Traditional Chinese medicine engineered core-shell structure gold nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine engineered core-shell structure gold nanoparticle and a preparation method and application thereof.The preparation method comprises the steps that D1-miR155 modified gold nanoparticles, D2-miR155 modified gold nanoparticles, polyethylene glycol modified norcantharidin and a tumor cell membrane are mixed to be uniform, extrusion is conducted through a lipidosome extruder, and the core-shell structure gold nanoparticle is obtained; the traditional Chinese medicine engineered core-shell structure gold nanoparticles are obtained. The prepared gold nanoparticles can capture oncogene miR155 overexpressed in tumor cells to activate the photothermal conversion effect, induce apoptosis of the tumor cells and cause the process of immunogenic death (ICD). Meanwhile, a key signal molecule STAT3 of a tumor can be regulated and controlled by reducing miR155 so as to enhance immunogenicity of the tumor, a Tregs inhibitor-norcantharidin (DMC and traditional Chinese medicine small molecules) is effectively delivered to inhibit immune escape of the tumor, and stronger photothermal-immunotherapy activity is achieved together with an ICD process caused by photothermal.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological nanomaterials and tumor photothermal-immunotherapy, and in particular to a traditional Chinese medicine engineered core-shell structure gold nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Cancer has become one of the most serious threats to human health, and traditional cancer treatment methods can only moderately prolong the life of patients, but the treatment effect is still unsatisfactory. In order to overcome the limitations of traditional treatment methods, a variety of emerging cancer treatment methods have been developed. Among them, photothermal therapy, especially photothermal therapy based on noble metal nanomaterials, has become a very effective tumor ablation method. Photothermal therapy not only can kill tumor cells by increasing tissue temperature, but also the apoptotic tumor fragments can become tumor-associated antigens, causing immunogenic cell death (ICD) and obtaining additional anti-tumor immune response.

[0003] However, the immune response efficiency of this tumor photothermal-immunotherapy strategy is limited, and it is difficult to achieve strong tumor treatment effect. Insufficient tumor immunogenicity and immunosuppressive microenvironment are considered to be the main obstacles limiting this treatment method. During the development of tumors, tumors are often in the state of immune desert, i.e. "cold" tumors. At this time, the distribution of effector T lymphocytes in the tumor is very small, and no adaptive immune response is established, and the response to immunotherapy is poor. In order to solve the problem of insufficient tumor immunogenicity, by controlling important regulatory proteins in the tumor, the "cold" tumor can be converted into an immune inflammatory "hot" tumor, which greatly increases the infiltration of effector T lymphocytes, so that the response to immunotherapy is more active. In addition, tumors can also regulate the immune microenvironment and evade the monitoring and attack of the immune system through various mechanisms to achieve tumor immune escape. The generation of regulatory T lymphocytes (Tregs) is one of the core mechanisms of tumor immune escape, and Tregs promote tumor growth by suppressing anti-tumor immune response. By using some Tregs inhibitors to inhibit the recruitment and activation of Tregs, the immune escape of tumors can be effectively reversed, and better immunotherapy results can be obtained. Therefore, it is urgent to construct a multifunctional tumor photothermal-immunotherapy strategy that can achieve precise photothermal therapy while overcoming the many obstacles of photothermal-immunotherapy. SUMMARY

[0004] In view of the problem that the insufficient tumor immunogenicity and immunosuppressive microenvironment in the prior art limit the application of photothermal-immunotherapy, the purpose of the present application is to provide a traditional Chinese medicine engineered core-shell structure gold nanoparticle (TCM DMCThe application discloses a kind of traditional Chinese medicine engineered core-shell structure gold nanoparticles and preparation method and application, the gold nanoparticles prepared by the method can capture oncogene miR155 overexpressed in tumor cell to activate photo-thermal conversion effect, to effectively remove tumor. Meanwhile, it can also regulate key signal molecules STAT3 of tumor and effectively deliver Tregs inhibitor-norcantharidin (DMC, small molecule of traditional Chinese medicine), to enhance the immunogenicity of tumor and inhibit the immune escape of tumor, and realize stronger photo-thermal-immunotherapy activity with the ICD process caused by photo-thermal.

[0005] To achieve the above object, the technical scheme adopted by the application is as follows:

[0006] A preparation method of traditional Chinese medicine engineered core-shell structure gold nanoparticles comprises the following steps:

[0007] The D1-miR155 modified gold nanoparticles, D2-miR155 modified gold nanoparticles, polyethylene glycol modified norcantharidin and tumor cell membrane are uniformly mixed, and are extruded by a liposome extruder to obtain the traditional Chinese medicine engineered core-shell structure gold nanoparticles.

[0008] Further, the preparation method of the D1-miR155 modified gold nanoparticles and the D2-miR155 modified gold nanoparticles is as follows:

[0009] The tetrachloroauric acid is mixed with sodium citrate, and then a reduction reaction is carried out to obtain a gold nanoparticle solution by using a sodium citrate reduction method;

[0010] The thiol-modified D1-miR155 is uniformly mixed with the gold nanoparticle solution, is frozen and then thawed to obtain the D1-miR155 modified gold nanoparticles;

[0011] The thiol-modified D2-miR155 is uniformly mixed with the gold nanoparticle solution, is frozen and then thawed to obtain the D2-miR155 modified gold nanoparticles.

[0012] Further, the nucleotide sequence of the thiol-modified D1-miR155 is shown in SEQ ID NO. 1;

[0013] The nucleotide sequence of the thiol-modified D2-miR155 is shown in SEQ ID NO. 2.

[0014] Further, the molar ratio of the tetrachloroauric acid to the sodium citrate is 1:5-10;

[0015] The reaction temperature of the reduction reaction is 110-140 DEG C, and the reaction time is 20-50 min;

[0016] The particle size of the gold nanoparticles is 10-30 nm;

[0017] The molar ratio of the thiol-modified D1-miR155 to the gold nanoparticles is (100-600):1;

[0018] The molar ratio of the thiol-modified D2-miR155 to the gold nanoparticles is (100-600):1;

[0019] The particle size of the thiol-modified D1-miR155 modified gold nanoparticles is 20-50 nm.

[0020] The particle size of the thiol-modified D2-miR155 modified gold nanoparticles is 20-50 nm.

[0021] Further, the polyethylene glycol modified norcantharidin is prepared by the following process:

[0022] The amino-containing phospholipid polyethylene glycol and the norcantharidin are added into dichloromethane, and then dimethylamino pyridine is added to perform a nucleophilic substitution reaction, so as to obtain the polyethylene glycol modified norcantharidin.

[0023] Further, the molar ratio of the amino-containing phospholipid polyethylene glycol, the norcantharidin and the dimethylamino pyridine is 2:5-50:1-10.

[0024] The temperature of the nucleophilic substitution reaction is 50-70 DEG C, and the reaction time is 12-24 h.

[0025] Further, the tumor cells are breast cancer tumor cells.

[0026] Further, the molar ratio of the D1-miR155 modified gold nanoparticles, the D2-miR155 modified gold nanoparticles and the polyethylene glycol modified norcantharidin is 1:1:(500-5000).

[0027] The mass ratio of the polyethylene glycol modified norcantharidin to the tumor cell membrane is 5 mg:(1-10) mg.

[0028] The pore size of the polycarbonate porous membrane is 100-400 nm.

[0029] The extrusion times are 10-50 times.

[0030] A traditional Chinese medicine engineered core-shell structure gold nanoparticle, wherein the particle size of the core-shell structure gold nanoparticle is 100-200 nm.

[0031] The application of a traditional Chinese medicine engineered core-shell structure gold nanoparticle in the preparation of an anti-tumor drug.

[0032] Further, the anti-tumor drug is an anti-breast cancer drug.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application mixes two DNA modified gold nanoparticles, PEG modified DMC molecules and tumor cell membranes, uses one-step extrusion method to self-assemble the DNA modified gold nanoparticles, tumor cell membranes and PEG modified traditional Chinese medicine molecules to obtain traditional Chinese medicine small molecule modified cell membrane coated gold nanoparticles, i.e. traditional Chinese medicine engineered core-shell structure gold nanoparticles (TCM DMC @SNAs), compared with the ultrasonic method, the self-assembly process is more compact, so that the material size is more uniform, the shape is more similar to spherical, and the repeatability is higher. The TCM DMC @SNAs prepared by the present application can release traditional Chinese medicine small molecules-norcantharidin (DMC) in the acidic tumor microenvironment by using the cleavage of acid-sensitive bonds to reduce the generation of Tregs and inhibit the immune escape of tumors. After the cleavage of the acid-sensitive bond, the TCM DMC @SNAs realizes the change of charge from negative to positive, accelerates the attraction between gold nanoparticles and tumor cells. And the TCM DMC @SNAs surface coated tumor cell membrane uses its biomimetic structure to carry out membrane-membrane fusion with tumor cells, which promotes the efficient uptake of gold nanoparticles. After entering the tumor cells, the TCM DMC @SNAs uses two DNA molecules (D1-miR155 and D2-miR155) to capture overexpressed oncogene miR155, realizes the aggregation of gold nanoparticles, and shows the effect of selective photothermal therapy. At the same time, the down-regulation of miR155 will inhibit the key signal molecule downstream-signal transduction and transcription activator 3 (STAT3), thereby increasing the immunogenicity of the tumor. Photothermal therapy will also cause tumor cell apoptosis, produce ICD process, further enhance the activation of the immune system, together with the decrease of Tregs and the increase of immunogenicity, make the tumor from "cold" to "hot", and obtain more excellent immunotherapy effect. In addition, the process of preparing TCM DMC @SNAs in the present application is simple, reproducible, green, pollution-free and stable.

[0035] Further, the molar ratio of DNA molecules to gold nanoparticles of the DNA modified gold nanoparticles prepared by the present application needs to be kept at 100-600:1, if it is lower than 100:1, it is difficult to keep stable in the freeze-thaw process, so that the gold nanoparticles aggregate, if it is higher than 600:1, the DNA on the surface of the gold nanoparticles is saturated, and it is difficult to load more DNA molecules. Two DNA molecules with unique sequences (D1-miR155 and D2-miR155) can respectively base-pair with half of miR155, so as to capture miR155, and at the same time cause the aggregation of gold nanoparticles, and play the effect of photothermal therapy.

[0036] Further, the application adopts the freeze-thaw method to modify the DNA to the surface of the gold nanoparticles, compared with the salt aging method, the operation is simple, the reproducibility is good, more DNA can be modified on the gold nanoparticles, and the obtained material is more stable, and the hybridization of miR155 and DNA is improved.

[0037] Further, the application adopts the amino-containing phospholipid polyethylene glycol, norcantharidin and dimethylaminopyridine to prepare PEG modified norcantharidin (DSPE-PEG-DMC), which is a functional application of traditional Chinese medicine small molecule-norcantharidin, and the unique anhydride structure can not only be released in an acid-responsive environment to play the function of an immunoregulator (inhibiting Tregs), but also can make the charge of the material reverse after release, further promote the uptake of tumor cells, and is a successful exploration of modernization of traditional Chinese medicine small molecules.

[0038] The gold nanoparticles prepared in the application can capture the overexpressed oncogene miR155 in tumor cells to activate the photothermal conversion effect, thereby effectively removing the tumor. Meanwhile, the key signal molecule STAT3 of the tumor can be regulated, and the norcantharidin (DMC, traditional Chinese medicine small molecule) as a Tregs inhibitor can be effectively delivered, so as to enhance the immunogenicity of the tumor and inhibit the immune escape of the tumor, and the ICD process caused by photothermal is combined to realize stronger photothermal-immunotherapy activity. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 TCM provided in example 1 DMC UV-visible spectra of SNAs, TCM@SNAs and DMC

[0040] Figure 2 TCM provided in example 1 DMC TEM image of SNAs

[0041] Figure 3 miR155 capture of D1-Au NPs and D2-Au NPs provided in example 2, wherein A is a different group of gel electrophoresis images, and B is corresponding quantitative statistics

[0042] Figure 4 miR155-dependent aggregation and photothermal of D1-Au NPs and D2-Au NPs provided in example 2, wherein A is a TEM image of miR155 causing D1-Au NPs and D2-Au NPs to aggregate, and B is a time-temperature change curve of miR155 causing D1-Au NPs and D2-Au NPs to aggregate

[0043] Figure 5 TCM provided in example 3 DMC DMC release curve of SNAs at different pH values

[0044] Figure 6 The 4T1 cells and MCF-10A cells provided in Example 4 were tested with PBS and D1-miR155. Cy5 +D2-miR155 Cy5 TCM@SNAs Cy5 and TCM DMC @SNAs Cy5 The uptake of cells is shown in Figure 1. A represents the flow cytometry analysis results of 4T1 cells uptake in different experimental groups, B represents the flow cytometry analysis results of MCF-10A cells uptake in different experimental groups, and C represents the corresponding quantitative statistics.

[0045] Figure 7 These are tumor growth curves for different treatment groups provided in Example 5;

[0046] Figure 8 This is the expression of STAT3 in tumor tissues of different treatment groups provided in Example 6, where A is the immunohistochemical section result of STAT3 in tumor tissues, and B is the quantitative statistics of the sections;

[0047] Figure 9 The different treatment groups provided in Example 7 show the inhibition of Tregs, where A is the immunofluorescence staining result of Foxp3 in tumor tissue and B is the quantitative statistics of the sections.

[0048] Figure 10 The different treatment groups provided in Example 8 are for CD8 + T cell recruitment data, where A represents the immunofluorescence staining results of CD8 in tumor tissue, and B represents the quantitative statistics of the sections. Detailed Implementation

[0049] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0050] The present invention discloses a method for preparing core-shell structured gold nanoparticles for engineering traditional Chinese medicine, comprising the following steps:

[0051] (1) The preparation methods of gold nanoparticles modified with D1-miR155 and D2-miR155 are as follows:

[0052] First, gold nanoparticles were prepared via the classic sodium citrate reduction method, which utilizes sodium citrate as both a reducing agent and a stabilizer to reduce the Au content in HAuCl4. 3+ Restore to Au 0, to obtain the gold nanoparticle solution. The specific process is as follows: using a hydrothermal method, the HAuCl4 solution is added to the sodium citrate solution, under the reduction of sodium citrate, a reduction reaction is carried out, Au in tetrachloroauric acid is reduced to Au 3+ 0 , by adjusting the concentration of the reactants, the reaction temperature and time, etc., the gold nanoparticle solution is prepared;

[0053] Then the thiol-modified D1-miR155 solution and the thiol-modified D2-miR155 solution are mixed with the gold nanoparticle solution, and are frozen at-20℃ for a certain period of time, and are thawed at room temperature, and the DNA is modified to the surface of the gold nanoparticles by the freeze-thaw method, to obtain D1-miR155 modified gold nanoparticles (D1-Au NPs) and D2-miR155 modified gold nanoparticles (D2-Au NPs). For convenience of description, the D1-Au NPs and the D2-Au NPs are mixed in a molar ratio of 1:1, and the obtained mixed solution is denoted as SNAs.

[0054] Further, the nucleotide sequence of the thiol-modified D1-miR155 is 5'-ATT AGC ATT AAT TTT TTTTTT-3' (as shown in SEQ ID NO. 1);

[0055] Further, the nucleotide sequence of the thiol-modified D2-miR155 is 5'-AAA AAA AAA AAC CCC TATCAC G-3' (as shown in SEQ ID NO. 2).

[0056] Further, the concentration of the sodium citrate solution in step (1) is 1-10 mmol / L.

[0057] Further, the concentration of the tetrachloroauric acid solution in step (1) is 25-250 mmol / L.

[0058] Further, the molar ratio of tetrachloroauric acid to sodium citrate in step (1) is 1:(5-10).

[0059] Further, the reaction temperature of the reduction reaction in step (1) is 110-140℃, and the reaction time of the reduction reaction is 20-50 min.

[0060] Further, the particle size of the gold nanoparticles prepared in step (1) is 10-30 nm.

[0061] Further, the concentration of the thiol-modified D1-miR155 solution in step (1) is 10-100 μmol / L.

[0062] ​Further, the concentration of the thiol-modified D2-miR155 solution in step (1) is 10-100 μmol / L.

[0063] Further, the molar ratio of the thiol-modified D1-miR155 to gold nanoparticles in step (1) is (100-600):1.

[0064] Further, the molar ratio of the thiol-modified D2-miR155 to gold nanoparticles in step (1) is (100-600):1.

[0065] Further, the freezing time of step (1) at -20℃ is 2-4 h.

[0066] Further, the particle size of the D1-Au NPs prepared in step (1) is 20-50 nm.

[0067] Further, the particle size of the D2-Au NPs prepared in step (1) is 20-50 nm.

[0068] (2) The preparation process of PEG-modified DMC is as follows: the amino-containing phospholipid PEG (DSPE-PEG-NH2) is directly mixed with DMC (Dimethyl Carbonate) in a reaction solvent, then DMAP (4-Dimethylaminopyridine) is added as a catalyst, a nucleophilic substitution reaction occurs, and by adjusting the reaction time and temperature, PEG-modified DMC, i.e. DSPE-PEG-DMC, is obtained.

[0069] Further, the concentration of DSPE-PEG-NH2 in the reaction solvent in step (2) is 2-20 mmol / L.

[0070] Further, the concentration of DMC in the reaction solvent in step (2) is 5-50 mmol / L.

[0071] Further, the concentration of DMAP in the reaction solvent in step (2) is 1-10 mmol / L.

[0072] Further, the molar ratio of DSPE-PEG-NH2, DMC and DMAP in step (2) is 2:(5-50):(1-10). Preferably, the molar ratio of DSPE-PEG-NH2: DMC: DMAP is 2:5:1.

[0073] Further, the temperature of the nucleophilic substitution reaction in step (2) is 50-70℃, and the reaction time is 12-24 h.

[0074] Further, the reaction solvent in step (2) is DCM (Dichloromethane).

[0075] (3) Using a small liposome extruder equipped with a polycarbonate porous membrane, D1-Au NPs, D2-Au NPs, DSPE-PEG-DMC and tumor cell membranes are mixed and extruded back and forth continuously, and the solution is passed through a polycarbonate porous membrane with a specific pore size to self-assemble into a traditional Chinese medicine engineered core-shell structure gold nanoparticle (TCM DMC @SNAs), i.e. a traditional Chinese medicine small molecule modified cell membrane coated gold nanoparticle.

[0076] Further, the tumor cell membrane is obtained by using a hypotonic lysis method, and the tumor cell is a 4T1 breast cancer tumor cell.

[0077] Further, the small liposome extruder is an Avanti micro-extruder.

[0078] Further, in step (3), the molar ratio of D1-Au NPs:D2-Au NPs:DSPE-PEG-DMC is 1:1:(500-5000).

[0079] Further, in step (3), when the amount of DSPE-PEG-DMC is 5 mg, the amount of tumor cell membrane added is 1-10 mg.

[0080] Further, in step (3), the membrane used in the liposome extruder is a polycarbonate porous membrane.

[0081] Further, in step (3), the pore size of the polycarbonate porous membrane is 100-400 nm.

[0082] Further, in step (3), the number of extrusions is 10-50 times.

[0083] Further, in step (3), the particle size of TCM DMC @SNAs is 100-200 nm.

[0084] A traditional Chinese medicine engineered core-shell structure gold nanoparticle in the preparation of an antitumor drug.

[0085] Further, the antitumor drug is an anti-breast cancer drug.

[0086] The following is a specific example.

[0087] Example 1: Preparation of a traditional Chinese medicine engineered core-shell structure gold nanoparticle (TCM DMC @SNAs)

[0088] (1) Preparation method of DNA1 and DNA2 modified gold nanoparticles:

[0089] First, gold nanoparticles solution was prepared. The specific process was as follows: 100 μL of HAuCl4-3H2O solution (0.25 mol / L) was added to 100 mL of deionized water, and stirred and boiled. Then, 15 mL of sodium citrate solution (10 mmol / L) was added, and the molar ratio of tetrachloroauric acid and sodium citrate was 1:6. The solution was continuously heated at 120 °C for 0.5 h, and the color of the solution finally changed to wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution with a concentration of 10 nmol / L, which was stored at 4 °C for standby.

[0090] DNA was modified to gold nanoparticles by freeze-thaw method. Specifically, 6 μL of thiol-modified D1-miR155 solution (100 μmol / L) was accurately measured by a pipette and added to 100 μL of gold nanoparticle solution (10 nmol / L), and the molar ratio of thiol-modified D1-miR155 and gold nanoparticles was 600:1. The mixture was vortexed thoroughly and stored in a refrigerator at -20 °C. After 2 h, the solution was thawed at room temperature to obtain D1-miR155 modified gold nanoparticles (D1-Au NPs).

[0091] The preparation method of D2-miR155 modified gold nanoparticles (D2-Au NPs) was similar to the preparation process of the aforementioned D1-miR155 modified gold nanoparticles, except that 6 μL of thiol-modified D1-miR155 solution was replaced by 6 μL of thiol-modified D2-miR155 solution.

[0092] (2) The preparation method of DSPE-PEG-DMC was as follows:

[0093] 20 mg of DSPE-PEG 2000 -NH2 (0.01 mmol) and 4.2 mg of DMC (0.025 mmol) were added to 5 mL of dichloromethane, followed by the addition of 0.61 mg of DMAP (0.005 mmol) as a catalyst. The mixed solution was transferred to a flask and refluxed at room temperature with continuous stirring. The solution was heated to 55 °C and reacted for 12 h. After the reaction was completed, the solution was cooled to room temperature, precipitated with excess ether, repeated three times, and finally vacuum dried to obtain DSPE-PEG-DMC.

[0094] (3) TCM DMC @SNAs were prepared as follows:

[0095] First, cell membranes were obtained using a hypotonic lysis method. A large number of 4T1 tumor cells were collected, and hypotonic lysis buffer was added. The cells were then incubated on ice for 30 minutes. Subsequently, three freeze-thaw cycles were performed to fully lyse the cells. The lysed cell solution was centrifuged at 800g for 10 minutes at 4°C, and the supernatant was collected and the precipitate discarded. Then, the cells were centrifuged at high speed (15000g) for 30 minutes at 4°C. The supernatant was carefully aspirated, and the precipitate was freeze-dried to obtain the tumor cell membrane.

[0096] Core-shell structured gold nanoparticles for engineering traditional Chinese medicine were then obtained using an extrusion method. 10 mg of 4T1 tumor cell membrane, 5 mg of DSPE-PEG-DMC, 0.5 mL of D1-Au NPs, and 0.5 mL of D2-Au NPs were mixed and thoroughly dissolved using ultrasonication in a water bath. The mixture was then extruded back and forth through an Avanti micro-extruder containing a 100 nm pore size polycarbonate porous membrane. After 30 extrusion cycles, core-shell structured gold nanoparticles (TCM) for engineering traditional Chinese medicine were finally obtained. DMC @SNAs.

[0097] Comparative Example 1

[0098] Using the same method as in Example 1, except that DSPE-PEG-DMC is not added, TCM@SNAs nanoparticles can be obtained.

[0099] Using ultraviolet-visible spectroscopy (UV-Vis) to study TCM DMC @SNAs, TCM@SNAs, and DMC were characterized, and TCM was analyzed using transmission electron microscopy (TEM). DMC The morphology of @SNAs is characterized.

[0100] like Figure 1 As shown, TCM DMC @SNAs exhibited a distinct characteristic absorption peak of DMC at 206 nm, while TCM@SNAs showed no absorption at the same location, proving that DMC was successfully modified onto the TCM surface. Meanwhile, TCM... DMC An absorption peak for DNA was observed at 260 nm in @SNAs, strongly suggesting that DNA was ligated to Au NPs. (TCM) DMC @SNAs still exhibit the characteristic absorption peak of Au NPs at 520 nm, and the optical photographs still show a wine-red color, indicating that the assembly of TCM and DSPE-PEG-DMC did not affect the monodisperse state of the SNAs.

[0101] like Figure 2 As shown in the TEM image, the TCM can be clearly seen. DMC@SNAs exhibit a spherical core-shell structure, approximately 140 nm in diameter. The inner layer encapsulates DNA-modified Au NPs in a monodisperse state, while the outer layer is attached with an organic layer. This demonstrates that the tumor cell membrane was successfully coated onto the surface of the DNA-modified Au NPs.

[0102] Example 2: Verification of the photothermal conversion effect induced by D1-Au NPs and D2-Au NPs

[0103] D1-Au NPs and D2-Au NPs can capture miR155, causing the aggregation of gold nanoparticles, which exhibit excellent photothermal conversion effects under 808 nm laser irradiation. First, agarose gel electrophoresis was used to separate miR155, D1-Au NPs+D2-Au NPs, D1-Au NPs+miR155, D2-Au NPs+miR155, and D1-Au NPs+D2-Au NPs+miR155, evaluating the miR155 capture effect of D1-Au NPs and D2-Au NPs. Next, D1-Au NPs and D2-Au NPs were mixed with miR155, and the morphology of the aggregated nanoparticles was characterized using TEM. Finally, under 808nm laser irradiation, the temperature rise of the four groups, D1-Au NPs+D2-Au NPs, D1-Au NPs+miR155, D2-Au NPs+miR155 and D1-Au NPs+D2-Au NPs+miR155, was recorded using a handheld thermal imager.

[0104] like Figure 3 As shown in Figure A, pure miR155 (Ⅰ) exhibits a distinct emission band, while D1-Au NPs + D2-Au NPs (Ⅱ) naturally shows no band. In D1-Au NPs + miR155 (Ⅲ) and D2-Au NPs + miR155 (Ⅳ), the band brightness is reduced. This is because D1-Au NPs and D2-Au NPs can hybridize with either the first or second half of miR155, leading to a decrease in the amount of miR155. Notably, when D1-Au NPs + D2-Au NPs are mixed with miR155 (Ⅴ), the emission band brightness is very weak. This indicates that D1-Au NPs + D2-Au NPs capture miR155 through base complementarity pairing, thus significantly reducing the miR155 content. Figure 3 As shown in Figure B, semi-quantitative statistics of the bands show that D1-AuNPs+D2-Au NPs+miR155(V) has the lowest relative brightness, which also indicates that D1-Au NPs+D2-Au NPs effectively captures miR155.

[0105] As Figure 4 shown in FIG. 3A, the aggregation of DNA-modified Au NPs was verified by TEM. By observing the TEM images of D1-Au NPs+D2-Au NPs+miR155, it can be found that there are a large number of aggregates containing nanoparticles, which proves that the DNA-modified Au NPs are in the aggregated state. As Figure 4 shown in FIG. 3B, under the irradiation of 808 nm laser, whether it is D1-Au NPs+D2-Au NPs, or D1-Au NPs+miR155, or D2-Au NPs+miR155, the temperature increase is very limited, and it is difficult to reach the required temperature for photothermal therapy. On the contrary, miR155 induces the aggregation of D1-Au NPs+D2-Au NPs, and the temperature rises by 30°C in 10 min of irradiation, showing excellent photothermal conversion efficiency.

[0106] Example 3 TCM DMC Verification of acid-responsive DMC release behavior of SNAs

[0107] In an acidic environment, the acid-sensitive bond on TCM DMC SNAs will be broken, the surface charge will change from positive to negative, and DMC will be released at the same time. First, TCM DMC SNAs and TCM@SNAs were incubated with PBS solutions at pH 7.4 and pH 6.8, respectively, and then the Zeta potentials in different samples were measured by a nanoparticle size and Zeta potential analyzer. For the release of DMC, TCM DMC SNAs were added to PBS solutions at pH 7.4 and pH 6.8, and the solutions were placed in a constant temperature incubator at 37°C and rotated at a speed of 100 rpm. At the preset time point, the solution in different samples was taken, and the concentration of DMC was determined by the standard curve method, and finally the release curve of TCM DMC SNAs at different pH was obtained.

[0108] As Figure 5 shown in FIG. 4, the release of DMC on the surface of TCM DMC SNAs under different pH conditions was explored by dialysis method. Obviously, under neutral conditions (pH 7.4), the release rate of DMC in TCM DMC SNAs is relatively low, only more than 20%. When TCM DMC SNAs were incubated in a pH 6.8 environment, DMC could be released continuously, and the final release rate was more than 80%, which also proved that the nanoparticles had pH-dependent DMC release ability.

[0109] Example 4 TCM DMCValidation of @SNAs in vitro cellular uptake behavior

[0110] Different samples were prepared by labeling D1-miR155 and D2-miR155 with Cy5 (D1-miR155). Cy5 +D2-miR155 Cy5 TCM@SNAs Cy5 and TCM DMC @SNAs Cy5 These samples were incubated with 4T1 cells, and cells were collected at 2 h and 8 h, respectively, and washed three times with PBS. Finally, the intracellular Cy5 fluorescence intensity was measured using flow cytometry.

[0111] like Figure 6 As shown, compared with the PBS group, D1-miR155 Cy5 +D2-miR155 Cy5 The enhanced fluorescence signal in 4T1 cells is attributed to the ability of D1-miR155+D2-miR155 to capture overexpressed miR155 in tumor cells, achieving accumulation within tumor cells and thus enhancing retention in 4T1 cells (see [link to relevant documentation]). Figure 6 (A) After TCM was coated on the surface of SNAs, TCM achieved membrane fusion with 4T1 cells, further enhancing the tumor cells' response to TCM@SNAs. Cy5 Uptake by DMCs. When DMCs are intercalated onto the surface of TCM, 4T1 cells uptake TCMs. DMC @SNAs Cy5 The intake of TCM has increased significantly. This is due to the fact that under acidic conditions, TCM... DMC @SNAs Cy5 The acid-sensitive bonds on the surface break, and the charge changes from negative to positive, drawing the TCM closer through electrostatic adsorption. DMC The distance between @SNAs and tumor cells is adjusted, and then membrane fusion occurs, thereby enhancing their membrane fusion ability through charge flipping. Quantitative statistical analysis of cellular uptake revealed that 4T1 cells exhibit increased uptake of TCM. DMC @SNAs Cy5 The uptake efficiency is significantly higher than that of TCM@SNAs Cy5 (see Figure 6 (C). Furthermore, MCF-10A normal cells respond to DNA1 Cy5 +DNA2 Cy5 TCM@SNAs Cy5 and TCM DMC @SNAs Cy5 The intake amounts are very small, indicating that these nanoparticles do not enter normal cells (see [reference]). Figure 6 (B)

[0112] Example 5 TCM DMC @In vivo validation of SNAs in inhibiting tumor growth

[0113] Firstly, 4T1 tumor-bearing mouse model was constructed. 4-6 weeks old female BALB / c nude mice were selected and acclimated for one week, then 100 μL of 4T1 cell suspension (1 x 10 8 cells / mL) was subcutaneously inoculated on the back of the right leg of the mice. The tumor volume of the mice was calculated using the following formula: V = (L x W 2 ) / 2, where L is the long side of the tumor and W is the short side of the tumor. When the tumor volume of the mice grew to 50-100 mm 3 , the mice were randomly divided into 5 groups and injected with different treatment groups: PBS, SNAs, TCM DMC @SNAs, SNAs+L and TCM DMC @SNAs+L. During the 21-day treatment period, the tumor volume was measured every two days, and the tumor growth curve was plotted.

[0114] As shown in Figure 7 , the tumor in the PBS group progressed very quickly, and the tumor volume increased rapidly, eventually exceeding 1500 mm 3 . The tumor volume in the SNAs group alone decreased to about 1100 mm 3 , thanks to the ability of SNAs to capture the oncogenic miR155, not only exerting the effect of gene therapy, but also inhibiting the immune escape of the tumor, showing some of the functions of immunotherapy. After being wrapped with TCM and intercalated with DMC, the tumor volume of the TCM DMC @SNAs treatment group of mice was further inhibited, with a tumor volume of only 870 mm 3 . This good inhibitory effect is because TCM DMC @SNAs can actively target tumor tissue, not only releasing DMC in the tumor microenvironment to inhibit the activity of Tregs, but also down-regulating the oncogenic miR155 after entering a large number of tumor cells to inhibit the immune escape of the tumor, thus achieving the synergistic effect of gene therapy and immunotherapy. Further NIR irradiation, the tumor burden of the SNAs+808 nm light group of mice was greatly relieved, with slow tumor growth and a tumor volume of only 580 mm 3 , indicating that laser irradiation caused a photothermal effect, which not only ablated the tumor, but also mediated the immune activation of the tumor, together with the inhibition of tumor immune escape caused by the decrease of miR155, showing excellent immunotherapy efficacy, and ultimately achieving the synergy of photothermal therapy, gene therapy and immunotherapy. Surprisingly, after TCM DMC @SNAs+808 nm light treatment, the tumor growth of the mice almost stopped, only slowly increasing to 283 mm3 It exhibited remarkably superior antitumor activity. This result is attributed to TCM. DMC @SNAs actively enrich in tumors, inhibit Tregs, capture more miR155, exhibit a stronger photothermal effect, significantly activate tumor immune responses and inhibit tumor immune escape, thus exerting a better synergistic effect of photothermal therapy, gene therapy and immunotherapy.

[0115] Example 6 TCM DMC Validation of @SNAs in vivo inhibiting the expression of the key signaling molecule STAT3

[0116] TCM DMC @SNAs can capture the overexpressed oncogene miR155 in tumor cells, inhibit the activation of the downstream key signaling molecule STAT3, and reduce the secretion of immunosuppressive factors, thereby promoting increased tumor immunogenicity. Therefore, mouse tumor tissues from Example 5 were collected, sections were prepared, and immunohistochemical staining was performed using a STAT3 antibody. Images of the tumor tissues were acquired using a microscope to determine the TCM. DMC The suppression of STAT3 by @SNAs.

[0117] like Figure 8 As shown in Figure A, STAT3 protein was highly expressed in the PBS group. However, STAT3 expression was reduced in the SNAs group because SNAs can capture miR155, preventing downstream STAT3 expression. Compared to the SNAs group, STAT3 expression did not change significantly in the SNAs + 808nm laser irradiation group, indicating that increased tumor tissue temperature does not affect miR155 capture, and therefore does not affect STAT3 expression. TCM DMC @SNAs can deliver more globular nucleic acids into tumor cells through membrane fusion, leading to a further decrease in STAT3 expression. Similarly, 808nm laser radiation does not affect the pairing of DNA and miR155, and therefore has no effect on STAT3 expression. Figure 8 As shown in Figure B, through semi-quantitative statistics on STAT3, TCM DMC In the @SNAs+808nm illumination group, STAT3 expression was reduced by 12.56-fold, indicating that STAT3 activity was greatly inhibited.

[0118] Example 7 TCM DMC Verification of @SNAs's Inhibition of Treg Formation in Vivo

[0119] In the acidic tumor microenvironment, TCM DMCThe acid-sensitive bonds on @SNAs break, releasing the small molecule DMC, a traditional Chinese medicine, which inhibits the formation of Tregs by suppressing the forkhead box protein (Foxp3), a marker molecule of Tregs. First, tumor tissue from mice in Example 5 was sectioned, incubated with Foxp3 primary antibody, and then immunofluorescence stained with a fluorescent secondary antibody. Finally, images were taken under a fluorescence microscope.

[0120] like Figure 9 As shown in Figure A, compared with the PBS group, the fluorescence of Foxp3 in the SNAs treatment group was significantly reduced, indicating inhibition of Tregs. This is related to the fact that SNAs capture miR155 and weaken STAT3 expression. However, 808nm laser irradiation does not affect miR155 capture, and therefore does not affect this inhibitory effect. As for TCM... DMC @SNAs can release DMC in the tumor microenvironment, further reducing Foxp3. This, along with the effect of SNAs in reducing Tregs by inhibiting STAT3, leads to a significant reduction in the proportion of Tregs. The same NIR illumination does not affect the inhibition of Tregs. Quantitative analysis of Foxp3 fluorescence (see [link to relevant documentation]) further supports this effect. Figure 9 In the middle B group, compared with the PBS group, TCM DMC The @SNAs+808nm laser irradiation group reduced the proportion of Tregs by 15.1 times, demonstrating a remarkable ability to inhibit tumor immune escape.

[0121] Example 8 TCM DMC @SNAs enhance CD8 in the body + Verification of T cell infiltration

[0122] TCM DMC @SNAs can enhance tumor immune escape by inhibiting the overexpression of miR155 and suppressing the downstream signaling molecule STAT3. Meanwhile, TCM... DMC @SNAs release small-molecule DMC from traditional Chinese medicine, reducing the formation of Tregs and thus inhibiting tumor immune escape. Ultimately, by enhancing tumor immunogenicity and inhibiting tumor immune escape, they jointly achieve a shift from a "cold" to a "hot" tumor response, TCM. DMC @SNAs promote the infiltration of a large number of effector T cells, enhancing the efficacy of anti-tumor immunotherapy. Therefore, tumor tissue from mice in Example 5 was obtained, sectioned, and fixed. Next, the tissue sections were incubated with anti-CD8 antibody, followed by incubation with fluorescently labeled secondary antibody in the dark. CD8 immunofluorescence staining was used to assess CD8 levels. + The infiltration of T cells was assessed. Finally, the fluorescence intensity of the samples was observed using a fluorescence microscope.

[0123] like Figure 10 As shown in Figure A, after treatment with SNAs, CD8 in the tumor... + The increase in T cells was attributed to the fact that SNAs can reduce STAT3 expression and decrease the secretion of immunosuppressive cytokines, thereby increasing CD8+ expression. + T cell recruitment. (TCM) DMC In the @SNAs treatment group, CD8 + The proportion of T cells was significantly higher, which was attributed to TCM. DMC @SNAs release DMC, reducing Tregs, which in turn increases CD8. + The number of T cells, and the entry of more SNAs into tumor cells, further increases CD8 by affecting STAT3. + T cell distribution. The SNAs+808nm light group achieved photothermal-mediated ICD, enhanced DC cell maturation, and, together with SNAs attenuating STAT3 expression, recruited a large number of CD8+ cells. + T cells. Even more exciting is the emergence of TCM... DMC Treatment with @SNAs+808nm light resulted in a more intense photothermal effect, leading to further maturation of dendritic cells (DCs), increased SNAs entry into cells, downregulation of STAT3, and the highest proportion of CD8+ T cell infiltration. Quantitative analysis (see [link to analysis]) further demonstrated this effect. Figure 10 (B), TCM DMC @SNAs+808nm illumination group will CD8 + The infiltration of T cells increased by 14.3 times, ultimately resulting in excellent anti-tumor immunotherapy effects.

[0124] Example 9

[0125] (1) Preparation method of gold nanoparticles modified with D1-miR155 and D2-miR155:

[0126] First, a gold nanoparticle solution was prepared. The specific procedure was as follows: 100 μL of HAuCl4·3H2O solution (0.25 mol / L) was added to 100 mL of deionized water, stirred thoroughly, and boiled. Then, 12.5 mL of sodium citrate solution (10 mmol / L) was added, with a molar ratio of tetrachloroauric acid to sodium citrate of 1:5. The solution was heated at 140 °C for 20 min until it turned wine-red. The solution was then cooled to room temperature to obtain a 10 nmol / L gold nanoparticle solution, which was stored at 4 °C for later use.

[0127] Accurately pipette 1 μL of thiol-modified D1-miR155 solution (100 μmol / L) into 100 μL of gold nanoparticle solution (10 nmol / L), and the molar ratio of thiol-modified D1-miR155 to gold nanoparticles is 100:1. Mix well by vortexing and place in a refrigerator at -20°C. After 2 h, thaw at room temperature to obtain D1-miR155-modified gold nanoparticles (D1-Au NPs).

[0128] The preparation method of D2-miR155-modified gold nanoparticles (D2-Au NPs) is similar to the preparation process of the aforementioned D1-miR155-modified gold nanoparticles, except that 1 μL of thiol-modified D2-miR155 solution is used instead of 1 μL of thiol-modified DNA1 solution.

[0129] (2) The preparation method of DSPE-PEG-DMC is as follows:

[0130] Weigh 20 mg of DSPE-PEG 2000 -NH2 (0.01 mmol) and DMC into 5 mL of dichloromethane, then add DMAP, and the molar ratio of DSPE-PEG-NH2, DMC and DMAP is 2:5:3. Transfer the mixed solution to a flask and reflux under continuous stirring. Heat the solution to 50°C and react for 24 h. After the reaction is completed, cool the solution to room temperature, precipitate with excess ether, repeat three times, and finally vacuum dry to obtain DSPE-PEG-DMC.

[0131] (3) TCM DMC @SNAs preparation process:

[0132] First, use the low-osmotic lysis method to obtain cell membranes. Collect a large number of 4T1 tumor cells and add low-osmotic lysis solution and ice bath for 30 min. Then, perform three freeze-thaw cycles to fully lyse the cells. Centrifuge the lysed cell solution at 800 g for 10 min at 4°C, collect the supernatant, and discard the precipitate. Then, perform high-speed centrifugation (15000 g) at 4°C for 30 min. Carefully aspirate the supernatant and freeze-dry the precipitate to obtain tumor cell membranes.

[0133] The traditional Chinese medicine engineered core-shell structure gold nanoparticles were obtained by reusing extrusion method. 1 mg of 4T1 tumor cell membrane, 5 mg of DSPE-PEG-DMC were mixed with D1-Au NPs and D2-Au NPs, and the molar ratio of D1-Au NPs:D2-Au NPs:DSPE-PEG-DMC was 1:1:500. The mixture was fully dissolved by water bath ultrasonic. Subsequently, the mixture was extruded back and forth through the Avanti micro-extruder, and a polycarbonate porous membrane with a pore size of 200 nm was installed in the middle of the extruder. After 50 times of back and forth extrusion, the traditional Chinese medicine engineered core-shell structure gold nanoparticles were finally obtained.

[0134] Example 10

[0135] (1) Preparation method of D1-miR155 and D2-miR155 modified gold nanoparticles:

[0136] First, gold nanoparticle solution was prepared. Specifically, 100 μL of HAuCl4·3H2O solution (0.25 mol / L) was added to 100 mL of deionized water, stirred thoroughly and boiled. Then, 25 mL of sodium citrate solution (10 mmol / L) was added, and the molar ratio of tetrachloroauric acid to sodium citrate was 1:10. The solution was continuously heated at 110°C for 50 min, and the color of the solution finally changed to wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution with a concentration of 10 nmol / L, which was stored at 4°C.

[0137] Accurately take 3 μL of thiol-modified D1-miR155 solution (100 μmol / L) with a pipette and add it to 100 μL of gold nanoparticle solution (10 nmol / L), and the molar ratio of thiol-modified D1-miR155 to gold nanoparticles is 300:1. Mix thoroughly by vortexing and store in a refrigerator at -20°C. After 3 h, thaw at room temperature to obtain D1-miR155 modified gold nanoparticles (D1-Au NPs).

[0138] The preparation method of D2-miR155 modified gold nanoparticles (D2-Au NPs) is similar to the preparation method of the aforementioned D1-miR155 modified gold nanoparticles, except that 3 μL of thiol-modified DNA1 solution is replaced by 3 μL of thiol-modified D2-miR155 solution.

[0139] (2) The preparation method of DSPE-PEG-DMC is as follows:

[0140] Weigh 20 mg of DSPE-PEG 2000-NH2(0.01 mmol) and DMC were added to 5 mL of dichloromethane, followed by the addition of DMAP, the molar ratio of DMC and DMAP to DSPE-PEG-NH2 was 50:1:2, the mixed solution was transferred to a flask, and the solution was heated to 70°C under continuous stirring and cooling reflux, and the reaction was carried out for 12 h. After the reaction was completed, the solution was cooled to room temperature, precipitated with excess ether, repeated three times, and finally dried under vacuum to obtain DSPE-PEG-DMC.

[0141] (3) TCM DMC @Preparation process of SNAs:

[0142] First, the cell membrane was obtained by low osmotic lysis. A large number of 4T1 tumor cells were collected and added with a low osmotic lysis solution, and then ice-bathed for 30 min. Subsequently, three freeze-thaw cycles were performed to fully lyse the cells. The lysed cell solution was centrifuged at 800 g for 10 min at 4°C, and the supernatant was collected and the precipitate was discarded. Subsequently, high-speed centrifugation (15000 g) was performed at 4°C for 30 min. The supernatant was carefully aspirated, and the precipitate was freeze-dried to obtain the tumor cell membrane.

[0143] Then, the traditional Chinese medicine engineered core-shell structure gold nanoparticles were obtained by extrusion method. 5 mg of 4T1 tumor cell membrane, 5 mg of DSPE-PEG-DMC, and D1-Au NPs and D2-Au NPs were mixed, and the molar ratio of D1-Au NPs:D2-Au NPs:DSPE-PEG-DMC was 1:1:2000. The mixture was fully dissolved by water bath ultrasonic. Subsequently, the mixture was extruded back and forth through an Avanti mini-extruder, and a polycarbonate porous membrane with a pore size of 300 nm was installed in the middle of the extruder. The mixture was extruded back and forth for 10 times, and finally the traditional Chinese medicine engineered core-shell structure gold nanoparticles were obtained.

[0144] Example 11

[0145] (1) Preparation method of D1-miR155 and D2-miR155 modified gold nanoparticles:

[0146] First, the gold nanoparticle solution was prepared as follows: 1000 μL of HAuCl4·3H2O solution (0.025 mol / L) was added to 100 mL of deionized water, and stirred and boiled. Then, 20 mL of sodium citrate solution (10 mmol / L) was added, and the molar ratio of tetrachloroauric acid to sodium citrate was 1:8. The solution was continuously heated at 120°C for 40 min, and the color of the solution finally changed to wine red. The solution was cooled to room temperature to obtain a gold nanoparticle solution with a concentration of 10 nmol / L, which was stored at 4°C.

[0147] Precisely pipette 4 μL of thiol-modified D1-miR155 solution (100 μmol / L) into 100 μL of gold nanoparticle solution (10 nmol / L), and the molar ratio of thiol-modified D1-miR155 to gold nanoparticles is 400:1. Mix well by vortexing and place in a refrigerator at -20°C. After 4 h, thaw at room temperature to obtain D1-miR155-modified gold nanoparticles (D1-Au NPs).

[0148] The preparation method of D2-miR155-modified gold nanoparticles (D2-Au NPs) is similar to the preparation of the aforementioned D1-miR155-modified gold nanoparticles, except that 4 μL of thiol-modified D2-miR155 solution is used instead of 4 μL of thiol-modified DNA1 solution.

[0149] (2) The preparation method of DSPE-PEG-DMC is as follows:

[0150] Weigh 20 mg of DSPE-PEG 2000 -NH2(0.01 mmol) and DMC into 5 mL of dichloromethane, then add DMAP, and the molar ratio of DSPE-PEG-NH2, DMC and DMAP is 2:30:10. Transfer the mixed solution to a flask and reflux under continuous stirring. Heat the solution to 60°C and react for 20 h. After the reaction is completed, cool the solution to room temperature, precipitate with excess ether, repeat three times, and finally vacuum dry to obtain DSPE-PEG-DMC.

[0151] (3) TCM DMC @SNAs preparation process:

[0152] First, use the low-osmotic lysis method to obtain cell membranes. Collect a large number of 4T1 tumor cells and add low-osmotic lysis solution and ice bath for 30 min. Then, perform three cycles of freeze-thawing to fully lyse the cells. Centrifuge the lysed cell solution at 800 g for 10 min at 4°C, collect the supernatant, and discard the precipitate. Then, perform high-speed centrifugation (15000 g) at 4°C for 30 min. Carefully aspirate the supernatant and freeze-dry the precipitate to obtain tumor cell membranes.

[0153] The traditional Chinese medicine engineered core-shell structure gold nanoparticles were obtained by reusing extrusion method. 8 mg of 4T1 tumor cell membrane, 5 mg of DSPE-PEG-DMC were mixed with D1-Au NPs and D2-Au NPs, and the molar ratio of D1-Au NPs:D2-Au NPs:DSPE-PEG-DMC was 1:1:5000, and the mixture was fully dissolved by water bath ultrasonic. Subsequently, the mixture was extruded back and forth through the Avanti micro-extruder, and a polycarbonate porous membrane with a pore size of 400 nm was installed in the middle of the extruder. After 20 times of back and forth extrusion, the traditional Chinese medicine engineered core-shell structure gold nanoparticles were finally obtained.

[0154] The above description is only for the best embodiments of the present application, but should not be understood as limiting the claims. The present application is not limited to the above embodiments, and the specific structure allows changes. Any changes made within the scope of the independent claims of the present application are within the scope of the present application.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

Claims

1. A preparation method of a traditional Chinese medicine engineered core-shell structure gold nanoparticle, characterized in that, The method comprises the following steps: The D1-miR155 modified gold nanoparticles, the D2-miR155 modified gold nanoparticles, the polyethylene glycol modified norcantharidin and the tumor cell membrane are uniformly mixed, and are extruded through a liposome extruder to obtain the traditional Chinese medicine engineered core-shell structure gold nanoparticles.

2. The method of claim 1, wherein the method is characterized by, The preparation method of the D1-miR155 modified gold nanoparticles and the D2-miR155 modified gold nanoparticles is as follows: The tetrachloroauric acid is mixed with sodium citrate, and then a reduction reaction is carried out to obtain a gold nanoparticle solution; The thiol-modified D1-miR155 is uniformly mixed with the gold nanoparticle solution, is frozen and then thawed to obtain the D1-miR155 modified gold nanoparticles; The thiol-modified D2-miR155 is uniformly mixed with the gold nanoparticle solution, is frozen and then thawed to obtain the D2-miR155 modified gold nanoparticles.

3. The method of claim 2, wherein the method is characterized by, The nucleotide sequence of the thiol-modified D1-miR155 is shown as SEQ ID NO. 1; The nucleotide sequence of the thiol-modified D2-miR155 is shown as SEQ ID NO.

2.

4. The method of claim 2, wherein the method is characterized by, The molar ratio of the tetrachloroauric acid to the sodium citrate is 1:(5-10); The reaction temperature of the reduction reaction is 110-140 DEG C, and the reaction time is 20-50 min; The particle size of the gold nanoparticles is 10-30 nm; The molar ratio of the thiol-modified D1-miR155 to the gold nanoparticles is (100-600):1; The molar ratio of the thiol-modified D2-miR155 to the gold nanoparticles is (100-600):1; The particle size of the thiol-modified D1-miR155 modified gold nanoparticles is 20-50 nm; The particle size of the thiol-modified D2-miR155 modified gold nanoparticles is 20-50 nm.

5. The method of claim 1, wherein the method is characterized by, The polyethylene glycol modified norcantharidin is prepared through the following process: The amino-containing phospholipid polyethylene glycol and the norcantharidin are added into dichloromethane, and then dimethylaminopyridine is added to carry out a nucleophilic substitution reaction to obtain the polyethylene glycol modified norcantharidin.

6. The method of claim 5, wherein the method is characterized by, The molar ratio of the amino-containing phospholipid polyethylene glycol, the norcantharidin and the dimethylaminopyridine is 2:5-50:1-10; The temperature of the nucleophilic substitution reaction is 50-70 DEG C, and the reaction time is 12-24 h.

7. The method of claim 1, wherein the method is characterized by, The tumor cell is a breast cancer tumor cell.

8. The method of claim 1, wherein the method is characterized by, The molar ratio of the D1-miR155 modified gold nanoparticles, the D2-miR155 modified gold nanoparticles and the polyethylene glycol modified norcantharidin is 1:1:(500-5000); The mass ratio of the polyethylene glycol modified norcantharidin to the tumor cell membrane is 5 mg:(1-10) mg; The pore size of the polycarbonate porous membrane is 100-400 nm; The extrusion frequency is 10-50 times.

9. The engineered core-shell gold nanoparticle of traditional Chinese medicine according to the method of any one of claims 1-8, wherein, The particle size of the core-shell structure gold nanoparticles is 100-200 nm. 10.A use of the traditional Chinese medicine engineered core-shell structure gold nanoparticles prepared by the method of any one of claims 1-8 in the preparation of an antitumor drug.

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