Anti-novel coronavirus nanosphere and preparation method and application thereof

Abstract

This invention discloses an anti-novel coronavirus nanosphere, which mainly consists of a core-shell structure nanomaterial formed by multi-layering chitosan with sulfated alginate polysaccharides of different molecular weights, creating a shell-core structure. This structure mimics the host cell surface HSPGs, interfering with the binding of novel coronavirus surface proteoglycans to host cell surface HSPGs and ACE2 receptors, thereby blocking the novel coronavirus from invading host cells. The preparation method of the anti-novel coronavirus nanospheres provided by this invention is simple, using natural, safe, and non-toxic raw materials. It can be directly applied to human skin and mucous membranes, exhibiting good neutralizing and inhibitory effects on the novel coronavirus. It can protect cells from novel coronavirus infection and also has a certain antiviral effect on cells already infected with the novel coronavirus. It can be used to prepare protective equipment for preventing novel coronavirus infection, or to prepare drugs for the prevention and / or treatment of novel coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-novel coronavirus nanosphere, its preparation method, and its application. Background Technology

[0002] The SARS-CoV-2 virus primarily enters cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface. Studies have shown that if the cell surface lacks heparan sulfate proteoglycan (HSPG), the SARS-CoV-2 virus cannot bind to ACE2; HSPG is a co-receptor for viral binding to cellular ACE2. Therefore, finding inhibitors that interfere with the affinity of the SARS-CoV-2 virus for HSPG, thus preventing the virus from effectively binding to ACE2, is an effective way to directly inhibit viral binding to ACE2 and prevent SARS-CoV-2 infection. Summary of the Invention

[0003] The purpose of this invention is to provide an anti-novel coronavirus nanosphere to solve at least one of the above-mentioned technical problems.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned anti-novel coronavirus nanospheres to solve at least one of the above-mentioned technical problems.

[0005] Another object of the present invention is to provide the application of the above-mentioned anti-novel coronavirus nanospheres in the preparation of protective products or drugs for the prevention of novel coronavirus infection and in the preparation of drugs for the treatment of novel coronavirus infection, so as to solve at least one of the above-mentioned technical problems.

[0006] According to one aspect of the present invention, an anti-novel coronavirus nanosphere is provided, which has a core-shell structure with sulfated seaweed polysaccharide as the shell and chitosan as the core, and the preparation method includes the following steps:

[0007] (1) Dissolve chitosan in an acidic solution with a concentration of 2.5-10% to prepare a chitosan solution with a concentration of 5-20 mg / mL; and dissolve sulfated alginate in water according to the mass ratio of chitosan to sulfated alginate polysaccharide of 1-2:6-10 to prepare a sulfated alginate polysaccharide solution with a concentration of 5-50 mg / mL.

[0008] (2) Mix the sulfated seaweed polysaccharide solution and the chitosan solution at a mass ratio of chitosan to sulfated seaweed polysaccharide of 1:(3-10), homogenize at high speed for 5-10 min, and ultrasonically disperse for 0.5-3 h to obtain sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0009] (3) Mix the sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite with the remaining sulfated seaweed polysaccharide solution, stir for 0.5-3h, ultrasonically disperse for 0.5-2h, and spray dry at a temperature of 125-150℃ to obtain anti-novel coronavirus nanospheres.

[0010] In some embodiments, the molecular weight of chitosan can be 3-10 kDa, and the molecular weight of sulfated seaweed polysaccharide can be 150-550 kDa.

[0011] In some embodiments, the sulfated seaweed polysaccharide comprises, by weight, 1-5 parts of sulfated seaweed polysaccharide with a molecular weight of 150-200 kDa, 1-5 parts of sulfated seaweed polysaccharide with a molecular weight of 300-350 kDa, and 2-10 parts of sulfated seaweed polysaccharide with a molecular weight of 500-550 kDa.

[0012] The novel coronavirus-resistant nanospheres provided by this invention use sulfated alginate and chitosan as the main components. They are encapsulated by a molecular weight clamping mechanism, with multiple layers of sulfated alginate of different molecular weights encapsulating chitosan to form a core-shell structure. The high molecular weight sulfated alginate exposes long sugar chains. (The synthesis process and structural diagram of the novel coronavirus-resistant nanospheres of this invention are shown in the figure.) Figure 1 (As shown).

[0013] The specific structure of the anti-novel coronavirus nanospheres of this invention can mimic the heparan sulfate proteoglycan (HSPG) on the surface of host cells, interfering with the binding of the novel coronavirus surface proteoglycan to the host cell surface heparan sulfate proteoglycan and ACE2 receptor, thereby blocking the invasion of host cells by the novel coronavirus. Neutralization and inhibition experiments on the novel coronavirus (SARS-CoV-2) show that the anti-novel coronavirus nanospheres provided by this invention have good neutralizing and inhibitory effects on the novel coronavirus, can protect cells from novel coronavirus infection, and also have a certain antiviral effect on cells already infected with the novel coronavirus.

[0014] In some embodiments, the sulfated alginate polysaccharide comprises, by weight parts, 2 parts of sulfated alginate polysaccharide with a molecular weight of 150-200 kDa, 3 parts of sulfated alginate polysaccharide with a molecular weight of 300-350 kDa, and 5 parts of sulfated alginate polysaccharide with a molecular weight of 500-550 kDa. Therefore, the prepared anti-novel coronavirus nanospheres exhibit the best neutralizing and inhibitory effects on the novel coronavirus.

[0015] In some embodiments, the main component of sulfated seaweed polysaccharide is κ-carrageenan, whose structural formula is shown below:

[0016]

[0017] In some embodiments, in step (1), the acid used to prepare the acidic solution may be selected from at least one of acetic acid, formic acid, and hydrochloric acid.

[0018] In some implementations, in step (2), the rotation speed of high-speed homogenization can be 3000-7000 rpm / min; in step (3), the stirring speed can be 250-500 rpm / min.

[0019] In some implementations, the conditions for ultrasonic dispersion may include: power 400-550W and frequency 30-50kHz.

[0020] The method for preparing anti-novel coronavirus nanospheres provided by this invention is simple, uses natural raw materials, is safe and non-toxic, and can be directly applied to human skin and mucous membranes. It has a clear anti-novel coronavirus effect and can be used to prepare protective equipment for preventing novel coronavirus infection, or to prepare drugs for preventing and / or treating novel coronavirus infection.

[0021] In some implementations, protective equipment for preventing novel coronavirus infection may include topical disinfectants for skin and mucous membranes, protective agents, cleaning agents, detergents, skin care products, personal care products, laundry detergent, masks, protective clothing, or medical face shields, etc.

[0022] In some embodiments, the dosage form of the medicament for preventing and / or treating novel coronavirus infection may be any one of tablets, capsules, gels, or sprays.

[0023] In some implementations, the medicament for preventing and / or treating novel coronavirus infection also includes pharmaceutically acceptable excipients.

[0024] In some embodiments, pharmaceutically acceptable excipients may be selected from one or more of flavoring agents, fillers, coating materials, lubricants, disintegrants, etc. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the synthesis process of the novel coronavirus-resistant nanospheres of the present invention.

[0026] Figure 2 These are scanning electron microscope (SEM) images of the nanospheres prepared in the embodiments and comparative examples 1-2 of this invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0028] In the examples, the chitosan used had a molecular weight of 3-10 kDa, and the ultrasonic dispersion power was 480 W with a frequency of 40 kHz.

[0029] Example 1: Preparation of Anti-Novel Coronavirus Nanospheres

[0030] In this embodiment, the sulfated seaweed polysaccharide used is mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 2:3:5, and the main component is κ-carrageenan.

[0031] The preparation method of anti-novel coronavirus nanospheres includes the following steps:

[0032] (1) Dissolve 10 mg of chitosan in 2 mL of 2.5% acetic acid solution, stir and swell to obtain a chitosan acetic acid solution with a concentration of 5 mg / mL;

[0033] (2) Dissolve 100 mg of sulfated seaweed polysaccharide in 20 mL of deionized water to prepare a 5 mg / mL sulfated seaweed polysaccharide solution;

[0034] (3) Add 10 mL of the sulfuric acid seaweed polysaccharide solution prepared in step (2) to the chitosan acetate solution prepared in step (1), homogenize at 5000 rpm / min for 10 min, and ultrasonically disperse for 30 min to obtain the sulfuric acid seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0035] (4) The sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite obtained in step (3) was added to 10 mL of sulfated seaweed polysaccharide solution obtained in step (2), stirred at 300 rpm / min for 2 hours, ultrasonically dispersed for 30 min, and spray-dried at 125-150℃ to obtain anti-novel coronavirus nanospheres.

[0036] Example 2: Preparation of Anti-Novel Coronavirus Nanospheres

[0037] In this embodiment, the sulfated seaweed polysaccharide used is mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 1:1:3, and the main component is κ-carrageenan.

[0038] The preparation method of anti-novel coronavirus nanospheres includes the following steps:

[0039] (1) Dissolve 10 mg of chitosan in 2 mL of 2.5% acetic acid solution, stir and swell to obtain a chitosan acetic acid solution with a concentration of 5 mg / mL;

[0040] (2) Dissolve 100 mg of sulfated seaweed polysaccharide in 20 mL of deionized water to prepare a 5 mg / mL sulfated seaweed polysaccharide solution;

[0041] (3) Add 10 mL of the sulfuric acid seaweed polysaccharide solution prepared in step (2) to the chitosan acetate solution prepared in step (1), homogenize at 5000 rpm / min for 10 min, and ultrasonically disperse for 30 min to obtain the sulfuric acid seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0042] (4) The sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite obtained in step (3) was added to 10 mL of sulfated seaweed polysaccharide solution obtained in step (2), stirred at 300 rpm / min for 2 hours, ultrasonically dispersed for 30 min, and spray-dried at 125-150℃ to obtain anti-novel coronavirus nanospheres.

[0043] Example 3: Preparation of Anti-Novel Coronavirus Nanospheres

[0044] In this embodiment, the sulfated seaweed polysaccharide used is mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 1:2:2, and the main component is κ-carrageenan.

[0045] The preparation method of anti-novel coronavirus nanospheres includes the following steps:

[0046] (1) Dissolve 10 mg of chitosan in 2 mL of 2.5% acetic acid solution, stir and swell to obtain a chitosan acetic acid solution with a concentration of 5 mg / mL;

[0047] (2) Dissolve 100 mg of sulfated seaweed polysaccharide in 20 mL of deionized water to prepare a 5 mg / mL sulfated seaweed polysaccharide solution;

[0048] (3) Add 10 mL of the sulfuric acid seaweed polysaccharide solution prepared in step (2) to the chitosan acetate solution prepared in step (1), homogenize at 5000 rpm / min for 10 min, and ultrasonically disperse for 30 min to obtain the sulfuric acid seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0049] (4) The sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite obtained in step (3) was added to 10 mL of sulfated seaweed polysaccharide solution obtained in step (2), stirred at 300 rpm / min for 2 hours, ultrasonically dispersed for 30 min, and spray-dried at 125-150℃ to obtain anti-novel coronavirus nanospheres.

[0050] Comparative Example 1: Preparation of sulfated seaweed polysaccharide-chitosan nanospheres

[0051] In this comparative example, the sulfated seaweed polysaccharide used is mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 2:3:5, and the main component is κ-carrageenan.

[0052] The preparation method of sulfated seaweed polysaccharide-chitosan nanospheres includes the following steps:

[0053] (1) Dissolve 10 mg of chitosan in 2 mL of 2.5% acetic acid solution, stir and swell to obtain a chitosan acetic acid solution with a concentration of 5 mg / mL;

[0054] (2) Dissolve 100 mg of sulfated seaweed polysaccharide in 20 mL of deionized water to prepare a 5 mg / mL sulfated seaweed polysaccharide solution;

[0055] (3) Add 10 mL of the sulfuric acid seaweed polysaccharide solution prepared in step (2) to the chitosan acetate solution prepared in step (1), stir at 500 rpm / min for 30 min, and ultrasonically disperse for 30 min to obtain the sulfuric acid seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0056] (4) The sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite obtained in step (3) was added to 10 mL of sulfated seaweed polysaccharide solution obtained in step (2), stirred at 300 rpm / min for 2 hours, ultrasonically dispersed for 30 min, and then spray-dried at 125-150℃ to obtain sulfated seaweed polysaccharide-chitosan nanospheres.

[0057] Comparative Example 2: Preparation of sulfated seaweed polysaccharide-chitosan nanospheres

[0058] In this comparative example, the sulfated seaweed polysaccharide used is mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 2:3:5, and the main component is κ-carrageenan.

[0059] The preparation method of sulfated seaweed polysaccharide-chitosan nanospheres includes the following steps:

[0060] (1) Dissolve 10 mg of chitosan in 2 mL of 2.5% acetic acid solution, stir and swell to obtain a chitosan acetic acid solution with a concentration of 5 mg / mL;

[0061] (2) Dissolve 100 mg of sulfated seaweed polysaccharide in 20 mL of deionized water to prepare a 5 mg / mL sulfated seaweed polysaccharide solution;

[0062] (3) Add 10 mL of the sulfuric acid seaweed polysaccharide solution prepared in step (2) to the chitosan acetate solution prepared in step (1), homogenize at 5000 rpm / min for 10 min, and ultrasonically disperse for 30 min to obtain the sulfuric acid seaweed polysaccharide-chitosan self-assembled nanocomposite.

[0063] (4) The sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite obtained in step (3) was added to 10 mL of sulfated seaweed polysaccharide solution obtained in step (2), stirred at 300 rpm / min for 2 hours, ultrasonically dispersed for 30 min, and freeze-dried to obtain sulfated seaweed polysaccharide-chitosan nanospheres.

[0064] Comparative Example 3: Sulphurized seaweed polysaccharide-chitosan composition

[0065] The comparative example of sulfated seaweed polysaccharide-chitosan composition was obtained by mixing chitosan and sulfated seaweed polysaccharide at a mass ratio of 1:10. The sulfated seaweed polysaccharide used was mainly composed of sulfated seaweed polysaccharides with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa at a mass ratio of 2:3:5, and the main component was κ-carrageenan.

[0066] Experimental Example 1

[0067] The nanomaterials obtained in Examples 1-3 and Comparative Examples 1-2 were tested using a desktop scanning electron microscope (TM3030, Hitach, Japan). Specifically, the nanomaterials were fixed onto the scanning electron microscope stage, sputtered with gold, and then observed within the microscope. The results are as follows: Figure 2 As shown.

[0068] like Figure 2 As shown, the nanospheres prepared in Example 1 have a uniform morphology and good dispersion; the nanospheres in Comparative Example 1 have a uniform morphology but are somewhat aggregated; the freeze-dried nanomaterials in Comparative Example 2 are flocculent, with a disordered and non-uniform morphology. Therefore, it can be concluded that the nanospheres with a multi-layered chitosan shell-core structure encapsulated by sulfated seaweed polysaccharide, prepared by high-speed homogenization and spray drying, exhibit better morphology and dispersibility.

[0069] Experiment Example 2: Antiviral Experiment

[0070] 1. Neutralization experiment with novel coronavirus (SARS-CoV-2)

[0071] 1.1 Materials

[0072] HEK293-ACE2 cells; SARS-CoV-2 pseudovirus (provided by Yisheng Biotechnology (Shanghai) Co., Ltd., experiments were conducted in a P2 laboratory); the drugs used in the drug group were the anti-novel coronavirus nanospheres prepared in Examples 1-3, sulfated seaweed polysaccharide of the same concentration (mainly composed of sulfated seaweed polysaccharide with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 2:3:5, the main component of which is κ-carrageenan), chitosan of the same concentration, sulfated seaweed polysaccharide-chitosan nanospheres prepared in Comparative Examples 1-2 and sulfated seaweed polysaccharide-chitosan composition prepared in Comparative Example 3.

[0073] 1.2 Experimental Methods

[0074] (1) The experimental group was divided into a blank group, a drug group and a virus group (only virus was added). Each reactant was added to a 96-well plate according to the dosage, with 3 parallel replicates for each group.

[0075] (2) Pseudovirus (0.37 μL, 1×10⁻⁶) 7 The mixture (TU / mL) was incubated with serially diluted drugs (4-fold serial dilutions) at room temperature for 1 hour. Then, the mixture was added to wells seeded with HEK293-ACE2 cells (1×10⁻⁶). 4 (Cells / well). The medium was replaced with fresh DMEM 6 hours after infection. A luciferase assay was performed 48 hours post-infection to determine the half-maximal inhibitory concentration (IC50). 50 (half maximum effective concentration).

[0076] 2. Inhibition experiment of novel coronavirus (SARS-CoV-2)

[0077] 2.1 Materials

[0078] Vero E6 cell line; SARS-CoV-2 virus (provided by Guangdong Provincial Center for Disease Control and Prevention, experiments were conducted in a P3 laboratory); the drugs used in the drug group were anti-novel coronavirus nanospheres prepared in Examples 1-3, sulfated seaweed polysaccharide of the same concentration (mainly composed of sulfated seaweed polysaccharide with molecular weights of 150-200kDa, 300-350kDa and 500-550kDa in a mass ratio of 2:3:5, the main component of which is κ-carrageenan), chitosan of the same concentration, sulfated seaweed polysaccharide-chitosan nanospheres prepared in Comparative Examples 1-2 and sulfated seaweed polysaccharide-chitosan composition prepared in Comparative Example 3.

[0079] 2.2 Experimental Methods

[0080] (1) Arrange Vero E6 cells at a density of 2 × 10⁶ cells per well. 5Cells were seeded in 96-well cell culture plates. After the cells adhered and grew uniformly, the medium was replaced with 2% FBS cell culture medium.

[0081] (2) After pretreating cells for 2 hours with 50 μL of different concentrations (2000, 1000, 500, 250, 125, 61.25, 30.63, 15.31 μg / mL) of drugs, virus (0.37 μL, 1×10⁻⁶) was added. 7 Cells were infected with TU / mL; cells without sample pretreatment were used as controls.

[0082] (3) After 48 hours, the cell culture supernatant was collected and the amount of antigen in the supernatant was detected. The amount of antigen in the control cells without sample treatment was 100%. The inhibition rate was obtained by calculating the amount of antigen in the cell supernatant after different sample pretreatment.

[0083] 3. Experimental Results

[0084] The results of the SARS-CoV-2 neutralization experiment are shown in Table 1. In the inhibition experiment, the highest inhibition rates against SARS-CoV-2 virus obtained by the same drug at the experimental concentrations (2000, 1000, 500, 250, 125, 61.25, 30.63, 15.31 μg / mL) and the corresponding drug concentrations are shown in Table 2. As can be seen from the results in Tables 1 and 2, the anti-novel coronavirus nanospheres provided by this invention have good neutralizing and inhibitory effects on the novel coronavirus, not only protecting cells from novel coronavirus infection but also exhibiting good antiviral activity against cells already infected with the novel coronavirus.

[0085] Table 1. Results of neutralization experiments against novel coronavirus (SARS-CoV-2)

[0086] drug group <![CDATA[IC 50 (ppm)]]> Example 1 39 Example 2 73 Example 3 65 Sulfated seaweed polysaccharide 106 Chitosan 157 Comparative Example 1 83 Comparative Example 2 79 Comparative Example 3 95

[0087] Table 2. Results of the inhibitory experiment against the novel coronavirus (SARS-CoV-2)

[0088] drug group Maximum inhibition rate (%) Corresponding drug concentration (μg / mL) Example 1 99.3 125 Example 2 90.7 250 Example 3 94.5 250 Sulfated seaweed polysaccharide 95.4 500 Chitosan 38.7 1000 Comparative Example 1 89.5 500 Comparative Example 2 92.6 500 Comparative Example 3 86.3 500

[0089] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing anti-novel coronavirus nanospheres, characterized in that, Includes the following steps: (1) Dissolve chitosan in an acidic solution with a concentration of 2.5-10% to prepare a chitosan solution with a concentration of 5-20 mg / mL; and dissolve sulfated alginate in water according to the mass ratio of chitosan to sulfated alginate polysaccharide of 1-2:6-10 to prepare a sulfated alginate polysaccharide solution with a concentration of 5-50 mg / mL. (2) Mix the sulfated seaweed polysaccharide solution and the chitosan solution at a mass ratio of chitosan to sulfated seaweed polysaccharide of 1:(3-10), homogenize at high speed for 5-10 min, and ultrasonically disperse for 0.5-3 h to obtain sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite. (3) Mix the sulfated seaweed polysaccharide-chitosan self-assembled nanocomposite with the remaining sulfated seaweed polysaccharide solution, stir for 0.5-3 h, ultrasonically disperse for 0.5-2 h, and spray dry at a temperature of 125-150℃ to obtain anti-novel coronavirus nanospheres. The main component of the sulfated seaweed polysaccharide is κ-carrageenan; The molecular weight of the chitosan is 3-10 kDa; By mass fraction, the sulfated seaweed polysaccharide comprises 2 parts of sulfated seaweed polysaccharide with a molecular weight of 150-200 kDa, 3 parts of sulfated seaweed polysaccharide with a molecular weight of 300-350 kDa, and 5 parts of sulfated seaweed polysaccharide with a molecular weight of 500-550 kDa. In step (2), the rotation speed of the high-speed homogenizer is 3000-7000 rpm / min; in step (3), the rotation speed of the stirrer is 250-500 rpm / min.

2. The preparation method according to claim 1, characterized in that, In step (1), the acid in the acidic solution is selected from at least one of acetic acid, formic acid, and hydrochloric acid.

3. Anti-novel coronavirus nanospheres prepared by the preparation method according to claim 1 or 2.

4. The application of the anti-novel coronavirus nanospheres according to claim 3 in the preparation of protective equipment for preventing novel coronavirus infection.

5. The use of the anti-novel coronavirus nanospheres according to claim 3 in the preparation of medicaments for the prevention and / or treatment of novel coronavirus infection.

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