way they bind to the trimeric spike conformation, was registered at 1 °C intervals, with a heating rate of protein, the reason for this similar activity remains uncertain; 0.5 °C min−1 from 20 to 80 °C in a Jasco J-815 CD spectropolarimeter still, the fact that the two domains dock with opposing (Jasco Corporation) equipped with a Peltier Jasco CDF-426S/15 orientations in the crevice created by the RBD motif suggests temperature controller and Jasco MCB-100 mini water circulation bath. that they might be used in combination. To determine the Tm, transition curves were normalized and fitted Importantly, the monomeric components of OligoBinders are using Kaleida Graph software. amenable to large-scale production in microbial cell factories, Intrinsic Tryptophan Fluorescence. Intrinsic tryptophan fluo- like E. coli, and their assembly occurs spontaneously after rescence spectra of Sup35-DHFR-LCB1 and Sup35-DHFR-LCB3 at 10 μM were analyzed at 25 °C in PBS pH 7.4 using a Jasco FP-8200 incubation, enabling their cost-effective manufacture; moreover, spectrofluorometer (Jasco Corporation). The average of 3 accumu- they might not require cold chain storage due to their stability. lations were measured in the range 300−400 nm every 0.5 nm, with 0.1 Altogether, the two novel functional protein-based nanoparticles s response time, using an excitation wavelength of 280 nm. we describe here might be potentially exploited for applications Thermal denaturation was followed by intrinsic fluorescence in biomedicine, such as self-administered nasal treatment, or emission of Trp recorded at 350 nm, after an excitation wavelength biotechnology, including the development of SARS-CoV-2 of 280 nm, with a heating rate of 0.5 °C min−1 from 25 to 90 °C and 0.5 diagnostic kits, or as potential prophylactic agents. °C intervals, in a Jasco FP-8200 spectrofluorometer (Jasco Corpo- In conclusion, our strategy is simple, modular, and can be ration) equipped with a Jasco ETC-814 Peltier temperature control system and Jasco MCB-100 water circulation minibath. Transition adapted to target any virus of interest by incorporating the curves were normalized and fitted using Kaleida Graph software to corresponding inhibitory domain in the fusion. Furthermore, it calculate the Tm. offers the possibility to engineer oligomeric nanoparticles Preparation of Oligomeric Particles. For the preparation of combining two or more functional domains that simultaneously oligomeric particles of Sup35-DHFR-LCB1 and Sup35-DHFR-LCB3, target different binders or diverse binding sites in the same proteins in 20 mM sodium phosphate buffer pH 8, at a final protein, to create potentiated antiviral molecules in the future. concentration of 150 μM, were filtered through 0.22 μm filters. Samples were incubated at 37 °C with agitation (600 rpm) for 4 days. ■ MATERIALS AND METHODS Protein Expression and Purification. The cDNA of LCB1 and After incubation, the solution was ultracentrifuged for 1 h at 400000g at 20 °C to eliminate any insoluble aggregates. Then, to remove remaining monomers, 500 μL of supernatant was collected into an LCB3 protein sequences16 were subcloned into a pET28a vector Amicon Ultra cellulose membrane centrifugal filter of 100 kDa (Addgene), containing the sequence for SAC Sup35-linker-DHFR with (Millipore) and centrifuged at 9600g for 2 min. The concentrated a 6x-His-tag (see the complete sequence in Figure 1). solution was then washed 3 times with 400 μL of 20 mM sodium The two plasmids were transformed into E. coli BL21 (DE3) strain. phosphate buffer and concentrated down to 100 μL. Then, the transformed cells were grown overnight in LB medium Buffer exchange of Oligobinder-1 and OligoBinder-3 was performed containing 50 μg/mL kanamycin at 37 °C with agitation. Then, 1:100 by diluting samples 20× in the corresponding buffer with different pH dilutions from overnight cultures were used to inoculate fresh LB values (6.5, 7.0, 7.5, and 8.0) and centrifuging them in Amicon Ultra medium containing 50 μg/mL kanamycin, and cultures were grown at cellulose membrane centrifugal filters of 100 kDa at 9600g for 75 s. This 37 °C with agitation until reaching an optical density (OD 600 nm) of procedure was repeated five times, obtaining a final volume of about 0.6 and induced using 1 mM IPTG at 20 °C for 16 h. Samples from 100 μL. noninduced and induced cultures were taken to analyze soluble and Native Polyacrylamide Gel Electrophoresis (Native-PAGE). A insoluble fractions by 15% SDS-PAGE. Cells were harvested by discontinuous Tris-glycine polyacrylamide gel system consisting of 4% centrifugation at 6238g for 20 min at 4 °C. The cell pellet was washed in stacking and 8% separation gel was used to check the purity and 30 mL of phosphate buffered saline (PBS, pH 7.4) by centrifugation at integrity of soluble oligomeric particles at different pH values. Based on 3220g for 20 min at 4 °C and kept at −80 °C. Then, the collected cell the isoelectric point of OligoBinder-1 and OligoBinder-3 (∼5.1 and 5.3, pellet was resuspended in lysis buffer [30 mM Tris-HCl pH 8.0, 300 respectively), the most suitable buffer system was Tris-HCl, with a 11453 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article separating gel of 0.375 M Tris-HCl (pH = 8.8) and a stacking gel of membrane was washed with TBST buffer (3 × 5 min), and YFP 0.375 mM Tris-HCl (pH = 6.8). The native gels were run in a Mini- fluorescence intensity was visualized using a ChemiDoc imaging system PROTEAN electrophoresis chamber (BioRad) in running buffer (25 (Bio-Rad) and quantified with ImageJ software (U.S. National mM Tris, 192 mM glycine, pH ∼8.8). Institutes of Health, Bethesda, MD). The mean and SD of fluorescence Dynamic Light Scattering. The size and molecular weight of intensity were calculated from three independent membrane blots, and oligomers were estimated using a Malvern Zetasizer Nano S90 values were plotted with GraphPad Prism software V6.01. For group- ZEN1690 instrument. 100 μL of the oligomeric samples were prepared wise comparisons, the two-sample (independent groups) t test was in 20 mM sodium phosphate buffer at different pH values (6.5, 7.0, 7.5, performed using IBM SPSS software, version 23.0 (SPSS Inc., Chicago, and 8.0) at 25 °C. The measurement for each sample corresponded to IL). 30 averaged acquisitions repeated 3 times. The estimated molecular The ability of OligoBinders to disrupt the spike RBD−ACE2r weight for OligoBinder-1 and OligoBinder-3 was calculated from the interaction was assessed with the Lumit SARS-CoV-2 spike mass distribution results using an empirical mass vs globular protein RBD:hACE2r immunoassay (Promega), which is a combination of calibration curve, provided by Malvern. immunodetection and NanoLuc binary technology (NanoBiT). Amyloid Dye Binding. The fluorescence signal of the thioflavin-T According to the manufacturer’s instructions, 10× oligomeric particles (Th-T) and absorbance changes of the Congo red (CR) dyes were were diluted in 1× immunoassay reaction buffer at a ratio of 10:1 and measured to determine the formation of amyloid assemblies. For the incubated with 1.5 nM SARS-CoV-2 RBD (Rabbit Fc) for 30 min at RT Th-T binding assay, aggregated and soluble proteins at a final prior to the addition of 1.5 nM hACE2r-Fc (Mouse Fc) and Lumit concentration of 20 μM were mixed with 25 μM Th-T. Samples were antibody mix. After 1 h of incubation at RT, 12.5 μL of Lumit detection excited at 445 nm, and emission fluorescence was recorded between reagent was added, followed by incubation at RT for 30 min. 460 and 600 nm with an emission bandwidth of 5 nm using a Jasco FP- Luminescence was recorded using a Spark Tecan fluorescence 8200 spectrofluorometer (Jasco Corporation). multimode microplate reader. Lumit reagent buffers with RBD/ To evaluate the CR spectral shift, aggregated and soluble proteins at ACE2r were used as positive control signal, and only buffer was used as a final concentration of 20 μM were incubated with 20 μM CR. negative controls to subtract the background signal contribution. Then, Absorbance spectra were recorded at the range from 375 to 700 nm in a signal fold changes were calculated by dividing sample luminescence by SPECORD 200 Plus spectrophotometer (Analytik Jena, Jena, positive control values and converted to % neutralization by subtracting Germany). Spectra of proteins alone were acquired to subtract their from 100. Concentrations in the range 0.2−40000 pM were log10 contribution to the CR signal. Buffers without protein were used as the transformed and final values represented by the mean of two baseline signal for Th-T and CR. independent experiments. IC50 values were calculated using a nonlinear Transmission Electron Microscopy. To obtain transmission regression (least-squares) fitting method using GraphPad Prism V6.01 electron microscopy images, 10 μL of aggregated samples in the (GraphPad Software, Inc.). presence of 20 mM sodium phosphate buffer with different pH values Virus Neutralization. To examine the capacity of OligoBinders to (6.5, 7.0, 7.5, and 8.0) was deposited into carbon-coated copper grids prevent the infection of cells by SARS-CoV-2, we employed a cell-based for 10 min. Grids were negatively stained with 10 μL of 2% (w/v) uranyl screening assay using HiBiT-tagged PsVLP from Promega Biotech. The acetate solution and wiped out after 1 min with filter paper strips. genome-free nature of SC2-VLPs eliminates the need for biosafety level Resulting grids were visualized using a JEOL 1400 (JEOL Ltd.) TEM 3 (BSL3) facilities during handling. HiBiT technology provides the instrument at 120 kV, and images were acquired with a CCD GATAN benefits of high sensitivity and convenience of a single-reagent-addition ES1000W Erlangshen camera (Gatan Inc.). Size measurements were step, all while overcoming the disadvantage of difficulty quantifying performed using ImageJ software (U.S. National Institutes of Health, Bethesda, MD), averaging 10 individual measurements for each PsVLP cell entry and membrane fusion. oligomeric nanoparticle in 20 mM sodium phosphate buffer pH 8. Serial dilutions of OligoBinder-1 and OligoBinder-3 were prepared Fourier Transform Infrared Spectroscopy. To obtain the IR in the range 0.2−40000 pM. Next, 4× OligoBinder-1 and OligoBinder- spectra, 10 μL of purified OligoBinder-1 and OligoBinder-3 was loaded 3 were incubated with SARS-CoV-2 S(G614) HiBiT-SC2-VLPs on the diamond crystal of a Bruker Tensor 27 FTIR spectrometer dissolved in assay buffer for 30 min at 37 °C. Next, SARS-CoV-2 (Bruker Optics) supplied with a Specac MKII Golden Gate ATR HEK293T (LgBiT) target cells were thawed and transferred to a 96- accessory, and solvent was evaporated in a stream of nitrogen. FTIR well plate containing HiBiT-SC2-VLPs and OligoBinders. After 3 h of spectra were recorded between 1700 and 1600 cm−1 in 32 scans at a incubation at 37 °C, 25 μL of 5× Nano-Glo live cell reagent was added resolution of 1 cm−1. Spectra were corrected for the background to each well, and luminescence was measured after 15 min of incubation absorption and normalized with the Min/Max normalization method at 37 °C. In the presence of inhibitory particles, SC2-VLP entry and using OPUS MIR Tensor 27 software (Bruker Optics). IR spectra were fusion with target cells were blocked; therefore, this prevented HiBiT fitted employing a nonlinear peak-fitting equation using PeakFit release, and no luminescent signal was produced. Assay buffer was used package v4.12 (Systat Software, San Jose, CA). The area for each to calculate the baseline signal. Next, maximum entry values of SC2- Gaussian curve was calculated in the amide I region from 1700 to 1600 VLPs into cells were calculated by dividing baseline-corrected values by cm−1 using the “AutoFit Peaks I Residuals” option, manually as the values from control samples containing no oligomeric particle. The starting condition as many peaks as minima were identified in the obtained luminescence values were normalized to neutralization%, second derivative. The resulting area, amplitude, and center values of where the highest luminescence value was defined as 0%, and the lowest the fitted bands were exported as a table and plotted. luminescence values were defined as 100%. GraphPad Prism V6.01 Functional Assessment of OligoBinders. The capacity of (GraphPad Software, Inc.) was used to fit the obtained values to a OligoBinders to bind SARS-CoV-2-S-RBD protein was explored by a nonlinear regression curve (least-squares fitting method) to determine dot blot assay. For this purpose, 10 μL of purified oligomeric particles of the IC50. 0.40, 0.20, 0.10, and 0.04 μg were spotted onto methanol activated Cytotoxicity Assay. HeLa and MRC-5 cell lines were acquired polyvinylidene difluoride membranes (PVDF) (Immobilon-P transfer from American Type Culture Collection (ATCC). Cells were membranes, Merck Millipore). Sup35-DHFR-Z oligomers at the same maintained in DMEM medium supplemented with 10% fetal bovine concentration range were used as a negative control. A positive control serum at 37 °C in a 5% CO2 atmosphere. HeLa cells were seeded onto a was performed by using rabbit anti-SARS-CoV-2 spike S1 subunit 96-well plate at a density of 3500 cells/well, and MRC-5 cells were primary antibody (Sino Biological, 40150-T62) at 1, 0.8, 0.4, and 0.2 seeded at 2000 cells/well with 100 μL of culture medium. After 24 h, μg. cells were incubated with OligoBinder-1 and OligoBinder-3 particles in The membrane was blocked with 5% BSA/TBST (20 mM Tris-HCl, the range 1−10 μM in triplicate. The control group received the same pH 7.4, and 150 mM NaCl, 1% Tween-20) buffer for 30 min at RT. volume of sterile PBS as vehicle. After 72 h of incubation, 10 μL of Next, the membrane was incubated with YFP-S-RBD protein (0.54 ng/ Prestoblue cell viability reagent (Invitrogen) was added for 30 min. The μL) dissolved in 0.1% BSA/TBST buffer for 30 min at RT. The fluorescence emission was recorded at 615 nm, with an excitation 11454 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article wavelength of 531 nm using a Victor III multilabel plate reader of oligomeric Sup35-DHFR-Z fusion protein; stability of (PerkinElmer). OligoBinders in plasma; and characterization of Oligo- Stability of OligoBinders in Plasma. OligoBinder nanoparticles Binders’ size and stability in plasma (PDF) were dissolved in 100 μL of plasma at a final concentration of 16 μM into each of four 0.5 mL low protein binding microtubes and incubated at 25 °C. Aliquots at 0, 24, and 48 h were removed, and 4× loading buffer was added into each sample and immediately stored frozen at ■ AUTHOR INFORMATION Corresponding Authors −20 °C until analyzed. Plasma without oligomeric particles was used as Susanna Navarro − Institut de Biotecnologia i de Biomedicina a control. To analyze samples, 7 μL of each sample was mixed with 5 μL of Milli (IBB) and Departament de Bioquímica i Biologia Molecular, Q water and incubated at 90 °C for 5 min, and 12 μL (9.3 μM) of the Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; incubated samples was loaded onto a 15% SDS-PAGE gel. The protein orcid.org/0000-0001-8160-9536; was electrophoretically transferred to a methanol activated PVDF Email: Susanna.Navarro.Cantero@uab.cat membrane (Immobilon-P transfer membranes, Merck Millipore) in a Salvador Ventura − Institut de Biotecnologia i de Biomedicina trans-blot cell (Bio-Rad) with transfer buffer 1× (0.05 M Tris-HCl, 0.04 (IBB) and Departament de Bioquímica i Biologia Molecular, M glycine, 0.04% SDS, 20% methanol) at 100 V for 1 h. The membrane Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; was then blocked for 1 h with 1X TBST buffer containing 10% nonfat orcid.org/0000-0002-9652-6351; dried milk. After blocking, the membrane was incubated with 1:2000 Email: salvador.ventura@uab.cat anti-His tag mouse monoclonal antibody (Invitrogen, MA1-21315) overnight at 4 °C. The membrane was then washed with TBST 1× (3 × Authors 10 min) and incubated with 1:10000 anti-mouse IgG conjugated to Molood Behbahanipour − Institut de Biotecnologia i de horseradish peroxidase (Bio-RAD, 170-6516) for 1 h. The membrane was revealed with Immobilon Forte Western HRP substrate (Millipore Biomedicina (IBB) and Departament de Bioquímica i Biologia Corporation) for 5 min. Images were captured using a ChemiDoc Molecular, Universitat Autònoma de Barcelona, 08193 imaging system−Bio-Rad with an exposure time of 500 s. Barcelona, Spain Analytical Size Exclusion Chromatography (SEC). OligoBinder Roger Benoit − Laboratory of Nanoscale Biology, Division of nanoparticles were incubated in the presence of human plasma at a final Biology and Chemistry, Paul Scherrer Institute, 5232 Villigen concentration of 16 μM for 24 h at room temperature. Next, 100 μL of PSI, Switzerland; orcid.org/0000-0001-9420-7739 OligoBinder-1 and OligoBinder-3 incubated in human plasma was injected separately into a Superdex 200 10/300 GL column using 20 Complete contact information is available at: mM sodium phosphate buffer, pH 8.0, at room temperature. In https://pubs.acs.org/10.1021/acsami.2c18305 addition, the elution profiles of human plasma alone, Oligobinder-1 and OligoBinder-3, and Sup35-DHFR-LCB1 and Sup35-DHFR-LCB3 in Author Contributions sodium phosphate buffer were analyzed. S.V. conceived the project. S.N. and S.V. designed the For size reference, an elution pattern was performed using different experiments. M.B. and S.N. performed the experiments. M.B. proteins as calibrators (carbonic anhydrase 29 kDa, β-amylase 200 kDa, and S.N. analyzed the data. R.B. produced YFP-S-RBD. M.B., ferritin 440 kDa, thyroglobuline 669 kDa) in a Superdex 200 10/300 S.N., and S.V. wrote the manuscript. All authors have given GL column (GE Healthcare), using a flow rate of 0.5 mL/min in 20 mM approval to the final version of the manuscript. sodium phosphate buffer, pH 8.0. Immunoblotting Analysis of SEC Samples. The elution Notes fractions corresponding to monomeric and oligomeric forms were The authors declare no competing financial interest. further detected by SDS-PAGE immunoblotting. For this purpose, collected SEC fractions were concentrated 100× for monomeric forms using 10,000 MWCO cellulose membrane centrifugal filters (Millipore) and 30× for oligomeric forms using 100,000 MWCO centrifugal filters ■ ACKNOWLEDGMENTS This work was funded by the Spanish Ministry of Science and (Millipore). 4× loading buffer was added into each sample, and 16 μL Innovation (PID2019-105017RB-I00) and by ICREA, ICREA- sample fractions were loaded on 15% SDS-PAGE and blotted onto a Academia 2020, to S.V. M.B. was supported by the Spanish PVDF membrane. Soluble monomeric Sup35-DHFR-LCB1, Sup35- Ministry of Science and Innovation (PRE2020-092634). We DHFR-LCB3, OligoBinder-1, and OligoBinder-3 in sodium phosphate thank Promega Biotech Ibérica SL for providing us with the buffer were used as positive controls, and plasma without added NanoBiT technology. OligoBinders was used as negative control. The antibodies employed were 1:1000 primary 6x-His Tag monoclonal antibody (Invitrogen, MA1-21315) and 1:10000 secondary anti-mouse IgG-HRP conjugate (Bio-RAD, 170-6516). The membranes were developed with ■ ABBREVIATIONS SARS-CoV-2, severe acute respiratory syndrome coronavirus Immobilon Forte Western HRP substrate (Millipore), and images 2 were captured using a ChemiDoc imaging system−Bio-Rad with an COVID-19, coronavirus disease 2019 exposure time of 500 s. RBD, receptor binding domain ACE2r, angiotensin-converting enzyme 2 receptor ■ ASSOCIATED CONTENT * Supporting Information sı mAb, monoclonal antibody SAC, soft amyloid core The Supporting Information is available free of charge at DHFR, dihydrofolate reductase https://pubs.acs.org/doi/10.1021/acsami.2c18305. IC50, half-maximal inhibitory concentration CD, circular dichroism Expression and purification of soluble and oligomeric Trp, tryptophan Sup35-DHFR-LCB1 fusion protein and Sup35-DHFR- Th-T, thioflavin-T LCB3 fusion protein; characterization of size distribution CR, congo red of OligoBinder-1 and OligoBinder-3 at different pHs at FTIR, Fourier-transform infrared time 0 and after 24 h of incubation; TEM images; TEM, transmission electron microscopy schematic representation and biophysical characterization DLS, dynamic light scattering 11455 https://doi.org/10.1021/acsami.2c18305 ACS Appl. Mater. Interfaces 2023, 15, 11444−11457 ACS Applied Materials & Interfaces www.acsami.org Research Article SC2-VLPs, SARS-CoV-2 virus-like particles Vanbever, R. Pegylation of Antibody Fragments Greatly Increases SD, standard deviation Their Local Residence Time Following Delivery to the Respiratory BSL3, biosafety level 3 Tract. J. Controlled Release 2014, 187, 91−100. PBS, phosphate buffered saline (15) Guillon, A.; Pardessus, J.; Lhommet, P.; Parent, C.; Respaud, R.; PVDF, polyvinylidene difluoride membranes Marchand, D.; Montharu, J.; De Monte, M.; Janiak, P.; Boixel, C.; Audat, H.; Huille, S.; Guillot, E.; Heuze-Vourc’h, N. Exploring the Fate RT, room temperature of Inhaled Monoclonal Antibody in the Lung Parenchyma by ■ REFERENCES (1) Santomauro, D. F.; Herrera, A. M. M.; Shadid, J.; Zheng, P.; Microdialysis. MAbs 2019, 11, 297−304. (16) Cao, L.; Goreshnik, I.; Coventry, B.; Case, J. B.; Miller, L.; Kozodoy, L.; Chen, R. E.; Carter, L.; Walls, A. C.; Park, Y. J.; Strauch, E. 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