Method for preparing high-viscosity system monodisperse liquid drops through in-situ concentration

Abstract

The invention belongs to the field of preparation of monodisperse liquid drops of a high-viscosity system, and provides a method for preparing monodisperse liquid drops of a high-viscosity system through in-situ concentration, which comprises the following steps: (1) dissolving internal reagents including a high polymer material and a cross-linking agent in water to form an internal phase fluid, the viscosity of which does not exceed 100mpa. S; dissolving an oil-soluble surfactant in a concentrating agent to form an external phase fluid or a collection liquid; and (2) preparing the internal phase fluid and the external phase fluid into a monodisperse water-in-oil emulsion through a microfluidic device, collecting the water-in-oil emulsion by adopting a collecting solution, and carrying out in-situ concentration on the water-in-oil emulsion in the process of moving in the microfluidic device and keeping in the collecting solution to obtain the monodisperse liquid drops of a high-viscosity system. According to the method, the monodispersity of the high-viscosity system liquid drops prepared based on the microfluidic device can be effectively improved, and then the monodispersity and biocompatibility of the cross-linked polymer microspheres prepared by taking the obtained liquid drops as the template can be improved.

Description

Technical Field

[0001] The invention belongs to the field of preparation of monodisperse droplets of high-viscosity systems, and relates to a method for preparing monodisperse droplets of high-viscosity systems by in-situ concentration. Background Art

[0002] Droplet microfluidics is a technology that controls the interfacial tension, flow rate and other parameters between two or more liquids to prepare droplets with highly uniform chemical composition and size. The droplets prepared by microfluidics can be solidified to obtain microspheres. For microspheres used for embolization, tissue filling, drug carriers, cell delivery, biological detection, etc., the size uniformity of the microspheres will directly affect the effects of embolism, tissue filling, drug loading or delivery, and biological detection. The size uniformity of microspheres mainly depends on the uniformity of the droplets used as templates, but not all liquids are suitable for preparation using droplet microfluidics and obtaining droplets with highly uniform size. For example, when a high-viscosity liquid is used as the internal phase fluid, it often causes problems such as uneven size of the droplets prepared by microfluidics and the appearance of satellite droplets, which greatly limits the application of microfluidics in the preparation of monodisperse droplets of high-viscosity systems. However, when preparing microspheres for in vivo applications such as embolization, tissue filling, drug delivery, and cell delivery, ensuring that the degradation and biocompatibility of the microspheres meet application requirements requires the use of high-molecular-weight polymers cross-linked at high polymer concentrations. However, the high molecular weight and high concentration of the polymers result in high viscosity of the internal phase, making it difficult to produce monodisperse droplets using existing microfluidic technologies. Therefore, how to prepare monodisperse droplets of high-viscosity systems using microfluidic technology is a major challenge facing this field.

[0003] Taking the preparation of cross-linked hyaluronic acid (HA) microspheres for human tissue filling as an example, the higher the molecular weight of HA, the stronger the anti-degradation ability of the cross-linked HA microspheres, and the longer they can last in the human body. Currently, most cross-linked HA microspheres on the market use 1,4-butanediol glycidyl ether (BDDE) as a cross-linker. BDDE is added to a solution of HA and NaOH to form an HA reaction solution, which is obtained by direct stirring or emulsion cross-linking reaction. This preparation process involves two reactions: the cross-linking reaction between HA and BDDE, and the degradation reaction of HA under alkaline conditions. In the preparation of cross-linked HA microspheres, the preparation of cross-linked HA microspheres with performance that meets the application requirements can only be achieved when at least one of HA and BDDE is at a high concentration. Under conditions of low HA concentration and low BDDE concentration, the HA reaction solution cannot undergo a cross-linking reaction to form a solid, and the viscosity of the reaction solution will even decrease further. Under the conditions of low HA concentration and high BDDE concentration, cross-linked HA microspheres can be successfully prepared, but the BDDE modification degree of the obtained cross-linked HA microspheres is too high, which can easily cause biocompatibility after injection into the body, leading to side effects such as delayed swelling. Under the conditions of high HA concentration and low BDDE concentration, the BDDE modification degree of the prepared HA microspheres is low and the biocompatibility is good. Most cross-linked HA microsphere products on the market are prepared under this condition. However, the higher the concentration of high molecular weight HA (for example, molecular weight above 1MDa) in the solution, the more viscous the HA reaction solution. Although the high viscosity HA reaction solution can be prepared into cross-linked HA microspheres by stirring, emulsifying and then cross-linking, the size uniformity of the cross-linked HA microspheres prepared in this way is poor. In general, although droplets of uniform size can be prepared by droplet microfluidics, when the viscosity of the internal phase fluid is too high, droplet microfluidics cannot prepare droplets of uniform size and monodisperse, but faces the problem of uneven droplet size and the appearance of satellite droplets. Many other cross-linking systems for polymers face similar challenges to those of HA. Addressing the difficulty of preparing monodisperse droplets of high-viscosity systems within microfluidic devices and improving the monodispersity and biocompatibility of cross-linked polymer microspheres using these droplets as templates remains a pressing technical challenge in this field. Summary of the Invention

[0004] In response to the current problem that it is difficult to prepare high-viscosity droplets of uniform size in droplet microfluidic devices, the present invention provides a method for preparing monodisperse droplets of high-viscosity systems by in situ concentration, so as to effectively improve the monodispersity of high-viscosity system droplets prepared based on microfluidic devices, and thereby improve the monodispersity and biocompatibility of cross-linked polymer microspheres prepared using the obtained droplets as templates.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0006] A method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, comprising the following steps:

[0007] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0008] Prepare the internal phase fluid: dissolve the polymer material and the crosslinking agent in water to form a crosslinking reaction precursor solution, and use the crosslinking reaction precursor solution as the internal phase fluid; the viscosity of the internal phase fluid does not exceed 100 mPa·s, and the concentration of the crosslinking agent in the internal phase fluid is 0.001 wt% to 10 wt%;

[0009] preparing an external phase fluid: dissolving an oil-soluble surfactant in a concentrate to obtain an external phase fluid; the concentration of the oil-soluble surfactant in the external phase fluid is 0.1 wt% to 5 wt%;

[0010] Preparation of a collection solution: dissolving an oil-soluble surfactant in a concentrator to obtain a collection solution; the concentration of the oil-soluble surfactant in the collection solution is 0.1 wt% to 5 wt%;

[0011] The concentrator is an organic solvent that is miscible with water but does not dissolve the polymer material in the internal phase fluid;

[0012] (2) In situ concentration to prepare monodisperse droplets of high viscosity systems

[0013] The inner phase fluid and the outer phase fluid are respectively injected into the injection tube and the collecting tube of the primary microfluidic device to form a monodisperse oil-in-water emulsion in the collecting tube. The oil-in-water emulsion is collected under stirring using a container containing the collecting liquid. During the movement of the oil-in-water emulsion in the channel of the primary microfluidic device and the retention in the collecting liquid, water in the oil-in-water emulsion droplets diffuses into the outer phase fluid and the collecting liquid, causing the components in the oil-in-water emulsion droplets to be concentrated. When the concentration of water in the oil-in-water emulsion droplets and the collecting liquid reaches equilibrium, monodisperse droplets of a high viscosity system are obtained.

[0014] In the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the concentrator is preferably one or more of ethyl acetate, isobutanol, ethanol, acetone, and methyl isobutyl ketone. In practical applications, the type of concentrator is mainly selected according to the specific polymer material used.

[0015] In step (2) of the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the volume of the collecting liquid used is at least 10 times the volume of the internal phase fluid used. For example, the volume of the collecting liquid used can be 10 to 100 times the volume of the internal phase fluid used. By adjusting the ratio of the volume of the collecting liquid used to the volume of the internal phase fluid used, the concentration degree of each component in the water-in-oil emulsion droplets can be adjusted.

[0016] In the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the polymer material includes at least one of hyaluronic acid, sodium hyaluronate, agarose, gelatin, chitosan, polyvinyl alcohol, and sodium alginate.

[0017] In the technical solution of the above-mentioned method for in-situ concentration to prepare monodisperse droplets of a high-viscosity system, the concentration of the polymer material in the internal phase fluid is determined according to the type and molecular weight of the polymer material, with the viscosity of the internal phase fluid not exceeding 100 mPa·s (for example, between 0.1 and 100 mPa·s) as a principle. Generally speaking, for the different polymer materials listed above, the concentration of the polymer material in the internal phase fluid can be selected in the range of 0.5 wt% to 15 wt%.

[0018] In the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the crosslinking agent includes any one of 1,4-butanediol glycidyl ether, divinyl sulfone, 1-bromo-3,4-epoxybutane, 1-chloro-2,3-epoxypropane, 1-bromo-2,3-epoxypropane, 2,5-dibromopentanol, 2,4-dibromobutanethiol, 2,5-dibromopentane-thiol epichlorohydrin, dimethylaminopropylcarbodiimide, formaldehyde, and glutaraldehyde. In practical applications, the type of crosslinking agent is mainly selected based on the specific polymer material used.

[0019] In the technical solution of the above-mentioned method for in-situ concentration preparation of monodisperse droplets of a high-viscosity system, the cross-linking reaction precursor in step (1) contains a reagent that enables the polymer material and the cross-linking agent to undergo a cross-linking reaction under appropriate conditions. After the monodisperse droplets of the high-viscosity system are prepared in step (2), the cross-linking reaction between the polymer material and the cross-linking agent can be initiated by applying appropriate conditions.

[0020] In the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the crosslinking reaction precursor solution further contains at least one of an acid-base modifier, a catalyst, and an initiator. Whether to use an acid-base modifier, a catalyst, or an initiator, and the type of acid-base modifier, catalyst, and initiator to use, depends primarily on the selected polymer material and crosslinker. In practical applications, the selection can be based on the specific polymer material and crosslinker used. For example, when the polymer material is hyaluronic acid and the cross-linking agent is 1,4-butanediol glycidyl ether, the internal phase fluid should also contain an acid-base regulator (such as sodium hydroxide or hydrochloric acid). Specifically, when the prepared high-viscosity droplets need to be used to prepare cross-linked hyaluronic acid microspheres, a feasible method for preparing the internal phase fluid is: dissolve the polymer material hyaluronic acid, the cross-linking agent 1,4-butanediol glycidyl ether and the acid-base regulator sodium hydroxide in water to obtain an internal phase fluid; in the internal phase fluid, the concentration of the polymer material is 0.5wt% to 5wt%, the concentration of the cross-linking agent does not exceed 1wt%, and the concentration of the acid-base regulator is 0.1wt% to 3wt%.

[0021] In the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the oil-soluble surfactant prevents the droplets of the water-in-oil emulsion from merging and maintains the stability of the water-in-oil emulsion. Suitable oil-soluble surfactants include any one of Span 20, Span 60, Span 80, glyceryl monostearate, polyglyceryl ricinoleate, and cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethicone.

[0022] In step (2) of the technical solution of the above-mentioned method for in situ concentration to prepare monodisperse droplets of a high-viscosity system, the external phase fluid flow rate and the internal phase fluid flow rate conditions adopted are based on the principle that the external phase fluid can stably shear the internal phase fluid into droplets. In practical applications, they can be determined based on the size of the oil-in-water emulsion droplets, the channel size of the primary microfluidic device adopted, etc.

[0023] In step (2) of the technical solution of the above-mentioned method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration, the primary microfluidic device used is an existing conventional primary microfluidic device. A feasible primary microfluidic device includes an injection tube and a collection tube, which are used in conjunction with a syringe pump. The outlet end of the injection tube is inserted into the inlet end of the collection tube. The injection tube and the collection tube are coaxially arranged and fixed to a glass slide by resin glue. A flat needle can be fixedly connected to the inlet end of the injection tube and the inlet end of the collection tube by resin glue to facilitate connection to the syringe pump. In the primary microfluidic device used, the inner diameter of the injection tube is usually 10 to 1000 μm, and the inner diameter of the collection tube is usually 50 to 2000 μm.

[0024] The present invention also provides monodisperse droplets of a high viscosity system prepared by the above-mentioned in-situ concentration method for preparing monodisperse droplets of a high viscosity system, wherein the coefficient of variation of the diameter of the droplets does not exceed 10%, and preferably, the coefficient of variation of the diameter of the droplets does not exceed 5%.

[0025] The present invention also provides a method for preparing monodisperse cross-linked polymer microspheres with excellent biocompatibility. The operation of the method is: applying conditions so that the polymer material in the monodisperse droplets of the high viscosity system prepared by the above-mentioned method of in situ concentration to prepare monodisperse droplets of the high viscosity system undergoes a cross-linking reaction with the cross-linking agent, and the monodisperse droplets are solidified, and then washed to remove the unreacted components to obtain.

[0026] The present invention also provides the monodisperse cross-linked polymer microspheres prepared by the aforementioned method for preparing monodisperse cross-linked polymer microspheres with excellent biocompatibility. The cross-linked polymer microspheres have uniform size and excellent biocompatibility. The coefficient of variation of the diameter of the cross-linked polymer microspheres does not exceed 10%. Furthermore, the coefficient of variation of the diameter of the cross-linked polymer microspheres does not exceed 5%.

[0027] The technical principles of the present invention are mainly as follows:

[0028] In order to solve the problem that a high viscosity solution cannot be prepared into a monodisperse droplet by using a microfluidic device when a high viscosity solution is used as the internal phase fluid, the present invention first prepares a low viscosity cross-linking reaction precursor solution with water as a solvent, uses the low viscosity cross-linking reaction precursor solution as the internal phase fluid, and uses a concentrator as a solvent to prepare an external phase fluid and a collection liquid, and then uses a microfluidic device to prepare a monodisperse oil-in-water emulsion. Since the water in the internal phase fluid is miscible with the concentrator and the polymer material is insoluble in the concentrator, when the oil During the movement of the water-in-oil emulsion in the channel of the droplet microfluidic device and the retention in the collecting liquid, the water in the oil-in-water emulsion droplets will diffuse into the external phase fluid and the collecting liquid, causing the components in the oil-in-water emulsion droplets to concentrate. When the concentration of water in the oil-in-water emulsion droplets and the collecting liquid reaches equilibrium, the concentration of the components in the oil-in-water emulsion droplets (such as polymer materials and crosslinking agents) will be significantly increased. At the same time, the concentration process will not significantly affect the monodispersity of the oil-in-water emulsion droplets, thereby obtaining monodisperse droplets of a high viscosity system. Since the concentration of the polymer material and crosslinking agent in the monodisperse droplets of the high viscosity system has been effectively increased, the polymer material that originally could not undergo crosslinking reaction under low concentration conditions can be smoothly crosslinked with the crosslinking agent, thereby preparing crosslinked polymer microspheres under the condition of low crosslinking agent dosage, avoiding excessive crosslinking agent dosage resulting in excessive crosslinking agent modification of the crosslinked polymer microspheres, which easily causes the problem of bioincompatibility of the crosslinked polymer microspheres. Since the droplets of the high viscosity system are monodisperse, the cross-linked polymer microspheres obtained by cross-linking and curing using it as a template also have good monodispersity, which can solve the problem that the stirring emulsification cross-linking method cannot produce monodisperse cross-linked polymer microspheres of uniform size.

[0029] Compared with the prior art, the technical solution of the present invention produces the following beneficial technical effects:

[0030] 1. The present invention provides a method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration. First, reagents including a polymer material and a crosslinking agent are prepared into a low-viscosity crosslinking reaction precursor using water as a solvent. The low-viscosity crosslinking reaction precursor is used as the internal phase fluid, and the external phase fluid and the collecting liquid are prepared using a concentrator as a solvent. Then, a monodisperse oil-in-water emulsion is prepared using a microfluidic device. Because the water in the internal phase fluid is miscible with the concentrator and the polymer material is insoluble in the concentrator, when the oil-in-water emulsion moves in the channel of the droplet microfluidic device and is maintained in the collecting liquid, the water in the oil-in-water emulsion will diffuse into the external phase fluid and the collecting liquid, causing the various components in the oil-in-water emulsion droplets to be concentrated. This concentration process will not significantly affect the monodispersity of the oil-in-water emulsion droplets. When the concentration of water in the oil-in-water emulsion droplets and the collecting liquid reaches equilibrium, monodisperse droplets of the high-viscosity system can be obtained. The present invention first uses a low-viscosity internal phase fluid to prepare a monodisperse oil-in-water emulsion by droplet microfluidics technology, and then concentrates the oil-in-water emulsion to prepare monodisperse droplets of a high-viscosity system, effectively solving the problem that the microfluidic device cannot prepare droplets of uniform size when a high-viscosity solution is used as the internal phase fluid.

[0031] 2. Based on the method of preparing monodisperse droplets of a high-viscosity system by in-situ concentration as described in the present invention, the present invention also provides a method for preparing monodisperse cross-linked polymer microspheres with excellent biocompatibility. After the monodisperse droplets of the high-viscosity system are prepared, conditions are applied to cause the polymer material therein to undergo a cross-linking reaction with a cross-linking agent to solidify, and the unreacted components are removed by washing. Because the concentrations of the polymer material and the cross-linking agent in the monodisperse droplets of the high-viscosity system are effectively increased, the polymer material that originally could not undergo a cross-linking reaction under low concentration conditions can be smoothly cross-linked with the cross-linking agent, thereby preparing cross-linked polymer microspheres under conditions of low cross-linking agent dosage, avoiding excessive cross-linking agent dosage that leads to excessive cross-linking agent modification of the cross-linked polymer microspheres, which easily causes the problem of bioincompatibility of the cross-linked polymer microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the preparation process of high-viscosity HA monodisperse droplets and monodisperse cross-linked HA microspheres in Example 1.

[0033] Figure 2 yes Figure 2 (A) shows an optical micrograph of the W / O emulsion droplets in Example 1 moving to the outlet of the outlet hose. Figure 2 Figure (B) is an optical micrograph of high viscosity HA monodisperse droplets prepared in Example 1. Figure 2 Figure (C) is an optical micrograph of the cross-linked HA microspheres prepared in Example 1 when they reached swelling equilibrium in PBS buffer.

[0034] Figure 3 This is an optical micrograph of the W / O emulsion prepared in Comparative Example 1.

[0035] Figure 4 Figure (A) is an optical micrograph of the W / O emulsion prepared in Comparative Example 1. Figure 4 Figure (B) is an optical micrograph of the W / O emulsion droplets prepared in the comparative example after cross-linking and washing.

[0036] Figure 5 This is an optical micrograph of the cross-linked HA microspheres prepared in Comparative Example 3. DETAILED DESCRIPTION

[0037] The following examples further illustrate the method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration provided by the present invention. It should be noted that the following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Based on the above disclosure, non-essential improvements and adjustments made by persons skilled in the art to the present invention and to further implement the present invention remain within the scope of the present invention.

[0038] In the following examples and comparative examples, the microfluidic device used is a primary microfluidic device, including an injection tube, a collecting tube and a lead-out hose. The outlet end of the injection tube is inserted into the inlet end of the collecting tube, and the outlet end of the collecting tube is connected to the lead-out hose. The injection tube and the collecting tube are coaxially arranged and fixed on a glass slide by resin glue. The injection tube is a glass tube or steel tube with a circular cross-section, and the inner diameter of the injection tube is 10 to 1000 μm and the outer diameter is 50 to 2000 μm. The collecting tube is a glass tube or steel tube with a square or circular cross-section, and the inner diameter of the collecting tube is 50 to 2000 μm and the outer diameter is 100 to 3000 μm. The cross-section of the lead-out hose is circular, and the material can be polytetrafluoroethylene (PTFE), polyethylene (PE) or silicone, etc. The inner diameter of the lead-out hose is 100 to 3000 μm and the outer diameter is 200 to 5000 μm. A flat-ended needle can be attached to the inlet of the injection tube using resin glue, and a flat-ended needle can be fixedly attached to the inlet of the collection tube using resin glue to facilitate connection to the injection pump. Each flat-ended needle can be connected to the injection pump via a tubing. The structure of the primary microfluidic device can be found in Hao Tan and Sooyoung Park, Poly(acrylic acid) hydrogel microspheres for a metal-ion sensor, ACS Sensors, 2021, 6, 3, 1039–1048.

[0039] Example 1

[0040] In this embodiment, a method for preparing monodisperse droplets of a high viscosity system by in situ concentration is described in detail. Specifically, the preparation of monodisperse droplets of high viscosity hyaluronic acid (HA) and monodisperse cross-linked HA microspheres is described as an example. The schematic diagram of the preparation process is shown in FIG. Figure 1 As shown, the steps are as follows:

[0041] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0042] Preparation of an internal phase fluid: HA with a molecular weight of 1 to 2 MDa, a cross-linking agent 1,4-butanediol glycidyl ether (BDDE), and sodium hydroxide were added to high-purity water and stirred until the components were completely dissolved to obtain an internal phase fluid; the concentration of HA in the internal phase fluid was 4 wt%, the concentration of BDDE was 0.4 wt%, and the concentration of NaOH was 1 wt%, and the viscosity of the internal phase fluid was 56 mPa·s.

[0043] The external phase fluid was prepared by dissolving the surfactant cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethylsiloxane (Abil EM90) in ethyl acetate (EA) to obtain the external phase fluid; the concentration of Abil EM90 in the external phase fluid was 3 wt %.

[0044] Preparation of collecting solution: dissolving surfactant Abil EM90 in EA to obtain collecting solution; the concentration of Abil EM90 in the collecting solution is 3 wt%.

[0045] (2) In situ concentration to prepare high-viscosity HA monodisperse droplets

[0046] The inner phase fluid is injected into the injection tube of the first-level microfluidic device through a syringe pump, and the outer phase fluid is injected into the collection tube of the first-level microfluidic device. A monodisperse water-in-oil (W / O) emulsion is formed at the outlet of the syringe in the collection tube. The monodisperse W / O emulsion is collected under stirring conditions using a container containing the collection liquid. After receiving, the emulsion is maintained in the collection liquid for a period of time and stirring is continuously applied during the process. During the movement of the W / O emulsion droplets in the collection tube and the outlet hose and the maintenance of the collection liquid, since the solubility of water in EA is 3% and HA is insoluble in EA, the water in the W / O emulsion droplets will continuously diffuse into the outer phase fluid and the collection liquid, causing the various components in the W / O emulsion droplets to concentrate until the concentration of water in the W / O emulsion droplets and the collection liquid reaches equilibrium, thereby obtaining high-viscosity HA monodisperse droplets.

[0047] In this step, the outer diameter of the outlet end of the injection tube of the primary microfluidic device used is 400 μm, the inner diameter of the collecting tube is 650 μm, the inner diameter of the outlet hose is 1000 μm, the flow rate of the internal phase fluid is controlled to be 200 μL / h, and the flow rate of the external phase fluid is 200 μL / min; the volume of the collecting liquid is controlled to be 100 times the volume of the internal phase fluid. After the receiving is completed, it is kept in the collecting liquid for 30 minutes until the concentration of water in the W / O emulsion droplets and the collecting liquid reaches equilibrium, and the stirring is stopped. After the high-viscosity HA monodisperse droplets settle to the bottom of the collecting container, the receiving liquid in the container is poured out to separate the high-viscosity HA monodisperse droplets.

[0048] (3) Preparation of monodisperse cross-linked HA microspheres

[0049] The container containing the high-viscosity HA monodisperse droplets was placed in an oven, and the monodisperse droplets were cross-linked and solidified into microspheres at 37°C. The cross-linking time was controlled to be 15 hours. The obtained microspheres were washed 3 times with ethanol and then washed 3 times with PBS buffer to obtain cross-linked HA microspheres, which were stored in PBS buffer.

[0050] In step (2) of this embodiment, when the W / O emulsion droplets move to the outlet of the outlet hose, the diameter of the W / O emulsion droplets is about 380 μm and the size is uniform. The optical micrograph thereof is as follows: Figure 2 As shown in Figure (A), when the W / O emulsion droplets are collected and kept in the collection liquid for 30 minutes, the diameter of the obtained high-viscosity HA monodisperse droplets is about 180 μm. The optical micrograph is shown in Figure 2 As shown in Figure (B), at this time, the concentration of HA in the high-viscosity HA monodisperse droplets is above 40%. The coefficient of variation (CV value) of the diameter of the high-viscosity HA monodisperse droplets is calculated based on the optical micrograph, and the result is 2.5%. After the cross-linked HA microspheres prepared in step (3) of this embodiment are dispersed in PBS buffer, the cross-linked HA microspheres can be stably present. The diameter of the cross-linked HA microspheres when they reach swelling equilibrium in PBS buffer is about 410 μm, and the optical micrograph thereof is shown in FIG. Figure 2 As shown in FIG. 5(C) , the CV value of the diameter of the cross-linked HA microspheres was calculated based on the optical microscopic image, and the result was 3.6%.

[0051] Comparative Example 1

[0052] This comparative example is used for comparison with Example 1 to examine the effect of the viscosity of the internal phase fluid on the microfluidic preparation of high-viscosity droplets.

[0053] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0054] Preparation of an internal phase fluid: HA with a molecular weight of 1 to 2 MDa, a crosslinker BDDE, and sodium hydroxide were added to high-purity water and stirred until the components were completely dissolved to obtain an internal phase fluid; the internal phase fluid had a concentration of 12 wt% HA, 1 wt% BDDE, and 1 wt% NaOH; and a viscosity of 571 mPa·s.

[0055] The external phase fluid was prepared by dissolving the surfactant Abil EM90 in liquid paraffin to obtain the external phase fluid; the concentration of Abil EM90 in the external phase fluid was 3 wt %.

[0056] Preparation of a collecting solution: dissolving the surfactant Abil EM90 in liquid paraffin to obtain a collecting solution; the concentration of Abil EM90 in the collecting solution is 3 wt %.

[0057] (2) Preparation of W / O emulsion

[0058] The inner phase fluid is injected into the injection tube of the primary microfluidic device through a syringe pump, and the outer phase fluid is injected into the collection tube of the primary microfluidic device. A W / O emulsion is formed at the injection tube outlet in the collection tube, and the W / O emulsion is collected in a container containing the collection liquid.

[0059] The optical micrograph of the W / O emulsion prepared in this comparative example is as follows: Figure 3 As shown by Figure 3 As can be seen, the W / O emulsion prepared in this comparative example exhibits extremely uneven droplet sizes, with numerous satellite droplets present. The larger droplets have diameters of approximately 240 μm, while the smaller droplets have diameters of less than 10 μm. This suggests that droplet microfluidics cannot shear the high-viscosity internal fluid into uniformly sized droplets.

[0060] Comparative Example 2

[0061] This comparative example is used for comparison with Example 1 to examine the effect of using a concentrator to concentrate the W / O emulsion droplets on the preparation of cross-linked HA microspheres.

[0062] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0063] Preparation of an internal phase fluid: HA with a molecular weight of 1 to 2 MDa, a crosslinker BDDE, and sodium hydroxide were added to high-purity water and stirred until the components were completely dissolved to obtain an internal phase fluid; the concentration of HA in the internal phase fluid was 4 wt%, the concentration of BDDE was 1.2 wt%, and the concentration of NaOH was 1 wt%, and the viscosity of the internal phase fluid was 57 mPa·s.

[0064] The external phase fluid was prepared by dissolving the surfactant Abil EM90 in liquid paraffin to obtain the external phase fluid; the concentration of Abil EM90 in the external phase fluid was 3 wt %.

[0065] Preparation of a collecting solution: dissolving the surfactant Abil EM90 in liquid paraffin to obtain a collecting solution; the concentration of Abil EM90 in the collecting solution is 3 wt %.

[0066] (2) Preparation of W / O emulsion

[0067] The inner phase fluid is injected into the injection tube of the primary microfluidic device through a syringe pump, and the outer phase fluid is injected into the collection tube of the primary microfluidic device. A W / O emulsion is formed at the injection tube outlet in the collection tube, and the W / O emulsion is collected in a container containing the collection liquid.

[0068] (3) Preparation of cross-linked HA microspheres

[0069] The container containing the W / O emulsion was placed in an oven and cross-linked at 37°C for 15 h. The cross-linked product was washed three times with ethanol and then three times with PBS buffer. Finally, the cross-linked product was stored in PBS buffer.

[0070] The optical micrograph of the W / O emulsion prepared in this comparative example is as follows: Figure 4 As shown in Figure (A), Figure 4 As shown in Figure (A), this comparative example successfully prepared a W / O emulsion with uniform size by microfluidic technology. However, after cross-linking and washing in step (3), no microspheres were observed under an optical microscope, as shown in Figure (A). Figure 4 As shown in Figure (B), this shows that in this comparative example, even if the concentration of BDDE in the internal phase flow is as high as 3 times the concentration of BDDE in the internal phase fluid in Example 1, HA still fails to crosslink.

[0071] Comparative Example 3

[0072] This comparative example is used for comparison with Example 1 to examine the effect of using a concentrator to concentrate the W / O emulsion droplets on the preparation of cross-linked HA microspheres.

[0073] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0074] Preparation of an internal phase fluid: HA with a molecular weight of 1 to 2 MDa, a crosslinker BDDE, and sodium hydroxide were added to high-purity water and stirred until the components were completely dissolved to obtain an internal phase fluid; the concentration of HA in the internal phase fluid was 4 wt%, the concentration of BDDE was 8 wt%, and the concentration of NaOH was 1 wt%, and the viscosity of the internal phase fluid was 55 mPa·s.

[0075] The external phase fluid was prepared by dissolving the surfactant Abil EM90 in liquid paraffin to obtain the external phase fluid; the concentration of Abil EM90 in the external phase fluid was 3 wt %.

[0076] Preparation of a collecting solution: dissolving the surfactant Abil EM90 in liquid paraffin to obtain a collecting solution; the concentration of Abil EM90 in the collecting solution is 3 wt %.

[0077] (2) Preparation of W / O emulsion

[0078] The inner phase fluid is injected into the injection tube of the primary microfluidic device through a syringe pump, and the outer phase fluid is injected into the collection tube of the primary microfluidic device. A W / O emulsion is formed at the injection tube outlet in the collection tube, and the W / O emulsion is collected in a container containing the collection liquid.

[0079] (3) Preparation of cross-linked HA microspheres

[0080] The container containing the W / O emulsion was placed in an oven and cross-linked at 37°C for 15 h. The cross-linked product was washed three times with ethanol and then three times with PBS buffer. Finally, the cross-linked product was stored in PBS buffer.

[0081] The W / O emulsion prepared in this comparative example has uniform size, and its optical micrograph is as follows Figure 5 As shown in Figure (A), after the cross-linked HA microspheres prepared in this comparative example were dispersed in PBS buffer, the optical microscopic picture thereof was as follows Figure 5 As shown in Figure (B), Figure 5 As shown in Figure (B), in this comparative example, when the concentration of BDDE in the internal phase flow increased to 20 times that of the internal phase fluid in Example 1, although the cross-linking of HA and BDDE was achieved, the edges of the obtained cross-linked HA microspheres were not clear, which indicates that the cross-linking effect of HA was not good and could only maintain the gel morphology.

[0082] Example 2

[0083] In this example, a method for preparing monodisperse droplets of a high viscosity system by in situ concentration is described in detail. Specifically, the preparation of high viscosity monodisperse gelatin droplets and monodisperse cross-linked gelatin microspheres is described as an example. The steps are as follows:

[0084] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0085] Preparation of an internal phase fluid: Gelatin with a molecular weight of 0.1 to 1 MDa and the cross-linking agent glutaraldehyde were added to high-purity water and stirred in a 50°C water bath until all components were completely dissolved to obtain an internal phase fluid; the concentration of gelatin in the internal phase fluid was 15 wt%, the concentration of glutaraldehyde was 0.01 wt%, and the viscosity of the internal phase fluid was 61 MPa·s.

[0086] Preparation of external phase fluid: Dissolve surfactant Abil EM90 in EA to obtain external phase fluid; the concentration of Abil EM90 in the external phase fluid is 5wt%.

[0087] Preparation of collecting solution: dissolving surfactant Abil EM90 in EA to obtain collecting solution; the concentration of Abil EM90 in the collecting solution is 5 wt%.

[0088] (2) In situ concentration to prepare high-viscosity gelatin monodisperse droplets

[0089] An inner phase fluid is injected into an injection tube of a primary microfluidic device through a syringe pump, and an outer phase fluid is injected into a collecting tube of the primary microfluidic device, forming a monodisperse W / O emulsion at an outlet of the injection tube in the collecting tube. The monodisperse W / O emulsion is collected under stirring using a container containing a collecting liquid. After receiving, the monodisperse W / O emulsion is maintained in the collecting liquid for a period of time and continuously stirred during the process. As the W / O emulsion droplets move in the collecting tube and the outlet hose and are maintained in the collecting liquid, water in the W / O emulsion droplets continuously diffuses into the outer phase fluid and the collecting liquid, causing the components in the W / O emulsion droplets to concentrate until the concentration of water in the W / O emulsion droplets and the collecting liquid reaches equilibrium, thereby obtaining high-viscosity monodisperse gelatin droplets.

[0090] In this step, the primary microfluidic device used is the same as that in Example 1, and the flow rate of the internal phase fluid is controlled to be 200 μL / h, and the flow rate of the external phase fluid is 200 μL / min; the volume of the collecting liquid is controlled to be 50 times the volume of the internal phase fluid. After the receiving is completed, it is kept in the collecting liquid for 30 minutes until the concentration of water in the W / O emulsion droplets and the collecting liquid reaches equilibrium. The stirring is stopped, and after the high-viscosity gelatin monodisperse droplets settle to the bottom of the collecting container, the receiving liquid in the container is poured out to separate the high-viscosity gelatin monodisperse droplets.

[0091] (3) Preparation of monodisperse cross-linked gelatin microspheres

[0092] The container containing high-viscosity gelatin monodisperse droplets was placed in an environment of 4°C to allow the monodisperse droplets to crosslink and solidify into microspheres. The crosslinking time was controlled to be 10 h. The obtained microspheres were washed 3 times with ethanol and then washed 3 times with PBS buffer to obtain monodisperse cross-linked gelatin microspheres, which were stored in PBS buffer.

[0093] The high-viscosity gelatin monodisperse droplets and monodisperse cross-linked gelatin microspheres prepared in this example were observed using an optical microscope, and the sizes were measured and the CV values of the diameters were calculated. The results showed that the CV value of the diameter of the high-viscosity gelatin monodisperse droplets was 2.4%, and the CV value of the diameter of the monodisperse cross-linked gelatin microspheres was 3.3%.

[0094] Example 3

[0095] In this example, a method for preparing monodisperse droplets of a high-viscosity system by in-situ concentration is described in detail. Specifically, the preparation of high-viscosity polyvinyl alcohol (PVA) monodisperse droplets and monodisperse cross-linked PVA microspheres is described as an example. The steps are as follows:

[0096] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0097] Preparation of an internal phase fluid: PVA with a molecular weight of 0.1 to 2 MDa and a cross-linking agent, glutaraldehyde, were added to high-purity water and stirred until all components were completely dissolved to obtain an internal phase fluid; the concentration of PVA in the internal phase fluid was 0.5 wt%, the concentration of glutaraldehyde was 1 wt%, and the viscosity of the internal phase fluid was 36 mPa·s.

[0098] The external phase fluid was prepared by dissolving the surfactant Span 80 in EA to obtain the external phase fluid; the concentration of Span 80 in the external phase fluid was 1 wt%.

[0099] Preparation of a collecting solution: dissolving the surfactant Span 80 in EA to obtain a collecting solution; the concentration of Span 80 in the collecting solution is 1 wt %.

[0100] (2) In situ concentration to prepare high-viscosity gelatin monodisperse droplets

[0101] The inner phase fluid is injected into the injection tube of the first-level microfluidic device through a syringe pump, and the outer phase fluid is injected into the collecting tube of the first-level microfluidic device. A monodisperse W / O emulsion is formed at the outlet of the injection tube in the collecting tube. The monodisperse W / O emulsion is collected under stirring conditions using a container containing the collecting liquid. After receiving, the monodisperse W / O emulsion is maintained in the collecting liquid for a period of time and stirring is continuously applied during the process. As the W / O emulsion droplets move in the collecting tube and the outlet hose and are maintained in the collecting liquid, water in the W / O emulsion droplets will continuously diffuse into the outer phase fluid and the collecting liquid, causing the various components in the W / O emulsion droplets to concentrate until the water concentration in the W / O emulsion droplets and the collecting liquid reaches equilibrium, thereby obtaining high-viscosity PVA monodisperse droplets.

[0102] In this step, the primary microfluidic device used is the same as that in Example 1, and the flow rate of the internal phase fluid is controlled to be 200 μL / h, and the flow rate of the external phase fluid is 200 μL / min; the volume of the collecting liquid is controlled to be 80 times the volume of the internal phase fluid. After receiving, it is kept in the collecting liquid for 30 minutes until the concentration of water in the W / O emulsion droplets and the collecting liquid reaches equilibrium. Stirring is stopped, and after the high-viscosity PVA monodisperse droplets settle to the bottom of the collecting container, the receiving liquid in the container is poured out to separate the high-viscosity PVA monodisperse droplets.

[0103] (3) Preparation of monodisperse cross-linked PVA microspheres

[0104] The container containing the high-viscosity PVA monodisperse droplets was placed in an environment of 70°C to allow the monodisperse droplets to crosslink and solidify into microspheres. The crosslinking time was controlled to be 6 hours. The obtained microspheres were washed 3 times with ethanol and then washed 3 times with PBS buffer to obtain monodisperse cross-linked PVA microspheres, which were stored in PBS buffer.

[0105] The high-viscosity PVA monodisperse droplets and monodisperse cross-linked PVA microspheres prepared in this example were observed using an optical microscope, and their sizes were measured and the CV values of their diameters were calculated. The results showed that the CV value of the diameter of the high-viscosity PVA monodisperse droplets was 2.1%, and the CV value of the diameter of the cross-linked PVA monodisperse microspheres was 3.1%.

[0106] Example 4

[0107] In this example, a method for preparing monodisperse droplets of a high viscosity system by in situ concentration is described in detail. Specifically, the preparation of high viscosity HA monodisperse droplets and monodisperse cross-linked HA microspheres is described as an example. The steps are as follows:

[0108] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0109] Preparation of an internal phase fluid: HA with a molecular weight of 1 to 2 MDa and a crosslinker, BDDE, were added to high-purity water and stirred until completely dissolved. The pH of the resulting solution was then adjusted to 3 with hydrochloric acid to obtain an internal phase fluid. The concentration of HA in the internal phase fluid was 1 wt%, the concentration of BDDE was 0.3 wt%, and the viscosity of the internal phase fluid was 91 MPa·s.

[0110] The external phase fluid was prepared by dissolving the surfactant Abil EM90 in isobutanol to obtain the external phase fluid; the concentration of Abil EM90 in the external phase fluid was 0.1 wt %.

[0111] Preparation of a collecting solution: dissolving the surfactant Abil EM90 in isobutanol to obtain a collecting solution; the concentration of Abil EM90 in the collecting solution is 0.1 wt %.

[0112] (2) In situ concentration to prepare high-viscosity HA monodisperse droplets

[0113] The inner phase fluid is injected into the injection tube of the first-level microfluidic device through a syringe pump, and the outer phase fluid is injected into the collecting tube of the first-level microfluidic device. A monodisperse W / O emulsion is formed at the outlet of the injection tube in the collecting tube. The monodisperse W / O emulsion is collected under stirring conditions using a container containing the collecting liquid. After receiving, the emulsion is kept in the collecting liquid for a period of time and stirring is continuously applied during the process. As the W / O emulsion droplets move in the collecting tube and the outlet hose and are kept in the collecting liquid, the water in the W / O emulsion droplets will continuously diffuse into the outer phase fluid and the collecting liquid, causing the various components in the W / O emulsion droplets to concentrate until the water concentration in the W / O emulsion droplets and the collecting liquid reaches equilibrium, thereby obtaining high-viscosity HA monodisperse droplets.

[0114] In this step, the primary microfluidic device used is the same as that in Example 1, and the flow rate of the internal phase fluid is controlled to be 200 μL / h, and the flow rate of the external phase fluid is 200 μL / min; the volume of the collecting liquid is controlled to be 10 times the volume of the internal phase fluid. After the receiving is completed, it is kept in the collecting liquid for 30 minutes until the concentration of water in the W / O emulsion droplets and the collecting liquid reaches equilibrium. The stirring is stopped, and after the high-viscosity HA monodisperse droplets settle to the bottom of the collecting container, the receiving liquid in the container is poured out to separate the high-viscosity HA monodisperse droplets.

[0115] (3) Preparation of monodisperse cross-linked HA microspheres

[0116] The container containing the high-viscosity HA monodisperse droplets was placed in an oven, and the monodisperse droplets were cross-linked and solidified into microspheres at 25°C. The cross-linking time was controlled to be 48 hours. The obtained microspheres were washed 3 times with ethanol and then washed 3 times with PBS buffer to obtain monodisperse cross-linked HA microspheres, which were stored in PBS buffer.

[0117] The high-viscosity HA monodisperse droplets and monodisperse cross-linked HA microspheres prepared in this example were observed using an optical microscope, and their sizes were measured and the CV values of their diameters were calculated. The results showed that the CV value of the diameter of the high-viscosity HA monodisperse droplets was 2.2%, and the CV value of the diameter of the monodisperse cross-linked HA microspheres was 3.3%.

Claims

1. A method for preparing monodisperse droplets of a high viscosity system by in-situ concentration, characterized in that: The following steps are involved: (1) Preparation of internal phase fluid, external phase fluid and collection fluid Prepare the internal phase fluid: dissolve the internal reagent including the polymer material and the cross-linking agent in water to form a cross-linking reaction precursor liquid, and use the cross-linking reaction precursor liquid as the internal phase fluid; The viscosity of the internal phase fluid does not exceed 100 mPa·s, and the concentration of the cross-linking agent in the internal phase fluid is 0.001 wt% to 10 wt%; Prepare the external phase fluid: dissolve the oil-soluble surfactant in the concentrate to obtain the external phase fluid; In the external phase fluid, the concentration of the oil-soluble surfactant is 0.1 wt% to 5 wt%; Preparation of a collection solution: dissolving an oil-soluble surfactant in a concentrator to obtain a collection solution; the concentration of the oil-soluble surfactant in the collection solution is 0.1 wt% to 5 wt%; The concentrator is an organic solvent that is miscible with water but does not dissolve the polymer material in the internal phase fluid; (2) In situ concentration to prepare monodisperse droplets of high viscosity systems The inner phase fluid and the outer phase fluid are respectively injected into the injection tube and the collecting tube of the first-level microfluidic device to form a monodisperse oil-in-water emulsion in the collecting tube. The oil-in-water emulsion is collected under stirring conditions using a container containing the collecting liquid. During the movement of the oil-in-water emulsion in the channel of the first-level microfluidic device and the retention in the collecting liquid, the water in the oil-in-water emulsion droplets will diffuse into the outer phase fluid and the collecting liquid, causing the various components in the oil-in-water emulsion droplets to be concentrated. When the concentration of water in the oil-in-water emulsion droplets and the collecting liquid reaches equilibrium, monodisperse droplets of a high viscosity system are obtained.

2. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to claim 1, characterized in that: The concentrator is one or more of ethyl acetate, isobutanol, ethanol, acetone, and methyl isobutyl ketone.

3. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to claim 1, characterized in that: In step (2), the volume of the collecting liquid is at least 10 times the volume of the internal phase fluid.

4. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to claim 1, characterized in that: The polymer material includes at least one of hyaluronic acid, sodium hyaluronate, agarose, gelatin, chitosan, polyvinyl alcohol, and sodium alginate.

5. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to claim 1, characterized in that: The cross-linking agent includes any one of 1,4-butanediol glycidyl ether, divinyl sulfone, 1-bromo-3,4-butylene oxide, 1-chloro-2,3-epoxypropane, 1-bromo-2,3-epoxypropane, 2,5-dibromopentanol, 2,4-dibromobutyl mercaptan, 2,5-dibromopentane-thiol epichlorohydrin, dimethylaminopropylcarbodiimide, formaldehyde and glutaraldehyde.

6. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to any one of claims 1 to 5, characterized in that: The cross-linking reaction precursor solution further contains at least one of an acid-base regulator, a catalyst, and an initiator.

7. The method for preparing monodisperse droplets of a high viscosity system by in-situ concentration according to any one of claims 1 to 5, characterized in that: The oil-soluble surfactant includes any one of Span 20, Span 60, Span 80, glyceryl monostearate, polyglyceryl ricinoleate, and cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethicone.

8. Monodisperse droplets of a high viscosity system prepared by the method according to any one of claims 1 to 7, characterized in that The coefficient of variation of the droplet diameter does not exceed 10%.

9. A method for preparing monodisperse cross-linked polymer microspheres with excellent biocompatibility, characterized in that: Conditions are applied to allow the polymer material in the monodisperse droplets of the high viscosity system prepared by the method of any one of claims 1 to 7 to undergo a cross-linking reaction with the cross-linking agent to solidify the monodisperse droplets, and then the unreacted components are removed by washing to obtain the product.

10. Monodisperse cross-linked polymer microspheres with excellent biocompatibility prepared by the method of claim 9, characterized in that: The coefficient of variation of the diameter of the cross-linked polymer microspheres does not exceed 10%.

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