A continuous flow method for the preparation of chitosan-walnut protein nanogel microspheres carriers

Abstract

This invention discloses a continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers. Utilizing ultrasonic microreactor technology combined with emulsion droplet aggregation, this invention successfully prepared chitosan-walnut protein composite hydrogel microspheres with submicron particle sizes. These microspheres form stable polymers through acid-base neutralization precipitation of polysaccharides and proteins, as well as non-covalent complexation. Stability is enhanced by electrostatic interactions, hydrophobic interactions, and hydrogen bonds. Simultaneously, the excellent internal network structure of the microspheres enables high drug loading, and ultrasonic emulsification improves the controllability of product size and particle uniformity. This invention provides a novel method for preparing nanogel carriers, which can expand the application of gel materials in biomedicine, drug delivery, and other fields.

Description

Technical Field

[0001] This invention relates to the field of carrier technology, and in particular to a continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers. Background Technology

[0002] Hydrogels have attracted much attention in the field of biomaterials due to their excellent biocompatibility, ease of modification, and properties similar to the intercellular matrix, making them an ideal choice for cell culture substrates and tissue engineering templates. Their unique internal nanomesh structure also offers great potential for drug delivery. However, traditional bulk hydrogels are limited by size in specific applications such as injection and drug delivery.

[0003] By preparing hydrogel microspheres at the micrometer or nanometer scale, the dual advantages of hydrogels and micro / nanoparticles can be fully utilized in the field of drug delivery. However, there is currently a lack of effective industrial-scale preparation methods for nanogel microspheres. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers.

[0005] Another objective of this invention is to provide a chitosan-walnut protein nanogel microsphere carrier.

[0006] Another object of the present invention is to provide the application of the above-mentioned chitosan-walnut protein nanogel microsphere carrier.

[0007] The objective of this invention is achieved through the following technical solution: A continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers includes the following steps: 1) Preparation of chitosan solution: Chitosan is dissolved in acetic acid solution to obtain chitosan solution; 2) Preparation of walnut protein solution: Walnut protein is dissolved in NaOH solution to obtain walnut protein solution; 3) Preparation of the oil phase: An emulsifier is added to liquid paraffin to obtain the oil phase; 4) Mix the chitosan solution from step 1) with the oil phase from step 3), and shear and stir the mixture in a high-speed homogenizer to obtain mixture A; 5) Mix the walnut protein solution from step 2) with the oil phase from step 3), first shear and stir in a high-speed homogenizer to mix them evenly, and then pass them into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump, to obtain emulsion A and emulsion B respectively; 7) Emulsion A and Emulsion B flow out of the ultrasonic microreactor and are directly fed into a T-type mixer for mixing to obtain Emulsion C; 8) The obtained emulsion C was allowed to stand and solidify to obtain the solidified product; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

[0008] Further, the concentration of the chitosan solution in step 1) is 0.5 wt% to 2 wt%, preferably 1.0 wt%.

[0009] Further, the concentration of the acetic acid solution in step 1) is 1 wt% to 3 wt%, preferably 2 wt%.

[0010] Further, the concentration of the walnut protein solution in step 2) is 0.5 wt% to 2 wt%, preferably 1.0 wt%.

[0011] Further, the concentration of the NaOH solution mentioned in step 2) is 0.1 to 1 mol / L, preferably 0.4 mol / L.

[0012] Further, the emulsifier mentioned in step 3) is at least one of glyceryl stearate, polyglycerol ester, and EM90, preferably EM90.

[0013] Further, the mass fraction of the emulsifier mentioned in step 3) is 5% to 15%, preferably 10%.

[0014] Further, the volume ratio of the chitosan solution to the oil phase in step 4) is 1:3 to 1:8, preferably 1:5.

[0015] Further, the volume ratio of the walnut protein solution to the oil phase in step 5) is 1:3 to 1:8, preferably 1:5.

[0016] Furthermore, the parameters of the high-speed homogenizer mentioned in steps 4) and 5) are 3500±500 rpm.

[0017] Furthermore, in step 6), both ultrasonic microreactor I and ultrasonic microreactor II are set to a flow rate of 6±1 mL / min and a power of 120±20 W.

[0018] Further, in step 7), the volume ratio of the walnut protein solution to the oil phase and the volume ratio of emulsion A to emulsion B are 1:2 to 1:10, preferably 1:5.

[0019] Furthermore, the curing time described in step 8) is 12 ± 2 h.

[0020] A chitosan-walnut protein nanogel microsphere carrier was obtained by the above preparation method.

[0021] The above-mentioned chitosan-walnut protein nanogel microsphere carriers are used in the preparation of drug carriers / drug delivery systems.

[0022] This invention utilizes ultrasonic microreactor (USMR) technology combined with emulsion droplet aggregation to successfully prepare chitosan-walnut protein composite hydrogel microspheres with submicron particle sizes. These microspheres form stable polymers through acid-base neutralization and non-covalent complexation of polysaccharides and proteins, with stability enhanced by electrostatic interactions, hydrophobic interactions, and hydrogen bonding. Simultaneously, the excellent internal network structure of the microspheres enables high drug loading, and ultrasonic emulsification improves the controllability of product size and particle uniformity. This invention provides a novel method for preparing nanogel carriers, which can expand the application of gel materials in biomedicine, drug delivery, and other fields.

[0023] The present invention has the following advantages and effects compared with the prior art: The chitosan-walnut protein nanogel microsphere carrier provided by this invention is suitable for encapsulating various drugs, exhibiting excellent biocompatibility and biodegradability, and a high drug loading space. The preparation process is a continuous synthesis process, resulting in highly uniform particle size and morphology of the product. The preparation process is safe, efficient, and easily scaled up. Attached Figure Description

[0024] Figure 1 This is a flowchart of the continuous flow preparation method of the chitosan-walnut protein nanogel microsphere carrier of the present invention; Figure 2 This is a diagram of the continuous flow preparation apparatus for the chitosan-walnut protein nanogel microsphere carrier of the present invention; Figure 3 This is a scanning electron microscope image (scale bar is 10 μm) of the chitosan-walnut protein nanogel microsphere carrier of the present invention. Figure 4 This is a scanning electron microscope image (1 μm) of the chitosan-walnut protein nanogel microsphere carrier of the present invention. Detailed Implementation

[0025] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0026] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0027] In the following examples, room temperature refers to 15–30°C.

[0028] Unless otherwise specified, all percentages in the following examples are by mass percentages.

[0029] The walnut protein in the following examples was prepared according to the method described in the literature "Mao Xiaoying, Zhu Xinrong, Wan Yinsong, et al. Compositional analysis and optimization of separation and extraction process of walnut protein [J]. Chinese Journal of Food Science, 2019, 19(3):195-205." The details are as follows: Take the kernels of thin-skinned walnuts from Hotan, Xinjiang, soak them in 2% NaOH for 4 min to remove the kernel skin, dry them at 45-48℃, pulverize them, extract them with n-hexane at a material-to-liquid ratio of 1:5 (w / v) for 1 h, then filter them. Collect the residue when the filtrate is colorless and transparent, and evaporate the solvent. Crush the residue with a pulverizer and pass it through a 150-mesh sieve to obtain walnut defatted powder, which is then stored in a refrigerator at 4℃ for later use.

[0030] Walnut defatted powder was washed with 95% ethanol at a ratio of 1:10 (w / v), filtered to obtain a filter cake, and the solvent was evaporated. Deionized water was added to adjust the pH of the solution to 11, the ratio of solid to liquid was 1:26 (w / v), the extraction temperature was 53℃, and the mixture was stirred for 1.5 h to obtain a protein extract. The extract was centrifuged at 7000 r / min for 15 min at 25℃, the supernatant was collected, and the pH of the supernatant was adjusted to 4.5 with 0.1 mol / L HCl. After the pH stabilized, the mixture was magnetically stirred for 1 h, centrifuged at 7000 r / min for 15 min at 25℃, the precipitate was collected, washed with water until neutral, and freeze-dried for later use.

[0031] The walnut protein isolate prepared according to the above process has a purity of 90.5%.

[0032] The ultrasonic microreactors in the following examples are products sold by Moge Microfluidics Technology (Shantou) Co., Ltd.

[0033] Example 1 A continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers includes the following steps: 1) Preparation of chitosan solution: Chitosan was dissolved in 2% acetic acid solution at room temperature to obtain a chitosan solution with a mass concentration of 0.6%; 2) Preparation of walnut protein solution: Walnut protein was dissolved in 0.4 mol / L NaOH solution at room temperature to obtain a walnut protein solution with a mass concentration of 1.0%; 3) Preparation of the oil phase: Add 5% by mass of emulsifier EM90 to the liquid paraffin; 4) Mix the chitosan solution from step 1) with the oil phase from step 3) at a volume ratio of 1:8, and first shear and stir the mixture in a high-speed homogenizer (3500 rpm) to obtain mixture A; 5) Mix the walnut protein solution in step 2) with the oil phase in step 3) at a volume ratio of 1:3. First, shear and stir the mixture in a high-speed homogenizer (3500 rpm) until homogenized, and then pass it into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump to obtain emulsion A and emulsion B respectively; the flow rate of ultrasonic microreactor I and ultrasonic microreactor II is set to 6 mL / min and the power is 120 W.

[0034] 7) Emulsion A and Emulsion B are mixed in a 1:2 volume ratio after flowing out of the ultrasonic microreactor and directly fed into a T-type mixer to obtain Emulsion C; 8) The resulting emulsion C was allowed to stand and solidify at room temperature for 12 h; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

[0035] Example 2 A continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers includes the following steps: 1) Preparation of chitosan solution: Chitosan is dissolved in 2% acetic acid solution at room temperature to obtain a chitosan solution with a mass concentration of 1.0%; 2) Preparation of walnut protein solution: Walnut protein was dissolved in 0.4 mol / L NaOH solution at room temperature to obtain a walnut protein solution with a mass concentration of 2.0%; 3) Preparation of the oil phase: Add 10% by mass of emulsifier EM90 to the liquid paraffin; 4) Mix the chitosan solution from step 1) with the oil phase from step 3) at a volume ratio of 1:3, and first shear and stir in a high-speed homogenizer (3500 rpm) to obtain mixture A; 5) Mix the walnut protein solution in step 2) with the oil phase in step 3) at a volume ratio of 1:6. First, shear and stir the mixture in a high-speed homogenizer (3500 rpm) until homogenized, and then pass it into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump to obtain emulsion A and emulsion B respectively; the flow rate of ultrasonic microreactor I and ultrasonic microreactor II is set to 6 mL / min and the power is 120 W.

[0036] 7) Emulsion A and Emulsion B, in a volume ratio of 1:6, flowed directly from the ultrasonic microreactor into a T-type mixer for mixing to obtain Emulsion C; 8) The resulting emulsion C was allowed to stand and solidify at room temperature for 12 h; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

[0037] Example 3 A continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers includes the following steps: 1) Preparation of chitosan solution: Chitosan was dissolved in 2% acetic acid solution at room temperature to obtain a chitosan solution with a mass concentration of 2.0%; 2) Preparation of walnut protein solution: Walnut protein was dissolved in 0.4 mol / L NaOH solution at room temperature to obtain a walnut protein solution with a mass concentration of 0.5%; 3) Preparation of the oil phase: Add 15% by mass of emulsifier EM90 to the liquid paraffin; 4) Mix the chitosan solution from step 1) with the oil phase from step 3) at a volume ratio of 1:5, and first shear and stir the mixture in a high-speed homogenizer (3500 rpm) to obtain mixture A; 5) Mix the walnut protein solution in step 2) with the oil phase in step 3) at a volume ratio of 1:8. First, shear and stir the mixture in a high-speed homogenizer (3500 rpm) until homogenized, and then pass it into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump to obtain emulsion A and emulsion B respectively; the flow rate of ultrasonic microreactor I and ultrasonic microreactor II is set to 6 mL / min and the power is 120 W.

[0038] 7) Emulsion A and Emulsion B are mixed at a volume ratio of 1:10 after flowing out of the ultrasonic microreactor, and are directly fed into a T-type mixer to obtain Emulsion C; 8) The resulting emulsion C was allowed to stand and solidify at room temperature for 12 h; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

[0039] Example 4 A continuous flow preparation method for chitosan-walnut protein nanogel microsphere carriers includes the following steps: 1) Preparation of chitosan solution: Chitosan is dissolved in 2% acetic acid solution at room temperature to obtain a chitosan solution with a mass concentration of 1.0%; 2) Preparation of walnut protein solution: Walnut protein was dissolved in 0.4 mol / L NaOH solution at room temperature to obtain a walnut protein solution with a mass concentration of 1.0%; 3) Preparation of the oil phase: Add 10% by mass of emulsifier EM90 to the liquid paraffin; 4) Mix the chitosan solution from step 1) with the oil phase from step 3) at a volume ratio of 1:5, and first shear and stir the mixture in a high-speed homogenizer (3500 rpm) to obtain mixture A; 5) Mix the walnut protein solution in step 2) with the oil phase in step 3) at a volume ratio of 1:5. First, shear and stir the mixture in a high-speed homogenizer (3500 rpm) until homogenized, and then pass it into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump to obtain emulsion A and emulsion B respectively; the flow rate of ultrasonic microreactor I and ultrasonic microreactor II is set to 6 mL / min and the power is 120 W.

[0040] 7) Emulsion A and Emulsion B, at a volume ratio of 1:5, flowed directly from the ultrasonic microreactor into a T-type mixer for mixing to obtain Emulsion C; 8) The resulting emulsion C was allowed to stand and solidify at room temperature for 12 h; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

[0041] Figure 3 , Figure 4 This is a scanning electron microscope (SEM) image of the chitosan-walnut protein nanogel microsphere carrier prepared in this embodiment. As can be seen from the image, this invention successfully prepared chitosan-walnut protein composite hydrogel microspheres with submicron particle sizes. The carrier has highly uniform particle size and morphology, and the microspheres possess a good internal mesh structure, enabling high drug loading.

[0042] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A continuous flow process for the preparation of chitosan-walnut protein nanogel microspheres carrier, characterized by: Includes the following steps: 1) Preparation of chitosan solution: Chitosan is dissolved in acetic acid solution to obtain chitosan solution; 2) Preparation of walnut protein solution: Walnut protein is dissolved in NaOH solution to obtain walnut protein solution; 3) Preparation of the oil phase: An emulsifier is added to liquid paraffin to obtain the oil phase; 4) Mix the chitosan solution from step 1) with the oil phase from step 3), and shear and stir the mixture in a high-speed homogenizer to obtain mixture A; 5) Mix the walnut protein solution from step 2) with the oil phase from step 3), first shear and stir in a high-speed homogenizer to mix them evenly, and then pass them into an ultrasonic microreactor for emulsification to obtain mixture B. 6) Mixture A is continuously fed into ultrasonic microreactor I through a pump, while mixture B is continuously fed into ultrasonic microreactor II through a pump, to obtain emulsion A and emulsion B respectively; 7) Emulsion A and Emulsion B flow out of the ultrasonic microreactor and are directly fed into a T-type mixer for mixing to obtain Emulsion C; 8) The obtained emulsion C was allowed to stand and solidify to obtain the solidified product; 9) The cured product was washed sequentially with petroleum ether, ethanol and water and then freeze-dried to obtain chitosan-walnut protein nanogel microsphere carrier.

2. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to claim 1, characterized in that: The concentration of the chitosan solution mentioned in step 1) is 0.5 wt% to 2 wt%; The concentration of the acetic acid solution mentioned in step 1) is 1 wt% to 3 wt%; The concentration of the walnut protein solution mentioned in step 2) is 0.5 wt% to 2 wt%; The concentration of the NaOH solution mentioned in step 2) is 0.1–1 mol / L.

3. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to claim 1, characterized in that: The emulsifier mentioned in step 3) is at least one of glyceryl stearate, polyglycerol ester, and EM90; The mass fraction of the emulsifier mentioned in step 3) is 5% to 15%.

4. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to claim 1, characterized in that: The volume ratio of the chitosan solution to the oil phase in step 4) is 1:3 to 1:8; The volume ratio of the walnut protein solution to the oil phase in step 5) is 1:3 to 1:8; The volume ratio of the walnut protein solution to the oil phase in step 7) is 1:2 to 1:10, which is also the volume ratio of emulsion A to emulsion B.

5. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to any one of claims 1-4, characterized in that: The concentration of the chitosan solution mentioned in step 1) is 1 wt% The concentration of the acetic acid solution mentioned in step 1) is 2 wt%; The concentration of the walnut protein solution mentioned in step 2) is 1 wt% The concentration of the NaOH solution mentioned in step 2) is 0.4 mol / L; The emulsifier mentioned in step 3) is EM90; The mass fraction of the emulsifier mentioned in step 3) is 10%.

6. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to any one of claims 1-4, characterized in that: The volume ratio of the chitosan solution to the oil phase in step 4) is 1:5; The volume ratio of the walnut protein solution to the oil phase in step 5) is 1:5; The volume ratio of the walnut protein solution to the oil phase in step 7) is 1:5, and the volume ratio of emulsion A to emulsion B is 1:

5.

7. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to any one of claims 1-4, characterized in that: The parameters of the high-speed homogenizer mentioned in steps 4) and 5) are 3500±500 rpm; The curing time mentioned in step 8) is 12 ± 2 h.

8. The continuous flow preparation method of chitosan-walnut protein nanogel microsphere carrier according to any one of claims 1-4, characterized in that: In step 6), both ultrasonic microreactors I and II are set to a flow rate of 6±1 mL / min and a power of 120±20 W.

9. A chitosan-walnut protein nanogel microsphere carrier characterized by: It is obtained by the preparation method described in any one of claims 1-8.

10. The application of the chitosan-walnut protein nanogel microsphere carrier as described in claim 9 in the preparation of drug carrier / drug delivery systems.

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