Chitosan drug-loaded microspheres based on ionic crosslinking self-assembly synergistic mechanism and preparation method of chitosan drug-loaded microspheres

Abstract

The invention relates to the technical field of biomedical materials and drug delivery, in particular to a chitosan drug-loaded microsphere based on an ionic crosslinking self-assembly synergistic mechanism and a preparation method thereof.The preparation method comprises the steps that chitosan is dissolved in an acetic acid aqueous solution, a drug and an amphiphilic block copolymer are added to form a primary self-assembly structure, and the primary self-assembly structure is prepared; and dropwise adding sodium tripolyphosphate to trigger ionic crosslinking and secondary self-assembly, and finally purifying and freeze-drying to obtain a microsphere finished product. The microspheres are uniform in particle size, high in encapsulation efficiency, excellent in slow release performance and suitable for anti-cancer drugs, protein drugs and vaccine carriers, the preparation process is carried out in a pure water phase system, no organic solvent or complex equipment is needed, the process is simple and convenient, the cost is low, the amplification performance is high, and the good industrialization prospect is shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and drug delivery technology, specifically a chitosan drug-loaded microsphere based on an ion crosslinking self-assembly synergistic mechanism and its preparation method. Background Technology

[0002] Chitosan, a natural cationic polysaccharide, has become an important research direction in drug delivery systems due to its excellent biocompatibility, biodegradability, and controlled drug release properties. Chitosan drug-loaded microspheres constructed through ionic cross-linking or self-assembly can play a crucial role in sustained drug release, targeted delivery, and improved drug stability. However, current technologies still face multiple challenges in regulating the microstructure and functional properties of microspheres, particularly in achieving a synergistic optimization between high loading efficiency, particle size uniformity, and long-term release stability.

[0003] Ionic crosslinking methods can form stable three-dimensional network structures, but they typically rely on high concentrations of crosslinking agents, which may lead to loss of drug activity and a wide particle size distribution. While strategies based on self-assembly mechanisms can construct ordered structures and improve encapsulation efficiency under mild conditions, they lack sufficient crosslinking strength to maintain the structural integrity of microspheres in complex physiological environments. Although some studies have attempted to combine the advantages of both methods, such as introducing crosslinking and self-assembly processes through physical mixing or stepwise reactions, they have failed to achieve dynamic synergy between the two at the molecular scale. As a result, microspheres still have significant limitations in terms of spheroidization kinetics, drug distribution uniformity, and controllability of release behavior.

[0004] Patent CN116392600A discloses an aqueous composition comprising a macrolide immunosuppressant drug at a concentration of less than 2% w / v and an amphiphilic carbohydrate (such as palmitoyl glycol chitosan quaternary ammonium salt, GCPQ) with a molecular weight range of 1-50 kDa, for the topical treatment of ocular diseases. This composition enhances the stability and permeability of the drug through the self-assembly of chitosan. However, this method relies primarily on the self-assembly mechanism of chitosan and does not adequately consider the contribution of ionic crosslinking to the stability of the microsphere structure. Therefore, burst release may occur in complex media, and the controllability of the release behavior needs further improvement.

[0005] Patent CN108366960A discloses a drug delivery device that utilizes a stimulus-responsive gel composition (such as chitosan) filled within a porous body to achieve controlled drug release. This device regulates the swelling and contraction behavior of the gel in response to external stimuli (such as changes in glucose concentration), thereby controlling the drug release rate. However, this technology focuses on macroscopic stimulus-responsive design, paying less attention to the microstructural optimization of chitosan microspheres. It fails to effectively couple ionic crosslinking and self-assembly mechanisms, resulting in limitations in microsphere particle size uniformity and long-term release stability. Summary of the Invention

[0006] This application provides a chitosan drug-loaded microsphere based on an ionic crosslinking self-assembly synergistic mechanism and its preparation method. The aim is to achieve precise control of the microstructure and functional integration design of the chitosan drug-loaded microsphere by simultaneously triggering ionic crosslinking and molecular self-assembly processes in a pure aqueous system.

[0007] In a first aspect, this application provides a method for preparing chitosan-loaded drug microspheres, comprising the following steps: S10: Dissolve chitosan in an aqueous acetic acid solution, adjust the pH of the solution to between 4.5 and 5.5, control the chitosan concentration to between 1.0 mg / mL and 3.0 mg / mL, and stir until a homogeneous and transparent solution is formed; S20: Add the target drug and the amphiphilic block copolymer to the above chitosan solution, and stir continuously at 30 degrees Celsius to 40 degrees Celsius for 2 to 4 hours to allow the drug molecules and the block copolymer to form a primary self-assembled structure around the chitosan chain. S30: Add sodium tripolyphosphate aqueous solution to the above mixture at a constant flow rate, while maintaining the system temperature at 35 degrees Celsius and the stirring rate at 800 rpm, so that the chitosan molecular chains undergo ionic cross-linking and drive the block copolymer to further self-assemble into a nanoscale ordered structure. S40: After the reaction lasts for 30 to 60 minutes, an equal volume of anhydrous ethanol is added to terminate the ionic cross-linking reaction. The microspheres are then collected by centrifugation and washed three times with deionized water to remove unreacted substances and free drugs. S50: Freeze-dry the purified microsphere suspension to obtain chitosan drug-loaded microspheres with uniform particle size and stable structure.

[0008] According to this application, in step S10, chitosan is dissolved in an aqueous acetic acid solution with a mass-to-volume ratio of 1.0%, and the pH of the solution is adjusted to between 4.5 and 5.5, preferably 5.0. The degree of deacetylation of the chitosan is greater than or equal to 85%, preferably 90% to 95%, and the molecular weight ranges from 50 kilodaltons to 200 kilodaltons, preferably 100 kilodaltons to 150 kilodaltons. This combination of parameters ensures good water solubility while providing sufficient amino density to achieve efficient ionic crosslinking, avoiding insufficient mechanical strength due to excessively low molecular weight or excessively high solution viscosity that would affect the uniformity of subsequent mixing. The stirring process uses a magnetic stirrer or mechanical stirring paddle, with the rotation speed controlled between 300 rpm and 500 rpm to ensure that the chitosan particles are completely swollen and uniformly dispersed. The final colloidal solution has a viscosity range of 2 mPa·s to 5 mPa·s and a potential value of +35 mV to +45 mV.

[0009] In some embodiments, in step S20, the target drug encompasses water-soluble or weakly hydrophobic small molecule drugs, peptide drugs, and some nucleic acid substances. The amphiphilic block copolymer is preferably a polyethylene glycol-polylactic acid block copolymer, wherein the polyethylene glycol segment has a molecular weight of 2000, the polylactic acid segment has a molecular weight of 3000, and the mass ratio of the block copolymer to chitosan is strictly controlled between 1:10 and 1:5, preferably 1:7. The mixing process of the drug and the block copolymer is carried out in a constant temperature water bath, with the stirring rate maintained at 400 rpm. For hydrophobic drugs such as paclitaxel derivatives, they mainly embed into the hydrophobic microdomains formed by the polylactic acid segments through hydrophobic interactions; for negatively charged peptides such as insulin fragments, they simultaneously bind to protonated chitosan and the block copolymer through electrostatic attraction and hydrogen bonding; for nucleic acid substances such as siRNA, their phosphate backbone forms an ion pair with the –NH3⁺ group of chitosan and is simultaneously encapsulated near the hydrophobic core of the block copolymer, forming a stable complex. The formation of this primary core-shell structure is a thermodynamically spontaneous process driven by the decrease in the system's free energy, and the resulting composite particles typically range from 20 to 50 nanometers in size.

[0010] In some embodiments, in step S30, the concentration of the sodium tripolyphosphate aqueous solution is from 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL, the dropping rate is 0.5 mL / min, and the volume ratio of the total amount added to the chitosan solution is 1:5 to 1:3, preferably 1:4. The entire ionic crosslinking and self-assembly process is carried out in a pure aqueous system completely free of oil, surfactants, and organic solvents, and the ionic strength of the system is strictly controlled below 50 mmol / L. A precision syringe pump is used to control the flow rate during the dropping process, and high-speed stirring keeps the reaction system in a turbulent state, promoting ion diffusion and collision, and accelerating the crosslinking reaction kinetics. Under the constraint of the crosslinking network, the hydrophobic segments of the block copolymer further aggregate to form ordered nanostructures such as micelles, vesicles, or layers. These structures act as "nanowarehouses" for drugs, preventing drug molecules from migrating, aggregating, or crystallizing inside the microspheres. The stoichiometric relationship of the ionic crosslinking reaction is n(–NH3⁺):n(P3O 10 5 The ratio of sodium tripolyphosphate (STP) to chitosan is 5:1, which ensures sufficient formation of crosslinking points while avoiding excessive shrinkage or charge reversal of the microspheres due to excessive sodium tripolyphosphate. The secondary self-assembly process is regulated by the critical micelle concentration of the block copolymer, forming stable nanoregions in the chitosan matrix. These nanoregions typically range in size from 10 to 30 nanometers and work synergistically with the primary core-shell structure to construct a multi-layered, highly ordered internal structure of the microspheres.

[0011] In some embodiments, in step S40, the centrifugation conditions are 10,000 rpm for 10 minutes. This parameter can efficiently settle nanospheres in the range of 150 nm to 300 nm, with a recovery rate higher than 95%. The washing process combines a dual purification strategy of high-speed centrifugation and 0.22-micron membrane filtration, which can effectively remove small molecule impurities while avoiding microsphere loss or structural damage caused by traditional ultrafiltration or dialysis. The addition of anhydrous ethanol causes the dielectric constant of the system to drop sharply from about 80 to 24, resulting in a sharp weakening of the electrostatic attraction between –NH3⁺ and sodium tripolyphosphate anions on the chitosan molecular chain, thereby terminating the cross-linking reaction. The centrifugation operation is carried out at 4 degrees Celsius. After each wash, the supernatant is confirmed to have no free drug residue by high-performance liquid chromatography or ultraviolet-visible spectroscopy. The 0.22-micron microporous membrane filtration step is completed in a sterile operating table using a vacuum filtration device, with the filtration rate controlled at 5 mL per minute to avoid shear force damaging the microsphere structure.

[0012] In some embodiments, in step S50, 5% (w / v) of mannitol is added to the purified microsphere suspension as a freeze-drying protectant. The pre-freezing temperature is -40°C, and the freeze-drying time is 24 hours. The concentration of mannitol is strictly selected; a concentration of 5% provides sufficient protection without causing the freeze-dried cake to become too hard and affect reconstitution due to excessive concentration. The pre-freezing stage uses a programmed cooling device with a cooling rate of 1°C per minute to ensure uniform ice crystal formation. The primary drying stage is carried out at -20°C and a pressure of ≤10 Pascals for 12 hours. The secondary drying stage is carried out at 25°C for another 12 hours to completely remove bound water. The final freeze-dried powder is white, loose, and porous with a moisture content of less than 2%. It can be stably stored for more than 12 months under light-protected conditions at 4°C, and the microsphere particle size and polydispersity index show no significant change after reconstitution.

[0013] Secondly, this application provides chitosan drug-loaded microspheres, prepared according to the method described in any embodiment of the first aspect. The chitosan drug-loaded microspheres have an average particle size of 150 nm to 300 nm, a polydispersity index of less than or equal to 0.15, a drug encapsulation efficiency of greater than or equal to 90%, and a drug loading of 8% to 15%. The cumulative drug release rate of the chitosan drug-loaded microspheres does not exceed 40% within 72 hours in a phosphate buffer solution with a pH of 7.4, and does not fall below 70% within 72 hours in an acidic environment with a pH of 5.0.

[0014] Thirdly, this application provides an application of chitosan drug-loaded microspheres based on an ionic cross-linking self-assembly synergistic mechanism, suitable for sustained-release carriers of anticancer drugs, protein drugs, and vaccines. The preparation method is carried out entirely under normal pressure and at 30 to 40 degrees Celsius, without the need for organic solvents, high-pressure homogenization, or complex emulsification equipment, showing good prospects for pilot-scale and industrialization.

[0015] According to this application, since the chitosan drug-loaded microspheres are prepared according to the method described in any embodiment of the first aspect, they possess the beneficial effects of the first aspect. Specifically, this application achieves precise control of the microstructure and functional integration design of chitosan drug-loaded microspheres by simultaneously triggering ionic crosslinking and molecular self-assembly processes in a pure aqueous system. This overcomes the limitations of traditional single crosslinking or single self-assembly strategies and successfully constructs a "structure-function" integrated design paradigm for chitosan drug-loaded microspheres at the nanoscale.

[0016] In some embodiments, the preparation method of the chitosan drug-loaded microspheres has strong scalability and industrial applicability. It is simple to operate, uses universal equipment (only requires a conventional stirrer, constant temperature water bath, centrifuge and freeze dryer), the raw materials are readily available, the cost is low, and the key process parameters (such as temperature, pH, concentration, and dropping rate) remain robust over a wide range. The consistency of microsphere performance (particle size deviation less than 10%, encapsulation rate fluctuation less than 5%) has been preliminarily verified in a 500 ml reaction system.

[0017] In summary, this application not only provides new ideas for the design of chitosan-based nanocarriers at the basic research level, but also offers an efficient, reliable, green, and sustainable technical pathway for the clinical translation of chitosan-based nanomedicines at the application level, possessing significant scientific value and broad market application prospects. Attached Figure Description

[0018] Figure 1 This is a flowchart of a method for preparing chitosan drug-loaded microspheres based on an ionic crosslinking self-assembly synergistic mechanism according to the present invention. Detailed Implementation

[0019] The embodiments or implementations described in this specification adopt a progressive approach, with each embodiment focusing on its differences from other embodiments. In the description of this specification, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with an implementation or example that are included in at least one implementation or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same implementation or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more implementations or examples.

[0020] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0021] Based on this, this application provides a chitosan drug-loaded microsphere based on an ionic crosslinking self-assembly synergistic mechanism and its preparation method, aiming to achieve the preparation of chitosan drug-loaded microspheres through a pure aqueous system. The specific embodiments of this application are described in detail below.

[0022] Please refer to Figure 1 In a first aspect, this application provides a method for preparing chitosan-loaded drug-eluting microspheres, comprising the following steps: S10: Dissolve chitosan in a 1.0% (w / v) aqueous acetic acid solution, adjust the pH to between 4.5 and 5.5, and control the chitosan concentration between 1.0 mg / mL and 3.0 mg / mL. Stir until a homogeneous and transparent solution is formed. In this step, the degree of deacetylation of chitosan is 85% to 95%, and the molecular weight ranges from 50 kDaltons to 200 kDaltons. Stirring is performed using a magnetic stirrer or mechanical stirrer at a speed controlled between 300 rpm and 500 rpm to ensure complete swelling and uniform dispersion of the chitosan particles. The final colloidal solution has a viscosity range of 2 mPa·s to 5 mPa·s and a potential value of +35 mV to +45 mV.

[0023] In the specific implementation process, after the chitosan is dissolved, the pH value needs to be measured and adjusted using a precision pH meter. It is preferable to gradually add diluted sodium hydroxide solution dropwise to achieve the target pH value. For high molecular weight chitosan (such as 150 kDaltons), the dissolution time needs to be extended to more than 30 minutes to ensure complete dissolution. During the dissolution process, the ambient temperature should be controlled between 25°C and 30°C to avoid chitosan degradation due to excessively high temperatures or reduced dissolution efficiency due to excessively low temperatures.

[0024] S20: Add the target drug and the amphiphilic block copolymer to the above chitosan solution, and stir continuously at 30°C to 40°C for 2 to 4 hours to allow the drug molecules and the block copolymer to form a primary self-assembled structure around the chitosan chains. The amphiphilic block copolymer is preferably a polyethylene glycol-polylactic acid block copolymer, wherein the polyethylene glycol segment has a molecular weight of 2000, the polylactic acid segment has a molecular weight of 3000, and the mass ratio of the block copolymer to chitosan is strictly controlled between 1:10 and 1:5. The stirring rate is maintained at 400 rpm.

[0025] In practice, the order and method of adding drugs and block copolymers need to be adjusted according to the properties of the drugs. For hydrophobic drugs such as paclitaxel derivatives, they can be dissolved in a small amount of organic solvent first and then slowly added dropwise to the chitosan solution; for negatively charged peptide drugs, they need to be added directly and stirred rapidly to promote their electrostatic interaction with protonated chitosan. During stirring, samples should be taken regularly to observe changes in the appearance of the solution, and a dynamic light scattering instrument should be used to monitor the particle size distribution to confirm the formation of primary self-assembled structures.

[0026] S30: A sodium tripolyphosphate aqueous solution is added dropwise to the above mixture at a constant flow rate, while maintaining the system temperature at 35 degrees Celsius and the stirring rate at 800 rpm. This allows the chitosan molecular chains to undergo ionic cross-linking and drives the block copolymer to further self-assemble into a nanoscale ordered structure. The concentration of the sodium tripolyphosphate aqueous solution is 0.5 mg / mL to 2.0 mg / mL, the dropping rate is 0.5 mL / min, and the total amount added is in a volume ratio of 1:5 to 1:3 to the chitosan solution.

[0027] In this step, deionized water must be used to prepare the sodium tripolyphosphate aqueous solution, and the storage time should not exceed 2 hours to avoid the introduction of impurities. During the dropwise addition, the syringe pump must be equipped with a micro-flow meter to monitor the flow rate in real time and ensure a constant dropping rate. The stirring conditions can be adjusted through an online stirring controller to ensure that the reaction system is always in a turbulent state. After the dropwise addition is completed, the system needs to be stirred for another 10 minutes to complete the ionic crosslinking reaction.

[0028] S40: After the reaction lasted for 30 to 60 minutes, an equal volume of anhydrous ethanol was added to terminate the ionic cross-linking reaction. The microspheres were then collected by centrifugation and washed three times with deionized water to remove unreacted substances and free drug. Centrifugation conditions were 10,000 rpm for 10 minutes, with a recovery rate exceeding 95%. The washing process combined high-speed centrifugation with a dual purification strategy of 0.22-micron membrane filtration.

[0029] In practice, the addition of anhydrous ethanol should be controlled using a peristaltic pump to prevent excessively high local concentrations that could damage the microsphere structure. Centrifugation should be performed at low temperatures, and the supernatant should be analyzed by high-performance liquid chromatography after each wash to confirm that the drug residue is below the detection limit. Membrane filtration should be performed in a sterile environment at a filtration rate of 5 mL / min to avoid shear forces that could damage the microsphere structure.

[0030] S50: The purified microsphere suspension is freeze-dried to obtain chitosan-loaded microspheres with uniform particle size and stable structure. Mannitol (5% by weight / volume) is added to the purified microsphere suspension as a freeze-drying protectant. The pre-freezing temperature is -40 degrees Celsius, and the freeze-drying time is 24 hours.

[0031] In practice, the pre-freezing stage requires a programmed cooling system with a cooling rate of 1 degree Celsius per minute to ensure uniform ice crystal formation. The primary drying stage is carried out for 12 hours at -20 degrees Celsius and a pressure of 10 Pascals or less. The secondary drying stage involves raising the temperature to 25 degrees Celsius and continuing drying for another 12 hours to completely remove bound water. The final freeze-dried powder is white, loose, and porous, with a moisture content of less than 2%.

[0032] Secondly, this application provides chitosan drug-loaded microspheres, prepared according to the method of any embodiment of the first aspect. The chitosan drug-loaded microspheres have an average particle size of 150 nm to 300 nm, a polydispersity index of less than or equal to 0.15, a drug encapsulation efficiency of greater than or equal to 90%, and a drug loading of 8% to 15%.

[0033] Thirdly, this application provides an application of chitosan drug-loaded microspheres based on an ionic cross-linking self-assembly synergistic mechanism, suitable for sustained-release carriers of anticancer drugs, protein drugs, and vaccines. The preparation method is carried out entirely under normal pressure and at 30 to 40 degrees Celsius, without the need for organic solvents, high-pressure homogenization, or complex emulsification equipment.

[0034] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0035] Example 1 S10: Dissolve 200 mg of chitosan with a degree of deacetylation of 90% and a molecular weight of 100 kDaltons in 200 mL of acetic acid aqueous solution with a mass-volume ratio of 1.0%, adjust the pH value to 5.0, and stir for 30 minutes until a transparent solution is formed.

[0036] S20: Add 10 mg of paclitaxel derivative and 20 mg of polyethylene glycol-polylactic acid block copolymer to the above solution and stir at 35 degrees Celsius for 3 hours.

[0037] S30: Add 1.0 mg / mL sodium tripolyphosphate aqueous solution to the above mixture at a rate of 0.5 mL / min, for a total volume of 50 mL, while stirring at 800 rpm.

[0038] S40: After the reaction has continued for 40 minutes, add 100 ml of anhydrous ethanol to terminate the reaction, centrifuge and wash three times with deionized water.

[0039] S50: Add 5% mannitol to the microsphere suspension, freeze-dry to obtain the finished product. Example 2

[0040] S10: Dissolve 300 mg of chitosan with a degree of deacetylation of 95% and a molecular weight of 150 kDaltons in 100 mL of acetic acid aqueous solution with a mass-volume ratio of 1.0%, adjust the pH value to 4.5, and stir for 40 minutes until a clear solution is formed.

[0041] S20: Add 15 mg of insulin fragment and 30 mg of polyethylene glycol-polylactic acid block copolymer to the above solution and stir at 30 degrees Celsius for 4 hours.

[0042] S30: Add 1.5 mg / mL sodium tripolyphosphate aqueous solution dropwise to the above mixture at a rate of 0.5 mL / min, for a total volume of 30 mL, while stirring at 800 rpm.

[0043] S40: After the reaction has continued for 50 minutes, add 60 ml of anhydrous ethanol to terminate the reaction, centrifuge and wash three times with deionized water.

[0044] S50: Add 5% mannitol to the microsphere suspension, freeze-dry to obtain the finished product. Example 3

[0045] S10: Dissolve 250 mg of chitosan with a degree of deacetylation of 85% and a molecular weight of 200 kDaltons in 150 mL of acetic acid aqueous solution with a mass-volume ratio of 1.0%, adjust the pH value to 5.5, and stir for 50 minutes until a clear solution is formed.

[0046] S20: Add 20 mg of siRNA and 25 mg of polyethylene glycol-polylactic acid block copolymer to the above solution and stir at 40 degrees Celsius for 2 hours.

[0047] S30: Add 2.0 mg / mL sodium tripolyphosphate aqueous solution dropwise to the above mixture at a rate of 0.5 mL / min, for a total volume of 40 mL, while stirring at 800 rpm.

[0048] S40: After the reaction has continued for 60 minutes, add 80 ml of anhydrous ethanol to terminate the reaction, centrifuge and wash three times with deionized water.

[0049] S50: Add 5% mannitol to the microsphere suspension, freeze-dry to obtain the finished product.

[0050] Comparative Example 1

[0051] S10: Dissolve 200 mg of chitosan with a degree of deacetylation of 90% and a molecular weight of 100 kDaltons in 200 mL of acetic acid aqueous solution with a mass-volume ratio of 1.0%, adjust the pH value to 5.0, and stir for 30 minutes until a transparent solution is formed.

[0052] S20: Add 10 mg of paclitaxel derivative and 20 mg of polyethylene glycol-polylactic acid block copolymer to the above solution and stir at 35 degrees Celsius for 3 hours.

[0053] S30: Microspheres are formed solely through physical cross-linking without the addition of sodium tripolyphosphate aqueous solution.

[0054] S40: Add 100 ml of anhydrous ethanol to terminate the reaction, centrifuge and wash 3 times with deionized water.

[0055] S50: Add 5% mannitol to the microsphere suspension, freeze-dry to obtain the finished product.

[0056] Comparative Example 2

[0057] S10: Dissolve 200 mg of chitosan with a degree of deacetylation of 90% and a molecular weight of 100 kDaltons in 200 mL of acetic acid aqueous solution with a mass-volume ratio of 1.0%, adjust the pH value to 5.0, and stir for 30 minutes until a transparent solution is formed.

[0058] S20: Add 10 mg of paclitaxel derivative to the above solution, without adding block copolymer, and stir at 35 degrees Celsius for 3 hours.

[0059] S30: Add 1.0 mg / mL sodium tripolyphosphate aqueous solution to the above mixture at a rate of 0.5 mL / min, for a total volume of 50 mL, while stirring at 800 rpm.

[0060] S40: After the reaction has continued for 40 minutes, add 100 ml of anhydrous ethanol to terminate the reaction, centrifuge and wash three times with deionized water.

[0061] S50: Add 5% mannitol to the microsphere suspension, freeze-dry to obtain the finished product.

[0062] The performance of the chitosan-loaded microspheres obtained in the above examples and comparative examples was tested, and the results are shown in the table below: Particle size (nanometers) Multi-dispersion index Drug encapsulation rate (%) Drug loading (%) Example 1 200 0.12 92 Example 2 250 0.10 95 Example 3 180 0.13 90 Comparative Example 1 400 0.25 70 Comparative Example 2 300 0.20 80 Based on the above results, the chitosan drug-loaded microspheres prepared in this application embodiment are superior to the comparative example in terms of particle size, polydispersity index, drug encapsulation efficiency, and drug loading, which verifies the effectiveness of the synergistic mechanism of ionic crosslinking and self-assembly.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing chitosan-loaded drug-eluting microspheres, characterized in that, Includes the following steps: S10: Dissolve chitosan in an aqueous acetic acid solution with a mass-volume ratio of 1.0%, adjust the pH of the solution to between 4.5 and 5.5, control the chitosan concentration to between 1.0 mg / mL and 3.0 mg / mL, and stir until a homogeneous and transparent solution is formed; S20: Add the target drug and the amphiphilic block copolymer to the above chitosan solution and stir continuously for 2 to 4 hours at 30 to 40 degrees Celsius. S30: Add sodium tripolyphosphate aqueous solution dropwise to the above mixture at a constant flow rate, while maintaining the system temperature at 35 degrees Celsius and the stirring speed at 800 rpm; S40: After the reaction lasts for 30 to 60 minutes, an equal volume of anhydrous ethanol is added to terminate the ionic crosslinking reaction. The microspheres are then collected by centrifugation and washed three times with deionized water. S50: Freeze-dry the purified microsphere suspension to obtain chitosan drug-loaded microspheres with uniform particle size and stable structure.

2. The method according to claim 1, characterized in that, In step S10, the degree of deacetylation of chitosan is greater than or equal to 85%, the molecular weight ranges from 50 kilodaltons to 200 kilodaltons, and the stirring rate is controlled between 300 rpm and 500 rpm.

3. The method according to claim 1, characterized in that, In step S20, the target drug includes water-soluble or weakly hydrophobic small molecule drugs, peptide drugs, and nucleic acid substances. The amphiphilic block copolymer is a polyethylene glycol-polylactic acid block copolymer, and the mass ratio of the block copolymer to chitosan is between 1:10 and 1:

5.

4. The method according to claim 1, characterized in that, In step S30, the concentration of the sodium tripolyphosphate aqueous solution is between 0.5 mg / mL and 2.0 mg / mL, the dropping rate is 0.5 mL / min, and the total amount added is in a volume ratio of 1:5 to 1:3 to the chitosan solution.

5. The method according to claim 1, characterized in that, In step S40, the centrifugation conditions are 10,000 rpm for 10 minutes, and the washing process combines a dual purification strategy of high-speed centrifugation and 0.22-micron membrane filtration.

6. The method according to claim 1, characterized in that, In step S50, 5% mannitol by mass volume is added to the purified microsphere suspension as a freeze-drying protectant, the pre-freezing temperature is -40 degrees Celsius, and the freeze-drying time is 24 hours.

7. The method according to claim 1, characterized in that, In step S10, the potential value of the chitosan solution is +35 mV to +45 mV, and the viscosity range is 2 mPa second to 5 mPa second.

8. The method according to claim 1, characterized in that, In step S30, the ionic strength of the system is controlled below 50 mmol / L, and the stoichiometric relationship of the ionic crosslinking reaction is n(–NH3⁺):n(P3O) 10 5 ⁻)=5:

1.

9. A chitosan-loaded drug-eluting microsphere, characterized in that, The chitosan drug-loaded microspheres prepared according to any one of claims 1 to 8 have an average particle size of 150 nm to 300 nm, a polydispersity index of less than or equal to 0.15, a drug encapsulation efficiency of greater than or equal to 90%, and a drug loading of 8% to 15%.

10. An application of chitosan drug-loaded microspheres based on an ionic crosslinking self-assembly synergistic mechanism, characterized in that, Suitable for sustained-release carriers of anticancer drugs, protein drugs and vaccines.

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