Nanometer delivery system of load activation module and identification module and preparation method and application of nanometer delivery system

Abstract

The invention discloses a nano delivery system loaded with an activation module and an identification module and a preparation method and application thereof, and relates to the technical field of functional nano materials. The nano delivery system comprises nano gold particles, and an activation module and a targeted recognition module which are loaded on the nano gold particles at the same time. The nano-gold is used as a bridge, on one hand, the nano-gold is loaded with an NK cell activation module IL-15, and on the other hand, a nano-delivery system IL15-AuNPs-SA of a targeted recognition module SA antibody (taking new coronavirus infection as a model) of virus infected cells is loaded, so that on the premise of safely activating an immune system, the immune function of the immune system is enhanced, and the immune function of the immune system is enhanced. The effective screening and removing efficiency of the NK cells on virus infected cells is improved to the maximum extent.

Description

Technical Field

[0001] The present invention relates to the technical field of functional nanomaterials, and in particular to a nano delivery system loaded with an activation module and a recognition module, and a preparation method and application thereof. Background Art

[0002] The threat and lethality caused by viral infections are enormous, bringing great disasters and heavy losses to people all over the world. For example, the coronavirus disease (COVID-19) pandemic caused by the highly pathogenic and contagious coronavirus SARS-CoV-2 has far exceeded the previous SARS and MERS in terms of transmission and number of infections, posing a huge threat to human health around the world. However, there is still no specific drug against viruses like antibiotics against bacteria. At this stage, antiviral drugs mainly start from inhibiting the replication and reproduction of the virus itself, but because the virus parasitizes the host cell and uses the host system to replicate, this means that unless a virus-specific target is found, antiviral drugs generally designed for viruses will usually cause serious damage to the host's normal cells and cause a variety of side effects. This is also one of the main reasons why there is still a lack of broad-spectrum and effective antiviral drugs.

[0003] As we all know, the immune system has a unique ability to identify and clear viruses and their infected cells, which is an innate characteristic of the body. Therefore, at present, viral infections in clinical practice still rely mainly on the human immune system to achieve self-healing. In addition, clinical findings show that the blood routine of patients with COVID-19 pneumonia shows a significant decrease in the number of lymphocytes, which indicates a significant decrease in the patient's immunity. Therefore, the immune system should be one of the key targets for the treatment of pneumonia caused by the new coronavirus infection and other viral infections. However, the overactivation of the immune system as a whole will produce a large number of inflammatory factors (cytokine storms). Since viruses and virus-infected cells cannot be accurately identified, these cytokines will also cause serious damage to normal cells and even endanger life. Therefore, how to efficiently and safely activate the immune system and effectively identify virus-infected cells is bound to be a key direction for anti-viral infection research. Natural killer cells (NK cells) are the core immune cells that the body has innately for defense against viral infections and tumor treatment. One of the biggest characteristics of NK cells, which is different from other immune cells, is that they rarely produce a large number of cytokines, avoiding abnormal attacks on normal tissues and having high safety. The latest research shows that NK cells can control the development of COVID-19 by directly killing virus-infected cells and producing antiviral cytokines such as IFN-γ. At the same time, clinical studies have shown that the increase in COVID-19 lung virus titers is associated with the decline of NK cell function. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned background technology, the present invention mainly solves the problem of how to maximize the killing activity of NK cells and their recognition of virus-infected cells. The present invention provides a nano-delivery system loaded with activation module and recognition module and its preparation method and application. The method uses the new coronavirus infection as a model, and designs a nano-delivery system IL15-AuNPs-SA that uses nano-gold as a bridge, loads the NK cell activation module IL-15 on the one hand, and loads the SA antibody of the targeting recognition module of virus-infected cells on the other hand. Under the premise of safely activating the immune system, the effective identification and removal efficiency of NK cells for virus-infected cells is maximized.

[0005] The first object of the present invention is to provide a nano-delivery system loaded with an activation module and a recognition module, wherein the nano-delivery system comprises nano-gold particles, and an activation module and a targeting recognition module simultaneously loaded on the nano-gold particles; The activation module is an IL-15 molecule that has an activation effect on NK cells.

[0006] Preferably, the targeting recognition module comprises an S protein antibody; the S protein antibody is capable of targeting A549s cells.

[0007] Preferably, the molar ratio of the nano-gold particles to the total amount of the activation module and the targeting recognition module is 1:13-1300.

[0008] Preferably, the molar ratio of the activation module to the targeting recognition module is 3:1-100.

[0009] Preferably, the particle size of the nano-gold particles is 10.20±0.1 nm.

[0010] The second object of the present invention is to provide a method for preparing a nano-delivery system loaded with an activation module and a recognition module, characterized in that it comprises the following steps: The gold nanoparticles are mixed with the activation module and the targeting recognition module by natural adsorption to obtain the gold nanoparticles loaded with both the activation module and the targeting recognition module, that is, the nano delivery system loaded with the activation module and the recognition module.

[0011] Preferably, the nano-gold particles are positively charged nano-gold particles prepared by a reduction method.

[0012] Preferably, after mixing the nanogold particles with the activation module and the targeting recognition module, incubate for 1 to 2 hours, and then centrifuge at 10,000 to 12,000 r / min for 20 to 30 minutes, the resulting precipitate is the nanogold particles loaded with both the activation module and the targeting recognition module.

[0013] Preferably, the activation module refers to the IL-15 molecule that has an activating effect on NK cells; the targeting recognition module refers to the S protein antibody that can recognize the surface of virus-infected cells.

[0014] The third object of the present invention is to provide a nano-delivery system loaded with an activation module and a recognition module for use in preparing drugs for treating viral infectious diseases.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a nano delivery system loaded with an activation module and a recognition module, and a preparation method and application thereof. The present invention uses the novel coronavirus infection as a model, uses nanogold particles as a medium, and organically combines the NK cell targeted activation module with the virus-infected cell recognition module in an electrostatic adsorption manner to prepare an immune antiviral system. The prepared dual-targeted nanosystem IL15-AuNPs-SA simultaneously achieves the improvement of the killing function of NK cells and the efficient targeting effect on virus-infected cells, thereby maximizing the clearance of virus-infected cells by NK cells. More importantly, the system can flexibly introduce various receptors, adhesion molecules, etc. with binding activity to viruses as virus recognition modules for different types of viruses. By replacing the virus recognition module, a high immune activity prevention and control system specifically targeting a certain type of virus is established, thereby achieving rapid response to various potential epidemics or viral infections in the future. This is not only of great significance for the research on the protection of novel coronavirus infection, but more importantly, it provides an important theoretical reference for the development of broad-spectrum antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression level of S protein in A549 cells and A549s cells is detected, wherein A: A549 cells; B: A549s cells; Figure 2 The in vitro recognition and binding ability test of SA antibody and A549 / A549s cells, wherein A: A549 cells; B: A549s cells; Figure 3 The preparation process of AuNPs (A) and transmission electron microscopy image (B); Figure 4 is the IL-15 and SA loading rate at different ratios ( ±s, n=3); * and ** indicate significant differences ( p <0.05, p <0.01); Figure 5 The particle size (A), zeta potential (B) and particle size distribution (C) of AuNPs, IL-15-AuNPs and IL-15-AuNPs-SA; Figure 6 To simulate the stability of the nanogold system in vivo and in vitro environments ( ±s, n=3), where A: IL-15 remaining in the nanosystem under 37°C serum environment; B: SA remaining in the nanosystem under 37°C serum environment; C: IL-15 remaining in the nanosystem under 4°C sterile water environment; D: SA remaining in the nanosystem under 4°C sterile water environment; Figure 7 The effect of the nanogold system on the proliferation activity of NK-92 cells ( ±s, n=3), where ns means no significant difference ( p >0.05; *** and **** indicate significant differences ( p <0.001 and p <0.0001); Figure 8 To study the activity of NK-92 killing A549 / A549s cells by nanogold system ( ±s, n=3), where ns means no significant difference ( p >0.05); *, ** and **** indicate significant differences ( p <0.05, p <0.01 and p <0.0001). DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0018] The purpose of the present invention is to maximize the killing activity of NK cells and their recognition of virus-infected cells.

[0019] In order to achieve the above-mentioned object, the present invention provides a nano-delivery system loaded with an activation module and a recognition module in a first aspect, wherein the nano-delivery system comprises nano-gold particles, and an activation module and a targeting recognition module simultaneously loaded on the nano-gold particles; The activation module is an IL-15 molecule that has an activation effect on NK cells.

[0020] The targeting recognition module includes an S protein antibody; the S protein antibody can target A549s cells.

[0021] The molar ratio of the nano-gold particles to the total amount of the activation module and the targeting recognition module is 1:13 to 1300. The molar ratio of the activation module to the targeting recognition module is 3:1 to 100. The particle size of the nano-gold particles is 10.20±0.1 nm.

[0022] In one embodiment, constructing a lung cancer A549 cell with high expression of S protein on its surface specifically comprises the following steps: (1) A549 cells were seeded into 6-well plates and transfected when the cell density reached 70-80%. The medium was changed after 24 hours and puromycin was added for selection to obtain A549 cells expressing S protein on the surface.

[0023] (2) Confocal laser scanning was used to verify the expression of S protein on the surface of A549s cells.

[0024] (3) Further confirm that SA antibody can target A549s cells.

[0025] The second aspect of the present invention provides a method for preparing a nano-delivery system loaded with an activation module and a recognition module, comprising the following steps: The nano-gold particles are mixed with the activation module and the targeting recognition module by natural adsorption, and the nano-gold particles loaded with both the activation module and the targeting recognition module are obtained, which is a nano-delivery system loaded with the activation module and the recognition module. The activation module refers to the IL-15 molecule that has an activating effect on NK cells; the targeting recognition module refers to the S protein antibody that can recognize the surface of virus-infected cells.

[0026] After mixing the gold nanoparticles with the activation module and the target recognition module, incubate for 1 to 2 hours, and then centrifuge at 10,000 to 12,000 r / min for 20 to 30 minutes. The resulting precipitate is the gold nanoparticles loaded with both the activation module and the target recognition module.

[0027] Specifically, the natural adsorption method is used to mix the gold nanoparticles with the NK cell activation module and the S protein antibody to obtain the gold nanoparticles that are loaded with both the NK cell activation module and the S protein antibody, that is, the nano-delivery system loaded with the activation module and the recognition module, referred to as the IL15-AuNPs-SA nano-delivery system.

[0028] The nano-gold particles are positively charged nano-gold particles prepared by a reduction method.

[0029] After mixing the gold nanoparticles with the NK cell activation module and the S protein antibody, incubate for 1 to 2 hours, and then centrifuge at 10,000 to 12,000 r / min for 20 to 30 minutes. The resulting precipitate is the gold nanoparticles loaded with both the NK cell activation module and the S protein antibody.

[0030] Among them, the NK cell activation module IL-15 and the recognition module S protein antibody are both commercial products.

[0031] In one embodiment, constructing an IL15-AuNPs-SA nano delivery system specifically comprises the following steps: (1) Gold nanoparticles were prepared by reduction method to obtain positively charged AuNPs.

[0032] (2) AuNPs were mixed with IL-15 and S protein antibody (SA) by natural adsorption method to obtain gold nanoparticles loaded with both IL-15 and SA (IL15-AuNPs-SA).

[0033] (3) At the same time, a single IL-15-loaded nanosystem IL15-AuNPs lacking viral infection targeting was constructed.

[0034] (4) Use ELISA detection kits to detect the loading rates of IL-15 and SA respectively to determine the optimal ratio between the two.

[0035] (5) Dynamic light scattering (DLS) was used to test the particle size and zeta potential of the nanosystem.

[0036] (6) The free IL-15 and SA in the supernatant were measured by ELISA to evaluate the stability of the IL15-AuNPs-SA nanosystem.

[0037] (7) IL15-AuNPs-SA enhanced the effect of NK cells in clearing virus-infected cells.

[0038] A third aspect of the present invention provides an application of a nano-delivery system loaded with an activation module and a recognition module in the preparation of a drug for treating viral infectious diseases.

[0039] The present invention constructs lung cancer A549 cells A549s with high expression of S protein on the surface, and verifies that SA antibody can target and bind to S protein on the surface of A549s cells. Positively charged AuNPs are prepared, and on this basis, a nanogold system IL15-AuNPs-SA loaded with IL-15 and SA antibody is constructed. This system can significantly improve the recognition and killing efficiency of NK cells for virus-infected cells.

[0040] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0041] Example 1 Construction and verification of A549 cells with high surface expression of S protein; 1.1 Lipofectamine transfection of A549 cells (1) Plate cells one day in advance. Digest A549 cells and count them at 6×10 per well. 5 Each well was inoculated with 4 mL of RPMI-1640 complete medium and cultured in a 37°C, 5% CO2 incubator.

[0042] (2) Take two sterile 1.5 mL EP tubes and add 125 μL of blank 1640 medium to each tube. Add 7.5 μL of Lipofectamine TM 3000 transfection reagent, add 10 μL of P3000 reagent and 5 μg of recombinant plasmid DNA to another tube and mix gently. Add the culture medium in the centrifuge tube containing the recombinant plasmid DNA to the medium containing Lipofectamine TM 3000 μL of transfection reagent in a centrifuge tube, mix well, and let stand at room temperature for 10-15 min.

[0043] (3) Lipofectamine TM 3000 μL of transfection reagent and plasmid DNA mixture were evenly added to the 6-well plate and mixed evenly. At the same time, blank wells without transfection reagent were set up as the control group.

[0044] (4) After overnight culture, carefully aspirate and collect the cell supernatant, add PBS to gently wash the cells, wash off the transfection reagent, add 1640 complete medium containing 10% fetal bovine serum and 1% antibiotics, add 10 μL of 1 mg / mL puromycin to each well for selective culture, and gradually screen to obtain A549s cells with high surface expression of S protein.

[0045] 1.2 Verify A549s cells with high expression of S protein on their surface; (1) Dry the A549 cell and A549s cell samples, immerse them in 4% paraformaldehyde fixative for 30 min, and then wash them with PBS for 3 min. Repeat this process three times.

[0046] (2) Add 2 drops of 3% H2O2-methanol solution, block at room temperature for 10 min, and wash three times with PBS. Add 100 μL of fetal bovine serum and incubate at room temperature for 20 min.

[0047] (3) Add 100 μL of primary antibody (1:100 dilution) and incubate at 37°C for 2 h. After washing with PBS three times, add 50-100 μL of secondary antibody (1:100 dilution) in tritc and incubate at 37°C for 1 h in the dark.

[0048] (4) Wash with PBS three times, add 100 μL of DAPI staining solution to each slide, and place at room temperature away from light for 5 min. Seal the slide with anti-fading mounting medium, and observe the protein expression in the cells under laser confocal microscope.

[0049] (5) The results are as follows, see Figure 1 As shown in the figure, the expression of S protein on the surface of A549s cells was verified by immunofluorescence. When S protein was expressed on the cell surface, its cell membrane would be labeled with red fluorescence. Figure 1 As shown in B, the red fluorescence around A549s cells indicates that the S protein is indeed highly expressed on their surface. Compared with the control group A549, Figure 1 A has no red fluorescence, which indicates that the cell A549s with high expression of S protein was successfully constructed.

[0050] 1.3 Verify the targeting effect of SA antibody on A549s cells; (1) 10,000 cells (A549 / A549s) were plated per well of a 96-well plate and allowed to adhere overnight.

[0051] (2) On the second day, replace the culture medium with new one, add 50 μL of 10 μg / ml SA antibody and mix well. Incubate at 37°C for 2 h, aspirate the liquid, add PBS and wash three times to remove free SA antibody.

[0052] (3) Add 100 μL FITC-labeled goat anti-human IgG antibody (1:400 dilution) and incubate for 1 h in the dark; wash three times with PBS to remove free FITC-labeled antibody and observe under an inverted fluorescence microscope.

[0053] (4) See Figure 2 As shown, compared with A549 cells (A), the surface of A549s cells (B) was bound to green fluorescent-labeled SA antibodies, which indicated that SA antibodies had no targeting effect on A549 cells, but could target and bind to A549s cells expressing S protein on their surface.

[0054] Example 2 Preparation of IL15-AuNPs-SA nanodelivery system; 2.1 See also Figure 3 As shown in (A), gold nanoparticles were prepared by reduction method: (1) Add 60 mL of 5 mM sodium citrate to a three-necked flask, place in a 120 °C oil bath, stir vigorously at 1000 r / min, and heat to boiling for 20 min, ensuring that the reaction temperature reaches 100 °C.

[0055] (2) Add 600 μL of 25 mM HAuCl4 and stir for 20 min. After cooling, take out the solution to obtain the gold nanoparticles solution.

[0056] (3) Take 5 mL of the prepared gold nanoparticles, add 1 mL of toluene containing 5 mM ODA (4,4'-diaminodiphenyl ether), shake, and take the organic phase after separation.

[0057] (4) Add 62.5 μL of 5 mM MUTAB, in which ethanol is used as the solvent, to the organic phase, and then add 1 mL of ultrapure water. Shake and separate the layers to obtain the aqueous phase. Rinse the aqueous phase repeatedly with toluene to remove excess ODA and MUTAB ((11-mercaptoundecane)-N,N,N-trimethylammonium bromide). Finally, positively charged AuNPs are obtained.

[0058] (5) See Figure 3 As shown in (B), the gold nanoparticles are regular in shape, spherical, and evenly dispersed. The particle size of the gold nanoparticles is measured to be 10.20 ± 0.1 nm.

[0059] 2.2 Preparation of dual-targeting IL15-AuNPs-SA system; AuNPs were mixed with IL-15 and S protein antibody (SA) by natural adsorption and incubated for 1 h. After centrifugation at 12000 r / min for 30 min, the precipitate was the gold nanoparticles loaded with IL-15 and SA (IL15-AuNPs-SA). At the same time, a nanosystem IL15-AuNPs with a single load of IL-15 that lacked viral infection targeting was constructed. Among them, the total control system was 100 μL, and the molar ratio between IL-15 and SA was set to 3:10 according to the molecular weight and charge of the loaded protein, and the molar ratio of gold nanoparticles to the total protein of IL-15 and SA was 1:130.

[0060] Example 3 Same as Example 2, except that The molar ratio of nanogold to the total protein of IL-15 and SA is 1:13.

[0061] Example 4 Same as Example 2, except that The molar ratio of nanogold to the total protein of IL-15 and SA is 1:260.

[0062] Example 5 Same as Example 2, except that The molar ratio of nanogold to the total protein of IL-15 and SA is 1:1300.

[0063] Example 6 Same as Example 2, except that The molar ratio between IL-15 and SA was 3:1.

[0064] Example 7 Same as Example 2, except that The molar ratio between IL-15 and SA was 3:100.

[0065] According to Examples 2 to 7, the present invention controls the total system to be 100 μL, first optimizes the ratio between nanogold and total protein, sets the molar ratio between IL-15 and SA to be 3:10 unchanged according to the molecular weight and charge of the loaded protein, changes the molar ratio of nanogold to the total amount of protein to be 1:13, 1:130, 1:260, 1:1300, respectively, and detects the loading rates of IL-15 and SA by ELISA to confirm the optimal ratio of nanogold to total protein. Then, fix this ratio, adjust the molar ratio between the two proteins IL-15 and SA, set IL-15:SA=3:1, 3:10, 3:100, respectively, and use ELISA detection kits to detect the loading rates of IL-15 and SA to determine the optimal ratio of the two.

[0066] See also Figure 4 As shown, the IL-15 and SA loading rates at different ratios ( ±s, n=3); * and ** indicate significant differences ( p <0.05, p <0.01); from Figure 4 It can be seen that when AuNPs:IL-15:SA=1:30:100, the loading rates of IL-15 and SA are 99.79 ± 0.21% and 81.78 ± 14.49%, respectively.

[0067] In order to illustrate the relevant performance of the nano-delivery system of the activation module and the recognition module provided by the present invention, the nano-delivery system of the activation module and the recognition module prepared when the molar ratio of AuNPs:IL-15:SA is 1:30:100 in Example 2 is used as an example for illustration. The control group mentioned below was PBS; The AuNPs group was the gold nanoparticles prepared in Example 2; The IL15-AuNPs group is a nanodelivery system containing only the payload activation module, and the preparation method is the same as Example 2, the only difference being that only the payload activation module is added; The IL15+SA group was a mixed activation module and recognition module in a free state; The IL15-AuNPs-SA group is a nano-delivery system provided by Example 2 that simultaneously carries an activation module and a recognition module.

[0068] The details are as follows: The IL15-AuNPs-SA nano-delivery system provided in Example 2 was characterized: (1) Dynamic light scattering (DLS) was used to test the particle size and zeta potential of the nanosystem. The sample was placed in a measuring dish, and the test conditions were 25°C and a scattering angle of 173°. Each sample was tested at least 3 times.

[0069] (2) See Figure 5 Particle size (A), zeta potential (B) and particle size distribution (C) of AuNPs, IL-15-AuNPs and IL-15-AuNPs-SA. Figure 5 It can be seen that the particle size of IL15-AuNPs is 172.23 ± 15.49 nm, the ζ potential is -11.77 ± 1.15 mV, and the particle size of IL15-AuNPs-SA is 204.88 ± 17.07 nm, and the ζ potential is -13.68 ± 1.45 mV.

[0070] The stability test of the IL15-AuNPs-SA nano delivery system provided in Example 2 was performed: (1) IL15-AuNPs-SA was placed in sterile water at 4 °C or culture medium containing 10% FBS at 37 °C. After 48 h, the supernatant was obtained by centrifugation.

[0071] (2) The free IL-15 and SA in the supernatant were measured by ELISA to evaluate the stability of the IL15-AuNPs-SA nanosystem.

[0072] (3) See Figure 6 As shown, the stability of the nanogold system in simulated in vivo and in vitro environments ( ±s, n=3); A: IL-15 remaining in the nanosystem under 37℃ serum environment; B: SA remaining in the nanosystem under 37℃ serum environment; C: IL-15 remaining in the nanosystem under 4℃ sterile water environment; D: SA remaining in the nanosystem under 4℃ sterile water environment. Figure 6 It can be seen that only about 26% of the protein was lost from the simulated serum environment within 48 hours, and there was basically no protein loss in the water environment.

[0073] In vitro proliferation activity of NK cells using the IL15-AuNPs-SA nano-delivery system provided in Example 2: (1) NK-92 cells were plated in a 96-well plate at a density of 10,000 per well, and a control group, AuNPs group, IL15-AuNPs group, IL15+SA group, and IL15-AuNPs-SA group were set up.

[0074] (2) After 24 h of culture, CCK-8 was added. After 2 h of incubation, the absorbance at 450 nm was measured to calculate the proliferation rate of NK cells.

[0075] (3) See Figure 7 As shown, AuNPs had no effect on the proliferation activity of NK cells, while free IL-15+SA, IL15-AuNPs and IL15-AuNPs-SA could significantly stimulate the proliferation of NK cells, increasing by 52.02 ± 8.39% (p<0.0001), 43.72 ± 9.79% (p<0.001) and 51.52 ± 5.88% (p<0.0001), respectively. Figure 7 The effect of the nanogold system on the proliferation activity of NK-92 cells ( ±s, n=3), ns means no significant difference ( p >0.05; *** and **** indicate significant differences ( p <0.001 and p <0.0001).

[0076] In vitro cytotoxic activity of NK cells using the IL15-AuNPs-SA nano-delivery system provided in Example 2: (1) NK-92 cells were used as effector cells and A549 / A549s as target cells. The cells were plated in a 96-well plate at a ratio of 300,000 NK-92 cells to 100,000 target cells. (2) The control group, AuNPs group, IL15-AuNPs group, IL15+SA group, and IL15-AuNPs-SA were added and incubated for 4 h. (3) CCK-8 was added, 20 μL per well, and after incubation for 2 h, the absorbance was measured at 450 nm to calculate the killing rate of NK cells.

[0077] See also Figure 8 As shown, the study of the activity of NK-92 killing A549 / A549s cells by the nanogold system ( ±s, n=3), ns means no significant difference ( p >0.05); *, ** and **** indicate significant differences ( p <0.05, p <0.01 and p <0.0001); from Figure 8It can be seen that compared with the control group and the pure AuNPs group, IL15-AuNPs, IL-15+SA, and IL15-AuNPs-SA can all increase the activity of NK cells in killing A549s, which is due to the activation of IL-15 on the killing function of NK cells. In the loading system, compared with the single loading system IL15-AuNPs, due to the targeting effect of SA on A549s cells, the double loading nanosystem IL15-AuNPs-SA further promoted the killing effect of NK cells on them. Compared with A549 cells that do not express S protein, the effect of IL15-AuNPs-SA on NK cell killing of A549s cells was not significantly different, but it was still slightly improved, which to a certain extent also shows that the targeting effect of SA antibody on A549s cells promotes the killing ability of NK cells.

[0078] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nano delivery system loaded with an activation module and a recognition module, characterized in that: The nano delivery system comprises nano gold particles, and an activation module and a targeting recognition module simultaneously loaded on the nano gold particles; The activation module is an IL-15 molecule that has an activation effect on NK cells.

2. The nano delivery system loaded with activation module and recognition module according to claim 1, characterized in that: The targeting recognition module includes an S protein antibody; the S protein antibody can target A549s cells.

3. The nano-delivery system loaded with activation modules and recognition modules according to claim 1, characterized in that: The molar ratio of the nano-gold particles to the total amount of the activation module and the targeting recognition module is 1:13-1300.

4. The nano-delivery system loaded with activation modules and recognition modules according to claim 1, characterized in that: The molar ratio of the activation module to the targeting recognition module is 3:1-100.

5. The nano-delivery system loaded with activation modules and recognition modules according to claim 1, characterized in that: The particle size of the nano-gold particles is 10.20±0.1 nm.

6. A method for preparing a nano delivery system loaded with an activation module and a recognition module according to any one of claims 1 to 5, characterized in that: The following steps are involved: The gold nanoparticles are mixed with the activation module and the targeting recognition module by natural adsorption to obtain the gold nanoparticles loaded with both the activation module and the targeting recognition module, that is, the nano delivery system loaded with the activation module and the recognition module.

7. The method for preparing a nano-delivery system loaded with an activation module and a recognition module according to claim 6, characterized in that: The nano gold particles are positively charged nano gold particles prepared by a reduction method.

8. The method for preparing a nano-delivery system loaded with an activation module and a recognition module according to claim 6, characterized in that: After mixing the gold nanoparticles with the activation module and the target recognition module, incubate for 1 to 2 hours, and then centrifuge at 10,000 to 12,000 r / min for 20 to 30 minutes. The resulting precipitate is the gold nanoparticles loaded with both the activation module and the target recognition module.

9. The method for preparing a nano-delivery system loaded with an activation module and a recognition module according to claim 6, characterized in that: The activation module refers to the IL-15 molecule that has an activation effect on NK cells; the targeting recognition module refers to the S protein antibody that can recognize the surface of virus-infected cells.

10. Use of a nano delivery system loaded with an activation module and a recognition module as claimed in any one of claims 1 to 5 in the preparation of a drug for treating viral infectious diseases.

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