Size‐Dependent Neutralization Efficacy of Nanodecoys Against SARS‐CoV‐2 Mimics in Mammalian Cell Infection Models

ABSTRACT

Nanodecoys that competitively inhibit viral entry represent a promising antiviral strategy, yet the influence of their physical properties on performance remains underexplored. In this work, we systematically investigate the size‐dependent antiviral activity of ACE2‐conjugated nanodecoys against a safe SARS‐CoV‐2 mimic. Our results demonstrate that the inhibition of mimic cellular uptake is highly size‐dependent. The 10 nm nanodecoys exhibited the most potent neutralization, followed by the comparable 5 and 20 nm, while the 50 and 100 nm nanodecoys demonstrated similar, lower efficacies. This work identifies nanodecoy size as a critical design principle, providing a rational framework for engineering high‐performance antiviral nanotherapeutics to combat SARS‐CoV‐2 and other viral pathogens.

ACE2‐conjugated nanodecoys offer a promising antiviral strategy. This study investigates their size‐dependent neutralization against SARS‐CoV‐2 mimics, revealing that 10 nm nanodecoys achieve the most potent cellular uptake inhibition. Conversely, 5 and 20 nm particles show moderate efficacy, while 50 and 100 nm nanodecoys perform poorly. These findings establish size as a critical design principle for antiviral nanotherapeutics.

1. Introduction

Viral infectious diseases are characterized by high transmissibility and pathogenicity, posing significant threats to global health security [1]. The SARS‐CoV‐2 pandemic has particularly underscored the exigency for effective countermeasures [2]. While vaccination remains a cornerstone for prophylaxis [3], antiviral therapeutics are indispensable for treating infected individuals. Conventional antivirals, including small molecules, proteins, and nucleic acids, are often limited by off‐target effects and the propensity to induce viral resistance [4, 5]. Nanotechnology has emerged as a transformative platform for antiviral innovation, offering advantages such as enhanced drug solubility, prolonged circulation, and targeted delivery [6, 7, 8, 9]. Furthermore, certain nanomaterials possess intrinsic antiviral properties.

Viral entry into host cells is a critical step for replication and propagation, making it an attractive therapeutic target [10, 11]. SARS‐CoV‐2 initiates infection by its spike (S) protein binding to the angiotensin‐converting enzyme 2 (ACE2) receptor on host cells [12, 13]. The S protein comprises S1 and S2 subunits, where S1 mediates receptor binding, and S2 facilitates membrane fusion [14, 15]. Strategies to disrupt this process have led to the development of nanodecoys—such as ACE2‐conjugated nanoparticles or cell membrane‐derived vesicles—that competitively inhibit viral entry [16, 17, 18, 19].

The efficacy of nanodecoys is influenced by material composition, morphology, and size [20]. Although our prior research highlighted the size‐dependent interactions of nanoparticles with biological systems [21, 22, 23, 24], systematic investigations into how size affects nanodecoy performance remain limited. To address this critical gap, we herein explore the interplay between nanodecoy size and antiviral activity (Figure 1). We fabricated a panel of nanodecoys of varying sizes and evaluated their interaction with a SARS‐CoV‐2 mimic—a structurally and functionally analogous model that circumvents the biosafety constraints of live virus. Our results demonstrate a highly size‐dependent inhibition of mimic cellular uptake by the nanodecoys. The 10 nm nanodecoys exhibited the highest potency, followed by 5 nm, while 20 and 50 nm showed comparable efficacy, and 100 nm was the least effective. This finding identifies nanodecoy size as a critical design principle for creating efficient antiviral therapeutics and provides a rational design framework for developing high‐performance nanodecoys to combat SARS‐CoV‐2 and other viral pathogens.

FIGURE 1.

Schematic illustration of the size‐dependent neutralization efficacy of nanodecoys (5–100 nm), highlighting the peak antiviral potency of 10 nm nanodecoys, establishing a critical size‐activity relationship for designing antiviral nanotherapeutics (PS NP: polystyrene nanoparticle, GNPs: gold nanoparticles).

2. Results and Discussion

2.1. Preparation and Characterization of Different‐Sized Nanodecoys

Gold nanoparticles (GNPs) were selected as the core scaffold of nanodecoys due to their tunable size, facile surface modification, and biocompatibility in biomedical applications [25, 26]. Uniform GNPs with core sizes of 5, 10, 20, 50, and 100 nm were synthesized using established protocols [27, 28]. Nitrilotriacetic acid‐modified thioctic acid (TA‐NTA) was then prepared and confirmed to facilitate ACE2 conjugation (Figures S1 and S2) [29]. Surface functionalization was characterized by a red shift in the GNPs’ maximum absorption wavelength (Figure S3). ACE2 proteins were immobilized via Ni^{2 +}‐His tag coordination [30]. The successful conjugation of ACE2 proteins on these nanodecoys was first observed by transmission electron microscopy (TEM). The images revealed protein coronas surrounding the GNPs post‐conjugation, absent in unmodified controls (Figure 2a,b). Dynamic light scattering (DLS) showed increased hydrodynamic diameters after ACE2 attachment (Figure 2c). For clarity and convenience of discussion, all sizes mentioned in the text refer to the core diameter of nanodecoys determined by TEM. Zeta potential measurements indicated reduced absolute surface charges, attributable to charge shielding by the protein layer (Figure 2d). Substantiating these results, agarose gel electrophoresis revealed that all nanoparticles, being well‐dispersed, migrated toward the anode, indicating a uniform negative surface charge (Figure 2e). The successful conjugation of ACE2 significantly altered the electrophoretic mobility of the nanodecoys, retarding their migration in a manner consistent with their increased hydrodynamic size and augmented negative charge.

FIGURE 2.

Preparation and characterization of different‐sized nanodecoys. Representative TEM images of different‐sized (a) GNPs as the core scaffold of nanodecoys and (b) ACE2‐conjugated nanodecoys. (c) Hydrodynamic diameter and (d) zeta potential of different‐sized GNPs and nanodecoys, confirming successful surface conjugation and colloidal stability. (e) Agarose gel (4%) electrophoresis validated the effective functionalization of GNPs (left lane) into nanodecoys (right lane). All measurements were performed in triplicate (mean ± SD).

To investigate the size‐dependent effects of the nanodecoys, it was essential to maintain a consistent surface density of ACE2 protein across all variants. This was achieved by first quantifying the nanoparticle concentrations using the Lambert‐Beer law. The amount of bound ACE2 was then determined using a BCA protein assay, where the bound fraction was calculated by subtracting the unbound protein from the total initial amount. The calculated coupling efficiency between the ACE2 protein and the GNPs‐Ni was approximately 65%–80% (Figure S4). This approach ensured a uniform ACE2 modification, which is critical for the subsequent experiments exploring the influence of nanodecoy size.

2.2. Fabrication and Characterization of SARS‐CoV‐2 Mimics

Given that the S1 subunit (specifically its RBD) is the sole domain responsible for ACE2 binding [31], we used S1‑conjugated polystyrene nanoparticles as a simplified model to evaluate the binding capabilities of the nanodecoys. The SARS‐CoV‐2 mimic was constructed by functionalizing green‐fluorescent polystyrene nanoparticles (PS NPs) with the spike S1 protein. As depicted in Figure S5, this was achieved by first modifying PS NPs with Ni‐NTA, followed by the conjugation of His‐tagged S1 proteins via Ni‐His affinity interaction, yielding PS‐S1 NPs. The successful fabrication of the SARS‐CoV‐2 mimic was first confirmed through TEM; the image revealed a distinct surface layer of approximately 15 nm on the PS‐S1 NPs, corresponding to the conjugated S1 protein (Figure 3a). DLS measurements showed an increase in hydrodynamic diameter from ∼124 nm (PS‐Ni) to ∼140 nm (SARS‐CoV‐2 mimic), a size comparable to the native SARS‐CoV‐2 virus (Figure 3b) [32, 33]. This protein conjugation also shifted the zeta potential from −24.5 to −20.4 mV (Figure 3c), a result consistent with the observed migration pattern in agarose gel electrophoresis (Figure 3d). The stability of the conjugation was further assessed by SDS‐PAGE. Silver staining demonstrated that the S1 protein remained bound after two washes with 250 mM imidazole but was efficiently eluted with 500 mM imidazole, confirming the robustness of the interaction (Figure 3e). To accurately mimic the S1 protein density on the actual virus, the coupling efficiency was optimized. An input NP/protein ratio of 1:200 yielded a coupling efficiency of ∼80% (Figure S6), resulting in approximately 160 S1 proteins per SARS‐CoV‐2 mimic, a density representative of the native virion [34, 35].

FIGURE 3.

Fabrication and characterization of SARS‐CoV‐2 mimics. (a) Representative TEM micrographs, (b)hydrodynamic diameter, and (c) zeta potential of the core PS NPs and SARS CoV‐2 mimics. (d) Agarose gel of PS NPs and SARS CoV‐2 mimics. Lane left: PS NPs; lane right: SARS‐CoV‐2 mimics. (e) SDS‐PAGE analysis of the purified SARS‐CoV‐2 mimics, indicating the presence of the S1 protein. (f) Hydrodynamic diameter of SARS‐CoV‐2 mimics upon incubation with increasing concentrations of ACE2. (g) Infection ability of PS NPs and SARS‐CoV‐2 mimics in different concentrations in high‐ACE2‐expressing 293T cells. (h) Infection ability of SARS‐CoV‐2 mimics (2 µg/mL) in 293T cells with different incubation times. (i) Representative CLSM images visualizing the time‐dependent infection efficacy of SARS‐CoV‐2 mimics (green) in high‐ACE2‐expressing 293T cells at different times. Scale bar, 50 µm. All measurements were performed in triplicate (mean ± SD).

Before infecting mammalian cells, the binding affinity of SARS‐CoV‐2 mimics for the ACE2 protein is investigated. The size changes of SARS‐CoV‐2 mimics are measured after incubating with different concentrations of ACE2 protein. The size of SARS‐CoV‐2 mimics increased gradually from an initial ∼140 to ∼155 nm with increasing ACE2 concentration (Figure 3f). When the treated ACE2 protein reached 1 µg/mL, the size was basically unchanged, indicating the saturated binding of ACE2 protein on the surface of SARS‐CoV‐2 mimics. Prior to assessing cell infection ability, we first confirmed that the SARS‐CoV‐2 mimics exhibited no significant cytotoxicity towards HEK 293T cells, even at high concentrations (Figure S7). The functional validity of the mimics was then evaluated using HEK 293T cells with low and high ACE2 expression. Compared with low‐ACE2‐expressing cells, high‐ACE2‐expressing cells demonstrated significantly greater infection of the mimics in a concentration‐dependent manner (Figures 3g and S8). The infectivity reached a plateau at a mimic concentration of 2 µg/mL, which is consistent with the saturation of ACE2 receptors on the cell surface. Subsequently, a time‐course analysis of cellular infection was performed using a mimic concentration of 2 µg/mL. The results showed that the SARS‐CoV‐2 mimics infection efficiency in high‐ACE2‐expressing cells exceeded 80% after a 12‐h incubation (Figure 3h). Finally, confocal laser scanning microscopy (CLSM) provided direct visual confirmation of the infection process of green‐fluorescent SARS‐CoV‐2 mimics, further validating that our constructed mimics effectively recapitulate the key steps of viral cell entry (Figure 3i).

2.3. In Vitro Interaction Between SARS‐CoV‐2 Mimics and Different‐Sized Nanodecoys

The morphological interaction between nanodecoys and the SARS‐CoV‐2 mimics was first investigated by TEM. As shown in Figure 4a, all ACE2‐modified nanodecoys bound more strongly to the mimics than the control nanoparticles. Notably, the unmodified 5 nm control NPs displayed a high degree of non‐specific adhesion, likely due to their ultrasmall size and high surface energy [36]. A clear inverse relationship was observed between nanodecoy size and binding affinity: the smaller the nanodecoy, the more effectively it bound the mimic. This indicates that steric hindrance is a critical factor governing the interaction and subsequent neutralization [37].

FIGURE 4.

In vitro interaction between SARS‐CoV‐2 mimics and different‐sized nanodecoys. (a) Representative TEM images showing the binding affinity of SARS‐CoV‐2 mimics with different‐sized nanodecoys. Scale bar, 100 nm. (b) Agarose gel electrophoresis was used to analyze the binding between SARS‐CoV‐2 mimics and different‐sized nanodecoys across a gradient of ACE2 concentrations. Quantitative ICP‐MS analysis of different‐sized nanodecoys binding to SARS‐CoV‐2 mimics. (c) Binding efficiency (bound/total nanodecoys). (d) Table of numbers of bound nanodecoys and ACE2 per SARS‐CoV‐2 mimic. All measurements were performed in quintuplicate (mean ± SD).

To semi‐quantitatively evaluate the multivalent cross‐linking capacity of these interactions, we employed agarose gel electrophoresis. In this assay, binding is indicated by the formation of large aggregates of nanodecoys and SARS‐CoV‐2 mimics that are retained in the wells, visible as green fluorescence (Figure 4b). The results revealed a distinct size‐dependent effect. The 5 nm nanodecoys only induced significant aggregation at the highest ACE2 density tested (2 µg/mL). In contrast, the 10 nm nanodecoys caused aggregation at a much lower ACE2 density (0.25 µg/mL). Similarly, the 20 and 50 nm nanodecoys showed progressively stronger binding effects as the ACE2 density increased, while the 100 nm nanodecoys required an intermediate ACE2 density of 0.5 µg/mL to exhibit apparent fluorescence retention.

To provide an absolute quantitative analysis and directly interrogate the impact of steric hindrance, we performed Inductively Coupled Plasma Mass Spectrometry (ICP‐MS) binding assays. As shown in Figure 4c, the 10 nm nanodecoys exhibited the highest binding efficiency (approximately 77%), substantially outperforming all other sizes (ranging from ∼16% to ∼34%). In absolute terms, each SARS‐CoV‐2 mimic was conjugated by approximately 22 particles of the 10 nm nanodecoys, and the total number of ACE2 molecules delivered to a single mimic is substantially greater for the 10 nm size than others (Figure 4d). Conversely, the larger nanodecoys (50 and 100 nm) exhibited a drastic reduction in both binding efficiency and particle counts. Crucially, this continuous, size‐dependent decline in affinity rules out spontaneous sedimentation as the cause of the reduced efficacy for large particles, instead providing direct quantitative evidence that severe steric hindrance restricts their ability to engage the densely distributed S1 proteins on the mimic surface.

Finally, SPR measurements were conducted to elucidate the underlying kinetic mechanisms of this size‐dependent multivalent avidity (Figure S9, and Table S1). Free ACE2 showed monovalent binding with a KD of 48.7 nM. The 10 nm nanodecoys demonstrated an exceptionally potent multivalent avidity (KD = 45.1 pM), representing an enhancement of nearly three orders of magnitude. This optimal kinetic profile was driven by a combination of a remarkably high association rate (ka) and a slow dissociation rate (kd) comparable to monovalent ACE2, indicating a perfect geometric match for rapid capture and stable retention. In contrast, the 5 nm nanodecoys suffered from a faster dissociation rate due to lower local ACE2 stoichiometry, whereas the 20, 50, and 100 nm nanodecoys were primarily penalized by rapid dissociation stemming from steric constraints that prevented the formation of stable, multivalent contact points. Collectively, these kinetic data definitively confirm that the 10 nm size represents the structural “sweet spot,” maximizing multivalent capture while minimizing the steric penalty.

2.4. Neutralization of SARS‐CoV‐2 Mimics by Different‐Sized Nanodecoys

Following the in vitro determination of size‐dependent binding efficiency, the neutralization efficacy of the nanodecoys was first qualitatively assessed in high‐ACE2‐expressing HEK 293T cells using CLSM. Direct infection of cells with SARS‐CoV‐2 mimics resulted in intense green fluorescence within the cells, indicating extensive infection uptake. At low nanodecoy concentrations (corresponding to 0.125 µg/mL ACE2), this intense fluorescence signal was readily detectable (Figure 5a). However, this uptake was inhibited in a dose‐dependent manner as the nanodecoy concentration increased. Notably, 10 nm nanodecoys achieved potent neutralization, with minimal fluorescence observed at a concentration of 2 µg/mL ACE2, suggesting that 10 nm may be an optimal size.

FIGURE 5.

Neutralization of SARS‐CoV‐2 mimics by different‐sized nanodecoys. (a) Representative CLSM images showing inhibition of SARS‐CoV‐2 mimic infection in mammalian cells by different‐sized nanodecoys under varying ACE2 protein concentrations. (b) Flow cytometry quantification of infection inhibition efficiency mediated by different‐sized nanodecoys. (c, d) Neutralization efficiency of SARS‐CoV‐2 mimics by different‐sized GNPs and nanodecoys. All measurements were performed in triplicate (mean ± SD).

For quantitative validation, flow cytometry was performed to analyze the size‐dependent neutralization trend (Figure 5b). The 10 nm nanodecoys exhibited the most potent inhibitory effect on cellular infection by SARS‐CoV‐2 mimics, followed by the 50 nm nanodecoys. The 5 and 100 nm nanodecoys showed comparable, intermediate efficacy, while the 20 nm nanodecoys were the least effective. The neutralization efficiency was further quantified by calculating the percentage of inhibited uptake (Figure 5c,d). At an ACE2 concentration of 2 µg/mL, 10 nm nanodecoys prevented nearly 60% of the SARS‐CoV‐2 mimics from being internalized by the cells. This was followed by 5 and 20 nm nanodecoys, which inhibited approximately 30% of uptake. The 50 and 100 nm nanodecoys demonstrated similar, lower efficacies, each preventing around 20% of cellular uptake. These cellular results are consistent with the in vitro binding data, confirming a significant size‐dependent effect. The superior neutralization capability of the 10 nm nanodecoys highlights their potential as an optimal design for antiviral nanomedicines.

3. Conclusion and Prospects

In conclusion, by pairing a safe SARS‐CoV‐2 mimic with a series of size‐tuned ACE2‐conjugated nanodecoys, we have elucidated a critical size‐dependent paradigm for nanodecoy‐mediated viral neutralization. Our findings explicitly demonstrate that the 10 nm nanodecoys possess the most potent neutralization capacity, serving as a structural “sweet spot” that optimizes multivalent capture while minimizing steric hindrance. In contrast, the 5 and 20 nm nanodecoys exhibit moderate efficacy, while the larger 50 and 100 nm particles suffer from severe spatial constraints that drastically limit their competitive binding.

The developed SARS‐CoV‐2 mimic safely and effectively models viral entry, providing a valuable platform for antiviral drug screening. Notably, its modular design offers a “plug‐and‐play” strategy; by simply swapping the conjugated surface protein, the platform can be readily adapted to simulate other respiratory viruses (Figure S10). This characteristic not only highlights the robustness and generalizability of the system but also provides a safe, convenient, BSL‐independent alternative for studying respiratory viruses in settings lacking high‐containment biosafety facilities—a particularly vital feature for responding to rapidly emerging pathogens.

Furthermore, because the nanodecoys function as natural receptor decoys, they create an “evolutionary trap” where mutations that weaken decoy binding would simultaneously compromise viral entry. This mechanism suggests our platform has the potential to neutralize a broader spectrum of ACE2‐dependent coronaviruses [38, 39]. Crucially, the core size‐dependent design principle is expected to remain valid even against high‐affinity variants such as Omicron, although an increased ACE2 surface density on the nanodecoys may be required to achieve equivalent neutralization efficacy [40, 41].

Despite these promising in vitro results, we recognize the necessity for translational investigations. Nanoparticles are known to exhibit size‐dependent lung clearance and alveolar macrophage uptake, which dictate their in vivo efficacy after respiratory delivery [42, 43]. Therefore, future efforts will focus on two main fronts: enhancing the in vivo stability of the nanodecoys and conducting comprehensive in vivo validations using pseudovirus and animal models. Ultimately, our findings establish a fundamental design principle for the development of highly efficient, broad‐spectrum nanodecoy‐based antiviral therapeutics.

Author Contributions

Nan Li and Xuejian Li: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – Original Draft, Visualization, Software. Yuchen Guo: Methodology, Investigation, Formal analysis, Data curation, Software. Daiwei Zhang, Xiangfu Du, Zihao Teng and Aijie Liu: Visualization, Validation, Software. Lizhi Zhou, Zhihuan Liao and Shuaidong Huo: Writing – Review & Editing, Supervision, Funding acquisition, Resources, Conceptualization.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll74751‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.