Use of caP / sti nanoprobe in preparation of product for distinguishing viruses dependent on sialic acid

Abstract

The application provides application of CaP / STI nanoprobes in preparation of virus products capable of distinguishing sialic acid dependence, and belongs to the technical field of biological medicines. The CaP / STI can accurately distinguish the sialic acid dependence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), porcine epidemic diarrhea virus (PEDV), rotavirus and influenza virus, and still has a significant effect compared with neuraminidase (NA). In addition, the CaP / STI significantly inhibits the replication of SARS-CoV-2 early epidemic strains, PEDV, influenza virus and most rotavirus epidemic strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological drugs, and particularly relates to application of a CaP / STI nanoprobe in preparation of a virus product distinguishing sialic acid dependence. BACKGROUND

[0002] Viral respiratory tract infections and the resulting gastroenteritis are prevalent worldwide, causing serious morbidity, mortality, and sequelae. Although most viruses use different receptors to attach and infect host cells, some viruses, including influenza virus, enterovirus D68, adenovirus, and Zika virus, all have a common receptor-sialic acid. Sialic acid addition to the non-reducing end of glycoproteins / glycolipids is catalyzed by 20 different sialyltransferases (STs), which are mainly distributed on the Golgi apparatus.

[0003] Currently, limited strategies are available for removing sialic acid from the surface of host cells. These strategies include treatment with neuraminidase (NA) and siRNA knockdown of specific genes involved in the sialic acid biosynthesis pathway. The currently widely used neuraminidase produced by Vibrio cholerae is not very efficient in removing biological macromolecules such as GM1 gangliosides. According to statistics, the neuraminidase produced by Vibrio cholerae can only strip 60-80% of the sialic acid on the surface of different cells. Therefore, when using NA to remove sialic acid, it is best to combine a complementary detection method and implement strong control to obtain relatively reliable results. SiRNA treatment is difficult to effectively block sialylation on the surface of cells, and the blocking process is only temporary, which seriously hinders the judgment of whether viruses rely on sialic acid to infect host cells. Therefore, it is of high application value to develop an effective tool to strip sialic acid from the surface of cells.

[0004] Currently, more and more nanoprobes with high sensitivity and low manufacturing cost have been approved for the diagnosis and treatment of cancer, metabolic diseases, cardiovascular diseases, and the like. Compared with the wide application of nanoprobes in cancer diagnosis and treatment, the development of nanoprobes in the field of virology has been slow. Therefore, there is an urgent need to develop an ideal nanoprobe platform to analyze, detect, and block viruses, enabling us to decipher the biological characteristics of newly emerging viruses in a timely manner, thereby achieving precise diagnosis and treatment. SUMMARY

[0005] Therefore, the application aims to provide application of a CaP / STI nanoprobe in preparation of a virus product distinguishing sialic acid dependence. The CaP / STI nanoprobe can effectively distinguish the dependence of coronavirus, rotavirus, and influenza virus on sialic acid, and has a significant inhibitory effect on the above viruses.

[0006] To solve the above technical problems, the application provides the following technical solutions:

[0007] The application provides application of the CaP / STI nanoprobe in preparation of a product for distinguishing viruses from sialic acid.

[0008] The application provides application of the CaP / STI nanoprobe in preparation of an antiviral product.

[0009] Preferably, the viruses include a coronavirus, a rotavirus and an influenza virus.

[0010] Preferably, the CaP / STI nanoprobe is released into cells by endocytosis to release Ca 2+ and an ST inhibitor.

[0011] Preferably, the CaP / STI nanoprobe is used at a concentration of 350-650 μg / mL.

[0012] Preferably, the CaP / STI nanoprobe is prepared by the following steps: dispersing n-hexanol and Triton X-100 in cyclohexane to obtain an oil phase system; mixing calcium chloride with the oil phase system to obtain a calcium microemulsion system; mixing an ST inhibitor with the oil phase system, and then adding chloroform to obtain a phosphate microemulsion system; mixing the calcium microemulsion system and the phosphate microemulsion system to obtain a core; and dispersing the core in a tetrahydrofuran mixed solution containing DOPC, DSPE-PEG 2000 and cholesterol to obtain the CaP / STI nanoprobe.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The application first finds that a core-shell calcium phosphate nanoprobe (CaP / STI) has the characteristics of distinguishing the dependence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), porcine epidemic diarrhea virus (PEDV), rotavirus and influenza virus on sialic acid, and still has a significant effect compared with neuraminidase (NA); meanwhile, the CaP / STI has the ability to continuously block sialylation, which avoids the false positive results that may occur due to the rapid recovery of sialic acid on the cell surface, and provides a new and attractive nanoprobe for accurate analysis and even prevention of viral infection.

[0015] The application also first finds that the inhibition rate of the CaP / STI on adsorption and replication of SARS-CoV-2, PEDV, influenza virus and most epidemic strains of rotavirus is as high as 50%, which lays a theoretical foundation for subsequent determination of the CaP / STI alone or in combination with existing antiviral drugs and vaccines, and effectively solves the problems of viral resistance and variability. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Schematic diagram of CaP / STI manufacturing process.

[0017] Figure 2 Characterization results of CaP / STI (A: Structure of ST inhibitor. B-D: Spherical morphology (B), particle size (C), and zeta potential (D) of CaP / STI measured by STEM and DLS (scale bar: 100 nm). E: EDX signature of CaP / STI core. F: Stability test results of CaP / STI (n = 3; mean ± SEM)).

[0018] Figure 3 Effects of different concentrations of CaP / STI on A549, 293T, Huh-7, and Vero cell viability (Data shown as mean ± SEM, n = 3).

[0019] Figure 4 Cellular internalization results of CaP / STI (A: Internalization of different concentrations of CaP / STI in A549 cells (n = 3; mean ± SEM). B: Internalization of 200 μg / ml CaP / STI in A549 cells at different time points (n = 3; mean ± SEM). C: Energy-dependent uptake results of CaP / STI evaluated by low temperature incubation (n = 3; mean ± SEM; ***P < 0.001). D: Internalization of CaP / STI in the presence of different endocytosis inhibitors (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001). E-F: Representative confocal images (E) and statistical analysis results (F) of Fluo-4 signal in A549 cells treated with PBS or CaP / STI for 3 h (n = 9-19 cells; mean ± SEM; **P < 0.01; scale bar: 50 μm).

[0020] Figure 5 Results of CaP / STI effectively blocking sialylation of A549 cells (A-B: CaP / STI concentration screening results (n = 3; mean ± SEM). C-D: CaP / STI substrate competition experiment results (n = 3; mean ± SEM; ***P < 0.001; ****P < 0.0001). E-F: Long-lasting results of CaP / STI blocking (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001).

[0021] Figure 6CaP / STI effectively blocked sialylation in Huh-7, 293T, and Vero cells (A: a-2,3-linked sialic acid. B: a-2,6-linked sialic acid. (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001; ****P < 0.0001)).

[0022] Figure 7 CaP / STI distinguished sialic acid dependency during infection of A549 cells with different coronaviruses (A-B: sialic acid dependency of different SARS-CoV-2 mutants (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001; ns, not significant). C: Effect of NA or CaP / STI on SARS-CoV-2 original strain adsorption (n = 3; mean ± SEM; **P < 0.01; ns, not significant). D-E: Effect of NA or CaP / STI on SARS-CoV-2 original strain replication (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant). F: Effect of NA or CaP / STI on SARS-CoV-2 original strain viral titer (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001). G-H: Immunofluorescence images of SARS-CoV-2 original strain infected cells (G) and quantification of infected areas (H) (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant). I: Effect of CaP / STI or NA on PEDV adsorption (n = 3; mean ± SEM; **P < 0.01; ns, not significant). J: Effect of NA or CaP / STI on PEDV replication (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0023] Figure 8CaP / STI distinguishes sialic acid dependence during infection of Huh-7 cells with different coronaviruses (A: Results of sialic acid dependence of different SARS-CoV-2 mutants treated with CaP / STI (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). B: Results of sialic acid dependence of SARS-CoV-2 wild-type treated with NA or CaP / STI (n = 3; mean ± SEM; **P < 0.01; ns, not significant). C-D: Results of the effect of NA or CaP / STI on replication of SARS-CoV-2 wild-type (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). E: Luciferase activity assay of SARS-CoV-2 pseudovirus infected cells treated with NA or CaP / STI (n = 3; mean ± SEM; ***P < 0.001; ****P < 0.0001).

[0024] Figure 9 CaP / STI distinguishes sialic acid dependence during infection of A549 cells with different rotaviruses (A-B: Results of the effect of CaP / STI on the replication performance of different subtypes of rotavirus (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001; ns, not significant). C: Effect of NA and CaP / STI on the adsorption performance of G3P[2] (n = 3; mean ± SEM; ***P < 0.001; ns, not significant). D-E: Results of viral RNA detection in cell lysates at 12, 24, and 48 hours post-infection (D) and viral titer measurement in cell supernatants at 24 hours post-infection (E) (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). F-G: Representative immunofluorescence images (F) and quantitative analysis results (G) of A549 cells infected with G3P[2] after different treatments (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0025] Figure 10Results of CaP / STI in differentiating sialic acid dependence during rotavirus infection of 293T cells (A: Effect of CaP / STI on the replication performance of different subtypes of rotavirus (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ns, not significant). B: Effect of NA and CaP / STI on the adsorption performance of G3P[2] (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001; ns, not significant). C: Effect of NA and CaP / STI on the replication of G3P[2] (n = 3; mean ± SEM; **P < 0.01; ****P < 0.0001).

[0026] Figure 11 Results of CaP / STI in differentiating sialic acid dependence during influenza virus infection of A549 cells (A-B: qRT-PCR (A) and Western blot (B) detection results of H9N2, H3N2, and H1N1 infection of A549 cell lysates pretreated with CaP / STI (n = 3; mean ± SEM; *P < 0.05; **P < 0.01). C: Effect of NA and CaP / STI on the adsorption of H9N2 (n = 3; mean ± SEM; *P < 0.05; ***P < 0.001; ****P < 0.0001). D-E: HA (D) and TCID50 (E) detection results of H9N2 infection of A549 cell culture supernatants pretreated with CaP / STI and NA (n = 3; mean ± SEM; **P < 0.01; ***P < 0.001; ****P < 0.0001). F-H: Infection cell immunofluorescence images (F), infection area quantification results (G), and viral RNA detection results in cell lysates (H) (n = 3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0027] Figure 12 Results of CaP / STI in differentiating sialic acid dependence during influenza virus infection of 293T cells (A: qRT-PCR detection results of H9N2, H3N2, and H1N1 infection of A549 cell lysates pretreated with CaP / STI (n = 3; mean ± SEM; ***P < 0.001; ****P < 0.0001). B: Effect of NA and CaP / STI on the adsorption performance of H9N2 (n = 3; mean ± SEM; *P < 0.05; **P < 0.01). C-D: HA (C) and TCID50 (D) detection results of H9N2 infection of A549 cell culture supernatants pretreated with CaP / STI and NA (n = 3; mean ± SEM; ****P < 0.0001).). DETAILED DESCRIPTION

[0028] The application provides application of the CaP / STI nanoprobe in preparation of a product for distinguishing viruses depending on sialic acid. The CaP / STI nanoprobe can effectively distinguish the characteristics of sialic acid dependence of various viruses (including subtypes), can deeply understand the transmission and pathogenic mechanism of viruses, and can find potential drug targets. The CaP / STI nanoprobe has the ability to continuously block sialylation, and can avoid false positive results that may occur due to rapid recovery of cell surface sialic acid.

[0029] The application also provides application of the CaP / STI nanoprobe in preparation of an antiviral product. The CaP / STI nanoprobe has a high inhibition rate of adsorption and replication of influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV) and most epidemic strains of rotavirus, up to 50 %, which lays a foundation for subsequent use of the CaP / STI alone or in combination with existing antiviral drugs and vaccines.

[0030] In the application, the viruses include coronavirus, rotavirus and influenza virus. The coronavirus includes SARS-CoV-2 original strain, SARS-CoV-2 mutant strain Delta, SARS-CoV-2 mutant strain Beta, SARS-CoV-2 mutant strain Omicron and PEDV; the rotavirus includes different subtypes of rotavirus G3P[2], G3P[3], G3P

[10] and G6P[1]; and the influenza virus includes different subtypes of IAV strains H9N2, H3N2 and H1N1.

[0031] In the application, the CaP / STI nanoprobe uses a concentration of 350-650 μg / mL, preferably 500 μg / mL. When the concentration of the CaP / STI nanoprobe is 500 μg / mL, the CaP / STI can obviously block α-2,3-sialylation and α-2,6-sialylation, and also reach a platform.

[0032] In the present application, the preparation method of the CaP / STI nanoprobe comprises the following steps: dispersing n-hexanol and Triton X-100 in cyclohexane to obtain an oil phase system; mixing calcium chloride with the oil phase system to obtain a calcium microemulsion system; mixing ST inhibitor with the oil phase system, and then adding chloroform to obtain a phosphate microemulsion system; mixing the calcium microemulsion system and the phosphate microemulsion system to obtain a core; dispersing the core in a tetrahydrofuran mixed solution containing DOPC, DSPE-PEG 2000 and cholesterol to obtain the CaP / STI nanoprobe. The CaP / STI nanoprobe disclosed in the present application comprises a core and a shell. The core disclosed in the present application is obtained by polymerization between the phosphate group of the ST inhibitor and Ca 2+ A. The structural formula of the ST inhibitor disclosed in the present application is as shown in Figure 2 A. The DSPE-PEG 2000, DOPC and cholesterol disclosed in the present application are self-assembled on the surface of the core through hydrophobic-hydrophobic interaction in the presence of DOPA, that is, the surface formed by the combination of DSPE-PEG 2000, DOPC and cholesterol is the shell.

[0033] In the embodiments of the present application, the A549, 293T, Huh7, Vero, Vero E6, MA104 and MDCK cells are all from ATCC; the cells are cultured in RPMI 1640 or DMEM (Gibco) culture medium containing 10% fetal bovine serum (Gibco); and the cells are grown in a culture box containing 5% carbon dioxide at 37°C.

[0034] In the present embodiments, all chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. DOPA (1,2-dioleoyl-sn-glycero-3-phosphate), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), cholesterol and DSPE-PEG 2000 (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine- poly(ethylene glycol) 2000) were purchased from Avanti Polar Lipids (Alabaster, AL, USA), the neuraminidase (NA) from Vibrio cholerae was purchased from Roche (Basel, Switzerland), the SARS-CoV-2 nucleocapsid (N) protein antibody and HA antibody were provided by Sino Biological Inc. (Beijing, China), the rotavirus VP6 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the freshly prepared chicken red blood cells (cRBCs) were from Source Leaf Biotech Co. Ltd. (Shanghai, China), the biotinylated lectins MAL II (Maackia amurensis), SNA-I (Sambucus nigra) and the non- sugar blocking solution (CFBS) were purchased from Vector Laboratories (Burlingame, CA, USA), the Fluo-4 AM, 0.3% Triton X-100, DAPI, 4% paraformaldehyde, Cy3- and HRP-conjugated secondary antibodies were purchased from Beyotime Biotech Inc. (Jiangsu, China).

[0035] In the embodiments of the present application, the SARS-CoV-2 wild type (strain IME-BJ01) was derived from GenBank No. MT291831, the Delta variant (patient isolate from India) was derived from CSTR: 16698.06.NPRC6.CCPM-B-V-049-2105-6, the Beta variant (patient isolate from South Africa) was derived from CSTR: 16698.06.NPRC2.062100001, and the Omicron variant was derived from CHN_CVRI-01 / 2022, the A / New Caledonia / 20 / 99 (H1N1), A / Chicken / Guangdong / SS / 1994 (H9N2), and A / Chicken / Guangdong / 1 / 2003 (H3N2) influenza were stored in the laboratory of the applicant, the G6P[1] and G3P[2] rotaviruses were kindly gifted by Professor Wu Yuzhang (Institute of Immunology, Army Medical University of the People's Liberation Army), the G3

[10] and G3P[3] rotaviruses were from the China Disease Prevention and Control Center (Beijing, China), and the SARS-CoV-2 pseudovirus was provided by Dali Biotech Co., Ltd. (Guangzhou, China). All experiments related to SARS-CoV-2 virus in the present application were performed in a biosafety level 3 facility.

[0036] In the embodiments of the present application, the RNA extraction and qRT-PCR assay: total RNA was extracted from cells using Trizol reagent (Sangon Biotech, Shanghai, China) according to the kit instructions; reverse transcription was performed using HiScript II QRT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer's manual; SYBR Green qPCR was performed using GoTaq® qPCR Master Mix (Promega, Beijing, China); RNA in the supernatant was extracted using the QIAamp Virus RNA Kit (QIAGEN) and then detected by absolute qPCR method using the Hiscript II One-Step RT-qPCR SYBR Green Reagent Kit (Vazyme). All primer and probe sequences in the present application are shown in Table 1.

[0037] Table 1 Primer and probe sequences

[0038]

[0039]

[0040] ​In the embodiments of the present application, the Western blot: the infected cells are lysed with RIPA lysis buffer (Beyotime); the proteins are mixed with loading buffer and denatured by boiling; after electrophoresis, they are transferred to a nitrocellulose filter, incubated with primary antibody at 4°C overnight. Then incubate with HRP-conjugated secondary antibody at room temperature for 50 min, and detect with Amersham Imager 600 (GE, USA).

[0041] In the embodiments of the present application, the virus titer determination: cells are plated in 96-well plates, serially diluted with serum-free DMEM, then infected with virus supernatant, 2 hours later, the original culture medium is replaced with DMEM containing 2% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2 for 5 days. The virus titer is measured by the Reed-Muench method. The cells used in the present application for SARS-CoV-2, rotavirus, and influenza virus titer determination are Vero E6, MA104, and MDCK, respectively.

[0042] In the embodiments of the present application, the hemagglutination assay (HA): A549 cells pretreated with NA, CaP / STI or PBS are infected with H9N2, and cell supernatant is collected 24 hours after infection. HA assay is performed according to standard procedures; gradient-diluted supernatant is added to V-bottom 96-well microplates, then 50 μL of red blood cells are added to each well; the mixture is incubated at room temperature for 30 minutes, then the cRBCs aggregation on the plate is observed.

[0043] In the embodiments of the present application, the immunofluorescence staining: infected cells are fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.3% Triton X-100; to examine different viral protein expression, cells are incubated with SARS-CoV-2 N protein antibody, rotavirus VP6 antibody, or HA antibody at room temperature for 1 hour, then incubated with Cy3-conjugated secondary antibody and DAPI at room temperature for 45 minutes. The images are taken with EVOS M5000 (Invitrogen).

[0044] In the embodiments of the present application, the statistical analysis: data are expressed as mean ± SEM; statistical differences are evaluated using GraphPad Prism 8, two-tailed unpaired Student's t-test or one-way ANOVA; differences are considered statistically significant when P < 0.05.

[0045] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Apparently, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative effort should fall into the protective scope of the present application.

[0046] Preparation and characterization of CaP / STI nanoprobe

[0047] The hydrophobic core of CaP / STI was prepared by reverse microemulsion method, and the schematic diagram of CaP / STI preparation process is as Figure 1 , and the specific steps are as follows:

[0048] (1) 100 μL of 25 mM ST inhibitor (chemical structure as Figure 2 A) was added to 8 mL of oil phase (1.5 M n-hexanol and 0.6 M Triton X-100 in cyclohexane), and then 320 μL of 20 mM chloroform (DOPA) was added to prepare a phosphate microemulsion.

[0049] (2) 100 μL of 500 mM CaCl2·2H2O was added dropwise into 8 mL of the same oil phase (1.5 M n-hexanol and 0.6 M Triton X-100 in cyclohexane) to produce a calcium microemulsion.

[0050] (3) The above two microemulsions were stirred at room temperature for 30 minutes, and then the phosphate microemulsion was added to the calcium microemulsion and stirred for another 30 minutes. Then, 32 mL of ethanol was added and stirred for 20 minutes. Centrifugation was performed at 12,000 rpm for 30 minutes, and the precipitate was collected and washed with ethanol and 50% THF / ethanol successively, and the core was harvested.

[0051] (4) The core was dispersed in a tetrahydrofuran mixture of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), and cholesterol to obtain a core-shell nanoparticle precursor; then 160 μL of the core-shell nanoparticle precursor was added dropwise into 1 mL of ethanol / PBS (V / V=3:7) at 50°C, and then stirred until the organic solvent was completely removed to obtain CaP / STI.

[0052] The preparation of Nile red-labeled CaP / STI was that Nile red dissolved in tetrahydrofuran was added to a tetrahydrofuran mixture containing the core, dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), and cholesterol to obtain a core-shell nanoparticle precursor, and the remaining steps were the same.

[0053] The particle size and zeta potential of CaP / STI were characterized by dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern, UK). The morphology of CaP / STI and the energy dispersive X-ray (EDX) spectrum of the core were observed under a scanning electron microscope (SEM) equipped with an EDX detector system (JSM-7900F, JOEL, Japan). To determine the content of ST inhibitor, CaP / STI was digested with 0.1 M hydrochloric acid overnight, and then an equal volume of chloroform was added, and the water phase was collected by centrifugation. The absorbance of the water phase at 280 nm was recorded with NanoDrop One c (Thermo Fisher Scientific, USA).

[0054] By STEM and DLS measurements, it can be seen that CaP / STI is monodisperse in H2O, with a diameter of about 45 nm Figure 2 B,C). The average zeta potential of CaP / STI is -16.3 ± 0.3 mV Figure 2 D). EDX analysis shows that calcium and phosphorus coexist well in the core of CaP / STI, which further confirms that the core is formed by the aggregation between them Figure 2 E). As shown in Figure 2 F, the particle size of CaP / STI does not change significantly in PBS at 37°C for 72 hours, indicating that the nano-probe has good stability. Finally, the drug loading of CaP / STI was determined to be 9.5 ± 0.2 wt% at a wavelength of 280 nm.

[0055] Example 2 Cytotoxicity, endocytosis and intracellular release of CaP / STI

[0056] (1) Cytotoxicity test of CaP / STI

[0057] On A549, 293T, Huh-7 and Vero cells. 2 x 10 3 cells were seeded in 96-well plates and incubated at 37°C for 24 hours. Subsequently, the cells were incubated with different concentrations of CaP / STI at 37°C for 2 days. The viability of the cells was further determined by CCK-8 detection. It was found that even if the concentration of CaP / STI was as high as 2000 μg / ml, it would not have an anti-proliferation effect on the cells, indicating that the nano-probe has good biological safety Figure 3 ).

[0058] (2) Endocytosis and intracellular release of CaP / STI

[0059] To carry out the cell uptake experiment, A549 cells were seeded at 3 x 10 5Cells were seeded in 6-well plates and incubated at 37°C for 24 h. Then, cells were treated with Nile red-labeled CaP / STI at different concentrations (20, 200, 500 and 1000 pg / mL), different time (2, 6 and 24 h), different temperature (4°C and 37°C) and different endocytosis inhibitors (MβCD, Ly294002, amiloride, CPZ and Filipin). Cells were collected and analyzed for mean fluorescence intensity (MFI) by flow cytometry (FACSCalibur, BD, USA).

[0060] Intracellular ST inhibitor release experiment was performed after 3 h treatment with 500 pg / mL CaP / STI, cells were washed with PBS and incubated with Fluo-4 AM at 37°C for 30 min. Subsequently, cells were fixed with 4% paraformaldehyde and stained with DAPI for 20 min. Fluorescent signals were observed under confocal laser scanning microscope (Nikon, Japan) and fluorescent images were quantified using ImageJ.

[0061] The results showed that the endocytosis of CaP / STI was concentration and time dependent Figure 4 A,B). In addition, the uptake of CaP / STI was reduced by 45% at 4°C compared to 37°C, indicating that the internalization of CaP / STI was energy dependent Figure 4 C). MβCD had about 55% inhibition on the internalization of CaP / STI, while Ly294002 and amiloride reduced the uptake of CaP / STI by about 30% Figure 4 D). MβCD is a classic lipid raft / caveolae-dependent endocytosis inhibitor. Ly294002 and amiloride are potent inhibitors of macropinocytosis. Therefore, the internalization of CaP / STI was mainly based on a lipid raft / caveolae-dependent manner, partially mediated by macropinocytosis pathway. The release of intracellular ST inhibitors was further evaluated with a commercial Ca 2+ indicator Fluo-4. After 3 h incubation of A549 cells with CaP / STI, the intracellular Ca 2+ signals were significantly enhanced, indicating that CaP / STI actively released Ca 2+ and ST inhibitors Figure 4 E,F) after endocytosis into cells.

[0062] Example 3 CaP / STI can effectively block the sialylation of cells

[0063] (1) CaP / STI concentration screening: To determine the effective dose of CaP / STI in subsequent experiments, A549 cells were cultured with 0 to 500 μg / mL CaP / STI for 3 days. Afterwards, the cells were incubated with biotinylated lectin MAL II or SNA-I for 50 minutes to detect the expression of α-2,3-linked sialic acid or α-2,6-linked sialic acid, respectively. Cells were washed twice with CFBS and then incubated with FITC-streptavidin for 40 minutes. Finally, the cells were washed again, and the MFI of FITC was measured by flow cytometry. The dose-response experiment showed that 500 μg / mL CaP / STI significantly blocked α-2,3-sialylation and α-2,6-sialylation, while also reaching a plateau ( Figure 5 (A, B). Therefore, the selected CaP / STI concentration was 500 μg / mL in subsequent experiments. Similarly, Huh-7, 293T, and Vero cells were cultured with 500 μg / mL CaP / STI for 3 days. The effect of desialylation was determined by flow cytometry using MAL II staining for α-2,3-linked sialic acid and SNA-I staining for α-2,6-linked sialic acid. The results showed that CaP / STI effectively inhibited the expression of sialic acid on the surface of Huh-7, 293T, and Vero cells. Figure 6 ).

[0064] (2) CaP / STI substrate competition assay: A549 cells were treated with CaP / STI in the presence or absence of 30-fold concentration of ManNAc (ManNAc is an effective sialyl transferase substrate). After 3 days, cells were harvested, and the glycosylation status was further assessed using the various biotinylated lectins mentioned above. The results showed that even a concentration of up to 30-fold ManNAc could not reverse the sialylation inhibition effect of CaP / STI. Figure 5 C,D).

[0065] To assess the long-term effectiveness of CaP / STI blockade, A549 cells were treated with 500 μg / mL CaP / STI or neuraminidase (NA) derived from Vibrio cholerae for 3 days, followed by thorough washing, and the recovery of sialylation on the cell surface was monitored in real time. Simultaneously, A549 cells were treated with neuraminidase derived from Vibrio cholerae, and the recovery of sialylation on the cell surface was monitored in real time to determine the normal turnover rate of sialic acid in A549 cells. Cell surface sialylation was assessed in real time using biotinylated lectin MAL II and SNA-I. At the start of the experiment, both CaP / STI and NA effectively cleared α-2,3-linked and α-2,6-linked sialic acid from the cell surface. The results showed that NA-treated cells fully recovered sialic acid levels after 1 day. For CaP / STI-treated cells, the expression of sialic acid on the cell surface remained at a low level for at least 3 days.Figure 5 E, F).

[0066] In summary, the above experimental data show that even in the presence of a large number of competitive sialyltransferase substrates, CaP / STI can still block sialylation of various cells in a sustained and potent manner.

[0067] Example 4 CaP / STI distinguishes the sialic acid dependence of different coronaviruses

[0068] (1) A549 cells or Huh-7 cells were pretreated with CaP / STI at a concentration of 500 pg / mL for 3 days to remove sialic acid on the cell surface, then incubated with SARS-CoV-2 wild-type and different SARS-CoV-2 mutants (Delta, Beta and Omicron), and harvested cell lysates 24 hours after infection for qRT-PCR (A) and Western blot (B) analysis.

[0069] The results show that as the SARS-CoV-2 epidemic progresses, its mutant strains gradually lose their dependence on sialic acid, and the Omicron mutant does not depend on sialic acid during infection Figure 7 A, B; Figure 8 A). It indicates that the dependence of SARS-CoV-2 wild-type on sialic acid is a necessary condition for its spread from animals to humans, but this ability is gradually abandoned during evolution to facilitate the spread of mutants in the human population.

[0070] (2) A549 cells or Huh-7 cells were pretreated with neuraminidase (NA) produced by Vibrio cholera or CaP / STI to remove sialic acid on the cell surface, then inoculated with SARS-CoV-2 wild-type at 4°C for 2 hours (for pretreated Huh-7 cells, SARS-CoV-2 pseudovirus was also inoculated), and cell lysates were collected for detection of viral RNA by qRT-PCR.

[0071] The results show that both NA and CaP / STI significantly inhibit the adsorption of SARS-CoV-2 wild-type, and there is no significant difference between them Figure 7 C; Figure 8 B).

[0072] (3) A549 cells pretreated with NA or CaP / STI were infected with SARS-CoV-2 wild-type. Cell lysates (D) and supernatants (E) were harvested at 12, 24 and 48 hours after infection for qRT-PCR analysis. The viral titer of the supernatant was determined by the TCID50 method at 24 hours after infection.

[0073] Results showed that there was a great difference in the replication of SARS-CoV-2 wild-type strain after NA and CaP / STI treatment. CaP / STI treatment significantly inhibited the replication of the virus, which was reflected in the sharp decrease of viral RNA in cell lysates and supernatants. Meanwhile, NA treatment partially reduced viral replication at the early stage of infection, but there was no significant difference in the measurement of viral RNA at 48 hours post-infection compared with the control group (untreated) Figure 7 D, E; Figure 8 C, D). Similar results were also observed in pretreated Huh-7 cells infected with SARS-CoV-2 pseudovirus Figure 8 E). The positive effect of CaP / STI on viral replication was further demonstrated by TCID50 assay, and the viral titer after CaP / STI treatment was reduced by 73% and 86% compared with the NA group and the control group, respectively Figure 7 F).

[0074] (4) The infected cells treated in step (3) were subjected to viral nucleocapsid (N) protein immunofluorescence detection (G) and quantitative analysis of the infection area (H) to observe the spread of SARS-CoV-2 wild-type strain after different treatments to evaluate the role of CaP / STI during the infection of SARS-CoV-2 wild-type strain.

[0075] The imaging results and corresponding quantitative analysis showed that the viral spread was more moderate after CaP / STI treatment Figure 7 G, H). This is because NA cannot achieve sustained sialic acid clearance. Therefore, CaP / STI can truly reflect the role of sialic acid in the process of SARS-CoV-2 infection.

[0076] (5) Vero cells treated with CaP / STI or NA were incubated with PEDV (porcine susceptible alpha coronavirus) at 4°C for 2 hours to allow viral-cell adsorption, and cell lysates were collected for qRT-PCR to quantify the adsorbed viral RNA. At the same time, Vero cells were pretreated with NA or CaP / STI to strip the sialic acid on the cell surface, then inoculated with PEDV, and cell lysates were collected at 12, 24 and 48 hours post-infection for qRT-PCR analysis.

[0077] Results showed that both NA and CaP / STI could inhibit about 45% of PEDV adsorption Figure 7 I). In addition, CaP / STI significantly inhibited the replication of PEDV at 12, 24 and 48 hours post-infection compared with NA Figure 7 J). These results collectively demonstrate the potential of CaP / STI and emphasize that sialic acid is a potential target for the development of new anti-PEDV drugs.

[0078] Example 5 CaP / STI distinguishes different rotavirus dependence on sialic acid

[0079] (1) Pretreatment of A549 cells or 293T cells with CaP / STI to strip the cell surface of sialic acid, followed by infection with different subtypes of rotavirus (G6P[1], G3P[2], G3P[3], G3P

[10] ), and quantification of viral RNA and VP6 protein in cell lysates 24 hours post-infection (B) to determine rotavirus replication.

[0080] The results show that there is a large difference in the replication of G3P[2], G3P[3], G3P

[10] and G6P[1] rotavirus subtypes after CaP / STI treatment. CaP / STI treatment significantly inhibited the replication of G6P[1], with a reduction of viral RNA in cell lysates of about 84%. CaP / STI treatment moderately reduced the production of infectious particles of G3P[2] and G3P[3] (about 50% reduction), indicating that G3P[2] and G3P[3] can partially overcome the restriction mediated by sialic acid compared to G6P[1]. In sharp contrast to G6P[1], CaP / STI treatment did not affect the replication of G3P

[10] (A). Figure 9 A; Figure 10 A). Western blot further confirmed the role of sialic acid in rotavirus infection (B). Figure 9 B).

[0081] (2) To explore whether CaP / STI can truly reveal the role of sialic acid in the adsorption of G3P[2], A549 cells or 293T cells pretreated with NA or CaP / STI were inoculated with G3P[2] at 4°C for 2 hours, washed thoroughly, and then lysed, and viral RNA was quantified by qRT-PCR.

[0082] The results show that NA and CaP / STI treatment have the same degree of inhibition of G3P[2] adsorption, and are significantly different from the control group (C). Figure 9 B). Figure 10 B).

[0083] (3) To further determine whether sialic acid plays an important role in G3P[2] infection of A549 cells or 293T cells, cells were pretreated with NA or CaP / STI, inoculated with G3P[2], and viral RNA in cell lysates at 12, 24 and 48 hours post-infection (D) and viral titer in cell supernatant at 24 hours post-infection (E) were detected to study the replication of G3P[2] after NA and CaP / STI treatment.

[0084] Results showed that CaP / STI treatment significantly inhibited the replication of G3P[2] at different time points after infection compared with NA and control groups Figure 9 D) By analyzing the virus titer by TCID50, it was found that CaP / STI treatment had more significant inhibition on the replication of G3P[2] compared with NA treatment Figure 9 E; Figure 10 C).

[0085] (4) G3P[2] -infected A549 cells at different time points after treatment in step (3) and their lysates were obtained subsequently for immunofluorescence observation and RNA quantification analysis.

[0086] Results showed that although NA treatment could moderately inhibit the replication of G3P[2], the inhibitory effect was more obvious after CaP / STI treatment Figure 9 F, G).

[0087] In summary, CaP / STI was used to find that different rotavirus subtypes have different dependencies on sialic acid, which is valuable for the precise development of anti-rotavirus drugs targeting sialic acid.

[0088] Example 6 CaP / STI distinguishes the dependence of influenza virus on sialic acid

[0089] (1) A549 or 293T cells were pretreated with CaP / STI to strip sialic acid from the cell surface, and then the pretreated A549 cells were infected with different subtypes of IAV strains H9N2, H3N2 and H1N1, respectively. Cell lysates were harvested 24 hours after infection for qRT-PCR (A) and Western blot (B).

[0090] Results showed that the inhibition of H9N2 replication was more obvious than that of H3N2 and H1N1 after CaP / STI treatment, indicating that H9N2 was more dependent on sialic acid during infection Figure 11 A, B; Figure 12 A).

[0091] (2) Next, the effect of CaP / STI on virus adsorption was studied. CaP / STI or NA-treated A549 cells or 293T cells were incubated with H9N2 at 4°C for 2 hours, and the virus RNA was quantified to detect the adsorption of the virus.

[0092] Results showed that CaP / STI treatment significantly reduced the adsorption of H9N2 to A549 and 293T cells compared with NA treatment Figure 11 C; Figure 12 B).

[0093] (3) To further verify the potential of CaP / STI in inhibiting H9N2 replication, A549 cells or 293T cells were pretreated with CaP / STI and NA, and then infected with H9N2. Culture supernatants were collected 24 hours post-infection for HA and TCID50 detection to determine the viral content therein.

[0094] The results showed that, consistent with the virus adsorption test, CaP / STI treatment effectively inhibited the production of H9N2 in the supernatant of host cells Figure 11 D, E; Figure 12 C, D).

[0095] (4) Finally, by observing H9N2-infected A549 cells in step (3) by immunofluorescence, quantifying the infected area of H9N2-infected A549 cells, and detecting viral RNA in the lysate of H9N2-infected A549 cells, the time dependence of H9N2 infection spread was fully investigated.

[0096] The results showed that the degree of H9N2 infection spread over time was lower after CaP / STI treatment Figure 11 F), which was quantitatively confirmed by detecting viral infection areas Figure 11 G) and viral RNA in cell lysates Figure 11 H).

[0097] In summary, the above results not only show that CaP / STI can accurately distinguish the sialic acid dependence of different influenza A virus (IAV) strains, but also can serve as an entry inhibitor to significantly inhibit virus adsorption and replication in vitro.

[0098] In summary, the above experiments show that CaP / STI is a self-assembled core-shell nanoprober carrying a high load of ST inhibitors, which can accurately distinguish the sialic acid dependence of a variety of viruses. A key factor in the performance of CaP / STI is its ability to continuously block sialylation, which avoids false positive results that may occur due to the rapid recovery of sialic acid on the cell surface.

[0099] Meanwhile, based on CaP / STI, it was found that the dependence on sialic acid of SARS-CoV-2 variants gradually decreased as SARS-CoV-2 spread rapidly worldwide. At the early stage of the SARS-CoV-2 pandemic, the prognosis of patients with severe pneumonia was significantly improved after treatment with nebulized DAS181. We speculate that the impact of DAS181 on the subsequent Beta, Delta, and Omicron variants will be quite limited, as our results show that the dependence on sialic acid of these variants gradually decreases. CaP / STI not only effectively distinguishes the characteristics of sialic acid dependence of various viruses (including subtypes), but also has a high inhibition rate of up to 50% for the adsorption and replication of IAV, PEDV, and most rotavirus epidemic strains, laying the foundation for the subsequent use of CaP / STI alone or in combination with existing antiviral drugs and vaccines, as well as effectively addressing the problem of viral resistance and variability.

[0100] Meanwhile, the characteristics of sialic acid dependence of different viruses, including SARS-CoV-2, PEDV, rotavirus, and IAV, were also revealed using CaP / STI, which was previously not systematically reported. These characteristics can further understand the mechanism of viral infection at the molecular level and provide an important basis for the subsequent development of new antiviral drugs. Considering the importance of sialic acid and the scalability of CaP / STI synthesis, we hope that CaP / STI can move towards clinical practice and become an effective tool for understanding or treating emerging viruses.

[0101] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. Use of CaP / STI nanoprobe in the preparation of a product for differentiating viruses that are dependent on sialic acid; characterized in that, The preparation method of the CaP / STI nanoprobe comprises the following steps: Disperse n-hexanol and Triton X-100 in cyclohexane to obtain an oil phase system; Mix calcium chloride with the oil phase system to obtain a calcium microemulsion system; Mix ST inhibitor with the oil phase system, and then add chloroform to obtain a phosphate microemulsion system; Mix the calcium microemulsion system and the phosphate microemulsion system to obtain a core; Disperse the core in a tetrahydrofuran mixed solution containing DOPC, DSPE-PEG2000 and cholesterol to obtain the CaP / STI nanoprobe; The ST inhibitor is 2. The use according to claim 1, characterized in that, The virus includes a coronavirus, a rotavirus and an influenza virus.

3. The use according to claim 1, characterized in that, The CaP / STI nanoprobe releases Ca 2+ and ST inhibitors.

4. The use according to claim 1, characterized in that, The CaP / STI nanoprobe uses a concentration of 350-650 μg / mL.

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