WO2019061790A1 - Nanoparticle, and preparation method thereof - Google Patents

Abstract

Disclosed in the present invention are a nanoparticle, and a preparation method thereof. The method comprises: mixing an organic solvent and a surfactant to obtain a solution A; mixing a first reactant solution with a functional substance solution to obtain a solution B; mixing a second reactant solution with a functional substance solution to obtain a solution C; enabling the second reactant to contact the first reactant to form precipitation; mixing solution A and solution B uniformly to obtain a solution D; mixing solution A and solution C uniformly, adding a DOPA solution to the resulting mixed solution, and mixing uniformly to obtain a solution E; adding solution E to solution D, and performing centrifugation on the mixture to collect a precipitated substance, so as to obtain nanoparticles having a single-layer film; and preparing a dispersion of the nanoparticles having a single-layer film, and adding, to the dispersion, a mixture of DOPC and cholesterol or a mixture of DOTAP and cholesterol, wherein particles in the resulting particle suspension are nanoparticles having a double-layer film.

Description

Nanoparticle and preparation method thereof Technical field

The invention relates to the field of life science technology, in particular to a nano particle and a preparation method thereof.

Background technique

Nanoparticles are nanomaterials with a cavity in the middle. They can be encapsulated in small cavities, siRNAs, antibodies, etc. into the cavity and introduced into the target cells. TIL cells (T cells) are lymphocytes that are cultured together with the patient's cancer tissue and the patient's T cells, and then the lymphocytes are expanded and returned to the patient. However, there is a protein called PD-L1 on the surface of cancer cells. This PD-L1 protein binds to the PD-1 protein of T cells and inhibits the killing of cancer cells by T cells. Therefore, if you want T cells to kill cancer cells efficiently, you need to find ways to reduce the PD-1 of T cells and the PD-L1 of cancer cells.

At present, functional substances that inhibit PD-L1 and PD-1 are mainly transfected into T cells and cancer cells by vectors, thereby realizing reduction of PD-1 of T cells and PD-L1 of cancer cells. However, current vectors generally have the disadvantage of low transfection efficiency.

Summary of the invention

Based on this, it is necessary to provide an efficient nanoparticle and a preparation method thereof for the problem that the existing nanoparticles are inefficient for transfecting suspension cells.

One of the objects of the present invention is to provide a method for preparing nanoparticles, comprising the steps of:

Mixing an organic solvent with a surfactant to obtain a solution A;

Mixing the first reactant solution with the functional substance solution to obtain liquid B;

Mixing the second reactant solution with the functional substance solution to obtain a liquid C; and forming a precipitate after the second reactant contacts the first reactant;

Mixing the liquid A and the liquid B to obtain a liquid D;

Mixing the liquid A, the liquid C, and adding chlorine of DOPA (dopamine) to the resulting mixed solution Mix the imitation solution to obtain E solution;

Adding the E liquid to the D liquid, centrifuging the obtained mixture, collecting the precipitate to obtain a single layer membrane nanoparticle;

A dispersion of the monolayer film nanoparticles is prepared, and a mixture of DOPC (1,2-dioleoylphosphatidylcholine), cholesterol, or DOTAP (2-dioleoylhydroxypropyl-) is added to the dispersion. A mixture of 3-N,N,N-trimethylammonium) and cholesterol, the particles in the resulting suspension particle solution are two-layer membrane nanoparticles.

In some embodiments, the first reactant solution is a calcium salt solution, the second reactant solution is a phosphate solution or a pyrophosphate solution, the first reactant, the second reactant calcium, The mass ratio of phosphorus is (25 to 400): 1. In the embodiment of the present invention, by controlling the ratio of calcium to phosphorus, a nanoparticle core can be obtained well, the uneven particle size is uniform, and the stability is good, and aggregation is not easy. If it is beyond the limits of the embodiments of the present application, either the nanoparticle core cannot be formed, or the formed particle core is agglomerated, and the subsequent coating operation is not easy.

In some of these embodiments, the calcium salt solution has a concentration of 4.5 to 5.5 M; and the phosphate solution or pyrophosphate solution has a concentration of 45 to 55 mM. In the embodiment of the present invention, by using a 4.5-5.5 M calcium chloride solution, the uniformity of the precipitate produced by the combination of the first reactant and the second reactant can be further improved, thereby improving the uniformity of the nanoparticles.

In some embodiments, the calcium salt solution comprises a calcium chloride solution, a calcium nitrate solution, a calcium gluconate solution; the phosphate solution comprises a dipotassium hydrogen phosphate solution, a diammonium hydrogen phosphate solution, and an ammonium dihydrogen phosphate solution. , a calcium hydrogen phosphate solution, a calcium phosphate solution, a sodium dihydrogen phosphate solution, a disodium hydrogen phosphate solution, a sodium phosphate solution; the pyrophosphate solution comprises a calcium pyrophosphate solution, an acid sodium pyrophosphate solution, and a sodium pyrophosphate solution.

In some of the embodiments, the concentration of DOPA in the DOPA solution is 15-25 mg/ml, and the volume ratio of the added volume of the DOPA solution to the mixed solution of the A liquid and the C liquid is (70-80): 1.

In some of these embodiments, the solvent of the DOPA solution includes chloroform, dichloromethane, ethyl acetate, tetrahydrofuran.

In some of the embodiments, the mass ratio of DOPC to cholesterol in the mixture of DOPC and cholesterol is 1: (2.5 to 3.5); in the mixture of DOTAP and cholesterol, the quality of DOTAP and cholesterol The ratio is 2: (2.5 to 3.5).

In some embodiments, in the preparation of the liquid A, the organic solvent is any one or more of cyclohexane, benzene, toluene, n-heptane, and carbon tetrachloride; and the surfactant includes polyoxygenation. Ethylene (5) nonylphenyl ether; the volume ratio of the organic solvent to the surfactant is from 80:20 to 50:50. The invention adopts organic solvent screening and surfactant screening, and aims to introduce an oil-water system. The oil-water system has the advantages of obtaining a precipitate which is combined with the second reactant by the high suspension and protection, and improves stability, so that the stability is improved. Coagulation does not easily occur between particles.

In some of the embodiments, the ratio of the amount of the liquid A and the liquid B is (50 to 200): 1; the ratio of the amount of the liquid A and the liquid C is (50 to 200): 1; the liquid of the E and the liquid D The dosage ratio is (1 to 3): (1 to 3).

In some of the embodiments, the dispersing the monolayer film nanoparticles is specifically dispersing the monolayer film nanoparticles in chloroform, dichloromethane, ethyl acetate or tetrahydrofuran.

In some of these embodiments, the functional substance in the functional substance solution includes a functional nucleic acid sequence (dsDNA, siRNA, etc.), a protein antibody, a drug molecule.

In some of these embodiments, the functional substance in the functional substance solution comprises a functional nucleic acid sequence.

Another object of the present invention is to provide a nanoparticle obtained by the above production method.

Compared with the prior art, the embodiment of the invention has the following beneficial effects:

In the embodiment of the present invention, the organic solvent, the surfactant, the first reactant solution, the functional substance solution, the second reactant solution, and then the double outer membrane are mixed stepwise, thereby improving the nanoparticle carrying the functional substance to the target. Transfection efficiency of cells. Specifically, in the embodiment of the present invention, the organic solvent, the surfactant, the first reactant solution, the functional substance solution, and the second reactant solution are mixed stepwise, so that the first reactant and the second reactant are formed after carrying the functional substance. The nanoparticle core is small and uniform, and it is not easy to agglomerate. After the core is coated with dopamine, DOPC or DOTAP, the particle size of the obtained nanoparticle product can be controlled to a small scale (about 20 nm), and the film is also improved while coating the film. The hydrophilicity on the outer side, the dispersibility and stability of the particles are increased, aggregation is less likely to occur, and the affinity with the cell surface is enhanced, and it is easily absorbed by the cells, thereby increasing the transfection efficiency.

DRAWINGS

1A is a transmission electron microscope (TEM) photograph of a nanoparticle prepared according to an embodiment of the present invention; FIG. 1B is a dynamic light scattering diagram of a nanoparticle prepared according to an embodiment of the present invention;

2A is a cell uptake rate of nanoparticles prepared at different concentrations according to an embodiment of the present invention; FIG. 2B is a cell uptake rate of nanoparticles prepared according to an embodiment of the present invention during advancement;

3A is a PD1 mRNA level of a cell after transfecting a TIL cell with a nanoparticle prepared according to an embodiment of the present invention; FIG. 3B is a PD1 protein level of the cell after transfecting the TIL cell with the nanoparticle prepared by the embodiment of the present invention; FIG. After the transfected cells of the nanoparticles prepared in the examples, the PD1-positive cells were detected by flow cytometry;

4A is a PDL1 mRNA transcription level of a cell after transfecting a nanoparticle into a breast cancer cell according to an embodiment of the present invention; FIG. 4B is a PDL1 protein expression level of the cell after transfecting the nanoparticle prepared by the embodiment of the present invention; FIG. After transfecting the nanoparticles of the nanoparticles prepared in the examples of the present invention, PDL1-positive cells were detected by flow cytometry;

Figure 5 is a test for the lethality of TIL cells of different ratios and different degrees of silence on breast cancer cells;

6A and 6B are graphs showing the results of test results of transfection on TIL cell subsets; FIG. 6C and FIG. 6D are graphs showing test results of cytokine production by TIL cells before and after transfection;

Figure 7 is a schematic view showing the structure of the obtained nanoparticles according to an embodiment of the present invention.

Detailed ways

The nanoparticles of the present invention and a method for preparing the same are described in further detail below in conjunction with specific examples.

In order to more clearly understand the technical content of the present invention, the following embodiments are specifically described. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Various common chemistry used in the examples Reagents are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning The terms used in the description of the present invention are for the purpose of describing the specific embodiments and are not intended to limit the invention. The term "and/or" used in the present invention includes any and all combinations of one or more of the associated listed items.

In order to make the technical solution of the present invention clearer and easier to understand, it will be exemplified. It should be noted that the scope of protection of the present invention is not limited to the contents described in the following examples.

Example 1. Nanoparticles containing silencing siRNA (PD1 silencing TIL cells) and preparation thereof method

1.1 Preparation of nanoparticles

This section provides a method for preparing nanoparticles, comprising the following steps:

Preparation of liquid A: mixing cyclohexane with polyoxyethylene (5) nonylphenyl ether, the ratio of the two is 70:30 volume ratio; cyclohexane can be replaced by benzene, toluene, n-glycan Alkane, carbon tetrachloride;

Prepare solution B: 5M calcium chloride solution (volume used is 75 μL) mixed with 100 μM siRNA solution (volume used is 100 μL), and 50 μl of ultrapure water is added; in other examples, the calcium chloride here can also be replaced. Calcium acid solution or calcium gluconate solution;

Prepare liquid C: 50 mM disodium hydrogen phosphate (volume used is 75 μL) mixed with 100 μM siRNA (volume used is 100 μL), and add 50 μl of ultrapure water; in other examples, the disodium hydrogen phosphate solution can be replaced with Dipotassium hydrogen phosphate solution, diammonium hydrogen phosphate solution, ammonium dihydrogen phosphate solution, calcium hydrogen phosphate solution, calcium phosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, sodium phosphate solution, calcium pyrophosphate solution, acid form Sodium pyrophosphate solution or sodium pyrophosphate solution;

Prepare solution D: take a portion of solution A (the volume used is 15ml), add 150μl of B solution, stir for 20min;

Prepare E solution: Take a portion of solution A (the volume used is 15 ml), add 150 μl of C solution, stir for 5 min, then add DOPA in chloroform solution (concentration of DOPA is 20 mg/ml, the volume of the solution is 200 μL), and continue stirring for 15 min. The chloroform of this step can also be replaced by dichloromethane, ethyl acetate or tetrahydrofuran;

Preparation of single-layer membrane particles: E-liquid was dropped into D solution one by one, and stirred for 20 minutes, and the resulting precipitate was a single-layer membrane nanoparticle, that is, the first reactant, the second reactant, and the functional siRNA formed only outside the core. Wrapped with a layer of DOPA; in this step, in order to make the final nanoparticle size more uniform, the drop rate of E liquid here is controlled at 1-3 ml/min; this step also includes cleaning and collecting the single-layer film nanoparticles. The method includes the following steps: the step of washing comprises: adding 10 ml of ethanol to the mixture of the E solution and the D solution in a volume ratio of 1:1, stirring for 5 minutes, centrifuging at 10,000 g for 20 min, and discarding. Supernatant, leaving precipitate; add 10 ml of ethanol to the precipitate, centrifuge at 10,000 g for 20 min, then discard the supernatant and leave a precipitate; the collection step includes: adding 1 ml of chloroform to collect the monolayer membrane nanoparticles;

Preparation of bilayer membrane particles: Dispersing the monolayer membrane nanoparticles prepared above into 1 ml of chloroform, mixing the resulting dispersion with 100 μl of DOPC, cholesterol (volume ratio of 1:3) or a mixture of DOTAP and cholesterol (volume In the ratio of 2:3), chloroform and unbound DOPC or DOTAP and cholesterol are evaporated to dryness in a rotary evaporator, and the remainder is a two-layer membrane particle, which is the target product of the present invention, using phosphate buffer (PBS). pH=7.4) Two-layer membrane particles, ie.

The structure of the two-layer membrane particles prepared in this embodiment is shown in Figure 7. The first reactant, the second reactant, the core of the nuclear nanoparticles formed by the functional siRNA, the DOPA formation includes the inner membrane outside the core, and DOPC and DOTAP are formed. Outer membrane.

In the above preparation method, PD1 siRNA:

Justice chain: 5’-AGACCUUGAUACUUUCAAAdTsdT-3',

Antisense strand: 5'-UUUGAAAGUAUCAAGGUCUdTsdT-3';

The nanoparticles obtained by the preparation method of the present embodiment have good morphology, the size is in a controllable range, the particles are not adhered together, and the dispersion is uniform, as shown in FIG. 1A; the size of the nano material particles is about 20 nm, and the dispersion can be uniformly dispersed. Again, the proper size and good dispersibility of the particles were verified, see Figure 1B.

1.2 Transfection of TIL cells

This section deals with the transfection of TIL cells with nanoparticles prepared in 1.1, including the following steps:

Step one, inoculate a 6-well plate at a density of 1.5×10 ⁵ TIL cells/well, place in an incubator (condition: 37° C., 5% CO ₂ ) overnight, and remove the cell culture medium;

Step 2: Add fresh cell culture medium containing 1.1 nm particles to the 6-well plate treated in the first step, and let stand at 37 ° C; wherein the nanoparticles are coated with 50 nM of siRNA (the amount is: added siRNA) The total amount minus the amount of siRNA not encapsulated into the nanoparticles);

Step 3. Discard the medium in the 6-well plate treated in the second step, wash it three times with fresh phosphate buffer (PBS), and wash away the nanoparticles that were not absorbed by the TIL cells.

In order to achieve optimal cell uptake in the present embodiment, the concentration of the fresh cell culture medium containing the nanoparticles of Example 1 in step 2 was set to three concentration levels, namely 20 nM, 40 nM, and 80 nM, and three nanoparticle concentrations were measured. The rate of extraction of nanoparticles by TIL cells.

The results are shown in Fig. 2A. It can be seen from the figure that when the content of the nanoparticles in the medium is 40 nM, the uptake rate of the nanoparticles by the TIL cells can reach about 65%.

This example also tests the preferred uptake time for this preferred concentration (nanoparticle content 40 nM) at a preferred nanoparticle concentration.

The results are shown in Fig. 2B. It can be seen from the figure that about 65% of TIL cells can be transfected in 2 hours.

1.3 Detection of mRNA transcription level and protein expression level of PD1 gene in transfected TIL cells

Refer to 1.2 above to set up the following TIL cell treatment:

a, normal state of TIL cells that have not been transfected, labeled "TILs";

b. TIL cells transfected with nanoparticles loaded with 40 nM control siRNA, labeled "TILs + LCP + C-siRNA-40 nM";

Wherein, the sequence of the control siRNA is: 5'-UUCUCCGAACGUGUCACGUTT-3';

c. TIL cells transfected with the nanoparticles of the present example at a concentration of 20 nM, labeled as "TILs + LCP + siRNA-20 nM";

d. TIL cells transfected with the nanoparticles of the present example at a concentration of 40 nM, labeled as "TILs + LCP + siRNA-40 nM";

e, TIL cells transfected with the nanoparticles of the present example at a concentration of 80 nM, labeled as "TILs + LCP + siRNA-80 nM";

f. TIL cells transfected with the nanoparticles of the present application at a concentration of 160 nM, labeled "TILs + LCP + siRNA - 160 nM".

The results of the mRNA transcription level are shown in Figure 3A. As the concentration of nanoparticles increases from 20 nM to 160 nM, the transcription of PD1 decreases from 86% to about 15%. It can be seen that the nanoparticles can efficiently transfer PD1 siRNA into TIL cells, thereby silencing the expression of PD1 in TIL cells.

The results of detecting the expression level of PD1 protein in TIL cells are shown in Fig. 3B. When the concentration of the nanoparticles was 40 nM, the expression level of the PD1 protein was significantly reduced. It can be seen that when the concentration of nanoparticles is 40 nM, the expression of PD1 protein in TIL cells can be effectively reduced.

The results of PD1-positive cells after transfection are shown in Figure 3C. According to the flow cytometry map, the position indicated by the arrow can be seen, and the peak shifts. This indicates that the PD1-positive cells in the TIL cells are reduced, thus indicating the nanoparticles. TIL cells can be transfected efficiently.

Example 2. Nanoparticles containing silencing siRNA (PDL1 silencing breast cancer cell MCF7) And preparation method thereof

2.1 Preparation of Nanoparticles (Refer to Example 1)

This section provides a method for preparing nanoparticles, comprising the following steps:

Preparation of liquid A: mixing cyclohexane with polyoxyethylene (5) nonylphenyl ether, the ratio of the two is 70:30 volume ratio;

Preparation liquid B: 5M calcium chloride solution (volume used is 75 μL) mixed with 100 μM siRNA solution (volume used is 100 μL), and added 50 μl of ultrapure water;

Preparation of liquid C: 50 mM disodium hydrogen phosphate (volume used is 75 μL) mixed with 100 μM siRNA (volume used is 100 μL), and added 50 μl of ultrapure water;

Prepare solution D: take a portion of solution A (the volume used is 15ml), add 150μl of B solution, stir for 20min;

Prepare E solution: Take a portion of solution A (the volume used is 15 ml), add 150 μl of C solution, stir for 5 min, then add DOPA in chloroform solution (concentration of DOPA is 20 mg/ml, the volume of the solution is 200 μL), and continue stirring for 15 min. ;

Preparation of single-layer membrane particles: E-liquid was dropped into D solution one by one, and stirred for 20 minutes, and the resulting precipitate was a single-layer membrane nanoparticle, that is, the first reactant, the second reactant, and the functional siRNA formed only outside the core. Wrapped with a layer of DOPA; in this step, in order to make the final nanoparticle size more uniform, the drop rate of E liquid here is controlled at 1-3 ml/min; this step also includes cleaning and collecting the single-layer film nanoparticles. The method includes the following steps: the step of washing comprises: adding 10 ml of ethanol to the mixture of the E liquid and the D liquid in a volume ratio of 1:1, stirring for 5 minutes, centrifuging at 10,000 g for 20 min, and discarding. Clear and leave a precipitate; add 10 ml of ethanol to the precipitate and centrifuge at 10,000 g for 20 min. Discarding the supernatant and leaving the precipitate; the collecting step includes: adding 1 ml of chloroform to collect the monolayer film nanoparticles;

Preparation of bilayer membrane particles: Dispersing the monolayer membrane nanoparticles prepared above into 1 ml of chloroform, mixing the resulting dispersion with 100 μl of DOPC, cholesterol (volume ratio of 1:3) or a mixture of DOTAP and cholesterol (volume In the ratio of 2:3), chloroform and unbound DOPC or DOTAP and cholesterol are evaporated to dryness in a rotary evaporator, and the remainder is a two-layer membrane particle, which is the target product of the present invention, using phosphate buffer (PBS). pH=7.4) Two-layer membrane particles, ie.

The structure of the two-layer membrane particles prepared in this embodiment is shown in Figure 7. The first reactant, the second reactant, the core of the nuclear nanoparticles formed by the functional siRNA, the DOPA formation includes the inner membrane outside the core, and DOPC and DOTAP are formed. Outer membrane.

In the above preparation method, the sequence of the siRNA is PD-L1 siRNA:

Justice chain: 5'-AGACGUAAGCAGUGUUGAAdTsdT-3',

Antisense strand: 5'-UUCAACACUGCUUACGUCUdTsdT-3';

The nanoparticles obtained in the preparation method of this part are the same as in the first embodiment: the morphology is very good, the size is in a controllable range, the particles are not adhered together, and the dispersion is uniform, as shown in FIG. 1A; the size of the nano material particles is about 20 nm. And it can be evenly dispersed, and the proper size and good dispersibility of the particles are verified again, as shown in Fig. 1B.

2.2 Transfection of breast cancer cells

Transfection of breast cancer cells with the nanoparticles prepared in 2.1 includes the following steps:

Step one, inoculate a 6-well plate at a density of 1.5×10 ⁵ breast cancer cells/well, place in an incubator (condition: 37° C., 5% CO ₂ ) overnight, and remove the cell culture medium;

Step 2: Adding fresh cell culture medium containing the nanoparticles prepared in 2.1 above to the 6-well plate treated in the first step, and allowing to stand at 37 ° C for 4 h; wherein the nanoparticles are coated with 40 nM of siRNA (the amount is : the total amount of siRNA added minus the amount of siRNA not encapsulated into the nanoparticles);

Step 3. Discard the medium in the 6-well plate treated in the second step and wash it three times with fresh phosphate buffer (PBS) to wash away the nanoparticles that were not absorbed by the breast cancer cells.

2.3 Detection of mRNA transcription level and protein expression level of PDL1 gene in transfected breast cancer cells

Refer to 2.2 above to set up breast cancer cell processing as follows:

a, normal state of breast cancer cells that have not been transfected, labeled "Blank Control";

b, breast cancer cells transfected with nanoparticles loaded with 40 nM control siRNA at a concentration of 40 nM nanoparticles, labeled "LCP + C-siRNA-40 nM";

Wherein, the sequence of the control siRNA is: 5'-UUCUCCGAACGUGUCACGUTT-3';

c. Breast cancer cells transfected with the nanoparticles of the present example at a concentration of 10 nM, labeled "10 nM";

d, breast cancer cells transfected with the nanoparticles of the present example at a concentration of 20 nM, labeled "20 nM";

e. Breast cancer cells transfected with the nanoparticles of this example at a concentration of 40 nM, labeled "40 nM".

The detection results of mRNA transcription levels are shown in Figure 4A. As the concentration of nanoparticles increases from 10 nM to 40 nM, the transcription of PD1 decreases from 80% to about 20%. It can be seen that the nanoparticles can efficiently transfer PDL1 siRNA into breast cancer cells, thereby silencing the expression of PDL1 in breast cancer cells.

The results of the detection of protein expression levels are shown in Fig. 4B. The inventors found that when the concentration of the nanoparticles was 40 nM, the expression level of PDL1 protein was significantly reduced. It can be seen that when the concentration of the nanoparticles is 40 nM, the expression of PDL1 protein in breast cancer cells can be effectively reduced.

The results of PDL1-positive cells after transfection are shown in Figure 4C. According to the flow cytometry map, the position indicated by the arrow can be seen, and the peak shifts, indicating that PDL1-positive cells in breast cancer cells are reduced, so Nanoparticles can be efficiently transfected into breast cancer cells.

Example 3, cell killing efficiency test

In this example, the TIL cells involved in Example 1 were mixed with the breast cancer cell MCF7 of Example 2 to test the lethality of TIL cells against breast cancer cell MCF7.

Selected TIL cells include: untransfected TIL cells that highly express PD1, labeled "PD1+"; TIL cells that are lowly expressed PD1 transfected with 40 nM nanoparticles, labeled "PD1-".

The selected breast cancer cells MCF7 include: untransfected breast cancer cells with high expression of PDL1, labeled as "PDL1+"; breast cancer cells with low expression of PDL1 transfected with 40 nM nanoparticles, labeled "PDL1-";

The selected TIL cells and the selected breast cancer cells MCF7 were mixed at different cell weight ratios, and a total of 12 different mixing treatments were included, including the following:

PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- with a ratio of 10:1;

PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- with a ratio of 30:1;

PD1+/PDL1+, PD1-/PDL1+, PD1+/PDL1-, PD1-/PDL1- with a ratio of 100:1;

The cell killing efficiency test is detected by a lactate dehydrogenase detection kit. Ie using CytoTox

Standard 4-hour lactate dehydrogenase release assay was performed by Non-Radioactive Cytotoxicity Assay (Promega, WI). Methods as below:

1 The breast cancer cells to be examined (silencing breast cancer cells MCF7 ^K and unsilent breast cancer cells MCF7) were inoculated into 96-well plates at a density of 1×10 ⁴ per well, and after 18 hours of culture, the medium was changed to phenol-free. Red, RPMI 1640 medium (Gibco-BRL) containing 5% fetal bovine serum, continued to culture for 6 hours.

2 pairs of TIL cells: TIL cells (silent TIL cells TIL ^K and unsiltained TIL cells TIL) were cultured in CNE-2 medium for 24 hours, and then the cells were collected, in phenol-free red, RPMI 1640 containing 5% fetal bovine serum. The cells were resuspended in medium (Gibco-BRL).

3 Take 50 μL of cell suspension, add TIL cells to the wells containing breast cancer cells according to the above different ratios. After incubating for 4 hours, centrifuge 96-well plates (250×g, 10 minutes), and add 50 μL of supernatant to another 96. In the orifice plate, follow the instructions provided by the manufacturer.

Formula for killing efficiency: % killing efficiency = (experimental result - breast cancer cell basal release value - TIL cell basal release value) / (peak killing cell release peak - breast cancer cell basal release value) x 100.

The test results are shown in Fig. 5. It can be seen from the figure that the PD1-/PDL1- treatment killing effect is the best at different ratios, and has a particularly high killing efficiency.

Example 4, cytokine test

First, in this example, the coated siRNA nanoparticles were prepared by referring to the preparation method of Example 1, and then the TIL cells were transfected with the nanoparticles at a concentration of 40 nM.

The pre-transfection TIL cells (pre-silent TIL cells, labeled as TILs) and the transfected TIL cells (ie, silenced TIL cells, labeled TILs ^K ) were detected, and the detection methods were routine methods in the art. . The results of the assay are shown in Figures 6A and 6B; Figure 6A shows TIL cells after silencing, and Figure 6B shows TIL cells before silencing. According to Figures 6A and 6B, there is no change in TIL cell subsets after and after PD1 silencing. This indicates that the entire silencing process has no effect on the cell subset of TIL cells.

Next, in this example, 40NM concentrated nanoparticle-transfected TIL cells were obtained by referring to step 1.1 and step 1.2 in Example 1, and then the transfected TIL cells (TILs ^K ) and MCF7, MCF7 ^K and CTL6 cells were used. Co-culture, and the amount of cytokine expression before and after transfection is tested before and after co-culture, and the method of testing the expression level of cytokines is a routine operation in the art. The results of the assay are shown in Figure 6C and Figure 6D. The expression levels of IL17, IL10, INFγ, and INFα were significantly increased in transfected TIL cells, especially INFγ, indicating that the killing ability of TIL cells after transfection was significantly enhanced.

It should be noted that, after repeated experiments, the above embodiments can be realized within the range defined by the following parameters: the first reactant solution is a calcium salt solution, and the second reactant solution is a phosphate solution or a coke. a phosphate solution, the first reactant, the second reactant has a calcium to phosphorus mass ratio of (25 to 400):1; the calcium salt solution has a concentration of 4.5 to 5.5 M; and the phosphate solution Or the concentration of the pyrophosphate solution is 45-55 mM; the concentration of the DOPA in the DOPA solution is 15-25 mg/ml, and the volume ratio of the added volume of the DOPA solution to the mixed solution of the A liquid and the C liquid is ( 70-80): 1; the mass ratio of DOPC and cholesterol in the mixture of DOPC and cholesterol is 1: (2.5 to 3.5); in the mixture of DOTAP and cholesterol, the mass ratio of DOTAP to cholesterol is 2: ( 2.5 to 3.5); in the preparation of the liquid A, the organic solvent is any one or more of cyclohexane, benzene, toluene, n-heptane, and carbon tetrachloride; and the surfactant includes polyoxyethylene ( 5) nonylphenyl ether; the volume ratio of the organic solvent mixed with the surfactant is 80:20 to 50:50; The ratio of the amount of the liquid A and the liquid B is (50 to 200):1; the ratio of the amount of the liquid A and the liquid C is (50 to 200):1; the ratio of the amount of the liquid E to the liquid D is (1 to 3): (1 to 3).

Comparative example 1

(1) Nanoparticles containing the silencing siRNA (PD1 silencing TIL cells) used in this comparative example were obtained by the following preparation methods:

Step 1, 10.5 ml of cyclohexane, 4.5 ml of polyoxyethylene (5) nonylphenyl ether, 5 M calcium chloride solution (volume 50 μL), 100 μM siRNA solution (volume used is 66.7 μL), ultrapure Mixing 33.3 μL of water to obtain a mixture I;

Step 2, 10.5 ml of cyclohexane, 4.5 ml of polyoxyethylene (5) nonylphenyl ether, 50 mM disodium hydrogen phosphate (volume 50 μL), 100 μM siRNA solution (volume used is 66.7 μL), ultrapure 33.3 μL of water was mixed to obtain a mixture II;

In the third step, the mixture I was dropped into the mixture II at a rate of 1 to 3 ml/min, and stirred for 20 minutes, and the resulting precipitate was a nanoparticle.

In step one, in step two, the volume ratio of cyclohexane to polyoxyethylene (5) nonylphenyl ether is 70:30, and the sequence and concentration (50 nM) of the encapsulated siRNA are the same as in the first embodiment.

The nanoparticles prepared in this comparative example have a single-layer membrane structure, the particle size is uneven, the particle diameter is larger than 20 nm, and the dispersibility is poor.

The concentration of the obtained nanoparticles was adjusted to 40 nM, and TIL cells were transfected with reference to section 1.2 in Example 1, and samples were taken at different time periods to measure transfection efficiency. The results are shown in Table 1. According to Table 1, it can be seen that the extraction rate of the nanoparticle of Comparative Example 1 does not change significantly with time, and the corresponding extraction rate is not high for 8 hours of transfection.

Table 1

	0.25h	2h	4h	6h	8h
Acquisition rate%	2.17±0.21	25.43±6.31	31.21±4.47	43.53±3.28	43.57±4.32

(2) A method for preparing nanoparticles coated with a silencing siRNA (PDL1 for silencing breast cancer cell MCF7) used in the present comparative example is referred to the above (1) in the comparative examples.

The concentration of the obtained nanoparticles was adjusted to 40 nM, and the breast cancer cells MCF7 were transfected with reference to section 2.2 in Example 1, and samples were taken at different time periods to measure the transfection efficiency. The results are shown in Table 2. According to Table 2, the extraction rate of the nanoparticle of Comparative Example 1 did not change significantly with time, and the corresponding extraction rate was not high for 8 hours of transfection.

Table 2

	0.25h	2h	4h	6h	8h
Acquisition rate%	2.93±0.41	24.63±5.43	26.11±4.34	34.53±2.73	34.71±5.33

(3) Killing efficiency test: Take the above (1) transfection for 2 h to obtain TIL cells, and (2) transfect 2 h to obtain breast cancer cells MCF7, and perform the lethality test according to the test method of the above Example 3, and set a 10:1. 30:1, 100:1 three ratios, the results are shown in Table 3. According to Table 3, since the transfection rate was not high, the killing efficiency of the two nanoparticles after transfection of TIL cells and breast cancer cells MCF7 was significantly lower than that of the examples.

table 3

Killing efficiency%	PD1-/PDL1-
10:1	22.11±1.50
30:1	30.06±4.40
100:1	48.09±6.84

(4) cytokines Test: The method of Example 4 with reference to embodiments, TIL cells were transfected with the above-mentioned (1) obtained in the MCF7, MCF7 ^K, CTL6 three cell co-cultures and tested before and after transfection in culture before and after The amount of cytokine expression. The test results are shown in Table 4.

Table 4

Unit pg/ml	IL17	IL10	INFγ	TNFα
TIL/CTL4	23±3.55	15.00±4.00	17.33±3.61	5.33±1.15
TIL/MCF7 K	309.67±90.73	56.33±7.51	772.67±10.50	81.22±6.11
TIL/MCF7	269.00±33.05	27.67±10.21	414.33±22.30	58.33±4.73
TIL K /CTL4	23.00±7.55	13.00±4.00	23.67±28.88	13.11±3.46
TIL K /MCF7 K	509.67±90.73	66.33±7.51	923.67±48.39	91.67±9.45
TIL K /MCF7	369.00±30.05	32.67±10.21	595.67±24.68	86.13±4.58

According to Table 4, since the transfection rate was not high, the expression of cytokines between cells obtained before and after transfection was not significant.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (10)

1. A method for preparing a nanoparticle, comprising the steps of:

   Mixing an organic solvent with a surfactant to obtain a solution A;

   Mixing the first reactant solution with the functional substance solution to obtain liquid B;

   Mixing the second reactant solution with the functional substance solution to obtain a liquid C; and forming a precipitate after the second reactant contacts the first reactant;

   Mixing the liquid A and the liquid B to obtain a liquid D;

   Mixing the liquid A and the liquid C, and adding DOPA solution to the obtained mixed solution to obtain an E liquid;

   Adding the E liquid to the D liquid, centrifuging the obtained mixture, collecting the precipitate to obtain a single layer membrane nanoparticle;

   A dispersion of the monolayer film nanoparticles is prepared, and a mixture of DOPC and cholesterol is added to the dispersion, or a mixture of DOTAP and cholesterol is added, and the particles in the obtained suspension particle solution are double-layered membrane nanoparticles.
2. The method for preparing a nanoparticle according to claim 1, wherein the first reactant solution is a calcium salt solution, and the second reactant solution is a phosphate solution or a pyrophosphate solution, the first The mass ratio of calcium to phosphorus of the reactant and the second reactant is (25 to 400):1.
3. The method for preparing a nanoparticle according to claim 2, wherein the concentration of the calcium salt solution is 4.5 to 5.5 M; and the concentration of the phosphate solution or the pyrophosphate solution is 45 to 55 mM.
4. The method for preparing a nanoparticle according to claim 1, wherein a concentration of DOPA in the DOPA solution is 15 to 25 mg/ml, and a mixed solution of the added volume of the DOPA solution and the liquid A and C is mixed. The volume ratio is (70-80): 1.
5. The method for preparing a nanoparticle according to claim 1, wherein a mass ratio of DOPC to cholesterol in the mixture of DOPC and cholesterol is 1: (2.5 to 3.5); in the mixture of DOTAP and cholesterol, The mass ratio of DOTAP and cholesterol is 2: (2.5 to 3.5).
6. The method for preparing a nanoparticle according to claim 1, wherein in the preparation of the liquid A, the organic solvent is any one of cyclohexane, benzene, toluene, n-heptane, and carbon tetrachloride. Several The surfactant includes polyoxyethylene (5) nonylphenyl ether; the volume ratio of the organic solvent to the surfactant is 80:20 to 50:50.
7. The method for preparing a nanoparticle according to claim 1, wherein the ratio of the amount of the liquid A and the liquid B is (50 to 200): 1; the ratio of the amount of the liquid A to the liquid C is (50 200): 1; the ratio of the amount of the E liquid to the D liquid is (1 to 3): (1 to 3).
8. The method for preparing a nanoparticle according to claim 1, wherein the functional substance in the functional substance solution comprises a functional nucleic acid sequence, a protein antibody, and a drug molecule.
9. The method of preparing a nanoparticle according to claim 8, wherein the functional substance in the functional substance solution comprises a functional nucleic acid sequence.
10. A nanoparticle obtained by the production method according to any one of claims 1-9.

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