Multi-active targeting tumor nano-drug with multimode imaging function and immune activation effect and preparation method of multi-active targeting tumor nano-drug

Abstract

The invention discloses a multi-active targeting tumor nano-drug with a multimode imaging function and an immune activation effect and a preparation method thereof, and belongs to the technical field of nano-drugs. The preparation method comprises the following steps: coupling inorganic metal-based nano particles / nano drugs with a multimode imaging function or / and targeting active organic biomolecules with specific functions and specific therapeutic drugs through a silane cross-linking agent or a combination of the silane cross-linking agent and a carbodiimide condensing agent; the active targeting type organic-inorganic composite nano-drug aggregate with a multi-mode imaging function and an immune activation effect is prepared. Wherein the inorganic component is a metal-based heterostructure nanoparticle with a multimode imaging function, and the organic component is a bioactive molecule or / and a specific therapeutic drug. The obtained nano-drug aggregate is stable in system, uniform and controllable in size and good in biocompatibility, and has specific targeting, such as specific targeting liver cancer cells.

Description

Technical Field

[0001] The present invention relates to an anti-tumor nano-drug having multiple active targeting (targeting tumor cell microenvironment to control decomposition and release, targeting specific tumor cells, targeting specific organelles, targeting specific biomolecular pathways and targeting specific biomolecular fragments (such as specific gene fragments and their expression)) and multi-modal imaging function and immune activation effect, and its microstructure design and preparation method, belonging to the technical field of anti-tumor drugs and their preparation. Background Art

[0002] The main problems with most current chemotherapy treatments are insufficient drug delivery to the tumor site, cell membrane penetration problems, drug degradation and dissolution problems, and off-target toxicity that leads to adverse side effects. The emerging field of nanomedicine attempts to address these issues by minimizing off-target events using drug molecule delivery vehicles that can protect drugs from degradation and safely and effectively deliver them to tumor lesions (OSTERRIETH JWM, FAIREN-JIMENEZ D. Metal–Organic Framework Composites for Theragnostics and Drug Delivery Applications [J]. Biotechnology Journal, 2021, 16(2): 2000005).

[0003] Typically, nanomedicines are administered intravenously or orally, and before reaching the tumor site, they must cross multiple lines of defense, including vascular barriers, active biomolecules such as proteins, immune systems such as macrophages, excretory systems such as kidneys, filtration and elimination, tumor cell microenvironment, and various cell barriers in the circulatory system. Under normal circumstances, these defense barriers are crucial to the drug resistance of pathogens, but they also prevent nanomedicines from being efficiently transported to specific tumor sites (CHENGZ, LI M, DEY R, et al. Nanomaterials for cancer therapy: current progress and perspectives [J]. Journal of Hematology & Oncology, 2021, 14 (1): 85). At the same time, the anti-tumor effect of nanomedicines must be strictly and comprehensively evaluated, especially the specific killing efficiency of tumor cells, as well as the biocompatibility and biodegradability of nanomedicines, and the physiological toxicity to organisms (LI Y, ZHENG X, CHU Q. Bio-based nanomaterials for cancer therapy [J]. Nano Today, 2021, 38: 101134.). Obviously, successful cancer treatment requires multi-faceted considerations to design nanomaterials and drugs. It is necessary to develop multi-attribute and multifunctional nanomaterials. Single inorganic or organic nanomaterials are difficult to meet these multifunctional requirements due to their single functions. It is essential to combine various organic and inorganic components to develop multifunctional composite organic-inorganic composite nanodrug prodrugs (PARK W, SHIN H, CHOI B, et al. Advanced hybrid nanomaterials for biomedical applications [J]. Progress in Materials Science, 2020, 114: 100686.). These organic-inorganic composite materials maintain the physical and chemical properties of organic and inorganic components, improve the biocompatibility of nanocomposites, and endow nanocomposites with optical, electrical, acoustic, magnetic and thermal properties.In addition, organic-inorganic nanocomposites also exhibit unique properties, such as enhancing the biocompatibility and safety of therapeutic drugs, improving the water solubility of hydrophobic drugs, maintaining colloidal stability in biological environments, and prolonging the circulation time of loaded drugs in the body (CHEN G, QIAN Y, ZHANG H, et al. Advances in cancer theranostics using organic-inorganic hybrid nanotechnology [J]. Applied Materials Today, 2021, 23: 101003.).

[0004] Nanomedicines with targeted delivery function have shown the potential to deliver large amounts of therapeutic drugs into cancer cells to effectively treat drug-resistant tumor cells (ECK W, CRAIG G, SIGDEL A, et al. PEGylated gold nanoparticles conjugated to monoclonal F19 antibodies as targeted labeling agents for human pancreatic carcinoma tissue [J]. ACS nano, 2008, 2 (11): 2263-2272.). Insulin-like growth factor 1 receptor (IGF1R) is highly expressed in tumor and stromal cells (OUBAN A, MURACA P, YEATMAN T, et al. Expression and distribution of insulin-like growth factor-1 receptor in human carcinomas [J]. Human Pathology, 2003, 34 (8): 803-808.). In contrast, its expression in normal cells is relatively low. In addition, since the expression level of IGF1R increases in drug-resistant tumor cell populations, it becomes a marker for drug-resistant tumor cells (PELED N, WYNES MW, IKEDA N, et al. Insulin-like growth factor-1 receptor (IGF-1R) as a biomarker for resistance to the tyrosine kinase inhibitor gefitinib in non-small cell lung cancer [J]. Cellular Oncology, 2013, 36 (4): 277-288.). Therefore, nanomedicine targeting IGF1R is a promising anti-tumor drug delivery nanocarrier that can be used to develop new targeted therapeutic drugs.

[0005] In addition, PD-1 (programmed death protein-1) is an immune checkpoint molecule present on the surface of T cells. PD-L1 (programmed death ligand-1) is a ligand of PD-1. It can significantly upregulate expression in tumor cells under the influence of cytokines secreted by T cells and intrinsic genetic mechanisms. When PD-L1 binds to PD-1, it inhibits T cell activation and reduces its cytokine production, thereby inhibiting the immune response of T cells to tumors and their mediated anti-tumor activity. In addition, due to the specific expression of PD-L1 on tumor cells, it can also be used as a target for active tumor targeting and as a target for nano-drug delivery. For example, PD-L1-related antibodies are conjugated with certain drugs to make them actively targeted and can specifically recognize PD-L1 in tumor cells. At the same time, PD-L1-related antibodies can also activate T cells by inhibiting PD-L1, so they have an immune activation effect. In addition to the above-mentioned targets or target molecules, anti-mesothelin antibodies, mesothelin antibodies, V-ATPase antibodies, etc. are also very promising tumor cell-specific recognition molecules. Summary of the invention

[0006] In order to improve the specificity, targeting and utilization rate of metal-based nanoparticles / nanomedicines, the present invention provides a multi-active targeted tumor nanomedicine with multi-modal imaging function and immune activation effect and a preparation method thereof.

[0007] A multi-active targeted tumor nanomedicine with multi-modal imaging function and immune activation effect, characterized in that inorganic metal-based nanoparticles / nanomedicines with multi-modal imaging function or / and targeted active organic biological molecules with specific functions and special therapeutic drugs are coupled by silane crosslinking agents or silane crosslinking agents combined with carbodiimide condensation agents (for nanoparticle surface modification and molecular activation conjugation coupling) to prepare active targeted organic-inorganic composite nanomedicine aggregates with multi-modal imaging function and immune activation effect. The obtained nanomedicine aggregate system is stable, has uniform and controllable size, good biocompatibility, and has specific targeting, such as specific targeting of liver cancer cells. The inorganic component is a metal-based heterostructured nanoparticle with multi-modal imaging function, and the organic component is a biologically active molecule or / and a special therapeutic drug.

[0008] According to the nanomedicine of the present invention, the metal-based heterostructured nanoparticles with multimodal imaging function are selected from one or more of NiFe, CoFeB, AuPt, AuCu, Au@CoFeB, Ag@NiFe, Fe@Fe3O4, AuPt@CoFeB, FePt@Fe3O4, Ag@CoFeB, Au@CoFeB-WOx and other nanoparticles (patent application numbers 201510395518.4, 202011462174.1).

[0009] According to the nanomedicine of the present invention, the bioactive molecule is an active biomolecule with amino and / or carboxyl groups, including monoclonal antibodies, bispecific antibodies, related functional proteins / peptides, gene drugs, etc., and is further selected from (1) antibodies against substances overexpressed by tumor cells or tissues: such as mesothelin antibodies, mesothelin antibodies, V-ATPase antibodies, ASO-modified ISIS-2503 and AEG35156, and oncogene protein hedgehog pathway antibodies; (2) antibodies against PD-L1 highly expressed in the tumor microenvironment (such as atezolizumab, etc.), antibodies against epithelial cell growth factor receptor (EGFR), antibodies against insulin-like growth factor 1 receptor (IGF1R) (such as tetutumab IBI311) or active substances that can bind thereto (such as insulin-like growth factor 1: IGF1); (3) intravascular targeting of the cell matrix (4) Immune checkpoint inhibitors that enhance anti-tumor immune responses, such as PD-L1 inhibitors (such as atezolizumab, durvalumab, etc.), PD-1 inhibitors (such as nivolumab, etc.), CTLA-4 inhibitors (such as ipilimumab), and bispecific antibodies (PD-1 / CLTA-4 dual antibody KN046, PD-1 / PD-L1 dual antibody IBI318, etc.); (5) Gene drugs, such as one or more of oncolytic DNA hairpin pairs (oHPs), small interfering RNA (siRNA), micro (miRNA), and small activating RNA (saRNA).

[0010] The specific therapeutic drugs are mainly small molecule anticancer drugs or / and small molecule targeted drugs: mainly selected from one or more of ginsenoside (Rg3), paclitaxel (PTX), vincristine (VCR), sea cucumber saponin (HTH), cytotoxic T-lysine (Tox-H), doxorubicin (ADM), gefitinib (Iressa), icotinib hydrochloride (Conmana), proliferating cell nuclear antigen inhibitor (AOH1996), erlotinib hydrochloride (Tarceva), ametinib mesylate (Almonertinib), and chidamide (Epidaza).

[0011] The above-mentioned multi-active targeted tumor nanomedicine with multimodal imaging function and immune activation effect can be an organic-inorganic composite component nanomedicine that actively targets tumor cells (such as liver cancer cells).

[0012] The method for preparing the multi-active targeted tumor nanomedicine with multi-mode imaging function and immune activation effect involves a variety of surface modifiers, cross-linking agents, condensing agents, activators and terminators.

[0013] According to the nanomedicine of the present invention, the surface modifier is selected from at least one of silane coupling agents with amino groups, such as 3-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, etc.; preferably 3-aminopropyltrimethoxysilane (APTMS).

[0014] According to the nanomedicine of the present invention, the crosslinking agent is selected from a crosslinking agent containing two amine-reactive NHS ester groups, such as disuccinimidyl suberate (DSS), di(sulfonic acid-succinimidyl) suberate (BS 3 ), disuccinimidyl glutarate (DSG), polyethylene glycol-bis(succinimidyl succinate) (SS-PEG-SS), ethylene glycol bis(succinimidyl succinate) (EGS), pegylated di(sulfosuccinimidyl) suberic acid (BS(PEG)9) and the like cross-linking agents.

[0015] According to the nanomedicine of the present invention, the condensing agent is selected from at least one of carbodiimide condensing agents, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0016] According to the nanomedicine of the present invention, the activator is selected from one or more of N-hydroxysulfosuccinimide (Sulfo-NHS), N-hydroxysuccinimide (NHS), ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EGS), iodoacetyl-N-hydroxysuccinimide, N-cyclohexyl-N'-(2-morpholinethyl)carbodiimide methyl p-toluenesulfonate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleimide, N-ethylmaleimide, p-formylbenzoic acid-N-hydroxysuccinimide ester, and succinic anhydride (dihydro-2,5-furandione).

[0017] According to the nanomedicine of the present invention, the reaction terminator is selected from one or more of 2-mercaptoethanol, glycine, alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, methionine, serine, threonine, aspartic acid, cysteine, histidine, arginine, tyrosine, lysine, asparagine, glutamic acid, proline, glutamine, selenocysteine, tris(hydroxymethyl)aminomethane hydrochloride) and the like.

[0018] The active targeted tumor nanomedicine described in the present invention mainly includes two preparation methods: one method (method one) is characterized in that the organic and inorganic components are surface modified and conjugated by the silane coupling method. The other method (method two) is characterized in that the organic and inorganic components are conjugated by the silane coupling method and the carbodiimide amidation method.

[0019] For method 1, in one specific embodiment, the method comprising the following steps is used for preparation:

[0020] (1) preparing or purchasing metal-based nanoparticles with multi-mode imaging function; dispersing the obtained nanoparticles into an organic solvent solution (such as anhydrous toluene), adding a surface modifier to obtain a mixed solution; stirring the mixed solution, and after the stirring is completed, transferring the mixed solution to a centrifuge tube for centrifugation, washing, removing the supernatant, and retaining the precipitate;

[0021] (2) dissolving the precipitate from (1) in dimethyl sulfoxide (DMSO) and adding a cross-linking agent thereto; stirring or incubating, transferring the mixture into a centrifuge tube and centrifuging the mixture in a centrifuge, removing the supernatant and retaining the precipitate;

[0022] (3) dissolving the precipitate from (2) in PBS and re-dispersing it, and washing it by centrifugation for 1 to 4 times; freeze-drying the final product, weighing it, and re-dispersing it in PBS to form a modified nanoparticle solution;

[0023] (4) adding an organic bioactive molecule with an amino group or an organic bioactive molecule with an amino group and a specific therapeutic drug as needed to the modified nanoparticle solution obtained in (3), mixing on a shaker, and incubating after the stirring is completed; taking the product and gently shaking it, centrifuging to remove the supernatant, and dissolving it in PBS to obtain the final product.

[0024] The amount of surfactant added in step (1) is preferably 0.5 to 20% of the mass of the metal-based nanoparticles.

[0025] The amount of cross-linking agent added in step (2) is preferably 0.2 to 2 times the mass of the nanoparticles.

[0026] In step (4), the amount of organic bioactive molecules added is 0.01 to 20% of the amount of the cross-linking agent. The specific therapeutic drug added as needed has a mass of 0.2 to 3 times that of the nanoparticles.

[0027] As a preferred embodiment, the nanoparticles in step (1) are metal-based compound nanoparticles composed mainly of metals and their metal alloys and all elements in the periodic table, and the size of the nanoparticles is below 10 nm, preferably below 5 nm, and more preferably below 3 nm; the preparation method adopts a hydrothermal method, an electrodeposition method, a magnetron sputtering method, a microfluidic synthesis method, and a large-scale continuous flow microfluidic preparation method; more preferably, the microfluidic synthesis method and the large-scale continuous flow microfluidic preparation method are used.

[0028] Specifically, the microfluidics method is a continuous flow reaction process constructed using a micrometer scale (sub-millimeter) to control the mixing of reactants, reaction nucleation, nanoparticle or drug growth and growth termination processes in the synthesis of nanomaterials or drugs within a reaction volume of μL to pL or even smaller. This device and method refer to the patent application number 201510395518.4.

[0029] The large-scale continuous preparation microfluidic method is a device and method for continuous microfluidics to synthesize low-cost metal nanoparticles in large quantities. The nanoparticles obtained by this device have regular morphology and uniform and controllable size. This device is referred to in the patent application number 202011462174.1, which will not be repeated here.

[0030] As a preferred embodiment, the stirring temperature in step (1) is preferably 15-70°C, the stirring time is preferably 12-36 hours, the centrifugal speed is preferably 10000rpm-13000rpm, the centrifugal time is preferably 3-15 minutes, and the washing number is preferably 2-4 times.

[0031] As a preferred embodiment, step (2) uses magnetic stirring, and the stirring or incubation temperature is preferably 5 to 36° C., and the time is preferably 1 to 5 hours.

[0032] As a preferred embodiment, the concentration of the modified nanoparticles after adding PBS in step (3) is preferably 0.01 to 3 mg / mL.

[0033] As a preferred embodiment, the shaking mixing time in step (4) is preferably 24 to 48 hours, and the incubation time is preferably 1 to 10 hours.

[0034] As a preferred embodiment, the centrifugal speed in step (4) is preferably 10000 rpm to 13000 rpm, the centrifugal time is preferably 3 to 15 minutes, the number of washes is preferably 2 to 4 times, and the drying method is preferably programmed freeze drying.

[0035] Further, after the stirring and incubation in step (4) are completed, a terminator solution is added to terminate the reaction.

[0036] In step (4), specific therapeutic drugs are added as needed. If the drug itself has a primary amine, it can be added directly. Otherwise, it needs to be activated and modified to have a primary amine group (preferably using the surfactant mentioned above) before being added.

[0037] For method 2, in one specific embodiment, the method comprising the following steps is used for preparation:

[0038] (1) preparing or purchasing metal-based nanoparticles with multi-mode imaging function; dispersing the obtained nanoparticles in an organic solvent (such as anhydrous toluene), adding a surface modifier to obtain a mixed solution, and stirring; after the stirring is completed, transferring the mixed solution to a centrifuge tube, centrifuging, washing, and finally obtaining modified nanoparticles; and then dispersing or dissolving the mixed solution in PBS to obtain an activated nanoparticle solution;

[0039] (2) preparing a solution of a bioactive molecule with a carboxyl group or / and a specific therapeutic drug with a carboxyl group using a PBS buffer;

[0040] (3) preparing a condensing agent solution with MES; taking a certain amount of the condensing agent solution and the solution of the biologically active molecule with a carboxyl group or / and the specific therapeutic drug with a carboxyl group obtained in (2);

[0041] (4) preparing an activator solution with PBS buffer; adding a certain amount of the activator solution to the mixed solution obtained in (3) to form a solution A;

[0042] (5) mixing the activated nanoparticle solution obtained in (1) with the solution A obtained in (4) as solution B, and incubating at low temperature overnight;

[0043] (6) Dissolve the terminator in PBS to prepare a terminator solution, and add the terminator solution to solution B obtained in (5) to terminate the reaction to obtain the product.

[0044] The amount of the surface modifier added in step (1) is preferably 0.5 to 20% of the mass of the metal-based nanoparticles;

[0045] The size and preparation method of the nanoparticles in step (1) are as described in method 1 and will not be repeated here.

[0046] In step (1), the stirring temperature is preferably 15 to 70° C., the stirring time is preferably 12 to 36 hours, the centrifugal speed is preferably 10,000 rpm to 13,000 rpm, the centrifugal time is preferably 3 to 15 minutes, and the washing number is preferably 2 to 4 times.

[0047] The concentration of the activated nanoparticle solution in step (1) is preferably 0.01 to 3 mg / mL.

[0048] In step (2), the concentration of the organic biomolecule solution is preferably 0.01 to 10 mg / mL, and the concentration of the specific therapeutic drug is preferably 0.05 to 3 mg / mL.

[0049] As a preferred embodiment, the pH value of MES in step (3) is preferably 7.5 to 7.5, the concentration of the condensing agent solution is preferably 0.05 to 1 mg / mL, and when the solutions are mixed, the amount of the added bioactive molecule substance is 0.01 to 50% of the amount of the condensing agent substance, and the amount of the added special therapeutic drug substance is 0.01 to 1 times the amount of the condensing agent substance.

[0050] As a preferred embodiment, the concentration of the activator solution in step (4) is preferably 0.05-1.5 mg / mL, and the amount of the activator substance in solution A is 1-50 times the amount of the bioactive molecule and / or the specific therapeutic drug substance.

[0051] As a preferred embodiment, the mixing temperature in step (4) is preferably 0 to 10° C., and the mixing time is preferably 10 to 40 min.

[0052] As a preferred embodiment, the incubation temperature in step (5) is preferably 0-15°C, more preferably 0-5°C, and the mass of the nanoparticles in solution B is 5-80 times the mass of the activator.

[0053] As a preferred solution, the concentration of the reaction terminator solution in step (6) is preferably 0.02 to 0.5 mg / mL, and the added volume is preferably 5 to 70% of the volume of the entire system.

[0054] The silane coupling method and carbodiimide amidation method provided by the present invention can couple small molecule drugs, functional active biological molecules, etc. with nanoparticles with multimodal imaging functions, thereby constructing active targeted tumor nanomedicines with multimodal imaging functions and immune activation effects, which can improve the specificity, targeting and utilization rate of metal-based nanoparticles / nanomedicines, improve drug internalization efficiency, and activate immune effects. In addition, its multimodal imaging function can be used to study drug metabolism processes and mechanisms of action online. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1The coupling roadmap for modifying nanoparticles and coupling bioactive molecules by silane coupling method. It is mainly divided into the following steps: when the easily oxidized metal elements in the nanoparticles are treated with the necessary aqueous solution (such as PBS buffer) during the alloying or hybridization process, many -OH groups are easily generated on their surface, and the functional groups on the surface modifier are connected to them (such as the methoxy group on APTMS condenses with -OH to achieve connection), and the amino functional groups are modified to the surface of the nanoparticles. (Xiaoxiong Z, Junmei W, Yujun S, et al. Synthesis of Nanomedicines by Nanohybrids Conjugating Ginsenosides with Auto-Targeting and Enhanced MRI Contrast for Liver Cancer Therapy. [J]. Drug Development & Industrial Pharmacy, 2018: 1-30) Then, the NHS ester group on one side of the cross-linking agent is used to form a stable amide bond with the amino group on the nanoparticle through activation, nucleophilic substitution and hydrolysis. Finally, the NHS ester group on the other side of the cross-linker is used to connect with the biologically active molecules with amino groups to achieve the coupling of nanoparticles and active biological molecules.

[0056] Figure 2 The activated coupling route of the carbodiimide amidation method is shown in Figure 1. First, the carboxyl group of the bioactive molecule reacts with a condensing agent (such as EDC) to produce a highly reactive unstable intermediate O-acylisourea active ester; then, an activating agent (such as Sulfo-NHS) reacts with the intermediate to form a stable activated ester; finally, the formed activated ester reacts with the amino group in the modified nanoparticle to form a stable amide bond.

[0057] Figure 3 FT-IR spectra of the nanoparticles and nanomedicines in Examples 1 and 2 of the present invention. Au@CoFeB was coupled with ATZ monoclonal antibody and IGF1 and then detected by FT-IR. The results are shown in the figure. Amino acids form peptide chains through amide bonds, so amide bonds are generally the characteristic peaks in proteins, especially band I (1680-1630cm -1 , C=O) and band II (1640-1550cm -1 According to the literature, the main characteristic peaks of ATZ monoclonal antibody and IGF1 are at 1550cm -1 (amide II) and 1660 cm -1 (Amide I) near (Compare Figure 3 Curve 1 in (a) (nanoparticles coupled with ATZ) and Figure 3Curve 3 in (b) (nanoparticles coupled with IGF1) and Figure 3 The two curves in (a) and Figure 3 Curve 4 in b (pure inorganic Au@CoFeB nanoparticles). Figure 3 In (a), 1 can clearly see 1547nm (amide I) and 1636cm -1 A new peak appeared at , proving that ATZ was successfully conjugated to the nanoparticles. Figure 3 In (b), 3, we can clearly see that 1547cm -1 (amide I) and 1643 cm -1 A clear new peak appeared at , proving that IGF1 was successfully conjugated to the nanoparticles. Figure 3 From Figure (a) and Figure (b), we can see that both Au@CoFeB-ATZ and Au@CoFeB-IGF1 have obvious peaks at 1547nm, and the peak of Au@CoFeB-ATZ is stronger here, which may be due to the different protein structures of the two coupled. This further illustrates that this method has a good coupling effect on both active molecules, and this method has universal conjugation coupling for biological molecules of different structures.

[0058] Figure 4 The absorbance (a) detected by ultraviolet spectrophotometer in Example 1 of the present invention and the standard curve (b) of protein concentration determination by Lowry method were used. Au@CoFeB (5 in a) and Au@CoFeB-ATZ (6 in a) were detected by ultraviolet spectrophotometer, and Au@CoFeB-ATZ showed obvious red shift, which may be due to the enlargement of particles caused by protein coupling. In addition, we used the standard curve of protein concentration determination by Lowry method (7 in b) and the protein content of the supernatant after coupling to calculate that the conjugated coupling encapsulation efficiency of Au@CoFeB-ATZ can reach more than 93%, and the drug loading rate can be as high as 1.5%. The simulation of the relationship curve between its concentration and absorbance coefficient (8 in b) found that, except for some fluctuations at low concentrations, it is basically a linear relationship. Based on this, the drug loading rate can be increased to more than 50% as needed, but the conjugated coupling encapsulation efficiency will decrease to about 80%.

[0059] Figure 5 LSPR images of Example 1 and Example 3 of the present invention taken under a dark field microscope. Figure 5 a in the middle is Au@CoFeB nanomedicine, Figure 5 b is the Au@CoFeB-ATZ nanomedicine prepared in Example 1. Figure 5 Figure c is Au@CoFeB-ATZ (2) prepared in Example 3. The Au@CoFeB-ATZ prepared by the two methods are relatively uniform, but Figure 5The nano drug group of Example 1 shown in b is smaller in size, which is conducive to transportation.

[0060] Figure 6 is a dark field / fluorescence dual-mode image of Example 2 of the present invention. Figure 6 The middle a is the dark field image of Au@CoFeB. Figure 6 Figure b is a dark field image of Au@CoFeB-IGF1. Figure 6 c is the fluorescence image of Au@CoFeB-IGF1, Figure 6 The dark field / fluorescence dual-mode composite image of Au@CoFeB-IGF1 is shown in Figure d. Due to the unique surface plasmon resonance optical scattering characteristics of Au@CoFeB particles, it can be seen under dark field observation that although there is a small amount of aggregation, its dispersion is still sufficiently uniform. In the study, we used FITC fluorescein isothiocyanate to label IGF1 so that it can be captured by a fluorescence microscope, making it easier to observe its coupling sites using surface plasmon resonance optics under a dark field microscope and fluorescence dual optical modes under a fluorescence microscope. Figure 6 Figures 6b and 6c show the dark field and fluorescence images of Au@CoFeB and its conjugated IGF1 in the same field of view. Figure 6 The middle d is a combined dark field and fluorescence image of the same field of view. After coupling with IGF1, the nanodrugs will agglomerate and the particle size will increase. Due to the different effects of coupling on the local dielectric properties of drug aggregates and the interaction between particles, the uniformly dispersed nanoparticles before coupling appear yellow-green ( Figure 6 In a), after coupling, the individual drug particles in the aggregate show colorful scattered light colors ( Figure 6 b) and the aggregation phenomenon is obvious, the size of the nano drug aggregates becomes larger, but the particle size analysis of electron microscopy and laser scattering is still at the submicron scale, and can still be delivered through the normal blood transport system. At the same time, after FITC fluorescence staining of IGF1, it can be observed under green fluorescence. Figure 6 The even distribution of IGF1 is shown in c, and the dark field image of the same area ( Figure 6 By comparing and integrating in (d), we can clearly see the coupling position of IGF1 in the nanomedicine. It is evenly distributed in the shell layer of the inorganic core (metal-based nanoparticles) surrounding the nanomedicine, and the coupling effect is very perfect.

[0061] Figure 7This is the cytotoxicity test of Example 1 and Example 2 of the present invention. The survival rates of normal liver cells QSG7701 (a)-(c) and liver cancer cells HCCLM3 (d)-(f) treated with different concentrations of Au@CoFeB (9 in a, 12 in b, 15 in 7c, 18 in 7d, 21 in 7e, 24 in 7f), Au@CoFeB-ATZ (10 in a, 13 in b, 16 in c, 19 in d, 22 in e, 25 in f) and Au@CoFeB-IGF1 (11 in a, 14 in b, 17 in c, 20 in d, 23 in e, 26 in f) at 24 hours, 48 ​​hours and 72 hours were compared. The toxicity to both cells increased with the increase of time and concentration. After unifying the coordinate axes, it can be seen that the toxicity of the three drugs to normal liver cells is slightly lower than that to human highly metastatic liver cancer cells, especially after 24 hours, which indicates that within a certain period of time, this group of drugs is more toxic to HCCLM3 and more friendly to normal cells.

[0062] Interestingly, the toxicity trends of the three drugs are similar in normal liver cells, but different in liver cancer cells. At 24h, Au@CoFeB-IGF1 showed higher toxicity than the other two drugs at a lower concentration, although this difference gradually weakened with the increase of time and concentration. This may be due to the high expression of IGF1 receptors in liver cancer cells, which makes it easier to bind to the IGF1-loaded drugs in the short term.

[0063] In addition, at 48h, the toxicity of both targeted drugs was stronger to tumor cells at low concentrations. This may be because ATZ monoclonal antibody gradually began to work over time. However, since the amount of ATZ substance used is lower than IGF1, there are relatively fewer binding sites, and it takes a longer time to achieve a better effect.

[0064] Figure 8 The survival trend fitting curves and half inhibition concentrations of cells in Example 1 and Example 2 of the present invention are compared. The survival trend fitting curves and half inhibition concentrations of normal liver cells QSG7701 (a)-(c) and liver cancer cells HCCLM3 (d)-(f) treated with different concentrations of Au@CoFeB (27 in a, 30 in b, 33 in c, 36 in d, 39 in e, 42 in f), Au@CoFeB-ATZ (28 in a, 31 in b, 34 in c, 37 in d, 40 in e, 43 in f) and Au@CoFeB-IGF1 (29 in a, 32 in b, 35 in c, 38 in d, 41 in e, 44 in f) at 24 hours, 48 ​​hours and 72 hours. The vertical dotted line in the (af) figure is the intersection of the survival fitting curve and the 50% cell survival rate (the horizontal black curve in the af figure), which describes the drug concentration of the corresponding color when the cell survival rate is 50%, that is, the IC50 value.

[0065] At 24 h (a and d), Au@CoFeB and Au@CoFeB-ATZ failed to reach half lethality in normal hepatocytes (a), but their IC50 values ​​in hepatoma cell HCCLM3 (d) were 243.5 μg / mL (black vertical dotted line in d) and 409.6 μg / mL (red vertical dotted line in d), respectively, indicating that the toxicity of Au@CoFeB and Au@CoFeB-ATZ to LM3 was significantly higher than that to normal hepatocytes; Au@CoFe The IC50 value of B-IGF1 in normal liver cells is 495μg / mL (blue vertical dashed line in a), and in HCCLM3 is 23.32μg / mL (blue vertical dashed line in d), which also shows its low toxicity to normal liver cells. In addition, its IC50 value in HCCLM3 is much lower than that of the other two drugs, indicating that Au@CoFeB-IGF1 can achieve a 50% mortality rate in HCCLM3 at a lower dose (about 23.32μg / mL). This may be due to the high expression of IGF1 receptors in liver cancer cells, which makes it easier to bind to the drug loaded with IGF1 in the short term, and when coupled with the same mass of ATZ monoclonal antibody, the amount of IGF1 substance is higher than ATZ, so there are relatively more binding sites, which can achieve a better effect.

[0066] At 48h (b and e), the IC50 values ​​of Au@CoFeB in normal liver cells QSG7701 (b) and liver cancer cells HCCLM3 (e) were 251.6μg / mL (black vertical dotted line in b) and 374.7μg / mL (black vertical dotted line in e), respectively, indicating that the toxicity of Au@CoFeB to normal cells is slightly higher than that to tumor cells, which may be caused by drug accumulation, but may also be affected by objective factors such as errors and cell states; the IC50 values ​​of Au@CoFeB-ATZ in normal liver cells QSG7701 (b) and liver cancer cells HCCLM3 (e) were 401.6μg / mL (red vertical dotted line in b) and 131.5μg / mL (red vertical dotted line in e), the toxicity to HCCLM3 is much greater than that to normal liver cells and higher than that to Au@CoFeB, indicating that Au@CoFeB-ATZ has a targeting effect on liver cancer cells at 48h; the IC50 values ​​of Au@CoFeB-IGF1 in normal liver cells QSG7701 (b) and liver cancer cells HCCLM3 (e) are 230.3μg / mL (blue vertical dotted line in b) and 115.2μg / mL (blue vertical dotted line in e), respectively. The toxicity to HCCLM3 is similar to that of Au@CoFeB-ATZ and much greater than that to normal liver cells and higher than that to Au@CoFeB, indicating that Au@CoFeB-IGF1 has a targeting effect on liver cancer cells at 48h.

[0067] At 72 h (c and f), the IC50 values ​​of Au@CoFeB in normal liver cells QSG7701 (c) and liver cancer cells HCCLM3 (f) were 162.7 μg / mL ( Figure 8 The IC50 values ​​of Au@CoFeB-ATZ in normal liver cells QSG7701 (c) and liver cancer cells HCCLM3 (f) were 335.6 μg / mL (red vertical dotted line in c) and 343.3 μg / mL (red vertical dotted line in f), respectively. The toxicity to liver cancer cells was similar to that of normal liver cells, but lower than that of Au@CoFeB, indicating that Au@CoFeB-ATZ has a higher toxicity to normal cells than to tumor cells. eB-ATZ reduced the toxicity of Au@CoFeB to liver cells and increased its toxicity to liver cancer cells, indicating that it has a targeting effect; the IC50 values ​​of Au@CoFeB-IGF1 in normal liver cells QSG7701 (c) and liver cancer cells HCCLM3 (f) were 228.3 μg / mL (blue vertical dotted line in c) and 186.2 μg / mL (blue vertical dotted line in f), respectively. The toxicity to liver cancer cells HCCLM3 was greater than that of normal liver cells, and higher than Au@CoFeB and Au@CoFeB-ATZ, indicating that Au@CoFeB-IGF1 has a targeting effect on liver cancer cells within 72 h.

[0068] comprehensive Figure 7 and Figure 8 , Example 1 and Example 2 are both targeted to the liver cancer cell HCCLM3. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with examples, but the present invention is not limited to the following examples.

[0070] Example 1

[0071] Preparation of targeted nanodrug Au@CoFeB-ATZ (method 1):

[0072] Au@CoFeB metal-based nanoparticles with multi-mode imaging function were prepared by a sequential microfluidic method. First, Au@CoFeB was prepared by programmed microfluidics. Then, 50 mL of 2 mmol / mL CoFeB nanoparticles were used as seed particles, and a certain amount of sodium borohydride was added to construct a reductant-stabilized CoFeB seed solution with a concentration of about 50 mM. 50 mL of 10 mM chloroauric acid solution was used as a gold source. The core-shell structured Au@CoFeB nanoparticles were prepared by programmed microfluidic technology at a flow channel inner diameter of 0.5 mm, a flow rate of 0.5 mL / min, and a mixing reaction temperature of 120 °C (reference preparation literature: Weiwei Zhang, Xiaoxiong Zhao, Yuan Yuan, Fenglin Miao, Wengang Li, Shaoxia Ji, Xing Huang, Xinhua Chen, Tianan Jiang, David A. Weitz, and Yujun Song, Microfluidic Synthesis of Multi-mode Au@CoFeB-Rg3 Nanomedicines and Their Cytotoxicity and Anti-Tumor Effects. Chem. Mater. 2020, 32, 5044-5056). Au@CoFeB nanoparticles were dispersed in anhydrous toluene solution and 1% APTMS was added. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, it was transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the mixture was repeated 3 times to obtain activated nanoparticles. DSS was then used to activate the amino groups on the surface of the nanoparticles. The activated nanoparticles were dissolved in DMSO, and an equal amount of DSS was weighed and added to the nanoparticles; at room temperature, the mixture solution was magnetically stirred or incubated for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the mixture was repeated 3 times to retain the precipitate. The final product was dried and quantified under vacuum conditions and stored in a desiccator for later use; it was dissolved in PBS to form a 0.1 mg / mL modified nanoparticle solution, 10 mL of this solution was taken, about 2 mg of atezolizumab (ATZ) was added, and the mixture was shaken at low temperature for 24 hours. After stirring, it was incubated at low temperature for about 1 hour, and 1 mL of 0.15 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au@CoFeB-ATZ.

[0073] Example 2

[0074] Preparation of targeted nanodrug Au@CoFeB-IGF1 (method 1):

[0075] The method in Example 1 was used to prepare Au@CoFeB metal-based nanoparticles with multi-mode imaging function. The Au@CoFeB nanoparticles were dispersed in an anhydrous toluene solution, and 1.5% APTMS was added. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 20 hours. After the stirring was completed, it was transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the process was repeated twice to finally obtain activated nanoparticles. The activated nanoparticles were dissolved in DMSO, and DSS with a mass of 0.8 times that of the nanoparticles was weighed and added thereto. At room temperature, the mixture solution was magnetically stirred or incubated for 1.5 hours, transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the process was repeated twice to retain the precipitate. The final product was dried and quantified under vacuum conditions and stored in a desiccator for later use. Dissolve in PBS to form a 1 mg / mL modified nanoparticle solution, take 10 mL of this solution, add about 10 mg of IGF1, shake and mix at low temperature for 24 hours, incubate at low temperature for about 1 hour after stirring, add 1 mL of 0.10 mg / mL lysine solution to terminate the reaction, and obtain the final product Au@CoFeB-IGF1; the rest is the same as Example 1.

[0076] Example 3

[0077] Preparation of targeted nanomedicine Au@CoFeB-ATZ(2) (Method 2):

[0078] The method in Example 1 was used to prepare Au@CoFeB metal-based nanoparticles with multi-mode imaging function. The Au@CoFeB nanoparticles were dispersed in an anhydrous toluene solution, and 1% of the surfactant 3-aminopropyltrimethoxysilane (APTMS) was added and stirred for 24 hours. After stirring, the mixture was transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, and the supernatant was removed. The mixture was repeated 3 times to finally obtain aminosilane-modified nanoparticles. The mixture was resuspended in PBS to form a 0.1mg / mL activated Au@CoFeB solution for use.

[0079] Take 2 mg of the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and dissolve it in 10 mL MES buffer to make a 0.2 mg / mL EDC solution; weigh 8 mg of the activating agent N-hydroxysulfosuccinimide (Sulfo-NHS) and dissolve it in 10 mL MES buffer to make a 0.8 mg / mL Sulfo-NHS solution. First, 20 μL of EDC solution was added to 200 μL (concentration was 10 mg / mL, containing 2 mg ATZ) of ATZ solution to form an ATZ-EDC mixed solution. Then, 20 μL of Sulfo-NHS solution was added to the ATZ-EDC mixed solution. 760 μL of MES buffer was added and mixed at room temperature for 15 min. 10 mL of the above 0.1 mg / mL activated Au@CoFeB nanoparticle solution was added and incubated at low temperature overnight. 1 mL of glycine (concentration was 0.11 mg / mL) was added to the above reaction to terminate the reaction and obtain the final product Au@CoFeB-ATZ (2).

[0080] Example 4

[0081] Preparation of targeted nanodrug Au@CoFeB-Rg3-ATZ (method 1):

[0082] Au@CoFeB metal-based nanoparticles with multi-mode imaging function were prepared by the method in Example 1. The Au@CoFeB nanoparticles were dispersed in an anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as a mixed solution 1. At the same time, Rg3 with the same mass as the metal nanoparticles was dispersed in an anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as a mixed solution 2. Mixed solutions 1 and 2 were placed on a magnetic stirrer respectively and magnetically stirred at room temperature for 24 hours. After the stirring was completed, the mixed solutions 1 and 2 were transferred to a centrifuge tube and centrifuged at 11000r for 4 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain aminosilane-activated nanoparticles and activated Rg3, respectively. The activated nanoparticles were dissolved in DMSO, and DSS equal to the amount of the nanoparticles was weighed and added thereto. At room temperature, the mixture solution was magnetically stirred or incubated for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the process was repeated 3 times. The precipitate was retained to obtain modified nanoparticles. The modified nanoparticles and activated Rg3 were centrifuged separately and resuspended with PBS to form a 1 mg / mL solution respectively. 10 mL of the modified nanoparticle solution was mixed with 5 mL of the activated Rg3 solution, and about 5 mg of atezolizumab (ATZ) was added. The mixture was mixed on a shaker at low temperature for 24 hours. After the stirring was completed, the mixture was incubated at low temperature for about 1 hour, and 1 mL of 0.15 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au@CoFeB-ATZ.

[0083] Example 5

[0084] Preparation of targeted nanodrug AuPt@CoFeB-ATZ (Method 2):

[0085] The AuPt@CoFeB nanoparticles were redispersed in anhydrous toluene solution and 1% APTMS was added. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After the stirring was completed, it was transferred to a centrifuge tube for centrifugation and washing to finally obtain aminosilane-activated nanoparticles. Dissolved in PBS to form a 0.1mg / mL activated nanoparticle solution. ATZ was prepared with MES buffer to a solution of about 50mmol / mL. 2mg ATZ monoclonal antibody was added to a 0.2mg / mL EDC solution prepared with 20uLMES buffer. On this basis, 0.8mg / mL Sulfo-NHS solution prepared with 20uLMES buffer was added and quantified to 1mL, mixed at 4℃ for 20min to form solution A. Solution A and 10mL activated nanoparticle solution were mixed as solution B and incubated at 4℃ overnight. After the incubation, 1mL 0.10mg / mL lysine solution was added to solution B to terminate the reaction, and the final product AuPt@CoFeB-ATZ was obtained.

[0086] Example 6

[0087] Preparation of targeted nanomedicine Au@FePt-mesothelin antibody (method 1):

[0088] Au@FePt nanoparticles were dispersed in anhydrous toluene solution and 2% APTMS was added. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, it was transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the reaction was repeated 3 times to finally obtain activated nanoparticles, and then the DSS activation reaction was used to activate the amino groups on the surface of the nanoparticles. The aminosilane-modified nanoparticles were dissolved in DMSO, and an equal amount of DSS as the nanoparticles was weighed and added thereto. At room temperature, the mixture solution was magnetically stirred or incubated for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the reaction was repeated 3 times to retain the precipitate. The final product was dried and quantified under vacuum conditions and stored in a desiccator for later use. Dissolved in PBS to form a 0.5mg / mL modified nanoparticle solution, 10mL of this solution was taken, and about 0.7*10 -4 mmol of mesothelin antibody was mixed on a shaking table at low temperature for 24 hours. After stirring, it was incubated at low temperature for about 1 hour. 0.5 mL of 0.11 mg / mL glycine solution was added to terminate the reaction to obtain the final product Au@FePt-mesothelin antibody.

[0089] Example 7

[0090] Preparation of targeted nanodrug Ag@NiFe-Rg3-IBI311 (Method 1):

[0091] Ag@NiFe nanoparticles and Rg3 were dispersed in anhydrous toluene solution, and 1.5% APTMS was added. Both were placed on a magnetic stirrer and magnetically stirred at room temperature for 20 hours. After stirring, they were transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated Rg3. The activated nanoparticles were dissolved in DMSO, and 0.8 times the mass of DSS of the nanoparticles was added thereto, magnetically stirred at room temperature for 1.5 hours, transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the mixture was repeated twice to retain the precipitate. The precipitate was vacuum freeze-dried and quantified for later use. The modified nanoparticles and activated Rg3 were dissolved in PBS to form a 1 mg / mL solution, 10 mL of the modified nanoparticle solution and 5 mL of activated Rg3 were mixed, and about 2.8*10 -4 mmol of IBI311 was mixed on a shaking table at low temperature for 24 h. After stirring, it was incubated at low temperature for about 1 h. 1 mL of 0.10 mg / mL lysine solution was added to terminate the reaction to obtain the final product Ag@NiFe–Rg3-IBI311.

[0092] Example 8

[0093] Preparation of targeted nanodrug Au@CoFeB-WOx-PTX-IGF1 (method 1):

[0094] Au@CoFeB-WOx onion-shaped multilayered metal-based nanoparticles were prepared by an efficient program microfluidic method. First, Au@CoFeB was prepared by using an efficient program microfluidic method. Then, 50 mL of Au@CoFeB nanoparticles with a concentration of 2 mmol / mL were used as seed particles, and a certain amount of sodium borohydride was added to construct a reducing agent-stabilized Au@CoFeB seed solution with a concentration of about 50 mM. 50 mL of ammonium tungstate ((NH4)6W7O 24·6H2O) aqueous solution as the tungsten source, and the onion-structured Au@CoFeB-WOx nanoparticles were prepared using a programmed microfluidic process (reference preparation method literature: Qiong Wu, Ran Liu, Fenglin Miao, Guangxing Zhang, Wengang Li, Yujun Song, Multimodal ultra-small CoFe-WOx nanohybrids synthesized by a pilot microfluidic system. Chemical Engineering Journal, 2023, 452 (2), 139355) at a flow channel inner diameter of 0.5 mm, a flow rate of 0.5 mL / min, and a mixing reaction temperature of 120 ° C. Au@CoFeB-WOx nanoparticles and PTX were dispersed in anhydrous toluene solution, and 2% APTMS was added to each solution. Both were placed on a magnetic stirrer and magnetically stirred at room temperature for 20 hours. After stirring, they were transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and repeated 3 times to finally obtain activated nanoparticles and activated PTX. The activated nanoparticles were dissolved in DMSO, and 0.8 times the mass of the nanoparticles was weighed and added to the DSS. The mixture was stirred magnetically at room temperature for 1.5 hours, transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, and the supernatant was removed. The mixture was repeated twice, and the precipitate was retained. The precipitate was vacuum freeze-dried and quantified for later use. The modified nanoparticles and activated PTX were dissolved in PBS to form a 1 mg / mL solution. 10 mL of the modified nanoparticle solution and 5 mL of activated PTX were mixed, and about 2*10 -4 mmol IGF1 was mixed on a shaking table at low temperature for 24 h. After stirring, it was incubated at low temperature for about 1 h. 1 mL of 0.10 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au@CoFeB-WOx-PTX-IGF1.

[0095] Example 9

[0096] Preparation of targeted nanodrug Au@CoFeB-WOx-Rg3-ATZ (method 1):

[0097] Au@CoFeB-WOx metal-based nanoparticles with multi-mode imaging function were prepared by the method in Example 8. The Au@CoFeB-WOx nanoparticles were dispersed in an anhydrous toluene solution, and 1% of the surface modifier APTMS was added as a mixed solution 1. At the same time, Rg3 with the same mass as the metal nanoparticles was dispersed in an anhydrous toluene solution, and 1% of the surface modifier APTMS was added as a mixed solution 2. Mixed solutions 1 and 2 were placed on a magnetic stirrer respectively and magnetically stirred at room temperature for 24 hours. After the stirring was completed, the mixed solutions 1 and 2 were transferred to a centrifuge tube and centrifuged at 11000r for 4 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain aminosilane-activated nanoparticles and activated Rg3, respectively. The activated nanoparticles were dissolved in DMSO, and DSS equal to the amount of the nanoparticles was weighed and added thereto. At room temperature, the mixture solution was magnetically stirred or incubated for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the process was repeated 3 times. The precipitate was retained to obtain modified nanoparticles. The modified nanoparticles and activated Rg3 were centrifuged separately and resuspended with PBS to form a 1 mg / mL solution respectively. 10 mL of the modified nanoparticle solution was mixed with 5 mL of the activated Rg3 solution, and about 5 mg of atezolizumab (ATZ) was added. The mixture was mixed on a shaker at low temperature for 24 hours. After the stirring was completed, the mixture was incubated at low temperature for about 1 hour, and 1 mL of 0.15 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au@CoFeB-ATZ.

[0098] Example 10

[0099] Preparation of targeted nanodrug Au@CoFeB-WOx-HTH-IGF1 (method 1):

[0100] Au@CoFeB-WOx nanoparticles were dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 1. At the same time, an equal amount of HTH was dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 2. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After stirring, the mixed solutions 1 and 2 were transferred to a centrifuge tube and centrifuged at 11000r for 4 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated HTH. The activated nanoparticles were dissolved in DMSO, and an equal amount of DSS as the nanoparticles was weighed and added thereto. The mixture was stirred magnetically at room temperature for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, and the supernatant was removed. The mixture was repeated 3 times, and the precipitate was retained to obtain modified nanoparticles. The modified nanoparticles and activated HTH were centrifuged separately and resuspended in PBS to form a 1 mg / mL solution. 10 mL of the activated nanoparticle solution was mixed with 5 mL of the modified HTH solution, and about 1.4*10 -4mmol of IGF1, shake and mix at low temperature for 24 hours, incubate at low temperature for about 1 hour after stirring, add 1mL of 0.15mg / mL lysine solution to terminate the reaction, and the final product Au@CoFeB-WOx-HTH-IGF1 was obtained.

[0101] Embodiment 11

[0102] Preparation of targeted nanodrug Au4Pt1@CoFeB-WOx-(ATZ-IGF1) (Method 2):

[0103] 1 / 5 of the raw material chloroauric acid (HAuCl4) in the step of preparing Au@CoFeB in the preparation of Au@CoFeB in Example 8 was replaced with chloroauric acid H2PtCl6 to prepare Au4Pt1@CoFeB as seed nanoparticles. The rest of the process was the same as Example 10 to prepare Au4Pt1@CoFeB-WOx nanoparticles, which were dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added, placed on a magnetic stirrer, and magnetically stirred at room temperature for 24 hours. After stirring, transfer to a centrifuge tube and centrifuge at 11000r for 4 minutes, remove the supernatant, and repeat twice to obtain activated nanoparticles. Dissolve in PBS to form a 0.1mg / mL activated Au4Pt1@CoFeB-WOx nanoparticle solution for use. Take 2mg EDC and dissolve it in 10mL MES buffer to form an EDC solution; weigh 8mg NHS and dissolve it in MES buffer to form an NHS solution. First, take 20 μL of LEDC solution and add it to a concentration of about 1.4×10 -4 mmol of IGF1 and 1.4×10 -5 mmol ATZ solution to form an IGF1-ATZ-EDC mixed solution, then 20 μL NHS solution was added to the IGF1-ATZ-EDC mixed solution, a certain amount of MES buffer was added to make the system 1.5 mL, mixed at room temperature for 15 min, 10 mL of modified Au4Pt1@CoFeB-WOx nanodrug solution was added, incubated at low temperature overnight, 1.5 mL of glycine (concentration of 0.11 mg / mL) was added to the above reaction to terminate the reaction, and the final product Au4Pt1@CoFeB-WOx-(ATZ-IGF1) was obtained.

[0104] Example 12

[0105] Preparation of targeted nanodrug Au4Pt1@CoFeB-WOx-Iressa-KN046) (Method 1):

[0106] Au4Pt1@CoFeB-WOx nanoparticles were prepared with reference to Example 11, dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 1. At the same time, an equal mass of Iressa was dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 2. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After the stirring was completed, the mixed solutions 1 and 2 were transferred to a centrifuge tube and centrifuged at 11000r for 4 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated Iressa. The activated nanoparticles were dissolved in DMSO, and an equal amount of DSS as the nanoparticles was weighed and added thereto. The mixture was stirred magnetically at room temperature for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, and the supernatant was removed. The mixture was repeated 3 times, and the precipitate was retained to obtain modified nanoparticles. The modified nanoparticles and activated Iressa were centrifuged separately and resuspended in PBS to form a 1 mg / mL solution. 10 mL of the activated nanoparticle solution was mixed with 5 mL of the modified Iressa solution, and about 0.7*10 -5 mmol of KN046 was mixed on a shaker at low temperature for 24 h. After stirring, it was incubated at low temperature for about 1 h. 1 mL of 0.15 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au4Pt1@CoFeB-WOx-Iressa-KN046.

[0107] Embodiment 13

[0108] Preparation of targeted nanodrug Au4Pt1@CoFeB-WOx-Iressa-siRNA (Method 1):

[0109] Au4Pt1@CoFeB-WOx nanoparticles were prepared with reference to Example 11, dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 1. At the same time, an equal mass of Iressa was dispersed in anhydrous toluene solution, and 1.5% of the surface modifier APTMS was added as mixed solution 2. The mixed solution was placed on a magnetic stirrer and magnetically stirred at room temperature for 24 hours. After the stirring was completed, the mixed solutions 1 and 2 were transferred to a centrifuge tube and centrifuged at 11000r for 4 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated Iressa. The activated nanoparticles were dissolved in DMSO, and an equal amount of DSS as the nanoparticles was weighed and added thereto. The mixture was stirred magnetically at room temperature for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, and the supernatant was removed. The mixture was repeated 3 times, and the precipitate was retained to obtain modified nanoparticles. The modified nanoparticles and activated Iressa were centrifuged separately and resuspended in PBS to form a 1 mg / mL solution. 10 mL of the activated nanoparticle solution was mixed with 5 mL of the modified Iressa solution, and about 1.4*10-4 mmol of siRNA was mixed on a shaker at low temperature for 24 h. After stirring, it was incubated at low temperature for about 1 h. 1 mL of 0.15 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au4Pt1@CoFeB-WOx-Iressa-siRNA.

[0110] Embodiment 14

[0111] Preparation of targeted nanodrug Au@CoFeB-ToxH-siRNA (Method 1):

[0112] The method in Example 1 was used to prepare Au@CoFeB metal-based nanoparticles with multi-mode imaging function. The Au@CoFeB nanoparticles and ToxH were dispersed in anhydrous toluene solution, and 1.5% APTMS was added. The two were placed on a magnetic stirrer and magnetically stirred at room temperature for 20 hours. After the stirring was completed, they were transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated ToxH. The activated nanoparticles were dissolved in DMSO, and DSS with a mass of 0.8 times that of the nanoparticles was weighed and added thereto, magnetically stirred at room temperature for 1.5 hours, transferred to a centrifuge tube and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the mixture was repeated twice to retain the precipitate. The precipitate was vacuum freeze-dried and quantified for later use. The modified nanoparticles and activated ToxH were dissolved in PBS to form a 1 mg / mL solution, 10 mL of the modified nanoparticle solution and 5 mL of activated ToxH were mixed, and about 2.8*10 -4 mmol siRNA was mixed on a shaker at low temperature for 24 h. After stirring, it was incubated at low temperature for about 1 h. 1 mL 0.10 mg / mL lysine solution was added to terminate the reaction to obtain the final product Au@CoFeB-ToxH-siRNA.

[0113] Embodiment 15

[0114] Preparation of targeted nanodrug Au@CoFeB-ToxH-(IGF1-AOH1996) (Method 2):

[0115] The method in Example 1 was used to prepare Au@CoFeB metal-based nanoparticles with multi-mode imaging function. Au@CoFeB nanoparticles and ToxH were dispersed in anhydrous toluene solution, and 1.5% APTMS was added. Both were placed on magnetic stirring and magnetically stirred at room temperature for 20 hours. After stirring, they were transferred to centrifuge tubes and centrifuged at 11000r for 10 minutes, the supernatant was removed, and the mixture was repeated twice to finally obtain activated nanoparticles and activated ToxH. The activated nanoparticles were dissolved in DMSO, and DSS equal to the nanoparticles was weighed and added thereto. At room temperature, the mixture solution was magnetically stirred or incubated for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, the supernatant was removed, and the mixture was repeated 3 times to retain the precipitate. The final product was dried and quantified under vacuum conditions and stored in a desiccator for later use. The modified nanoparticles and activated ToxH were dissolved in PBS to form 1 mg / mL modified solutions. Take 2 mg of EDC and dissolve it in 10 mL of MES buffer to form an EDC solution; weigh 8 mg of NHS and dissolve it in MES buffer to form an NHS solution. First, take 20 μL of EDC solution and add it to a volume of about 1.4×10 -5 mmol of AOH1996 and 1.4×10 - 4 mmol IGF1 solution to form an AOH1996-IGF1-EDC mixed solution, then 20 μL NHS solution was added to the AOH1996-IGF1-EDC mixed solution, MES buffer was added to make the whole system 1 mL, mixed at room temperature for 15 min, 10 mL of modified Au@CoFeB nanoparticles and 5 mL of activated ToxH solution were added, incubated at low temperature overnight, 1 mL of glycine (concentration of 0.11 mg / mL) was added to the above reaction to terminate the reaction, and the final product Au@CoFeB-ToxH-(IGF1-AOH1996) was obtained.

[0116] Example 16

[0117] Preparation of targeted nanodrug Au4Pt1@CoFeB-WOx-oHPs-IGF1 (method 1):

[0118] Refer to the Au4Pt1@CoFeB-WOx nanoparticles prepared in Example 11, disperse them in anhydrous toluene solution, add 1.5% by mass of surface modifier APTMS, and stir magnetically at room temperature for 24 hours. After stirring, transfer to a centrifuge tube and centrifuge at 11000r for 4 minutes, remove the supernatant, repeat twice, and finally obtain activated nanoparticles. The activated nanoparticles are dissolved in DMSO, and an equal amount of DSS as the nanoparticles is weighed and added thereto, magnetically stirred at room temperature for 2 hours, transferred to a centrifuge tube and centrifuged at 12000r for 5 minutes, remove the supernatant, repeat 3 times, retain the precipitate, obtain modified nanoparticles, resuspend with PBS to form a 1 mg / mL solution, take 10mL of the activated nanoparticle solution, add about 0.7*10 -4 mmol of IGF1 and 0.7*10 -4 mmol oHPs, shake and mix at low temperature for 24 h, incubate at low temperature for about 1 h after stirring, add 1 mL 0.15 mg / mL lysine solution to terminate the reaction, and the final product Au4Pt1@CoFeB-WOx-oHPs-IGF1 was obtained.

[0119] Embodiment 17

[0120] Preparation of targeted nanodrug Au3Cu1@CoFeB-WOx-(ATZ-IGF1) (Method 2):

[0121] 1 / 4 of the raw material chloroauric acid (HAuCl4) in the step of preparing Au@CoFeB in Example 8 was replaced with copper nitrate Cu(NO3)2 to prepare Au3Cu1@CoFeB as seed nanoparticles. The rest of the process was the same as Example 8 to prepare Au3Cu1@CoFeB-WOx nanoparticles. The nanoparticles were dispersed in anhydrous toluene solution, and 1% of the surface modifier APTMS was added, placed on a magnetic stirrer, and magnetically stirred at room temperature for 24 hours. After stirring, transfer to a centrifuge tube and centrifuge at 11000r for 4 minutes, remove the supernatant, repeat 3 times, and obtain activated nanoparticles, which were dissolved in PBS to form a 0.1mg / mL activated Au3Cu1@CoFeB-WOx nanoparticle solution for use. Take 2mg EDC and dissolve it in 10mMES buffer to form an EDC solution; weigh 8mg NHS and dissolve it in MES buffer to form an NHS solution. First, take 20μL of EDC solution and add it to a solution containing about 1.4×10 - 4 mmol of IGF1 and 1.4×10 -5mmol ATZ solution to form an IGF1-ATZ-EDC mixed solution, then 20 μL NHS solution was added to the IGF1-ATZ-EDC mixed solution, a certain amount of MES buffer was added to make the system 1.5 mL, mixed at room temperature for 15 min, 10 mL of active Au3Cu1@CoFeB-WOx nanodrug solution was added, incubated at low temperature overnight, 1.5 mL of glycine (concentration of 0.11 mg / mL) was added to the above reaction to terminate the reaction, and the final product Au3Cu1@CoFeB-WOx-(ATZ-IGF1) was obtained.

Claims

1. A multi-active targeted tumor nanomedicine with multimodal imaging function and immune activation effect, characterized in that: Actively targeted organic-inorganic composite nanodrug aggregates with multimodal imaging functions and immune activation effects are prepared by coupling inorganic metal-based nanoparticles / nanomedicines with multimodal imaging functions or / and targeted active organic biomolecules and special therapeutic drugs with specific functions through silane crosslinkers or silane crosslinkers combined with carbodiimide condensation agents (for nanoparticle surface modification and molecular activation conjugation crosslinking); The silane crosslinking agent is selected from silane coupling agents with amino groups, and is further selected from at least one of silane coupling agents such as 3-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, and N-(β-aminoethyl)-N-(β-aminoethyl)-γ-aminopropyltriethoxysilane; preferably 3-aminopropyltrimethoxysilane (APTMS).

2. A multi-active targeted tumor nanomedicine with multimodal imaging function and immune activation effect according to claim 1, characterized in that: The metal-based heterostructured nanoparticles with multi-mode imaging function are selected from NiFe, CoFeB, AuPt, AuCu, Au@CoFeB, Ag@NiFe, Fe@Fe3O4, AuPt@CoFeB, FePt@Fe3O4, Ag@CoFeB, and Au@CoFeB-WOx nanoparticles. The biologically active molecules, including monoclonal antibodies, bispecific antibodies, related functional proteins / peptides, and gene drugs, are further selected from (1) antibodies against substances overexpressed by tumor cells or tissues, such as mesothelin antibodies, mesothelin antibodies, V-ATPase antibodies, ASO-modified ISIS-2503 and AEG35156, and oncogene protein hedgehog pathway antibodies; (2) antibodies against PD-L1 highly expressed in the tumor microenvironment (such as atezolizumab, etc.), antibodies against epithelial cell growth factor receptor (EGFR), antibodies against insulin-like growth factor 1 receptor (IGF1R) (such as tetulumab IBI311) R) or active substances that can bind to it (such as insulin-like growth factor 1: IGF1); (3) antibodies targeting vascular endothelial growth factor (VEGFR) and cyclooxygenase 2 (COX-2) of the cell matrix, antibodies against matrix metalloproteinases (MMPs) and urokinase receptor (uPAR), plectin-1 and mucin (MUC1); (4) immune checkpoint inhibitors that enhance anti-tumor immune responses, such as PD-L1 inhibitors (such as atezolizumab, durvalumab, etc.), PD-1 inhibitors (such as nivolumab, etc.), CTLA-4 inhibitors (such as ipilimumab), bispecific antibodies (PD-1 / CLTA-4 bispecific antibody KN046, PD-1 / PD-L1 bispecific antibody IBI318, etc.); (5) gene drugs, such as one or more of oncolytic DNA hairpin pairs (oHPs), small interfering RNA (siRNA), micro RNA (miRNA), small activating RNA (saRNA), etc.; The specific therapeutic drugs are mainly small molecule anticancer drugs or / and small molecule targeted drugs: mainly selected from one or more of ginsenoside (Rg3), paclitaxel (PTX), vincristine (VCR), sea cucumber saponin (HTH), cytotoxic T-lysine (Tox-H), doxorubicin (ADM), gefitinib (Iressa), icotinib hydrochloride (Conmana), proliferating cell nuclear antigen inhibitor (AOH1996), erlotinib hydrochloride (Tarceva), ametinib mesylate (Almonertinib), and chidamide (Epidaza).

3. A multi-active targeted tumor nanomedicine with multimodal imaging function and immune activation effect according to claim 1 or 2, characterized in that: It is an organic-inorganic composite nanomedicine that actively targets cells, especially tumor cells (such as liver cancer cells).

4. A method for preparing a multi-active targeted tumor nanomedicine with multimodal imaging function and immune activation effect according to claim 1 or 2, characterized in that: There are mainly two preparation methods, one of which is method 1, which uses silane coupling method to carry out surface modification and conjugation coupling of organic and inorganic components; the other method is method 2, which uses silane coupling method and carbodiimide amidation method to carry out conjugation coupling of organic and inorganic components; For method 1, a method comprising the following steps is used for preparation: (1) preparing or purchasing metal-based nanoparticles with multi-mode imaging function; dispersing the obtained nanoparticles into an organic solvent solution (such as anhydrous toluene), adding a surface modifier to obtain a mixed solution; stirring the mixed solution, and after the stirring is completed, transferring the mixed solution to a centrifuge tube for centrifugation, washing, removing the supernatant, and retaining the precipitate; (2) dissolving the precipitate from (1) in dimethyl sulfoxide (DMSO) and adding a cross-linking agent thereto; After stirring or incubating, transfer the mixture into a centrifuge tube and centrifuge it, remove the supernatant and retain the precipitate; (3) dissolving the precipitate from (2) in PBS and re-dispersing it, and washing it by centrifugation for 1 to 4 times; freeze-drying the final product, weighing it, and re-dispersing it in PBS to form a modified nanoparticle solution; (4) adding an organic bioactive molecule with an amino group or an organic bioactive molecule with an amino group and a specific therapeutic drug as needed to the modified nanoparticle solution obtained in (3), mixing on a shaker, and incubating after the stirring is completed; taking the product and gently shaking it, centrifuging it to remove the supernatant, and dissolving it in PBS to obtain the final product; For method 2, a method comprising the following steps is used for preparation: (1) preparing or purchasing metal-based nanoparticles with multi-mode imaging function; dispersing the obtained nanoparticles in an organic solvent, adding a surface modifier to obtain a mixed solution, and stirring; after the stirring is completed, transferring the solution to a centrifuge tube for centrifugation, washing, and finally obtaining modified nanoparticles; Redisperse or dissolve in PBS to obtain activated nanoparticle solution; (2) preparing a solution of a bioactive molecule with a carboxyl group or / and a specific therapeutic drug with a carboxyl group using a PBS buffer; (3) preparing a condensing agent solution with MES; taking a certain amount of the condensing agent solution and the solution of the biologically active molecule with a carboxyl group or / and the specific therapeutic drug with a carboxyl group obtained in (2); (4) preparing an activator solution with PBS buffer; adding a certain amount of the activator solution to the mixed solution obtained in (3) to form a solution A; (5) mixing the activated nanoparticle solution obtained in (1) with the solution A obtained in (4) as solution B, and incubating at low temperature overnight; (6) Dissolve the terminator in PBS to prepare a terminator solution, and add the terminator solution to the solution B obtained in (5) to terminate the reaction and obtain the product; The surface modifier is selected from silane coupling agents with amino groups; wherein the cross-linking agent is selected from a cross-linking agent containing two amine-reactive NHS ester groups; The condensing agent is selected from carbodiimide condensing agents.

5. The method according to claim 4, characterized in that The surface modifier is at least one selected from silane coupling agents such as 3-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane, and N-(β-aminoethyl)-N-(β-aminoethyl)-γ-aminopropyltriethoxysilane; preferably 3-aminopropyltrimethoxysilane (APTMS); The crosslinking agent is selected from disuccinimidyl suberate (DSS), di(sulfonic acid-succinimidyl) suberate (BS 3 ), disuccinimidyl glutarate (DSG), polyethylene glycol-bis(succinimidyl succinate) (SS-PEG-SS), ethylene glycol bis(succinimidyl succinate) (EGS), pegylated di(sulfosuccinimidyl) suberic acid (BS(PEG)9) and the like cross-linking agents; The condensing agent is selected from at least one of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); The activator is selected from one or more of N-hydroxysulfosuccinimide (Sulfo-NHS), N-hydroxysuccinimide (NHS), ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) (EGS), iodoacetyl-N-hydroxysuccinimide, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, maleimide, N-ethylmaleimide, p-formylbenzoic acid-N-hydroxysuccinimide ester, and succinic anhydride (dihydro-2,5-furandione); The reaction terminator is selected from one or more of 2-mercaptoethanol, glycine, tris(hydroxymethyl)aminomethane hydrochloride, histidine and lysine.

6. The method according to claim 4, characterized in that In method 1 and method 2, the size of the nanoparticles is less than 10 nm, preferably less than 5 nm, and more preferably less than 3 nm.

7. The method according to claim 4, characterized in that For method 1, the amount of surfactant added in step (1) is preferably 0.5 to 20% of the mass of the metal-based nanoparticles; The amount of crosslinking agent added in step (2) is preferably 0.2 to 2 times the mass of the nanoparticles; In step (4), the amount of organic bioactive molecules added is 0.01 to 20% of the amount of the cross-linking agent, and the specific therapeutic drug added as needed has a mass of 0.2 to 3 times that of the nanoparticles; In step (4), a specific therapeutic drug is added as needed. If the drug itself has a primary amine, it can be added directly. Otherwise, it needs to be activated and modified to have a primary amine group before being added (preferably, the modification can be performed using the surfactant described above).

8. The method according to claim 4, characterized in that For method 1, the stirring temperature in step (1) is preferably 15 to 70° C., the stirring time is preferably 12 to 36 hours, the centrifugal speed is preferably 10000 rpm to 13000 rpm, the centrifugal time is preferably 3 to 15 minutes, and the number of washing times is preferably 2 to 4 times; In step (2), magnetic stirring is used, and the stirring or incubation temperature is preferably 5 to 36° C., and the time is preferably 1 to 5 hours. In step (3), the concentration of the modified nanoparticles after adding PBS is preferably 0.01 to 3 mg / mL; The shaking mixing time in step (4) is preferably 24 to 48 hours, and the incubation time is preferably 1 to 10 hours; In step (4), the centrifugal speed is preferably 10000 rpm to 13000 rpm, the centrifugal time is preferably 3 to 15 minutes, the number of washing times is preferably 2 to 4 times, and the drying method is preferably programmed freeze drying; After the stirring and incubation in step (4) are completed, a terminator solution is added to terminate the reaction.

9. The method according to claim 4, characterized in that For method 2, the amount of the surface modifier added in step (1) is preferably 0.5 to 20% of the mass of the metal-based nanoparticles; In step (1), the stirring temperature is preferably 15 to 70° C., the stirring time is preferably 12 to 36 hours, the centrifugal speed is preferably 10000 rpm to 13000 rpm, the centrifugal time is preferably 3 to 15 minutes, and the washing number is preferably 2 to 4 times; The concentration of the activated nanoparticle solution in step (1) is preferably 0.01 to 3 mg / mL; In step (2), the concentration of the organic biomolecule solution is preferably 0.01 to 10 mg / mL, and the concentration of the specific therapeutic drug is preferably 0.05 to 3 mg / mL; In step (3), the pH value of MES is preferably 7.5 to 7.5, the concentration of the condensing agent solution is preferably 0.05 to 1 mg / mL, and when the solutions are mixed, the amount of the added bioactive molecule substance is 0.01 to 50% of the amount of the condensing agent substance, and the amount of the added specific therapeutic drug substance is 0.01 to 1 times the amount of the condensing agent substance; The concentration of the activator solution in step (4) is preferably 0.05 to 1.5 mg / mL, and the amount of the activator substance in solution A is 1 to 50 times the amount of the bioactive molecule or / and the specific therapeutic drug substance; The mixing temperature in step (4) is preferably 0 to 10° C., and the mixing time is preferably 10 to 40 min; The incubation temperature in step (5) is preferably 0-15°C, more preferably 0-5°C, and the mass of the nanoparticles in solution B is 5-80 times the mass of the activator. The concentration of the reaction terminator solution in step (6) is preferably 0.02 to 0.5 mg / mL, and the added volume is preferably 5 to 70% of the volume of the entire system.

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