key: cord-355931-3mvmetuv
authors: Nie, Chuanxiong; Ma, Lang; Luo, Hongrong; Bao, Jinku; Cheng, Chong
title: Spiky nanostructures for virus inhibition and infection prevention
date: 2020-07-16
journal: nan
DOI: 10.1016/j.smaim.2020.07.004
sha: 
doc_id: 355931
cord_uid: 3mvmetuv

Abstract The outbreak of a novel highly infectious virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has aroused people’s concern about public health. The lack of ready-to-use vaccines and therapeutics makes the fight with these pathogens extremely difficult. To this point, rationally designed virus entry inhibitors that block the viral interaction with its receptor can be novel strategies to prevent virus infection. For ideal inhibition of the virus, the virus-inhibitor interaction has to outperform the virus-host interaction. In our view, the morphology of the inhibitor should be carefully designed to benefit virus-inhibitor binding, especially that the surfaces of viruses are mostly rough due to the existence of surface proteins for receptor-binding. In this perspective article, we would like to discuss the recent progress of designing inhibitors with spiky topography to maximize the interactions between viruses and inhibitors. We also would like to share our idea for the future study of inhibitors to prevent virus infection.

flu' in 1918, the 'H2N2' outbreak in 1957, and the recent 'H1N1' outbreak in 2009, each of them has caused massive damage to the human society. [3] Flu vaccines have been developed to prevent the infection, but the high error rate of RNA polymerase enables IAV to mutate with unpredictable antigen shifts and drifts, which makes it challenging to prepare the correct vaccine for the coming flu outbreak. [4] Therefore, inhibitors that prevent virus infection are urgently needed.

The infection cycle of IAV starts with binding to the sialic acid receptors on the host cell surface. [5] Blocking the virus binding with a decoy has been realized as an effective strategy to inhibit the virus infection at the beginning. [6] [7] [8] [9] [10] [11] To design such an inhibitor, maximizing the inhibitor-virus interaction to compete virus-cell interaction is the key to achieve potent virus inhibition. To this point, functional group valency, inhibitor size, and scaffold stiffness are reported to be the main parameters to match the receptor binding sites of targeted virions. [12, 13] Additionally, the morphological matching of two subjects also benefits the binding, e. g. most of the plant weeds have evolved a spiky surface to adhere to a host for long-distance spreading. [14] As the virus-inhibitor binding is the key for inhibition, this morphological matching principle can be considered for the design of inhibitors to robustly bind to the virions and compete with the virus-host binding as shown in Scheme 1.

The general concepts of using spiky nanostructures for inhibiting virus binding to cell membranes.

In a recent report by Nie et al., [15] the principle of morphology-matching for IAV inhibition was introduced. They firstly obtained the morphological details from cryogenic transmission electron microscopy (cryo-TEM) images as shown in Figure 1 . Despite the difference between spherical and filament morphologies, the virions of IAV exhibit similar surface nanostructure. The hemagglutinin (HA) and neuraminidase (NA) generate a rough surface as shown in Figure 1a . From the reconstruction of the geometry models, they found a gap around 10 nm for the surface proteins of IAV. In order to match the surface nanostructure of IAV virion, geometry models of spiky nanoparticles are generated with similar size but different spiky length and space as shown in Figure 1b ,c. Compared with smooth nanoparticles, these spikes increase the interface area by inserting into the gap of the proteins as shown in Figure 1b . The binding patterns for the spiky nanoparticles are also different for different spike structures as shown in Figure 1c . If the spikes are too long, the virion interacts only with the tip, resulting in decreased area. When the spikes are too dense, they no longer fit the gap, which decreases the interface area. At a suitable spike length and spacing (10 nm length, 10 nm spacing), the nanoparticle can tightly bind to IAV virion with a maximized contact area. Interaction between IAV virion and spiky nanoparticles with different geometry parameters. Reprinted with permission from [15] . Copyright (2020) American Chemical Society.

As an experimental proof to this idea, spiky nanostructures (SNS) with similar morphologies with bioinert SiO 2 cores are synthesized as shown in Figure 2a . Nanoparticles with a ~120 nm core and spike length of 0-30 nm are synthesized. The virion-binding abilities are compared via a centrifuge-western blot assay as shown in Figure 2b . It is clear that the spikes enhance the binding between the nanoparticle and IAV virion. When the spikes are 5-10 nm, maximized virus binding is achieved, which is in agreement with the geometry simulation results in Figure 1c . To check the binding patterns between the spiky nanoparticles and IAV virion, cryo-TEM images are also acquired (Figure 2c ). For the smooth nanoparticle (SNS-0), the virion interacts with the nanoparticle with a very limited area. As expected, for SNS with 10 nm spikes (SNS-10), the spikes insert into the gap of the protein, forming a conjunction to benefit the binding. However, for SNS with 30 nm spikes (SNS-30), tip-to-tip interactions are noticed, for which the interaction area is smaller than SNS-10. However, it is noticed that even the spiky nanoparticles show viral binding abilities, they are not able to inhibit the infection of IAV. This is probably because the virus-inhibitor binding is not strong enough to compete with the virus-host binding. To achieve potent virus inhibition, the surface of the nanoparticles should be functionalized with binding motifs towards IAV. Erythrocyte has a natural display of sialic acid as the binding target for several subgroups of IAV (Figure 3a) . As a proof of concept, the authors coat the spiky nanostructures with erythrocyte membrane (EM) as a targeting shell towards IAV virion. During the coating process, all the membrane components are transferred to the surface of spiky nanoparticles. With EM coating, the viral binding is further enhanced, and an inhibitor that can effectively reduce the viral binding to the host cells is obtained. In the cellular infection assay, all the inhibitors show a reduced number of infected cells as shown in Figure 3b , c. SNS-10 is the best inhibitor, by which >90% of cellular infection is inhibited. They also check if the inhibitors can prevent virus replication by plaque assay (Figure 3d, e) . 99.9% inhibition is achieved with 1 mg/mL dose of the inhibitor. Be co-used with NA inhibitor, more potent (>99.99%) inhibition of virus replication is achieved. HA and NA regulate IAV binding in mucus and to host cells. Briefly speaking, HA interacts with sialic acid to trigger virus entry, and NA cleaves the HA-sialic acidbinding for virus release after the replication. [16] To achieve a better inhibition with sialic acid-based compounds, the negative effects of NA on the binding should be minimized. In another report by this group, a heteromultivalent IAV inhibitor based on the spiky nanoparticles is developed. The surface of the spiky nanoparticle is functionalized covalently with sialyllactose (SAL) and zanamivir (Zanamivir), via a linear polyglycerol linker as shown in Figure 4a , b. [17] With zanamivir on the surface, the activity of NA is inhibited (Figure 4e) , the virus-inhibitor binding is further enhanced (Figure 4c, d) , which results in nearly 100% blocking of virion interaction with the host cells. In a cellular infection assay, it is noticed that in the presence of the spiky inhibitors, there are no infected cells. Even being used after the first cycle of infection, the inhibitor, VLNP-SAL/Zan, still shows a >99.9999% inhibition of virus titre (Figure 4f ). They also report that the inhibitor is active against three human IAV strains, which are A/X31 (H3N2), A/PR/8/34 (H1N1) and A/Panama/2007/1999 (H3N2), as shown in Figure 4g . [17] . Copyright (2020) Wiley.

As most of the virus starts the replication with the binding to the receptors and most of the virus exhibit similar spiky morphology as IAV, it is envisioned that such a strategy can also be used for other viral strains, e. g. coronaviruses. For a potent virus inhibition, the concept of geometry matching can be incorporated with different antiviral strategies. The nanostructures in these studies are hollow mesoporous structures, which are capable of loading antiviral cargos as delivery systems to further increase the virus inhibitor performance. On the other hand, enzyme-mimicking nanostructures have also been developed as a powerful tool to combat pathogen infections. These nanostructures exhibit the ability to produce highly 'toxic' reactive oxygen species, including OH , O 2 -, and etc., which show the ability to prevent wound infections. [18] [19] [20] These enzyme-mimic nanostructures have been fabricated into a self-disinfection mouth mask for the combating of the respiratory pathogen infections. [21] It is envisioned that combining functional materials cores with the spiky surfaces, a more potent virus inhibitor with multiple modes of action can be produced.

However, the drawback of such spiky inhibitors is also clear: the non-specific interaction with the biological molecules will facilitate the uptake and clearance by immune systems. [22] Therefore, the surface of the inhibitor should be also be functionalized with highly antifouling or bio-stealth groups to avoid the rapid clearance after the intaking. Cellular membrane coating can be a good solution, which enables the nanoparticle to bypass the immune system for long-term circulation. In such a system, the source of host cells needs to be carefully selected to avoid the side effects of cellular membrane antigens. [23] [24] [25] Another solution can be smart nanostructures that are able to change the morphology upon stimuli. [26] Spiky nanostructures can also be obtained via other approaches, e. g. coating of a small particle onto an existing core material, which offers more possibilities to control the surface morphology and functionalization. [27] Nevertheless, the geometry matching can be a universal approach to benefit the binding between the two subjects. Not only for virus inhibition, this idea can also be used as a general approach for the combat with bacteria and tumors. [28] [29] [30] [31] 

The authors declare that they have no conflict of interest.

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Spiky nanoparticles that match the surface topography of virions are believed to be ideal scaffolds for virus inhibitors