Preparation method of PH-triggered composite hydrogel microparticles

Abstract

The invention discloses a preparation method of composite hydrogel microparticles triggered by PH (Potential of Hydrogen). According to the method disclosed by the invention, emulsion templates in different forms are constructed, and acetic acid molecules in an oil phase are diffused to enter water-phase liquid drops so as to lower the pH value of a chitosan-alginic acid homogeneous solution, so that chitosan is protonized and is subjected to ionic crosslinking with alginic acid carboxyl; further, the preparation of chitosan-alginic acid composite microspheres, microcapsules and core-shell hydrogel microparticles is realized. The prepared composite hydrogel microparticles show good morphology stability, monodispersity and a certain autofluorescence characteristic. Meanwhile, no organic solvent or harmful cross-linking agent is used for microsphere forming, and the prepared microparticles have good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of hydrogel microparticles. Specifically, it relates to a method for preparing pH-triggered composite hydrogel microparticles. Background Art

[0002] The content of this part only provides background information related to the present invention, which may not constitute prior art.

[0003] Hydrogel microparticles are three-dimensional network particles with highly hydrated properties formed by cross-linking reactions using polymer solutions as precursors, and are widely used in fields such as cell culture, drug delivery, tissue engineering, wound healing on the body surface, and antibacterial dressings.

[0004] Chitosan is a natural polysaccharide mainly extracted from the shells of marine arthropods, mollusk shells, and bones. It has good biocompatibility and is widely used in the preparation of hydrogels. Developing chitosan and its composite hydrogel microparticles that can be used in the fields of food engineering, biomedicine, and cosmetics has become a research hotspot and frontier in the current academic and industrial circles.

[0005] Traditional methods for preparing chitosan-based hydrogel microparticles mainly include spray drying, electrospraying, and ultrasonic emulsification. There are still significant limitations in the particle structure, morphology control, and particle size control of the hydrogel microparticles prepared using these methods. Microfluidics technology is a technology for precisely manipulating fluids at the microscale and has been widely used in the controllable preparation of microparticles based on the droplet template method. However, at present, the hydrogel microparticles prepared based on chitosan usually require Schiff base reactions with organic reagents such as terephthalaldehyde and glutaraldehyde for cross-linking, or complex modification operations on chitosan molecules and then molding through photo-crosslinking or chemical crosslinking. These have greatly restricted the application scenarios of the prepared chitosan-based hydrogel microparticles, and there is an urgent need to develop new microfluidic preparation methods for the preparation of chitosan-based microparticles. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method that combines a pH-triggered cross-linking mechanism and microfluidics technology, uses chitosan and sodium alginate as raw materials, and can controllably prepare hydrogel microparticles by regulating the pH to control the gelation state of chitosan-alginate without using organic solvents or harmful cross-linking agents. The preparation method of the present invention is environmentally friendly and easy to operate, and can prepare chitosan-alginate composite hydrogel microparticles with precisely regulated particle size and good monodispersity. The present invention can strongly promote the broader application of chitosan materials.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention discloses a method for preparing pH-triggered composite hydrogel microparticles, comprising the following steps:

[0009] Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate;

[0010] Dissolve 10 wt% of PGPR in soybean oil to obtain a first oil phase solution O1;

[0011] Add an additional 0.5% v / v of acetic acid to the first oil phase solution O1 to obtain a collection phase solution O3;

[0012] Inject the crosslinked main body homogeneous solution W1 as the inner phase and the first oil phase solution O1 as the outer phase into a W / O microfluidic device, and control the flow rates of the crosslinked main body homogeneous solution W1 and the first oil phase solution O1 in the W / O microfluidic device to obtain W / O single emulsion droplets, and let the W / O single emulsion droplets enter the collection phase solution O3;

[0013] After the W / O single emulsion droplets enter the collection phase solution O3, let it stand for a predetermined time; subsequently, wash the excess mixed oil phase of the first oil phase solution O1 and the collection phase solution O3 with n-hexane; finally, wash away the n-hexane with 1 mM PBS solution to obtain hydrogel microparticles.

[0014] Optionally, the flow rate of the crosslinked main body homogeneous solution W1 in the W / O microfluidic device is 200 μl / h, and the flow rates of the first oil phase solution O1 in the W / O microfluidic device are 400, 800, 1200, 1600 or 2000 μl / h.

[0015] Optionally, preparing the crosslinked main body homogeneous solution W1 using chitosan and sodium alginate specifically includes:

[0016] Dissolve 2 wt% of sodium alginate and 2 wt% of chitosan in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution;

[0017] Adjust the pH value of the chitosan solution to 7; dissolve 0.5% of poloxamer F-127 in the chitosan solution, and appropriately add NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6;

[0018] Inject the sodium alginate solution into the chitosan solution with a pH value of 7.4 - 7.6 and mix to obtain the crosslinked main body homogeneous solution W1.

[0019] Optionally, in the crosslinked main body homogeneous solution W1, the volume ratio of sodium alginate to chitosan is: 2:1.

[0020] Optionally, the pH value of the PBS solution is 3 - 9.

[0021] In a second aspect, the present invention discloses a method for preparing a pH-triggered composite hydrogel microparticle, comprising the following steps:

[0022] Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate;

[0023] Dissolve 10 wt% of PGPR in soybean oil to serve as the first oil phase solution O1;

[0024] Dissolve a non-crosslinked polymer in ultrapure water to prepare an aqueous phase solution W2;

[0025] Add 0.5% v / v of acetic acid to the first oil phase solution O1 to serve as the collection phase solution O3;

[0026] Inject the aqueous phase solution W2 as the inner phase, the crosslinked main body homogeneous solution W1 as the intermediate phase, and the first oil phase solution O1 as the outer phase into a W / W / O type microfluidic device, and control the flow rates of the aqueous phase solution W2, the crosslinked main body homogeneous solution W1, and the first oil phase solution O1 in the W / W / O type microfluidic device to obtain W / W / O type double emulsion droplets, and let the W / W / O type double emulsion droplets enter the collection phase solution O3;

[0027] After the W / W / O type double emulsion droplets enter the collection phase solution O3, let it stand for a predetermined time; subsequently, wash the mixed oil phase of the excess first oil phase solution O1 and the collection phase solution O3 with n-hexane; finally, wash away the n-hexane with 1 mM PBS solution to obtain hydrogel microparticles.

[0028] Optionally, the non-crosslinked polymer is 2 wt% of PEG6000, 2 wt% of Dextran, or 2 wt% of PVA.

[0029] Optionally, the flow rate of the aqueous phase solution W2 in the W / W / O type microfluidic device is 100 μl / h, the flow rate of the crosslinked main body homogeneous solution W1 in the W / W / O type microfluidic device is 100 μl / h, and the flow rate of the first oil phase solution O1 in the W / W / O type microfluidic device is 2000 μl / h.

[0030] Optionally, preparing the crosslinked main body homogeneous solution W1 using chitosan and sodium alginate specifically includes:

[0031] Dissolve 2 wt% of sodium alginate and 2 wt% of chitosan in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution;

[0032] Adjust the pH value of the chitosan solution to 7; dissolve 0.5% of poloxamer F-127 in the chitosan solution, and appropriately add NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6;

[0033] Inject the sodium alginate solution into the chitosan solution with a pH value of 7.4 - 7.6 and mix to obtain the crosslinked main body homogeneous liquid W1.

[0034] Optionally, in the crosslinked main body homogeneous liquid W1, the volume ratio of sodium alginate to chitosan is: 2:1.

[0035] Optionally, the pH value of the PBS solution is 3 - 9.

[0036] Thirdly, the present invention discloses a preparation method of pH-triggered composite hydrogel microparticles, comprising the following steps:

[0037] Prepare the crosslinked main body homogeneous liquid W1 by using chitosan and sodium alginate;

[0038] Dissolve 10 wt% of PGPR in soybean oil to serve as the first oil phase solution O1;

[0039] Use dimethyl silicone oil PMX-200 with a viscosity of 10 mPa·s as the second oil phase solution O2;

[0040] Add 0.5% v / v of acetic acid additionally to the first oil phase solution O1 to serve as the collection phase solution O3;

[0041] Inject the second oil phase solution O2 as the inner phase, the crosslinked main body homogeneous liquid W1 as the middle phase, and the first oil phase solution O1 as the outer phase into an O / W / O type microfluidic device, and control the flow rates of the second oil phase solution O2, the crosslinked main body homogeneous liquid W1, and the first oil phase solution O1 in the O / W / O type microfluidic device to obtain O / W / O type multiple emulsion droplets, and let the O / W / O type multiple emulsion droplets enter the collection phase solution O3;

[0042] After the O / W / O type multiple emulsion droplets enter the collection phase solution O3, let it stand for a predetermined time; subsequently, wash the mixed oil phase of the excess first oil phase solution O1 and the collection phase solution O3 with n-hexane; finally, wash away the n-hexane with 1 mM PBS solution to obtain the hydrogel microparticles.

[0043] Optionally, the flow rate of the second oil phase solution O2 in the O / W / O type microfluidic device is 300 μl / h, the flow rate of the crosslinked main body homogeneous liquid W1 in the O / W / O type microfluidic device is 400, 500, 600, 700, 800 μl / h, and the flow rate of the first oil phase solution O1 in the O / W / O type microfluidic device is 300 - 1500 μl / h.

[0044] Optionally, the specific steps of preparing the crosslinked main body homogeneous liquid W1 by using chitosan and sodium alginate include:

[0045] Dissolve 2 wt% of sodium alginate and 2 wt% of chitosan in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution;

[0046] Adjust the pH value of the chitosan solution to 7; dissolve 0.5% of poloxamer F-127 in the chitosan solution and appropriately add NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6;

[0047] Inject the sodium alginate solution into the chitosan solution with a pH value of 7.4 - 7.6 and mix to obtain a cross-linked main body homogeneous liquid W1.

[0048] Optionally, in the cross-linked main body homogeneous liquid W1, the volume ratio of sodium alginate to chitosan is: 2:1.

[0049] Optionally, the pH value of the PBS solution is 3 - 9.

[0050] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0051] The present invention proposes a new green and environmentally friendly method combining a pH-triggered mechanism and microfluidic technology for preparing chitosan-alginate composite hydrogel microparticles. This method constructs emulsion templates in different forms, and uses acetic acid molecules in the oil phase to diffuse into the aqueous droplets, thereby lowering the pH of the chitosan-alginate homogeneous liquid, causing the protonation of chitosan and the ionic cross-linking of the carboxyl groups of alginic acid, and then realizing the preparation of chitosan-alginate composite microspheres, microcapsules, and core-shell hydrogel microparticles. The prepared composite hydrogel microparticles exhibit good morphological stability, monodispersity, and certain spontaneous fluorescence characteristics. At the same time, the present invention does not require the use of organic solvents or harmful cross-linking agents, avoiding problems of biological toxicity and environmental pollution, and the prepared hydrogel microparticles have broad application prospects in multiple fields such as the encapsulation of functional substances, drug delivery, food nutrition delivery, and three-dimensional cell culture, providing a new microfluidic method for the controllable preparation of chitosan composite hydrogel microparticles, and providing new ideas and solutions for intelligent delivery and encapsulation applications. Description of the Drawings

[0052] Figure 1 It is a schematic diagram of the cross-linking process of the hydrogel microparticles provided in Example 1 of the present invention;

[0053] Figure 2 It is a schematic diagram of the W / O type microfluidic device and the preparation of W / O type single emulsion droplets provided in Example 1 of the present invention;

[0054] Figure 3 For the first oil phase solution O1 with different flow rates Q provided in Example 1 of the present invention OUnder certain conditions, the morphology and particle size diagrams of W / O single emulsion droplets and hydrogel microparticles are obtained; among them, Figure 3 (a) is the inverted microscope field view of W / O single emulsion droplets; Figure 3 (b) is the inverted microscope field view of hydrogel microparticles; Figure 3 (c1) is the size statistical curve diagram of W / O single emulsion droplets; Figure 3 (c2) is the size statistical curve diagram of hydrogel microparticles;

[0055] Figure 4 For Example 2 of the present invention, under the condition of different pH values of PBS solution, the morphology change and particle size statistical chart of hydrogel microparticles; among them, Figure 4 (a) is the diagram of the morphology change of hydrogel microparticles under the inverted microscope before and after being transferred to PBS solutions with different pH values; Figure 4 (b) is the particle size statistical chart of hydrogel microparticles before and after being transferred to PBS solutions with different pH values;

[0056] Figure 5 For Example 3 of the present invention, hydrogel microparticles are dispersed in PBS solution with a pH value of 3, and time points 0, 4, 8, 12, 24, 36 h are taken as observation points, and the particle size and fluorescence change diagram of hydrogel microparticles observed under a laser confocal field of view are obtained; among them, Figure 5 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 5 (b1) is the particle size change diagram of hydrogel microparticles; Figure 5 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0057] Figure 6 For Example 3 of the present invention, hydrogel microparticles are dispersed in PBS solution with a pH value of 4, and time points 0, 4, 8, 12, 24, 36 h are taken as observation points, and the particle size and fluorescence change diagram of hydrogel microparticles observed under a laser confocal field of view are obtained; among them, Figure 6 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 6 (b1) is the particle size change diagram of hydrogel microparticles; Figure 6 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0058] Figure 7 For Example 3 of the present invention, hydrogel microparticles are dispersed in PBS solution with a pH value of 5, and time points 0, 4, 8, 12, 24, 36 h are taken as observation points, and the particle size and fluorescence change diagram of hydrogel microparticles observed under a laser confocal field of view are obtained; among them, Figure 7 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 7(b1) is the particle size change diagram of hydrogel microparticles; Figure 7 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0059] Figure 8 This is the particle size and fluorescence change diagram of hydrogel microparticles obtained by dispersing hydrogel microparticles in PBS solution with a pH value of 6 according to Example 3 of the present invention, and taking time points 0, 4, 8, 12, 24, and 36 h as observation points and observing under a laser confocal field of view; among them, Figure 8 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 8 (b1) is the particle size change diagram of hydrogel microparticles; Figure 8 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0060] Figure 9 This is the particle size and fluorescence change diagram of hydrogel microparticles obtained by dispersing hydrogel microparticles in PBS solution with a pH value of 7 according to Example 3 of the present invention, and taking time points 0, 4, 8, 12, 24, and 36 h as observation points and observing under a laser confocal field of view; among them, Figure 9 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 9 (b1) is the particle size change diagram of hydrogel microparticles; Figure 9 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0061] Figure 10 This is the particle size and fluorescence change diagram of hydrogel microparticles obtained by dispersing hydrogel microparticles in PBS solution with a pH value of 8 according to Example 3 of the present invention, and taking time points 0, 4, 8, 12, 24, and 36 h as observation points and observing under a laser confocal field of view; among them, Figure 10 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 10 (b1) is the particle size change diagram of hydrogel microparticles; Figure 10 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0062] Figure 11 This is the particle size and fluorescence change diagram of hydrogel microparticles obtained by dispersing hydrogel microparticles in PBS solution with a pH value of 9 according to Example 3 of the present invention, and taking time points 0, 4, 8, 12, 24, and 36 h as observation points and observing under a laser confocal field of view; among them, Figure 11 (a) is the fluorescence change diagram of hydrogel microparticles; Figure 11 (b1) is the particle size change diagram of hydrogel microparticles; Figure 11 (b2) is the fluorescence value change diagram of hydrogel microparticles;

[0063] Figure 12Schematic diagram of the W / W / O microfluidic device provided in Example 4 of the present invention and its preparation of W / W / O double emulsion droplets;

[0064] Figure 13 Morphology and particle size diagrams of the W / W / O double emulsion droplets and hydrogel microparticles prepared in Example 4 of the present invention; wherein, Figure 13 (a) Inverted microscope field of view diagram of the prepared W / W / O double emulsion droplets; Figure 13 (b) Inverted microscope field of view diagram of the prepared hydrogel microparticles; Figure 13 (c) Particle size frequency distribution diagram of the prepared double emulsion droplets (Droplets) and hydrogel microparticles (MP);

[0065] Figure 14 (a) Laser confocal field of view diagram of the hydrogel microparticles prepared in Example 4 of the present invention under an excitation wavelength of 488 nm, bright field BF, and superposition field MG; Figure 14 (b) Green fluorescence profile diagram of the hydrogel microparticles under a laser confocal excitation wavelength of 488 nm.

[0066] Figure 15 Schematic diagram of the O / W / O microfluidic device provided in Example 5 of the present invention and its preparation of O / W / O double emulsion droplets;

[0067] Figure 16 For Example 5 of the present invention, at different flow rates Q of the crosslinked main body homogeneous liquid W1 M Under the conditions, inverted microscope field of view diagrams of the prepared O / W / O double emulsion droplets (In oil) and hydrogel microparticles (In pbs) with different thicknesses;

[0068] Figure 17 For Example 5 of the present invention, at different flow rates Q of the crosslinked main body homogeneous liquid W1 M Under the conditions, fitting curve diagrams of the droplet size, internal oil phase particle size, and particle wall thickness of the prepared hydrogel microparticles with different thicknesses; wherein, Figure 17 (a) Fitting curve diagram of the droplet size of hydrogel microparticles with different thicknesses, Figure 17 (b) Fitting curve diagram of the internal oil phase particle size of hydrogel microparticles with different thicknesses, Figure 17 (c) Fitting curve diagram of the particle wall thickness of hydrogel microparticles with different thicknesses, Figure 17 (d) Fitting curve diagram of the overall particle size of hydrogel microparticles with different thicknesses;

[0069] Figure 18 For Example 5 of the present invention, at different flow rates Q of the crosslinked main body homogeneous liquid W1 MUnder the conditions, the confocal laser scanning microscopy images of the hydrogel microparticles prepared under the excitation wavelength of 488 nm, bright field BF, and superposition field MG;

[0070] Figure 19 For Example 6 of the present invention, at different flow rates Q of the crosslinked main body homogeneous solution W1 M Under the conditions, by adjusting the flow rate Q of the first oil phase solution O1 O , the inverted microscopy images of the multiple-loaded O / W / O type double emulsion droplets and multiple-loaded hydrogel microparticles prepared;

[0071] Figure 20 For Example 6 of the present invention, at different flow rates Q of the crosslinked main body homogeneous solution W1 M Under the conditions, by adjusting the flow rate Q of the first oil phase solution O1 O , the confocal laser scanning microscopy images of the multiple-loaded hydrogel microparticles prepared under the excitation wavelength of 488 nm, bright field BF, and superposition field MG. Detailed implementation manners

[0072] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the detailed implementation manners. It should be noted that the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0073] Example 1

[0074] Example 1 of the present invention discloses a preparation method of pH-triggered composite hydrogel microparticles, especially a preparation method of pH-triggered chitosan-alginate microspheres. The preparation method includes the following steps:

[0075] Prepare the crosslinked main body homogeneous solution W1 by using chitosan and sodium alginate. Specifically, dissolve 2 wt% of sodium alginate (SA) and 2 wt% of chitosan (CS) in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution; adjust the pH value of the chitosan solution to 7; dissolve an appropriate amount of 0.5% poloxamer F-127 in the chitosan solution, and appropriately add an NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6; finally, inject the sodium alginate solution into the stirring chitosan solution with a pH value of 7.4 - 7.6 through an injection pump and mix to obtain the crosslinked main body homogeneous solution W1.

[0076] Dissolve 10 wt% of PGPR (polyglycerol polyricinoleate) in Jinlongyu soybean oil to serve as the first oil phase solution O1.

[0077] 0.5% v / v of acetic acid was additionally added to the first oil phase solution O1 as the collection phase solution O3.

[0078] Based on microfluidic technology, the crosslinked matrix homogeneous liquid W1 was used as the internal phase and the first oil phase solution O1 was used as the external phase, and they were injected into a W / O microfluidic device. The flow rates of the crosslinked matrix homogeneous liquid W1 and the first oil phase solution O1 in the W / O microfluidic device were controlled. In the W / O microfluidic device, under the shearing action of the first oil phase solution O1, the crosslinked matrix homogeneous liquid W1 was sheared into W / O single emulsion droplets with uniform particle size and controllable size. The obtained W / O single emulsion droplets entered the collection phase solution O3 through the output port of the W / O microfluidic device to complete the preparation of the W / O emulsion template.

[0079] Exemplarily, Figure 2 The schematic diagram of the W / O microfluidic device and the preparation of the above-mentioned W / O single emulsion droplets is shown. In Figure 2 In the shown embodiment, the crosslinked matrix homogeneous liquid W1 as the internal phase was injected through the No. 1 input port (i.e., the water phase sampling port) of the W / O microfluidic device, and the first oil phase solution O1 as the external phase was injected through the No. 2 input port (i.e., the oil phase sampling port) of the W / O microfluidic device. The obtained W / O single emulsion droplets entered the collection phase solution O3 through the No. 3 output port of the W / O microfluidic device. Among them, the first oil phase solution O1 was injected into the W / O microfluidic device earlier than the crosslinked matrix homogeneous liquid W1.

[0080] Among them, the W / O microfluidic device described in this embodiment can be a two-phase coaxial flow type microfluidic device known in the prior art, and its specific structure can refer to the two-phase coaxial flow type microfluidic device known in the prior art, and will not be elaborated here.

[0081] After the above-prepared W / O single emulsion droplets entered the collection phase solution O3, they were left standing for a predetermined time, such as 10 min. Subsequently, the mixed oil phase of the excess first oil phase solution O1 and the collection phase solution O3 was washed with n-hexane; finally, the n-hexane was washed off with 1 mM PBS solution (phosphate buffer solution) to obtain hydrogel microparticles, that is, chitosan-alginate microspheres, and the hydrogel microparticles were dispersed in the PBS solution for storage.

[0082] In this embodiment, in the crosslinked matrix homogeneous liquid W1, the volume ratio of sodium alginate to chitosan can be: 2:1.

[0083] In this embodiment, the flow rate of the crosslinked main body homogeneous liquid W1 in the W / O microfluidic device can be 200 μl / h, and the flow rate of the first oil-phase solution O1 in the W / O microfluidic device can be 400, 800, 1200, 1600 or 2000 μl / h.

[0084] In this embodiment, the PBS solution can be arbitrarily weakly acidic, neutral or weakly basic. For example, the pH value of the PBS solution can be 3-9. It should be noted that by allowing the prepared hydrogel microparticles to exist in PBS solutions with different pH values for a long time, hydrogel microparticles with different particle sizes can be washed out.

[0085] It is worth noting that in the present invention, the first oil-phase solution O1 added with acetic acid is used as the collecting-phase solution O3, and its purpose is to provide an acidic environment for the prepared W / O single emulsion droplets. Under the acidic environment, as shown in Figure 1 shown, acidic ions in the collecting-phase solution O3, such as H + will diffuse from the oil phase to the water phase, that is, the crosslinked main body homogeneous liquid W1, so as to lower the pH of the chitosan-alginate mixed homogeneous solution, thereby promoting the crosslinking of chitosan and sodium alginate in the crosslinked main body homogeneous liquid W1, and then forming hydrogel microparticles, especially hydrogel microparticles with controllable particle size and good monodispersity, that is, chitosan-alginate microspheres.

[0086] It can be understood that in actual preparation, only by controlling the flow rates of the crosslinked main body homogeneous liquid W1 and the first oil-phase solution O1 in the W / O microfluidic device according to different flow rate ratios, hydrogel microparticles with different sizes and good monodispersity can be obtained.

[0087] Figure 3 shows the morphology and particle size of the W / O single emulsion droplets and hydrogel microparticles prepared in this embodiment under different flow rates Q of the first oil-phase solution O1 O conditions. Among them, Figure 3 (a) is an inverted microscope field view of the prepared W / O single emulsion droplets under different flow rate conditions of the first oil-phase solution O1; Figure 3 (b) is an inverted microscope field view of the prepared hydrogel microparticles under different flow rate conditions of the first oil-phase solution O1; Figure 3 (c1) is a size statistical curve graph of the W / O single emulsion droplets under different flow rate conditions of the first oil-phase solution O1; Figure 3 (c2) is a size statistical curve graph of the hydrogel microparticles under different flow rate conditions of the first oil-phase solution O1.

[0088] From Figure 3 (a) and (c1), it can be seen that as the flow rate Q of the first oil-phase solution O1 in the W / O deviceO Gradually increasing from 400 μl / h to 2000 μl / h, the size of the shear-prepared W / O single emulsion droplets gradually decreases from about 130 microns to about 60 microns (the coefficient of variation CV values of the droplet size corresponding to the first oil phase flow rates Q O = 400, 800, 1200, 1600, 2000 μl / h are 0.75%, 0.75%, 1.0%, 1.4%, 1.5%); as can be seen from Figure 3 (b) and (c2), as the flow rate Q of the first oil phase solution O1 in the W / O device O gradually increases from 400 μl / h to 2000 μl / h, the particle size of the shear-prepared hydrogel microparticles gradually decreases from about 110 microns to about 60 microns (the coefficient of variation CV values of the microparticle size corresponding to the first oil phase flow rates Qo = 400, 800, 1200, 1600, 2000 μl / h are 3.7%, 3.6%, 3.5%, 3.8%, 4.1%).

[0089] Example 2

[0090] On the basis of Example 1, this Example 2 discloses a method for preparing pH-triggered composite hydrogel microparticles. In the preparation method disclosed in this Example 2, the preparation process of the W / O single emulsion droplets is basically the same as that of the foregoing Example 1. Different from Example 1, in this Example 2, the flow rates of the cross-linking main body homogeneous liquid W1 and the first oil phase solution O1 in the W / O microfluidic device are limited to 200 μl / h and 1200 μl / h respectively. At the same time, when washing away n-hexane with a 1 mM PBS solution, the pH value of the PBS solution is 5.4; and, after obtaining the hydrogel microparticles, the hydrogel microparticles are transferred from the PBS solution with a pH value of 5.4 to 1 mM PBS solutions with pH values of 3, 4, 5, 6, 7, 8, and 9 respectively, to observe the morphological changes of the hydrogel microparticles and to count the morphology and size of the hydrogel microparticles.

[0091] Figure 4 Shows the statistical chart of the morphological changes and particle sizes of the hydrogel microparticles under different pH conditions of the PBS solution.

[0092] Among them, Figure 4 (a) is the morphological change diagram of the hydrogel microparticles before and after being transferred to PBS solutions with different pH values under an inverted microscope; Figure 4 (b) is the statistical chart of the particle sizes of the hydrogel microparticles before and after being transferred to PBS solutions with different pH values.

[0093] As can be seen from Figure 4It can be seen that when the hydrogel microparticles are transferred from a 1 mM PBS solution with a pH value of 5.4 to 1 mM PBS solutions with pH values of 3, 4, 5, 6, 7, 8, and 9 respectively, the change in pH value before and after the transfer will directly affect the particle size of the hydrogel microparticles. Based on the cross-linking mechanism of the present invention: in an acidic environment, chitosan is protonated and undergoes ionic cross-linking with carboxyl groups. In environments with different pH values, the cross-linking of chitosan and alginic acid will be affected, which will further lead to changes in the composite hydrogel network and thus changes in the particle size of the microparticles.

[0094] Example 3

[0095] On the basis of Example 1, this Example 3 discloses a method for preparing pH-triggered composite hydrogel microparticles. In the preparation method disclosed in this Example 3, the preparation process of the W / O single emulsion droplets is basically the same as that of the aforementioned Example 1. Different from Example 1, in this Example 3, the flow rates of the cross-linking main body homogeneous solution W1 and the first oil phase solution O1 in the W / O microfluidic device are limited to be 200 μl / h and 1200 μl / h respectively. At the same time, when washing away n-hexane with a 1 mM PBS solution, PBS solutions with various different pH values are used for washing, and after washing, the hydrogel microparticles are directly dispersed in the PBS solution with the corresponding pH value. Among them, the pH values of the PBS solutions are 3, 4, 5, 6, 7, 8, and 9 respectively.

[0096] After obtaining the hydrogel microparticles, laser confocal microscopy is used to observe the situation of the hydrogel microparticles under different pH values of the PBS solution, and time points 0, 4, 8, 12, 24, and 36 h are taken as the observation points respectively to statistically analyze the particle size and fluorescence changes of the hydrogel microparticles. Among them, the excitation wavelength during laser confocal microscopy is 488 nm.

[0097] Specifically, as can be seen from the content shown in Figures 5 to 11 , the hydrogel microparticles are dispersed in 1 mM PBS solutions with pH values of 3, 4, 5, 6, 7, 8, and 9. Taking the observation time points as 0, 8, 12, 24, and 36 h, within the observed time range, the morphology of the hydrogel microparticles dispersed in pH = 3 is stable, as shown in Figure 5 (a), the average particle size is 95 ± 5 μm, as shown in Figure 5 (b1), the fluorescence intensity gradually increases and reaches the maximum at 24 h, and then decreases, as shown in Figure 5 (b2); the morphology of the hydrogel microparticles dispersed in pH = 4 is stable, as shown in Figure 6 (a), the average particle size is 95 ± 5 μm, as shown in Figure 6 (b1), the fluorescence intensity gradually increases and reaches the maximum at 24 h, and then decreases, as shown in Figure 6 (b2); the morphology of the hydrogel microparticles dispersed in pH = 5 is stable, as shown in Figure 7(a), with an average particle size of 95 ± 5 μm, see Figure 7 (b1), the fluorescence intensity gradually increases and reaches the maximum at 24 h, and then decreases, see Figure 7 (b2); the morphology of the hydrogel microparticles dispersed in water with pH = 6 is stable, see Figure 8 (a), with an average particle size of 105 ± 5 μm, see Figure 8 (b1), the fluorescence intensity gradually increases and reaches the maximum at 24 h, and then decreases, see Figure 8 (b2); the morphology of the hydrogel microparticles dispersed in water with pH = 7 is stable, see Figure 9 (a), with an average particle size of 130 ± 10 μm, see Figure 9 (b1), the fluorescence intensity gradually increases and reaches the maximum at 12 h, and then decreases, see Figure 9 (b2); the morphology of the hydrogel microparticles dispersed in water with pH = 8 is stable, see Figure 10 (a), with an average particle size of 105 ± 5 μm, see Figure 10 (b1), the fluorescence intensity gradually increases and reaches the maximum at 12 h, and then decreases Figure 10 (b2); the morphology of the hydrogel microparticles dispersed in water with pH = 9 is stable, see Figure 11 (a), with an average particle size of 115 ± 5 μm, see Figure 11 (b1), the fluorescence intensity gradually increases and reaches the maximum at 12 h, and then decreases, see Figure 11 (b2).

[0098] Example 4

[0099] Example 4 of the present invention discloses a preparation method of pH-triggered composite hydrogel microparticles, especially a preparation method of pH-triggered chitosan-alginate microcapsules. The preparation method includes the following steps:

[0100] Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate. Specifically, dissolve 2 wt% of sodium alginate (SA) and 2 wt% of chitosan (CS) in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution; adjust the pH value of the chitosan solution to 7; dissolve an appropriate amount of poloxamer F-127 with a concentration of 0.5% in the chitosan solution, and appropriately add NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6; finally, inject the sodium alginate solution into the stirring chitosan solution with a pH value of 7.4 - 7.6 through an injection pump and mix to obtain the crosslinked main body homogeneous solution W1.

[0101] Dissolve 10 wt% of PGPR (polyglycerol polyricinoleate) in Jinlongyu soybean oil to obtain a first oil phase solution O1.

[0102] A non-crosslinked polymer is dissolved in ultrapure water to prepare an aqueous solution W2. Among them, the non-crosslinked polymer can be 2 wt% PEG6000 (polyethylene glycol 6000), 2 wt% Dextran, 2 wt% PVA (polyvinyl alcohol), etc.

[0103] 0.5% v / v of acetic acid is additionally added to the first oil phase solution O1 to serve as the collection phase solution O3.

[0104] Based on microfluidic technology, the aqueous solution W2 is used as the inner phase, the crosslinked main body homogeneous solution W1 is used as the middle phase, and the first oil phase solution O1 is used as the outer phase, and they are injected into a W / W / O type microfluidic device, and the flow rates of the aqueous solution W2, the crosslinked main body homogeneous solution W1, and the first oil phase solution O1 in the W / W / O type microfluidic device are controlled. In the W / W / O type microfluidic device, the aqueous solution W2 and the crosslinked main body homogeneous solution W1 form a laminar flow of the inner aqueous phase (the middle is the aqueous solution W2, surrounded by the crosslinked main body homogeneous solution W1 on all sides) and are simultaneously sheared by the first oil phase solution O1 to obtain W / W / O type double emulsion droplets with uniform particle size and controllable size. The obtained W / W / O type double emulsion droplets enter the collection phase solution O3 through the output port of the W / W / O type microfluidic device to complete the preparation of the W / W / O type emulsion template.

[0105] Exemplarily, Figure 12 shows a schematic diagram of a W / W / O type microfluidic device and the preparation of the above-mentioned W / W / O type double emulsion droplets. In Figure 12 the shown embodiment, the aqueous solution W2 as the inner phase is injected through the No. 2 input port (i.e., the first aqueous phase injection port) of the W / W / O type microfluidic device, the crosslinked main body homogeneous solution W1 as the middle phase is injected through the No. 1 input port (i.e., the second aqueous phase injection port) of the W / W / O type microfluidic device, the first oil phase solution O1 as the outer phase is injected through the No. 3 input port (i.e., the oil phase injection port) of the W / W / O type microfluidic device, and the obtained W / W / O type double emulsion droplets enter the collection phase solution O3 through the No. 4 output port of the W / W / O type microfluidic device. Among them, the first oil phase solution O1, the crosslinked main body homogeneous solution W1, and the aqueous solution W2 are injected into the W / W / O type microfluidic device in sequence in order: first inject the first oil phase solution O1, then inject the crosslinked main body homogeneous solution W1, and finally inject the aqueous solution W2.

[0106] Among them, the W / W / O type microfluidic device described in this embodiment can be modified from the W / O type microfluidic device described in Embodiment 1, that is, the two coaxial flow type microfluidic devices known in the prior art. Specifically, the W / W / O type microfluidic device is based on the known two coaxial flow type microfluidic devices. Inside the pulling needle capillary used to construct the aqueous phase injection port (that is, the No. 2 input port of the W / W / O type microfluidic device described above) of the two coaxial flow type microfluidic devices, a coaxial and smaller-sized pulling needle capillary is added to construct an independent inner aqueous phase laminar flow channel as another aqueous phase injection port, that is, the No. 1 input port of the W / W / O type microfluidic device described above. And the pulling needle end of the smaller-sized pulling needle capillary extends to a position flush with the pulling needle end of the pulling needle capillary used to construct the aqueous phase injection port, so as to realize that at the same shearing position as the two coaxial flow type microfluidic devices, the first oil phase solution O1 shears the aqueous phase solution W2 in the form of inner aqueous phase laminar flow and the crosslinked main body homogeneous solution W1 at the same time.

[0107] After the W / W / O type double emulsion droplets prepared above enter the collecting phase solution O3, they are left standing for a predetermined time, such as 10 min. Subsequently, the mixed oil phase of the excess first oil phase solution O1 and the collecting phase solution O3 is washed away with n-hexane; finally, the n-hexane is washed away with a 1 mM PBS solution (phosphate buffer solution) to obtain hydrogel microparticles, that is, chitosan-alginate microcapsules, especially chitosan-alginate hydrophilic inclusion core-shell type microcapsules, and the hydrogel microparticles are dispersed in the PBS solution for storage.

[0108] In this embodiment, in the crosslinked main body homogeneous solution W1, the volume ratio of sodium alginate to chitosan can be: 2:1.

[0109] In this embodiment, the flow rate of the aqueous phase solution W2 in the W / W / O type microfluidic device can be 100 μl / h, the flow rate of the crosslinked main body homogeneous solution W1 in the W / W / O type microfluidic device can be 100 μl / h, and the flow rate of the first oil phase solution O1 in the W / W / O type microfluidic device can be 2000 μl / h.

[0110] In this embodiment, the PBS solution can be arbitrarily weakly acidic, neutral or weakly basic. For example, the pH value of the PBS solution can be 3-9. It should be noted that by allowing the prepared hydrogel microparticles to exist in PBS solutions with different pH values for a long time, hydrogel microparticles with different particle sizes can be washed out.

[0111] It should be noted that in the present invention, the first oil-phase solution O1 added with acetic acid is used as the collecting-phase solution O3, aiming to provide an acidic environment for the prepared W / W / O type double emulsion droplets. In the acidic environment, the acidic ions in the collecting-phase solution O3 will diffuse from the oil phase to the water phase, that is, the cross-linking main homogeneous liquid W1, so as to lower the pH of the chitosan-alginate mixed homogeneous solution, thereby promoting the cross-linking of chitosan and sodium alginate in the cross-linking main homogeneous liquid W1, and then forming hydrogel microparticles, especially hydrogel microparticles with controllable particle size and good monodispersity, that is, chitosan-alginate microcapsules.

[0112] Meanwhile, by using the aqueous solution W2 as the inner phase, core-shell microparticles are constructed. Based on the different properties of the inner core and the outer shell, different release rates of the inner core inclusion (including slow-release degradation, burst release, gradient release, and the release sequence of two water-soluble inclusions) are achieved. In the present invention, PEG6000 is used as the aqueous solution W2 to prepare chitosan-alginate core-shell microparticles, providing a new preparation strategy for drug carriers with different structures.

[0113] In addition, this embodiment innovatively proposes a new double emulsion droplet structure, namely W / W / O type (water-in-water-in-oil) double emulsion droplets, filling the blank in the prior art which is limited to O / W / O type (oil-in-water-in-oil) and W / O / W type (water-in-oil-in-water) double emulsion droplets.

[0114] It can be understood that during actual preparation, only by controlling the flow rates of the aqueous solution W2, the cross-linking main homogeneous liquid W1, and the first oil-phase solution O1 in the W / W / O type microfluidic device according to different flow rate ratios, hydrogel microparticles with different sizes and good monodispersity can be obtained.

[0115] Figure 13 The morphologies and particle sizes of the W / W / O type double emulsion droplets and hydrogel microparticles prepared in this embodiment are shown. Among them, Figure 13 (a) is an inverted microscope field view of the prepared W / W / O type double emulsion droplets; Figure 13 (b) is an inverted microscope field view of the prepared hydrogel microparticles; Figure 13 (c) is a particle size frequency distribution diagram of the prepared double emulsion droplets (Droplets) and hydrogel microparticles (MP).

[0116] From Figure 13 It can be seen that by introducing PEG6000 and the cross-linking main homogeneous liquid W1 into the W / W / O type microfluidic device, the uncharged PEG6000 and the cross-linking main homogeneous liquid W1 do not cross-link and are easy to form a laminar flow, and are stably sheared at the same position, that is, using the aqueous solution W2 as the inner core, the preparation of W / W / O microparticles is successfully realized, and the prepared microparticles have good monodispersity, controllable particle size, and regular morphology.

[0117] To further prove the core-shell structure of the prepared W / W / O type double emulsion droplets, laser confocal microscopy was used to observe the hydrogel microparticles under an excitation wavelength of 488 nm, bright field BF, and merged field MG, respectively.

[0118] Specifically, as Figure 14 shown, chitosan produces green fluorescence under a 488 nm wavelength field of view. Laser confocal fluorescence analysis was performed on the hydrogel microparticles, and the fluorescence intensity distribution of its cross-section showed a double peak and was distributed at the edge of the microparticle cross-section, indicating that it was a hydrogel microparticle with a core-shell structure.

[0119] Example 5

[0120] Example 5 of the present invention discloses a method for preparing pH-triggered composite hydrogel microparticles, especially a method for preparing pH-triggered chitosan-alginate microcapsules. The preparation method includes the following steps:

[0121] Prepare a cross-linked main body homogeneous solution W1 using chitosan and sodium alginate. Specifically, it includes: dissolving 2 wt% of sodium alginate (SA) and 2 wt% of chitosan (CS) in ultrapure water respectively to obtain a sodium alginate solution and a chitosan solution; adjusting the pH value of the chitosan solution to 7; dissolving an appropriate amount of 0.5% poloxamer F-127 in the chitosan solution, and appropriately adding an NaOH solution to adjust the pH value of the chitosan solution to 7.4 - 7.6; finally, injecting the sodium alginate solution into the stirring chitosan solution with a pH value of 7.4 - 7.6 through an injection pump and mixing to obtain the cross-linked main body homogeneous solution W1.

[0122] Dissolve 10 wt% of PGPR (polyglycerol polyricinoleate) in Golden Dragon fish soybean oil to serve as the first oil phase solution O1.

[0123] Use dimethyl silicone oil PMX-200 with a viscosity of 10 mPa·s as the second oil phase solution O2.

[0124] Add an additional 0.5% v / v of acetic acid to the first oil phase solution O1 to serve as the collection phase solution O3.

[0125] Based on microfluidic technology, the second oil phase solution O2 is used as the inner phase, the crosslinked main body homogeneous solution W1 is used as the middle phase, and the first oil phase solution O1 is used as the outer phase, and they are injected into an O / W / O type microfluidic device, that is, a three-phase coaxial flow type microfluidic device, and the flow rates of the second oil phase solution O2, the crosslinked main body homogeneous solution W1, and the first oil phase solution O1 in the O / W / O type microfluidic device are controlled. In the O / W / O type microfluidic device, the second oil phase solution O2 is first used to prepare O / W type droplets under the shearing action of the crosslinked main body homogeneous solution W1, and then O / W / O type double emulsion droplets with uniform particle size and controllable size are prepared under the shearing action of the first oil phase solution O1. The obtained O / W / O type double emulsion droplets enter the collecting phase solution O3 through the output port of the O / W / O type microfluidic device to complete the preparation of the O / W / O type emulsion template.

[0126] Exemplarily, Figure 15 The schematic diagram of the O / W / O type microfluidic device and the preparation of the above-mentioned O / W / O type double emulsion droplets is shown. In Figure 15 In the illustrated embodiment, the second oil phase solution O2 as the inner phase is injected through the No. 1 input port (i.e., the first oil phase injection port) of the O / W / O type microfluidic device, the crosslinked main body homogeneous solution W1 as the middle phase is injected through the No. 2 input port of the O / W / O type microfluidic device (i.e., the water phase injection port), and the first oil phase solution O1 as the outer phase is injected through the No. 3 input port (i.e., the second oil phase injection port) of the O / W / O type microfluidic device. The obtained O / W / O type double emulsion droplets enter the collecting phase solution O3 through the No. 4 output port of the O / W / O type microfluidic device. Among them, the second oil phase solution O2, the crosslinked main body homogeneous solution W1, and the first oil phase solution O1 are injected into the O / W / O type microfluidic device in sequence in order, and the injection order is: first inject the second oil phase solution O2, then inject the crosslinked main body homogeneous solution W1, and finally inject the first oil phase solution O1.

[0127] Among them, the O / W / O type microfluidic device described in this embodiment can be a three-phase coaxial flow type microfluidic device known in the prior art, and its specific structure can refer to the three-phase coaxial flow type microfluidic device known in the prior art, and will not be elaborated here too much.

[0128] After the above-prepared O / W / O type double emulsion droplets enter the collecting phase solution O3, they are left standing for a predetermined time, such as 10 min. Subsequently, the mixed oil phase of the excess first oil phase solution O1 and the collecting phase solution O3 is washed with n-hexane; finally, the n-hexane is washed away with 1 mM PBS solution (phosphate buffer solution) to obtain hydrogel microparticles, that is, chitosan-alginate microcapsules, and the hydrogel microparticles are dispersed in the PBS solution for storage.

[0129] In this embodiment, in the crosslinked main body homogeneous liquid W1, the volume ratio of sodium alginate to chitosan can be: 2:1.

[0130] In this embodiment, the flow rate of the second oil phase solution O2 in the O / W / O type microfluidic device can be 300 μl / h, the flow rate of the crosslinked main body homogeneous liquid W1 in the O / W / O type microfluidic device can be 400, 500, 600, 700, 800 μl / h, and the flow rate of the first oil phase solution O1 in the O / W / O type microfluidic device can be 300 - 1500 μl / h, particularly can be 330, 600, 1000 μl / h as described in Example 6.

[0131] In this embodiment, the PBS solution can be arbitrarily weakly acidic, neutral or weakly basic. For example, the pH value of the PBS solution can be 3 - 9. It should be noted that by allowing the prepared hydrogel microparticles to exist in PBS solutions with different pH values for a long time, hydrogel microparticles with different particle sizes can be washed out.

[0132] It is worth noting that in the present invention, the first oil phase solution O1 added with acetic acid is used as the collecting phase solution O3, the purpose of which is to provide an acidic environment for the prepared O / W / O type double emulsion droplets. In the acidic environment, the acidic ions in the collecting phase solution O3 will diffuse from the oil phase to the water phase, that is, the crosslinked main body homogeneous liquid W1, so as to lower the pH of the chitosan - alginic acid mixed homogeneous solution, thereby promoting the crosslinking of chitosan and sodium alginate in the crosslinked main body homogeneous liquid W1 to form a hydrogel capsule wall, and then obtaining hydrogel microcapsules with controllable particle size and good monodispersity, that is, chitosan - sodium alginate microcapsules.

[0133] At the same time, by using the second oil phase solution O2 as the inner phase, effective encapsulation of fat-soluble carriers or essential oils and traditional Chinese medicine oils with medicinal value by themselves can be achieved.

[0134] It can be understood that in actual preparation, only by controlling the flow rates of the second oil phase solution O2, the crosslinked main body homogeneous liquid W1, and the first oil phase solution O1 in the O / W / O type microfluidic device according to different flow rate ratios, O / W / O type double emulsion droplets with different wall thicknesses and different numbers of oil droplet encapsulations can be obtained, and then monodisperse chitosan - alginic acid microcapsules with different wall thicknesses and single encapsulation or multi - encapsulation can be prepared.

[0135] Figure 16 Shows different flow rates Q of the crosslinked main body homogeneous liquid W1 at the middle intersection M And under the condition that the flow rates of other phases are fixed, the inverted microscope field - of - view diagrams of O / W / O type double emulsion droplets with different thicknesses (In oil) and hydrogel microparticles (In PBS) prepared.

[0136] ByFigure 16 It can be seen that the O / W / O double emulsion droplets appear as microparticles with a composite hydrogel capsule wall under the inverted microscope field of view. The inner oil phase structure is encapsulated inside, and the hydrogel microparticles dispersed in 1 mM PBS solution (pH = 5.4) after washing away the oil phase have regular morphology and good monodispersity.

[0137] Figure 17 Shows the different flow rates Q of the crosslinked main homogeneous liquid W1 in the middle M And under the condition that the flow rates of other phases are fixed, the particle size-flow rate relationship diagrams of the droplet size, inner oil phase particle size, and particle wall thickness of the prepared hydrogel microparticles with different thicknesses; among them, Figure 17 (a) is the droplet size-flow rate relationship diagram of hydrogel microparticles with different thicknesses, Figure 17 (b) is the inner oil phase particle size-flow rate relationship diagram of hydrogel microparticles with different thicknesses, Figure 17 (c) is the particle wall thickness-flow rate relationship diagram of hydrogel microparticles with different thicknesses, Figure 17 (d) is the microparticle size-flow rate relationship diagram of hydrogel microparticles with different thicknesses.

[0138] From Figure 17 (a), it can be seen that as the flow rate of the crosslinked main homogeneous liquid W1 in the O / W / O device increases from 400 μl / h to 800 μl / h, the droplet size of the prepared hydrogel microparticles gradually increases from 270 microns to 285 microns. At 400, 500, 600, 700, and 800 μl / h, their coefficient of variation (CV) values of monodispersity are 0.67%, 0.93%, 0.43%, 0.50%, and 0.62% respectively. From Figure 17 (b), it can be seen that as the flow rate of the crosslinked main homogeneous liquid W1 in the O / W / O device increases from 400 μl / h to 800 μl / h, the inner oil phase particle size of the prepared hydrogel microparticles gradually decreases from 185 microns to 165 microns; at 400, 500, 600, 700, and 800 μl / h, their coefficient of variation (CV) values of monodispersity are 0.55%, 1.2%, 1.1%, 0.69%, and 0.96% respectively. From Figure 17 (c), it can be seen that as the flow rate of the crosslinked main homogeneous liquid W1 in the O / W / O device increases from 400 μl / h to 800 μl / h, the wall thickness of the prepared hydrogel microparticles gradually increases from 45 microns to 60 microns; at 400, 500, 600, 700, and 800 μl / h, their coefficient of variation (CV) values of monodispersity are 2.4%, 4.0%, 1.2%, 1.5%, and 2.1% respectively. From Figure 17(d) It can be seen that as the flow rate of the crosslinked matrix homogeneous solution W1 in the O / W / O device increases from 400 μl / h to 800 μl / h, the particle size of the prepared hydrogel microparticles gradually increases from 270 microns to 285 microns; at 400, 500, 600, 700, and 800 μl / h, their coefficient of variation (CV) values of monodispersity are 2.3%, 0.94%, 1.0%, 2.0%, and 1.4% respectively.

[0139] Figure 18 Shows the confocal laser scanning microscopy images of the hydrogel microparticles prepared under different flow rates Q of the crosslinked matrix homogeneous solution W1 M under the excitation wavelength of 488 nm, bright field BF, and merged field MG.

[0140] From Figure 18 it can be seen that based on the green fluorescence of chitosan at the excitation wavelength of 488 nm, it is suggested that coaxial multiple shearing by sequentially introducing the oil phase - water phase - oil phase into the O / W / O double emulsion device can form composite microcapsules with a chitosan - alginic acid hydrogel outer shell wall and an inner core containing oil droplets.

[0141] Example 6

[0142] On the basis of Example 5, in order to explore the encapsulation ability of the hydrogel microparticles prepared in Example 5, that is, the chitosan - alginic acid microcapsules, in this Example 6, single - encapsulation, double - encapsulation, and triple - encapsulation matching were achieved by adjusting the flow rate ratio. Specifically, by adjusting the flow rate of the first oil phase solution O1 as the outer phase, chitosan - alginic acid microcapsules with different encapsulation amounts were successfully prepared.

[0143] Among them, the flow rates of the first oil phase solution O1 are 330, 600, and 1000 μl / h respectively.

[0144] Combined with Figure 19 and Figure 20 it can be seen that by regulating the flow rate Q of the first oil phase solution O1 introduced into the O / W / O microfluidic device, multi - chamber microcapsules can be flexibly prepared. For example, by regulating the device to simultaneously introduce multiple oils other than the second oil phase solution dimethyl silicone oil O2, multiple different lipophilic encapsulates, two traditional Chinese medicine oils and essential oils with medicinal value can be encapsulated into the same microcapsule, increasing the encapsulation efficiency of the microparticles. O

[0145] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of PH-triggered composite hydrogel microsphere microparticles, characterized in that, Including the following steps: Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate; Dissolve 10 wt% of PGPR in soybean oil to obtain a first oil phase solution O1; Add 0.5% v / v of acetic acid to the first oil phase solution O1 to obtain a collection phase solution O3; Use the crosslinked main body homogeneous solution W1 as the internal phase and the first oil phase solution O1 as the external phase, inject them into a W / O type microfluidic device, and control the flow rates of the crosslinked main body homogeneous solution W1 and the first oil phase solution O1 in the W / O type microfluidic device to obtain W / O type single emulsion droplets, and let the W / O type single emulsion droplets enter the collection phase solution O3; After the W / O type single emulsion droplets enter the collection phase solution O3, let it stand for a predetermined time; Subsequently, wash the mixed oil phase of the excess first oil phase solution O1 and the collection phase solution O3 with n-hexane; finally, wash away the n-hexane with 1 mM PBS solution to obtain hydrogel microparticles.

2. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 1, characterized in that, The flow rate of the crosslinked main body homogeneous solution W1 in the W / O type microfluidic device is 200 μl / h, and the flow rates of the first oil phase solution O1 in the W / O type microfluidic device are 400, 800, 1200, 1600, or 2000 μl / h.

3. A preparation method of a pH-triggered composite hydrogel core-shell microparticle, characterized in that, Including the following steps: Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate; Dissolve 10 wt% of PGPR in soybean oil to obtain a first oil phase solution O1; Dissolve a non-crosslinked polymer in ultrapure water to obtain an aqueous phase solution W2; Add 0.5% v / v of acetic acid to the first oil phase solution O1 to obtain a collection phase solution O3; Use the aqueous phase solution W2 as the internal phase, the crosslinked main body homogeneous solution W1 as the intermediate phase, and the first oil phase solution O1 as the external phase, inject them into a W / W / O type microfluidic device, and control the flow rates of the aqueous phase solution W2, the crosslinked main body homogeneous solution W1, and the first oil phase solution O1 in the W / W / O type microfluidic device to obtain W / W / O type double emulsion droplets, and let the W / W / O type double emulsion droplets enter the collection phase solution O3; After the W / W / O type double emulsion droplets enter the collection phase solution O3, let it stand for a predetermined time; Subsequently, wash the mixed oil phase of the excess first oil phase solution O1 and the collection phase solution O3 with n-hexane; finally, wash away the n-hexane with 1 mM PBS solution to obtain hydrogel microparticles.

4. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 3, characterized in that, The non-crosslinked polymer is 2 wt% of PEG6000, 2 wt% of Dextran, or 2 wt% of PVA.

5. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 3, characterized in that, The flow rate of the aqueous phase solution W2 in the W / W / O type microfluidic device is 100 μl / h, the flow rate of the crosslinked main body homogeneous solution W1 in the W / W / O type microfluidic device is 100 μl / h, and the flow rate of the first oil phase solution O1 in the W / W / O type microfluidic device is 2000 μl / h.

6. A preparation method of a pH-triggered composite hydrogel microcapsule-type microparticle, characterized in that, Including the following steps: Prepare a crosslinked main body homogeneous solution W1 using chitosan and sodium alginate; Dissolve 10 wt% of PGPR in soybean oil to obtain a first oil phase solution O1; Use dimethyl silicone oil PMX-200 with a viscosity of 10 mPa·s as the second oil phase solution O2; An additional 0.5% v / v acetic acid was added to the first oil phase solution O1 to serve as the collecting phase solution O3; The second oil phase solution O2 was used as the internal phase, the crosslinking matrix homogeneous solution W1 was used as the middle phase, and the first oil phase solution O1 was used as the external phase, which were injected into the O / W / O type microfluidic device. The flow rates of the second oil phase solution O2, the crosslinking matrix homogeneous solution W1, and the first oil phase solution O1 in the O / W / O type microfluidic device were controlled to obtain O / W / O type double emulsion droplets, and the O / W / O type double emulsion droplets were allowed to enter the collecting phase solution O3; After the O / W / O type double emulsion droplets entered the collecting phase solution O3, they were left standing for a predetermined time; Subsequently, the mixed oil phase of the excess first oil phase solution O1 and the collecting phase solution O3 was washed with n-hexane; finally, the n-hexane was washed off with 1 mM PBS solution to obtain hydrogel microparticles.

7. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 6, characterized in that The flow rate of the second oil phase solution O2 in the O / W / O type microfluidic device was 300 μl / h, the flow rates of the crosslinking matrix homogeneous solution W1 in the O / W / O type microfluidic device were 400, 500, 600, 700, 800 μl / h, and the flow rate of the first oil phase solution O1 in the O / W / O type microfluidic device was 300 - 1500 μl / h.

8. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 1, 3 or 6, characterized in that, Preparing the crosslinking matrix homogeneous solution W1 using chitosan and sodium alginate specifically includes: 2 wt% sodium alginate and 2 wt% chitosan were respectively dissolved in ultrapure water to obtain a sodium alginate solution and a chitosan solution; The pH value of the chitosan solution was adjusted to 7; 0.5% poloxamer F-127 was dissolved in the chitosan solution, and an appropriate amount of NaOH solution was added to adjust the pH value of the chitosan solution to 7.4 - 7.6; The sodium alginate solution was injected into the chitosan solution with a pH value of 7.4 - 7.6 and mixed to obtain the crosslinking matrix homogeneous solution W1.

9. The preparation method of the pH-triggered composite hydrogel microparticles according to claim 8, wherein, In the crosslinking matrix homogeneous solution W1, the volume ratio of sodium alginate to chitosan was: 2:

1.

10. The method for preparing a pH-triggered composite hydrogel microparticle according to claim 1, 3 or 6, characterized in that The pH value of the PBS solution was 3 - 9.

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