CN113509546A

CN113509546A - Nano trapping agent for inhibiting SARS-CoV-2

Info

Publication number: CN113509546A
Application number: CN202110422601.1A
Authority: CN
Inventors: 刘庄; 陈倩; 张晗
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-10-19
Also published as: CN113842453B; CN113842453A; WO2022217966A1

Abstract

The invention relates to a nano trapping agent for inhibiting SARS-CoV-2, which comprises a nano vesicle containing hACE2, wherein the nano vesicle containing hACE2 is prepared by the following steps: transfecting 293T cells with lentivirus encoding hACE2 to construct 293T cells stably expressing hACE 2; extracting cell membranes of the obtained cells to prepare the nano vesicles containing hACE 2. The invention protects the host cells from being infected by competing and combining the virus with the host cells, has the neutralization titer not influenced by virus mutation, is easy to store and transport, has low cost and can realize rapid mass production.

Description

Nano trapping agent for inhibiting SARS-CoV-2

Technical Field

The invention relates to the field of functional materials, in particular to a nano trapping agent for inhibiting SARS-CoV-2.

Background

The 2019 coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the surface of which has spike (S) glycoprotein that enters cells and infects by binding with human angiotensin converting enzyme II (hACE2) on the surface of recipient cells. The S protein is constantly mutated, with increased affinity for the hACE2 receptor, enhancing infectivity and transmission. Wherein the D614G mutant is the main body of the SARS-CoV-2 virus, the D614G mutant has obviously improved binding efficiency with hACE2 receptor in the virus infection process. The S protein affinity of the novel SARS-CoV-2 variant B.1.1.7 with a large number of mutant genes is improved by about 1000 times, which is about 70% higher than the spreading ability of SARS-CoV-2 found before.

Since the outbreak of COVID-19, vaccines have received much attention. Currently, there are over 160 of the world's SARS-CoV-2 vaccines in development, of which 4 have been clinically approved, including mRNA vaccines (BNT162 and mRNA-1273), viral vector vaccines (ChAdOx1-2), and inactivated virus vaccines (BBIBP-CorV), which bring about the presence of eosin against COVID-19.

Current vaccines protect the host from infection primarily by generating neutralizing antibodies to the surface of the S protein. However, mutations in the S protein may reduce the effectiveness of these vaccines. For example, sera from volunteers vaccinated with modern (mRNA-1273) or Pfizer-BioNTech (BNT162b2) showed a significant reduction in neutralizing activity against the south African mutant (B.1.351). Therefore, it is urgent to develop new strategies for efficiently and rapidly preventing SARS-CoV-2 mutant infection.

Disclosure of Invention

In order to solve the technical problems, the invention provides a nano vesicle containing hACE2, which competes with host cells for binding viruses, and the neutralization titer is not influenced by virus mutation.

The invention relates to a nano trapping agent for inhibiting SARS-CoV-2, which comprises a nano vesicle containing hACE2, wherein the nano vesicle containing hACE2 is prepared by the following steps:

(1) human embryonic kidney epithelial cell 293T cells were transfected with lentivirus encoding hACE2 to construct 293T cells stably expressing hACE2 (hACE2-293T cells);

(2) extracting cell membranes of the cells obtained in the step (1), and preparing the nano vesicles (NCs) containing hACE2 by using the cell membranes as raw materials.

The SARS-CoV-2 virus infects host through hACE2 receptor, the nano vesicle containing hACE2 of the invention competes with host cell to combine with SARS-CoV-2 virus to protect host cell from infection, but not generate neutralizing antibody on S protein surface, therefore, the invention is not affected by S protein mutation and is effective to different virus mutant strains, thereby improving the neutralizing efficiency of nano vesicle to pseudovirus and realizing the purpose of high-efficiency treatment.

Further, in step (1), 293T cells stably expressing hACE2 were screened with puromycin.

Further, in step (2), nanovesicles containing hACE2 were prepared by a miniliposome extruder.

Further, the particle size of the nano vesicle containing hACE2 is 100-400 nm.

Further, the dosage form of the nano trapping agent is a powder dosage form or an aerosol dosage form. The ultrapure water redissolved freeze-dried powder can be prepared into inhalable aerosol dosage forms through a nasal spray bottle.

Further, the nano trapping agent also comprises one or two of a freeze-drying protective agent and a mucous membrane adhesion auxiliary material. The existing vaccine has harsh transportation and storage conditions, and the existing vaccine has limited productivity and cannot meet the requirement of mass inoculation in a short time. By introducing the freeze-drying protective agent, the stability and the transportation convenience of the nano capture agent in the storage process are obviously improved, so that the feasibility of the clinical application of the nano capture agent is greatly improved. The mucosa adhesion auxiliary material can obviously prolong the detention of the nano capture agent in the lung and enhance the virus inhibition effect.

Further, the mass ratio of the nano vesicle containing hACE2 to the lyoprotectant is 0.2-5:1, wherein the mass concentration ratio of the transmembrane protein of hACE2 to the lyoprotectant is 1: 25-100.

Furthermore, the mass ratio of the nano vesicle containing hACE2 to the mucous membrane adhesion auxiliary material is 0.2-5: 1-5.

Further, the lyoprotectant includes one or both of sucrose and trehalose, preferably sucrose.

Further, the mucoadhesion adjuvant comprises Hyaluronic Acid (HA).

The invention also claims the application of the nano trapping agent in preparing the medicine for protecting lung tissues from SARS-CoV-2 virus infection. By inhaling the nano capture agent/freeze-drying protective agent/mucous membrane adhesion auxiliary material aerosol, the lung tissue can be protected from SARS-CoV-2 pseudovirus infection in a non-invasive and high-efficient manner.

By the scheme, the invention at least has the following advantages:

(1) the present invention protects host cells from infection by competing with host cells for binding to the virus, and this strategy can be extended to other potential epidemics, with profound impact on vaccine development.

(2) The invention has high safety, easy storage and transportation, low cost, rapid volume production, convenient timely and effective response to frequently-occurring mutant strains and good clinical transformation prospect.

(3) The traditional antiviral vaccine has reduced protective effect on SARS-CoV-2 mutant strain, and the vaccine needs to be updated in time, but the nano trapping agent of the invention is different from antibody vaccine, can not cause lasting immunological memory, and can realize high-efficiency protection.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a Western blot result of hACE2-293T cells prepared in example 1 and negative control 293T cells (1a) and a transmission electron micrograph (1b) of NCs;

FIG. 2 is a graph of the neutralization of pseudoviruses of nanovesicles extracted from hACE2-293T cells and nanovesicles extracted from negative control 293T cells;

FIG. 3 is a graph showing the residence time and residence location of NCs after mixing different mucoadhesive excipients with the NCs;

FIG. 4 is a graph of particle size and potential after reconstitution of powder formulations with NCs containing different lyoprotectants;

FIG. 5 is a graph showing the neutralization effect of different lyoprotectants with mixed NCs solutions on pseudoviruses;

FIG. 6 is a graph showing the expression of hACE2 in mouse lung tissue;

FIG. 7 is a graph of LUCI expression in mouse lung tissue;

FIG. 8 is a graph showing the results of the biosafety of NCs/HA/sucrose in vivo.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Preparation of nano vesicles (NCs) containing hACE2 on surface

First, 293T cells (hACE2-293T) stably expressing hACE2 were constructed by transfecting human embryonic kidney epithelial cells 293T cells with a lentivirus encoding the transmembrane protein hACE2 and incubating the cells with 2. mu.g/ml puromycin. Next, hACE2-293T cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1mM EDTA, 10mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted by a 200W power sonicator while in an ice bath. The suspension was then centrifuged at 3000rpm for 10min to remove the nuclei and cytoplasm. The resulting cell membranes were washed twice with cold homogeneous medium. Thereafter, the cell membranes were collected by centrifugation at 14800rpm for 30 min. Finally, the resulting suspension was extruded sequentially through 400nm and 200nm polycarbonate porous membranes 10 times each using a miniliposome extruder. The results are shown in FIG. 1.

FIG. 1a is a Western blot result of hACE2-293T cells and negative control 293T cells (with beta-actin as an internal reference to ensure consistent protein loading), which indicates that hACE2-293T cells express a large amount of hACE2 on the surface and have the potential to neutralize new coronavirus; FIG. 1b is a transmission electron micrograph of the nanovesicles prepared in example 1, from which it can be seen that NCs having hACE2 on the surface and a particle size of about 200nm were successfully prepared.

Example 2

In vitro virus neutralization capacity of hACE2 nano vesicles (NCs)

A pseudovirus based on Vesicular Stomatitis Virus (VSV) is used, comprising the SARS-CoV-2 virus S protein and carrying a Luciferase (LUCI) reporter gene. The neutralization of pseudoviruses by NCs was assessed by observing the infection of hACE2-293T cells. First, hACE2-293T cells were cultured at 10⁴The density of individual cells/well was plated in 96-well plates and cultured for 16 h. Next, different concentrations of NCs (50. mu.L) were combined with equal volumes of pseudovirus (500 TCID)₅₀) After incubation at 37 ℃ for 1h, the mixed solution was transferred to a monolayer of hACE2-293T for infection for 48 hours. And finally, counting the fluorescence intensity of luciferase in the cell lysate, and calculating the neutralization efficiency. Neutralization efficiency (%) - [ 1- [ (fluorescence intensity value of sample-average value of background fluorescence intensity value)/(average value of fluorescence intensity value of control virus only-average value of background fluorescence intensity value)]]×100％。TCID_{50 refers to}Half of the tissue culture infectious dose. The results are shown in FIG. 2.

FIG. 2 is a neutralization curve of nano-vesicle NCs extracted from hACE2-293T cells and nano-vesicle NVs extracted from negative control 293T cells against pseudoviruses, with half inhibitory concentration IC on the horizontal axis₅₀Values (in. mu.g/ml) and the vertical axis is the neutralization efficiency. It can be seen that NCs exhibit a strong pseudovirus neutralizing capacity, compared to NVs, with a half-inhibitory concentration IC₅₀The value was 9.8. mu.g/ml.

Example 3

Mucoadhesive adjuvants enhance pulmonary retention of NCs

Firstly, three mucoadhesion auxiliary materials, namely polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and Hyaluronic Acid (HA) (1mg/ml), are respectively selected and mixed with cyanine dye Cy5.5 labeled NCs (2 mg/ml). Then, the mouse aeroma is inhaled more than 50ul of the mixed solution by using an aerosol device capable of atomizing, the mouth of the mouse is opened, an atomizing needle head is extended to the bronchus of the mouse, and the aerosol containing NCs and auxiliary materials is sprayed out. Finally, the fluorescence intensity of the major organs was observed and counted at different time points after inhalation administration. The results are shown in FIG. 3.

FIG. 3a is a statistical plot of fluorescence intensity of lung tissue at 6, 12 and 24h after mice were inhaled with mixtures of different excipients with NCs. Wherein, the lung tissues of the mice inhaled with NCs/HA all show the strongest fluorescence intensity values at different time points, and the lung treatment effect is the best; FIG. 3b is the biodistribution of NCs in each major organ at different time points after mice were inhaled with NCs/HA. The fluorescence intensity statistical graph shows that the lung tissue HAs the strongest fluorescence signals at different time points compared with other organs, and further shows that HA prolongs the lung retention time of NCs and improves the bioavailability.

Example 4

Preparation of NCs powder dosage forms

Three commonly used lyoprotectants, sucrose, trehalose and mannitol, were chosen for the preparation of NCs powder dosage forms. First, a mixed solution of NCs (2mg/ml) and lyoprotectant (2.5mg/ml) was prepared. Next, the mixed solution (1ml) was flash-lyophilized in liquid nitrogen for 10min and freeze-dried using a lyophilizer for 24 h. The lyophilized powder was weighed as HA powder (2.5mg) and stored at 4 ℃. When in use, the aerosol is dissolved in molecular biological grade ultrapure water, and is provided with a matched spray nose head to spray the aerosol containing NCs. The correlation results are shown in FIG. 4.

Fig. 4a is a graph of particle size and potential after reconstitution of a powder dosage form containing a lyoprotectant. Wherein the NCs of the control group and the mannitol group are increased in size after being dissolved in water, obvious aggregation occurs, and the size and zeta potential of the NCs of the sucrose and trehalose groups are basically kept unchanged; FIG. 4b is a schematic of a sample used for lyophilized powder. Dissolving the NCs-containing lyophilized powder in water, and installing the nasal spray head to spray aerosol. The lyophilized powder is convenient for long-term storage and transportation.

Example 5

In vitro neutralization capacity of NCs powder dosage forms

First, the lyophilized powder prepared in example 4 was dissolved in biological grade ultrapure water, and the dilutions and pseudoviruses of different concentrations were neutralized according to the method in example 2, and the neutralization efficiency was calculated. Then, after the freshly prepared NCs solution and the optimized NCs/sucrose lyophilized powder are stored in a refrigerator at 4 ℃ for 1 month, a pseudovirus neutralization experiment is performed under the same conditions. The correlation results are shown in FIG. 5.

FIG. 5a is a graph showing the neutralization effect of different lyoprotectants and NCs mixed solutions on pseudoviruses. Compared with fresh NCs, the freeze-dried powder added with sucrose shows the optimal pseudovirus neutralization effect after being dissolved; FIG. 5b is a graph showing the neutralization effect of NCs solution and optimized NCs/sucrose lyophilized powder after being stored at 4 ℃ for one month on pseudoviruses. The titer of NCs after reconstitution remained about 90%. The result shows that the virus neutralization capacity of the NCs is better reserved by the NCs/HA/sucrose freeze-dried powder.

Example 6

Establishment of hACE2 mouse model

A mouse model expressing hACE2 was constructed by anesthetizing male immunodeficient NSG mice and expressing hACE2 in mouse lung tissue by bronchial administration of replication-deficient adenovirus encoding hACE2 (AdV-hACE2) (1X1010 PFU, 50. mu.L). After 5 days, mouse lung tissue was removed, a portion was used to prepare single cell suspensions for flow antibody staining of hACE2, and another portion was used to prepare cell lysates for detecting expression of hACE2 by western blotting. The results are shown in FIG. 6.

FIG. 6 shows the expression of hACE2 in mouse lung tissue, where AdV-Empty indicates a replication-deficient adenovirus that does not carry genetic information. Both the flow chart of FIG. 6a and the Western blot result chart of FIG. 6b demonstrate that AdV encoding hACE2 successfully induced expression of hACE 2. The above results indicate that the hACE2 mouse model was successfully established.

Example 7

Ability of inhalable NCs to inhibit viral infection in vivo

The ability of NCs/HA/sucrose optimized in example 4 to inhibit viral infection in vivo was evaluated using the hACE2 mouse model. Mice expressing hACE2 were divided into three groups, 50. mu.L of Phosphate Buffered Saline (PBS), NCs/sucrose and NCs/HA/sucrose, respectively, and pseudovirus containing the SARS-CoV-2 virus S protein coat encoding LUCI was inhaled twice after 4h and 8 h. LUCI expression in mouse lung tissue is shown in FIG. 7.

FIG. 7a flow cytometry results for the LUCI-positive cell percentages of the PBS control, NCs/sucrose, and NCs/HA/sucrose groups at 6.5%, 2.1%, and 0%, respectively; FIG. 7b Western blot analysis of the PBS group with the highest LUCI expression. The above results indicate that inhalation of NCs and HA containing hACE2 shows potent pseudoviral inhibition in a mouse model expressing hACE2 by prolonging pulmonary retention.

Example 8

In vivo biosafety of inhalable NCs

Higher doses of NCs/HA/sucrose (200 μ g mass of NCs membrane protein) were inhaled and mouse serum and whole blood samples were collected on days 1 and 7 for serum biochemistry and whole blood analysis. The concentration of inflammatory cytokines (tumor necrosis factor α, interleukin 6, interleukin 12) in the serum of mice was determined by ELISA kit. The biosafety results of NCs/HA/sucrose in vivo are shown in FIG. 8.

Figure 8a shows that all blood markers did not differ significantly from the PBS treatment group; FIG. 8b serum inflammatory cytokine concentrations are all at baseline levels. All these results indicate that the NCs/HA/sucrose complex HAs excellent biocompatibility.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A nano trapping agent for inhibiting SARS-CoV-2, wherein the nano trapping agent comprises a nano vesicle containing hACE2, and the nano vesicle containing hACE2 is prepared by the following steps:

(1) transfecting 293T cells with lentivirus encoding hACE2 to construct 293T cells stably expressing hACE 2;

(2) extracting cell membranes of the cells obtained in the step (1), and preparing the nano vesicles containing hACE2 by using the cell membranes as raw materials.

2. The nanotrap according to claim 1, characterized in that: the particle size of the nano vesicle containing hACE2 is 100-400 nm.

3. The nanotrap according to claim 1, characterized in that: the dosage form of the nano trapping agent is a powder dosage form or an aerosol dosage form.

4. The nanotrap according to claim 1, characterized in that: the nano trapping agent also comprises one or two of a freeze-drying protective agent and a mucous membrane adhesion auxiliary material.

5. The nanotrap according to claim 4, characterized in that: the mass ratio of the nano vesicle containing hACE2 to the freeze-drying protective agent is 0.2-5: 1.

6. The nanotrap according to claim 4, characterized in that: the mass ratio of the nano vesicle containing hACE2 to the mucous membrane adhesion auxiliary material is 0.2-5: 1-5.

7. The nanotrap according to claim 4, characterized in that: the lyoprotectant includes sucrose.

8. The nanotrap according to claim 4, characterized in that: the mucosal adhesion adjuvant comprises hyaluronic acid.

9. The nanotrap according to claim 1, characterized in that: 293T cells stably expressing hACE2 were screened for puromycin.

10. Use of a nanocapture agent according to any one of claims 1 to 9 for the preparation of a medicament for protecting lung tissue against infection by SARS-CoV-2 virus.