A nano-targeting chimera, and a preparation method and application thereof

Abstract

This invention provides a nano-targeted chimera, its preparation method, and its application. It is obtained by in-situ polymerization of acrylamide serum albumin, polymeric monomers, cross-linking agents, and PD-L1 inhibitors in an organic solvent or water. The polymeric monomers include one or both of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride. This invention forms a cross-linked polymer network through in-situ polymerization of acrylamide serum albumin. This network integrates functional polymers that target the target protein. These polymers bind to the target protein and, without relying on lysosomal targeting receptors on the cell surface, are taken up by the cell via non-specific endocytosis and efficiently transported to lysosomes, thereby achieving lysosomal degradation of the target protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nano-targeted chimera, its preparation method, and its application. Background Technology

[0002] Trageted protein degradation (TPD) technology is developing rapidly. Utilizing the ubiquitin-proteasome system (UPS), lysosomes, and autophagy, it selectively degrades target proteins (POIs) through intracellular protein cycling mechanisms, demonstrating great potential in the treatment of various refractory diseases, including cancer. Proteolytic targeted chimeras (PROTACs) and molecular glue technology can achieve intracellular protein clearance. Furthermore, lysosome-targeted chimeras (LYTACs) are being developed to target non-cytoplasmic degradation sites. These chimeras bind to membrane-associated POIs and guide them to lysosomes, thereby completing the TPD process.

[0003] LYTACs, as bifunctional molecules, contain two key binding domains: one end is an oligosaccharide structure that binds to lysosomal targeting receptors on the cell surface, and the other end is an antibody, peptide, or small molecule that targets the target protein. Through this structure, lysosomal targeting receptors can transport the POI complex captured by the antibody or small molecule to the lysosome, thereby initiating the degradation of disease-related extracellular or membrane proteins.

[0004] However, the dependence of LYTACs on cell surface lysosomal targeting receptors makes them cell type specific, which greatly limits their application in cancers with significant intertumor heterogeneity. Furthermore, some lysosomal targeting receptors (such as integrin αvβ3) are closely related to tumor progression, and their overactivation may reduce therapeutic efficacy and induce side effects. Tumor cells themselves possess the ability to take up extracellular substances and transport them to lysosomes for degradation through various non-specific endocytic pathways (such as clathrin-mediated endocytosis and macropinocytosis), which provides a theoretical possibility for bypassing lysosomal targeting receptor dependence.

[0005] Therefore, existing LYTACs rely on limited and occupied lysosomal targeting receptors on the cell surface, resulting in low degradation efficiency and instability. Drugs prepared using existing LYTACs exhibit insufficient accumulation and limited penetration in solid tumors. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to improve the degradation efficiency of the target protein.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of the present invention provides a nano-targeted chimera, which is obtained by mixing acrylamide serum albumin, a polymeric monomer, a crosslinking agent and a PD-L1 inhibitor in an organic solvent or water under conditions of in-situ initiated polymerization; the polymeric monomer includes one or two of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride.

[0009] Beneficial Effects: This invention achieves a nano-targeting chimera by in-situ polymerization of acrylamide serum albumin to form a cross-linked polymer network, which integrates functional monomers (PD-L1 inhibitors) targeting the target protein. The nano-targeting chimera of this invention can bind to the target protein and, without relying on lysosomal targeting receptors on the cell surface, is taken up by the cell via non-specific endocytosis and efficiently transported to lysosomes, thereby achieving lysosomal degradation of the target protein.

[0010] Preferably, the molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 10:0 to 0:10.

[0011] Preferably, the molar ratio of acrylamide serum albumin, polymeric monomer, and crosslinking agent is 1:1000:25.

[0012] Preferably, the molar ratio of acrylamide serum albumin to PD-L1 inhibitor is 1:20 to 1:30.

[0013] Preferably, the crosslinking agent is N,N'-methylenebisacrylamide.

[0014] Preferably, the PD-L1 inhibitor is BMS-8.

[0015] Preferably, the initiator for the in-situ polymerization reaction is ammonium persulfate, and the catalyst for the in-situ polymerization reaction is tetramethylethylenediamine.

[0016] Preferably, the molar ratio of ammonium persulfate to acrylated serum albumin is 1:300.

[0017] Preferably, acrylamide serum albumin is prepared from bovine serum albumin and N-acryloyloxysuccinimide in solution.

[0018] Preferably, the molar ratio of bovine serum albumin to N-acryloyloxysuccinimide is 1:3 to 1:10.

[0019] A second aspect of the present invention provides a method for preparing the above-mentioned nano-targeted chimera, comprising the following steps: Bovine serum albumin was dissolved in PBS, and N was added to it. Acryloxysuccinimide dimethyl sulfoxide solution was reacted to yield acrylylated serum albumin; Acrylated serum albumin, aqueous solution of polymeric monomer, crosslinking agent dimethyl sulfoxide solution and PD-L1 inhibitor dimethyl sulfoxide solution are mixed, and ammonium persulfate and tetramethylethylenediamine are added to initiate an in-situ polymerization reaction to obtain the product.

[0020] Preferably, the PD-L1 inhibitor dimethyl sulfoxide solution is an Ac-BMS solution.

[0021] Preferably, the Ac-BMS solution is obtained by the following method: BMS-8 is dissolved in dichloromethane, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and dimethylaminopyridine are added under ice bath conditions and stirred, followed by the addition of N-(3-aminopropyl)methacrylamide; the solvent is removed by vacuum concentration to obtain a crude product, which is purified by C18 reversed-phase column chromatography to obtain Ac-BMS; Ac-BMS is dissolved in dimethyl sulfoxide to obtain an Ac-BMS solution.

[0022] The third aspect of this invention provides the application of the above-mentioned nano-targeting chimera in drugs for treating solid tumors.

[0023] Beneficial effects: The nano-targeting chimera of the present invention binds to the tumor cell membrane protein PD-L1 and, in the absence of cell surface lysosomal targeting receptors, is taken up by cells via non-specific endocytosis and efficiently transported to lysosomes for degradation of the target protein.

[0024] Preferably, the solid tumor is breast cancer.

[0025] The fourth aspect of the present invention provides the application of the above-mentioned nano-targeting chimera in the hybrid drug delivery for the treatment of breast cancer, wherein the nano-targeting chimera is coupled to the surface of engineered bacteria to obtain a hybrid drug delivery.

[0026] Preferably, the nano-targeting chimera is coupled to the surface of engineered bacteria via COOH-TK-COOH to obtain a hybrid drug delivery system.

[0027] Preferably, COOH-TK-COOH is dissolved in water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added for activation. Then, a nano-targeting chimera is added for reaction. After that, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added again for activation. After activation, engineered bacteria in the logarithmic growth phase are added for reaction to obtain a hybrid drug delivery system.

[0028] Preferably, the molar ratio of COOH-TK-COOH to the nano-targeted chimera is 5:1.

[0029] Preferably, the engineered bacterial cells include engineered Escherichia coli cells.

[0030] Beneficial Effects: Bacteria, as natural tumor-targeting carriers, possess unique advantages in cancer treatment. Their inherent ability to colonize hypoxic tumor regions and their strong deep penetration into tumor tissue, combined with their immunogenic pathogen-associated molecular patterns (PAMPs), make them effective immune adjuvants. This invention combines engineered bacteria with a nano-targeting chimera; the complementary advantages of both enhance cancer treatment efficacy and reduce side effects.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The degradation mechanism of this invention utilizes a nano-targeting chimera that targets receptors via lysosomes on the cell surface without relying on limited lysosomes. It achieves total tumor drug delivery (TPD) by leveraging and regulating the inherent lysosomal targeting and endocytosis properties of nanomaterials. The design is simple, versatile, and eliminates the need to synthesize complex lysosomal targeting ligands (such as oligosaccharides), simplifying the material design and preparation process. This invention yields a hybrid drug delivery system that combines the active tumor-targeting penetration capability of engineered bacteria with ROS-responsive linkage, enabling spatiotemporally controlled drug release and synergistically enhancing anti-tumor immunotherapeutic efficacy. Attached Figure Description

[0032] Figure 1 This is a flowchart illustrating the preparation of the nano-targeted chimera and hybrid drug delivery system, and a schematic diagram illustrating its anti-tumor effects, as described in the embodiments of the present invention. Figure 2 The particle size diagram (a) and potential diagram (b) of the nano-targeted chimeras in Examples 1-7 of the present invention are shown. Figure 3 These are transmission electron microscopy (TEM) images of the nano-targeted chimeras in Examples 1-7 of this invention, with a scale bar of 50 nm; where ag represents Examples 1-7 respectively. Figure 4 The results of the toxicity experiment of the nano-targeted chimera on 4T1 cells in Example 3 of this invention; Figure 5 This is a WB image of the degradation of PD-L1 by the nano-targeted chimera in Example 3 of the present invention; Figure 6 This is an immunofluorescence image of the degradation of PD-L1 by the nano-targeted chimera in Example 3 of the present invention; Figure 7 The image shows a transmission electron microscope (TEM) image of the naked bacteria and EcN@BLTC prepared in Example 8 of this invention. The scale bar is 1 μm. Figure 8 This figure shows the results of the successful construction of the EcN@BLTC hybrid drug based on the FRET effect verification of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0035] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0036] Example 1 This embodiment provides a nano-targeted chimera and its preparation method, based on... Figure 1 As shown, the specific steps include: (1) Preparation of Acrylylated Serum Albumin Bovine serum albumin (BSA) was dissolved in PBS (pH 7.4) to prepare a 5 mg / mL solution. 10.24 μl of a 50 mg / mL solution of N-acryloyloxysuccinimide (NAS) in dimethyl sulfoxide was added, with a BSA to N-acryloyloxysuccinimide molar ratio of 1:5. The mixture was stirred at room temperature for 2 h. The reaction solution was transferred to a dialysis bag (MWCO: 2000 Da) and dialyzed against PBS for 24 hours to remove unreacted NAS, yielding an acrylated serum albumin solution.

[0037] (2) Preparation of nano-targeted chimeras (BLTC) Acrylamide-modified serum albumin solution was placed in a 1.5 mL centrifuge tube, and 15.39 μl of acrylamide aqueous solution (70 mg / mL), 18.69 μl of N,N'-methylenebisacrylamide dimethyl sulfoxide solution (50 mg / mL), and 23.48 μl of Ac-BMS solution were added sequentially. The volume was then adjusted to 1 mL with PBS, and the mixture was incubated for 5 min. The Ac-BMS solution was obtained using the following method: BMS-8 (986 mg) was dissolved in dichloromethane (20 ml), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (479 mg) and dimethylaminopyridine (305 mg) were added under ice bath conditions. The mixture was stirred for 1.5 h, followed by the addition of N-(3-aminopropyl)methacrylamide (355 mg) and the reaction was continued for 2 h. The solvent was removed by vacuum concentration to obtain the crude product, which was purified by C18 reversed-phase column chromatography (MeOH / DCM = 1 / 20) to obtain a white solid Ac-BMS. Ac-BMS was dissolved in dimethyl sulfoxide to obtain an Ac-BMS solution.

[0038] The molar ratio of acrylamide (AAM) to N-(3-aminopropyl)methacrylamide hydrochloride (APM) is 10:0; the molar ratio of acrylated serum albumin, acrylamide, and N,N'-methylenebisacrylamide is 1:1000:25; and the molar ratio of acrylated serum albumin to BMS-8 is 1:25.

[0039] 10.37 μL of ammonium persulfate aqueous solution (100 mg / mL) and 2.68 μL of tetramethylethylenediamine aqueous solution (775 mg / mL) were added to the incubated solution, and the mixture was kept at room temperature for 2 h. Dialysis (MWCO: 10 kDa) was performed for 24 h to remove unreacted monomers and byproducts, yielding the nano-targeted chimera.

[0040] Example 2 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is in step (2), where 15.39 μl of acrylamide aqueous solution (70 mg / ml) is replaced with 12.31 μl; and 10.83 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) is added. All other steps are the same as in Example 1.

[0041] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 8:2; the molar ratio of acrylated serum albumin, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:800:200:25.

[0042] Example 3 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is in step (2), where 15.39 μl of acrylamide aqueous solution (70 mg / ml) is replaced with 9.23 μl; and 21.66 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) is added. All other steps are the same as in Example 1.

[0043] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 6:4; the molar ratio of acrylated serum albumin, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:600:400:25.

[0044] Example 4 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is in step (2), where 15.39 μl of acrylamide aqueous solution (70 mg / ml) is replaced with 7.69 μl; and 27.07 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) is added. All other steps are the same as in Example 1.

[0045] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 5:5; the molar ratio of acrylated serum albumin, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:500:500:25.

[0046] Example 5 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is in step (2), where 15.39 μl of acrylamide aqueous solution (70 mg / ml) is replaced with 6.15 μl; and 32.48 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) is added. All other steps are the same as in Example 1.

[0047] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 4:6; the molar ratio of acrylated serum albumin, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:400:600:25.

[0048] Example 6 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is in step (2), where 15.39 μl of acrylamide aqueous solution (70 mg / ml) is replaced with 3.08 μl; and 43.31 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) is added. All other steps are the same as in Example 1.

[0049] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 2:8; the molar ratio of acrylated serum albumin, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:200:800:25.

[0050] Example 7 This embodiment provides a nano-targeted chimera and its preparation method. The difference between this embodiment and Example 1 is that in step (2), no acrylamide aqueous solution (70 mg / ml) was added; only 54.14 μl of N-(3-aminopropyl)methacrylamide hydrochloride aqueous solution (50 mg / ml) was added. All other steps are the same as in Example 1.

[0051] The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 0:10; the molar ratio of acrylated serum albumin, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:1000:25.

[0052] Example 8 This embodiment provides the application of nano-targeting chimeras in the preparation of hybrid drug delivery systems, based on... Figure 1 As shown, the details are as follows: 1 mg of COOH-TK-COOH was dissolved in 1 ml of pure water, and 0.85 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.26 mg of N-hydroxysuccinimide were added. After activation for 30 min, a mixture was obtained. 100 μl of the mixture was added to 1 ml of BLTC solution (1 mg / ml). The molar ratio of COOH-TK-COOH to BLTC was 5:1. After reacting for 30 min, 8.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2.6 mg of N-hydroxysuccinimide were added. After activation for another 30 min, engineered Escherichia coli Nissle 1917 (EcN) cells in the logarithmic growth phase were added. After reacting for 1 h, the cells were collected by centrifugation (5000 rpm, 10 min) and washed three times with PBS to remove uncoupled BLTC, yielding the hybrid delivery drug EcN@BLTC.

[0053] Experimental Example 1. According to Figure 2 As shown, particle size measurements of the nano-targeted chimeras prepared in Examples 1-7 confirmed their successful polymerization, and the surface charge of the nano-targeted chimeras was closely related to the polymerized monomers. Subsequently, the morphology of the nano-targeted chimeras was characterized by transmission electron microscopy, as shown... Figure 3As shown, the nano-targeted chimera has a spherical structure with a size of about 30 nm.

[0054] 2. Cytotoxicity experiments of the nano-targeted chimera are as follows: The nano-targeting chimera was prepared using Example 3, and 4T1 cells were distributed at 5 × 10⁶ cells per well. 3 Cells were seeded at a density in 96-well plates and incubated overnight in 0.1 mL of RAPI-1640 (v / v) containing 10% fetal bovine serum. Different molar amounts of BLTC (12 nM to 1600 nM) were added to the 4T1 cells.

[0055] Then, CCK reagent and RAPI-1640 were mixed at a volume ratio of 1:9 to obtain the CCK working solution. The CCK working solution was added to 96-well plates and incubated with 4T1 cells for 24 hours. The cells were then washed with PBS (pH 7.4, 10 mM), followed by the addition of 100 μL of the CCK working solution and incubation for another 2 hours. Cell viability was quantitatively determined by measuring absorbance (λ = 450 nm) using a microplate reader. Cell viability was calculated based on the viability of untreated cells.

[0056] Experimental results are as follows Figure 4 As shown, the nano-targeting chimera with an incubation concentration of less than 200 nM did not have significant toxicity to tumor cells (4T1 cells), indicating that the nano-targeting chimera has good safety and few side effects on 4T1 cells.

[0057] 3. Experiment on the degradation of tumor cell membrane protein PD-L1 by nano-targeted chimeras, details are as follows: 4T1 cells were seeded in 6-well plates (1.0 × 10⁻⁶). 5 Cells / well were incubated for 24 h. Subsequently, 4T1 cells were treated for 48 h with the nano-targeting chimera (BLTC) prepared in Example 3, free BSA, nBSA polymerized from Ac-BMS, and Ac-BMS, respectively. After treatment, the cells were washed three times with ice-cold PBS and lysed for 30 min on ice in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM EGTA, and a mixture of 1% protease inhibitors). The lysates were collected, centrifuged at 12000 rpm for 30 min, and the protein concentration in the supernatant was measured using a BCA protein quantification kit.

[0058] Protein samples were mixed with 5× Loading buffer (4:1 ratio), denatured at 100 °C for 10 min, and separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm pores) using transfer buffer, blocked with high-performance blocking buffer in 1×Tris-buffered saline containing Tween-20 (TBST) for 20 min, and incubated overnight at 4 °C with primary antibody (anti-PD-L1 antibody). After washing three times with TBST for 5 min each time, the PVDF membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 h, washed with TBST, and visualized using chemiluminescent HRP substrate and a ChemiDoc XRS imaging system (Bio-Rad). Binding strength was quantified using ImageJ software and normalized to a loading control.

[0059] Immunofluorescence analysis: 4T1 cells were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 nM in 35 mm confocal culture dishes (Ф=15 mm) and incubated overnight for cell attachment. 4T1 cells were then exposed to BLTC (100 nM) containing different molecular weights of Ac-BMS. After 48 h of incubation, cells were washed with PBS and fixed with 4% (w / v) paraformaldehyde at room temperature for 15 min. PD-L1 cells were stained with anti-mouse PD-L1 (1:200 dilution) for 2 h, washed three times with PBS, and further stained with Alexa594-conjugated monoclonal secondary antibody (1:20 dilution) for 1 h. Finally, cells were counterstained with DAPI and imaged using an LSM 800.

[0060] Experimental results are as follows Figure 5 , Figure 6 As shown, Western blotting analysis and confocal imaging demonstrate that BLTC can significantly degrade PD-L1. Compared to other groups, the nano-targeted chimera exhibits highly efficient PD-L1 degradation capabilities.

[0061] 4. The hybrid delivery drug prepared in Example 8 was characterized and its effectiveness was verified, as follows: (1) The morphology of EcN@BLTC obtained in Example 8 was characterized by TEM. The details are as follows: 10 μL of EcN@BLTC solution was added to a copper grid. After standing for 15 min, the EcN@BLTC droplets were removed from the edge of the copper grid using filter paper. Then, 10 μL of 1% phosphotungstic acid was added for staining for 10 s. After removing the staining solution with filter paper, the grid was vacuum dried for 48 h and observed using TEM.

[0062] Experimental results are as follows Figure 7As shown, the surface of the engineered bacteria changes from "smooth" to "rough," with a layer of material tightly attached, indicating that BLTC is coupled to the surface of the engineered bacteria.

[0063] (2) To verify the successful construction of EcN@BLTC and its ROS-responsive release capability, the nano-targeting chimera was stained with FITC and EcN was stained with rhodamine. The hybrid delivery drug was prepared according to the preparation method in Example 8 and FRET analysis was performed.

[0064] The excitation wavelength was fixed at 490 nm (primarily for FITC excitation), and the emission spectrum was scanned from 500 to 700 nm. The fluorescence spectra of EcN@BLTC, BLTC, and EcN@BLTC under 1 mM H2O2 conditions were measured.

[0065] Experimental results are as follows Figure 8 As shown, when BLTC and EcN were labeled with the donor (FITC) and acceptor (Rh) respectively, EcN@BLTC was successfully prepared. Upon excitation of the donor (490 nm), a significant decrease in donor fluorescence and strong acceptor-sensitized emission (at 575 nm) were observed, indicating that in the hybrid drug delivery system, BLTC and the EcN surface are tightly connected by chemical bonds, with an average distance within 10 nm, thus successfully constructing a structurally stable hybrid drug delivery system. EcN@BLTC then releases BLTC under H2O2 conditions, losing its FRET effect.

[0066] according to Figure 1 As shown in the figure, a novel nano-targeted chimera BLTC capable of degrading PD-L1 was prepared by in-situ polymerization of monomers. The amino group of BLTC was covalently coupled to the surface of engineered bacteria via an amidation reaction using the ROS-responsive linker COOH-TK-COOH. After intravenous injection, the bacteria reached the tumor tissue by relying on their deep tumor penetration characteristics. Under the high ROS environment of the tumor, the bacteria achieved controlled release and degraded the tumor cell surface membrane protein PD-L1. At the same time, the engineered bacteria can secrete XCL1 protein, promote dendritic cell recruitment, and improve the tumor immune microenvironment.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nano-targeting chimera, characterized in that, Acrylamide serum albumin, polymeric monomers, crosslinking agents, and PD-L1 inhibitors are mixed in an organic solvent or water and subjected to in-situ initiated polymerization. The polymeric monomers include one or both of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride.

2. The nano-targeted chimera according to claim 1, characterized in that, The molar ratio of acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 10:0 to 0:

10.

3. The nano-targeted chimera according to claim 1, characterized in that, The molar ratio of acrylamide serum albumin, polymeric monomer, and crosslinking agent is 1:1000:

25.

4. The nano-targeted chimera according to claim 1, characterized in that, The molar ratio of acrylamide serum albumin to PD-L1 inhibitor is 1:20 to 1:

30.

5. The nano-targeted chimera according to claim 1, characterized in that, The initiator for the in-situ polymerization reaction is ammonium persulfate, and the catalyst for the in-situ polymerization reaction is tetramethylethylenediamine.

6. The nano-targeted chimera according to claim 1, characterized in that, Acrylamide serum albumin is composed of bovine serum albumin and N Acryloyloxysuccinimide is prepared in solution.

7. The nano-targeted chimera according to claim 6, characterized in that, Bovine serum albumin and N The molar ratio of acryloyloxysuccinimide is 1:3 to 1:

10.

8. A method for preparing a nano-targeted chimera as described in any one of claims 1-7, characterized in that, Includes the following steps: Bovine serum albumin was dissolved in PBS, and N was added to it. Acryloxysuccinimide dimethyl sulfoxide solution was reacted to yield acrylylated serum albumin; Acrylated serum albumin, aqueous solution of polymeric monomer, crosslinking agent dimethyl sulfoxide solution and PD-L1 inhibitor dimethyl sulfoxide solution are mixed, and ammonium persulfate and tetramethylethylenediamine are added to initiate an in-situ polymerization reaction to obtain the product.

9. The use of a nano-targeting chimera as described in any one of claims 1-7 in a drug for treating solid tumors.

10. The use of a nano-targeting chimera as described in any one of claims 1-7 in hybrid drug delivery for the treatment of breast cancer, characterized in that, Nanoparticle-targeted chimeras are coupled to the surface of engineered bacteria to achieve hybrid drug delivery.

Images