Lung-targeted drug-loaded micromotor and preparation method and application thereof

Abstract

The invention relates to a lung-targeted drug-loaded micromotor as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing mPEG (Polyethylene Glycol), N-t-Boc-glycine, DMAP (Dimethylaminopyridine), DCC (Dendritic Cellulose) and a solvent for reaction, and sequentially obtaining an intermediate product preparation process of mPEG-CH2CH2NH-Boc, CH3OPEG-NH2 and mPEG-NH2; performing mixed reaction on NHS, EDC and a sodium alginate solution in a buffer system to obtain a carboxyl activation process of an activated product; the mPEG-NH2 and the activated product are mixed and subjected to a reaction, and a precursor material of Alg-g-mPEG is obtained; a saturated alpha-CD aqueous solution of an FOF1-ATPase molecular motor is mixed with the Alg-g-mPEG aqueous solution, and the Alg-g-mPEG self-assembled micro-capsule with the FOF1-ATPase molecular motor coating is obtained in the carrier preparation process; mixing a drug with the self-assembled micro-capsule to obtain a lung-targeted drug-loading micro-motor; the invention further discloses application of the lung-targeted drug-loaded micromotor prepared according to the preparation method in preparation of a drug delivery system for treating lung adenocarcinoma. The double-site high-drug-loading-capacity hydrogel has the advantages of being high in double-site high drug loading capacity, high in biological safety, high in pH response self-driving capacity, good in lung tumor targeting capacity, good in lung retention capacity and the like.

Description

Technical Field

[0001] This invention relates to the technical field of drug delivery systems, and in particular to a lung-targeting drug-carrying micromotor, its preparation method, and its application. Background Technology

[0002] Lung cancer, a major global disease, poses a serious threat to both men and women. It is not only one of the most common malignant tumors but also the deadliest type of cancer. Recent statistics show that lung cancer accounts for 18.7% of all cancer-related deaths, making it the leading cause of death among all cancers. Among the different subtypes of lung cancer, lung adenocarcinoma is the most common subtype of non-small cell lung cancer (NSCLC). Current medical practice primarily utilizes surgical resection, chemotherapy, radiotherapy, and molecular targeted therapy.

[0003] In recent years, with the continuous advancement and innovation of medical technology, novel therapies such as immunotherapy for lung adenocarcinoma have gradually matured, bringing new hope to patients. However, in areas with relatively scarce medical resources, surgery and chemotherapy remain the primary treatment options for the general patient population, and traditional treatment methods still dominate. Although surgery is highly effective in controlling disease progression in early-stage lung adenocarcinoma patients, it often fails to achieve ideal clinical outcomes for advanced-stage patients. At this point, the importance of chemotherapy becomes apparent, as its efficacy is more significant and it can slow the progression of the disease.

[0004] However, currently used chemotherapy drugs have many drawbacks, such as poor targeting, wide distribution in the body, low bioavailability, and significant toxic side effects. These problems seriously affect patients' treatment tolerance and quality of life. Furthermore, insufficient accumulation of chemotherapy drugs in tumor tissue easily leads to drug resistance, thus limiting the clinical efficacy of chemotherapy. In this context, how to design and develop a novel targeted formulation system to increase drug concentration at the tumor site, reduce systemic toxicity, and enhance treatment efficacy has become a key problem that researchers in this field urgently need to solve. This is not only related to prolonging the survival of lung adenocarcinoma patients, but also to improving their quality of life and treatment experience. Therefore, future research should focus on overcoming the shortcomings of existing chemotherapy drugs and exploring more precise and efficient treatment strategies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the first objective of this invention is to provide a method for preparing lung-targeted drug-loaded micromotors, which utilizes the water-insoluble nature of the crystals formed after mPEG and α-CD binding, in the presence of F... OThe self-assembly of F1-ATPase molecular motors in a pure water system can prepare self-assembled microcapsules with a cavity structure, which can simultaneously endow the carrier with multiple functions such as high drug loading capacity at two sites, high biosafety, pH-responsive self-driving ability, good lung tumor targeting ability and lung retention ability.

[0006] The second objective of this invention is to provide a lung-targeting drug-carrying micromotor that has the advantages of being suitable for intelligent nano-drug delivery systems targeting lung tumors.

[0007] The third objective of this invention is to provide an application of a lung-targeting drug-carrying micromotor, which has the advantages of being suitable for intelligent nano-drug delivery systems targeting lung tumors.

[0008] To achieve the first objective mentioned above, the present invention provides the following technical solution: A method for preparing a lung-targeted drug-loaded micromotor, comprising, The process of preparing intermediate products mPEG-CH2CH2NH-Boc, CH3OPEG-NH2, and mPEG-NH2 by mixing mPEG, Nt-Boc-glycine, DMAP, DCC with solvent and reacting them sequentially. The process of carboxyl activation of NHS, EDC and sodium alginate solution in a buffer system to react and obtain the activated product; The process of preparing Alg-g-mPEG precursor material by mixing and reacting the mPEG-NH2 and the activation product; F O A saturated α-CD aqueous solution of the F1-ATPase molecular motor is mixed with the Alg-g-mPEG aqueous solution to obtain a product with F1-ATPase molecular motor. O Preparation process of carriers for Alg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; The process of mixing the drug with the self-assembled microcapsules to obtain a lung-targeted drug-loaded micromotor.

[0009] Further, in the preparation of the intermediate product, mPEG, Nt-Boc-glycine, DMAP, DCC and dichloromethane solvent are mixed. After the reaction is completed, post-treatment is performed to obtain mPEG-CH2CH2NH-Boc. The molar ratio of mPEG, Nt-Boc-glycine, DMAP and DCC is controlled to be 1:(2.0~2.4):(0.8~1.2):(2.2~2.6). The amount of mPEG added in dichloromethane solvent is 0.5~1.0M. The reaction temperature of mPEG-CH2CH2NH-Boc is 20~30℃ and the reaction time is 18~28h. After the reaction is completed, it is dissolved in acetone, cooled overnight, filtered to remove precipitated byproducts, and the filtrate is collected and vacuum dried at room temperature for 20~25h.

[0010] Further, in the preparation of the intermediate product, mPEG-CH2CH2NH-Boc is mixed with dichloromethane / tetrahydrofuran solvent, and after the reaction is completed, it is post-treated to obtain CH3OPEG-NH2; the volume ratio of dichloromethane to tetrahydrofuran in the dichloromethane / tetrahydrofuran solvent is controlled to be (1~2):(1~2), the reaction temperature of CH3OPEG-NH2 is -5~5℃, the reaction time is 1.5~2.5h, and the product is evaporated and dried after the reaction is completed.

[0011] Further, in the process of obtaining the lung-targeted drug-loaded micromotor, CH3OPEG-NH2 is mixed with NaCl / water solvent and extracted to obtain mPEG-NH2; the NaCl concentration in the NaCl / water solvent is controlled to be 10~20%w / v, the pH is adjusted to 4.0~6.0, and the extraction is performed by extracting the aqueous phase with chloroform 3~5 times. The organic phases are combined, anhydrous sodium sulfate is added to remove the residual aqueous phase, and the sodium sulfate is removed by filtration. After the organic solvent evaporates, the remaining solid is vacuum dried at room temperature for 20~28h.

[0012] Furthermore, during the carboxyl activation process, the mass-to-volume ratio of NHS, EDC, and sodium alginate is controlled to be (280~320) mg: (95~100) mg: 100 mL, the concentration of sodium alginate in the buffer system is 0.5~2.0 wt%, the buffer system is a MES / NaCl buffer solution with pH=5.5~6.5, the molar ratio of MES to NaCl in the buffer system is (0.05~0.15):(0.40~0.60), the reaction temperature is 20~30℃, and the reaction time is 1~3 h.

[0013] Furthermore, during the preparation of the precursor material, mPEG-NH2 is dissolved in 2% w / v activated product, the reaction temperature is 20~30℃, the reaction time is 5~15h, after the reaction is completed, the mixture is dialyzed with pure water for 70~75h, the dialysis bag MWCO = 8000~14000 to remove unreacted compounds, and then transferred to a vacuum drying oven for drying for 70~75h.

[0014] Furthermore, during the carrier preparation process, F is controlled O The volumes of the α-CD saturated aqueous solution and the Alg-g-mPEG aqueous solution of the F1-ATPase molecular motor were (2~4): 2, F OThe concentration of Alg-g-mPEG in the α-CD saturated aqueous solution of F1-ATPase molecular motor was 0.5~2.0% w / v, the reaction temperature was 20~30℃, the reaction time was 1.5~2.5h, the reaction stirring speed was 300~500rpm, and after the reaction was completed, the mixture was dialyzed with pure water for 70~75h with a MWCO of 8000~14000 in the dialysis bag to remove excess α-CD.

[0015] Furthermore, in the process of obtaining the lung-targeted drug-loaded micromotor, the drug and self-assembled microcapsules are dissolved in an α-CD aqueous solution, the pH is adjusted to 6.0~8.0, and the reaction is carried out at 20~30℃ for 5~15h. After the reaction is completed, the mixture is dialyzed with pure water for 70~75h, with the MWCO of the dialysis bag being 8000~14000, in order to remove excess α-CD.

[0016] To achieve the second objective mentioned above, the present invention provides the following technical solution: A lung-targeted drug-carrying micromotor was prepared according to the above-described preparation method.

[0017] To achieve the third objective mentioned above, the present invention provides the following technical solution: Application of a lung-targeted drug-loaded micromotor: The lung-targeted drug-loaded micromotor prepared according to the above preparation method is used in the preparation of a drug delivery system for treating lung adenocarcinoma.

[0018] In summary, the beneficial technical effects of the present invention are as follows: 1. Sodium alginate is a naturally derived polysaccharide, primarily extracted from the cell walls of brown algae and other seaweeds. It is commonly used in gel carriers, drug delivery systems, and food additives. Due to its molecular structure composed of irregularly alternating β-D-mannuronic acid and α-L-guluronic acid units, sodium alginate possesses several key characteristics: its molecular chain is rich in carboxyl groups, exhibiting strong hydrophilicity; the carboxyl groups are highly reactive, readily undergoing chemical reactions and providing modification sites for functional molecules such as targeting ligands; and it exhibits excellent biocompatibility and biodegradability. 2. Methoxy polyethylene glycol (mPEG) is a linear polyether polymer. mPEG possesses the following key characteristics: its molecular chain exhibits high flexibility and hydration capacity, effectively shielding the surface of the target material through steric hindrance, reducing protein adsorption and immune cell recognition, thus achieving long-term circulation in vivo; the terminal methyl groups are highly reactive and easily functionalized; and it exhibits good biocompatibility and low immunogenicity. Based on these characteristics, mPEG plays an important role in protein modification, nanomedicine functionalization, and the development of long-acting formulations.

[0019] 2. This invention uses sodium alginate as the main component and performs a grafting reaction with mPEG. The resulting graft retains the advantages of high hydrophilicity, biocompatibility, biodegradability, and low immunogenicity. The graft solution is mixed with a saturated α-CD solution. Utilizing the property that the crystals formed after the binding of mPEG and α-CD are insoluble in water, the solution is then used in a solution containing F... O Cavity-structured coated microcapsules were prepared by self-assembly of the F1-ATPase molecular motor in a pure water system. Specifically, F... O The F1-ATPase molecular motor can specifically target the weakly acidic environment of the tumor site, thus the present invention is designed to target lung tumors.

[0020] 3. This invention creatively utilizes the water-insoluble nature of the crystals formed by the combination of mPEG and α-CD to self-assemble coated microcapsules with a cavity structure in a pure water system containing a FOF1-ATPase molecular motor, thus obtaining a drug carrier. This carrier simultaneously endows it with multiple functions: ① high drug loading capacity at two sites; ② high biocompatibility; ③ pH-responsive self-driving ability; ④ good lung tumor targeting and lung retention capabilities. This drug carrier provides a new approach for the design of intelligent nanomedicine delivery systems targeting lung tumors.

[0021] 4. The F obtained by any method in this invention O F1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules can all be used as drug carriers, but the size of the drug carriers prepared by different methods is different. Using the preparation method described above in this invention, the parameters are coordinated with each other, and the drug carrier obtained is better than that prepared by other methods. Attached Figure Description

[0022] Figure 1 This is a drug loading detection diagram of the lung-targeted drug-loaded micromotor prepared in Example 2 of the present invention.

[0023] Figure 2 The diagram shows the biosafety test results of the lung-targeted drug-loaded micromotor prepared in Example 2 of this invention for HUVEC; where (A) is the test result at 12h; and (B) is the test result at 24h.

[0024] Figure 3 This is a biosafety test diagram of the lung-targeted drug-loaded micromotor prepared in Example 2 of the present invention for L929; wherein, (A) is the test result at 12h; and (B) is the test result at 24h.

[0025] Figure 4 This is a 0-10s motion trajectory diagram of the lung-targeted drug-loaded micromotor prepared in Example 2 of the present invention in different pH environments.

[0026] Figure 5This is a pH response self-driving capability test diagram of the lung-targeted drug-carrying micromotor prepared in Example 2 of the present invention.

[0027] Figure 6 This is an in vivo distribution diagram of the lung-targeting drug-loaded micromotor prepared in Example 2 of the present invention after intratracheal administration in nude mice.

[0028] Figure 7 This is an in vitro tissue fluorescence distribution map of the lung-targeting drug-loaded micromotor prepared in Example 2 of the present invention.

[0029] Figure 8 This is a fluorescence quantitative analysis image of lung tissue of the lung-targeted drug-loaded micromotor prepared in Example 2 of the present invention. Detailed Implementation

[0030] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] Example 1: A method for preparing a lung-targeted drug-loaded micromotor disclosed in this invention, comprising, The process of preparing intermediate products mPEG-CH2CH2NH-Boc, CH3OPEG-NH2, and mPEG-NH2 by mixing mPEG, Nt-Boc-glycine, DMAP, DCC with solvent and reacting them sequentially. The process of carboxyl activation of NHS, EDC and sodium alginate solution in a buffer system to react and obtain the activated product; The process of preparing Alg-g-mPEG precursor material by mixing and reacting mPEG-NH2 and activation products to obtain Alg-g-mPEG; F O A saturated α-CD aqueous solution of the F1-ATPase molecular motor was mixed with an Alg-g-mPEG aqueous solution to obtain a motor with F1-ATPase molecular motor. O Preparation process of carriers for Alg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; The process of mixing drugs with self-assembled microcapsules to obtain lung-targeted drug-loaded micromotors.

[0032] Example 2: This invention discloses a method for preparing a lung-targeted drug-loaded micromotor, which differs from Example 1 in that it includes the following steps: S1 dissolved 2.5 mmol of methoxy polyethylene glycol (mPEG), 5.5 mmol of Nt-Boc-glycine and 2.8 mmol of 4-(dimethylamino)pyridine (DMAP) in 30 mL of dichloromethane (CH2Cl2) solvent, and then slowly added 6 mmol of dicyclohexylcarbodiimide (DCC). The reaction was stirred at 0 °C for 24 h. After the reaction was completed, the mixture was dissolved in acetone, cooled overnight, filtered to remove precipitated byproducts, and the filtrate was collected and dried under vacuum at room temperature for 24 h to obtain a white paste-like semi-solid, which is mPEG-CH2CH2NH-Boc. S2 dissolves mPEG-CH2CH2NH-Boc in dichloromethane / tetrahydrofuran solvent, controlling the volume ratio of dichloromethane to tetrahydrofuran (THF) in the dichloromethane / tetrahydrofuran solvent to be 1:1, and stirs the reaction at 0℃ for 2h. After the reaction is completed, evaporate and dry to obtain a white powder solid, which is the deprotected product CH3OPEG-NH2. S3. CH3OPEG-NH2 was mixed with NaCl / water solvent, and the NaCl concentration in the NaCl / water solvent was controlled at 15% w / v. The pH was adjusted to 5.0. The aqueous phase was extracted three times with chloroform. The organic phases were combined, anhydrous sodium sulfate was added to remove the residual aqueous phase, and the sodium sulfate was removed by filtration. After the organic solvent evaporated, the remaining solid was vacuum dried at room temperature for 24 h to obtain mPEG-NH2. S4 prepared a 1% w / v sodium alginate solution using a buffer solution containing 0.1M MES and 0.5M NaCl, adjusted the pH to 6.0, and dissolved 292.2 mg of N-hydroxysuccinimide (NHS) and 98.8 mg of ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) in 100 mL of sodium alginate solution. The mixture was stirred at 25 °C for 2 h to fully activate the carboxylic acid groups on the polymer backbone, thus obtaining the activated product. S5 dissolves mPEG-NH2 in 2% w / v activated product, reacts at 25℃ for 10 h, dialyzes with pure water for 72 h after the reaction, MWCO=10000 in the dialysis bag to remove unreacted compounds, and then transfers to a vacuum drying oven to dry for 72 h to obtain Alg-g-mPEG. S6. 2 mL of a 1% w / v, pH 8.0 Alg-g-mPEG aqueous solution was added dropwise to 3 mL of F solution while stirring. O In a saturated α-CD aqueous solution containing the F1-ATPase molecular motor, the solution gradually became turbid with the addition of Alg-g-mPEG, indicating the formation of microcapsules. The reaction was carried out at 25°C for 2.0 h with a stirring speed of 400 rpm. After the reaction, the solution was dialyzed with pure water for 72 h with a MWCO of 10000 in the dialysis bag to remove excess α-CD, yielding the F1-ATPase molecular motor. OAlg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; S7 dissolves the drug and self-assembled microcapsules in an α-CD aqueous solution, adjusts the pH to 7.0, reacts at 25°C for 10 h, and after the reaction is complete, dialyzes with pure water for 72 h with a dialysis bag MWCO = 10000 to remove excess α-CD, thus obtaining a lung-targeted drug-loaded micromotor.

[0033] Example 3: This is a method for preparing a lung-targeted drug-loaded micromotor disclosed in this invention. The difference from Example 2 is that it includes the following steps: S1 dissolves mPEG, Nt-Boc-glycine, and DMAP in dichloromethane solvent, then slowly adds DCC dropwise, controlling the molar ratio of mPEG, Nt-Boc-glycine, DMAP, and DCC to be 1:2.0:0.8:2.2. The amount of mPEG added in dichloromethane solvent is 0.5M. The reaction is carried out at 20℃ for 18h. After the reaction is completed, the mPEG is dissolved in acetone, cooled overnight, filtered to remove precipitated byproducts, and the filtrate is collected and vacuum dried at room temperature for 24h to obtain a white paste-like semi-solid, which is mPEG-CH2CH2NH-Boc. S2 mixes mPEG-CH2CH2NH-Boc with dichloromethane / tetrahydrofuran solvent, controlling the volume ratio of dichloromethane to tetrahydrofuran in the dichloromethane / tetrahydrofuran solvent to be 1:1, reacts at -5℃ for 1.5h, and evaporates and dries after the reaction to obtain a white powder solid, which is the deprotected product CH3OPEG-NH2; S3. CH3OPEG-NH2 was mixed with NaCl / water solvent, and the NaCl concentration in the NaCl / water solvent was controlled to be 10% w / v. The pH was adjusted to 4.0. The extraction was performed by extracting the aqueous phase three times with chloroform. The organic phases were combined, anhydrous sodium sulfate was added to remove the residual aqueous phase, and the sodium sulfate was removed by filtration. After the organic solvent evaporated, the remaining solid was vacuum dried at room temperature for 20 h to obtain mPEG-NH2. S4 involves mixing NHS, EDC, and sodium alginate solution in a buffer system, controlling the mass-to-volume ratio of NHS, EDC, and sodium alginate to be 280 mg: 95 mg: 100 mL, the concentration of sodium alginate in the buffer system to be 0.5 wt%, and the buffer system to be a MES / NaCl buffer solution with pH=5.5, and the molar ratio of MES to NaCl in the buffer system to be 0.05: 0.40. The reaction is carried out at 20 °C for 1 h to obtain the activated product. S5 dissolves mPEG-NH2 in 2% w / v activated product and reacts at 20℃ for 5 h. After the reaction is complete, dialyze with pure water for 72 h with MWCO = 800000 of the dialysis bag to remove unreacted compounds. Then, transfer the solution to a vacuum drying oven and dry for 72 h to obtain Alg-g-mPEG. S6 will FO A saturated α-CD aqueous solution of the F1-ATPase molecular motor was mixed with an Alg-g-mPEG aqueous solution to control F O The volume ratio of the α-CD saturated aqueous solution and the Alg-g-mPEG aqueous solution of the F1-ATPase molecular motor was 2:2. O The concentration of Alg-g-mPEG in the α-CD saturated aqueous solution of F1-ATPase molecular motor was 0.5% w / v. The reaction was carried out at 20°C for 1.5 h with a stirring speed of 300 rpm. After the reaction, the mixture was dialyzed with pure water for 72 h with a MWCO of 8000 in the dialysis bag to remove excess α-CD, thus obtaining F1-ATPase molecular motor. O Alg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; S7 dissolves the drug and self-assembled microcapsules in an α-CD aqueous solution, adjusts the pH to 6.0, reacts at 20°C for 5 hours, and after the reaction is complete, dialyzes with pure water for 72 hours with a dialysis bag MWCO = 8000 to remove excess α-CD, thus obtaining a lung-targeted drug-loaded micromotor.

[0034] Example 4: This is a method for preparing a lung-targeted drug-loaded micromotor disclosed in this invention. The difference from Example 2 is that it includes the following steps: S1 dissolves mPEG, Nt-Boc-glycine, and DMAP in dichloromethane solvent, then slowly adds DCC dropwise, controlling the molar ratio of mPEG, Nt-Boc-glycine, DMAP, and DCC to be 1:2.4:1.2:2.6. The amount of mPEG added in dichloromethane solvent is 1.0 M. The reaction is carried out at 30 °C for 28 h. After the reaction is completed, the mPEG is dissolved in acetone, cooled overnight, filtered to remove precipitated byproducts, and the filtrate is collected and vacuum dried at room temperature for 24 h to obtain a white paste-like semi-solid, which is mPEG-CH2CH2NH-Boc. S2 mixes mPEG-CH2CH2NH-Boc with dichloromethane / tetrahydrofuran solvent, controlling the volume ratio of dichloromethane to tetrahydrofuran in the dichloromethane / tetrahydrofuran solvent to be 1:1, reacts at -5℃ for 2.5h, and evaporates and dries after the reaction to obtain a white powder solid, which is the deprotected product CH3OPEG-NH2; S3: CH3OPEG-NH2 was mixed with NaCl / water solvent, the NaCl concentration in the NaCl / water solvent was controlled at 20% w / v, the pH was adjusted to 6.0, and the aqueous phase was extracted three times with chloroform. The organic phases were combined, anhydrous sodium sulfate was added to remove the residual aqueous phase, and the sodium sulfate was removed by filtration. After the organic solvent evaporated, the remaining solid was vacuum dried at room temperature for 28 h to obtain mPEG-NH2. S4 involves mixing NHS, EDC, and sodium alginate solution in a buffer system, controlling the mass-to-volume ratio of NHS, EDC, and sodium alginate to be 320 mg: 100 mg: 100 mL, the concentration of sodium alginate in the buffer system to be 2.0 wt%, and the buffer system to be a MES / NaCl buffer solution with pH=6.5, and the molar ratio of MES to NaCl in the buffer system to be 0.15:0.60. The reaction is carried out at 30 °C for 3 h to obtain the activated product. S5 dissolves mPEG-NH2 in 2% w / v activated product and reacts at 30℃ for 15 h. After the reaction is complete, dialyze with pure water for 72 h with MWCO = 814000 in the dialysis bag to remove unreacted compounds. Then, transfer the product to a vacuum drying oven and dry for 72 h to obtain Alg-g-mPEG. S6 will F O A saturated α-CD aqueous solution of the F1-ATPase molecular motor was mixed with an Alg-g-mPEG aqueous solution to control F O The volume ratio of the α-CD saturated aqueous solution of F1-ATPase molecular motor to the Alg-g-mPEG aqueous solution is 4:2. O The concentration of Alg-g-mPEG in the α-CD saturated aqueous solution of F1-ATPase molecular motor was 2.0% w / v. The reaction was carried out at 30°C for 2.5 h with a stirring speed of 500 rpm. After the reaction, the mixture was dialyzed with pure water for 72 h with a MWCO of 14000 in the dialysis bag to remove excess α-CD, thus obtaining F1-ATPase. O Alg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; S7 dissolves the drug and self-assembled microcapsules in an α-CD aqueous solution, adjusts the pH to 8.0, reacts at 30°C for 15 h, and after the reaction is complete, dialyzes with pure water for 72 h with a dialysis bag MWCO = 14000 to remove excess α-CD, thus obtaining a lung-targeted drug-loaded micromotor.

[0035] Example 5: A lung-targeted drug-loaded micromotor disclosed in this invention was prepared according to any one of the preparation methods in Examples 1 to 4.

[0036] Example 6: This invention discloses the application of a lung-targeted drug-loaded micromotor. The lung-targeted drug-loaded micromotor prepared according to any of the preparation methods in Examples 1 to 4 is used in the preparation of a drug delivery system for treating lung adenocarcinoma. The performance of the lung-targeted drug-loaded micromotor prepared in Example 2 is tested below.

[0037] (1) F O Drug loading capacity of F1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules The prepared F OF1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules and an appropriate amount of doxorubicin hydrochloride (DOX) were added to α-CD solution. After complete dissolution, the reaction was carried out for 12 hours. The sample was then dialyzed (dialysis bag MWCO = 8000-14000, which needs to be activated in advance) for 24 hours to obtain drug-loaded molecular motor coated microcapsules (DOX-CHR-MC).

[0038] Plotting the standard curve: DOX standard solutions with concentrations of 4, 5, 10, 15, 20, 30, and 40 μg / mL were precisely prepared. The absorbance of DOX was measured at 479 nm using a UV spectrophotometer. A standard curve was plotted with DOX concentration on the x-axis and absorbance on the y-axis. The final standard curve was y = 0.0156x + 0.0149 (R²). 2 =0.9997).

[0039] Drug loading of DOX was investigated: The drug loading was calculated using the standard curve of DOX and the formula "Drug loading rate = Drug mass / Total mass". The results are as follows: Figure 1 As shown, compared to the molecular motor assembly and microcapsule assembly, the drug loading capacity of the molecular motor coated microcapsule assembly is significantly higher, reaching 21.73%. Since the molecular motor coated microcapsules have multiple drug loading sites, they can increase the drug loading capacity of this drug carrier, which fully demonstrates the effectiveness of F... O F1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules have good drug loading capacity.

[0040] (2) F O Biocompatibility of F1-ATPase Molecular Motor Coated Alg-g-mPEG Self-Assembled Microcapsules Cells were seeded into 96-well plates, and the number of cells seeded in each well was determined by cell counting. Different concentrations of Alg-g-mPEG were added to co-culture the cells. At 12 h and 24 h, 10 μL MTT (5 mg / mL) was added to different groups of samples, and the cells were cultured for another 4 h. The internal liquid was discarded, and 150 μL LDMSO was added to each well. The plates were then incubated on a shaker for 5 min. The OD value was measured using a microplate reader, and cell viability was calculated based on the OD value. Experiments were performed on both HUVEC and L929 cell lines.

[0041] The results are as follows Figure 2 and Figure 3As shown, co-culturing HUVEC and L929 cells with the microencapsulation component Alg-g-mPEG for 12h and 24h did not significantly affect the survival rate of HUVEC and L929 cells, which remained above 80%. This demonstrates that the concentration of the microencapsulation component Alg-g-mPEG between 31.25-500 μg / mL has good biocompatibility.

[0042] (3) F O pH-responsive self-driving capability of F1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules Three groups of aqueous solutions with pH values ​​of 5.5, 6.0, and 7.4 were prepared. The coated microcapsules were added to the three groups of aqueous solutions respectively, and the motility of the molecular motor coated microcapsules at different pH values ​​was observed under a microscope with a 10× eyepiece and a 40× objective lens.

[0043] The results are as follows Figure 4 As shown, the movement of the coated microcapsule motors gradually decreases with increasing pH, and only slight vibration occurs when the pH reaches 7.4. This demonstrates that the drug carrier's motility changes with environmental pH, further promoting its development for drug delivery in weakly acidic environments. Trajectory measurements and calculations using ImageJ were used to obtain... Figure 5 The experimental results showed that as the pH decreased, the movement speed of the coated microcapsules increased from 0.16±0.03 μm / s to 0.25±0.02 μm / s, proving that the coated microcapsules have pH-responsive self-driving ability.

[0044] (4) F O F1-ATPase molecular motor coating Alg-g-mPEG self-assembled microcapsules' targeting and retention capabilities Nine nude mice with orthotopic lung adenocarcinoma were established using A549-luc cells and randomly divided into three groups (n=3). All three groups were injected via endotracheal intubation with DiR solution, DiR-stained drug-loaded molecular motor solution (DiR-DOX-CHR), and DiR-stained drug-loaded molecular motor coated microcapsule solution (DiR-DOX-CHR-MC). The DOX concentration was controlled at 1 mg / kg. In vivo biodistribution fluorescence images of DiR were acquired at 4, 8, 12, 24, 48, and 72 hours after injection using a small animal in vivo imaging system. The mice were euthanized 72 hours after injection for dissection to observe the distribution of DiR fluorescence in major ex vivo organs.

[0045] The results are as follows Figure 6As shown, 48 hours after injection, the free DiR group exhibited weaker fluorescence intensity in vivo, while the DiR-DOX-CHR and DiR-DOX-CHR-MC groups showed stronger fluorescence intensity, indicating that the drug-loaded molecular motor coated microcapsules and the drug-loaded molecular motors possess good lung tissue enrichment capabilities. 72 hours after injection, the fluorescence intensity of the DiR-DOX-CHR-MC group was higher than that of the DiR-DOX-CHR group, indicating that the drug-loaded molecular motor coated microcapsules have better lung tissue retention capabilities than the drug-loaded molecular motors. (Comparison of fluorescence distribution in isolated major organs, such as...) Figure 7 and Figure 8 As shown, compared to the DiR group and the DiR-DOX-CHR group, the fluorescence intensity of the isolated lung tissue in the DiR-DOX-CHR-MC group was stronger, verifying the results of the aforementioned in vivo biodistribution fluorescence images, i.e., F... O F1-ATPase molecular motor coated Alg-g-mPEG self-assembled microcapsules exhibit good in vivo targeting and retention capabilities.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a lung-targeted drug-loaded micromotor, characterized in that: include, The process of preparing intermediate products mPEG-CH2CH2NH-Boc, CH3OPEG-NH2, and mPEG-NH2 by mixing mPEG, Nt-Boc-glycine, DMAP, DCC with solvent and reacting them sequentially. The process of carboxyl activation of NHS, EDC and sodium alginate solution in a buffer system to react and obtain the activated product; The process of preparing Alg-g-mPEG precursor material by mixing and reacting the mPEG-NH2 and the activation product; F O A saturated α-CD aqueous solution of the F1-ATPase molecular motor is mixed with the Alg-g-mPEG aqueous solution to obtain a product with F1-ATPase molecular motor. O Preparation process of carriers for Alg-g-mPEG self-assembled microcapsules coated with F1-ATPase molecular motor; The process of mixing the drug with the self-assembled microcapsules to obtain a lung-targeted drug-loaded micromotor.

2. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the preparation of the intermediate product, mPEG, Nt-Boc-glycine, DMAP, DCC and dichloromethane solvent were mixed. After the reaction was completed, post-treatment was performed to obtain mPEG-CH2CH2NH-Boc. The molar ratio of mPEG, Nt-Boc-glycine, DMAP and DCC was controlled to be 1:(2.0~2.4):(0.8~1.2):(2.2~2.6). The amount of mPEG added in dichloromethane solvent was 0.5~1.0M. The reaction temperature of mPEG-CH2CH2NH-Boc was 20~30℃ and the reaction time was 18~28h. After the reaction was completed, it was dissolved in acetone, cooled overnight, filtered to remove precipitated byproducts, and the filtrate was collected and vacuum dried at room temperature for 20~25h.

3. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the preparation of the intermediate product, mPEG-CH2CH2NH-Boc was mixed with dichloromethane / tetrahydrofuran solvent. After the reaction was completed, CH3OPEG-NH2 was obtained through post-treatment. The volume ratio of dichloromethane to tetrahydrofuran in the dichloromethane / tetrahydrofuran solvent was controlled to be (1~2):(1~2). The reaction temperature of CH3OPEG-NH2 was -5~5℃ and the reaction time was 1.5~2.5h. After the reaction was completed, the product was evaporated and dried.

4. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the process of obtaining the lung-targeted drug-loaded micromotor, CH3OPEG-NH2 was mixed with NaCl / water solvent and extracted to obtain mPEG-NH2; the NaCl concentration in the NaCl / water solvent was controlled at 10~20%w / v, the pH was adjusted to 4.0~6.0, and the extraction was performed by extracting the aqueous phase with chloroform 3~5 times. The organic phases were combined, anhydrous sodium sulfate was added to remove the residual aqueous phase, and the sodium sulfate was removed by filtration. After the organic solvent evaporated, the remaining solid was vacuum dried at room temperature for 20~28h.

5. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: During the carboxyl activation process, the mass-to-volume ratio of NHS, EDC, and sodium alginate was controlled at (280~320) mg: (95~100) mg: 100 mL. The concentration of sodium alginate in the buffer system was 0.5~2.0 wt%. The buffer system was a MES / NaCl buffer solution with pH=5.5~6.

5. The molar ratio of MES to NaCl in the buffer system was (0.05~0.15): (0.40~0.60). The reaction temperature was 20~30℃, and the reaction time was 1~3 h.

6. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the preparation process of the precursor material, mPEG-NH2 is dissolved in 2% w / v activation product, the reaction temperature is 20~30℃, the reaction time is 5~15h, after the reaction is completed, the mixture is dialyzed with pure water for 70~75h, the dialysis bag MWCO = 8000~14000 to remove unreacted compounds, and then transferred to a vacuum drying oven for drying for 70~75h.

7. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the carrier preparation process, F is controlled O The volumes of the α-CD saturated aqueous solution and the Alg-g-mPEG aqueous solution of the F1-ATPase molecular motor were (2~4): 2, F O The concentration of Alg-g-mPEG in the α-CD saturated aqueous solution of F1-ATPase molecular motor was 0.5~2.0% w / v, the reaction temperature was 20~30℃, the reaction time was 1.5~2.5h, the reaction stirring speed was 300~500rpm, and after the reaction was completed, the mixture was dialyzed with pure water for 70~75h with a MWCO of 8000~14000 in the dialysis bag to remove excess α-CD.

8. The method for preparing a lung-targeted drug-loaded micromotor according to claim 1, characterized in that: In the process of obtaining the lung-targeted drug-loaded micromotor, the drug and self-assembled microcapsules are dissolved in an α-CD aqueous solution, the pH is adjusted to 6.0~8.0, and the reaction is carried out at 20~30℃ for 5~15h. After the reaction is completed, the mixture is dialyzed with pure water for 70~75h, and the MWCO of the dialysis bag is 8000~14000 to remove excess α-CD.

9. A lung-targeted drug-carrying micromotor, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. An application of a lung-targeted drug-carrying micromotor, characterized in that: The application of the lung-targeting drug-loaded micromotor prepared by any one of claims 1 to 8 in the preparation of a drug delivery system for treating lung adenocarcinoma.

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