Folic acid and phenylboronic acid modified albumin nanocarriers and uses thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a folate and phenylboronic acid modified albumin nano-carrier and use thereof. The present application aims to solve the technical problem of the single targeting of the existing delivery system and the low intracellular delivery efficiency. The technical solution for solving the technical problem of the present application is to provide an albumin nano-carrier, which comprises nanoparticles formed by serum albumin and / or a functional variant thereof, and folate and phenylboronic acid modified on the serum albumin and / or the functional variant thereof. The albumin nano-carrier of the present application has a dual targeting effect, can significantly improve the enrichment efficiency of the nano-carrier at the tumor site, especially in the over-sialylated tumor, can achieve more accurate and efficient cascade targeting, and has a lysosome escape effect, can improve the intracellular bioavailability of the drug, thereby providing a new effective treatment strategy for tumors, especially those refractory tumors which are not sensitive to the existing therapy or have metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a folic acid and phenylboronic acid modified albumin nanocarrier and its application. Background Technology

[0002] Nanomedicine delivery systems are a key strategy for improving drug efficacy and reducing toxic side effects. Among them, serum albumin-based nanocarriers (such as bovine serum albumin, BSA) have become a research hotspot due to their good biocompatibility, biodegradability, non-immunogenicity, and ease of functionalization. Natural albumin can target tumor tissues through gp60 receptor-mediated transcellular endocytosis and other pathways, but its targeting specificity and delivery efficiency still need to be improved.

[0003] To improve targeting, researchers often attach specific ligands to albumin carriers. Folic acid (FA) is a classic targeting molecule, and its receptor is highly expressed on the surface of various epithelial tumor cells (such as ovarian cancer, breast cancer, lung cancer, and colorectal cancer). FA modification can effectively promote the uptake of nanocarriers by tumor cells. However, single FA targeting mainly relies on receptor overexpression on the cell surface and lacks the ability to recognize other important targets in the tumor microenvironment (such as over-sialyzed glycoproteins). Furthermore, its targeting efficiency is limited for certain tumors (such as some liver cancers and pancreatic cancers), and it is difficult to effectively solve the problem of nanocarriers being trapped in lysosomes after endocytosis, leading to drug degradation and ineffective release into the cytoplasm.

[0004] The phenylboronic acid (PBA) group shows potential in tumor targeting and responsive drug release due to its ability to form reversible boronic ester bonds with sialic acid overexpressed on cell surfaces or in the tumor microenvironment. PBA modification facilitates the accumulation of nanocarriers in tumor vascular endothelium and on the surface of certain tumor cells. However, single PBA modification also suffers from relatively broad target range and insufficient specificity, and its ability to actively promote endocytosis and subsequent intracellular fate regulation is weak.

[0005] Therefore, developing a novel albumin nanocarrier that can integrate different targeting mechanisms, overcome the limitations of single ligands, and synergistically enhance intracellular drug delivery efficiency to improve intracellular drug utilization is of great significance in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that existing delivery systems have single targeting and low intracellular delivery efficiency.

[0007] The technical solution of this invention is to provide an albumin nanocarrier with dual targeting and lysosomal escape functions.

[0008] The albumin nanocarrier provided by the present invention comprises nanoparticles formed from serum albumin and / or its functional variants, and folic acid (FA) and / or folic acid derivatives modified on said serum albumin and / or its functional variants, as well as phenylboronic acid (PBA) and / or phenylboronic acid derivatives.

[0009] In this embodiment, the folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, described in the albumin nanocarrier above are modified by being covalently or non-covalently linked to the serum albumin or its functional variants. The serum albumin is bovine serum albumin, human serum albumin, recombinant serum albumin, or its modified derivatives.

[0010] Furthermore, the modification ratio of folic acid and / or folic acid derivatives on albumin in the above-mentioned albumin nanocarrier is 1:0.5~5 molar ratio of albumin to folic acid; preferably, the molar ratio of albumin to folic acid is 1:1~2.

[0011] Furthermore, the modification ratio of phenylboronic acid and / or phenylboronic acid derivatives on albumin in the above-mentioned albumin nanocarrier is 1:5 to 40 in a molar ratio of albumin to phenylboronic acid. Preferably, the molar ratio of albumin to phenylboronic acid is 1:15 to 25.

[0012] In the aforementioned albumin nanocarrier, the folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, are linked to the corresponding functional groups on the serum albumin via their carboxyl and / or amino groups. Further, the linkage between folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, and the corresponding functional groups on the serum albumin is achieved through at least one of the following: amide bond formation via carboxyl-amino condensation reaction, Schiff base bond formation via aldehyde-amino condensation, or borate ester bond formation via borate group-cis-diol condensation.

[0013] Furthermore, the folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, are linked to albumin by forming an amide bond between the carboxyl group of folic acid and / or folic acid derivatives, as well as the amino group of phenylboronic acid and / or phenylboronic acid derivatives, and the amino group of albumin.

[0014] Furthermore, when linked by forming an amide bond, the phenylboronic acid derivative is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3-carboxyphenylboronic acid.

[0015] Furthermore, the linkage reaction is carried out in the presence of a coupling agent, which is a carbodiimide, an acylurea onium salt, or other condensing agent capable of activating the carboxyl group. Preferably, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and / or N-hydroxysuccinimide (NHS).

[0016] The albumin nanocarriers mentioned above were prepared by the following method:

[0017] a. Dissolve serum albumin in a buffer solution to obtain a serum albumin solution;

[0018] b. Take folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, dissolve them in a solvent, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature in the dark to obtain an activated PBA / FA mixed solution.

[0019] c. The activated PBA / FA mixed solution was slowly added to the serum albumin solution, and the resulting mixed solution was continuously stirred under light-protected conditions. After the reaction was completed, the resulting solution was dialyzed in ultrapure water. After dialyzing, it was filtered through a microporous membrane and freeze-dried to obtain the folic acid and phenylboronic acid modified albumin nanocarrier. The obtained folic acid and phenylboronic acid modified albumin nanocarrier was a pale yellow flocculent solid product.

[0020] The solvent mentioned in step b is N,N-dimethylformamide (DMF).

[0021] Preferably, the phenylboronic acid derivative mentioned in step b is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3-carboxyphenylboronic acid.

[0022] The present invention also provides a nanoparticle. This nanoparticle is obtained by loading a therapeutic agent onto the albumin nanocarrier described above.

[0023] The loading of the therapeutic agent into the aforementioned nanoparticles is achieved through at least one of the following methods: electrostatic interaction, hydrophobic interaction, π-π stacking, covalent linkage, or encapsulation. The therapeutic agent can be loaded within the nanocarrier or adsorbed onto its surface.

[0024] The therapeutic agent in the aforementioned nanoparticles is selected from one or more of small molecule chemical drugs, nucleic acid drugs, peptide or protein drugs, or contrast agents. The therapeutic agent includes anti-angiogenic drugs and / or anti-tumor drugs. Further, the anti-angiogenic drug includes a drug acting on the vascular endothelial growth factor signaling pathway or a tyrosine kinase inhibitor. The aforementioned anti-tumor drug includes one or more of chemotherapy drugs, molecularly targeted drugs, or immunotherapy drugs. Preferably, the therapeutic agent is at least one of regorafenib, paclitaxel, doxorubicin, curcumin, or sofospilanib.

[0025] Furthermore, the above-mentioned nanoparticles were prepared by the following method:

[0026] a. Weigh the drug to be treated and dissolve it in a solvent to prepare a stock solution, thus obtaining the drug phase;

[0027] b. Take the albumin nanocarrier described in any one of claims 7 to 9, and disperse it together with a stabilizer in ultrapure water to obtain the carrier aqueous phase;

[0028] c. The drug phase is slowly added dropwise to the aqueous phase of the carrier to obtain a mixture. The mixture is then homogenized using an ultrasonic cell disruptor. Subsequently, the resulting suspension is transferred to a dialysis bag and dialyzed in ultrapure water to remove organic solvents. The dialysate is concentrated by ultrafiltration and then filtered through a 0.22 μm sterile filter membrane to obtain a nanoparticle suspension.

[0029] In step a, the solvent is at least one of DMSO, water, or methanol.

[0030] The stabilizer mentioned in step b is polyvinylpyrrolidone.

[0031] In step c, the mixture is placed in an ice bath and treated with an ultrasonic cell disruptor for 3-10 minutes to homogenize it.

[0032] The nanoparticles obtained above have an average hydrated particle size between 10 nm and 300 nm and a zeta potential between -50 mV and +30 mV. The prepared nanoparticle suspension can be stored at -20°C for later use, or it can be prepared into a lyophilized form or a pharmaceutically acceptable dosage form.

[0033] The present invention also provides a pharmaceutical composition. The pharmaceutical composition comprises a therapeutically effective amount of the aforementioned nanoparticles, and one or more pharmaceutically acceptable carriers, excipients, stabilizers, or diluents. The pharmaceutical composition may be formulated as an injection, a lyophilized formulation, or a gel.

[0034] Based on this, the present invention also provides the use of the above-mentioned albumin nanocarrier, the above-mentioned nanoparticles, or the above-mentioned pharmaceutical composition in the preparation of medicaments for the treatment and / or prevention of cancer.

[0035] Furthermore, the cancer is a tumor that overexpresses folic acid receptors and / or is excessively sialylated.

[0036] Furthermore, the cancer is at least one of the following: epithelial tumors, digestive system tumors, respiratory system tumors, reproductive system tumors, or urinary system tumors. Specifically, the digestive system tumor is liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, or metastatic tumors of these.

[0037] The treatment and / or prevention of tumors described above include one or more of the following: inhibiting tumor growth, inhibiting tumor metastasis, modulating the tumor microenvironment, enhancing anti-tumor immune responses, and / or reducing systemic drug toxicity. Further, the modulation of the tumor microenvironment includes promoting tumor angiogenesis, reprogramming tumor-associated macrophages, reducing immunosuppressive cells, and / or increasing effector immune cell infiltration.

[0038] The present invention also provides the use of the above-described albumin nanocarrier in the preparation of a delivery system for facilitating the escape of delivered therapeutic agents from intracellular vesicles.

[0039] This invention offers the following advantages: It innovatively modifies serum albumin with folic acid and phenylboronic acid, yielding a dual-ligand-modified albumin nanocarrier. This albumin nanocarrier exhibits dual targeting capabilities, significantly improving its accumulation efficiency at tumor sites, particularly in hypersialylated tumors, enabling more precise and efficient cascade targeting. Simultaneously, the unique lysosomal escape mechanism of this nanocarrier synergistically enhances intracellular drug bioavailability, overcoming a key bottleneck in intracellular drug release. This provides a new and effective treatment strategy for malignant tumors, especially refractory tumors insensitive to existing therapies or those that have metastasized. The albumin nanocarrier of this invention, and the nanoparticles prepared based on it and loaded with therapeutic agents, possess excellent biocompatibility and degradability, high safety, and can achieve synergistic delivery of multiple therapeutic agents, providing an effective platform for combination therapy and demonstrating promising application prospects. Attached Figure Description

[0040] Figure 1 Structural characterization of BPF, an albumin nanocarrier modified with folic acid and phenylboronic acid. Figure 1 A is the UV-Vis absorption spectrum of BPF. Figure 1 B is the infrared spectrum of BPF. Figure 1 C is the NMR spectrum of BPF.

[0041] Figure 2 Performance parameters of Reg@BPF nanoparticles. Among them, Figure 2 A is the particle size distribution of Reg@BPF nanoparticles. Figure 2 B represents the polydispersity index (PDI) of Reg@BPF nanoparticles. Figure 2 C represents the potential of the Reg@BPF nanoparticles. Figure 2 D is a TEM image of the Reg@BSA nanoparticles. Figure 2 E is a TEM image of Reg@BPF nanoparticles. Figure 2 E represents the XRD patterns of Reg and Reg@BPF nanoparticles.

[0042] Figure 3 In vitro uptake of BPF. Figure 3 A is a flow cytometry plot of BPF uptake in CT26-luc cells. Figure 3 B is a statistical graph of the mean fluorescence intensity (MFI) of BPF uptake in CT26-luc cells. Figure 3 C is a flow cytometry plot of BPF uptake in HUVECs cells. Figure 3 D is a statistical graph of the average fluorescence intensity of BPF uptake in HUVECs cells. Figure 3 E is a statistical chart of the Pearson coefficient (PCC). Figure 3 F is a statistical graph of the average fluorescence intensity of the flow cytometry APC detection channel after HUVECs cells were stained with acridine orange.

[0043] Figure 4 The in vitro antitumor properties of Reg@BPF nanoparticles. Among them, Figure 4 A represents the cell viability of CT26-luc cells after treatment with different concentrations of Reg@BPF for 48 hours. Figure 4 B represents the cell viability of CT26-luc cells after treatment with different concentrations of BPF for 48 hours. Figure 4 C represents the quantitative analysis of apoptosis in CT26-luc cells using flow cytometry. Figure 4 D represents the statistical count of the number of nodes and network structures in the angiogenesis experiment.

[0044] Figure 5 The efficacy of Reg@BPF nanoparticles in in vivo treatment of peritoneal metastases of colorectal cancer. Among them, Figure 5 A represents the tumor weight of mice in each treatment group. Figure 5 B represents the ascites volume of mice in each treatment group. Figure 5 C represents the survival status of mice in different treatment groups. Figure 5 D represents the change in mouse body weight during treatment. Detailed Implementation

[0045] Current albumin delivery systems suffer from single-targeting and low intracellular delivery efficiency. To improve targeting, albumin nanocarriers are modified by linking folic acid ligands, which may enhance their targeting in epithelial tumor cells. However, single-targeting with folic acid primarily relies on receptor overexpression on the cell surface, lacking the ability to recognize other important targets in the tumor microenvironment, and exhibiting limited targeting efficiency for certain tumors (such as some liver and pancreatic cancers). This invention introduces phenylboronic acid, enabling it to be combined with folic acid for dual-ligand modification of the albumin nanocarrier, thereby increasing the accumulation of the nanocarrier on tumor vascular endothelium and the surface of certain hypersialylated tumor cells. Simultaneously, it enhances the ability to actively promote endocytosis and translocation into tumor cells.

[0046] Building upon this foundation, this invention has developed an albumin carrier modified with folic acid (FA) and phenylboronic acid (PBA) dual ligands. This carrier exhibits dual targeting capabilities, significantly improving the enrichment efficiency of nanocarriers at various tumor sites, particularly enabling more precise and efficient cascade targeting in hypersialylated tumors; it also significantly enhances cellular uptake of nanoparticles. Even more surprisingly, the nanocarrier of this invention possesses a lysosomal escape mechanism, effectively improving intracellular bioavailability after drug loading and overcoming a key bottleneck in intracellular drug release. Due to the reaction requirements of ligand modification, derivatives of folic acid and phenylboronic acid are sometimes required to complete the modification.

[0047] The albumin nanocarrier provided by the present invention comprises nanoparticles formed from serum albumin and / or its functional variants, as well as folic acid and / or folic acid derivatives modified on said serum albumin and / or its functional variants, and phenylboronic acid and / or phenylboronic acid derivatives.

[0048] The serum albumin is selected from one or more of bovine serum albumin, human serum albumin, recombinant serum albumin, and their modified derivatives. Preferably, the serum albumin is bovine serum albumin.

[0049] The connection between folic acid and / or folic acid derivatives, and phenylboronic acid and / or phenylboronic acid derivatives, and albumin includes, but is not limited to, the formation of an amide bond between the carboxyl group of folic acid and / or folic acid derivatives, and the amino group of phenylboronic acid and / or phenylboronic acid derivatives, and the amino group of albumin in the presence of a condensing agent. The condensing agent is selected from carbodiimides, acylurea onium salts, or other carboxyl-activating agents. Preferably, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) are used as condensing agents, the reaction pH is 8-10, and the reaction time is 16-24 hours, preferably 20 hours.

[0050] The molar ratio of folic acid and / or folic acid derivatives on albumin is from albumin to folic acid of 1:0.5 to 1:5. Preferably, the molar ratio of albumin to folic acid is 1:1 to 1:2. The molar ratio of phenylboronic acid and / or phenylboronic acid derivatives on albumin is from albumin to phenylboronic acid of 1:5 to 1:40. Preferably, the molar ratio of albumin to phenylboronic acid is 1:15 to 1:25.

[0051] Specifically, the serum albumin nanocarrier of the present invention can be prepared by the following method:

[0052] a. Dissolve serum albumin in a buffer solution to obtain a serum albumin solution;

[0053] b. Take folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, dissolve them in N,N-dimethylformamide (DMF), add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature in the dark to obtain the activated mixed solution;

[0054] c. The activated mixed solution was slowly added to the serum albumin solution, and the resulting mixed solution was continuously stirred under light-protected conditions. After the reaction was completed, the resulting solution was dialyzed in ultrapure water. After dialyzed, it was filtered through a microporous membrane and freeze-dried to obtain the folic acid and phenylboronic acid modified albumin nanocarrier. The obtained folic acid and phenylboronic acid modified albumin nanocarrier was a pale yellow flocculent solid product.

[0055] The solvent mentioned in step b is N,N-dimethylformamide (DMF).

[0056] Preferably, the phenylboronic acid derivative mentioned in step b is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3-carboxyphenylboronic acid. More preferably, the phenylboronic acid derivative is 4-carboxyphenylboronic acid. The carboxyl group of the phenylboronic acid derivative is used to link the carboxyl group to the amino group of albumin by forming an amide bond in the presence of a condensing agent.

[0057] In one embodiment of the present invention, 5.0 mg of 4-carboxyphenylboronic acid and 3.0 mg of folic acid were dissolved in 1 mL of N,N-dimethylformamide (DMF), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 18.4 mg) and N-hydroxysuccinimide (NHS, 11.6 mg) were added. The mixture was activated at room temperature in the dark for 60 minutes. The activated mixture was then added dropwise to a continuously stirred BSA solution. The mixture was stirred continuously at 25°C in the dark for 20 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in ultrapure water for 48 hours (the dialysate was changed every 8 hours). After dialysis, the solution was filtered through a 0.8 μm microporous membrane and freeze-dried to obtain a pale yellow flocculent solid product, which was an albumin nanocarrier modified with folic acid and phenylboronic acid, and stored at -20°C.

[0058] The present invention also provides nanoparticles loaded with therapeutic agents, which are composed of albumin nanocarriers modified with the above-mentioned dual ligands and therapeutic agents through one or more of the following methods: loading is achieved through electrostatic interaction, hydrophobic interaction, π-π stacking, covalent connection or embedding.

[0059] The therapeutic agent includes one or more of small molecule chemical drugs, nucleic acid drugs, peptide or protein drugs. Preferably, the small molecule chemical drug is an anti-angiogenic drug and / or an anti-tumor drug, wherein the anti-angiogenic drug is selected from drugs acting on the vascular endothelial growth factor signaling pathway or tyrosine kinase inhibitors, and the anti-tumor drug is selected from chemotherapy drugs, molecularly targeted drugs or immunotherapy drugs.

[0060] More preferably, the therapeutic agent comprises a combination of two or more drugs with synergistic therapeutic effects, including the combination of anti-angiogenic drugs and anti-tumor drugs, or the combination of chemotherapy drugs with different mechanisms of action.

[0061] The encapsulation efficiency of the therapeutic agent in the nanoparticles is 50%-95%, and the drug loading is 5%-20%. Preferably, the encapsulation efficiency is greater than 70%, and the drug loading is 5%-15%.

[0062] The nanoparticles have an average hydrated particle size of 10-300 nm and a Zeta potential of -50 mV to +30 mV. Preferably, the particle size is 50-200 nm and the Zeta potential is -25 mV to -15 mV.

[0063] Furthermore, the above-mentioned nanoparticles can be prepared by the following methods:

[0064] a. Weigh the stock solution prepared by dissolving the agent to be treated in a solvent to obtain the drug phase;

[0065] b. Take the albumin nanocarrier described in any one of claims 7 to 9, and disperse it together with a stabilizer in ultrapure water to obtain the carrier aqueous phase;

[0066] c. The drug phase is slowly added dropwise to the aqueous phase of the carrier to obtain a mixture. The mixture is then homogenized using an ultrasonic cell disruptor. Subsequently, the resulting suspension is transferred to a dialysis bag and dialyzed in ultrapure water to remove organic solvents. The dialysate is concentrated by ultrafiltration and then filtered through a 0.22 μm sterile filter membrane to obtain the nanoparticle suspension. The prepared nanoparticle suspension can be stored at -20℃ for later use, or it can be prepared into a lyophilized formulation or a pharmaceutically acceptable dosage form using appropriate methods.

[0067] In step a, the solvent is at least one of DMSO, water, or methanol.

[0068] The stabilizer mentioned in step b is polyvinylpyrrolidone.

[0069] In step c, the mixture is placed in an ice bath and treated with an ultrasonic cell disruptor for 3-10 minutes to homogenize it.

[0070] In one example of this invention, Reg@BPF NPs loaded with regorafenib were prepared using the aforementioned nanoprecipitation-dialysis method. Specifically, 5.0 mg of regorafenib was accurately weighed and dissolved in 0.5 mL of DMSO to prepare a 10 mg / mL stock solution (drug phase). 50 mg of the BPF carrier prepared in Example 1 and 10 mg of the stabilizer polyvinylpyrrolidone (PVP) were weighed and dispersed together in 25 mL of ultrapure water to obtain the carrier aqueous phase. Under magnetic stirring (800 rpm), the drug phase was slowly added dropwise to the carrier aqueous phase at a rate of 0.1 mL / min using a microinjection pump. After the addition was complete, the mixture was placed in an ice bath and homogenized using an ultrasonic cell disruptor for 5 minutes. Subsequently, the resulting suspension was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in ultrapure water for 24 hours to remove organic solvents. The dialysate was concentrated using a 100 kDa ultrafiltration centrifuge tube and then filtered through a 0.22 μm sterile filter membrane to obtain a suspension of Reg@BPF NPs.

[0071] In another embodiment of the present invention, nanoparticles loaded with paclitaxel, doxorubicin, curcumin, and sofilranil were also prepared using the above method.

[0072] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described nanoparticles and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated as an injection, a lyophilized formulation, or a gel.

[0073] The present invention also relates to the use of the nanocarrier or pharmaceutical composition in the preparation of medicaments for the treatment and / or prevention of cancer.

[0074] Experiments show that the folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives dual-ligand modified serum albumin nanocarriers of this invention, significantly enhance cellular uptake of serum albumin nanocarrier particles in tumor cells and HUVECs through an active targeting mechanism. Simultaneously, the BPF nanoparticles can effectively escape from lysosomes into the cytoplasm; this unexpected characteristic can better synergistically address the key bottleneck of intracellular drug release, effectively improving drug utilization efficiency. In further animal experiments, the Reg@BPF NPs prepared in this invention showed significantly improved efficacy in a peritoneal metastatic colorectal cancer model. This demonstrates that the synergistic effect of the dual-ligand enhancing targeting and the unique lysosomal escape effect of the nanocarriers of this invention can effectively improve intracellular drug utilization, thereby enhancing therapeutic efficacy and providing a new and effective treatment for malignant tumors, especially those refractory tumors that are insensitive to existing therapies or have metastasized.

[0075] The present invention will be explained and described below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0076] Example 1: Synthesis and Characterization of BPF Nanocarriers

[0077] 1. Synthesis Method

[0078] 100 mg of bovine serum albumin (BSA, purchased from Albemarle (Shanghai) Biotechnology Co., Ltd.) was dissolved in 10 mL of sodium carbonate / sodium bicarbonate buffer at pH 10.0. Separately, 5.0 mg of 4-carboxyphenylboronic acid (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 3.0 mg of folic acid (FA, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were dissolved in 1 mL of N,N-dimethylformamide (DMF), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 18.4 mg) and N-hydroxysuccinimide (NHS, 11.6 mg) were added. The mixture was activated at room temperature in the dark for 60 minutes. The activated 4-carboxyphenylboronic acid / FA mixture was then added dropwise to the continuously stirred BSA solution. The mixture was reacted at 25°C in the dark with continuous stirring for 20 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in ultrapure water for 48 hours (changing the dialysate every 8 hours). After dialysis, the solution was filtered through a 0.8 μm microporous membrane and freeze-dried to obtain a pale yellow flocculent solid product, which was a folic acid and phenylboronic acid modified albumin nanocarrier. This was designated as BPF nanocarrier and stored at -20℃. Unmodified BSA nanocarriers (BSA NPs) were prepared using the same method and stored at -20℃.

[0079] 2. Characterization Results

[0080] The structure of the obtained BPF nanocarrier was characterized as follows:

[0081] UV-Vis absorption spectroscopy: The absorption peak intensity of BPF at 280 nm is significantly enhanced compared to BSA (see...). Figure 1 A), this enhancement originates from the π-π* electron transitions of the aromatic rings of phenylboronic acid and folic acid.

[0082] Fourier transform infrared (FTIR) spectroscopy: The absorption peaks of BPF at 1650 cm⁻¹ (amide I band, C=O stretching vibration) and 1530 cm⁻¹ (amide II band, NH bending vibration) are significantly stronger than those of BSA (see [link to FTIR]). Figure 1 B) indicates that a new amide bond has been formed.

[0083] Modification ratio determination: Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the elemental ratios, and it was calculated that each BSA molecule was linked to approximately 15 PBA molecules on average. Molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and it was calculated that each BSA molecule was linked to approximately 2 FA molecules on average. Therefore, the molar ratio of FA to PBA in this support is approximately 1:7.5.

[0084] Example 2: Preparation and characterization of Reg@BPF nanoparticles

[0085] 1. Preparation method

[0086] Regorafenib (Reg)-loaded nanoparticles were prepared using a nanoprecipitation-dialysis method. 5.0 mg of regorafenib was accurately weighed and dissolved in 0.5 mL of DMSO to prepare a 10 mg / mL stock solution (drug phase). 50 mg of the BPF carrier prepared in Example 1 and 10 mg of the stabilizer polyvinylpyrrolidone (PVP) were weighed and dispersed together in 25 mL of ultrapure water to obtain the carrier aqueous phase. Under magnetic stirring (800 rpm), the drug phase was slowly added dropwise to the carrier aqueous phase at a rate of 0.1 mL / min using a microinjection pump. After the addition was complete, the mixture was placed in an ice bath and homogenized using an ultrasonic cell disruptor for 5 minutes. Subsequently, the resulting suspension was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in ultrapure water for 24 hours to remove organic solvents. The dialysate was concentrated using a 100 kDa ultrafiltration centrifuge tube and finally filtered through a 0.22 μm sterile filter membrane to obtain the Reg@BPF NPs suspension. Using the same method, but without adding regorafenib, blank BPF nanoparticles were prepared as a control.

[0087] 2. Characterization Results

[0088] Physicochemical properties of Reg@BPF NPs were characterized:

[0089] Particle size, polydispersity index (PDI), and zeta potential: determined by dynamic light scattering (DLS), the average hydration kinetic diameter of Reg@BPF NPs was 117.5 ± 2.5 nm, the PDI was 0.14 ± 0.02, and the zeta potential was -20.9 ± 0.8 mV (see [reference]). Figure 2 A, 2B, 2C) indicate that the particle size is uniform and the system is stable. The particle size of Reg@BPF NPs showed no significant change after being stored at -20℃ for six months.

[0090] Morphological observation: Transmission electron microscopy (TEM) images showed that Reg@BPF NPs were spherical with a core-shell structure, well-dispersed, and their particle size was consistent with the DLS results (see [link to TEM image]). Figure 2 D, 2E).

[0091] Drug crystal form analysis: X-ray diffraction (XRD) patterns showed that the sharp crystal diffraction peaks of regorafenib active pharmaceutical ingredient completely disappeared in the Reg@BPF NPs pattern, presenting as an amorphous diffuse mass (see [link to XRD pattern]). Figure 2 F) proves that the drug is highly dispersed in the nanocarrier in an amorphous state.

[0092] Drug loading performance: The encapsulation efficiency of Reg@BPF NPs was (77.57 ± 1.20)% and the drug loading was (7.60 ± 0.10)%, as determined by ultraviolet spectrophotometry.

[0093] Example 3: Preparation of nanoparticles by loading other drugs onto BPF nanocarriers

[0094] Paclitaxel loaded onto BPF nanocarriers: 5.0 mg of paclitaxel (PTX) was weighed and dissolved in 0.5 mL of DMSO to prepare a stock solution of 10 mg / mL. Subsequent preparation steps, including carrier dispersion, dropwise addition, sonication, dialysis, concentration, and filtration, were identical to the preparation method of Reg@BPF NPs in Example 2. The resulting product was designated as PTX@BPF NPs. This example demonstrates that the BPF carrier platform is suitable for loading other hydrophobic antitumor drugs such as paclitaxel. Using UV spectrophotometry, the encapsulation efficiency of PTX@BPF NPs was (70.78 ± 2.40)%, and the drug loading was (6.93 ± 0.16)%.

[0095] Doxorubicin loaded onto BPF nanocarriers: Loading was achieved through a combination of borate bonding and electrostatic adsorption. Specifically, 5.0 mg of doxorubicin was accurately weighed and dissolved in 0.5 mL of ultrapure water / methanol mixture to prepare a drug stock solution. 50 mg of BPF carrier and 10 mg of PVP were co-dispersed in 25 mL of ultrapure water to prepare an aqueous phase. Under magnetic stirring at 800 rpm, the drug stock solution was slowly added dropwise to the aqueous phase at 0.1 mL / min using a micro-injection pump. Homogenization was performed by sonication in an ice bath for 5 min, followed by dialyzing through a 3.5 kDa dialysis bag for 24 h to remove residual organic solvent, yielding stable doxorubicin@BPF NPs nanoparticles.

[0096] Curcumin was loaded onto BPF nanocarriers through a combination of coordination with vicinal diol of phenylboronic acid and hydrophobic interactions. The method involved accurately weighing 5.0 mg of curcumin and dissolving it in 0.5 mL of DMSO to prepare a stock solution. Subsequent dropwise addition, sonication, and dialysis processes were identical to those used in the regorafenib loading system in Example 2, resulting in stable curcumin@BPF NPs nanoparticles.

[0097] Sorafenib loaded onto BPF nanocarriers: Loading is mainly accomplished through hydrophobic interactions. The specific method is the same as the aforementioned paclitaxel drug loading preparation process, but with sorafenib replacing the hydrophobic antitumor drug paclitaxel. The same ratio and preparation parameters are used to prepare a stable drug-loaded nanosystem, sorafenib@BPF NPs nanoparticles.

[0098] Example 4: Evaluation of cellular uptake and lysosomal escape capabilities

[0099] 1. Cellular uptake experiment

[0100] Using Cy5-NHS-labeled nanoparticles (BPF-Cy5) as probes, the uptake efficiency of these nanoparticles by CT26-luc colon cancer cells or human umbilical vein endothelial cells (HUVECs) was quantitatively assessed by flow cytometry. Cells were seeded in 12-well plates, and after adhesion, the medium was replaced with serum-free medium containing either equal amounts of BPF-Cy5 or ligand-free BSA-Cy5, and incubated at 37°C for 4 hours. After incubation, cells were washed with PBS, trypsinized, and resuspended. The mean fluorescence intensity (MFI) of the cells was immediately measured by flow cytometry. A free Cy5-NHS group was included as a control.

[0101] Flow cytometry results showed that, compared with the BSA-Cy5 group, the mean fluorescence intensity (MFI) of CT26 cells in the BPF-Cy5 treatment group was increased by approximately 1.5 times (p < 0.05) (see [link to relevant documentation]). Figure 3 A, Figure 3 B). Pre-incubation of cells with excess free folic acid and phenylboronic acid to block the corresponding receptor significantly inhibited BPF-Cy5 uptake. This result demonstrates that FA and PBA dual-ligand modification significantly enhanced cellular uptake of nanoparticles through an active targeting mechanism. Increased BPF uptake was also observed in HUVECs (see [link to study]). Figure 3 C, Figure 3 D).

[0102] 2. Lysosomal escape experiment

[0103] Human umbilical vein endothelial cells (HUVECs) were co-incubated with coumarin 6 (Cou6)-labeled nanoparticles (Cou6@BPF NPs or Cou6@BSA NPs) for different times (2, 4, 8 h). After incubation, lysosomes were labeled with LysoTracker Red, and cell nuclei were labeled with Hoechst 33342. Z-Stack sequence images were observed and captured using confocal microscopy. The Pearson correlation coefficient (PCC) between Cou6 fluorescence and LysoTracker fluorescence in the cytoplasmic region (excluding the nucleus) was calculated using ImageJ software to quantitatively analyze the degree of co-localization.

[0104] Confocal microscopy observation and quantitative analysis showed (see...) Figure 3E), Cou6@BSA NPs were highly colocalized with lysosomes throughout the incubation period (PCC > 0.5 at 8h). Cou6@BPF NPs, on the other hand, were partially localized to lysosomes in the early stages of incubation (2h, 4h), but their colocalization coefficient with lysosomes decreased significantly at 8h (PCC dropped to approximately 0.27). These results indicate that BPF nanoparticles can effectively escape from lysosomes into the cytoplasm, and the mechanism may be related to the pH-responsive lysosomal membrane disruption effect of phenylboronic acid (PBA).

[0105] 3. Lysosomal membrane permeability test

[0106] To assess the impact of nanoparticles on lysosomal membrane integrity, changes in acridine orange (AO) fluorescence intensity were detected by flow cytometry. HUVECs were treated with PBS, BSA, or BPF, respectively. After treatment, cells were stained with AO at 37°C in the dark for 20 minutes, washed with PBS, digested, resuspended, and analyzed by flow cytometry (APC channel). The decrease in mean fluorescence intensity (MFI) compared to the untreated control group indicated increased lysosomal membrane permeability.

[0107] Experimental results show that ( Figure 3 (F) The AO fluorescence signal of HUVECs treated with BPF was significantly weakened. This indicates that the BPF nanocarrier leads to increased lysosomal membrane permeability, i.e., disruption of lysosomal integrity. This mechanism provides direct evidence for the efficient "lysosomal escape" of BPF nanoparticles observed above.

[0108] Example 5: Evaluation of in vitro anti-tumor cell proliferation and anti-angiogenic effects

[0109] 1. Toxicity to tumor cells

[0110] The cytotoxicity of Reg@BPF NPs to CT26 cells was evaluated using the CCK-8 assay. Cells were co-cultured with different concentrations of Reg@BPF NPs for 48 hours, and cell viability was assessed. Results showed that Reg@BPF NPs exhibited dose-dependent inhibitory effects on the proliferation of CT26 cells, while the blank BPF vector showed no significant cytotoxicity within the tested concentration range (see [link to study]). Figure 4 A, 4B).

[0111] 2. Inducing tumor cell apoptosis

[0112] Apoptosis was detected by flow cytometry using Annexin V-FITC / PI double staining. After treating CT26 cells with 10 μM Reg@BPF NPs for 24 hours, the total apoptosis rate (including early and late apoptosis) reached 82.7 ± 1.5% (see Annexin V-FITC / PI double staining). Figure 4 C).

[0113] 3. Anti-angiogenic effect (in vitro lumen formation experiment)

[0114] The effect of Reg@BPF NPs on the formation of vascular-like structures in human umbilical vein endothelial cells (HUVECs) was evaluated. HUVECs were seeded in Matrigel-coated well plates and treated with medium containing different concentrations of Reg@BPF NPs (5, 7.5, 10 μM). After 12 hours of incubation, Calcein-AM staining was performed, and the cells were observed and photographed under a fluorescence microscope. The number of luminal junctions was quantitatively analyzed using ImageJ software. The results showed that 10 μM Reg@BPF NPs significantly inhibited the formation of luminal networks in HUVECs, reducing the network structure by 80.3% (compared to the PBS control group) (see [link to relevant documentation]). Figure 4 D).

[0115] Example 6: In vivo efficacy evaluation of drugs against peritoneal metastatic colorectal cancer

[0116] 1. Animal model establishment and drug administration

[0117] A peritoneal metastatic cancer model was established in female BALB / c mice by intraperitoneal injection of luciferase-labeled CT26 colon cancer cells (CT26-luc). On day 4 post-inoculation, tumor-bearing mice were randomly divided into 5 groups (n=6 / group): (1) PBS control group; (2) BPF vector group (100 mg / kg); (3) free regorafenib group (8 mg / kg); (4) Reg@BSA NPs (8 mg / kg based on regorafenib); (5) Reg@BPF NPs group (8 mg / kg based on regorafenib). All groups were administered the drugs via intraperitoneal injection every 2 days for a total of 3 times.

[0118] 2. Efficacy evaluation results

[0119] Tumor weight and ascites: Mice were sacrificed after the last administration, and all tumor nodules in the peritoneal cavity were collected and weighed. The mean tumor weight in the Reg@BPF NPs group was 0.45 ± 0.09 g, significantly lower than that in the Reg@BSA NPs group (0.72 ± 0.11 g, p < 0.05), the free Reg group (0.94 ± 0.25 g, p < 0.01), and the PBS control group (1.55 ± 0.38 g) (see [reference]). Figure 5 A). Meanwhile, all mice in the PBS control group produced large amounts of ascites (1.73 ± 0.07 mL), while the Reg@BPF NPs group almost completely inhibited ascites production (0.25 ± 0.08 mL) (see [link]). Figure 5 B).

[0120] Survival: Mouse survival was monitored and Kaplan-Meier survival curves were plotted. Reg@BPF NPs treatment significantly prolonged the median survival of tumor-bearing mice to 38 days, superior to the Reg@BSA NPs group (30 days), the free Reg group (25 days), and the PBS control group (20 days) (see [link to relevant documentation]). Figure 5 C).

[0121] Safety observation: Mouse body weight was monitored regularly throughout the treatment period. The body weight of mice in the Reg@BPF NPs group remained stable and showed no significant difference compared to the control group (see [link]). Figure 5 (D) indicates that the nano-formulation has low systemic toxicity at effective doses.

[0122] The above experimental results demonstrate that this invention successfully prepared serum albumin nanocarriers modified with FA and PBA dual ligands, as well as nanoparticles loaded with drugs such as Regorafenib (Reg) and paclitaxel. The obtained nanoparticles are uniform in size, the system is stable, and the dispersibility is good, with excellent drug loading and encapsulation efficiency. X-ray diffraction showed that the loaded drugs were highly dispersed within the nanocarriers.

[0123] Cellular experiments showed that serum albumin nanocarriers modified with FA and PBA dual ligands significantly enhanced the uptake of nanocarrier particles in tumor cells and HUVECs through an active targeting mechanism. Simultaneously, BPF nanoparticles could effectively escape from lysosomes into the cytoplasm, a mechanism possibly related to the lysosomal membrane disruption caused by the pH-responsiveness of phenylboronic acid (PBA). This characteristic can better address the key bottleneck of intracellular drug release.

[0124] In further animal experiments, the Reg@BPF NPs nanoparticles prepared in this invention showed significantly improved efficacy in a peritoneal metastatic colorectal cancer (PMC) model, thus providing a new and effective treatment strategy for malignant tumors, especially those refractory tumors that are insensitive to existing therapies or have metastasized, and has special application value.

Claims

1. An albumin nanocarrier, characterized in that: The albumin nanocarrier comprises nanoparticles formed from serum albumin and / or its functional variants, as well as folic acid and / or folic acid derivatives modified on the serum albumin and / or its functional variants, and phenylboronic acid and / or phenylboronic acid derivatives.

2. The albumin nanocarrier according to claim 1, characterized in that: The folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, are modified by being covalently or non-covalently linked to the serum albumin or its functional variants; further, the serum albumin is bovine serum albumin, human serum albumin, recombinant serum albumin, or its modified derivatives; further, the modification ratio of folic acid and / or folic acid derivatives on albumin is 1:0.5-5 molar ratio of albumin to folic acid; preferably, the molar ratio of albumin to folic acid is 1:1-2; further, the modification ratio of folic acid and / or phenylboronic acid derivatives on albumin is 1:5-40 molar ratio of albumin to phenylboronic acid. Preferably, the molar ratio of albumin to phenylboronic acid is 1:15-25.

3. The albumin nanocarrier according to claim 1, characterized in that: The folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, are linked to the corresponding functional groups on the serum albumin via their carboxyl and / or amino groups; further, the linkage is achieved by at least one of the following: forming an amide bond through a condensation reaction of a carboxyl group and an amino group, forming a Schiff base bond through an aldehyde group and an amino group, or forming a borate ester bond through a borate group and a cis-diol.

4. The albumin nanocarrier according to claim 3, characterized in that: The folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, are linked to albumin by forming an amide bond between the carboxyl group of folic acid and / or folic acid derivatives, and the amino group of phenylboronic acid and / or phenylboronic acid derivatives and the amino group of albumin; further, the phenylboronic acid derivative is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, or 3-carboxyphenylboronic acid.

5. The albumin nanocarrier according to claim 4, characterized in that: The connection reaction is carried out in the presence of a coupling agent, which is a carbodiimide, an acylurea salt, or other condensing agent capable of activating the carboxyl group; preferably, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

6. The albumin nanocarrier according to any one of claims 1 to 5, characterized in that... Prepared by the following method: a. Dissolve serum albumin in a buffer solution to obtain a serum albumin solution; b. Take folic acid and / or folic acid derivatives, as well as phenylboronic acid and / or phenylboronic acid derivatives, dissolve them in a solvent, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activate at room temperature in the dark to obtain the activated mixed solution; c. Slowly add the activated mixed solution to the serum albumin solution, and continuously stir the resulting mixed solution under light-protected conditions. After the reaction is completed, dialyze the resulting solution in ultrapure water. After dialyzing, filter through a microporous membrane and freeze-dry to obtain the folic acid and phenylboronic acid modified albumin nanocarrier.

7. A nanoparticle, characterized in that: The therapeutic agent is loaded onto the albumin nanocarrier as described in any one of claims 1 to 6.

8. The nanoparticles according to claim 7, characterized in that: The loading is achieved through at least one of electrostatic interaction, hydrophobic interaction, π-π stacking, covalent connection, or embedding.

9. The nanoparticles according to claim 7, characterized in that, The therapeutic agent is selected from one or more of small molecule chemical drugs, nucleic acid drugs, peptide or protein drugs, or contrast agents; further, the therapeutic agent includes anti-angiogenic drugs and / or anti-tumor drugs; even further, the anti-angiogenic drug includes drugs acting on the vascular endothelial growth factor signaling pathway or tyrosine kinase inhibitors; even further, the anti-tumor drug includes one or more of chemotherapy drugs, molecularly targeted drugs, or immunotherapy drugs; preferably, the therapeutic agent is regorafenib and / or paclitaxel.

10. The nanoparticles according to any one of claims 7 to 9, characterized in that... It is prepared by the following method: a. Weigh the drug to be treated and dissolve it in a solvent to prepare a stock solution, thus obtaining the drug phase; b. Take the albumin nanocarrier described in any one of claims 7 to 9, and disperse it together with a stabilizer in ultrapure water to obtain the carrier aqueous phase; c. The drug phase is slowly added dropwise to the aqueous phase of the carrier to obtain a mixture. The mixture is homogenized by ultrasonic cell disruptor. Then, the resulting suspension is transferred to a dialysis bag and dialyzed in ultrapure water to remove organic solvents. The dialysate is concentrated by ultrafiltration and then filtered through a 0.22 μm sterile filter membrane to obtain a nanoparticle suspension. Furthermore, the solvent in step a is at least one of DMSO, water, or methanol; Furthermore, the stabilizer mentioned in step b is polyvinylpyrrolidone; Furthermore, in step c, the mixture is placed in an ice bath and treated with an ultrasonic cell disruptor for 3-10 minutes to homogenize it.

11. The nanoparticles according to any one of claims 7 to 10, characterized in that, The average hydrated particle size of the nanoparticles is between 10 nm and 300 nm, and the zeta potential is between -50 mV and +30 mV.

12. A pharmaceutical composition, characterized in that, The nanoparticles comprising a therapeutically effective amount of any one of claims 7 to 11, and one or more pharmaceutically acceptable carriers, excipients, stabilizers, or diluents.

13. The use of the albumin nanocarrier according to any one of claims 1 to 6, the nanoparticles according to any one of claims 7 to 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating and / or preventing cancer; further, the cancer is a tumor that overexpresses folic acid receptors and / or is excessively sialylated; even further, the cancer is at least one of an epithelial tumor, a digestive system tumor, a respiratory system tumor, a reproductive system tumor, or a urinary system tumor; and still further, the digestive system tumor is liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, or metastatic tumors thereof.

14. The application according to claim 13, characterized in that: The treatment and / or prevention of tumors includes one or more of the following: inhibiting tumor growth, inhibiting tumor metastasis, modulating the tumor microenvironment, enhancing anti-tumor immune responses, and / or reducing systemic drug toxicity; further, the modulation of the tumor microenvironment includes promoting tumor angiogenesis, reprogramming tumor-associated macrophages, reducing immunosuppressive cells, and / or increasing effector immune cell infiltration.

15. The use of the albumin nanocarrier according to any one of claims 1 to 6 in the preparation of a delivery system for facilitating the escape of delivered therapeutic agents from intracellular vesicles.

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