A method for scalable preparation of 5-fluorouracil drug-loaded lyophilized nanoparticles

Abstract

This invention discloses a method for scalable preparation of 5-fluorouracil-loaded lyophilized nanoparticles. The method first involves self-assembling 5-fluorouracil into a polymer carrier to form core-shell nanoparticles. Then, the hydrophobic core-shell nanoparticles undergo a double emulsification-isothermal evaporation process to form hydrophilic micro / nanoparticles. Finally, D-mannose is assembled onto the surface of the micro / nanoparticles, followed by gradient freeze-drying to prepare PLGA / 5-FU-loaded lyophilized nanoparticles. This method offers advantages such as simple operation, scalability, and good reproducibility. The 5-FU drug in the prepared lyophilized nanoparticles exhibits good crystallinity. During the inhibition of pancreatic cancer cell growth, it effectively improves the bioavailability, biocompatibility, and physical stability of 5-fluorouracil, significantly inhibiting pancreatic cancer cell growth and demonstrating high anti-tumor immunomodulatory properties.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical formulation technology and relates to a method for scalable preparation of 5-fluorouracil drug-loaded lyophilized nanoparticles. Background Technology

[0002] 5-Fluorouracil (5-FU), a classic antimetabolite broad-spectrum antitumor drug, is currently the first-line treatment for digestive system tumors and is widely used in the treatment of gastrointestinal malignancies such as colorectal cancer, gastric cancer, and pancreatic cancer. Natural killer (NK) cells are lymphocyte-like cytotoxic innate immune cells that play an important role in antitumor responses. Recent studies have shown that 5-FU has immunomodulatory effects and can serve as an effective inducer of tumor-infiltrating NK cell activity. However, similar to most chemotherapy drugs, 5-FU is not completely absorbed orally, resulting in low bioavailability and low effective concentrations of 5-FU at the tumor site. Furthermore, 5-FU has a short half-life (only 10–20 minutes), requiring frequent administration, leading to significant toxic side effects, increased burden on the liver and kidneys, and ultimately reduced patient compliance and drug resistance. These drawbacks limit its widespread clinical application. Therefore, improving the bioavailability of 5-FU, increasing drug targeting, prolonging the drug's half-life, reducing its drug resistance, and enhancing its anti-tumor immune activity are key challenges that urgently need to be addressed to improve the efficacy of 5-FU.

[0003] The development of amphiphilic block copolymer drug-loaded nanotechnology has shown excellent potential in improving the bioavailability of chemotherapeutic drugs. Chinese Patent 201710859517.X uses a hydrophilic aqueous solution to disperse 5-FU, resulting in drug-loaded microspheres that are sensitive to environmental pH and exhibit rapid swelling response in alkaline environments, enabling rapid release of the active ingredient. Chinese Patent 201110312636.6 uses an oil-phase hydrophobic dichloromethane solvent to disperse 5-FU, producing drug-loaded microspheres with uniform particle size, high drug loading and encapsulation efficiency, and good drug release performance. However, both methods struggle to improve the purity and crystallinity of 5-FU, leading to a decrease in its activity. Furthermore, the resulting microspheres are either too small (50-70 nm) or too large (30-80 μm), failing to meet the 100-200 nm size requirement for high permeability retention (EPR) effects. Chinese Patent 201010616596.X describes a method for preparing drug-loaded nanoparticles using polylactic acid-polyethylene glycol block copolymer (PLLA-PEG) as a carrier to improve the targeting of 5-FU and reduce its toxicity. However, this method is insufficient to effectively prolong the drug's half-life, reduce drug resistance, and enhance its anti-tumor immune activity. In addition, existing technologies (such as Chinese Patent 201110312636.6 and Chinese Patent 201710859517.X) have improved the drug delivery efficiency of 5-FU by selecting different carriers (such as chitosan, lactose, sodium alginate, etc.). However, the preparation process is complex, and the traditional high-pressure homogenization method combined with the double emulsion-solvent evaporation method for preparing 5-FU drug-loaded nanoparticles is difficult to scale up.

[0004] In addition, drug-loaded nanoparticles prepared by traditional thermal processing methods such as spray drying and microwave drying have small particle sizes and high surface activity, making them prone to aggregation, which in turn affects the improvement of the bioavailability of chemotherapy drugs. Furthermore, thermal drying can easily lead to the inactivation of active ingredients.

[0005] Freeze-drying technology is considered an ideal preservation method for maintaining the activity of active ingredients. It primarily involves physical sublimation to remove moisture from drug-loaded nanoparticle solutions under low temperature and vacuum conditions, resulting in drug-loaded lyophilized nanoparticle powders with a water content of less than 1%. Lyophilized nanoparticle powders can better improve the stability of chemotherapy drugs. The freeze-dried particles are small, uniform, and regularly shaped, with a loose morphology, allowing for rapid dissolution and restoration of the original aqueous solution's physicochemical properties and biological activity, reducing the chance of contamination. This not only facilitates the transportation of chemotherapy drugs but also extends their shelf life. Typically, during the freeze-drying process of drug-loaded nanoparticles, preparations such as sucrose, glucose, gelatin, mannitol, trehalose, lactose, and sorbitol are used as freeze-drying protectants to safeguard the structural stability and activity of the active drug molecules. D-Mannose is an alcohol-sugar compound that induces molecular glycosylation in various cellular processes and plays an important role in mediating the O-glycosylation of the helper T cell-derived cytokine interlukin-17A. However, D-mannose is rarely used as a lyophilization protectant for chemotherapy drugs to enhance their antitumor immune activity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the large-scale preparation of 5-fluorouracil drug-loaded lyophilized nanoparticles. This method uses polyethylene glycol-polylactic acid-hydroxyglycolic acid copolymer (PEG-PLGA) as a carrier, and 5-FU is purified and crystallized using a dimethyl sulfoxide-water (DMSO-H2O) mixed solvent. With D-mannose as a lyophilization protectant, the PLGA / 5-FU drug-loaded lyophilized nanoparticles are prepared through a combination of reemulsification-solvent evaporation and gradient freeze-drying processes.

[0007] The technical solution for achieving the objective of this invention is as follows:

[0008] A method for scalable preparation of 5-fluorouracil drug-loaded lyophilized nanoparticles involves first assembling 5-fluorouracil into a polymer carrier to form core-shell nanoparticles. Then, the hydrophobic core-shell nanoparticles undergo a double emulsion-isothermal evaporation process to form hydrophilic micro-nanoparticles. Finally, D-mannose is assembled onto the surface of the micro-nanoparticles, followed by gradient freeze-drying to prepare PLGA / 5-FU drug-loaded lyophilized nanoparticles. The specific steps are as follows:

[0009] S1. 5-FU is dispersed in a DMSO-H2O mixed solvent, and the resulting 5-FU DMSO-H2O solution is added dropwise to a PEG-PLGA dichloromethane solution at a constant temperature. After pulsed ultrasonic emulsification, the solution self-assembles to form a hydrophobic nano-core-shell particle emulsion.

[0010] S2. The hydrophobic nano-core-shell particle emulsion was added dropwise to a polyvinyl alcohol (PVA) complex emulsion aqueous solution at a constant temperature. After pulse ultrasonic emulsification, it was dispersed in PBS buffer solution. Then, under stirring conditions, the dichloromethane was evaporated at a constant temperature to remove the hydrophilic micro-nano particle solution.

[0011] S3. Under constant temperature stirring, D-mannose aqueous solution is added to the solution of surface hydrophilic micro-nano particles to form surface-assembled D-mannose micro-nano particles. After gradient freeze-drying, PLGA / 5-FU drug-loaded nano-lyophilized powder is obtained.

[0012] Further, in S1, the PEG-PLGA has a methoxyl active group at the PEG end; the mass ratio of 5-FU to PEG-PLGA is 1:5 to 1:1; in the DMSO-H2O mixed solvent, the volume ratio of DMSO to H2O is 4:1 to 1:4; the isothermal condition is 20 to 30°C; and the pulsed ultrasonic emulsification conditions are: ultrasonic frequency of 40 to 1000 kHz, ultrasonic power of 100 to 500 W, ultrasonic duty cycle of 50%, and ultrasonic time of 30 to 120 s. Preferably, in the DMSO-H2O mixed solvent, the volume ratio of DMSO to H2O is 4:1, the mass ratio of 5-FU to PEG-PLGA is 1:2; the isothermal condition is 20°C; and the pulsed ultrasonic emulsification conditions are: ultrasonic frequency of 40 kHz, ultrasonic power of 100 W, ultrasonic duty cycle of 50%, and ultrasonic time of 60 s.

[0013] Further, in S2, the mass ratio of PVA re-emulsion to PEG-PLGA is 1:20 to 5:20, the volume ratio of PBS buffer solution to dichloromethane is 1:1 to 10:1, and the isothermal evaporation temperature is 35 to 60°C. Preferably, the mass ratio of PVA re-emulsion to PEG-PLGA is 1:10, the volume ratio of PBS buffer solution to dichloromethane is 10:1, and the isothermal evaporation temperature is 37°C.

[0014] Further, in S3, the mass ratio of D-mannose to PEG-PLGA is 20:1 to 1:1, and the constant temperature stirring temperature is 10 to 25°C. Preferably, the mass ratio of D-mannose to PEG-PLGA is 10:1, and the constant temperature stirring temperature is 15°C.

[0015] Furthermore, in S3, the gradient freeze-drying employs a three-step gradient cooling process: the first step involves a cooling rate of 5℃ / min, decreasing the temperature from 35℃ to 0℃; the second step involves a cooling rate of 2–20℃ / h, decreasing the temperature to -10℃, with a vacuum of 1–100 Pa, and holding for 0.5–2 h; the third step involves a cooling rate of 5–15℃ / min, decreasing the temperature to -40℃, with a vacuum of 100–300 Pa, and holding for 10–20 h, until the freeze-drying is complete. Preferably, the first step involves a cooling rate of 5℃ / min, decreasing the temperature from 35℃ to 0℃; the second step involves a cooling rate of 10℃ / h, decreasing the temperature to -10℃, with a vacuum of 60 Pa, and holding for 1 h; the third step involves a cooling rate of 5℃ / min, decreasing the temperature to -40℃, with a vacuum of 180 Pa, and holding for 16 h, until the freeze-drying is complete.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) This invention makes full use of the specificity of the equivalent dielectric constant of the DMSO-H2O mixed solvent, which improves the stability of 5-FU drug in the reemulsion and solvent evaporation stages, and maintains the crystallinity of 5-FU in the carrier to a certain extent.

[0018] (2) The present invention selects D-mannose molecules as freeze-drying protectant and modifies the surface of drug-loaded nanoparticles. On the one hand, it improves the biocompatibility and uniform dispersion of nanoparticles and regulates the size of nanoparticles in the range of 100-200nm; on the other hand, it can enhance the anti-tumor immune activity of 5-FU.

[0019] (3) The present invention uses a process combining double emulsion-solvent evaporation and gradient freeze drying to prepare drug-loaded nano-lyophilized powder. During this process, the molecular structure of 5-FU drug does not change and its crystallinity is improved. The freeze-dried particles are small, uniform, regular in shape and loose in morphology, and can quickly dissolve and restore the physicochemical properties and biological activity of the original aqueous solution.

[0020] (4) This invention replaces the cumbersome ultrafiltration purification step in the traditional preparation of drug-loaded nanoparticles, while improving the crystallinity of the drug. This method has the advantages of simple operation, mild conditions, scalability, and good reproducibility. The prepared drug-loaded lyophilized nanoparticles can better improve the stability of 5-FU chemotherapy drugs and exert excellent sustained-release function in the process of inhibiting the growth of pancreatic cancer cells, and effectively improve the bioavailability of 5-FU, significantly inhibit the growth of pancreatic cancer cells, and show high anti-tumor immune performance. Attached Figure Description

[0021] Figure 1 This is a transmission electron microscope image of the drug-loaded nanoparticles prepared in Example 1;

[0022] Figure 2This is a scanning electron microscope image of the drug-loaded nanoparticles prepared in Example 1;

[0023] Figure 3 This is a comparison of the performance of the drug-loaded nanoparticles prepared in Example 1 and free 5-FU in inducing pyroptosis and release of lactate dehydrogenase (LDH) in pancreatic cancer cells;

[0024] Figure 4 The stability and sustained-release properties of the drug-loaded nanoparticles prepared in Example 1;

[0025] Figure 5 The image shows a transmission electron microscope (TEM) image of the drug-loaded nanoparticles prepared in Comparative Example 2.

[0026] Figure 6 The image shows a transmission electron microscope (TEM) image of the drug-loaded nanoparticles prepared in Comparative Example 4. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0028] All reagents used in this invention are commercially available. In the following examples and comparative examples, the PEG-PLGA used has a methoxy terminus in its PEG-terminus.

[0029] Example 1

[0030] S1. 5-Fluorouracil is self-assembled into a polymer carrier to form nano-core-shell particles:

[0031] Accurately weigh 20 mg of PEG-PLGA and disperse it in 5 mL of dichloromethane to obtain a dichloromethane solution of PEG-PLGA; weigh 10 mg of 5-FU and disperse it in 5 mL of dimethyl sulfoxide-water (V... 二甲基亚砜 :V 水 A DMSO-H2O solution of 5-FU was obtained in a mixed solvent of 4:1. The DMSO-H2O solution of 5-FU was added dropwise to a dichloromethane solution of PEG-PLGA at a mass ratio of 1:2 under constant temperature of 20℃. After pulsed ultrasonic emulsification, a hydrophobic nano-core-shell particle emulsion was prepared. The ultrasonic conditions were: 40kHz, 100W, duty cycle 50%, and time 60s.

[0032] S2. Re-emulsification of core-shell nanoparticles to form micro / nanoparticles:

[0033] Under constant temperature of 20℃, 2 mL of 1 mg / mL PVA emulsion aqueous solution was added dropwise to the above hydrophobic nano-core-shell particle emulsion at a mass ratio of PVA emulsion to PEG-PLGA of 2:20. After pulse ultrasonic emulsification, the emulsion was dispersed into 20 mL of PBS buffer solution. The ultrasonic conditions were: 40 kHz, 100 W, duty cycle of 50%, and time of 60 s. The volume ratio of PBS buffer solution to dichloromethane was 4:1. Dichloromethane was removed by constant temperature evaporation under stirring to prepare a solution of hydrophilic micro-nano particles.

[0034] S3. Assemble D-mannose onto the surface of micro / nano particles to prepare nano-lyophilized powder:

[0035] 200 mg of D-mannose was accurately weighed and dispersed in 5 mL of water. At a mass ratio of D-mannose to PEG-PLGA of 10:1, D-mannose was added to the hydrophilic micro / nano particle solution under constant temperature stirring at 15 °C. 5-FU-loaded nanoparticles with a loading rate of 3.63% and an encapsulation efficiency of 86% were prepared by a three-step gradient freeze-drying process. The specific conditions for the gradient freeze-drying were as follows: Step 1: Cooling rate of 5 °C / min, temperature decreasing from 35 °C to 0 °C; Step 2: Cooling rate of 10 °C / h, decreasing to -10 °C, vacuum degree of 60 Pa, held for 1 h; Step 3: Cooling rate of 5 °C / min, decreasing to -40 °C, vacuum degree of 180 Pa, held for 16 h.

[0036] The prepared 5-FU drug-loaded nanoparticles were characterized by transmission electron microscopy, such as... Figure 1 and Figure 2 As shown, relatively uniform nanoparticles with a particle size of approximately 100 nm can be observed.

[0037] The pyroptosis induced by lactate dehydrogenase (LDH) release assay was detected. The specific steps were as follows: SW1990 and Pan02c pancreatic cancer cells in logarithmic growth phase were seeded into 24-well cell culture plates. 24 hours after seeding, the culture medium was aspirated, and the cells were washed once with PBS. Drug was added or fresh culture medium was replaced, and the cells were divided into the following groups: untreated wells (spontaneous release control wells), untreated wells used for subsequent lysis (sample maximum enzyme activity control wells), and wells treated with free 5-FU or 5-FU-loaded nanoparticles (effective 5-FU concentration 65 mg / mL, incubation time 72 h). One hour before the predetermined detection time, LDH release reagent was added to the "sample maximum enzyme activity control wells," at a volume of 10% of the original culture medium. The mixture was repeatedly pipetted several times to mix thoroughly, and the cells were incubated in a cell culture incubator. After the predetermined time, the culture supernatant from each group was collected, centrifuged at 400g for 5 min, and the supernatant was transferred to a new tube. Take 120 μL of supernatant from each tube and add it to a 96-well plate. Add 60 μL of working solution to each well, mix well, and incubate at room temperature in the dark for 30 min. Perform dual-wavelength measurements at 490 nm and 630 nm to calculate the LDH release rate.

[0038] LDH release experiment results are as follows: Figure 3 and 4 As shown, by comparing with free 5-FU, when evaluating the cytotoxicity of two different cell lines, SW1990 and Pan02, it was found that the prepared 5-FU-loaded nanoparticles not only effectively delayed the sustained-release effect of 5-FU and still had efficacy after 72 hours of treatment; but also, compared with free 5-FU, the 5-FU-loaded nanoparticles significantly enhanced the activity of inducing pyroptosis and releasing LDH, thereby inhibiting the growth of pancreatic cancer cells.

[0039] Example 2

[0040] S1. 5-Fluorouracil is self-assembled into a polymer carrier to form nano-core-shell particles:

[0041] Accurately weigh 20 mg of PEG-PLGA and disperse it in 5 mL of dichloromethane to obtain a dichloromethane solution of PEG-PLGA; weigh 20 mg of 5-FU and disperse it in 5 mL of dimethyl sulfoxide-water (V... 二甲基亚砜 :V 水 A DMSO-H2O solution of 5-FU was obtained in a mixed solvent of 1:4. The DMSO-H2O solution of 5-FU was added dropwise to a dichloromethane solution of PEG-PLGA at a mass ratio of 1:1 under constant temperature of 20℃. After pulsed ultrasonic emulsification, a hydrophobic nano-core-shell particle emulsion was prepared. The ultrasonic conditions were: 40kHz, 100W, duty cycle 50%, and time 60s.

[0042] S2: Re-emulsification of core-shell nanoparticles to form micro / nanoparticles.

[0043] Under constant temperature of 20℃, 5 mL of 1 mg / mL PVA emulsion aqueous solution was added dropwise to the above hydrophobic nano-core-shell particle emulsion at a mass ratio of PVA emulsion to PEG-PLGA of 5:20. After pulse ultrasonic emulsification, the emulsion was dispersed in 50 mL of PBS buffer solution. The ultrasonic conditions were: 40 kHz, 100 W, duty cycle of 50%, and time of 60 s. The volume ratio of PBS buffer solution to dichloromethane was 10:1. Dichloromethane was removed by constant temperature evaporation under stirring to prepare a solution of hydrophilic micro-nano particles.

[0044] S3. Assemble D-mannose onto the surface of micro / nano particles to prepare nano-lyophilized powder:

[0045] 20 mg of D-mannose was accurately weighed and dispersed in 5 mL of water. At a mass ratio of D-mannose to PEG-PLGA of 1:1, the D-mannose was added to the hydrophilic micro / nano particle solution under constant temperature stirring at 15 °C. 5-FU-loaded nanoparticles with a drug loading of 3.28% and an encapsulation efficiency of 81.2% were prepared by a three-step gradient freeze-drying process. The specific conditions for the gradient freeze-drying were as follows: First, the cooling rate was 5 °C / min, with the temperature decreasing from 35 °C to 0 °C; second, the cooling rate was 10 °C / h, decreasing to -10 °C under a vacuum of 60 Pa, and held for 1 h; third, the cooling rate was 5 °C / min, decreasing to -40 °C under a vacuum of 180 Pa, and held for 16 h.

[0046] The 5-FU drug-loaded nanoparticles prepared in this embodiment have a particle size of approximately 150 nm, as shown in Table 1. Table 1 Comparison of physical properties and sustained-release effects of 5-FU drug-loaded nanoparticles prepared in Example 2 and Comparative Examples 1-4

[0047]

[0048] Comparative Example 1

[0049] This comparative example is basically the same as Example 1, except that in S1, 5-FU is dispersed in 5 mL of dimethyl sulfoxide solution.

[0050] The 5-FU-loaded nanoparticles prepared in this comparative example have a particle size of approximately 100 nm, a 5-FU loading rate of 3.2%, and an encapsulation efficiency of 76.7%. Compared with Example 1, this demonstrates that the equivalent dielectric constant of the dimethyl sulfoxide-water mixed solvent specifically promotes 5-FU loading, improves the stability of the 5-FU drug during reemulsification and solvent evaporation, and maintains the effectiveness of 5-FU in the carrier to a certain extent. Furthermore, analysis of the time required for complete drug release revealed that the sustained-release effect of the 5-FU-loaded nanoparticles prepared in this comparative example can only be extended to 60 h; specific data are shown in Table 1.

[0051] Comparative Example 2

[0052] This comparative example is basically the same as Example 1, except that in S1, 5-FU is dispersed in 5 mL of pure water.

[0053] The 5-FU drug-loaded nanoparticles prepared in this comparative example have a particle size of approximately 70 nm, a 5-FU drug loading rate of 2.5%, and an encapsulation efficiency of 70%. Compared with Example 1, the 5-FU drug-loaded nanoparticles prepared in this comparative example have lower crystallinity of 5-FU drug. Figure 5 As shown, pure water is not conducive to the effective loading of 5-FU into the polymer. Furthermore, analysis of the time required for complete drug release revealed that the sustained-release effect of the drug-loaded particles in this comparative example could only be extended to 36 hours; specific data are shown in Table 1.

[0054] Comparative Example 3

[0055] This comparative example is basically the same as Example 1, except that in S3, the three-step gradient freeze drying is replaced with vacuum drying at 35°C.

[0056] The 5-FU-loaded nanoparticles prepared in this comparative example had a 5-FU loading rate of 3.4%, an encapsulation efficiency of 60.5%, and a particle size of approximately 200 nm. These results indicate that the drug-loaded lyophilized nanoparticles prepared using a combination of double emulsion-solvent evaporation and gradient freeze-drying produce fine, uniform, regularly shaped, and loosely morphologically dispersed particles that can rapidly dissolve and restore the physicochemical properties and biological activity of the original aqueous solution. Furthermore, analysis of the time required for complete drug release revealed that the sustained-release effect of the 5-FU-loaded nanoparticles in this comparative example can be extended to 72 hours. Specific data are shown in Table 1.

[0057] Comparative Example 4

[0058] This comparative example is basically the same as Example 1, except that in S3, the three-step gradient freeze drying is replaced with a one-step freeze drying at -40°C.

[0059] The 5-FU-loaded nanoparticles prepared in this comparative example had a 5-FU loading rate of 2.8%, an encapsulation efficiency of 71.8%, and a particle size of approximately 100 nm. However, the nanoparticles exhibited breakage. Figure 6 As shown in the figure. The above results indicate that the drug-loaded lyophilized nanoparticles prepared by the process of combining double emulsion-solvent evaporation and gradient freeze drying are small, uniform, regularly shaped and loosely morphological. They can quickly dissolve and restore the physicochemical properties and biological activity of the original aqueous solution. At the same time, by analyzing the time required for complete drug release, it was found that the sustained release of the drug-loaded particles in this comparative example can be extended to 72 hours. The specific data are shown in Table 1.

Claims

1. A method for scalable preparation of 5-fluorouracil drug-loaded lyophilized nanoparticles, characterized in that, The specific steps are as follows: S1. 5-FU was dispersed in a DMSO-H2O mixed solvent, and the resulting 5-FU DMSO-H2O solution was added dropwise to a PEG-PLGA dichloromethane solution at a constant temperature. After pulsed ultrasonic emulsification, the mixture self-assembled to form a hydrophobic nano-core-shell particle emulsion. In the DMSO-H2O mixed solvent, the volume ratio of DMSO to H2O was 4:1, and the mass ratio of 5-FU to PEG-PLGA was 1:

2. The constant temperature condition was 20℃. The pulsed ultrasonic emulsification conditions were: ultrasonic frequency 40 kHz, ultrasonic power 100W, ultrasonic duty cycle 50%, and ultrasonic time 60s. S2. The hydrophobic nano-core-shell particle emulsion was added dropwise to an aqueous solution of PVA re-emulsifier at a constant temperature. After pulsed ultrasonic emulsification, it was dispersed in a PBS buffer solution. Then, under stirring conditions, the dichloromethane was evaporated at a constant temperature to remove it, resulting in a solution of hydrophilic micro-nano particles. The mass ratio of PVA re-emulsifier to PEG-PLGA was 1:10, the volume ratio of PBS buffer solution to dichloromethane was 10:1, and the constant temperature evaporation temperature was 37℃. S3. Under constant temperature stirring, D-mannose aqueous solution was added to the solution of hydrophilic micro / nano particles to form micro / nano particles with D-mannose assembled on the surface. After gradient freeze-drying, PLGA / 5-FU drug-loaded nano-lyophilized powder was obtained. The mass ratio of D-mannose to PEG-PLGA was 10:1, and the constant temperature stirring temperature was 15℃. The gradient freeze-drying adopted a three-step gradient cooling: the first step was a cooling rate of 5℃ / min, with the temperature dropping from 35℃ to 0℃; the second step was a cooling rate of 10℃ / h, dropping to -10℃, with a vacuum degree of 60 Pa, and held for 1h; the third step was a cooling rate of 5℃ / min, dropping to -40℃, with a vacuum degree of 180 Pa, and held for 16h, until the freeze-drying was completed.

2. The method according to claim 1, characterized in that, In S1, the PEG-PLGA has a methoxy active group at the PEG end; the isothermal condition is 20~30℃.

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