A method for preparing monodisperse multi-chamber microspheres based on protein-based high internal phase emulsion template method

Abstract

The application discloses a preparation method of monodisperse multi-chamber microspheres, characterized by using protein-based high internal phase emulsion (HIPEs) as a template to provide an internal multi-chamber structure and preparing monodisperse microspheres through a pneumatic shearing method. Specifically, bovine serum albumin is used as a stabilizer, an oil-in-water high internal phase emulsion is prepared first, and then mixed with a sodium alginate solution, and then monodisperse microspheres with an internal multi-chamber are prepared through pneumatic shearing. The application is characterized in that: (1) the size of the internal chamber can be adjusted by regulating the protein concentration; (2) the particle size and monodispersity of the microspheres can be adjusted by regulating the nitrogen flow rate; (3) the multiphase (water phase, oil phase and interface phase) of the monodisperse multi-chamber microspheres can be used as a multi-drug loading platform; (4) the microspheres have an intestinal targeting function, are suitable for intestinal targeting drug delivery, functional materials and other fields, and have high technical advantages and wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of food and pharmaceutical delivery, specifically relating to a method for preparing monodisperse multi-chamber microspheres and their applications. Background Technology

[0002] Microspheres, as spherical carriers at the micro- and nano-scale, play an important role in modern drug delivery systems. With their excellent biocompatibility, high specific surface area, and ability to encapsulate and protect sensitive drugs, microspheres have been widely used in long-acting sustained-release formulations, tumor-targeted delivery, and the protection of protein / peptide drugs, greatly improving drug delivery efficiency in complex clinical environments.

[0003] However, despite the enormous application potential of microsphere technology, bottlenecks hindering its clinical translation in actual production include the extensive use of organic solvents, difficulty in controlling particle size distribution, early burst release of drugs, poor batch-to-batch reproducibility, and limitations in production equipment. Therefore, overcoming these technological limitations to develop a novel microsphere system that achieves both monodispersity and avoids early release while ensuring stable preparation has become a critical issue urgently needing to be addressed in current pharmaceutical research.

[0004] In traditional microsphere preparation, the product often suffers from uneven particle size distribution. Monodisperse microspheres, on the other hand, refer to tiny spheres with uniform particle size and extremely narrow size distribution. Monodisperse multi-chamber microspheres, based on monodisperse microspheres, consist of numerous independent and uniformly distributed micro-cavities within precise micro-scale particles. By combining a narrow size distribution with a high-porosity honeycomb structure, they achieve efficient loading, precise protection, and predictable programmed release of multi-component drugs. Summary of the Invention

[0005] Purpose of the invention: To address the aforementioned problems and shortcomings, the purpose of this invention is to provide a method for preparing monodisperse multi-chamber microspheres. This method is characterized by a protein-based high internal phase emulsion template method. By optimizing the protein concentration, the size and uniformity of the internal chambers of the microspheres can be controlled, thereby achieving control over the size of the internal chambers. Furthermore, by controlling the flow rate of nitrogen gas in the outer needle of the coaxial needle, the dispersibility and particle size of the microspheres can be controlled.

[0006] Technical solution: The monodisperse multi-chamber microspheres of the present invention are prepared by mixing a protein-based high internal phase emulsion with sodium alginate to obtain a mixed emulsion, using an inert atmosphere as a carrier gas, and preparing monodisperse microspheres by pneumatic shearing technology, and then cross-linking and curing with a curing solution.

[0007] The monodisperse multi-chamber microspheres and the protein-based high internal phase emulsion are bovine serum albumin high internal phase emulsions; the inert atmosphere includes nitrogen, argon, and helium.

[0008] The monodisperse multi-chamber microspheres have a particle size range of 200-500 μm and a monodispersity CV of less than or equal to 10%.

[0009] The monodisperse multi-chamber microspheres and protein-based high internal phase emulsion are prepared through the following steps: (1) Add bovine serum albumin powder to water and disperse until the protein is completely hydrated into an aqueous phase; (2) Mix the oil phase with the aqueous solution obtained in step (1); (3) The solution obtained in step (2) was sheared using a high-speed shear disperser to obtain a semi-solid protein-based high internal phase emulsion.

[0010] In the monodisperse multi-chamber microspheres, in step (1), when the volume of water used is 10 mL, the corresponding amount of BSA powder added is 0.03 to 3.46 g; when the volume of water used is not 10 mL, the corresponding amount of BSA powder added is calculated according to the specific volume of water; in step (1), bovine serum albumin powder is added to water and dispersed, and then it can be refrigerated at 2-8 ℃ for more than 6 hours to ensure complete protein hydration; When the volume of water used in step (1) is 10 mL, the mass of the oil phase in step (2) is 31.96 to 42.30 g; preferably, the internal phase volume of the protein-based high internal phase emulsion is 0.68 to 0.90 g.

[0011] The oil phase in step (2) is one or both of medium-chain triglycerides and cyclohexane; In step (3), the high-speed shearing disperser operates at a rotation speed of 5000~15000 rpm and a shearing time of 1~10 minutes.

[0012] The method for preparing the monodisperse multi-chamber microspheres includes the following steps: S1. Mix the protein-based high internal phase emulsion and the sodium alginate aqueous solution to obtain a mixed emulsion; S2. Use a syringe to draw up the mixed emulsion and use a syringe pump to inject the liquid into the inner needle of the coaxial needle; S3. Nitrogen gas is introduced into the outer needle of the coaxial needle. The pneumatic shearing effect generated by the nitrogen gas is used to form uniform droplets of the mixed emulsion. S4. The droplets are injected into the curing solution for cross-linking and curing, thereby obtaining monodisperse microspheres with a multi-chamber structure.

[0013] In the method for preparing monodisperse multi-chamber microspheres, the concentration of sodium alginate in the sodium alginate aqueous solution in step S1 is 0.2–2.0 wt%; the mass ratio of the protein-based high internal phase emulsion to the sodium alginate aqueous solution is 2:1 to 1:5.

[0014] In the method for preparing monodisperse multi-chamber microspheres, the injection pump delivery rate in step S2 is less than or equal to 0.1 mL / min; and the gas pressure of the nitrogen cylinder in step S3 is 0.1~0.7 MPa.

[0015] In the preparation method of the monodisperse multi-chamber microspheres, the curing solution in step S4 is a calcium chloride solution with added anhydrous ethanol.

[0016] The application of the monodisperse multi-chamber microspheres in the preparation of drug intestinal targeted delivery materials.

[0017] Preferably, the inner and outer needles of the coaxial needle have dimensions of 20G and 28G, respectively.

[0018] Further preferably, this invention discloses a method for preparing monodisperse multi-chamber microspheres. The specific preparation process is as follows: using bovine serum albumin (BSA) as an emulsifying stabilizer, an aqueous solution is prepared with ultrapure water and refrigerated at 4 ℃ to allow the protein to fully hydrate; using medium-chain triglycerides or cyclohexane as the oil phase, the oil phase is mixed with the BSA solution and sheared at 10000 rpm for 2 min to obtain protein-based high internal phase emulsions (HIPEs); sodium alginate solution is mixed with HIPEs according to a preset mass ratio, and the system is uniformly mixed by repeated blowing and suction; the above mixed emulsion is pumped into the inner needle of a coaxial needle, while nitrogen gas is introduced into the outer needle, forming microdroplets by pneumatic shearing; the microdroplets are then dropped into a curing solution to undergo cross-linking and curing, and after washing and drying, they are stored in an ultrapure aqueous solution to obtain monodisperse microspheres with a dense surface and a multi-chamber structure inside. Among these methods, by adjusting the concentration of bovine serum albumin, precise control of the pore size and structure of the internal chambers of the high internal phase emulsion can be achieved; and by adjusting the nitrogen flow rate, the particle size of the microspheres can be controlled.

[0019] The method for preparing the protein-based high internal phase emulsion includes the following steps: (1) Accurately weigh BSA, add 10 mL of ultrapure water, and refrigerate at 2~8 ℃ for more than 8 hours to ensure that BSA is fully hydrated.

[0020] (2) Take the hydrated BSA solution and slowly add the corresponding mass of medium-chain triglycerides into it, and mix well.

[0021] (3) A white paste-like HIPEs was obtained by shearing at 10,000 rpm for 2 min using a high-speed shear disperser. In the preparation method of HIPEs, in step (1), the amount of BSA powder added is 0.03 to 3.46 g, and the concentration of BSA solution is 0.05 to 5 mM. Different concentrations of BSA can adjust the particle size of HIPEs, thereby changing the internal chamber size of the microspheres. In the preparation method of the HIPEs, in step (2), the mass of the added medium-chain triglycerides is 31.96 to 42.00 g, thereby forming HIPEs with different internal phase volume fractions, with internal phase volume fraction F = 0.68 to 0.90. The method for preparing monodisperse multi-chamber microspheres based on HIPEs templates includes the following steps: (1) Mix the HIPEs prepared above with sodium alginate solution to obtain a mixed emulsion.

[0022] (2) The mixed emulsion is pumped into the inner needle of the coaxial needle, while nitrogen gas is introduced into the outer needle. Microdroplets are obtained by pneumatic shearing. The microdroplets then fall into the curing solution and are cross-linked and cured to obtain monodisperse microspheres with a multi-chamber structure inside.

[0023] The method for preparing monodisperse multi-chamber microspheres based on HIPEs templates, specifically the preparation method of sodium alginate solution in step (1) is as follows: sodium alginate powder is slowly added to ultrapure water at 40 °C, and stirred continuously for 2 h until the sodium alginate powder is completely dissolved to obtain sodium alginate solution, which is then stored at 4 °C.

[0024] The method for preparing monodisperse multi-chamber microspheres based on HIPEs templates, wherein the concentration of sodium alginate solution used in step (1) is 0.2 to 2.0 wt%; and the mass ratio of HIPEs to sodium alginate solution is 2:1 to 1:5.

[0025] In the method for preparing monodisperse multi-chamber microspheres based on HIPEs templates, the inner and outer needles of the coaxial needle used in step (2) are 20 and 28G respectively; the injection pump push rate shall not exceed 0.1 mL / min; the nitrogen cylinder output pressure is 0.2 MPa; and the curing solution is 5% (w / v) calcium chloride solution with 10% (w / w) anhydrous ethanol added.

[0026] This invention uses protein-based high internal phase emulsions (HIPEs) (ZL 2022 1 0583838.2) as a template, combining pneumatic shearing technology with the pH-sensitive properties of sodium alginate to prepare a monodisperse multi-compartment microsphere multiphase drug delivery platform. Leveraging the high internal phase ratio of HIPEs, the system utilizes the oil, surface, and aqueous phases as drug enrichment spaces, significantly improving the encapsulation capacity of hydrophobic drugs and overcoming the challenge of low loading capacity for poorly soluble drugs. Simultaneously, precise control of the fluid state through pneumatic shearing yields monodisperse multi-compartment microspheres with uniform particle size, ensuring stable and controllable monomeric drug loading and providing good predictability of drug release behavior. With the pH-responsive encapsulation effect of sodium alginate, the microspheres remain stable in the gastric environment, achieving zero drug release, and can trigger drug release in the intestinal microenvironment, ultimately achieving oral intestinal targeted delivery.

[0027] Beneficial effects: Compared with the existing technology, it has the following significant advantages: (1) The HIPEs use BSA as a stabilizer, which can form a dense and stable film structure at the oil-water interface. Without the addition of additional surfactants, it can form an oil-in-water emulsion with an oil ratio of greater than or equal to 80%. At the same time, by adjusting the concentration of BSA, the structure and size of the internal chamber of the monodisperse microspheres can be precisely controlled, which can provide a structural basis for the targeted delivery of hydrophobic drugs. (2) The pneumatic shearing method is used to replace the traditional extrusion method or microfluidic preparation process. By adjusting the nitrogen flow rate, the particle size and monodispersity of the microspheres can be precisely controlled. The obtained particle size is significantly smaller than that of the microspheres prepared by the traditional extrusion method (≥ 500 µm). The equipment requirements are simple, requiring only an injection pump, nitrogen and its matching pressure control valve and coaxial needle. The preparation cost is low, and the batch preparation volume is large. It is easy to operate and easy to scale up. The process has strong continuous operation stability, realizing the integration of microsphere collection and cross-linking curing, avoiding the technical problems of channel blockage and interface contamination commonly found in microfluidic processes, and effectively improving the preparation efficiency and product quality. (3) The monodisperse multi-chamber microspheres prepared by the present invention can be used as a drug delivery platform for multiple drugs, especially as a targeted delivery formulation for hydrophobic drugs, to achieve intestinal targeted delivery of hydrophobic drugs, thereby improving drug delivery efficiency and achieving sustained drug release. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the microsphere preparation device;

[0029] Figure 2 This is a schematic diagram of the preparation process for the examples and comparative examples;

[0030] Figure 3Figure 1 shows an inverted fluorescence microscope image of a high internal phase emulsion with stable bovine serum albumin concentration; Figure 2a shows the microstructure and particle size distribution of the high internal phase emulsion with stable bovine serum albumin concentration at different internal phase volumes (F) of 0.80 (scale bar: 20 μm); Figure 3b shows a comparison of the average particle size of the high internal phase emulsion with stable bovine serum albumin concentration at different internal phase volumes.

[0031] Figure 4 Laser confocal microscopy images of high internal phase emulsions prepared with different bovine serum albumin concentrations when the internal phase volume (F) is 0.80.

[0032] Figure 5 Particle size distribution of the mixed emulsions in the examples and comparative examples with different protein concentrations (scale bar: 20 μm).

[0033] Figure 6 Stability tests were conducted on the mixed emulsions of different protein concentrations in the examples and comparative examples.

[0034] Figure 7 Thixotropic properties of mixed emulsions of high internal phase emulsions with different mass ratios and 2% wt. sodium alginate;

[0035] Figure 8 Microscopic morphology and particle size distribution of microspheres prepared from high internal phase emulsions of 0.5 mM bovine serum albumin at different nitrogen flow rates (0.3, 0.5, 0.7 L / min) and 2% wt sodium alginate in different proportions and pure sodium alginate (scale bar: 200 μm).

[0036] Figure 9 Laser confocal microscopy images of microspheres prepared from high internal phase emulsions with a protein concentration of 0.5 mM at different nitrogen flow rates (0.3, 0.5 L / min) and 2% wt sodium alginate in different proportions;

[0037] Figure 10 The image shows a scanning electron microscope (SEM) image of microspheres prepared with cyclohexane as the oil phase after vacuum drying.

[0038] Figure 11 Figure 1 shows the release trend of free fatty acids from microspheres; Figure 2 shows the release trend of free fatty acids from microspheres at different concentrations during the in vitro release process, where Examples 0.2, 0.5, and 1.0 refer to examples with bovine serum albumin concentrations of 0.2, 0.5, and 1.0 mM; Figure 3 shows the release trend of free fatty acids from microspheres at different concentrations, where Comparative Examples 0.2, 0.5, and 1.0 refer to comparative examples with bovine serum albumin concentrations of 0.2, 0.5, and 1.0 mM.

[0039] Figure 12Figure 1 shows the in vitro digestion simulation release curves of the drug-loaded microspheres; Figure 2 shows the in vitro digestion simulation release curves of the prepared curcumin microspheres and the examples, where 0.2, 0.5, and 1.0 in the examples refer to examples with bovine serum albumin concentrations of 0.2, 0.5, and 1.0 mM; Figure 3 shows the in vitro digestion simulation release curves of the prepared curcumin microspheres in the comparative examples, where 0.2, 0.5, and 1.0 in the comparative examples refer to comparative examples with bovine serum albumin concentrations of 0.2, 0.5, and 1.0 mM.

[0040] Figure 13 In vitro microscopic morphology of the microspheres prepared for the examples and comparative examples as digestion time in simulated gastric juice (SGF) and simulated intestinal juice (SIF). Detailed Implementation

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent forms also fall within the scope defined in this application. The apparatus used in this invention is shown in the attached figure. Figure 1 As shown. Schematic diagrams of the preparation processes for the examples and comparative examples are shown below. Figure 2 As shown.

[0042] Example 1: A method for preparing monodisperse multi-compartment microspheres (bovine serum albumin concentration of 0.2 mM) 1. Aqueous phase preparation: Weigh 0.14g of bovine serum albumin powder and dissolve it in 10 mL of ultrapure water. Use ultrasound to assist in dissolving until the bovine serum albumin powder is completely dissolved. Place the prepared bovine serum albumin solution in a 4°C environment and refrigerate overnight to allow the bovine serum albumin to fully hydrate.

[0043] 2. Preparation of protein-based high internal phase emulsions: Take 37.6 g of medium-chain triglycerides as the oil phase, and add the bovine serum albumin aqueous solution prepared in step 1 to it; use a high-speed shear disperser, adjust the speed to 10000 rpm, and shear the above for 2 min to obtain a protein-based high internal phase emulsion.

[0044] 3. Preparation of mixed emulsions of high internal phase emulsion and sodium alginate: A 2% wt. sodium alginate solution was mixed with a degassed protein-based high internal phase emulsion at a mass ratio of 1:2. The mixture was then repeatedly blown and aspirated with a syringe to ensure thorough mixing of the sodium alginate solution and the high internal phase emulsion, thus obtaining the mixed emulsion.

[0045] 4. Preparation of monodisperse microspheres: The prepared high internal phase emulsion was mixed with sodium alginate emulsion, and microdroplets were obtained by coaxial needle pneumatic shearing. These microdroplets were then dropped into a curing solution consisting of 5% (w / v) calcium chloride solution (containing 10% (v / v) anhydrous ethanol). After cross-linking and curing for 30 min, the resulting microspheres were washed three times with ultrapure water to obtain monodisperse multi-chamber microspheres. The microsphere preparation apparatus is shown in the attached figure. Figure 1 As shown.

[0046] Example 2: A method for preparing monodisperse multi-compartment microspheres (bovine serum albumin concentration of 0.5 mM) The preparation method in this embodiment is basically the same as that in Example 1, except that the amount of bovine serum albumin is 0.35g in the aqueous phase preparation in step 1. The remaining steps (including the amount of medium-chain triglycerides, shear parameters, mixing ratio, and cross-linking and curing conditions) are completely consistent with those in Example 1, and monodisperse multi-chamber microspheres are finally obtained. Taking the process of Example 2 (0.5mM) as an example, cyclohexane is used instead of medium-chain triglycerides as the oil phase, and other conditions remain unchanged. Microspheres are prepared and scanned electron microscope images of the microspheres are taken after vacuum drying at room temperature. It can be seen that the microspheres exhibit a porous structure, which verifies that the microspheres have a typical multi-chamber structure with high internal phase emulsion template.

[0047] Example 3: A method for preparing monodisperse multi-compartment microspheres (bovine serum albumin concentration of 1.0 mM). The preparation method of this embodiment is basically the same as that of Example 1. The only difference is that the amount of bovine serum albumin is 0.69g in the aqueous phase preparation in step 1. The remaining steps (including the amount of medium-chain triglycerides, shear parameters, mixing ratio and cross-linking curing conditions, etc.) are completely consistent with those of Example 1, and monodisperse multi-chamber microspheres are finally obtained.

[0048] Comparative Example 1: Microspheres were prepared by direct mixing and shearing without using a high internal phase emulsion template and then cross-linked (bovine serum albumin concentration was 0.2 mM). 1. Preparation of aqueous phase: Weigh 0.14 g of bovine serum albumin and dissolve it in 10 mL of ultrapure water, followed by ultrasonic-assisted dissolution; refrigerate the resulting bovine serum albumin solution at 4°C overnight to allow it to fully hydrate.

[0049] 2. Preparation of direct mixed emulsion: 37.6 g of medium-chain triglycerides, the bovine serum albumin aqueous solution obtained in step 1, and sodium alginate solution with a mass fraction of 2% (wt) were added to a container according to the set mass fractions; the mixture was sheared at 10,000 rpm for 2 min using a high-speed shear disperser to obtain a mixed emulsion.

[0050] 3. Crosslinking and curing: The obtained mixed emulsion was slowly dripped into a curing solution containing 5% (w / v) calcium chloride solution (containing 10% (v / v) anhydrous ethanol) using a coaxial needle pneumatic shearing method to carry out the crosslinking and curing reaction. After the crosslinking and curing continued for 30 min, the obtained microspheres were washed three times with ultrapure water to remove excess calcium chloride and oil phase components adhering to the surface, and finally control microspheres were obtained.

[0051] Comparative Example 2: Microspheres were prepared by direct mixing and shearing without using a high internal phase emulsion template and then cross-linked (bovine serum albumin concentration was 0.5 mM). The preparation methods of this comparative example are basically the same as those of comparative example 1. The only difference is that the amount of bovine serum albumin in the aqueous phase preparation in step 1 is 0.35 g. The other steps (including the amount of medium-chain triglycerides, the mixing ratio, the shear parameters, the cross-linking conditions, etc.) are completely consistent with those of comparative example 1.

[0052] Comparative Example 3: Microspheres were prepared by direct mixing and shearing without using a high internal phase emulsion template and then cross-linked (bovine serum albumin concentration was 1.0 mM). The preparation methods of this comparative example are basically the same as those of comparative example 1. The only difference is that the amount of bovine serum albumin in the aqueous phase preparation in step 1 is 0.69 g. The other steps (including the amount of medium-chain triglycerides, the mixing ratio, the shear parameters, the cross-linking conditions, etc.) are completely consistent with those of comparative example 1.

[0053] Experimental Example 1: Emulsion Performance Test 1. Particle size and morphology observation: The emulsions prepared in each example and comparative example were observed using an inverted fluorescence microscope, and droplet morphology photographs were taken; the average particle size and particle size distribution were statistically analyzed using ImageJ software. (See attached image.) Figure 3 As shown in a and 3b, in the embodiments, by increasing the protein concentration, the droplet size in the high internal phase emulsion can be controlled, and the droplet size decreases with increasing protein concentration. The results of laser confocal microscopy imaging of the emulsion are shown in the attached figure. Figure 4 As shown in the attached figure, the above conclusion is further verified. Figure 5 As shown, the comparative mixed emulsion prepared without using protein-based high internal phase emulsion as a template could not effectively control the particle size when adjusting the bovine serum albumin concentration, and there was an over-fine particle size phenomenon.

[0054] 2. Static Stability Determination: Static stability was characterized using a Turbiscan multiple light scattering instrument. An appropriate amount of the emulsion stock solution was placed in a sample tube (no dilution required), and tested at 25℃ for 120 min. The sample tube was scanned every 1 min along its 0-50 mm height, and the rate of change of backscattered light intensity relative to the initial time, ΔBS (%), was recorded. Stability was quantitatively evaluated by the magnitude and trend of ΔBS (%) deviation from the baseline over time; the smaller the fluctuation of ΔBS (%) and the closer it is to 0, the better the stability. Results are attached. Figure 6 As shown, the overall static stability of the mixed emulsions in each embodiment and comparative example is similar, and all exhibit good short-term static stability.

[0055] 3. Rheological recovery performance testing: Three-stage continuous step shear tests were conducted on mixed emulsions with different mixing ratios of high internal phase emulsions (HIPEs) and sodium alginate (ALG) (2:1, 1:1, 1:2, 1:5) using a rotational rheometer. The results are shown in the attached figure. Figure 7 As shown, in the low shear rate stage, all mixed emulsions exhibited high initial viscosity, which gradually decreased with increasing sodium alginate ratio. When a high shear rate was applied, the viscosity of each system rapidly decreased, exhibiting significant shear-thinning behavior. Subsequently, upon returning to the initial low shear rate, the viscosity of each emulsion instantly recovered to near its initial level. These results demonstrate that the mixed emulsion network structure prepared in this invention possesses excellent shear failure recovery capability, meeting the technical requirements of subsequent processing.

[0056] Experimental Example 2: Screening and Optimization of Preparation Process Parameters To optimize the process, this invention established a cross-experimental matrix consisting of different emulsion mixing ratios (2:1, 1:1, 1:2, 1:5 and pure sodium alginate) and nitrogen flow rates (0.3, 0.5, 0.7 L / min), as shown in the attached figure. Figure 8 As shown.

[0057] The results showed that with increasing sodium alginate content, the microsphere morphology changed from rough and irregular to uniform and regular spheres, the surface became smoother, and the particle size distribution narrowed (monodispersibility improved). With increasing nitrogen flow rate, the microsphere particle size decreased significantly. Laser confocal microscopy observations are shown in the attached figure. Figure 9 As shown, it is confirmed that during the cross-linking and curing process, the oil and water phases of the high internal phase emulsion are uniformly distributed inside the sodium alginate microspheres, and no macroscopic phase separation or droplet aggregation occurs.

[0058] Taking into account drug loading, particle size distribution, and microsphere morphology, the optimal process parameters determined in this invention are: a mixing ratio of 1:2 and a nitrogen flow rate of 0.5 L / min. Microspheres prepared under these conditions achieve both high drug loading and excellent monodispersity and regular geometric morphology.

[0059] Experiment Example 3: Verification of the Multi-chamber Structure of Microspheres To verify the multi-chamber structure of the microspheres prepared by the present invention through high internal phase emulsion template, cyclohexane was used instead of medium-chain triglycerides as the oil phase to prepare a high internal phase emulsion, which was then mixed with sodium alginate for crosslinking and curing to obtain microspheres. The obtained microspheres were vacuum dried and characterized by scanning electron microscopy; the results are shown in the attached figure. Figure 10 As shown, the microspheres are dense on the outside and exhibit a distinct porous structure on the inside, which directly confirms that the microspheres have a typical high internal phase emulsion templated multi-chamber configuration.

[0060] Experiment Example 4: Validation of the release performance of microspheres in an in vitro simulated gastrointestinal tract To verify the stability and targeted release capability of the microspheres in the gastrointestinal tract, the present invention conducts an in vitro digestion simulation experiment on the prepared microspheres.

[0061] 1. Preparation of simulated digestive fluid: Simulated gastric juice (SGF): Based on simulated gastric juice electrolyte solution, pepsin was added to achieve a final activity of 2000 U / mL; Simulated intestinal fluid (SIF): Based on simulated intestinal fluid electrolyte solution, bile salts and pancreatic enzymes are added to achieve final concentrations of 10 mM and 100 U / mL, respectively.

[0062] 2. Investigation into the release performance of free fatty acids in microspheres: After mixing the microsphere sample with the simulated digestive fluid, the microspheres maintained their morphology intact during the gastric juice stage, with no release of free fatty acids. During the intestinal juice stage, as digestion progressed, the microspheres disintegrated, and triglycerides were broken down into free fatty acids, leading to a decrease in pH. The digestive fluid was titrated with sodium hydroxide every 10 minutes to bring the pH to 7. The concentration and amount of sodium hydroxide used at each time point were recorded, the cumulative titration amount of sodium hydroxide at each time point was calculated, and a cumulative free fatty acid release curve was plotted. (See attached diagram) Figure 11 As shown in Figure a, in the example samples, the release rate of free fatty acids increased with increasing protein concentration from 0.2 to 1.0 mM. Combined with the emulsion particle size statistics, high protein concentration resulted in smaller droplets, and the larger specific surface area promoted the adsorption and hydrolysis of lipids by lipases, thereby improving lipid digestion efficiency. In contrast, as shown in the attached figure… Figure 11 As shown in b, the release rates of free fatty acids at different concentrations in the comparative samples showed little difference and did not exhibit a regular trend consistent with concentration changes. This indicates that protein-based high internal phase emulsion templates can achieve control over the internal particle size of microspheres.

[0063] The microspheres prepared in the embodiments and comparative examples of this invention were cultured in simulated gastric juice (SGF) for 120 min, as shown in the attached figure. Figure 13As shown, the spheres remained intact, and no obvious swelling or degradation was observed, demonstrating their excellent acid resistance. However, after entering simulated intestinal fluid (SIF), the microspheres rapidly disintegrated, with their structure essentially disappearing at 60 min and being released almost completely at 120 min.

[0064] 3. Study on drug release performance in microspheres: To further evaluate the application potential of protein-based microspheres as delivery carriers, curcumin was used as a model hydrophobic active substance. The curcumin release characteristics of microspheres with different protein concentrations in simulated gastrointestinal digestion processes were investigated, as shown in the attached figures. Figure 12 Microspheres were mixed with simulated gastric juice, and the pH was adjusted to 3.0. The reaction vessel was placed at 37°C and incubated with shaking for 2 hours. After the gastric digestion reaction was completed, an equal volume of simulated intestinal juice was added directly to the system, and the system was incubated at 37°C with shaking for another 2 hours. The pH of the system was monitored and maintained constant at 7.0 throughout the entire intestinal digestion process. Samples were taken every 20 minutes during digestion, and an equal volume of isothermal digestion solution was added simultaneously. After centrifugation at 12000 rpm for 2 minutes, the intermediate micelle layer was collected, and its absorbance was measured at 425 nm using a UV spectrophotometer.

[0065]

[0066] The Korsmeyer-Peppas model was used to perform kinetic fitting on the in vitro release data of microspheres during the intestinal digestion stage for each group (parameters are shown in Table 1, curves are shown in Appendix 1). Figure 12 The results showed that the release index n of each group of samples was less than 0.43, indicating that their drug release mechanism was controlled by Fickian diffusion. Based on this, the release rate constant k of the microspheres in the examples exhibited a significant protein concentration dependence. As the protein concentration increased from 0.5 mM to 1.0 mM, the release rate constant k significantly increased, and the drug release rate accelerated significantly, achieving effective control of release performance. In contrast, the release rate constant k and release curve of the comparative group did not change substantially when the concentration was changed. These results demonstrate that the monodisperse multi-compartment microspheres prepared by the protein-based high internal phase emulsion template method of this invention can regulate the drug release rate by adjusting the protein concentration.

Claims

1. A monodisperse multi-chamber microsphere, characterized in that: It is prepared by mixing protein-based high internal phase emulsion with sodium alginate to obtain a mixed emulsion, using an inert atmosphere as the carrier gas, preparing monodisperse microspheres by pneumatic shearing technology, and then cross-linking and curing them with a curing solution.

2. The monodisperse multi-chamber microspheres according to claim 1, characterized in that, The protein-based high internal phase emulsion is a high internal phase emulsion of bovine serum albumin; the inert atmosphere includes nitrogen, argon, and helium.

3. The monodisperse multi-chamber microspheres according to claim 1, characterized in that, The microspheres have a particle size range of 200-500 μm and a monodispersity CV of less than or equal to 10%.

4. The monodisperse multi-chamber microspheres according to claim 1, characterized in that, Protein-based high internal phase emulsions are prepared through the following steps: (1) Add bovine serum albumin powder to water and disperse until the protein is completely hydrated into an aqueous phase; (2) Mix the oil phase with the aqueous solution obtained in step (1); (3) The solution obtained in step (2) was sheared using a high-speed shear disperser to obtain a semi-solid protein-based high internal phase emulsion.

5. The monodisperse multi-chamber microspheres according to claim 4, characterized in that, In step (1), when the volume of water used is 10 mL, the corresponding amount of BSA powder added is 0.03 to 3.46 g; when the volume of water used is not 10 mL, the corresponding amount of BSA powder added is calculated according to the specific volume of water; in step (1), bovine serum albumin powder is added to water and dispersed, and then it can be refrigerated at 2-8 ℃ for more than 6 hours to ensure complete hydration of the protein; When the volume of water used in step (1) is 10 mL, the mass of the oil phase in step (2) is 31.96–42.30 g; The oil phase in step (2) is one or both of medium-chain triglycerides and cyclohexane; In step (3), the high-speed shearing disperser operates at a rotation speed of 5000~15000 rpm and a shearing time of 1~10 minutes.

6. A method for preparing monodisperse multi-chamber microspheres according to claim 1, characterized in that, Includes the following steps: S1. Mix the protein-based high internal phase emulsion and the sodium alginate aqueous solution to obtain a mixed emulsion; S2. Use a syringe to draw up the mixed emulsion and use a syringe pump to inject the liquid into the inner needle of the coaxial needle; S3. Nitrogen gas is introduced into the outer needle of the coaxial needle. The pneumatic shearing effect generated by the nitrogen gas is used to form uniform droplets of the mixed emulsion. S4. The droplets are injected into the curing solution for cross-linking and curing, thereby obtaining monodisperse microspheres with a multi-chamber structure.

7. The method for preparing monodisperse multi-chamber microspheres according to claim 6, characterized in that, In step S1, the concentration of sodium alginate in the sodium alginate aqueous solution is 0.2–2.0 wt%; the mass ratio of the protein-based high internal phase emulsion to the sodium alginate aqueous solution is 2:1 to 1:

5.

8. The method for preparing monodisperse multi-chamber microspheres according to claim 6, characterized in that, The injection pump push rate in step S2 is less than or equal to 0.1 mL / min; the nitrogen cylinder pressure in step S3 is 0.1~0.7 MPa.

9. The method for preparing monodisperse multi-chamber microspheres according to claim 6, characterized in that, The curing solution mentioned in step S4 is a calcium chloride solution with added anhydrous ethanol.

10. The use of the monodisperse multi-chamber microspheres according to any one of claims 1 to 5 in the preparation of drug enteric delivery materials.

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