Exosome engineered targeting drug delivery system and preparation method and application thereof

Abstract

The invention discloses an exosome engineered targeted drug delivery system and a preparation method and application thereof, and belongs to the field of biomedices.The exosome engineered targeted drug delivery system is prepared by mixing and reacting surface modified exosome and pH response type drug controlled release nanoparticles; the pH response type drug controlled release nanoparticles react with the surface modified exosome through a click chemical reaction; the surface of the membrane of which the surface is modified with the exosome is modified with a maleimide group and an azide group; the novel nano-drug has pH responsiveness; the click chemical reaction is thiol-ene click reaction. Bifunctional groups (MAL and N3) are directionally grafted on the surface of an exosome membrane, uniform drug particles are prepared in combination with a micro-fluidic technology, an efficient bimodal targeting drug delivery system is successfully constructed, and remarkable technical advantages are shown in the aspects of improving targeting precision, enhancing antitumor activity, controlling drug release, inhibiting tumor metastasis and the like; and a new technical approach is provided for precise treatment of adenoid cystic carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to an engineered exosome-targeted drug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Adenoid cystic carcinoma (ACC) is a common malignant tumor in the salivary gland, with characteristics such as strong local invasiveness and high distant metastasis rate. Among them, the lung is the most common metastatic site of ACC. Clinical statistical data shows that about 40% - 60% of ACC patients will develop lung metastasis within 5 - 20 years after diagnosis, seriously affecting the prognosis and quality of life of patients. In recent years, with the in-depth study of the tumor microenvironment, exosomes, as a key medium for intercellular communication, have increasingly attracted attention for their important role in the "seed - soil" theory of tumor metastasis.

[0003] Exosomes are a type of membranous vesicle with a diameter of about 30 - 150 nm, which are released into the extracellular environment after the fusion of intracellular multivesicular bodies and the plasma membrane. As the "messengers" for intercellular information transmission, exosomes can carry bioactive molecules such as proteins and nucleic acids, and play an important role in tumor microenvironment regulation, immune regulation, tumor metastasis, etc. Research shows that exosomes derived from tumor cells can serve as metastatic precursor substances and provide a suitable "soil" for distant metastasis, namely the so-called "pre-metastatic niche".

[0004] Heparan Sulfate Proteoglycans (HSPGs) are an important class of cell surface molecules, which consist of a core protein and covalently linked heparan sulfate glycosaminoglycan chains. The members of the HSPGs family mainly include Syndecans (SDCs) and Glypicans (GPCs). During tumor progression, SDC4 and GPC5 have been proven to be closely related to the invasion and metastasis of various tumors.

[0005] In the prior art, there has been research on isolating and purifying exosomes from adenoid cystic carcinoma high-metastasis potential cell lines (ACC-M) and low-metastasis potential cell lines (SACC-83, ACC-2) using the exoEasy Maxi Kit (Qiagen). Through Westernblot technology analysis, it was found that the expression levels of HSPGs (especially SDC4 and GPC5) in exosomes derived from ACC-M were significantly higher than those in exosomes derived from low-metastasis cell lines. Transwell migration and invasion experiments showed that exosomes derived from ACC-M could significantly promote the migration and invasion abilities of low-metastasis cell lines SACC-83 and ACC-2.

[0006] Immunohistochemistry and Real-time PCR results showed that SDC4 protein was highly expressed in adenoid cystic carcinoma tissues and cell lines, and was closely related to perineural invasion. The expression of GPC5 in the highly metastatic cell line (ACC-M) was significantly higher than that in the low metastatic cell line, and the expression of GPC5 in adenoid cystic carcinoma tissues with lung metastasis was significantly higher than that in cases without lung metastasis.

[0007] Through RNA interference technology, the researchers constructed shRNA interference plasmids targeting the silencing of HSPGs (SDC4, GPC5) gene expression and successfully established the ACC-M-silenced cell line with gene silencing. Experiments confirmed that after inhibiting the expression of HSPGs genes, the ability of ACC-M-derived exosomes to promote the migration and invasion of SACC-83 and ACC-2 cells decreased significantly, and the uptake efficiency of these exosomes by vascular endothelial cells also decreased significantly.

[0008] Through the search of the Therapeutic Target Database drug target database, it has been found that GPC-3298306 has a targeting effect on the GPC gene. Literature search showed that trastuzumab and panitumumab could inhibit the expression of SDC4 gene in other tumors or cell lines. However, the application research of these targeted drugs in the treatment of adenoid cystic carcinoma metastasis is still in its initial stage.

[0009] Although the above studies have shown that exosomal HSPGs play an important role in the lung metastasis of adenoid cystic carcinoma and inhibiting the expression of HSPGs can block the metastasis process, the existing technology still has significant limitations in the following aspects: When exosomes are used as carriers currently, there is a lack of effective targeting modification strategies, resulting in low targeting specificity. Research has shown that the uptake rate of unmodified ACC-M-derived exosomes by vascular endothelial cells is only 38.7%, and a large number of exosomes are absorbed by non-target tissues such as the liver and spleen during in vivo circulation, seriously reducing the treatment efficiency.

[0010] The existing technology mainly passively loads drugs into exosomes through simple co-incubation or sonication. Not only is the loading efficiency low (usually <30%), but the release process is difficult to precisely control, resulting in premature release of drugs in non-target tissues or slow release in target tissues.

[0011] Traditional exosome surface modification mostly relies on single chemical cross-linking methods, lacking multifunctional and controllable modification strategies, and it is difficult to achieve multi-target recognition and stimulus-responsive drug release.

[0012] Existing targeted drugs such as trastuzumab and GPC-3298306 lack an effective delivery system, resulting in a half-maximal inhibitory concentration (IC50) as high as 12.5 μM, which not only reduces the therapeutic effect but may also cause serious systemic toxic side effects.

[0013] The heterogeneity of the tumor microenvironment (such as changes in pH value and enzyme activity) poses challenges to the targeted delivery and drug release of exosomes, and the existing technology lacks the design of an intelligent response mechanism for microenvironment characteristics. Summary of the Invention

[0014] The present invention discloses an exosome-engineered targeted drug delivery system, a preparation method thereof, and an application thereof, which solve the technical problems of insufficient exosome targeting, low precision of drug loading and release, low efficiency of the targeted drug delivery system, and insufficient response to the complexity of the in vivo microenvironment in the prior art.

[0015] The exosome-engineered targeted drug delivery system disclosed by the present invention is prepared by mixing and reacting surface-modified exosomes with pH-responsive drug-controlled release nanoparticles; Preferably, the pH-responsive drug-controlled release nanoparticles react with the surface-modified exosomes through click chemistry reaction.

[0016] Preferably, the membrane surface of the surface-modified exosomes is modified with maleimide groups and azide groups.

[0017] Preferably, the novel nano-drug has pH responsiveness.

[0018] Preferably, the click chemistry reaction is a thiol-ene click reaction.

[0019] Preferably, the pH-responsive drug-controlled release nanoparticles contain a metal-organic framework and a targeted drug, and the decomposition rate of the metal-organic framework in an environment with a pH value of 4.5 - 5.5 is higher than that in a physiological pH environment.

[0020] Preferably, the maleimide group is inserted into the exosome lipid bilayer membrane through phosphatidylethanolamine-polyethylene glycol-maleimide; the azide group modifies the extracellular domain of exosome membrane protein CD63 through NHS-polyethylene glycol-azide.

[0021] Preferably, the molecular weight of polyethylene glycol in the phosphatidylethanolamine-polyethylene glycol-maleimide is 1000 - 3000, and the insertion density of the phosphatidylethanolamine-polyethylene glycol-maleimide on the exosome membrane surface is 0.5 - 1.5 molecules per square nanometer.

[0022] Preferably, the molecular weight of polyethylene glycol in the NHS-polyethylene glycol-azide is 200-600, and the molar ratio of the NHS-polyethylene glycol-azide to CD63 protein is 1:1.0-1.5.

[0023] Preferably, the metal-organic framework is zeolitic imidazolate framework-8, the targeted drug is GPC-3298306, and the drug loading amount of the zeolitic imidazolate framework-8 is 15-30 wt%.

[0024] Preferably, the preparation method of the zeolitic imidazolate framework-8 comprises the following steps: Dissolve a zinc salt and 2-methylimidazole in water at a molar ratio of 1:2, and stir at room temperature for 0.5-2 hours to obtain a precursor solution; Mix the precursor solution and a dimethyl sulfoxide solution of GPC-3298306 through a microfluidic device at a flow rate ratio of 1:3, with a total flow rate of 8-15 mL / min, and collect the particles; Centrifuge, wash, and dry the particles to obtain the drug-loaded nanoparticles of zeolitic imidazolate framework-8.

[0025] Preferably, the thiol-ene click reaction is carried out under ultraviolet light irradiation, the wavelength of the ultraviolet light is 350-380 nm, the light intensity is 5-15 mW / cm², and the irradiation time is 20-40 s.

[0026] The preparation method of the above-mentioned targeted drug delivery system disclosed by the present invention comprises the following steps: Extract exosomes; Modify maleimide groups and azide groups on the surface of the exosomes to obtain surface-modified exosomes; Prepare pH-responsive drug-controlled release nanoparticles; Connect the surface-modified exosomes and the nanoparticles through click chemistry reaction to obtain the engineered targeted drug delivery system of exosomes.

[0027] Preferably, the step of modifying maleimide groups and azide groups on the surface of the exosomes comprises: Incubate the exosomes with phosphatidylethanolamine-polyethylene glycol-maleimide at a concentration of 0.05-0.15 mg / mL at 20-30 °C for 1-3 hours, with a magnetic stirring rate of 100-140 rpm, to obtain maleimide-modified exosomes; Activate the surface of the maleimide-modified exosomes with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then react with NHS-polyethylene glycol-azide at a molar ratio of 1:1.0-1.5 for 3-5 hours, and purify by gel chromatography to obtain the surface-modified exosomes.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: (1) By constructing a bimodal targeted drug delivery system through the directional grafting of bifunctional groups (MAL and N3) on the surface of exosome membranes, the present invention significantly improves the targeting accuracy and drug delivery efficiency. Performance test data shows that compared with the click efficiency of 56.7% in Comparative Example 1 using only a single modification, the click efficiency of the examples of the present invention is as high as 88.5% - 94.7%, an increase of more than 36.4%, indicating that the bimodal orthogonal modification strategy of the present invention improves the precision of drug delivery.

[0029] (2)The present invention combines microfluidic technology to prepare uniform drug particles, effectively solving the problem of uneven particle distribution caused by the traditional solvent displacement method.

[0030] (3)The pH-responsive drug controlled release system designed by the present invention exhibits excellent pH-responsive release ability, with a drug release rate exceeding 90% under the conditions of simulating the tumor microenvironment (pH 5.0), while the release rate within 24 hours is less than 13% under physiological pH (7.4) conditions.

[0031] (4)The present invention shows significant advantages in the in vivo anti-tumor effect. The treatment group of Example 5 achieved a 91.3% reduction rate in the number of lung metastases in the mouse lung metastasis model, which is 148.1% higher than the positive control (36.8%). At the same time, it achieved an 87.2% reduction rate in tumor volume and a 92.5% extension rate of survival period. Especially in inhibiting exosome-mediated metastasis, Example 5 reached an inhibition rate of 90.6%.

[0032] (5)The targeted drug delivery system of the present invention exhibits excellent performance in terms of intracellular drug enrichment and tumor tissue penetration ability. The drug concentration in the SACC-83 cells of the example group is significantly higher than that of the control group. Especially, the highest drug concentration in Example 5 reached 1205.4 pmol / 10 6 cells, which is 4.24 times that of the positive control. In terms of the tumor penetration depth, the example group far exceeds the comparative example group and the positive control. The penetration depth of Example 5 reached 274 μm, which is 182.5% higher than that of the positive control.

[0033] In summary, by the directional grafting of bifunctional groups (MAL and N3) on the surface of exosome membranes and combining microfluidic technology to prepare uniform drug particles, the present invention successfully constructs an efficient bimodal targeted drug delivery system, which shows significant technical advantages in improving targeting accuracy, enhancing anti-tumor activity, controlling drug release and inhibiting tumor metastasis, providing a new technical approach for the precision treatment of adenoid cystic carcinoma. Detailed implementation manners

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0035] Example 1 Take the exoEasy Maxi Kit (Qiagen), and treat the ACC-M cell supernatant at 4°C. First, perform gradient centrifugation, and the centrifugation parameters are 300g×10min, 2000g×30min, and 10000g×1h in sequence. Subsequently, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation, and the parameters are 110,000g×2h to obtain purified exosomes.

[0036] Suspend the exosomes in PBS buffer (pH 7.4), adjust the concentration to 0.5mg / mL, and perform surface modification at a constant temperature of 25°C. Incubate the exosomes with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.08mg / mL for 2h, control the magnetic stirring rate at 120rpm, and then remove the unbound modifier through an ultrafiltration tube (MWCO 100kDa).

[0037] Use EDC (1-ethyl-3-3-dimethylaminopropyl) carbodiimide hydrochloride, 10mM) and NHS (N-hydroxysuccinimide, 15mM) to activate the extracellular domain of the exosome surface membrane protein CD63 in MES buffer (pH 6.0) for 15min, and then react with NHS-PEG4-N3 at a molar ratio of 1:1.2 for 4h. Purify and remove the unreacted reagents through a Sephadex G-25 gel chromatography column.

[0038] In the drug loading step, mix GPC-3298306 (10mM DMSO solution) with Zn(NO3)2 (25mM) and 2-MIM (50mM) at a molar ratio of 1:2:4, use a NanoAssemblr Ignite microfluidic device for assembly, with a flow rate ratio of 1:3 and a total flow rate of 12mL / min, and collect particles with a CV value of the particle size distribution <8%. Finally, perform a click coupling reaction, mix the modified exosomes with the drug particles at a mass ratio of 1:50, add the photoinitiator Irgacure 2959 (0.1wt%), and complete the reaction using 365nm UV light irradiation (10mW / cm²×30s) to obtain an exosome engineered targeted drug delivery system.

[0039] The particle size of the final product was determined to be 80 - 90 nm using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90), the Zeta potential was -14.6 mV, the MAL insertion density was determined to be 0.85 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with the FITC labeling rate reaching 89.2%.

[0040] Example 2 Take the exoEasy Maxi Kit (Qiagen) and treat the ACC-M cell supernatant at 3°C. First, perform gradient centrifugation with the centrifugation parameters being 300 g × 12 min, 2000 g × 35 min, and 10000 g × 70 min in sequence. Subsequently, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation with the parameter of 110,000 g × 2.5 h to obtain purified exosomes.

[0041] Suspend the exosomes in PBS buffer (pH 7.5), adjust the concentration to 0.6 mg / mL, and perform surface modification at a constant temperature of 24°C. Incubate the exosomes with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.10 mg / mL for 2.5 h, control the magnetic stirring rate at 130 rpm, and then remove the unbound modifier through an ultrafiltration tube (MWCO 100 kDa).

[0042] Use EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 12 mM) and NHS (N-hydroxysuccinimide, 18 mM) to activate the extracellular domain of the exosome surface membrane protein CD63 in MES buffer (pH 6.1) for 20 min, and then react with NHS-PEG4-N3 (molar ratio 1:1.3) for 4.5 h. Purify and remove the unreacted reagents through a Sephadex G-25 gel chromatography column.

[0043] In the drug loading step, GPC-3298306 (12 mM DMSO solution) was mixed with Zn(NO3)2 (30 mM) and 2-MIM (60 mM) at a molar ratio of 1:2.5:5, and assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:3.5 and a total flow rate of 14 mL / min. Particles with a CV value of the particle size distribution < 7% were collected. Finally, a click coupling reaction was carried out, and the modified exosomes and drug particles were mixed at a ratio of 1:55. Photoinitiator Irgacure 2959 (0.12 wt%) was added, and the reaction was completed using 365 nm UV light irradiation (12 mW / cm² × 35 s) to obtain an exosome-engineered targeted drug delivery system.

[0044] The particle size of the final product was measured to be 86 - 94 nm, the Zeta potential was -16.2 mV using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was determined to be 1.0 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with a FITC labeling rate of 91.5%.

[0045] Example 3 Take the exoEasy Maxi Kit (Qiagen), and treat the supernatant of ACC-M cells at 2°C. First, perform gradient centrifugation with centrifugation parameters of 300 g × 15 min, 2000 g × 40 min, and 10000 g × 90 min in sequence. Subsequently, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation with parameters of 110,000 g × 3 h to obtain purified exosomes.

[0046] The exosomes were suspended in PBS buffer (pH 7.2), and the concentration was adjusted to 0.7 mg / mL. Surface modification was carried out at a constant temperature of 26°C. The exosomes were incubated with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.12 mg / mL for 3 h, and the magnetic stirring rate was controlled at 140 rpm. Then, unbound modifiers were removed through an ultrafiltration tube (MWCO 100 kDa).

[0047] The extracellular domain of exosome surface membrane protein CD63 was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 15 mM) and NHS (N-hydroxysuccinimide, 20 mM) in MES buffer (pH 5.8) for 25 min, and then reacted with NHS-PEG4-N3 (molar ratio 1:1.4) for 5 h. Unreacted reagents were removed by purification through a Sephadex G-25 gel chromatography column.

[0048] In the drug loading step, GPC-3298306 (15 mM DMSO solution) was mixed with Zn(NO3)2 (35 mM) and 2-MIM (70 mM) at a molar ratio of 1:2.3:4.7, and assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:2.5 and a total flow rate of 16 mL / min. Particles with a CV value of the particle size distribution <6% were collected. Finally, a click coupling reaction was carried out by mixing the modified exosomes with the drug particles at a ratio of 1:60, adding the photoinitiator Irgacure 2959 (0.15 wt%), and completing the reaction under 365 nm UV light irradiation (15 mW / cm²×40 s) to obtain an exosome-engineered targeted drug delivery system.

[0049] The particle size of the final product was measured to be 92 - 98 nm, the Zeta potential was -17.8 mV using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was measured to be 1.15 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with a FITC labeling rate of 93.8%.

[0050] Example 4 Take the exoEasy Maxi Kit (Qiagen), and treat the supernatant of ACC-M cells at 1°C. First, perform gradient centrifugation with the centrifugation parameters of 300 g×8 min, 2000 g×25 min, and 10000 g×55 min in sequence. Then, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation with the parameter of 110,000 g×1.8 h to obtain purified exosomes.

[0051] Suspend the exosomes in PBS buffer (pH 7.3), adjust the concentration to 0.4 mg / mL, and perform surface modification at a constant temperature of 23°C. Incubate the exosomes with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.05 mg / mL for 1.8 h, control the magnetic stirring rate at 110 rpm, and then remove the unbound modifier through an ultrafiltration tube (MWCO 100 kDa).

[0052] The extracellular domain of the exosomal surface membrane protein CD63 was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 8 mM) and NHS (N-hydroxysuccinimide, 12 mM) in MES buffer (pH 6.2) for 12 min, and then reacted with NHS-PEG4-N3 (molar ratio 1:1.1) for 3.5 h. Unreacted reagents were removed by purification through a Sephadex G-25 gel chromatography column.

[0053] In the drug loading step, GPC-3298306 (8 mM DMSO solution), Zn(NO3)2 (20 mM), and 2-MIM (40 mM) were mixed at a molar ratio of 1:2:5, and assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:3.8 and a total flow rate of 10 mL / min. Particles with a CV value of the particle size distribution <9% were collected. Finally, a click coupling reaction was carried out. The modified exosomes and drug particles were mixed at a ratio of 1:45, and the photoinitiator Irgacure 2959 (0.08 wt%) was added. The reaction was completed by 365 nm UV light irradiation (8 mW / cm²×25 s) to obtain an exosome-engineered targeted drug delivery system.

[0054] The final product was measured to have a particle size of 74 - 86 nm, a Zeta potential of -13.9 mV using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was determined to be 0.8 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with an FITC labeling rate of 88.5%.

[0055] Example 5 Take the exoEasy Maxi Kit (Qiagen), and treat the supernatant of ACC-M cells at 2°C. First, perform gradient centrifugation with centrifugation parameters of 300 g×13 min, 2000 g×32 min, and 10000 g×65 min in sequence. Then, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation with parameters of 110,000 g×2.2 h to obtain purified exosomes.

[0056] The exosomes were suspended in PBS buffer (pH 7.6) and the concentration was adjusted to 0.8 mg / mL. Surface modification was carried out at a constant temperature of 27°C. The exosomes were incubated with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.15 mg / mL for 3.2 h, and the magnetic stirring rate was controlled at 150 rpm. Then, unbound modifiers were removed through an ultrafiltration tube (MWCO 100 kDa).

[0057] The extracellular domain of the exosomal surface membrane protein CD63 was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 18 mM) and NHS (N-hydroxysuccinimide, 22 mM) in MES buffer (pH 5.9) for 30 min, and then reacted with NHS-PEG4-N3 (molar ratio 1:1.5) for 5.5 h. Unreacted reagents were removed by purification through a Sephadex G-25 gel chromatography column.

[0058] In the drug loading step, GPC-3298306 (18 mM DMSO solution), Zn(NO3)2 (40 mM), and 2-MIM (80 mM) were mixed at a molar ratio of 1:2.2:4.5 and assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:2 and a total flow rate of 18 mL / min. Particles with a CV value of the particle size distribution < 5% were collected. Finally, a click coupling reaction was carried out by mixing the modified exosomes and the drug particles at a ratio of 1:65, adding the photoinitiator Irgacure 2959 (0.18 wt%), and completing the reaction with 365 nm UV light irradiation (18 mW / cm² × 45 s) to obtain an exosome-engineered targeted drug delivery system.

[0059] The final product had a particle size of 98 - 102 nm, a Zeta potential of -19.5 mV measured using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was measured to be 1.2 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with a FITC labeling rate of 94.7%.

[0060] Comparative Example 1 The exoEasy Maxi Kit (Qiagen) was used to treat the supernatant of ACC-M cells at 4°C. First, gradient centrifugation was performed with centrifugation parameters of 300 g × 10 min, 2000 g × 30 min, and 10000 g × 1 h in sequence. Subsequently, ultracentrifugation was carried out using an Optima XE-90 ultracentrifuge with a Type70 Ti rotor at a parameter of 110,000 g × 2 h to obtain purified exosomes.

[0061] The exosomes were suspended in PBS buffer (pH 7.4), and the concentration was adjusted to 0.5 mg / mL. Surface modification was carried out at a constant temperature of 25 °C. The exosomes were incubated with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.08 mg / mL for 2 h, and the magnetic stirring rate was controlled at 120 rpm. Then, the unbound modifier was removed through an ultrafiltration tube (MWCO 100 kDa).

[0062] In the drug loading step, GPC-3298306 (10 mM DMSO solution) was mixed with Zn(NO3)2 (25 mM) and 2-MIM (50 mM) at a molar ratio of 1:2:4, and assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:3 and a total flow rate of 12 mL / min. Particles with a CV value of the particle size distribution <8% were collected. Finally, a click coupling reaction was carried out. The modified exosomes and drug particles were mixed at a ratio of 1:50, and the photoinitiator Irgacure 2959 (0.1 wt%) was added. The reaction was completed by 365 nm UV light irradiation (10 mW / cm²×30 s) to obtain an exosome-engineered targeted drug delivery system.

[0063] The final product was measured to have a particle size of 80 - 94 nm, a Zeta potential of -18.3 mV using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was determined to be 0.82 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with an FITC labeling rate of 56.7%.

[0064] Compared with Example 1, this comparative example lacked the modification step of the azide group (N3) of the CD63 extracellular domain, resulting in the final product lacking dual-modal targeting ability, a 36.4% reduction in click efficiency, and a significant reduction in targeting specificity. In the in vitro Transwell experiment, the migration inhibition rate of SACC-83 cells was only 52.3%, which was 36.8% lower than 82.7% in Example 1.

[0065] Comparative Example 2 Take the exoEasy Maxi Kit (Qiagen), and treat the supernatant of ACC-M cells at 4 °C. First, perform gradient centrifugation with centrifugation parameters of 300 g×10 min, 2000 g×30 min, and 10000 g×1 h in sequence. Then, use an Optima XE-90 ultracentrifuge with a Type70 Ti rotor for ultracentrifugation with parameters of 110,000 g×2 h to obtain purified exosomes.

[0066] The exosomes were suspended in PBS buffer (pH 7.4), and the concentration was adjusted to 0.5 mg / mL. Surface modification was carried out under constant temperature conditions at 25 °C. The exosomes were incubated with DSPE-PEG-MAL (PEG molecular weight 2000, Sigma-Aldrich 900245) at a concentration of 0.08 mg / mL for 2 h, and the magnetic stirring rate was controlled at 120 rpm. Then, the unbound modifier was removed through an ultrafiltration tube (MWCO 100 kDa).

[0067] The extracellular domain of the exosome surface membrane protein CD63 was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 10 mM) and NHS (N-hydroxysuccinimide, 15 mM) in MES buffer (pH 6.0) for 15 min, and then reacted with NHS-PEG4-N3 (molar ratio 1:1.2) for 4 h. Unreacted reagents were removed by purification through a Sephadex G-25 gel chromatography column. In the drug loading step, the traditional solvent displacement method rather than the microfluidic technology was adopted. GPC-3298306 (10 mM DMSO solution) was mixed with Zn(NO3)2 (25 mM) and 2-MIM (50 mM) in a molar ratio of 1:2:4, and was added dropwise to an aqueous solution containing 70% methanol under magnetic stirring at 500 rpm. The product was collected after reacting at room temperature for 4 h. Finally, a click coupling reaction was carried out. The modified exosomes and drug particles were mixed at a ratio of 1:50, and photoinitiator Irgacure 2959 (0.1 wt%) was added. The reaction was completed by 365 nm UV light irradiation (10 mW / cm²×30 s) to obtain an exosome-engineered targeted drug delivery system.

[0068] The final product particle size was measured to be 97 - 133 nm, the Zeta potential was -16.5 mV using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90). The MAL insertion density was determined to be 0.85 molecules / nm² by fluorescence quenching method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with an FITC labeling rate of 85.7%.

[0069] Compared with Example 1, in Comparative Example 2, the traditional solvent replacement method was used instead of the microfluidic technology, resulting in a wider particle size distribution of the drug carrier (CV value of 22.6%, while that of Example 1 was only 5.9%), a significant decrease in particle size uniformity, and at the same time, the drug loading efficiency decreased from the original 97.2% to 76.5%. In the in vivo drug release curve test, an obvious burst release phenomenon occurred in Comparative Example 2, with the release amount in the first 1 h reaching 42.3%, while that of Example 1 was 13.5%, indicating that the microfluidic technology has a significant improvement effect on the drug sustained release effect. In the mouse lung metastasis model, the number of lung metastases in the treatment group of Comparative Example 2 decreased by 54.3%, while that of Example 1 was 81.6%, and the treatment effects were significantly different (p<0.01).

[0070] Comparative Example 3 Take the exoEasy Maxi Kit (Qiagen), and treat the supernatant of ACC-M cells at 4°C. First, perform gradient centrifugation, and the centrifugation parameters are 300g×10 min, 2000g×30 min, and 10000g×1 h in sequence. Subsequently, use an Optima XE-90 ultracentrifuge with a Type 70 Ti rotor for ultracentrifugation, and the parameters are 110,000g×2 h to obtain purified exosomes.

[0071] Suspend the exosomes in PBS buffer (pH 7.4), and adjust the concentration to 0.5 mg / mL. Perform surface modification under a constant temperature condition of 25±0.5°C. Incubate the exosomes with DSPE-PEG-NH2 (PEG molecular weight 2000, Sigma-Aldrich 880128) at a concentration of 0.08 mg / mL for 2 h, and control the magnetic stirring rate at 120 rpm. Then, remove the unbound modifier through an ultrafiltration tube (MWCO 100 kDa).

[0072] The extracellular domain of the exosome surface membrane protein CD63 was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 10 mM) and NHS (N-hydroxysuccinimide, 15 mM) in MES buffer (pH 6.0) for 15 min, and then reacted with NHS-PEG4-N3 (molar ratio 1:1.2) for 4 h. Unreacted reagents were removed by purification through a Sephadex G-25 gel chromatography column. In the drug loading step, GPC-3298306 (10 mM DMSO solution) was mixed with Zn(NO3)2 (25 mM) and 2-MIM (50 mM) at a molar ratio of 1:2:4, assembled using a NanoAssemblr Ignite microfluidic device with a flow rate ratio of 1:3 and a total flow rate of 12 mL / min, and particles with a CV value of the particle size distribution <8% were collected. Finally, a click coupling reaction was carried out, the modified exosomes and drug particles were mixed at a ratio of 1:50, photoinitiator Irgacure 2959 (0.1 wt%) was added, and the reaction was completed by 365 nm UV light irradiation (10 mW / cm²×30 s) to obtain an exosome-engineered targeted drug delivery system.

[0073] The particle size of the final product was measured to be 76 - 88 nm using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS90), the Zeta potential was -17.2 mV, the NH2 insertion density was determined to be 0.81 molecules / nm² by fluorescence labeling method, and the click efficiency was detected by flow cytometry (BD FACSAria III) with the FITC labeling rate being only 13.2%.

[0074] Compared with Example 1, in Comparative Example 3, DSPE-PEG-NH2 was used instead of DSPE-PEG-MAL, lacking the maleimide group required for the thiol-ene click reaction, resulting in a significant decrease in the coupling efficiency of drug particles, a decrease in click efficiency by 85.2%, and the in vitro cell uptake rate decreased from the original 94.3% to 21.8%. In immunohistochemical staining, the drug distribution in the tumor tissue of the treatment group in Comparative Example 3 was uneven, mainly concentrated in the perivascular area, and the penetration depth was only 78 μm, while that in Example 1 was 217 μm, indicating that the MAL group has a significant impact on the spatial distribution of drug delivery. In addition, in the serum stability test of Comparative Example 3, obvious degradation started after 4 h, and the integrity remained only 31.7% after 24 h, while that in Example 1 remained at 87.4%, showing the important role of the bioorthogonal modification strategy in the stability of the drug delivery system.

[0075] Performance detection 1. Determination of the particle size and stability of the drug delivery system Testing equipment: Malvern Zetasizer Nano ZS90 dynamic light scattering instrument.

[0076] Specific steps: Dilute the samples of each example and comparative example to a concentration of 0.1 mg / mL (PBS buffer, pH 7.4). Take 1 mL of the sample and place it in a special colorimetric cuvette. Equilibrate at 20 °C for 5 min. Set the measurement parameters: scattering angle 173°, average of 3 measurements, and 15 scans each time.

[0077] Measure the average particle size (Z-average), particle size distribution width (PDI), and Zeta potential.

[0078] Store the sample at 4 °C and repeat the measurement on days 0, 1, 3, 7, 14, 21, and 28 respectively.

[0079] 2. Test for the surface modification efficiency of exosomes Test equipment: flow cytometry (BD FACSAria III), fluorescence spectrometer (Hitachi F-7000).

[0080] Specific steps: Determination of MAL insertion density: React the sample with FITC-SH (5 μM) in PBS for 1 h. Remove the unreacted fluorescent molecules by ultrafiltration (MWCO 100 kDa). Determine the fluorescence positive rate by flow cytometry and calculate the insertion density by fluorescence quenching method.

[0081] Evaluation of azide group (N3) modification: React the sample with DBCO-Cy5.5 (10 μM) in PBS for 30 min. Purify by gel permeation chromatography (Sephadex G-25). Measure the fluorescence intensity (Ex / Em = 675 / 710 nm) and calculate the modification rate by comparing with the standard curve.

[0082] 3. Determination of drug loading and release Test equipment: UV-Vis spectrophotometer (Shimadzu UV-2600), Franz diffusion cell (Hanson Research).

[0083] Specific steps: Determination of drug loading amount and encapsulation efficiency: Take 1 mL of the sample, add 2 mL of methanol, sonicate for 15 min to destroy the carrier structure. Centrifuge at 12000 rpm for 10 min and take the supernatant. Determine the content of GPC-3298306 by high performance liquid chromatography (HPLC).

[0084] Calculate the drug loading efficiency (DLE%) and encapsulation efficiency (EE%).

[0085] Determination of pH-responsive release curve: Add 1 mL of the sample into a dialysis bag (MWCO 3.5 kDa), and place it in PBS buffer solutions at pH 7.4 and pH 5.0 (37 °C) respectively. Samples are taken at preset time points (0.5, 1, 2, 4, 8, 12, 24, 48 h), the drug content in the release medium is measured, the cumulative release curve is plotted, and the drug release kinetic model is fitted.

[0086] 4. In vitro cell uptake and targeting tests Testing equipment: Confocal laser scanning microscope (Leica SP8), flow cytometry (BD FACSAria III) Specific steps: Qualitative observation of cell uptake: Inoculate SACC-83 cells (high GPC5 expression) and MCF-7 cells (low GPC5 expression) at 5×10 4 cells / well into a confocal special culture dish. After culturing for 24 h, add various DiD-labeled samples (50 μg / mL), incubate at 37 °C for 2 h, wash 3 times with PBS, fix with 4% paraformaldehyde for 15 min, stain the cell nuclei with DAPI, and observe the intracellular fluorescence distribution and take pictures under a confocal microscope (DiD: Ex / Em = 644 / 665 nm).

[0087] Quantitative analysis of cell uptake: Inoculate the above cell lines at 2×10 5 cells / well into a 6-well plate, culture for 24 h, add various DiO-labeled samples (50 μg / mL), incubate at 37 °C for different times (0.5, 1, 2, 4 h), digest with trypsin, wash 2 times with PBS, and measure the fluorescence positive rate and mean fluorescence intensity (MFI) by flow cytometry.

[0088] Calculate the cell uptake index (CUI = MFI × positive rate%).

[0089] 5. In vitro anti-tumor activity tests Testing equipment: Microplate reader (BioTek Synergy HTX), Transwell device (Corning) Specific steps: Cell proliferation inhibition assay (CCK-8 method): Inoculate SACC-83 cells at 5×10³ cells / well into a 96-well plate, culture for 24 h, add samples with different concentrations (0.1 - 100 μM drug equivalent concentration), culture for 48 h, add 10 μL of CCK-8 reagent to each well, after incubating at 37 °C for 2 h, measure the absorbance at 450 nm, calculate the cell survival rate, fit the dose-response curve, and determine the IC50 value.

[0090] Cell migration inhibition assay (Transwell method): SACC-83 cells (5×10 4 / well) that had been starved for 24 h were seeded in the upper chamber of a Transwell (8 μm pore size). Serum-free medium and various samples (20 μg / mL) were added to the upper chamber, and complete medium containing 10% FBS was added to the lower chamber. After culturing at 37 °C for 24 h, the non-migrated cells in the upper chamber were removed, and the cells on the lower membrane surface were stained with crystal violet. Five random fields of view were selected under a microscope to count the migrated cells, and the migration inhibition rate was calculated.

[0091] Test data table Table 1. Physicochemical properties of the drug delivery system

[0092] * Comparative example 3 is the NH2 insertion density instead of the MAL insertion density.

[0093] Table 2. Test table for drug loading and release performance of the drug delivery system

[0094] Table 3. Test table for cell uptake and targeting

[0095] Table 4. Test table for in vitro antitumor activity

[0096] * The positive control is the free GPC-3298306 drug.

[0097] Table 5. Test table for intracellular drug enrichment and tumor penetration depth

[0098] * The drug retention time refers to the time required for the intracellular drug concentration to decrease to 50% of the initial value, and ** the solid tumor uniformity coefficient refers to the consistency of drug distribution from the tumor edge to the center (1 means completely uniform).

[0099] Table 6. Test table for in vivo model of mouse lung metastasis

[0100] Data analysis As can be seen from Table 1, the particle size ranges of the drug-loaded systems in Examples 1-5 are evenly distributed between 80-100 nm, with an extremely low polydispersity index (PDI < 0.1), indicating that the prepared nanoparticles are uniformly distributed. In particular, the PDI of Example 5 is only 0.036, and the particle size is 100 ± 2 nm, showing the most excellent uniformity. In contrast, although the particle size of Comparative Example 1 (87 ± 7 nm) is similar to that of Example 1, the lack of N3 modification (0%) results in a click efficiency of only 56.7%, which is 36.4% lower than 89.2% of Example 1; in Comparative Example 2, due to the use of the traditional solvent displacement method instead of the microfluidic technology, the PDI is as high as 0.226, and the particle size distribution is wide (115 ± 18 nm), with significantly insufficient uniformity; although Comparative Example 3 has similar particle size characteristics, the use of NH2 to replace the MAL group leads to a sharp drop in the click efficiency to 13.2%, which is quite different from 88.5% - 94.7% of the Example group. These results demonstrate the crucial role of the dual-modal orthogonal modification strategy and microfluidic technology in maintaining the uniformity and click efficiency of the drug-loaded system.

[0101] As can be seen from Table 2, the Example group shows significant advantages in terms of drug loading and release characteristics. The release rate of all Examples is less than 13% at physiological pH (7.4) for 24 h, but the release rate exceeds 90% under the condition of simulating the pH of the tumor microenvironment (5.0), indicating excellent pH responsiveness. In particular, the pH response magnification of Example 5 is as high as 11.09, which means that the drug release rate in the tumor environment is more than 11 times that in the physiological environment. In contrast, the drug release control performance of the Comparative Example group is significantly reduced. Especially in Comparative Example 2, due to the lack of the microfluidic process, the encapsulation efficiency is only 76.5%, and the release rate at pH 7.4 for 24 h is as high as 23.4%, showing an obvious burst release phenomenon, and the pH response magnification drops to 3.73, only 33.6% of Example 5. These data indicate that the present invention effectively improves the drug encapsulation efficiency and the ability of stimulus-responsive release through the precisely controlled microfluidic process and dual-modal modification strategy.

[0102] As can be seen from Table 3, the example group has significant targeting selectivity for SACC-83 cells with high GPC5 expression. The SACC-83 cell uptake rates of Examples 3 and 5 reached 97.5% and 98.2% respectively, while the uptake rates for MCF-7 cells with low GPC5 expression were only 18.4% and 17.9%, and the targeting selectivity indexes were as high as 5.30 and 5.49. This high selectivity provides excellent targeting efficiency with a 4-hour cell uptake index (CUI) as high as 19,845 - 20,963. In contrast, in Comparative Example 1, due to the lack of N3 modification of the extracellular domain of CD63, the targeting selectivity index was only 2.57; in Comparative Example 2, although the dual-modification strategy was retained, the selectivity index decreased to 2.15 due to insufficient particle size uniformity; in Comparative Example 3, due to low click efficiency, its CUI value was only 5,486, which was 26.2% of that in Example 5. These results fully demonstrate the importance of the spatial orthogonal distribution of MAL and N3 bifunctional groups in avoiding modification site competition and improving targeting specificity.

[0103] As can be seen from Table 4, the example group showed significantly enhanced in vitro anti-tumor activity. The IC50 value of Example 5 was the lowest, only 3.1 μM, which was 75.2% lower than that of the positive control (12.5 μM), and at the same time induced the highest apoptosis rate (67.4%) and migration inhibition rate (91.5%). With the increase in the MAL insertion density and N3 modification rate, the anti-tumor activities of Examples 1 - 5 showed an obvious increasing trend, which was consistent with the physicochemical property data in Table 1. In contrast, the anti-tumor activities of the comparative example group were significantly reduced. Especially in Comparative Example 3, its IC50 value (10.4 μM) was close to that of the positive control, and the migration inhibition rate was only 41.4%, indicating that a single surface modification strategy could not effectively improve the drug delivery efficiency and anti-tumor activity. The trend of the colony formation inhibition rate further confirmed that the example group (75.3% - 88.2%) was superior to the comparative example group (38.3% - 52.5%) in the ability to inhibit the proliferation of tumor cells, demonstrating the potential of the present invention in long-term inhibition of tumor growth.

[0104] As can be seen from Table 5, the example group has significant advantages in terms of intracellular drug enrichment and tumor tissue penetration ability. Example 5 reached the highest drug concentration of 1205.4 pmol / 10 6 cells in SACC-83 cells, which was the positive control (284.3 pmol / 10 64.24 times that of (cells), and the drug retention time was extended to 38.2 h, indicating that the present invention significantly improved the accumulation and retention ability of the drug in target cells. In terms of tumor penetration depth, the example group (203 - 274 μm) far exceeded the comparative example group (136 - 194 μm) and the positive control (97 μm). In particular, Example 5 reached a penetration depth of 274 μm, which was 182.5% higher than that of the positive control. The data of the solid tumor uniformity coefficient further showed that the drug distribution in the tumor tissue of the example group (0.79 - 0.92) was more uniform, while obvious non-uniform distribution occurred in the comparative example group (0.48 - 0.63) and the positive control (0.32). These results confirmed that the present invention significantly improved the penetration ability and distribution uniformity of the drug in tumor tissue through a dual-modal targeting strategy, providing an effective solution to overcome the drug delivery barrier of solid tumors.

[0105] As can be seen from Table 6, the example group showed excellent efficacy in the in vivo anti-tumor effect. In the mouse lung metastasis model, the reduction rate of the number of lung metastases in the treatment group of Example 5 was as high as 91.3%, which was 148.1% higher than that of the positive control (36.8%). At the same time, a tumor volume reduction rate of 87.2% and a survival period extension rate of 92.5% were achieved. It is particularly worth noting that the example group performed excellently in inhibiting exosome-mediated metastasis. The exosome-mediated metastasis inhibition rate of Example 5 reached 90.6%, which was highly consistent with the original design intention of the engineering transformation of exosomes in the present invention. Although the comparative example group was improved compared with the positive control, its effect was still significantly lower than that of the example group. In particular, for Comparative Example 3, each index was only about half of that of Example 5. Based on the data in Tables 1 - 6, it can be concluded that the present invention successfully constructed an efficient dual-modal targeted drug delivery system by directionally grafting bifunctional groups (MAL and N3) on the surface of the exosome membrane and combining microfluidic technology to prepare uniform drug particles, showing significant advantages in improving targeting accuracy, enhancing anti-tumor activity, inhibiting tumor metastasis, etc., providing a new technical approach for the precision treatment of adenoid cystic carcinoma.

[0106] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention shall still fall within the scope covered by the patent of the present invention.

Claims

1. An exosome-engineered targeted drug delivery system, characterized in that: It is prepared by the mixed reaction of surface-modified exosomes and pH-responsive drug-controlled release nanoparticles; The pH-responsive drug controlled-release nanoparticles react with the surface-modified exosomes through a click chemistry reaction; The membrane surface of the surface-modified exosomes is modified with maleimide groups and azide groups; The novel nanomedicine is pH responsive; The click chemistry reaction is a thiol-ene click reaction.

2. The targeted drug delivery system according to claim 1, characterized in that: The pH-responsive drug controlled-release nanoparticles contain a metal organic framework and a targeted drug. The decomposition rate of the metal organic framework in an environment with a pH value of 4.5-5.5 is higher than that in a physiological pH environment.

3. The targeted drug delivery system according to claim 1, characterized in that: The maleimide group is inserted into the exosome lipid bilayer membrane through phosphatidylethanolamine-polyethylene glycol-maleimide; the azide group modifies the extracellular domain of the exosome membrane protein CD63 through NHS-polyethylene glycol-azide.

4. The targeted drug delivery system according to claim 3, characterized in that: The molecular weight of polyethylene glycol in the phosphatidylethanolamine-polyethylene glycol-maleimide is 1000-3000, and the insertion density of the phosphatidylethanolamine-polyethylene glycol-maleimide on the surface of the exosome membrane is 0.5-1.5 molecules / square nanometer.

5. The targeted drug delivery system according to claim 3, characterized in that: The molecular weight of polyethylene glycol in the NHS-polyethylene glycol-azide is 200-600, and the molar ratio of the NHS-polyethylene glycol-azide to the CD63 protein is 1:1.0-1.

5.

6. The targeted drug delivery system according to claim 1, characterized in that: The metal organic framework is zeolite imidazolate framework-8, the targeted drug is GPC-3298306, and the drug loading amount of the zeolite imidazolate framework-8 is 15-30 weight percent.

7. The targeted drug delivery system according to claim 6, characterized in that: The preparation method of the zeolite imidazolate framework-8 comprises the following steps: Dissolve zinc salt and 2-methylimidazole in water at a molar ratio of 1:2, and stir at room temperature for 0.5-2 hours to obtain a precursor solution; The precursor solution and the dimethyl sulfoxide solution of GPC-3298306 were mixed through a microfluidic device at a flow rate ratio of 1:3, with a total flow rate of 8-15 ml / min, and particles were collected; The particles are centrifuged, washed and dried to obtain the zeolite imidazolate framework-8 drug-loaded nanoparticles.

8. The targeted drug delivery system according to claim 1, characterized in that: The thiol-ene click reaction is carried out under ultraviolet light irradiation conditions, the wavelength of the ultraviolet light is 350-380 nanometers, the light intensity is 5-15 milliwatts / square centimeter, and the irradiation time is 20-40 seconds.

9. A method for preparing the targeted drug delivery system according to any one of claims 1 to 8, characterized in that: The following steps are involved: Extracting exosomes; Modifying the surface of the exosomes with maleimide groups and azide groups to obtain surface-modified exosomes; Preparation of pH-responsive drug controlled-release nanoparticles; The surface-modified exosomes are connected to the nanoparticles through a click chemistry reaction to obtain the exosome-engineered targeted drug delivery system.

10. The preparation method according to claim 9, characterized in that: The step of modifying the maleimide group and the azide group on the surface of the exosomes comprises: Incubating the exosomes with phosphatidylethanolamine-polyethylene glycol-maleimide at a concentration of 0.05-0.15 mg / ml at 20-30° C. for 1-3 hours with a magnetic stirring rate of 100-140 rpm to obtain maleimide-modified exosomes; The maleimide-modified exosome surface is activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and then reacted with NHS-polyethylene glycol-azide at a molar ratio of 1:1.0-1.5 for 3-5 hours, and purified by gel chromatography to obtain the surface-modified exosomes.

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