Albumin derivative with dual targeting function of tumor cells and macrophages and application thereof

Abstract

The present application relates to an anisic acid modified albumin dual targeting function of tumor cells and macrophages. In view of the technical bottleneck that the existing drug delivery system is difficult to simultaneously target tumor cells and tumor-associated macrophages, the present application modifies the surface group of albumin molecules, so as to simultaneously target tumor cells and macrophages on the basis of retaining drug loading capacity to realize dual targeting function. The albumin modification strategy of the present application breaks through the limitation of the traditional single targeting drug delivery system, and through the synergistic regulation of tumor cells and macrophage double cell groups, a new technical path and idea are provided for the development of a drug delivery system targeting tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine delivery, specifically involving the application of anisic acid-modified albumin in simultaneously targeting tumor cells and macrophages. Background Technology

[0002] Macrophages, as core effector cells of the body's innate immune system, are widely distributed in blood, tissues, and organs, possessing multiple functions including phagocytosis and clearance, antigen presentation, and immune regulation. Their unique heterogeneity allows them to dynamically differentiate into functionally diverse subtypes in response to microenvironment signals, playing an irreplaceable role in maintaining tissue homeostasis, defending against pathogen invasion, and regulating inflammatory responses. This functional plasticity makes macrophages a key target for the treatment of many major diseases, and their clinical translational value has been validated in multiple fields, including cancer, autoimmune diseases, metabolic diseases, and neurodegenerative diseases.

[0003] Cancer progression is jointly determined by tumor cells and their surrounding microenvironment. Within the tumor microenvironment, tumor-associated macrophages (MAMs) are the dominant cell population, accounting for up to 50% of some solid tumors. MAMs directly drive tumor cell invasion and metastasis by secreting pro-cancer and growth factors. Clinical studies have shown that MAMs are present in almost all tumors, and their abundance is positively correlated with tumor progression and negatively correlated with patient prognosis.

[0004] With a deeper understanding of the interaction mechanisms between tumor cells and macrophages, directly killing tumor cells and targeting and regulating the function of tumor-associated macrophages have become important strategies for cancer treatment. Currently, various strategies for targeted drug delivery to tumor cells or macrophages have been developed. Among these, the most widely studied is the active targeted nanodelivery system based on ligand-receptor specific recognition. This system modifies the surface of a nanocarrier with ligands that match receptors highly expressed on the surface of tumor cells (such as folic acid receptors, Sigma receptors, and sodium-dependent multivitamin transporters) or macrophages (such as CD44, CD206, and scavenger receptors). Utilizing the strong specific interaction between the ligand and receptor, drugs are efficiently delivered to tumor cells or macrophages via receptor-mediated endocytosis.

[0005] Albumin, as a highly promising drug delivery platform, has shown great promise in the treatment of tumor-related diseases such as cervical cancer, bladder cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, glioma, and melanoma due to its excellent biocompatibility, modifiable surface properties, and efficient drug loading capacity. Targeted modification of albumin drug delivery systems is key to improving their efficacy, reducing toxicity, and adapting them to different tumor biological characteristics. However, their clinical application is still limited by issues such as protein crown masking effect, insufficient in vivo stability, and poor targeting efficiency. Therefore, constructing albumin nanodelivery systems with both high stability and efficient active targeting capabilities is a crucial challenge that urgently needs to be overcome in the field of pharmaceutical science.

[0006] While strategies targeting tumor cells or tumor-associated macrophages (MAMs) for cancer treatment have made some progress in drug delivery, they still face bottlenecks such as off-target toxicity, insufficient target coverage, and single-pathway drug resistance. Currently, dual-targeting drug delivery systems that can simultaneously target tumor cells and MAMs are extremely rare, as they cannot synergistically regulate tumor cell proliferation pathways and MAM-mediated immunosuppression. To address this, this invention focuses on novel dual-targeting albumin nanoformulations, aiming to overcome the limitations and drug resistance bottlenecks of single-target therapy by synergistically targeting tumor cells and MAMs, providing a more efficient and less toxic treatment strategy for solid tumors. Summary of the Invention

[0007] The purpose of this invention is to provide albumin derivatives with dual targeting capabilities for tumor cells and macrophages. Studies have shown that anisic acid-modified albumin can simultaneously target both tumor cells and macrophages. These albumin derivatives overcome the limitations of existing single-target drug delivery strategies, providing more feasible strategies for developing novel dual-cell population drug delivery systems that synergistically regulate tumor cell proliferation pathways and the immunosuppressive function of tumor-associated macrophages.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution: The present invention provides an albumin derivative, characterized in that the derivative is formed by coupling an active derivative of anisic acid with an albumin molecule, wherein the active derivative is selected from active esters or intermediates formed by in-situ activation through carbodiimide condensing agents.

[0009] Furthermore, the albumin is human serum albumin or bovine serum albumin.

[0010] Furthermore, the albumin derivative is formed by coupling an active derivative of anisic acid with a primary amine group on the surface of the albumin molecule via an amide bond.

[0011] This invention provides a method for preparing albumin derivatives, characterized in that the albumin derivatives are obtained by coupling an active derivative of anisic acid with a primary amine group on an albumin molecule through an amide condensation reaction to form an amide bond.

[0012] The albumin derivative can be prepared by the following method: 1) Dissolve anisic acid, N-hydroxysuccinimide (NHS), EDCI and DMAP in an organic solvent to form a reaction system, and stir the reaction system at room temperature for 10-24 hours; 2) After the reaction is complete, the reaction system is washed and the washed organic phase is concentrated to obtain the NHS activated ester of anisic acid (AA-NHS). 3) Dissolve albumin in a buffer salt solution and adjust its pH to 8.0~9.0. Dissolve AA-NHS in an organic solvent. The molar ratio of AA-NHS to albumin is 10:1~50:1. React at 4~60ºC for 10~30 hours. 4) Dialyze for 48-72 hours, centrifuge and discard the precipitate, freeze-dry for at least 24 hours to obtain anisic acid modified albumin; Preferably, the organic solvent in step 1) is selected from one or more of dichloromethane, trichloromethane, and tetrahydrofuran; Preferably, in step 1), the dissolution temperature of the reaction system formed by dissolving anisic acid, NHS, EDCI and DMAP in dichloromethane is 0~4ºC; Preferably, the step of washing the reaction system after the reaction in step 2) includes washing with 1M hydrochloric acid aqueous solution and saturated sodium bicarbonate solution in sequence; Preferably, the buffer solution in step 3) is selected from one or more of carbonate buffer, phosphate buffer, and borate buffer; Preferably, the organic solvent in step 3) is selected from N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Preferably, the molar ratio of AA-NHS to albumin in step 3) is 20:1 to 30:1.

[0013] This invention also provides the dual-targeting application of albumin derivatives on tumor cells and macrophages. The method by which the albumin derivatives achieve dual targeting of tumor cells and macrophages includes, but is not limited to, achieving dual targeting of tumor cells and macrophages by forming a complex of albumin derivatives and active ingredients; achieving dual targeting of tumor cells and macrophages by using albumin derivatives as a carrier component and forming nanoparticles with active ingredients; and achieving dual targeting of tumor cells and macrophages by using albumin derivatives as a target encapsulated on the surface of drug-loaded liposomes, micelles, polymeric nanoparticles, or biomimetic nanoparticles.

[0014] The anisic acid-modified albumin drug delivery system for dual targeting of tumor cells and macrophages is characterized in that the drug delivery system contains an albumin derivative with dual targeting effects on tumor cells and macrophages, modified or loaded active ingredients, and other pharmaceutically acceptable excipients.

[0015] Further, the pharmaceutically acceptable excipients include carrier materials and injectable adjuvants; preferably, the carrier material is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, chitosan, injectable oil, and injectable phospholipid; the injectable adjuvants include one or more of sodium chloride, sucrose, maltose, lactose, glucose, mannitol, trehalose, sodium sulfite, sodium bisulfite, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyethylene glycol.

[0016] Furthermore, the active ingredient is selected from one or more diagnostic agents or therapeutic agents; wherein the diagnostic agents include one or more fluorescent dyes and contrast agents, and the therapeutic agents include one or more anticancer drugs, anti-inflammatory drugs, and immunomodulatory drugs. The diagnostic agents are used for tracing and imaging studies of the delivery system, and the selection criteria for the therapeutic agents mainly include: ① Chemical modification compatibility: the drug must be compatible with the chemical modification of anisic acid, not competing for key binding sites or causing adverse interactions, to ensure that the targeting function is not affected; ② Functional targeting: the drug should be able to exert its effect on tumor cells or macrophages to achieve therapeutic efficacy.

[0017] Further, the fluorescent dye is selected from one or more of indocyanine green, FITC, Cy3, Cy5, Cy7, DiD, DiO, DiR, and DiL; the anticancer drug is selected from one or more of cytotoxic chemotherapy drugs, molecularly targeted drugs, and natural products with anticancer activity; the anti-inflammatory drug is selected from one or more of nonsteroidal anti-inflammatory drugs and glucocorticoids; the immunomodulatory drug is selected from immune adjuvants; more preferably, the anticancer drug is selected from one or more of doxorubicin, paclitaxel, mitoxantrone, cisplatin, fluorouracil, sorafenib, gefitinib, trans-retinoic acid, tripterygium wilfordii, curcumin, resveratrol, ginsenosides, diosgenin, and silymarin; the anti-inflammatory drug is selected from one or more of ibuprofen, indomethacin, meloxicam, dexamethasone, prednisone, and methylprednisolone; and the immunomodulatory drug is selected from one or more of R848, CpG, and MSA-2.

[0018] Furthermore, the active ingredient is selected from one or more of active ingredients, pharmaceutically acceptable salts, esters, prodrugs, and derivatives.

[0019] Furthermore, the drug delivery system has a particle size of 50-200 nm, preferably 100-150 nm.

[0020] Furthermore, the above-mentioned albumin derivative or the above-mentioned drug delivery system can be used to prepare antitumor drugs. Preferably, the tumor is selected from cervical cancer, bladder cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, glioma, and melanoma.

[0021] The albumin derivative disclosed in this invention has the ability to simultaneously target tumor cells and macrophages, providing a new technical solution for constructing a drug delivery system that targets the tumor microenvironment, and is expected to achieve synergistic regulation of tumor cell proliferation pathways and tumor-associated macrophage immunosuppressive function. Attached Figure Description

[0022] Figure 1 Fluorescence excitation spectra of HSA and a series of AHs.

[0023] Figure 2 Fluorescence emission spectra of HSA and a series of AHs.

[0024] Figure 3 Uptake of HSA nanoparticles and a series of AH nanoparticles in TC-1 cells (n = 3, ).

[0025] Figure 4 Uptake of HSA nanoparticles and a series of AH nanoparticles in Raw 264.7 cells (n = 3, ).

[0026] Figure 5 The mechanism of HSA nanoparticle uptake by TC-1 cells (n = 3, , (ns: no significant difference).

[0027] Figure 6 The mechanism of AH20 nanoparticle uptake by TC-1 cells (n = 3, , (ns: no significant difference).

[0028] Figure 7 The mechanism of HSA nanoparticle uptake by raw 264.7 cells (n = 3, , , (ns: no significant difference).

[0029] Figure 8 The mechanism of AH20 nanoparticle uptake by raw 264.7 cells (n = 3, , , (ns: no significant difference).

[0030] Figure 9 Representative images of isolated mouse organs and tumor tissues.

[0031] Figure 10 Semi-quantitative analysis of nanoparticle accumulation in isolated mouse organs and tumor tissues (n = 3, ).

[0032] Figure 11 Colocalization of HSA and AH20 nanoparticles with tumor cells (scale bar: 20 μm).

[0033] Figure 12 Colocalization of HSA nanoparticles and AH20 nanoparticles with tumor-associated macrophages (scale bar: 20 μm). Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field and are all commercially available.

[0035] Example 1 Synthesis of N-hydroxysuccinimide (NHS) reactive ester of anisolic acid (AA-NHS). At 0ºC, 8 mmol of anisolic acid, 12 mmol of NHS, 12 mmol of EDCI, and 16 mmol of DMAP were dissolved in 32 mL of dichloromethane to form a reaction system. The reaction system was stirred at room temperature for 16 hours. After the reaction was completed, the reaction system was washed successively with 1 M hydrochloric acid aqueous solution and saturated sodium bicarbonate solution. The organic solvent was removed by rotary evaporation of the washed organic phase to obtain AA-NHS.

[0036] Example 2 Synthesis of anisic acid-modified bovine serum albumin (AA-BSA). 2 mmol of anisic acid, 4 mmol of NHS, and 4 mmol of EDCI were dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) and activated by stirring at room temperature for 12 hours to obtain an anisic acid activation solution. 0.05 mmol of BSA powder was dissolved in 20 mL of carbonate buffer at pH 8.5 to obtain a BSA carbonate buffer. The anisic acid activation solution was rapidly added to the BSA carbonate buffer, and the reaction was carried out at 37ºC with stirring in an oil bath for 24 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (molecular weight cutoff of 8kDa-14kDa) and dialyzed with deionized water at room temperature with stirring for at least 48 hours. After dialysis, insoluble matter was removed by centrifugation, and the product was freeze-dried for at least 24 hours to obtain AA-BSA.

[0037] Example 3 AH10, an anisic acid-modified human serum albumin (AH) synthesized by reacting AA-NHS with human serum albumin (HSA) in a molar ratio of 10:1, was synthesized. 0.05 mmol of lyophilized HSA powder was completely dissolved in 10 mL of sodium bicarbonate solution, and sodium carbonate solution was added dropwise to adjust the pH to 8.4–8.6, yielding a carbonate buffer for HSA. 0.5 mmol of AA-NHS was completely dissolved in an appropriate amount of DMF, yielding a DMF solution of AA-NHS. The DMF solution of AA-NHS was rapidly added to the carbonate buffer for HSA, and the reaction was carried out at 37ºC with stirring in an oil bath for 24 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (molecular weight cutoff of 8 kDa–14 kDa) and dialyzed with deionized water at room temperature with stirring for at least 48 hours. After dialysis, insoluble matter was removed by centrifugation, and the solution was freeze-dried for at least 24 hours to obtain AH10.

[0038] Example 4 AH20, which is the AH synthesized by reacting AA-NHS and HSA in a molar ratio of 20:1, was obtained by replacing 0.5 mmol of AA-NHS in Example 3 with 1 mmol of AA-NHS, while keeping the rest of the operation the same as in Example 3.

[0039] Example 5 AH30, which is AH synthesized by reacting AA-NHS and HSA in a molar ratio of 30:1, was obtained by replacing 0.5 mmol of AA-NHS in Example 3 with 1.5 mmol of AA-NHS, and performing the remaining operations as in Example 3.

[0040] Example 6 AH40 was synthesized, which is AH synthesized by reacting AA-NHS and HSA in a molar ratio of 40:1. The 0.5 mmol of AA-NHS in Example 3 was replaced with 2 mmol of AA-NHS, and the remaining operations were the same as in Example 3, thus obtaining AH40.

[0041] Example 7 AH50 was synthesized, which is AH synthesized by reacting AA-NHS and HSA in a molar ratio of 50:1. The 0.5 mmol of AA-NHS in Example 3 was replaced with 2.5 mmol of AA-NHS, and the remaining operations were the same as in Example 3, thus obtaining AH50.

[0042] Example 8 Preparation of indocyanine green albumin complex (ICG-AH20). 300 mg of AH20 prepared in Example 4 was completely dissolved in 10 mL of water for injection to obtain an aqueous solution of AH20. 10 mg of indocyanine green and 10 mg of HS15 were completely dissolved in 5 mL of methanol. After removing the methanol by rotary evaporation, the aqueous solution of AH20 was added, and the mixture was sonicated in a water bath for 10 minutes. ICG-AH20 was successfully prepared with a binding rate of 97%.

[0043] Example 9 Preparation of AH20 nanoparticles encapsulated with pirarubicin (THP@AH20). 60 mg of AH20 prepared in Example 4 was completely dissolved in 3 mL of water for injection to obtain an aqueous phase. 4 mg of pirarubicin and 18 mg of PLGA were dissolved in 600 μL of chloroform to obtain an oil phase. The aqueous and oil phases were mixed and subjected to probe sonication (250 W power, 5 seconds on, 5 seconds off) for 10 minutes under ice bath conditions. The organic solvent was removed by rotary evaporation, successfully preparing THP@AH20. The particle size was uniform, approximately 124.8 nm, with an encapsulation efficiency of 99%.

[0044] Example 10 Preparation of AH20-modified liposomes (D@AH20-Lipo) loaded with the fluorescent dye DiD. 20 mg of AH20 prepared in Example 4 was completely dissolved in 2 mL of water for injection to obtain an AH20 solution. 12.5 μg DiD, 20 mg soybean lecithin S100, 5 mg cholesterol, and 2.5 mg mPEG2000-DSPE were completely dissolved in 5.5 mL of chloroform. The organic solvent was removed by rotary evaporation to form a thin film. After hydration of the film with the AH20 solution under ultrasonication in a water bath, the film was then ultrasonicated with a probe under ice bath conditions (250 W power, 5 seconds on, 5 seconds off) for 10 minutes, successfully preparing D@AH20-Lipo. The particle size was uniform, approximately 135.3 nm, with an encapsulation efficiency of 97%.

[0045] Example 11 Preparation of AH10 nanoparticles (D@AH10) coated with the fluorescent dye DiD. 60 mg of AH10 prepared in Example 3 was completely dissolved in 2 mL of water for injection to obtain an aqueous phase. Simultaneously, 25 μg of DiD and 16 mg of soybean oil were completely dissolved in 200 μL of chloroform to obtain an oil phase. The aqueous and oil phases were mixed and stirred. The mixture was then sonicated under ice bath conditions (230 W power, 5 seconds on, 5 seconds off) for 10 minutes. The organic solvent was removed by rotary evaporation, successfully preparing D@AH10. The particle size was uniform, approximately 111.2 nm, with an encapsulation efficiency of 99%.

[0046] Example 12 Preparation of AH20 nanoparticles (D@AH20) coated with the fluorescent dye DiD. The AH10 prepared in Example 3 was replaced with the AH20 prepared in Example 4 in the formulation of Example 11, while all other conditions remained exactly the same as in Example 11, and D@AH20 was successfully prepared. The particle size was uniform, approximately 117.1 nm, with an encapsulation efficiency of 99%.

[0047] Example 13 Preparation of AH30 nanoparticles (D@AH30) coated with the fluorescent dye DiD. The AH10 prepared in Example 3 was replaced with the AH30 prepared in Example 5 in the formulation of Example 11, while all other conditions remained exactly the same as in Example 11, and D@AH30 was successfully prepared. The particle size was uniform, approximately 131.1 nm, with an encapsulation efficiency of 99%.

[0048] Example 14 Preparation of AH40 nanoparticles (D@AH40) coated with the fluorescent dye DiD. The AH10 prepared in Example 3 was replaced with the AH40 prepared in Example 6 in the formulation of Example 11, while all other conditions remained exactly the same as in Example 11. D@AH40 was successfully prepared. The particle size was uniform, approximately 153.6 nm, and the encapsulation efficiency was 97%.

[0049] Example 15 Preparation of AH50 nanoparticles (D@AH50) coated with the fluorescent dye DiD. The AH10 prepared in Example 3 was replaced with the AH50 prepared in Example 7 in the formulation of Example 11, while all other conditions remained exactly the same as in Example 11, and D@AH50 was successfully prepared. The particle size was uniform, approximately 158.9 nm, and the encapsulation efficiency was 98%.

[0050] Comparative Example Preparation of HSA nanoparticles (D@H) loaded with the fluorescent dye DiD. D@H was successfully prepared by replacing AH10 prepared in Example 3 with HSA in the formulation of Example 11, while maintaining the same conditions as in Example 11. The particles had a uniform size of approximately 101.3 nm and an encapsulation efficiency of 99%.

[0051] Experimental Example 1 AH was qualitatively characterized using fluorescence spectroscopy. HSA, AH10 from Example 3, AH20 from Example 4, AH30 from Example 5, AH40 from Example 6, and AH50 from Example 7 were dissolved in PBS buffer at pH 7.2–7.4 to obtain HSA, AH10, AH20, AH30, AH40, and AH50 solutions with concentrations of approximately 53 μg / mL. The fluorescence excitation and emission spectra of each solution were scanned using a fluorescence spectrophotometer. The results are shown below. Figure 1 and Figure 2 The results showed that the maximum excitation wavelength of HSA was 280 nm. The maximum excitation wavelengths of AH10, AH20, and AH30 remained unchanged compared to HSA, while AH40 and AH50 did not exhibit a maximum excitation wavelength at 280 nm. Meanwhile, the maximum emission wavelength of HSA was 333 nm, while AH10, AH20, AH30, AH40, and AH50 all exhibited a blue shift to approximately 304 nm. This indicates that as the degree of modification of anisic acid on HSA increases, the maximum excitation wavelength at 280 nm gradually quenches, while the maximum emission wavelength shows a significant blue shift, proving the successful preparation of AH10, AH20, AH30, AH40, and AH50.

[0052] Experiment Example 2 AH was quantitatively characterized using the ninhydrin method. Appropriate amounts of sodium acetate and glacial acetic acid were weighed and dissolved in deionized water. The pH of the solution was adjusted to 5.4 with hydrochloric acid or sodium hydroxide to obtain an acetate buffer. An appropriate amount of acetate buffer was measured and mixed with twice the volume of DMSO to obtain a mixed solution of acetate buffer and DMSO. An appropriate amount of ninhydrin was weighed and added to the mixed solution of acetate buffer and DMSO to prepare a ninhydrin colorimetric solution with a concentration of 16.7 mg / mL. Appropriate amounts of HSA, AH10 from Example 3, AH20 from Example 4, AH30 from Example 5, AH40 from Example 6, and AH50 from Example 7 were weighed, dissolved in deionized water, and then serially diluted to obtain a series of HSA, AH10, AH20, AH30, AH40, and AH50 sample solutions with concentrations ranging from 0 to 5 mg / mL. Take 1 mL of each of the above sample solutions, add 1 mL of acetate buffer and 1 mL of ninhydrin colorimetric solution to each sample solution in sequence, mix well, heat in a 100ºC water bath for 15 minutes, cool to room temperature, add 3 mL of ethanol (60%, v / v) to each sample, mix well, and measure the UV absorption of each sample at a wavelength of 570 nm using an ELISA reader. Calculate the degree of modification of lysine residues on AH10, AH20, AH30, AH40, and AH50 according to formula (1), and calculate the molecular weight of AH10, AH20, AH30, AH40, and AH50 according to formula (2). The results are shown in Table 1.

[0053] Formula (1)

[0054] in, The degree of modification of the lysine residues on AH is represented by [value]. The concentration of lysine residues corresponding to B mg of HSA dissolved in A mL of deionized water. The concentration of lysine residues corresponding to B mg of AH dissolved in A mL of deionized water. ,in, The value is 59 (the number of lysine residues in one HSA molecule). This represents the molecular weight of anisolic acid. This represents the molecular weight of HSA.

[0055] Formula (2)

[0056] in, The molecular weight of AH is... This represents the molecular weight of HSA. The value is 59 (the number of lysine residues in one HSA molecule). The degree of modification of the lysine residues on AH is represented by [value]. This represents the molecular weight of anisolic acid.

[0057] Table 1. Modification degree and molecular weight of a series of AHs.

[0058]

[0059] It can be seen that AH10, AH20, AH30, AH40 and AH50 were all successfully modified with anisic acid, the difference being the degree of modification of anisic acid. It can also be found that during the synthesis of AH, as the molar ratio of AA-NHS to HSA increased from 10:1 to 50:1, the number of anisic acid successfully modified on HSA also gradually increased.

[0060] Experimental Example 3 Uptake of HSA nanoparticles and a series of AH nanoparticles by mouse cervical cancer cells (TC-1 cells). The concentration of TC-1 cells was adjusted to 5 × 10⁻⁶. 5 Cells / mL were seeded into each well of a 12-well flat-bottom cell culture plate with 1 mL of cell suspension and cultured overnight at 5% CO2 and 37ºC. The D@H cells prepared according to the formulation in the comparative example, D@AH10 prepared according to the formulation in Example 11, D@AH20 prepared according to the formulation in Example 12, D@AH30 prepared according to the formulation in Example 13, D@AH40 prepared according to the formulation in Example 14, and D@AH50 prepared according to the formulation in Example 15 were diluted with serum-free medium to a final DiD concentration of 400 ng / mL, yielding six drug-containing culture media. Once the cells reached a density of approximately 80%, the culture medium was discarded, and the cells were washed twice with sterile PBS buffer to completely remove the medium. 1 mL of the corresponding drug-containing culture medium was added to each well of each group. A blank control group without the drug was also included. Cells were cultured for another 2 hours at 5% CO2 and 37ºC. Afterward, the culture medium was discarded, and the cells were washed twice with sterile PBS buffer to completely remove any untaken nanoparticles. Cells were digested with trypsin containing EDTA, and the digestion was stopped with complete culture medium and followed by washing with PBS. Cells were collected and resuspended in 200–400 μL of PBS buffer to obtain a cell suspension. Flow cytometry was used to detect the uptake of nanoparticles by the cells. Results are shown below. Figure 3 The results showed that, except for AH10 nanoparticles, the uptake of the other anisic acid-modified albumin nanoparticles was significantly increased in TC-1 cells.

[0061] Experiment Example 4 Uptake of HSA nanoparticles and a series of AH nanoparticles by mouse macrophages (Raw 264.7 cells). The TC-1 cells used in Experiment 3 were replaced with Raw 264.7 cells, and trypsin digestion was not required during cell collection; all other procedures were the same as in Experiment 3. Results are shown below. Figure 4The results showed that, compared with HSA nanoparticles, AH nanoparticles with different degrees of anisic acid modification significantly increased uptake in macrophages, but the degree of anisic acid modification had little effect on uptake.

[0062] Experimental Example 5 Mechanism of D@H uptake by TC-1 cells. The TC-1 cell concentration was adjusted to 5 × 10⁻⁶. 5 Cells were seeded at a rate of 1 mL / well in 12-well flat-bottom cell culture plates and cultured overnight at 37°C with 5% CO2. The D@H prepared according to the formulation in the comparative example was diluted with serum-free medium to a final DiD concentration of 400 ng / mL to obtain drug-containing medium. After the cells reached 80% confluence, the medium was discarded, and the cells were washed once with sterile PBS buffer and discarded. For each inhibitor group, 1 mL of the inhibitor corresponding to the one listed in Table 2 was added to each well to inhibit the specific uptake pathway (no dextran sulfate group was set up). A control group without inhibitors was also set up. Cells were cultured at 37°C with 5% CO2 for 1 hour. After pretreatment with inhibitors, the culture medium was discarded, and 1 mL of drug-containing culture medium was added to each well of each group. A blank control group without drug administration was also set up. After culturing for 2 hours at 5% CO2 and 37ºC, the cells were washed twice with sterile PBS buffer, digested with trypsin, and the treatment was terminated with complete culture medium. After washing with PBS, the cells were collected and detected by flow cytometry. The relative uptake rate was calculated according to formula (3). The results are shown in [reference]. Figure 5 The results showed that the uptake of HSA nanoparticles on TC-1 cells was energy-dependent and mainly mediated by cholesterol-related lipid rafts.

[0063] Formula (3)

[0064] Table 2. Inhibitors of cellular uptake pathways and their working concentrations.

[0065]

[0066] Experimental Example 6 The mechanism of D@AH20 uptake by TC-1 cells. The specific experimental procedures are the same as in Example 5, except that the D@H20 prepared according to the formulation in the comparative example was replaced with D@AH20 prepared according to the formulation in Example 12. All other procedures were the same as in Example 5. Results are shown below. Figure 6 The results showed that the uptake of AH20 nanoparticles on TC-1 cells was energy-dependent and mainly mediated by sigma receptors and cholesterol-related lipid rafts.

[0067] Experimental Example 7 The mechanism of D@H uptake in Raw 264.7 cells. The specific experimental procedures are the same as in Experiment 5, except that the TC-1 cells used in Experiment 5 were replaced with Raw 264.7 cells, and the addition of 1 mL of the inhibitor corresponding to Table 2 (without the dextran sulfate group) was replaced with the addition of 1 mL of the inhibitor corresponding to Table 2 (without the haloperidol group). Furthermore, trypsin digestion was not required during cell collection; all other procedures were the same as in Experiment 5. Results are shown below. Figure 7 The results showed that the uptake of HSA nanoparticles in Raw264.7 cells was energy-dependent and mainly mediated by clathrin, caverin, and macropinocytosis.

[0068] Experimental Example 8 The mechanism of D@AH20 uptake in Raw 264.7 cells. The specific experimental procedures are the same as in Example 7, except that the D@H20 prepared according to the formulation in the comparative example was replaced with the D@AH20 prepared according to the formulation in Example 12. Results are shown below. Figure 8 The results showed that the uptake of AH20 nanoparticles on Raw 264.7 cells was energy-dependent and mainly mediated by clathrin and scavenger receptor-A.

[0069] Experimental Example 9 Distribution of D@H, D@AH20, and D@AH30 in TC-1 tumor-bearing mice. TC-1 cells in good growth condition were prepared to a concentration of 1×10⁻⁶. 7 100 μL of TC-1 cell suspension per mL was subcutaneously injected into the right back of each female C57 mouse. The inoculation continued until the tumor volume reached approximately 200 mm². 3 Tumor-bearing mice were randomly divided into three groups and administered D@H (prepared according to the formulation in the comparative example), D@AH20 (prepared according to the formulation in Example 12), and D@AH30 (prepared according to the formulation in Example 13) via tail vein, respectively. The DiD dosage for each mouse was 100 μg / kg. Thirty hours after administration, the tumor-bearing mice were euthanized, and the heart, liver, spleen, lung, kidney, and tumor tissues were dissected, washed in PBS buffer, and dried with filter paper. In vitro tissue imaging and semi-quantitative analysis were performed using a small animal in vivo optical imaging system. The results are shown in [link to results]. Figure 9 and Figure 10 The results showed that, compared with unmodified albumin nanoparticles (D@H), the accumulation of anisic acid-modified albumin nanoparticles (D@AH20 and D@AH30) at tumor sites was significantly increased. This indicates that AH nanoparticles have good tumor-targeting ability and are expected to achieve more efficient anti-tumor effects after drug loading.

[0070] Experimental Example 10 Targeting ability of D@H and D@AH20 on tumor cells and macrophages. Tumor tissue from Experiment 9 was washed with PBS buffer and fixed in approximately 10 times its volume of 4% paraformaldehyde solution for 48 hours. The fixed tissue was dehydrated using an automated dehydrator and embedded in paraffin before sectioning. After dewaxing to water and antigen retrieval, the paraffin sections were placed in 3% hydrogen peroxide and incubated at room temperature in the dark to block endogenous peroxidase. After washing with PBS buffer, serum blocking was performed by adding BSA solution. Primary antibody working solution (Sigma or F4 / 80) was added, and the sections were incubated overnight at 4ºC. After washing with PBS buffer, secondary antibody working solution (FITC-labeled goat anti-rabbit) was added, and the sections were incubated at 37ºC for 30 min. After washing with PBS buffer, DAPI was added, and the sections were incubated at room temperature for 10 min. After washing with PBS buffer, anti-fluorescence quenching mounting medium was added for mounting. The sections were observed and photographed under a fluorescence microscope. The results are shown in [link to results]. Figure 11 and Figure 12 The results showed that D@H and Sigma exhibited a certain co-localization effect, with D@AH showing a more pronounced co-localization effect, indicating that the ability of HSA nanoparticles to target TC-1 tumor cells is further enhanced after modification with anisic acid. Meanwhile, D@H and F4 / 80 showed almost no overlap, while D@AH and F4 / 80 exhibited some overlap, suggesting that HSA nanoparticles modified with anisic acid possess a certain macrophage targeting ability. These results further demonstrate that AH nanoparticles have dual targeting capabilities for both tumor cells and macrophages.

Claims

1. An albumin derivative, characterized in that, The derivative is formed by coupling an active derivative of anisolic acid with albumin molecules, wherein the active derivative is selected from active esters or intermediates formed by in-situ activation through carbodiimide condensing agents.

2. The albumin derivative according to claim 1, characterized in that, The albumin is human serum albumin or bovine serum albumin.

3. The albumin derivative according to claim 1, characterized in that, The albumin derivative is formed by coupling an active derivative of anisic acid with a primary amine group on the surface of the albumin molecule via an amide bond.

4. The albumin derivative according to any one of claims 1-3, characterized in that, The albumin derivative is prepared by a method comprising the following steps: 1) Dissolve anisic acid, N-hydroxysuccinimide (NHS), EDCI and DMAP in an organic solvent to form a reaction system. Stir the reaction system at room temperature for 10-24 hours. 2) After the reaction is complete, the reaction system is washed and the washed organic phase is concentrated to obtain the NHS activated ester of anisic acid (AA-NHS). 3) Dissolve albumin in a buffer salt solution and adjust its pH to 8.0~9.

0. Dissolve AA-NHS in an organic solvent. The molar ratio of AA-NHS to albumin is 10:1~50:

1. React at 4~60ºC for 10~30 hours. 4) Dialyze for 48-72 hours, centrifuge and discard the precipitate, freeze-dry for at least 24 hours to obtain anisic acid modified albumin; Preferably, the organic solvent in step 1) is selected from one or more of dichloromethane, trichloromethane, and tetrahydrofuran; Preferably, in step 1), the dissolution temperature of the reaction system formed by dissolving anisic acid, NHS, EDCI and DMAP in dichloromethane is 0~4ºC; Preferably, the step of washing the reaction system after the reaction in step 2) includes washing with 1M hydrochloric acid aqueous solution and saturated sodium bicarbonate solution in sequence; Preferably, the buffer salt solution in step 3) is selected from one or more of carbonate buffer, phosphate buffer, and borate buffer; Preferably, the organic solvent in step 3) is selected from N,N-dimethylformamide (DMF) and / or dimethyl sulfoxide (DMSO). Preferably, the molar ratio of AA-NHS to albumin in step 3) is 20:1 to 30:

1.

5. A drug delivery system with dual targeting functions for tumor cells and macrophages, characterized in that, The drug delivery system comprises the albumin derivative of claim 1.

6. The drug delivery system according to claim 5, characterized in that, The drug delivery system is selected from one or more of the following forms: (1) a complex formed by albumin derivative and active ingredient; (2) a nanoparticle formed by albumin derivative as carrier component and active ingredient; (3) a drug-loaded liposome, micelle, polymer nanoparticle or biomimetic nanoparticle with albumin derivative as target.

7. The drug delivery system according to claim 5, characterized in that, The delivery system contains the albumin derivative of claim 1, the active ingredient, and other pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include a carrier material and an injectable adjuvant; preferably, the carrier material is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, chitosan, injectable oil, and injectable phospholipid; the injectable adjuvant includes one or more of sodium chloride, sucrose, maltose, lactose, glucose, mannitol, trehalose, sodium sulfite, sodium bisulfite, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyethylene glycol.

8. The drug delivery system according to claim 6 or 7, characterized in that, The active ingredient is selected from one or more diagnostic agents or therapeutic agents; wherein the diagnostic agent includes one or more fluorescent dyes and contrast agents, and the therapeutic agent includes one or more anticancer drugs, anti-inflammatory drugs, and immunomodulatory drugs; preferably, the fluorescent dye is selected from one or more of indocyanine green, FITC, Cy3, Cy5, Cy7, DiD, DiO, DiR, and DiL; the anticancer drug is selected from one or more of cytotoxic chemotherapy drugs, molecularly targeted drugs, and natural products with anticancer activity; the anti-inflammatory drug is selected from nonsteroidal anti-inflammatory drugs. The anti-inflammatory drug is selected from one or more of the following: anti-inflammatory drugs and glucocorticoids; the immunomodulatory drug is selected from immune adjuvants; more preferably, the anticancer drug is selected from one or more of the following: doxorubicin, paclitaxel, mitoxantrone, cisplatin, fluorouracil, sorafenib, gefitinib, trans-retinoic acid, tripterygium wilfordii, curcumin, resveratrol, ginsenosides, diosgenin, and silymarin; the anti-inflammatory drug is selected from one or more of the following: ibuprofen, indomethacin, meloxicam, dexamethasone, prednisone, and methylprednisolone; the immunomodulatory drug is selected from one or more of the following: R848, CpG, and MSA-2.

9. The drug delivery system according to claim 8, characterized in that, The active ingredient is selected from one or more of the following: active ingredients, pharmaceutically acceptable salts, esters, prodrugs, and derivatives.

10. Use of the albumin derivative according to any one of claims 1-4 or the drug delivery system according to any one of claims 5-9 in the preparation of an antitumor drug, preferably, the tumor is selected from cervical cancer, bladder cancer, colon cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, glioma, and melanoma.

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