WO2022217966A1 - Nano-trapping agent that inhibits sars-cov-2 - Google Patents

Nano-trapping agent that inhibits sars-cov-2 Download PDF

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WO2022217966A1
WO2022217966A1 PCT/CN2021/141286 CN2021141286W WO2022217966A1 WO 2022217966 A1 WO2022217966 A1 WO 2022217966A1 CN 2021141286 W CN2021141286 W CN 2021141286W WO 2022217966 A1 WO2022217966 A1 WO 2022217966A1
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hace2
cells
nano
nanovesicles
ncs
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刘庄
陈倩
张晗
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苏州大学
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of functional materials, in particular to a nano-capturer for inhibiting SARS-CoV-2.
  • Severe acute respiratory syndrome coronavirus 2 causes coronavirus disease 2019 (COVID-19), and the SARS-CoV-2 virus has a spike (S) glycoprotein on the surface of the Human angiotensin-converting enzyme II (hACE2) binds, enters cells and infects.
  • S protein is continually mutated and its affinity for the hACE2 receptor increases, enhancing infectivity and transmission.
  • the D614G mutant strain is the main body of the current SARS-CoV-2 virus, and the D614G mutant strain has significantly improved binding efficiency to the hACE2 receptor during virus infection.
  • the novel SARS-CoV-2 variant B.1.1.7 with a large number of mutated genes has a 1000-fold increased affinity of the S protein for the hACE2 receptor, which is 70% more transmissible than previously discovered SARS-CoV-2.
  • SARS-CoV-2 vaccines have received a lot of attention since the outbreak of COVID-19.
  • SARS-CoV-2 vaccines there are more than 160 SARS-CoV-2 vaccines in the research and development stage around the world, of which 4 vaccines have been clinically approved, including mRNA vaccines (BNT162 and mRNA-1273), viral vector vaccines (ChAdOx1-2) and inactivated virus Vaccines (BBIBP-CorV), they bring light in the fight against COVID-19.
  • mRNA vaccines BNT162 and mRNA-1273
  • viral vector vaccines ChAdOx1-2
  • BBIBP-CorV inactivated virus Vaccines
  • the present invention provides a suitable freeze-drying protective agent and a mucoadhesive auxiliary material for the nanovesicles containing hACE2, and provides a safer and more effective storage for the inhalable nano-capturing agent that inhibits SARS-CoV-2.
  • the neutralization efficiency of nanovesicles against pseudoviruses can be significantly improved.
  • a nanometer trapping agent for inhibiting SARS-CoV-2 of the present invention includes nanovesicles containing hACE2, and one or both of a freeze-drying protective agent and a mucoadhesion adjuvant;
  • Nanovesicles containing hACE2 were prepared by the following steps:
  • cells (1) Transfect cells with hACE2-encoding lentivirus, hACE2-encoding plasmid or cationic liposomes carrying hACE2 genetic information to construct cells stably expressing hACE2
  • cells refer to 293T cells, Vero cells, L929 cells, Hela cells, DC2.4 cells, Raw cells, etc.;
  • the SARS-CoV-2 virus infects the host through the hACE2 receptor, and the hACE2-containing nanovesicles of the present invention compete with the host cell to bind the SARS-CoV-2 virus to protect the host cell from infection, instead of producing neutralization on the surface of the S protein Antibodies, therefore not affected by S protein mutation, are effective against different virus mutant strains, thereby improving the neutralization efficiency of nanovesicles against pseudoviruses and achieving the purpose of efficient treatment.
  • the transportation and storage conditions of existing vaccines are harsh, and the productivity of existing vaccines is limited, which cannot meet the requirements of large-scale vaccination in a short period of time.
  • the present invention introduces freeze-drying protective agent and mucoadhesive auxiliary materials for the nano-capturing agent.
  • the freeze-drying protective agent significantly improves the stability of the nano-capturing agent during storage and the convenience of transportation.
  • the neutralization titer of the vesicles remained above 90%, thus greatly increasing the feasibility of its clinical application; the mucoadhesive excipients can significantly prolong the retention of nanocaptures in the lungs and enhance the virus inhibition effect.
  • the lyoprotectant is sucrose, or sucrose and lactose, or sucrose and trehalose, or trehalose and mannitol.
  • the mucoadhesive adjuvant includes hyaluronic acid or polyvinyl alcohol.
  • the freeze-dried protective agent is mixed with the nanovesicles containing hACE2, the obtained mixed solution is freeze-dried, and finally the mucoadhesive auxiliary material is added to the freeze-dried powder. , to obtain nanometer collectors.
  • step (1) puromycin was used to select 293T cells stably expressing hACE2.
  • step (2) nanovesicles containing hACE2 are prepared by a micro-liposome extruder.
  • the particle size of the hACE2-containing nanovesicles is 100-400 nm.
  • the dosage form of the nano-collecting agent is a powder dosage form or an aerosol dosage form.
  • the lyophilized powder reconstituted in ultrapure water can be used to prepare an inhalable aerosol dosage form via a nasal spray bottle.
  • the mass ratio of the hACE2-containing nanovesicles and the lyoprotectant is 0.2-5:1, wherein the mass concentration ratio of the hACE2 transmembrane protein and the lyoprotectant is 1:25-100.
  • the mass ratio of the hACE2-containing nanovesicles and the mucoadhesive excipients is 0.2-5:1-5.
  • the present invention also claims the application of the above-mentioned nano-capturing agent in the preparation of a medicine for protecting lung tissue from SARS-CoV-2 virus infection.
  • Lung tissue can be non-invasively and efficiently protected from SARS-CoV-2 pseudovirus infection by inhaling nano-capturing agent/lyophilized protective agent/mucoadhesive excipient aerosol.
  • the present invention has at least the following advantages:
  • the nano-capturing agent of the invention has high safety, is easy to store and transport, has low cost, and can be mass-produced quickly, so as to timely and effectively deal with frequently occurring mutant strains, and has good clinical transformation prospects.
  • Figure 1 shows the results of Western blotting of hACE2-293T cells prepared in Example 1 and negative control 293T cells (1a) and the transmission electron micrograph (1b) of NCs;
  • Figure 2 is a graph showing the neutralization curves of nanovesicles extracted from hACE2-293T cells and nanovesicles extracted from negative control 293T cells to pseudoviruses;
  • Figure 3 is a comparison chart of the retention time and retention site of NCs after mixing different mucoadhesive excipients with NCs;
  • Figure 4 is the particle size and potential diagram of NCs containing different lyoprotectant powder formulations after reconstitution
  • Figure 5 is a graph showing the neutralization effect of different lyophilized protective agents and NCs mixed solutions on pseudoviruses
  • Figure 6 is a graph showing the expression of hACE2 in mouse lung tissue
  • Figure 7 is a graph showing the expression of LUCI in mouse lung tissue
  • Figure 8 is a graph showing the biosafety results of NCs/HA/sucrose in vivo
  • Fig. 9 is the transmission electron microscope image of the NCs prepared in Example 2.
  • Figure 10 shows the results of the reconstituted nanovesicle lyophilized powder obtained by lyophilization after adding HA.
  • NCs nanovesicles
  • human embryonic kidney epithelial cell 293T cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 ⁇ g/ml puromycin to construct 293T (hACE2-293T) cells stably expressing hACE2.
  • hACE2-293T cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath.
  • Figure 1a shows the Western blot results of hACE2-293T cells and negative control 293T cells (with ⁇ -actin protein as an internal reference to ensure the same amount of protein loaded), the figure shows that hACE2-293T cells express a large amount of hACE2 on the surface, with neutralization
  • Figure 1b is a transmission electron micrograph of the nanovesicles prepared in Example 1. It can be seen from the figure that NCs containing hACE2 on the surface and having a particle size of about 200 nm were successfully prepared.
  • Hela cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 ⁇ g/ml puromycin to construct Hela (hACE2-Hela) cells stably expressing hACE2.
  • hACE2-Hela cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath. Then, the suspension was centrifuged at 3000 rpm for 10 min to remove nuclei and cytoplasm.
  • the resulting cell membranes were washed twice with cold homogeneous medium. After that, cell membranes were collected by centrifugation at 14800 rpm for 30 min. Finally, the obtained suspension was sequentially extruded through a 400-nm and 200-nm polycarbonate porous membrane for 10 times using a micro-liposome extruder to obtain hACE2-containing nanovesicles. Transmission electron microscope image.
  • the inventors also tried other cells to express hACE2 through lentivirus transfection, and then prepared nanovesicles containing different hACE2 on the surface by a similar method.
  • the efficiency of lentivirus transfecting different cells is different, and the 293T cell in Example 1 is preferred, and its transfection efficiency is higher.
  • the nanovesicles in the present invention can be derived from different cells to obtain specific nanovesicles accordingly.
  • NCs nanovesicles
  • VSV vesicular stomatitis virus
  • LUCI luciferase reporter gene
  • 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 the tissue culture infectious dose. The results are shown in Figure 2.
  • Figure 2 is the neutralization curve of nanovesicle NCs extracted from hACE2-293T cells and nanovesicle NVs extracted from negative control 293T cells against pseudoviruses.
  • the axis is for neutralizing efficiency. It can be seen that compared with NVs, NCs showed strong pseudovirus neutralization ability, and its half-inhibitory concentration IC 50 value was 9.8 ⁇ g/ml.
  • Figure 3a is a graph showing the fluorescence intensity statistics of lung tissue at 6 and 24 h after inhaling a mixture of different excipients and NCs in mice.
  • the lung tissue of mice inhaled with NCs/HA showed the strongest fluorescence intensity values at different time points, and its lung treatment effect was the best. It is also significantly better than inhaling NCs/PVP and NCs/CD;
  • Figure 3b shows the biodistribution of NCs in various major organs at different time points after inhaling the mixture of different excipients and NCs in mice.
  • Figure 3c further shows the biodistribution of NCs in lung, liver, spleen, kidney, heart and other organs at different time points after mice inhaled a mixture of different excipients and NCs, and the fluorescence intensity statistics
  • the figure shows that, compared with other organs, the lung tissue has the strongest fluorescence signal at different time points, which further indicates that HA, PVA, CS and PLL all prolong the lung retention time of NCs and improve the bioavailability; The survival rate of mice after the drug is completed.
  • the figure shows that the cationic CS and PLL are highly toxic. Although they have a good therapeutic effect, they are not suitable for practical application. In conclusion, HA has a good therapeutic effect and is safe, followed by PVA.
  • the lyophilized protective agents sucrose, trehalose, mannitol, lactose and their mixtures were selected to prepare the NCs powder dosage form.
  • a mixed solution of NCs (2 mg/ml) and lyoprotectant (2.5 mg/ml) was prepared.
  • the mixed solution (1 ml) was quickly lyophilized in liquid nitrogen for 10 min and lyophilized using a lyophilizer for 24 h.
  • HA powder 2.5 mg was added to the lyophilized powder and stored at 4°C.
  • dissolve with molecular biological grade ultrapure water, and install the matching nozzle to spray the aerosol containing NCs The related results are shown in Figure 4.
  • Figure 4a shows the particle size and potential diagram of nanovesicles after lyophilization and reconstitution with different lyoprotectants.
  • the size of the NCs in the control group and the mannitol group increased after being dissolved in water, and obvious aggregation occurred
  • the size of the NCs in the lactose group decreased after being dissolved in water, and the size of the other groups did not change significantly, indicating that The homogeneity of vesicles after lyophilization and reconstitution with these lyophilized protective agents was better; in terms of potential, the size and zeta potential of NCs in other groups remained basically unchanged
  • Figure 4b is a schematic diagram of the samples used in lyophilized powder. Dissolve the lyophilized powder containing NCs with water, install the nose tip and spray the aerosol. The lyophilized powder dosage form facilitates long-term storage and transportation.
  • the lyophilized powder of nanovesicles can only be obtained by lyophilization After mixing the HA solid powder and dissolving it at the time of use, the ideal spray formulation effect can be achieved. This is different from the conventional preparation of freeze-dried powder preparations. In actual production, freeze-dried powder preparations are freeze-dried and packaged in the final step, which is more conducive to controlling parameters such as water content in freeze-dried powder. Applicable to the present invention, in order to better obtain a lyophilized powder formulation, the nanovesicles and the lyophilized protective agent must be mixed and lyophilized, and then mixed with dried auxiliary materials to obtain a usable formulation.
  • Example 4 the lyophilized powder prepared in Example 4 was dissolved in biological-grade ultrapure water, and the diluents of different concentrations were neutralized with pseudoviruses according to the method in Example 2, and the neutralization efficiency was calculated. Then, the freshly prepared NCs solution and the optimized NCs/HA/sucrose lyophilized powder were stored in a 4°C refrigerator for 1 month and then reconstituted. The pseudovirus neutralization experiment was performed under the same conditions. The related results are shown in Figure 5.
  • Figure 5a is a graph showing the neutralization effect of different lyoprotectants and NCs mixed solutions on pseudoviruses.
  • the pseudovirus neutralization efficiency is the most important indicator. The higher the pseudovirus neutralization efficiency, the better the lyophilization protection effect. It can be seen from the figure that the NCs/HA/sucrose group, NCs/HA/lactose and sucrose, NCs/ There was no significant difference between the HA/sucrose and trehalose, NCs/HA/trehalose and mannitol groups and the newly prepared nanovesicles, and they all maintained a high level;
  • Figure 5b shows the NCs solution and the optimized NCs/HA/sucrose jelly The neutralization effect of dry powder on pseudovirus after being stored at 4°C for one month. The titer of the reconstituted NCs retained approximately 90%. The results indicated that the NCs/HA/sucrose lyophilized powder better retained the virus neutralization ability of NCs.
  • mice Male immunodeficient NSG mice were anesthetized, and hACE2-encoding replication-deficient adenovirus (AdV-hACE2) (1 ⁇ 1010 PFU, 50 ⁇ L) was administered through the bronchi to express hACE2 in mouse lung tissue to establish a hACE2-expressing mouse model. After 5 days, the mouse lung tissue was removed, and part of it was used to prepare single cell suspension for hACE2 flow antibody staining, and the other part was used to prepare cell lysate to detect the expression of hACE2 by Western blotting. The results are shown in Figure 6.
  • AdV-hACE2 replication-deficient adenovirus
  • Figure 6 shows the expression of hACE2 in mouse lung tissue, wherein AdV-Empty represents a replication-deficient adenovirus that does not carry genetic information.
  • mice expressing hACE2 were divided into three groups, inhaled 50 ⁇ L of phosphate buffered saline (PBS), NCs/sucrose, and NCs/HA/sucrose, respectively, and inhaled twice after 4 h and 8 h of SARS-CoV-2 encoding LUCI containing SARS-CoV-2 Pseudovirus with viral S protein coat.
  • PBS phosphate buffered saline
  • NCs/sucrose NCs/HA/sucrose
  • Figure 7a In the flow cytometry results, the percentages of LUCI-positive cells in the PBS control group, NCs/sucrose and NCs/HA/sucrose groups were 6.5%, 2.1% and 0%, respectively; Figure 7b Western blot analysis of LUCI expression in the PBS group Highest. The above results suggest that inhalation of hACE2-containing NCs and HA exhibited potent pseudovirus inhibition in a hACE2-expressing mouse model by prolonging pulmonary retention.
  • NCs/HA/sucrose 200 ⁇ g of NCs membrane protein
  • mouse serum and whole blood samples were collected on days 1 and 7 for serum biochemistry and whole blood analysis.
  • concentrations of inflammatory cytokines (tumor necrosis factor alpha, interleukin 6, interleukin 12) in mouse serum were determined by ELISA kit.
  • the biosafety results of NCs/HA/sucrose in vivo are shown in Figure 8.
  • Figure 8a shows that all blood markers were not significantly different from the PBS treated group;
  • Figure 8b serum inflammatory cytokine concentrations were at baseline levels. All these results indicate that the NCs/HA/sucrose complex has excellent biocompatibility.

Abstract

The present invention relates to a nano-trapping agent that inhibits SARS-CoV-2, comprising nanovesicles containing hACE2, and one or two among a freeze-drying protective agent and a mucoadhesive auxiliary material, the nanovesicles containing hACE2 being prepared and obtained by the following steps: transfecting 293T cells by using lentivirus that codes hACE2, to construct 293T cells that stably express hACE2; and extracting cell membranes of the obtained cells to prepare nanovesicles containing hACE2. The freeze-drying protective agent is sucrose, sucrose and lactose, sucrose and trehalose, or trehalose and mannitol; and the mucoadhesive auxiliary material comprises hyaluronic acid or polyvinyl alcohol.

Description

一种抑制SARS-CoV-2的纳米捕集剂A nano trapping agent that inhibits SARS-CoV-2 技术领域technical field
本发明涉及功能材料领域，尤其涉及一种抑制SARS-CoV-2的纳米捕集剂。The invention relates to the field of functional materials, in particular to a nano-capturer for inhibiting SARS-CoV-2.
背景技术Background technique
严重急性呼吸系统综合征冠状病毒2(SARS-CoV-2)引起了2019冠状病毒病(COVID-19)，SARS-CoV-2病毒表面具有刺突(S)糖蛋白通过与受体细胞表面的人血管紧张素转换酶II(hACE2)结合，进入细胞并感染。S蛋白不断地发生突变，其与hACE2受体的亲和力增加，增强了感染性和传播能力。其中D614G突变株是目前SARS-CoV-2病毒的主体，D614G突变株在病毒感染过程中与hACE2受体结合效率明显提高。具有大量突变基因的新型SARS-CoV-2变异株B.1.1.7，其S蛋白与hACE2受体的亲和力提高了1000倍，比之前发现的SARS-CoV-2的传播能力高出70％。Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), and the SARS-CoV-2 virus has a spike (S) glycoprotein on the surface of the Human angiotensin-converting enzyme II (hACE2) binds, enters cells and infects. The S protein is continually mutated and its affinity for the hACE2 receptor increases, enhancing infectivity and transmission. Among them, the D614G mutant strain is the main body of the current SARS-CoV-2 virus, and the D614G mutant strain has significantly improved binding efficiency to the hACE2 receptor during virus infection. The novel SARS-CoV-2 variant B.1.1.7 with a large number of mutated genes has a 1000-fold increased affinity of the S protein for the hACE2 receptor, which is 70% more transmissible than previously discovered SARS-CoV-2.
自COVID-19爆发以来，疫苗受到广泛关注。目前，全球处于研发阶段的SARS-CoV-2疫苗有160多种，其中4种疫苗已经获得临床批准，包括mRNA疫苗(BNT162和mRNA-1273)、病毒载体疫苗(ChAdOx1-2)和灭活病毒疫苗(BBIBP-CorV)，它们为抗击COVID-19带来曙光。Vaccines have received a lot of attention since the outbreak of COVID-19. At present, there are more than 160 SARS-CoV-2 vaccines in the research and development stage around the world, of which 4 vaccines have been clinically approved, including mRNA vaccines (BNT162 and mRNA-1273), viral vector vaccines (ChAdOx1-2) and inactivated virus Vaccines (BBIBP-CorV), they bring light in the fight against COVID-19.
目前的疫苗主要通过产生S蛋白表面的中和抗体来保护宿主免受感染。然而，S蛋白的突变可能会降低这些疫苗的有效性。例如，接种了Moderna(mRNA-1273)或Pfizer-BioNTech(BNT162b2)疫苗志愿者的血清对南非突变株(B.1.351)的中和活性明显降低。另外，现有疫苗运输和存储条件苛刻，因此，仍需一种能保持纳米囊泡对假病毒的中和效率且便于运输和保存的疫苗。Current vaccines protect the host from infection mainly by producing neutralizing antibodies on the surface of the S protein. However, mutations in the S protein may reduce the effectiveness of these vaccines. For example, the neutralizing activity of sera from volunteers vaccinated with Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against the South African mutant (B.1.351) was significantly reduced. In addition, the transportation and storage conditions of existing vaccines are harsh, therefore, there is still a need for a vaccine that can maintain the neutralization efficiency of nanovesicles against pseudoviruses and is convenient for transportation and storage.
发明内容SUMMARY OF THE INVENTION
为解决上述技术问题，本发明针对含hACE2的纳米囊泡提供了合适的冻干保护剂和黏膜黏附辅料，为抑制SARS-CoV-2的可吸入型纳米捕集剂提供了更安全有效的存储方法，相对于传统保护剂可显著提升纳米囊泡对假病毒的中和效率。In order to solve the above technical problems, the present invention provides a suitable freeze-drying protective agent and a mucoadhesive auxiliary material for the nanovesicles containing hACE2, and provides a safer and more effective storage for the inhalable nano-capturing agent that inhibits SARS-CoV-2. Compared with traditional protective agents, the neutralization efficiency of nanovesicles against pseudoviruses can be significantly improved.
本发明的一种抑制SARS-CoV-2的纳米捕集剂，包括含hACE2的纳米囊泡，还包括冻干保护剂和黏膜黏附辅料中的一种或两种；A nanometer trapping agent for inhibiting SARS-CoV-2 of the present invention includes nanovesicles containing hACE2, and one or both of a freeze-drying protective agent and a mucoadhesion adjuvant;
含hACE2的纳米囊泡由以下步骤制备得到：Nanovesicles containing hACE2 were prepared by the following steps:
(1)用编码hACE2的慢病毒、编码hACE2的质粒或携带hACE2遗传信息的阳离子脂质体转染细胞，构筑稳定表达hACE2的细胞此处细胞指293T细胞、Vero细胞、L929细胞、Hela细胞、DC2.4细胞、Raw细胞等；(1) Transfect cells with hACE2-encoding lentivirus, hACE2-encoding plasmid or cationic liposomes carrying hACE2 genetic information to construct cells stably expressing hACE2 Here cells refer to 293T cells, Vero cells, L929 cells, Hela cells, DC2.4 cells, Raw cells, etc.;
(2)提取步骤(1)所得细胞的细胞膜，以细胞膜为原料制备含hACE2的纳米囊泡(NCs)。(2) Extracting the cell membrane of the cells obtained in step (1), and using the cell membrane as a raw material to prepare hACE2-containing nanovesicles (NCs).
SARS-CoV-2病毒通过hACE2受体感染宿主，本发明的含hACE2的纳米囊泡与宿主细胞竞争结合SARS-CoV-2病毒来保护宿主细胞免受感染，而不是产生S蛋白表面的中和抗体，因此不受S蛋白突变的影响，对不同的病毒突变株均有效，从而提高纳米囊泡对假病毒的中和效率，实现高效治疗的目的。现有疫苗运输和存储条件苛刻，且现有疫苗生产力有限，不能在短期满足大量接种要求。本发明针对此纳米捕集剂引入冻干保护剂和黏膜黏附辅料，冻干保护剂显著提高了纳米捕获剂储存过程中的稳定性和运输的便利性，保存较长时间后复溶，纳米囊泡的中和效价仍保持90％以上，从而大大增加了其临床应用的可行性；黏膜黏附辅料可以显著延长纳米捕获剂在肺中的滞留，增强病毒抑制作用。The SARS-CoV-2 virus infects the host through the hACE2 receptor, and the hACE2-containing nanovesicles of the present invention compete with the host cell to bind the SARS-CoV-2 virus to protect the host cell from infection, instead of producing neutralization on the surface of the S protein Antibodies, therefore not affected by S protein mutation, are effective against different virus mutant strains, thereby improving the neutralization efficiency of nanovesicles against pseudoviruses and achieving the purpose of efficient treatment. The transportation and storage conditions of existing vaccines are harsh, and the productivity of existing vaccines is limited, which cannot meet the requirements of large-scale vaccination in a short period of time. The present invention introduces freeze-drying protective agent and mucoadhesive auxiliary materials for the nano-capturing agent. The freeze-drying protective agent significantly improves the stability of the nano-capturing agent during storage and the convenience of transportation. The neutralization titer of the vesicles remained above 90%, thus greatly increasing the feasibility of its clinical application; the mucoadhesive excipients can significantly prolong the retention of nanocaptures in the lungs and enhance the virus inhibition effect.
进一步地，冻干保护剂为蔗糖，或蔗糖和乳糖，或蔗糖和海藻糖，或海藻糖和甘露醇。Further, the lyoprotectant is sucrose, or sucrose and lactose, or sucrose and trehalose, or trehalose and mannitol.
进一步地，黏膜黏附辅料包括透明质酸或聚乙烯醇。Further, the mucoadhesive adjuvant includes hyaluronic acid or polyvinyl alcohol.
进一步地，本发明的纳米捕集剂在制备过程中，先将冻干保护剂与含hACE2的纳米囊泡混合，将得到的混合溶液冷冻干燥，最后向冻干后的粉末中加入黏膜黏附辅料，得到纳米捕集剂。Further, in the preparation process of the nano-capturing agent of the present invention, firstly, the freeze-dried protective agent is mixed with the nanovesicles containing hACE2, the obtained mixed solution is freeze-dried, and finally the mucoadhesive auxiliary material is added to the freeze-dried powder. , to obtain nanometer collectors.
进一步地，在步骤(1)中，用嘌呤霉素筛选稳定表达hACE2的293T细胞。Further, in step (1), puromycin was used to select 293T cells stably expressing hACE2.
进一步地，在步骤(2)中，通过微型脂质体挤出器制备含hACE2的纳米囊泡。Further, in step (2), nanovesicles containing hACE2 are prepared by a micro-liposome extruder.
进一步地，含hACE2的纳米囊泡的粒径为100-400nm。Further, the particle size of the hACE2-containing nanovesicles is 100-400 nm.
进一步地，纳米捕集剂的剂型为粉末剂型或气溶胶剂型。超纯水复溶的冻干粉可通过喷鼻瓶制备可吸入的气溶胶剂型。Further, the dosage form of the nano-collecting agent is a powder dosage form or an aerosol dosage form. The lyophilized powder reconstituted in ultrapure water can be used to prepare an inhalable aerosol dosage form via a nasal spray bottle.
进一步地，含hACE2的纳米囊泡和冻干保护剂的质量比为0.2-5:1，其中，hACE2跨膜蛋白和冻干保护剂的质量浓度比为1:25-100。Further, the mass ratio of the hACE2-containing nanovesicles and the lyoprotectant is 0.2-5:1, wherein the mass concentration ratio of the hACE2 transmembrane protein and the lyoprotectant is 1:25-100.
进一步地，含hACE2的纳米囊泡和黏膜黏附辅料的质量比为0.2-5:1-5。Further, the mass ratio of the hACE2-containing nanovesicles and the mucoadhesive excipients is 0.2-5:1-5.
本发明的还要求保护上述纳米捕集剂在制备保护肺组织免受SARS-CoV-2病毒感染的药物中的应用。通过吸入纳米捕集剂/冻干保护剂/黏膜黏附辅料气溶胶，可以无创、高效地保护肺组织免受SARS-CoV-2假病毒的感染。The present invention also claims the application of the above-mentioned nano-capturing agent in the preparation of a medicine for protecting lung tissue from SARS-CoV-2 virus infection. Lung tissue can be non-invasively and efficiently protected from SARS-CoV-2 pseudovirus infection by inhaling nano-capturing agent/lyophilized protective agent/mucoadhesive excipient aerosol.
借由上述方案，本发明至少具有以下优点：By means of the above scheme, the present invention has at least the following advantages:
本发明的纳米捕集剂安全性高，易于存储与运输，成本低廉，能够快速量产，以便及时、有效的应对频繁出现的突变毒株，具有良好的临床转化前景。The nano-capturing agent of the invention has high safety, is easy to store and transport, has low cost, and can be mass-produced quickly, so as to timely and effectively deal with frequently occurring mutant strains, and has good clinical transformation prospects.
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the description, the following description is given with the preferred embodiments of the present invention and the detailed drawings.
附图说明Description of drawings
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below according to specific embodiments of the present invention and in conjunction with the accompanying drawings.
图1为实施例1制备的hACE2-293T细胞与阴性对照293T细胞的蛋白印迹结果图(1a)和NCs的透射电子显微图(1b)；Figure 1 shows the results of Western blotting of hACE2-293T cells prepared in Example 1 and negative control 293T cells (1a) and the transmission electron micrograph (1b) of NCs;
图2为hACE2-293T细胞提取的纳米囊泡与阴性对照293T细胞提取的纳米囊泡对假病毒的中和曲线图；Figure 2 is a graph showing the neutralization curves of nanovesicles extracted from hACE2-293T cells and nanovesicles extracted from negative control 293T cells to pseudoviruses;
图3为不同黏膜黏附辅料与NCs混合后NCs的滞留时间和滞留部位对比图；Figure 3 is a comparison chart of the retention time and retention site of NCs after mixing different mucoadhesive excipients with NCs;
图4为NCs包含不同冻干保护剂的粉末剂型复溶后的粒径和电位图；Figure 4 is the particle size and potential diagram of NCs containing different lyoprotectant powder formulations after reconstitution;
图5为不同冻干保护剂与NCs混合溶液对假病毒的中和效果图；Figure 5 is a graph showing the neutralization effect of different lyophilized protective agents and NCs mixed solutions on pseudoviruses;
图6为小鼠肺组织hACE2的表达情况图；Figure 6 is a graph showing the expression of hACE2 in mouse lung tissue;
图7为小鼠肺组织的LUCI表达情况图；Figure 7 is a graph showing the expression of LUCI in mouse lung tissue;
图8为NCs/HA/蔗糖在体内的生物安全性结果图；Figure 8 is a graph showing the biosafety results of NCs/HA/sucrose in vivo;
图9为实施例2制备的NCs的透射电镜图；Fig. 9 is the transmission electron microscope image of the NCs prepared in Example 2;
图10为加入HA后冻干得到的纳米囊泡冻干粉复溶时的结果。Figure 10 shows the results of the reconstituted nanovesicle lyophilized powder obtained by lyophilization after adding HA.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
实施例1Example 1
表面含有hACE2纳米囊泡(NCs)的制备Preparation of surface-containing hACE2 nanovesicles (NCs)
首先，利用编码跨膜蛋白hACE2的慢病毒转染人胚胎肾上皮细胞293T细胞，与2μg/ml嘌呤霉素共孵育构筑稳定表达hACE2的293T(hACE2-293T)细胞。其次，用胰蛋白酶消化收集hACE2-293T细胞，并用含有0.25M蔗糖，1mM EDTA，10mM Hepes(pH 7.4)和蛋白酶抑制剂的均质培养基重 悬。在冰浴下用功率为200W的超声仪破坏细胞。然后，将悬浮液以3000rpm离心10min以去除细胞核和细胞质。所得的细胞膜用冷的均质培养基洗涤两次。之后，以14800rpm离心30min收集细胞膜。最后，使用微型脂质体挤出器将所得悬浊液依次挤出400nm、200nm聚碳酸酯多孔膜各10次。结果见图1。First, human embryonic kidney epithelial cell 293T cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 μg/ml puromycin to construct 293T (hACE2-293T) cells stably expressing hACE2. Next, hACE2-293T cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath. Then, the suspension was centrifuged at 3000 rpm for 10 min to remove nuclei and cytoplasm. The resulting cell membranes were washed twice with cold homogeneous medium. After that, cell membranes were collected by centrifugation at 14800 rpm for 30 min. Finally, the obtained suspension was sequentially extruded 10 times each of 400 nm and 200 nm polycarbonate porous membranes using a micro-liposome extruder. The results are shown in Figure 1.
图1a为hACE2-293T细胞与阴性对照293T细胞的蛋白印迹结果图(以β-actin蛋白为内参，确保蛋白上样量一致)，该图表明hACE2-293T细胞表面表达大量的hACE2，具有中和新冠病毒的潜力；图1b为实施例1制备的纳米囊泡的透射电子显微图，从图中可见成功制备表面含有hACE2，粒径200nm左右的NCs。Figure 1a shows the Western blot results of hACE2-293T cells and negative control 293T cells (with β-actin protein as an internal reference to ensure the same amount of protein loaded), the figure shows that hACE2-293T cells express a large amount of hACE2 on the surface, with neutralization The potential of the new coronavirus; Figure 1b is a transmission electron micrograph of the nanovesicles prepared in Example 1. It can be seen from the figure that NCs containing hACE2 on the surface and having a particle size of about 200 nm were successfully prepared.
实施例2Example 2
首先，利用编码跨膜蛋白hACE2的慢病毒转染Hela细胞，与2μg/ml嘌呤霉素共孵育构筑稳定表达hACE2的Hela(hACE2-Hela)细胞。其次，用胰蛋白酶消化收集hACE2-Hela细胞，并用含有0.25M蔗糖，1mM EDTA，10mM Hepes(pH 7.4)和蛋白酶抑制剂的均质培养基重悬。在冰浴下用功率为200W的超声仪破坏细胞。然后，将悬浮液以3000rpm离心10min以去除细胞核和细胞质。所得的细胞膜用冷的均质培养基洗涤两次。之后，以14800rpm离心30min收集细胞膜。最后，使用微型脂质体挤出器将所得悬浊液依次挤出400nm、200nm聚碳酸酯多孔膜各10次，得到含hACE2的纳米囊泡，图9为本实施例制备的纳米囊泡的透射电镜图。First, Hela cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 μg/ml puromycin to construct Hela (hACE2-Hela) cells stably expressing hACE2. Next, hACE2-Hela cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath. Then, the suspension was centrifuged at 3000 rpm for 10 min to remove nuclei and cytoplasm. The resulting cell membranes were washed twice with cold homogeneous medium. After that, cell membranes were collected by centrifugation at 14800 rpm for 30 min. Finally, the obtained suspension was sequentially extruded through a 400-nm and 200-nm polycarbonate porous membrane for 10 times using a micro-liposome extruder to obtain hACE2-containing nanovesicles. Transmission electron microscope image.
发明人又尝试了其他细胞通过慢病毒转染表达hACE2，然后通过相似的方法制备得到表面含有不同hACE2的纳米囊泡。慢病毒转染不同细胞的效率不同，优选实施例1中293T细胞，其转染效率更高。但，本发明中的纳米囊泡可来自于不同细胞，以相应的得到特异性的纳米囊泡。The inventors also tried other cells to express hACE2 through lentivirus transfection, and then prepared nanovesicles containing different hACE2 on the surface by a similar method. The efficiency of lentivirus transfecting different cells is different, and the 293T cell in Example 1 is preferred, and its transfection efficiency is higher. However, the nanovesicles in the present invention can be derived from different cells to obtain specific nanovesicles accordingly.
实施例3Example 3
含hACE2纳米囊泡(NCs)的体外病毒中和能力In vitro virus neutralization ability of hACE2-containing nanovesicles (NCs)
所使用的基于水泡性口炎病毒(VSV)的假病毒，包含SARS-CoV-2病毒 S蛋白并携带荧光素酶(LUCI)报告基因。通过观察hACE2-293T细胞的感染情况评估NCs对假病毒的中和能力。首先，将hACE2-293T细胞以10 4个细胞/孔的密度接种于96孔板中，培养16h。其次，将不同浓度的NCs(50μL)与等体积的假病毒(500TCID 50)在37℃下孵育1h，然后将混合溶液转移到单层hACE2-293T中感染48小时。最后，统计细胞裂解液中荧光素酶的荧光强度，计算中和效率。中和效率(％)＝[1–[(样品的荧光强度值–背景荧光强度值的平均值)/(仅对照病毒荧光强度值的平均值–背景荧光强度值的平均值)]]×100％。TCID 50指半数组织培养感染剂量。结果见图2。 The vesicular stomatitis virus (VSV)-based pseudovirus used contains the SARS-CoV-2 virus S protein and carries a luciferase (LUCI) reporter gene. The ability of NCs to neutralize pseudoviruses was evaluated by observing the infection of hACE2-293T cells. First, hACE2-293T cells were seeded in a 96-well plate at a density of 10 4 cells/well and cultured for 16 hours. Second, different concentrations of NCs (50 μL) were incubated with an equal volume of pseudovirus (500TCID 50 ) for 1 h at 37°C, and then the mixed solution was transferred to a monolayer of hACE2-293T for infection for 48 h. Finally, the fluorescence intensity of luciferase in the cell lysate was counted to calculate 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 the tissue culture infectious dose. The results are shown in Figure 2.
图2是hACE2-293T细胞提取的纳米囊泡NCs与阴性对照293T细胞提取的纳米囊泡NVs对假病毒的中和曲线，横轴为半抑制浓度IC 50值(单位为μg/ml)，纵轴为中和效率。可见与NVs对比，NCs表现出强大的假病毒中和能力，其半抑制浓度IC 50值为9.8μg/ml。 Figure 2 is the neutralization curve of nanovesicle NCs extracted from hACE2-293T cells and nanovesicle NVs extracted from negative control 293T cells against pseudoviruses. The axis is for neutralizing efficiency. It can be seen that compared with NVs, NCs showed strong pseudovirus neutralization ability, and its half-inhibitory concentration IC 50 value was 9.8 μg/ml.
实施例4Example 4
黏膜黏附辅料增强NCs的肺部滞留Mucoadhesive excipients enhance pulmonary retention of NCs
首先，选取三种黏膜黏附辅料聚乙烯醇(PVA)、聚乙烯醇吡咯烷酮(PVP)、透明质酸(HA)、环糊精(CD)、壳聚糖(CS)和聚赖氨酸(PLL)(1mg/ml)分别与菁染料Cy5.5标记的NCs(2mg/ml)混合。然后，将小鼠气麻，使用可雾化的气溶胶装置吸入50ul以上混合溶液，打开小鼠口腔，将雾化针头伸置小鼠支气管口处，喷出包含NCs和辅料的气溶胶。最后，在吸入给药后的不同时间点观察并统计主要器官的荧光强度。结果见图3。First, three mucoadhesive excipients, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), hyaluronic acid (HA), cyclodextrin (CD), chitosan (CS) and polylysine (PLL) were selected. ) (1 mg/ml) were mixed with the cyanine dye Cy5.5-labeled NCs (2 mg/ml), respectively. Then, the mice were anesthetized, and more than 50 ul of the mixed solution was inhaled using an atomizable aerosol device. The oral cavity of the mice was opened, and the aerosol needle was placed at the bronchial orifice of the mice, and an aerosol containing NCs and excipients was sprayed out. Finally, the fluorescence intensity of major organs was observed and counted at different time points after inhalation administration. The results are shown in Figure 3.
图3a是小鼠吸入不同辅料与NCs的混合物，肺组织在6和24h的荧光强度统计图。其中吸入NCs/HA的小鼠肺组织在不同时间点均显示最强的荧光强度值，其肺部治疗效果最好，吸入NCs/PVA、NCs/CS和NCs/PLL的小鼠肺部治疗效果也显著优于吸入NCs/PVP和NCs/CD；图3b是小鼠吸入不同辅料与NCs的混合物后不同时间点NCs在各主要器官的生物分布，从图中可知HA、PVA、CS和PLL可显著延长NCs在主要器官中的滞留时间；图3c进一步显示小鼠吸入不同辅料与NCs的混合物后，不同时间点NCs 在肺、肝、脾、肾、心等器官中的生物分布，荧光强度统计图表明，相对于其他器官，肺组织在不同时间点均具有最强的荧光信号，进一步表明HA、PVA、CS和PLL均延长了NCs肺部滞留时间，提高了生物利用度；图3d为给药完成后的小鼠存活率，图中显示，阳离子的CS、PLL的毒性很大，虽然具有较好的治疗效果，但是不适于实际应用，综上HA治疗效果好且安全，PVA次之。Figure 3a is a graph showing the fluorescence intensity statistics of lung tissue at 6 and 24 h after inhaling a mixture of different excipients and NCs in mice. Among them, the lung tissue of mice inhaled with NCs/HA showed the strongest fluorescence intensity values at different time points, and its lung treatment effect was the best. It is also significantly better than inhaling NCs/PVP and NCs/CD; Figure 3b shows the biodistribution of NCs in various major organs at different time points after inhaling the mixture of different excipients and NCs in mice. Significantly prolong the retention time of NCs in major organs; Figure 3c further shows the biodistribution of NCs in lung, liver, spleen, kidney, heart and other organs at different time points after mice inhaled a mixture of different excipients and NCs, and the fluorescence intensity statistics The figure shows that, compared with other organs, the lung tissue has the strongest fluorescence signal at different time points, which further indicates that HA, PVA, CS and PLL all prolong the lung retention time of NCs and improve the bioavailability; The survival rate of mice after the drug is completed. The figure shows that the cationic CS and PLL are highly toxic. Although they have a good therapeutic effect, they are not suitable for practical application. In conclusion, HA has a good therapeutic effect and is safe, followed by PVA.
实施例5Example 5
NCs粉末剂型的制备Preparation of NCs Powder Dosage Form
选取冻干保护剂蔗糖、海藻糖、甘露醇、乳糖及其混合物来制备NCs粉末剂型。首先，制备NCs(2mg/ml)和冻干保护剂(2.5mg/ml)的混合溶液。其次，将混合溶液(1ml)置于液氮中10min快速冻干并使用冻干机冷冻干燥24h。冻干后的粉末中加入HA粉末(2.5mg)，于4℃保存。使用时用分子生物级超纯水溶解，并安装配套的喷鼻头，喷出含NCs的气溶胶。相关结果见图4。The lyophilized protective agents sucrose, trehalose, mannitol, lactose and their mixtures were selected to prepare the NCs powder dosage form. First, a mixed solution of NCs (2 mg/ml) and lyoprotectant (2.5 mg/ml) was prepared. Next, the mixed solution (1 ml) was quickly lyophilized in liquid nitrogen for 10 min and lyophilized using a lyophilizer for 24 h. HA powder (2.5 mg) was added to the lyophilized powder and stored at 4°C. When using, dissolve with molecular biological grade ultrapure water, and install the matching nozzle to spray the aerosol containing NCs. The related results are shown in Figure 4.
图4a为纳米囊泡与不同冻干保护剂冻干复溶后的粒径和电位图。其中，从粒径来看，对照组和甘露醇组的NCs溶于水后尺寸增大，出现明显的聚集，乳糖组的NCs溶于水后尺寸变小，其他组的尺寸无明显变化，说明采用这些冻干保护剂冻干复溶后的囊泡均一性较好；从电位来看，其他组NCs的大小和zeta电位基本保持不变；图4b为冻干粉使用的样品示意图。用水溶解含NCs的冻干粉，安装喷鼻头随即喷出气溶胶。该冻干粉剂型有利于长期存储和运输。Figure 4a shows the particle size and potential diagram of nanovesicles after lyophilization and reconstitution with different lyoprotectants. Among them, in terms of particle size, the size of the NCs in the control group and the mannitol group increased after being dissolved in water, and obvious aggregation occurred, the size of the NCs in the lactose group decreased after being dissolved in water, and the size of the other groups did not change significantly, indicating that The homogeneity of vesicles after lyophilization and reconstitution with these lyophilized protective agents was better; in terms of potential, the size and zeta potential of NCs in other groups remained basically unchanged; Figure 4b is a schematic diagram of the samples used in lyophilized powder. Dissolve the lyophilized powder containing NCs with water, install the nose tip and spray the aerosol. The lyophilized powder dosage form facilitates long-term storage and transportation.
在实验中发现，如果加入HA之后进行冻干，冻干产物不能很好的复溶，出现肉眼可见的颗粒团聚，如图10所示，因此只能在冻干得到纳米囊泡的冻干粉后混合HA固体粉末，并在使用时溶解，才能达到理想的喷雾制剂效果。这与常规的冻干粉制剂制备思路不同，实际生产中，冻干粉制剂在最后步骤进行冻干和封装，能更有利于控制冻干粉中的含水量等参数指标，而这种思路不适用于本发明，为了更好的得到冻干粉制剂，必须在纳米囊泡与冻 干保护剂混合冻干后，再与干燥的辅料混合，以得到可用的制剂。In the experiment, it was found that if lyophilized after adding HA, the lyophilized product could not be reconstituted well, and the particles agglomerated visible to the naked eye, as shown in Figure 10. Therefore, the lyophilized powder of nanovesicles can only be obtained by lyophilization After mixing the HA solid powder and dissolving it at the time of use, the ideal spray formulation effect can be achieved. This is different from the conventional preparation of freeze-dried powder preparations. In actual production, freeze-dried powder preparations are freeze-dried and packaged in the final step, which is more conducive to controlling parameters such as water content in freeze-dried powder. Applicable to the present invention, in order to better obtain a lyophilized powder formulation, the nanovesicles and the lyophilized protective agent must be mixed and lyophilized, and then mixed with dried auxiliary materials to obtain a usable formulation.
实施例6Example 6
NCs粉末剂型的体外中和能力In vitro neutralization ability of NCs powder dosage form
首先，将实施例4中制备的冻干粉用生物级超纯水溶解，按照实施例2中的方法将不同浓度的稀释液与假病毒进行中和，计算中和效率。然后，将新鲜制备的NCs溶液与优化的NCs/HA/蔗糖冻干粉置于4℃冰箱保存1个月后复溶，同样条件下进行假病毒中和实验。相关结果见图5。First, the lyophilized powder prepared in Example 4 was dissolved in biological-grade ultrapure water, and the diluents of different concentrations were neutralized with pseudoviruses according to the method in Example 2, and the neutralization efficiency was calculated. Then, the freshly prepared NCs solution and the optimized NCs/HA/sucrose lyophilized powder were stored in a 4°C refrigerator for 1 month and then reconstituted. The pseudovirus neutralization experiment was performed under the same conditions. The related results are shown in Figure 5.
图5a为不同冻干保护剂与NCs混合溶液对假病毒的中和效果图。假病毒中和效率作为最重要的指标，假病毒中和效率越高，视为冻干保护效果越好，从图中可知，NCs/HA/蔗糖组、NCs/HA/乳糖和蔗糖、NCs/HA/蔗糖和海藻糖、NCs/HA/海藻糖和甘露醇组与新制备的纳米囊泡效率无明显差异，均保持较高的水平；图5b为NCs溶液与优化的NCs/HA/蔗糖冻干粉4℃保存一个月后对假病毒的中和效果图。复溶后的NCs的效价保留了大约90％。该结果表明NCs/HA/蔗糖冻干粉较好的保留了NCs的病毒中和能力。Figure 5a is a graph showing the neutralization effect of different lyoprotectants and NCs mixed solutions on pseudoviruses. The pseudovirus neutralization efficiency is the most important indicator. The higher the pseudovirus neutralization efficiency, the better the lyophilization protection effect. It can be seen from the figure that the NCs/HA/sucrose group, NCs/HA/lactose and sucrose, NCs/ There was no significant difference between the HA/sucrose and trehalose, NCs/HA/trehalose and mannitol groups and the newly prepared nanovesicles, and they all maintained a high level; Figure 5b shows the NCs solution and the optimized NCs/HA/sucrose jelly The neutralization effect of dry powder on pseudovirus after being stored at 4°C for one month. The titer of the reconstituted NCs retained approximately 90%. The results indicated that the NCs/HA/sucrose lyophilized powder better retained the virus neutralization ability of NCs.
综合图4和图5的结果，NCs/HA/蔗糖组、NCs/HA/乳糖和蔗糖、NCs/HA/蔗糖和海藻糖、NCs/HA/海藻糖和甘露醇这四种组合可大大提升对纳米囊泡的保护作用。Combining the results in Figures 4 and 5, the four combinations of NCs/HA/sucrose group, NCs/HA/lactose and sucrose, NCs/HA/sucrose and trehalose, NCs/HA/trehalose and mannitol can greatly improve the effect of Protective effects of nanovesicles.
实施例7Example 7
hACE2小鼠模型的建立Establishment of hACE2 mouse model
麻醉雄性的免疫缺陷的NSG小鼠，通过支气管给予编码hACE2的复制缺陷型腺病毒(AdV-hACE2)(1x1010PFU，50μL)使小鼠肺组织表达hACE2，建立表达hACE2的小鼠模型。5天后，将小鼠肺组织取出，部分用于制备单细胞悬浮液进行hACE2流式抗体染色，另一部分制备细胞裂解液通过蛋白质印迹法检测hACE2的表达。结果见图6。Male immunodeficient NSG mice were anesthetized, and hACE2-encoding replication-deficient adenovirus (AdV-hACE2) (1×1010 PFU, 50 μL) was administered through the bronchi to express hACE2 in mouse lung tissue to establish a hACE2-expressing mouse model. After 5 days, the mouse lung tissue was removed, and part of it was used to prepare single cell suspension for hACE2 flow antibody staining, and the other part was used to prepare cell lysate to detect the expression of hACE2 by Western blotting. The results are shown in Figure 6.
图6为小鼠肺组织hACE2的表达情况，其中，AdV-Empty表示不携带遗传信息的复制缺陷型腺病毒。图6a的流式结果图与图6b的蛋白印迹结果 图均说明编码hACE2的AdV成功诱导了小鼠肺组织中hACE2的表达。以上结果表明hACE2小鼠模型成功建立。Figure 6 shows the expression of hACE2 in mouse lung tissue, wherein AdV-Empty represents a replication-deficient adenovirus that does not carry genetic information. The flow cytometry results in Figure 6a and the Western blot results in Figure 6b both indicate that AdV encoding hACE2 successfully induced the expression of hACE2 in mouse lung tissue. The above results indicated that the hACE2 mouse model was successfully established.
实施例8Example 8
可吸入NCs在体内抑制病毒感染的能力The ability of inhalable NCs to inhibit viral infection in vivo
利用hACE2小鼠模型评估实施例4中优化的NCs/HA/蔗糖在体内抑制病毒感染的能力。将表达hACE2的小鼠分为三组，分别吸入50μL磷酸盐缓冲液(PBS)、NCs/蔗糖和NCs/HA/蔗糖，并于4h和8h后吸入两次编码LUCI的含有SARS-CoV-2病毒S蛋白外壳的假病毒。小鼠肺组织的LUCI表达情况见图7。The ability of the NCs/HA/sucrose optimized in Example 4 to inhibit viral infection in vivo was assessed using the hACE2 mouse model. Mice expressing hACE2 were divided into three groups, inhaled 50 μL of phosphate buffered saline (PBS), NCs/sucrose, and NCs/HA/sucrose, respectively, and inhaled twice after 4 h and 8 h of SARS-CoV-2 encoding LUCI containing SARS-CoV-2 Pseudovirus with viral S protein coat. The expression of LUCI in mouse lung tissue is shown in Figure 7.
图7a流式细胞结果中，PBS对照组、NCs/蔗糖和NCs/HA/蔗糖组的LUCI阳性细胞百分比分别为6.5％，2.1％和0％；图7b蛋白质印迹分析中PBS组的LUCI表达量最高。以上结果说明吸入含hACE2的NCs和HA通过延长肺部滞留，在表达hACE2的小鼠模型中表现出有效的假病毒抑制作用。Figure 7a In the flow cytometry results, the percentages of LUCI-positive cells in the PBS control group, NCs/sucrose and NCs/HA/sucrose groups were 6.5%, 2.1% and 0%, respectively; Figure 7b Western blot analysis of LUCI expression in the PBS group Highest. The above results suggest that inhalation of hACE2-containing NCs and HA exhibited potent pseudovirus inhibition in a hACE2-expressing mouse model by prolonging pulmonary retention.
实施例9Example 9
可吸入NCs体内的生物安全性Biosafety of inhalable NCs in vivo
吸入较高剂量的NCs/HA/蔗糖(NCs膜蛋白质量为200μg)，在第1天和7天收集小鼠血清和全血样品进行血清生物化学和全血分析。通过ELISA试剂盒确定小鼠血清中炎性细胞因子(肿瘤坏死因子α，白介素6，白介素12)的浓度。NCs/HA/蔗糖在体内的生物安全性结果见图8。Higher doses of NCs/HA/sucrose (200 μg 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 concentrations of inflammatory cytokines (tumor necrosis factor alpha, interleukin 6, interleukin 12) in mouse serum were determined by ELISA kit. The biosafety results of NCs/HA/sucrose in vivo are shown in Figure 8.
图8a表明所有的血液标志物与PBS治疗组均无显著差异；图8b血清炎性细胞因子的浓度均处于基线水平。所有这些结果表明，NCs/HA/蔗糖复合物具有优异的生物相容性。Figure 8a shows that all blood markers were not significantly different from the PBS treated group; Figure 8b serum inflammatory cytokine concentrations were at baseline levels. All these results indicate that the NCs/HA/sucrose complex has excellent biocompatibility.
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, other different forms of changes or modifications can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. However, the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (10)

  1. 一种抑制SARS-CoV-2的纳米捕集剂，其特征在于，所述纳米捕集剂包括含hACE2的纳米囊泡，还包括冻干保护剂和黏膜黏附辅料中的一种或两种；A nano-capturing agent for inhibiting SARS-CoV-2, characterized in that, the nano-capturing agent comprises hACE2-containing nanovesicles, and also includes one or both of a freeze-drying protective agent and a mucoadhesion adjuvant;
    所述含hACE2的纳米囊泡由以下步骤制备得到：The hACE2-containing nanovesicles are prepared by the following steps:
    (1)用编码hACE2的慢病毒、编码hACE2的质粒或携带hACE2遗传信息的阳离子脂质体转染细胞，构筑稳定表达hACE2的细胞；所述细胞为293T细胞、Vero细胞、L929细胞、Hela细胞、DC2.4细胞和Raw细胞中的一种或几种；(1) Transfect cells with lentivirus encoding hACE2, plasmid encoding hACE2 or cationic liposomes carrying hACE2 genetic information to construct cells stably expressing hACE2; the cells are 293T cells, Vero cells, L929 cells, Hela cells , one or more of DC2.4 cells and Raw cells;
    (2)提取步骤(1)所得细胞的细胞膜，以细胞膜为原料制备含hACE2的纳米囊泡。(2) extracting the cell membrane of the cells obtained in step (1), and using the cell membrane as a raw material to prepare nanovesicles containing hACE2.
  2. 根据权利要求1所述的纳米捕集剂，其特征在于：所述冻干保护剂为蔗糖，或蔗糖和乳糖，或蔗糖和海藻糖，或海藻糖和甘露醇。The nanometer collector according to claim 1, wherein the freeze-drying protective agent is sucrose, or sucrose and lactose, or sucrose and trehalose, or trehalose and mannitol.
  3. 根据权利要求1所述的纳米捕集剂，其特征在于：所述黏膜黏附辅料包括透明质酸或聚乙烯醇。The nano-capturing agent according to claim 1, wherein the mucoadhesive adjuvant comprises hyaluronic acid or polyvinyl alcohol.
  4. 根据权利要求1所述的纳米捕集剂，其特征在于，由以下步骤制备得到：将冻干保护剂与含hACE2的纳米囊泡混合得到混合溶液，将所述混合溶液冷冻干燥，向冻干后的粉末中加入黏膜黏附辅料，得到所述纳米捕集剂。The nano-capturing agent according to claim 1 is characterized in that, it is prepared by the following steps: mixing the lyophilized protective agent with the nanovesicles containing hACE2 to obtain a mixed solution, freeze-drying the mixed solution, and lyophilizing the Mucoadhesive auxiliary materials are added to the obtained powder to obtain the nano-capturing agent.
  5. 根据权利要求1所述的纳米捕集剂，其特征在于：所述含hACE2的纳米囊泡和冻干保护剂的质量比为0.2-5:1。The nano-capturing agent according to claim 1, wherein the mass ratio of the hACE2-containing nanovesicles to the lyoprotectant is 0.2-5:1.
  6. 根据权利要求1所述的纳米捕集剂，其特征在于：所述含hACE2的纳米囊泡和黏膜黏附辅料的质量比为0.2-5:1-5。The nano-capturing agent according to claim 1, wherein the mass ratio of the hACE2-containing nanovesicles and the mucoadhesion auxiliary material is 0.2-5:1-5.
  7. 根据权利要求1所述的纳米捕集剂，其特征在于：所述含hACE2的纳米囊泡的粒径为100-400nm。The nano-capturing agent according to claim 1, wherein the particle size of the hACE2-containing nanovesicles is 100-400 nm.
  8. 根据权利要求1所述的纳米捕集剂，其特征在于：所述纳米捕集剂的 剂型为粉末剂型或气溶胶剂型。The nanometer collector according to claim 1, wherein the dosage form of the nanometer collector is a powder dosage form or an aerosol dosage form.
  9. 根据权利要求1所述的纳米捕集剂，其特征在于：用嘌呤霉素筛选稳定表达hACE2的293T细胞。The nano-capturing agent according to claim 1, characterized in that: 293T cells stably expressing hACE2 are screened with puromycin.
  10. 一种权利要求1-9任一项所述的纳米捕集剂在制备保护肺组织免受SARS-CoV-2病毒感染的药物中的应用。An application of the nano-capturing agent according to any one of claims 1-9 in the preparation of a medicine for protecting lung tissue from SARS-CoV-2 virus infection.
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