Preparation system and preparation method of sodium alginate hydrogel microspheres
Through a preparation system based on flow focus, using nested capillaries and crosslinking reactions, the problems of uneven particle size and complex preparation of sodium alginate hydrogel microspheres were successfully solved, achieving an efficient, economical and simple preparation process.
Patent Information
- Application Number
- CN202411683062.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-18
AI Technical Summary
In the prior art, when preparing sodium alginate hydrogel microspheres, the particle size range varies greatly, the surface is rough, and the preparation method is complex, and the operation is dangerous, making it difficult to achieve efficient and economical preparation.
Using a preparation system based on flow focus, the sodium alginate hydrogel microspheres are formed by nested capillary structures, and the sodium alginate hydrogel microspheres are formed by using an oil-phase mixed solution, combining the cross-linking reaction of citric acid and calcium carbonate.
It realizes efficient preparation of sodium alginate hydrogel microspheres, uniform particle size, smooth surface, simple operation, avoids external pollution and improves preparation efficiency.
Smart Images
Figure CN119455834A_ABST
Abstract
Description
Technical Field
[0001] The invention provides a preparation system and a preparation method of sodium alginate hydrogel microspheres, and relates to the technical fields of drug targeted delivery, cell tissue culture, active ingredient carrier packaging, and the like. Background Art
[0002] Sodium alginate is a commonly used biomaterial and a natural polysaccharide carbohydrate that can be extracted from brown seaweed and has been widely used in the biomedical field. As it is an algae derivative, it has excellent biocompatibility, stability, and non-toxicity, so it is often used in bone tissue repair treatment and hemostasis and drug delivery of damaged tissues.
[0003] Sodium alginate microstructures represented by microspheres can be used to prolong the duration of drug action to enhance drug efficacy. Controllable targeted drug delivery can be achieved by modifying specific drug molecules on the surface of sodium alginate microstructures. Various new types of cell and tissue interactive alginate hydrogels can be widely used in tissue engineering research.
[0004] At present, the most common method for preparing hydrogel microstructures is the extrusion method, in which a dropper containing sodium alginate solution or a syringe with a needle is squeezed against each other by external force, so that it drips into a cross-linking agent containing metal cations to produce sodium alginate microspheres. Although this method is simple to operate, the particle size range of the hydrogel microparticles prepared by it varies greatly and the surface is rough. The main reason is that the hydrogel microparticles prepared by this method are affected by the droplet falling distance, needle size, solution concentration and cross-linking agent concentration.
[0005] Another mainstream preparation method is the electrostatic spray method, which mainly relies on the effect of an external electric field on the flowing droplets. As the electric field increases, the change in the shape of the droplets can be observed. Using this principle, when the intensity of the electric field is sufficient to overcome the surface tension of the sodium alginate solution, the droplets of the needle will form jet-like microdroplets, and the particle size of the droplets can be controlled by voltage. Although this method can obtain sodium alginate microspheres with smaller particle sizes in a controllable manner, it has high requirements for the preparation instruments and there are certain risks in the operation process, which brings difficulties to the efficient and economical preparation of sodium alginate hydrogel microspheres. Summary of the invention
[0006] In order to solve the above problems, the present invention provides a preparation system and method of sodium alginate hydrogel microspheres. The specific technical scheme is as follows:
[0007] A preparation system for sodium alginate hydrogel microspheres, wherein the preparation system comprises a substrate, a first capillary arranged on the substrate, a second capillary partially sleeved in the first capillary, a third capillary partially sleeved in the second capillary, and a collecting tube partially sleeved in the first capillary, the droplet generation end of the second capillary and the droplet generation end of the third capillary are adjacent to each other, and there is a certain distance between the droplet generation end of the second capillary and the collecting tube in the axial direction.
[0008] Preferably, the second capillary tube and the third capillary tube are both circular tubes, and the second capillary tube and the third capillary tube are coaxially arranged.
[0009] Preferably, the droplet generation end of the second capillary tube and the droplet generation end of the third capillary tube are both configured to be tapered.
[0010] Preferably, the first capillary tube is a square tube.
[0011] Preferably, the preparation system further comprises a first needle and a second needle both disposed on the base, the first needle being connected to the first capillary, and the second needle being connected to the second capillary.
[0012] A method for preparing the preparation system described in the above technical solution, wherein the preparation method comprises the following steps:
[0013] The oil phase mixed solution is introduced between the first capillary and the second capillary,
[0014] A sodium alginate mixed solution is introduced between the second capillary and the third capillary,
[0015] A citric acid mixed solution is introduced into the third capillary, and the sodium alginate mixed solution is sheared into microspheres by the oil phase mixed solution. At the same time, the citric acid mixed solution and sodium alginate undergo a cross-linking reaction and solidify to form sodium alginate hydrogel microspheres, which are collected through the collection tube.
[0016] Preferably, the oil phase mixed solution comprises polydimethylsiloxane, dimethyl silicone oil and a surface film-forming agent.
[0017] Preferably, the sodium alginate mixed solution comprises sodium alginate, deionized water, calcium carbonate and polyvinyl alcohol.
[0018] Preferably, the citric acid mixed solution comprises citric acid monohydrate, deionized water and orange pigment.
[0019] Preferably, before assembling the first capillary, the first capillary is firstly subjected to a hydrophobic treatment by first preparing a toluene solution containing 1% trichlorooctadecylsilane, then passing the prepared toluene solution into the first capillary to soak its inner wall surface for 2 minutes, and then placing the soaked first capillary on a hot plate at 160° C. and heating it for 30 minutes.
[0020] Beneficial effect: The present invention proposes a preparation system and method for sodium alginate hydrogel microspheres, which can complete the formation of sodium alginate microspheres and hydrogel cross-linking molding after one solution filling. The overall chip is mainly composed of three nested capillaries, and the generated sodium alginate hydrogel microspheres are finally collected by a collecting tube. Specifically, it is composed of coaxial second capillaries and third capillaries of different thicknesses, and a square tube with matching inner diameter, namely the first capillary. The thinner third capillary of the inner layer is passed through a citric acid solution, and the thicker second capillary of the outer layer is passed through a mixed solution of sodium alginate and calcium carbonate containing a surfactant. When the inner and outer layer solutions are sheared by the continuous phase oil phase solution in the outer phase square tube at the discharge port, while forming microspheres, citric acid reacts with calcium carbonate inside to generate calcium ions and undergoes a cross-linking reaction in the sodium alginate, solidifying to form hydrogel microspheres. Compared with the traditional sodium alginate hydrogel microsphere preparation scheme, this scheme has a simple structure and avoids tedious intermediate processes such as solution transfer and drying treatment. The manipulation process can be controlled by fluid force, which greatly reduces the probability of the prepared samples being contaminated by the outside world and is more efficient. In addition, sodium alginate hydrogel microstructures of different forms can be prepared by adjusting the flow rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.
[0022] Figure 1 It is a three-dimensional schematic diagram of a preparation system of a sodium alginate hydrogel microstructure provided in a specific embodiment of the present application.
[0023] Figure 2 yes Figure 1 Schematic diagram of the three-dimensional decomposition of the preparation system.
[0024] Figure 3 yes Figure 1 Top view of the preparation system.
[0025] Figure 4 yes Figure 3 AA section view of the preparation system.
[0026] Figure 5 yes Figure 4 A partial enlarged schematic diagram is shown in the figure.
[0027] Figure 6 It's under the microscope Figure 1 Schematic diagram of the preparation of sodium alginate hydrogel microstructures in the preparation system.
[0028] Figure 7 It is a schematic diagram of the morphology of sodium alginate hydrogel microspheres generated in different stages of the preparation system. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.
[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.
[0032] like Figures 1 to 7 As shown, the present application discloses a preparation system of sodium alginate hydrogel microspheres, wherein the preparation system includes a substrate 10, a first capillary 12 arranged on the substrate 10, a second capillary 14 partially sleeved in the first capillary 12, a third capillary 16 partially sleeved in the second capillary 14, and a collecting tube 18 partially sleeved in the first capillary 12, the droplet generating end of the second capillary 14 and the droplet generating end of the third capillary 16 are adjacent to each other, and there is a certain distance between the droplet generating end of the second capillary 14 and the collecting tube 18 in the axial direction.
[0033] The preparation system and preparation method of sodium alginate hydrogel microspheres proposed in the present application can complete the microsphere formation and hydrogel cross-linking molding of sodium alginate after one solution filling. The overall chip is mainly composed of three nested capillaries, and the generated sodium alginate hydrogel microspheres are finally collected by the collecting tube 18. Specifically, it is composed of coaxial second capillaries 14 and third capillaries 16 of different thicknesses, and a square tube with matching inner diameter, namely the first capillary 12. The citric acid solution is passed through the thinner third capillary 16 of the inner layer, and the sodium alginate and calcium carbonate mixed solution containing a surfactant is passed through the thicker second capillary 14 of the outer layer. When the inner and outer layer solutions are sheared by the continuous phase oil phase solution in the outer phase square tube at the discharge port, the citric acid and calcium carbonate react inside the microspheres to generate calcium ions and undergo cross-linking reaction in the sodium alginate, and solidify to form hydrogel microspheres. Compared with the traditional sodium alginate hydrogel microsphere preparation scheme, this scheme has a simple structure and avoids tedious intermediate processes such as solution transfer and drying treatment. The manipulation process can be controlled by fluid force, which greatly reduces the probability of the prepared samples being contaminated by the outside world and is more efficient. In addition, sodium alginate hydrogel microstructures of different forms can be prepared by adjusting the flow rate.
[0034] The present invention adopts a method based on flow focusing droplet generation to achieve integrated preparation of hydrogel microstructures, aiming to improve the accuracy and convenience of preparing hydrogel microstructures, and effectively overcome the limitations and risks existing in traditional methods. This method uses droplet microfluidics to control the generation of hydrogel microstructures. According to the relative viscosity and relative speed adjustment of the continuous phase and the discrete phase, hydrogel microstructures of different sizes and shapes can be generated in a controllable and integrated manner, providing a more reliable and efficient technical platform for the efficient preparation of hydrogel microstructures and biomedical research.
[0035] Preferably, the second capillary tube 14 and the third capillary tube 16 are both circular tubes, and the second capillary tube 14 and the third capillary tube 16 are coaxially arranged.
[0036] Preferably, the droplet generation end of the second capillary tube 14 and the droplet generation end of the third capillary tube 16 are both configured to be tapered. The end of the collecting tube 18 that is closer to the third capillary tube 16 is also configured to be tapered.
[0037] Preferably, the first capillary tube 12 is a square tube.
[0038] In addition, the preparation system further includes a first needle 20 and a second needle 22 both disposed on the substrate 10 . The first needle 20 is connected to the inside of the first capillary 12 , and the second needle 22 is connected to the inside of the second capillary 14 .
[0039] The present application also discloses a preparation method, wherein the preparation method comprises the following steps:
[0040] The oil phase mixed solution is passed between the first capillary 12 and the first capillary 12.
[0041] A sodium alginate mixed solution is introduced between the second capillary 14 and the third capillary 16.
[0042] The citric acid mixed solution is introduced into the third capillary 16 , and the sodium alginate mixed solution is sheared into microspheres by the oil phase mixed solution. At the same time, the citric acid mixed solution and sodium alginate undergo a cross-linking reaction and solidify to form sodium alginate hydrogel microspheres, which are collected through the collection tube 18 .
[0043] Specifically, the oil phase mixed solution includes polydimethylsiloxane, dimethyl silicone oil and a surface film-forming agent.
[0044] Preferably, the sodium alginate mixed solution comprises sodium alginate, deionized water, calcium carbonate and polyvinyl alcohol.
[0045] Preferably, the citric acid mixed solution comprises citric acid monohydrate, deionized water and orange pigment.
[0046] The specific preparation of the oil phase mixed solution is: weigh about 10g of polydimethylsiloxane (PDMS) and 10g of dimethyl silicone oil, mix the two thoroughly, and then add an appropriate amount of cyclopentasiloxane / trimethylsiloxysilicate (RSN-0749) surface film-forming agent.
[0047] The specific preparation of the sodium alginate and calcium carbonate mixed solution is as follows: weigh about 0.5-1.5g of sodium alginate (analytical grade), add it to about 100ml of deionized water, then add 1-2g of calcium carbonate (analytical grade) and dropwise add about 100-300mg of polyvinyl alcohol (PVA), stir thoroughly and put it into an ultrasonic vibrator, and take it out for use after the reagents are fully mixed.
[0048] The specific preparation of the citric acid mixed solution is as follows: weigh about 0.5-2g of citric acid monohydrate (analytical grade), add it to about 100ml of deionized water and put it into an ultrasonic vibrator to mix it thoroughly. Finally, add 2 drops (about 1ml) of orange pigment to the prepared solution to facilitate the observation of the mixing condition in subsequent experiments.
[0049] In addition, specifically in this embodiment, the first capillary 12, the second capillary 14 and the third capillary 16 are all glass capillaries, and the substrate 10 is also a dry glass substrate. The tapered structures of the droplet generating ends of the second capillary 14 and the third capillary 16 are both drawn by a glass tube drawing instrument, and the second capillary 14 and the third capillary 16 are assembled in a coaxial manner.
[0050] In addition, the tapered opening of the third capillary 16 with a thinner inner portion needs to slightly protrude from the tapered opening of the thicker second capillary 14, so as to ensure that the two discrete phase liquids are fully mixed when the subsequent droplets are generated. The two discrete phase liquids are the oil phase mixed solution and the sodium alginate mixed solution. After the assembly is completed, the assembled coaxial second capillary 14 and third capillary 16 are inserted into the inner side of the square first capillary 12. A collecting tube 18 with a matching inner diameter is inserted into the rear end of the square first capillary 12 to receive the generated hydrogel droplets.
[0051] Since sodium alginate will react with calcium ions to generate calcium alginate hydrogel, when the two discrete phase liquids are respectively introduced into the second capillary 14 and the third capillary 16, calcium carbonate and citric acid will react to release calcium ions, and the sodium alginate wrapped in the shell will form gel droplets. After the continuous phase and discrete phase liquids are respectively introduced, the relative flow rate of the two phases is adjusted by a microinjection pump. After the adjustment is completed, the discrete phase gel liquid is sheared into droplets by the continuous phase liquid due to the influence of flow focusing.
[0052] By using the coaxial flow focusing technology and nested second capillary 14 and third capillary 16, the integrated molding of multiple hydrogel droplets in one package is realized, which solves the problems of low efficiency and easy damage of hydrogel droplet step-by-step molding in traditional hydrogel microstructure preparation technology, reduces the complexity of operation and preparation difficulty, and improves the efficiency of hydrogel microstructure preparation and raw material utilization.
[0053] The preparation system also includes three syringes, namely the first syringe, the second syringe and the third syringe. The first syringe is loaded with the prepared oil phase for easy mixing, the second syringe is loaded with the prepared sodium alginate mixed solution, and the third syringe is loaded with the prepared citric acid mixed solution. The three syringes are respectively installed on the corresponding microinjection pumps. The preparation system also includes a microscope, a camera and a computer, and the microscope and the camera are connected to the computer. The hydrogel microspheres are observed through a microscope.
[0054] The key structural parameters of this specific embodiment are as follows:
[0055] The inner diameter of the square first capillary 12 is about 1.14 mm, and its length is about 20 mm; the inner diameter of the circular second capillary 14 is about 0.98 mm, and its length is about 12 mm; the inner diameter of the circular third capillary 16 is about 0.46 mm, and its length is about 22 mm; the inner diameter of the collecting tube 18 is about 1 mm, and its length is about 20 mm; the size of the glass slide as the substrate 10 is about 30 mm long and about 10 mm wide.
[0056] The processing of the first capillary 12, the second capillary 14, the third capillary 16 and the collecting tube 18 is as follows:
[0057] By reasonably setting the parameters of the tube drawing instrument such as temperature, pressure and stretching time, the second capillary 14 and the third capillary 16 are heated, pressurized and stretched to obtain a conical structure that meets the requirements. Usually, the capillary conical section drawn is closed or the opening size is extremely small, and sandpaper needs to be used for grinding. The second capillary 14, the third capillary 16 and the collecting tube 18 are all prepared by this method. During grinding, in order to ensure that the size meets the requirements, the caliber needs to be observed under a microscope after each grinding. The caliber can be quickly measured by the supporting software window of the microscope. The caliber of the conical mouth of the second capillary 14 is about 300-500um; the caliber of the conical mouth of the third capillary 16 is about 100-200um; the caliber of the conical mouth of the collecting tube 18 is about 600-800um. For the part that exceeds the length, it is slowly cut and removed by a glass cutter. The first capillary 12 is directly cut by a glass cutter to obtain a suitable length and is reserved for standby. To prevent glass residue from remaining in the tube, all processed capillaries need to be ultrasonically cleaned with ethanol and then blown dry with nitrogen.
[0058] Preferably, before assembling the first capillary 12, the first capillary 12 is firstly subjected to a hydrophobic treatment, wherein a toluene solution containing 1% trichlorooctadecylsilane (OTS) is first prepared, and then the prepared toluene solution is passed into the first capillary 12 to soak its inner wall surface for 2 minutes, and the soaked first capillary 12 is placed on a hot plate at 160° C. and heated for 30 minutes. In this way, since the OTS molecules self-assemble into a film on the wall surface of the glass tube to achieve a hydrophobic treatment of the inner wall of the tube, it can be ensured that the oil phase is always in contact with the wall surface during the flow process in the first capillary 12, and the problem of unstable flow of the oil phase caused by water droplets adhering to the inner wall is avoided. In addition, the remaining capillaries do not need to be subjected to a hydrophobic treatment.
[0059] In the specific assembly process, the first capillary 12, the second capillary 14, the third capillary 16 and the collecting tube 18 that have been processed and surface treated are taken out, and the assembly process needs to be carried out in an orderly manner under a microscope. First, a clean glass slide (length 30 mm, width 10 mm) is selected as the substrate 10, and the positions of each tube are determined and marked on the glass slide according to the designed structure. The first capillary 12 is first fixed at the specified position and then fixed with UV light curing glue. Then, insert the second capillary 14 from one end of the first capillary 12, and then insert the third capillary 16 into the second capillary 14. Under a microscope, adjust the positions of the second capillary 14 and the third capillary 16 so that they are aligned on the same axis and the distance between the tapered ends is about 10-50um, and the third capillary 16 protrudes from the second capillary 14. The alignment of the second capillary 14 and the third capillary 16 on the horizontal plane (xy plane) can be ensured by the cross-sectional distance measurement of the visualization software provided by the microscope. The alignment in the vertical direction (z direction) can be determined by adjusting the focal length of the microscope. When the cone holes of the two tubes are focused at the same time, it means that they are at the same height in the z-axis direction. After determining the position, the position can be fixed with AB glue that dries quickly in 5 minutes. In the same way, install and fix the collecting tube 18 at a distance of about 50-150mm from the other end of the first capillary 12. Finally, the first needle 20 and the second needle 22 are respectively installed and fixed at the joints of the second capillary 14 and the first capillary 12 and the joints of the second capillary 14 and the third capillary 16 for passing the solution. At this point, the main assembly of the integrated sodium alginate hydrogel microsphere preparation system is completed. The newly manufactured main structure is generally placed for 12 hours to ensure that the AB glue is completely cured, thereby ensuring that the position of each tube is fixed, so as to avoid problems such as solution leakage or misalignment of the tube during the experiment of preparing sodium alginate hydrogel.
[0060] The experimental operation process of the preparation system is as follows:
[0061] 1) The three groups of prepared mixed solutions are respectively loaded into three syringes and connected to the main structure through hoses, and then the three syringes are installed on the corresponding microinjection pumps. The microscope, computer, and CCD camera are turned on in sequence, and the processed main structure is placed on the stage of the inverted microscope. The objective lens is reasonably adjusted according to the experimental needs, and the position and focal length of the main structure are fixed.
[0062] 2) Adjust the flow rates of the three micro-injection pumps to stably generate sodium alginate hydrogel droplets in the first capillary 12. The specific adjustment steps are: first turn on the oil phase solution, and after the flow is stable, turn on the injection pump of the sodium alginate mixed solution. Due to the focusing effect of liquid flow, the sodium alginate will be sheared into droplets by the oil phase solution. After the droplet generation is stable, finally turn on the injection pump of the citric acid mixed solution to observe the state of droplet mixing. After stabilization, use a culture dish to pick up the generated sodium alginate hydrogel microspheres and place them under a microscope for observation.
[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
[0064] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.
Claims
1. A system for preparing sodium alginate hydrogel microspheres, characterized in that: The preparation system includes a substrate, a first capillary arranged on the substrate, a second capillary partially arranged in the first capillary, a third capillary partially arranged in the second capillary, and a collecting tube partially arranged in the first capillary, the droplet generation end of the second capillary and the droplet generation end of the third capillary are adjacent to each other, and there is a certain distance between the droplet generation end of the second capillary and the collecting tube in the axial direction.
2. The preparation system according to claim 1, characterized in that: The second capillary tube and the third capillary tube are both circular tubes, and the second capillary tube and the third capillary tube are coaxially arranged.
3. The preparation system according to claim 1, characterized in that: The droplet generation end of the second capillary tube and the droplet generation end of the third capillary tube are both configured to be tapered.
4. The preparation system according to claim 1, characterized in that: The first capillary tube is a square tube.
5. The preparation system according to claim 1, characterized in that: The preparation system further includes a first needle and a second needle both disposed on the base, the first needle being connected to the first capillary, and the second needle being connected to the second capillary.
6. A method for preparing the preparation system according to claim 1, characterized in that: The preparation method comprises the following steps: The oil phase mixed solution is introduced between the first capillary and the second capillary, A sodium alginate mixed solution is introduced between the second capillary and the third capillary, A citric acid mixed solution is introduced into the third capillary, and the sodium alginate mixed solution is sheared into microspheres by the oil phase mixed solution. At the same time, the citric acid mixed solution and sodium alginate undergo a cross-linking reaction and solidify to form sodium alginate hydrogel microspheres, which are collected through the collection tube.
7. The preparation method according to claim 6, characterized in that: The oil phase mixed solution comprises polydimethylsiloxane, dimethyl silicone oil and a surface film-forming agent.
8. The preparation method according to claim 6, characterized in that: The sodium alginate mixed solution comprises sodium alginate, deionized water, calcium carbonate and polyvinyl alcohol.
9. The preparation method according to claim 6, characterized in that: The citric acid mixed solution comprises citric acid monohydrate, deionized water and orange pigment.
10. The preparation method according to claim 6, characterized in that: Before assembling the first capillary, the first capillary is firstly subjected to hydrophobic treatment by preparing a toluene solution containing 1% trichlorooctadecylsilane, then passing the prepared toluene solution into the first capillary to soak its inner wall surface for 2 minutes, and then placing the soaked first capillary on a hot plate at 160° C. and heating it for 30 minutes.