WO2019091145A1

WO2019091145A1 - Microfluidic controlling technology for single-step, continuous preparation of calcium alginate microgel

Info

Publication number: WO2019091145A1
Application number: PCT/CN2018/097726
Authority: WO
Inventors: 王华楠; 张丽媛
Original assignee: 深圳华诺生物科技有限公司
Priority date: 2017-11-13
Filing date: 2018-07-30
Publication date: 2019-05-16
Also published as: CN107930542B; CN107930542A

Abstract

The invention relates to a single-step, continuous preparation method of a multi-void calcium alginate microgel based on microfluidic droplet technology. The calcium alginate microgel can be used to immobilize bioactive substances. The method can achieve high-throughput and continuous production of a microgel material. The method includes: forming an aqueous solution having a multi-phase parallel flow by injecting a plurality of hydrogel prepolymer solutions into a microfluidic chip channel, in which the hydrogel prepolymer solutions form a parallel, steady flow field in the microfluidic chip channel and constitute a plurality of voids in the subsequently formed multi-void microgel, forming a water-in-oil emulsion droplet by mixing the aqueous solution with an immiscible fluid (oil phase) by means of a T-shaped microfluidic channel or a hydrodynamic focusing microfluidic channel, immediately initiating crosslinking of the calcium alginate in the water-in-oil emulsion droplet to obtain a multi-void microgel; and cleaning the water-in-oil emulsion droplet in the microfluidic chip, such that the multi-void microgel is rapidly transported to the aqueous phase. The method achieves single-step preparation of a calcium alginate microgel capable of immobilizing bioactive substances and is suitable for industrial application.

Description

Microfluidic Technology for Continuous Preparation of Calcium Alginate Microgel by One-Step Method

Technical field

The invention belongs to the field of material micro-processing, the field of biomaterial preparation technology, tissue engineering and cell therapy, and particularly relates to a micro-gel for preparing a solid-loaded living cell or a bioactive molecule drug by using a microfluidic device.

Background technique

In recent years, hydrogels have been widely used in many biomedical fields, especially in the field of tissue engineering, hydrogels have become very important biological materials. A hydrogel is a cross-linked network composed of a polymer that is insoluble in water but swellable by water. Similar to human tissues and organs, it is composed of a porous network rich in water. Such a porous structure resembles the tissues and organs of the human body. Conducive to the effective exchange of substances. At the same time, some hydrogels composed of natural polymers have good biocompatibility and biodegradability, and mechanical properties similar to those of human tissues. Therefore, they can be used as tissue engineering scaffold materials to support cell growth and tissue regeneration. . One branch of the hydrogel material is composed of biomacromolecular prepolymers, which have good biocompatibility and mild gelation conditions, and thus such materials are widely used for three-dimensional encapsulation of cells. Among them, many natural polymer materials including collagen, fibrin, or alginate belong to such materials and are widely used for cell embedding. Although hydrogels have been extensively studied and used in bioengineering materials in the field of tissue engineering, traditional hydrogel-embedded cell technologies still have problems to be solved in tissue engineering applications: 1) Block hydrogels Larger size (>1cm), the size of the polymer network is on the nanometer scale, and the biomacromolecules survive due to low diffusion rate and distance, so living cells embedded in the block gel survive due to low exchange efficiency of nutrients and metabolites. The rate of decline; 2) hydrogel as a bioactive substance delivery carrier is not injectable, can only be implanted, can not be implanted by minimally invasive intervention; 3) block hydrogel for In the process of macromolecule or cell embedding, it is difficult to ensure uniform distribution of the load material in the gel system. In contrast, the use of micro-scale microgels as carriers for biologically active substances provides an effective solution to the above problems. Because the microgel has a small scale, it is beneficial for the rapid diffusion of the substance, and the cells enclosed therein can be effectively exchanged with the external nutrients, signal factors and excretions. At the same time, microgel particles can be injected directly, providing an effective way for direct injection of active macromolecular drugs and living cells. Therefore, achieving high-throughput preparation of cell-loaded microgels will facilitate the development and clinical application of tissue engineering and cell therapy technologies.

Emulsion technology is widely used to prepare microgels that encapsulate biologically active substances such as bioactive macromolecular drugs or living cells. The method generally utilizes a water-oil two-phase non-blended fluid to form a dispersed phase and a continuous phase under shear. A typical preparation method is: first, a biologically active substance (for example, a biologically active macromolecule or a living cell) is dispersed in a hydrogel prepolymer aqueous solution, and the aqueous solution is blended with a continuous oil phase to form a water-in-oil single. The emulsion, followed by inducing a polymerization or crosslinking reaction, gels the hydrogel prepolymer within the droplets to give a cured microgel. These conventional emulsion methods have the following problems: 1) The living cells need to be dispersed in the dispersed microemulsion for a long time, and the cells cannot exchange nutrients and gases with the droplets, so the metabolic activity of the cells is gradually weakened for a long time. In the emulsion, it will cause a decrease in cell viability; 2) in order to form a stable emulsion, it is often necessary to add a surfactant to the oil phase or the aqueous phase, and the surfactant tends to damage the cell membrane to produce cytotoxicity, so the cells are in contact for a long time. Surfactants also have potential cytotoxicity problems; 3) In order to initiate polymerization or cross-linking of hydrogels, it is usually necessary to use cytotoxic cross-linking agents or cross-linking reaction initiators, and long-term exposure of cells to these components is also cytotoxic. The problem. Therefore, the traditional emulsion method for preparing cell-loaded microgels is difficult to maintain high cell survival rate and metabolic activity of cells, and requires additional time-consuming and labor-intensive oil phase cleaning after immobilization, so it cannot be used in the industry. Continuous processing.

Recently, a number of micro-processing technologies for cell immobilization, including photolithography, micro-template technology, and emulsion technology, have emerged, and there are problems in how to maintain the survival rate of the immobilized cells, so it is difficult to use it for cell-loading micro Industrial scale production of gels. Moreover, these conventional techniques are based on batch production processes, which are unable to achieve high-throughput continuous preparation, and there are differences in product performance between batches.

Microfluidic droplet technology is a micromachining technology based on microfluidic chip for precise control of immiscible multiphase fluids. This technology enables continuous injection and rapid production of monodisperse, precisely sized microgels or microcapsules. Compared with traditional water-in-oil (W/O) or oil-in-water (O/W) single emulsion droplet technology, it can be prepared with a uniform size by a microfluidic device with a T-shaped flow path or fluid focusing structure. Single emulsion droplets. And this can be used as a template to prepare monodisperse microgels by different polymerization methods. However, as with conventional emulsion methods, the microfluidic single emulsion droplet technique is not suitable for continuous processing of cell-loaded microgels. This is because during the preparation of the sample, the immobilized cells are exposed to the oil phase, surfactant, cross-linking agent, etc. for a long time to cause cytotoxicity. Therefore, it is necessary to intermittently collect the product and rapidly break the emulsion, and transfer the cells to the biocompatible aqueous phase solution to maintain the cell survival rate in the prepared microgel, which is time consuming and laborious. Therefore, how to achieve cell solidification in the microgel and rapid separation from the oil phase will greatly ensure cell activity, and can achieve continuous preparation and improve production efficiency.

In addition, water-in-water (W/W) emulsion technology can also be used to prepare microgels. The method uses an aqueous two-phase fluid, which forms droplets by non-blending between the two phases, and uses droplets as a template to produce microgel particles in an aqueous solution. This method avoids the extra step of breaking the emulsion and enables one-step cell entrapment. However, this system is limited to a combination of specific unblended aqueous solutes, such as dextran and polyethylene glycol, and has a high concentration of aqueous solute, thereby limiting the wide application of this method in cell immobilization.

In summary, although the cell-loaded microgel technology has important application value in the biomedical field, how to achieve cell immobilization embedded in the microgel while maintaining the cell survival rate is still urgent in this field. Solved technical problems. This has prompted researchers to develop new technologies that are simpler, more efficient, and that enable industrial production. Although microgel materials have important application potential in the field of biomedicine, their functions and structures are relatively simple, making it difficult to perform more functional expansion. Therefore, multi-chamber microgel materials have recently become a hotspot in biomedical applications and research. The multi-chamber microgel particles are microgel materials composed of two or more different chambers, and the different chambers may be the same or different materials. Compared to traditional single-chamber microgels, multi-chamber microgels have multiple structures that can impart different functions. Due to its complex structure and function, multi-chamber microgels have important application potential in the field of biomedicine, especially in the field of bioactive substance immobilization and delivery. Existing methods for preparing multi-chamber microgel materials usually involve emulsification of several different hydrogel monomers or aqueous solutions of prepolymers and rapid initiation of polymerization or crosslinking to obtain a stable multi-chamber structure. Microgel particles. Commonly used materials for preparing multi-chamber microgels are hydrogel prepolymers which can be crosslinked by photoinitiation, or alginate which can be rapidly ionically crosslinked. However, these rapid and severe cross-linking reactions usually have strong biological toxicity, which will affect the biological activity of the immobilized substances. Especially when embedding cells, these methods are generally cytotoxic, thus significantly reducing cell survival rate. Its development and application in this field. Due to its complex structure and function, multi-chamber microgels have important application potential in the field of biomedicine, especially in the field of bioactive substance immobilization and delivery.

Existing techniques for preparing multi-chamber microgels include microfluidic chip technology [Y. Du, E. Lo, S. Ali, A. Khademhosseini, P. Natl Acad. Sci. 2008, 105, 9522-9527; S. Seiffert, Angew. Chem. Int. Ed 2013, 52, 11462-11468], Fluid Imprinting Technology [D. Dendukuri, DC Pregibon, J. Collins, TA Hatton, PS Doyle, Nat. Mater. 2006, 5, 365 -369;b) D.Dendukuri, SSGu, DC Pregibon, TAHatton, PSDoyle, Lab. Chip 2007, 7, 818-828.], and centrifugal preparation process [K. Maeda, H. Onoe, M. Takinoue , S. Takeuchi, Adv. Mater. 2012, 24, 1340-1346].

Fluid Imprinting Technology [D. Dendukuri, DC Pregibon, J. Collins, TA Hatton, PS Doyle, Nat. Mater. 2006, 5, 365-369; b) D. Dendukuri, SSGu, DC Pregibon, TAHatton, PS Doyle , Lab.Chip 2007, 7, 818-828.] The preparation of multi-chamber microgel technology is to use photopolymerized hydrogel monomer or prepolymer as a fluid solution constituting different chambers of the microgel, through the light mask Membrane structure controls the structure of microgels, usually preparing non-spherical microgel particles. This technique can only control multi-chamber structure in two dimensions, but can not control the dimension of microgel in three dimensions; and the method is prepared The medium prepolymer solution, encapsulated drug or cell loss is large and the encapsulation is typically less than 50%.

Centrifugal preparation process [K. Maeda, H. Onoe, M. Takinoue, S. Takeuchi, Adv. Mater. 2012, 24, 1340-1346] by adding an aqueous solution of alginate as a hydrogel prepolymer to a multi-channel In the capillary, the liquid in the capillary is injected into the solution containing calcium ions by centrifugation. However, due to the high structural design and processing cost of the multi-channel capillary, the method is costly; and because the amount of prepolymer in the capillary is limited, the sample cannot be continuously loaded, so continuous processing cannot be realized, and it is not suitable for industrial production; This technique is difficult to use for cell encapsulation because cross-linked alginic acid requires the use of a high concentration of calcium ion solution, which is not conducive to cell survival.

Microfluidic chip technology [Y. Du, E. Lo, S. Ali, A. Khademhosseini, P. Natl Acad. Sci. 2008, 105, 9522-9527; S. Seiffert, Angew. Chem. Int. Ed 2013, 52, 11462-11468] Multi-chamber microgel preparation is achieved by fluid-focused microfluidic chip design by forming emulsion droplets and initiating rapid gelation reactions. The method generally requires a vigorous gelation reaction to rapidly fix the multi-chamber structure. When applied to the bioactive substance embedding, the protein molecules are liable to lose biological activity and the cell survival rate is decreased. Meanwhile, the existing process first utilizes the micro-flow. The microgel particles are prepared by the chip, and after the collection, the second phase cleaning is performed to elute the oil phase, so that the microgel is redispersed in the aqueous solution, so continuous injection preparation cannot be realized, and it is not suitable for industrial application.

Summary of the invention

The invention is based on the microfluidic droplet technology to realize the one-step continuous preparation of the immobilizable bioactive substance and the multi-chamber calcium alginate microgel, which can realize high-throughput and continuous production of the microgel material. Firstly, different hydrogel prepolymer solutions in the microfluidic channel can be used to form a parallel, stable flow field, thereby injecting the hydrogel prepolymer solution constituting the different chambers of the microgel into the microfluidic chip to form the water of the multiphase parallel fluid. The phase solution is then mixed with the unblended fluid (oil phase) through a T-channel or fluid-focusing microfluidic channel to form a water-in-oil emulsion droplet, which immediately initiates alginic acid in the droplet after formation Cross-linking to achieve multi-chamber microgel preparation; then through the cleaning of the emulsion droplets on the microfluidic chip, so that the microgel is quickly transported into the water phase, to achieve one-step preparation of immobilized bioactive substances Calcium alginate microgel. The preparation method has good biocompatibility and can well maintain the biological activity of the embedded active substance (such as biologically active macromolecule or living cell), and is an effective method for high-throughput and continuous preparation of microgel products for biomedicine.

The invention discloses a microfluidic technology for continuously preparing calcium alginate microgel by one-step method, which comprises the following steps:

(1) Preparation solution

1 using water-soluble alginate as raw material to prepare an aqueous solution of alginic acid in water, followed by adding a crosslinking reaction initiator, and then dispersing the biologically active substance and/or the nanoparticle in the aqueous solution to obtain a hydrogel prepolymer solution, An aqueous phase solution for preparing a water-in-oil emulsion system, wherein:

The crosslinking reaction initiator is selected from the group consisting of a chelate aqueous solution of calcium-ethylenediaminetetraacetic acid, a chelate aqueous solution of calcium-aminotriacetic acid, calcium carbonate nanoparticles, calcium sulfate nanoparticles, and calcium phosphate nanoparticles. Or a combination of several; the final concentration of the crosslinking reaction initiator is recorded as 10-1000 mM calcium; preferably 20-500 mM, more preferably 25-100 mM;

2 blending fluorinated oil, fluorinated surfactant and acidic substance as the first heavy oil phase of the water-in-oil emulsion system;

3 mixing the fluorinated oil with perfluoro alcohol or perfluoric acid to obtain a second heavy oil phase;

(2) injecting the aqueous phase solution obtained in step 1 into the microfluidic chip at a first flow rate, injecting the first heavy oil phase solution from the second input port into the microfluidic chip at a second flow rate, and emulsification of the aqueous phase solution and the first heavy oil phase The channel is blended, and the unblended two phases are blended through the emulsification channel to form a water-in-oil emulsion droplet; downstream of the emulsification channel, the alginic acid in the single emulsion droplet gels and rapidly solidifies (time is 0.00001) -1 second) forming alginate microgel;

(3) injecting the second heavy oil phase into the microfluidic chip from the third input port at a third flow rate, the second heavy oil phase and the emulsion obtained in the step (2) are thoroughly blended in the mixing channel disposed downstream of the emulsification channel, and the seaweed is dispersed The oil-water mixture of the acid microgel flows out of the chip from the output channel; the output solution is input into the collecting phase aqueous solution through the catheter, and the calcium alginate microgel carrying the biologically active substance and/or the nanoparticle is dispersed in the aqueous solution of the collecting phase. The oil phase and the aqueous phase are automatically separated and submerged in the bottom of the aqueous solution, and the alginic acid microgel is automatically dispersed in the aqueous solution; the aqueous phase solution is collected to obtain the final product.

In the above technical solution, in particular, the microfluidic chip has a fluid focusing structure, a T-shaped hybrid structure, a co-flow type or a cross-structure microfluidic channel, and the microfluidic chip further has at least 3-phase liquid. Input port, as well as emulsification channel and output channel. The 3-phase liquid input port comprises: an aqueous phase solution input port, a first heavy oil phase input port and a second heavy oil phase input port; and the inner surface of the microchannel is subjected to a hydrophobic treatment.

In the above-described technical solution, specifically, the alginic acid raw material is a mixture of alginic acid, alginic acid, aqueous alginate, or a mixture of alginic acid and a water-soluble polymer. The water-soluble polymer is selected from the group consisting of collagen, gelatin, hyaluronic acid, polyethylene glycol, polyvinyl alcohol, polyacrylamide, dextran, chitosan, agarose, or a combination of several.

In the technical solution described above, specifically, the total concentration of all alginic acid raw materials in the prepolymer solution is from 0.1 to 8 w/v%.

In the above technical solution, specifically, the embedded biologically active substance and/or the nanoparticle described in the step (1) is selected from one or a combination of the following: living cells, water-soluble Active protein drug molecules, nanoparticles; those skilled in the art can choose according to different implementation purposes, for example:

The living cells are generally one or more of primary cultured cells, subcultured cells, cell culture cells, and hybrids;

The water-soluble active protein drug molecule is generally one or more of a protein drug, a polypeptide drug, an enzyme drug, and a cell growth factor.

Protein drugs, such as: serum albumin, gamma globulin, insulin;

Peptide drugs, such as: oxytocin, glucagon;

Enzymes such as digestive enzymes (pepsin, trypsin, malt amylase), anti-inflammatory enzymes (lysozyme, trypsin), cardiovascular disease therapeutic enzymes (kinin releasing enzyme to dilate blood vessels to lower blood pressure), etc.;

Cell growth factors such as interferon, interleukin, tumor necrosis factor, bone formation protein-2, bone formation protein-7, fibroblast growth factor, angiogenic growth factor, etc.

The nanoparticles are generally: metal or metal oxide nanoparticles such as nano gold, nano silver, nano iron oxide, high molecular polymer nanometer such as polyethylene, polypropylene, polystyrene, polymethyl methacrylate or polylactic acid. One or more of particles, fat-soluble vitamins, quinolone water-insoluble drug nanoparticles, hydroxyapatite, silica, calcium phosphate, carbon nanotubes, and graphene inorganic non-metal material nanoparticles. The nanoparticle size is 5-1000 nm in diameter.

In the above technical solution, specifically, the fluorinated oil is generally selected from one or a combination of the following: perfluoropentane, perfluorohexane, perfluoroheptane, perfluorobutyl - methyl ether, perfluorooctane, perfluorodecane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane, perfluorotetradecane, perfluoropentadecane, Perfluorohexadecane, perfluoroheptadecane, perfluorodecalin.

In the above technical solution, specifically, the fluorinated surfactant is generally selected from one or a combination of the following: perfluorinated ethers (PFPE), perfluoroalkyl acid perfluoro Ether-polyvinyl alcohol block copolymer, perfluoroether-polyvinyl alcohol-perfluoroether block copolymer surfactant; wherein the concentration of the fluorinated surfactant is from 0.1 to 10% by weight, preferably from 0.5 to 5% by weight.

In the above technical solution, specifically, the acidic substance is selected from one or a combination of the following: sulfuric acid, nitric acid, hydrochloric acid, carbonic acid, phosphoric acid, acetic acid or citric acid, and the acidic substance is mixed. The concentration in the solution is 0.001-20 v/v%;

In the above technical solution, in particular, the perfluoroalcohol in the second heavy oil phase is generally selected from one or a combination of the following: 22, 33, 444-heptafluoro-1-butanol , perfluoroundecyl alcohol, 1H, 1H-perfluoro-1-dodecanol, 1H, 1H-perfluoro-1-heptanol, 1H, 1H, 2H, 2H-perfluorooctyl alcohol, 1H, 1H- Perfluorooctyl-1-ol, 1H, 1H-perfluoro-1-nonanol, 1H, 1H, 2H, 2H-perfluoro-1-dodecanol, 1H, 1H, 2H, 2H-perfluoro-1 - decyl alcohol, 1H, 1H-perfluoro-1-tetradecyl alcohol, perfluoropentanol, perfluorohexanol, perfluoroheptanol, perfluorooctyl alcohol, perfluorononanol, perfluorononanol;

The perfluoric acid is generally selected from one or a combination of the following: perfluorododecanoic acid, n-perfluoropentanoic acid, 5H-perfluoropentanoic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorodecanoic acid, Perfluoroheptanoic acid, perfluorohexanoic acid, perfluorobutyric acid, perfluoroundecanoic acid; the concentration of perfluoroalcohol or perfluoric acid in the second heavy oil phase in the fluorinated oil is 1-100 v/v%, preferably 10-50v/v%.

In the technical solution described above, specifically, the aqueous phase collecting solution described in the step (4) is non-cytotoxic, suitable for cell culture, buffer solution having a pH buffer range of 6-8, and can be diluted and medium. And a solution of a cross-linking agent and a surfactant in the aqueous phase, which can be specifically selected by a person skilled in the art according to the sample condition, and is generally selected from one or a combination of the following: buffering of HEPES buffer, cell culture medium, etc. Liquid, phosphate buffer (PBS), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, PBS buffer, disodium hydrogen phosphate-citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-lemon Sodium acetate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, barbital sodium-hydrochloric acid buffer, Tris-hydrochloric acid buffer, boric acid-borax buffer cell culture medium; wherein the buffer ion concentration is 10-2000 mM Preferably, 100-200 mM.

In the above technical solution, specifically, the internal solution of the multiple fluids, the first heavy oil phase and the second heavy oil phase are respectively passed through a micro pump or a microsyringe at a first flow rate of 5 to 2000 μL/hr. The second flow rate is 200-20000 μL/hr, and the third flow rate is 200-20000 μL/hr. The flow rate is delivered to the corresponding microchannel of the microfluidic device to form a monodisperse water/oil/water double emulsion.

Preferably the first flow rate is 10-500 μL/hr, more preferably 20-100 μL/hr;

Preferably the second flow rate is 500-3000 μL / hr, more preferably 800-1000 μL / hr;

A preferred third flow rate is from 500 to 3000 μL/hr, more preferably from 800 to 1000 μL/hr.

In the technical solution described above, specifically, the formed aqueous phase droplets are from 0.1 to 30 s, preferably from 0.1 to 10 s, from the upstream water-oil two-phase intersection channel to the second heavy oil phase blending channel; to ensure alginic acid The prepolymer is gelled in the chip channel to obtain an alginate microgel. The time during which the microgel particles are blended with the second heavy oil to the aqueous solution phase is 0.1-30 s, preferably 1-10 s; thereby ensuring sufficient mixing of the two phases while avoiding the residence time of the microgel particles under crosslinking conditions. Long resulting in cytotoxicity.

In the above technical solution, specifically, the ratio of the flow rate of the aqueous phase to the flow rate of the first heavy oil phase is between 0.01 and 1, preferably between 0.1 and 0.5; the ratio of the first heavy oil phase flow rate to the second heavy oil phase flow rate It is 1:0.5 to 50, preferably 1:0.5 to 10.

Another object of the present invention is to disclose the microgel product prepared by the microfluidic technology of the one-step continuous preparation of calcium alginate microgel as described above; the microgel has a diameter of 5 to 1000 μm; The dispersion coefficient of the particle size distribution is from 1 to 6%. The microgel prepared by the method of the invention is prepared from a microfluidic chip into a water-in-oil emulsion dispersed in the oil phase, and is injected into the aqueous solution through the outlet to spontaneously disperse in the aqueous solution. The microgel is dispersed in the oil phase for 1 to 60 seconds; when the living cells are embedded in the microgel, the cells stay in the emulsion for 1 to 60 seconds, and the cell survival rate is >85%. .

Another object of the present invention is to disclose a microfluidic technique for continuously preparing multi-chamber calcium alginate microgel particles in a one-step process, which is capable of continuously preparing a microfluidic calcium alginate microgel by the one-step method described above. The technical difference is: increasing the type of hydrogel prepolymer solution and correspondingly increasing the number of water phase input ports on the microfluidic chip;

Usually, the microcavity preparation of a single chamber requires 3 input ports (1 water phase input, 2 oil phase input); multi-chamber microgel preparation requires more than 2 aqueous phase solution input ports, the other unchanged . Specifically, the aqueous phase solution input port is used to input the hydrogel prepolymer solution, and therefore, when a person skilled in the art prepares a multi-chamber calcium alginate microgel as needed, the water needs to be added. The type of gel prepolymer solution, correspondingly, also requires an increase in the number of aqueous phase solution inlets.

When preparing the multi-chamber calcium alginate microgel particles, according to the method of step 1, a plurality of biologically active substances and/or nanoparticles are used to prepare a hydrogel prepolymer solution for constituting different chambers of the microgel. Different hydrogel prepolymer solutions are injected as two-dimensional, three-dimensional or multi-dimensional parallel fluids from multiple water phase input ports on the microfluidic chip, and after being concentrated into an aqueous phase solution, follow

steps

2, 3, (2), ( The method of 3) prepares multi-chamber microgel particles.

In the above technical solution for preparing multi-chamber microgel particles, specifically, the total flow velocity between the aqueous fluids of the plurality of input alginic acid hydrogel prepolymers is 5-2000 μL/hr; The difference between the input flow rates of the prepolymer aqueous solution is 1-100 times and is related to the size of the multi-chamber microgel chamber.

In the above technical solution for preparing the multi-chamber microgel particles, in the specific crosslinking process, the gelatinization reaction of the plurality of biologically active substances and/or the nanoparticle-containing alginic acid solution contained in the droplets simultaneously occurs. Rapid solidification forms alginic acid microgels with a multi-chamber structure; the concentration of alginic acid in each phase of the hydrogel prepolymer solution constituting the different chambers of the microgel is 1-10 times the concentration difference ( That is, the difference in concentration of the alginic acid raw materials in each chamber).

Beneficial effect

1) One-step preparation of microgels for immobilizing living cells and biologically active substances, continuous preparation of multi-chamber microgels, suitable for industrial applications.

2) The method is based on alginate microgel material, has good biocompatibility, low cytotoxicity, and the microgel stays in the emulsion for less than 30 seconds, and is quickly collected in an aqueous solution, which is beneficial to the encapsulated material organism. The retention of activity, the survival rate of the immobilized cells is high.

3) The micro-flow chip channel design can realize the preparation of complex three-dimensional microgels, and the number of microgel chambers can reach more than 6; continuous, high-throughput preparation of carrier-loaded or drug-loaded microgel particles can be realized.

4) Through the microfluidic chip design, the microgel has a size range of 10-800 μm, and the particle size distribution is narrow, and the dispersion is less than 5%. The size of the droplet is determined by the scale of the channel. The larger the channel size, the larger the droplet. At the same time, the ratio of the two flow rates of water and oil also affects the droplet size. The larger the internal phase velocity, the larger the droplet.

5) Preparation of microgels that can achieve different cross-linking modes of hydrogel materials.

6) The encapsulation efficiency of the bioactive substance reaches 100%.

7) Microgels can act as extracellular matrices, supporting the survival and function of cells in microgels.

8) The microgel particles can be crosslinked as basic structural units to obtain a cell-loaded scaffold having a macroscopic structure, or used as an injectable material for cell transplantation.

DRAWINGS

Figure 1 is a schematic diagram showing the preparation of alginate microgels by a one-step microfluidic chip. 5 in Fig. 1 means that alginic acid in the droplets starts to be crosslinked to form microgel particles.

2 is a schematic structural view of a typical T-type mixing channel described in Embodiment 1. 1 is an aqueous phase containing a biologically active substance, and 2 is an oil phase of a continuous phase.

3 is a schematic structural view of a typical fluid focusing mechanism mixing passage described in Embodiment 1. 1 is an aqueous phase containing a biologically active substance, and 2 is an oil phase of a continuous phase.

Figure 4 shows microgel particles dispersed in an aqueous phase solution by a one-step process using a two-layer fluid focusing microfluidic chip. (a) is a phase-separated aqueous phase after the emulsion is destroyed, which contains a large amount of microgel spheres (c), which are encapsulated with a green fluorescently-labeled dextran (molecular weight 5 kDa) due to a small molecular weight. It can be quickly infiltrated from the microgel; (b) is the oil phase of the continuous phase.

Figure 5 is a graph showing the size distribution of microgel particles prepared by the method described in Example 1.

Figure 6 is a schematic diagram showing the design of a microfluidic chip for preparing a water-in-oil emulsion droplet in Comparative Example 1. 4 in Fig. 6 means that alginic acid in the droplets starts to be crosslinked to form microgel particles.

Figure 7 is a microgel droplet dispersed in an oil phase prepared by a fluid focusing microfluidic chip as described in Comparative Example 1. (a) is an emulsion droplet prepared using a microfluidic chip (since the water phase is less dense than the oil phase, so it is deposited in the upper layer), (c) the droplet contains crosslinked microgel particles in which green fluorescence is encapsulated. Labeled dextran (molecular weight 5 kDa); (b) is the oil phase of the continuous phase.

Figure 8 shows the microgel particle size formed at different flow ratios.

Figure 9 shows the preparation of cell-loaded microgel particles dispersed in an aqueous phase solution by a one-step process using a two-layer fluid focusing microfluidic chip.

Figure 10 is a graph showing the viability of cells immobilized by microgels by different microfluidic chip methods.

Figure 11 is an alginate gel prepared using different crosslinkers. Wherein, A is an alginate microgel prepared by using the method described in Comparative Example 1, using Ca-NTA as a crosslinking reaction initiator, and B is the method described in Comparative Example 2, using CaCO ₃ nanoparticles as a crosslinking reaction. Alginate gel prepared by the agent.

Figure 12 is a schematic diagram of the preparation of a multi-chamber alginate microgel by a one-step microfluidic chip process, wherein 1, 2, 3, 4, n- constitute a hydrogel prepolymer of different chambers of the microgel. Solution, A-water phase, B-fluorinated oil, C-oil phase containing perfluoroalcohol, 4D output channel, E- is the alginic acid in the droplets begins to be crosslinked to form microgel particles; hydrogel pre- The polymer excites the cross-linking, and the multiple internal phase blending zone M1 forms a water-in-oil emulsion droplet blend to remove the M2 and the third heavy oil phase blending zone M3.

Figure 13 is a view showing the microgel particles of two chambers prepared by the preparation method described in Example 4.

Figure 14 is a view showing the microgel particles of three chambers prepared by the preparation method described in Example 5.

Figure 15 is a view showing the microgel particles of four chambers prepared by the preparation method described in Example 6.

Figure 16 is a microgel microparticle dispersed in an aqueous phase solution prepared by a two-layer fluid focusing microfluidic chip in a method as shown in Example 7. Figure A is the use of different two-phase aqueous alginic acid prepolymer solutions as input to control the size of the dual chamber microgel chamber by adjusting the flow ratio between the two input phases. In Fig. B, a, b, c, and d are fluorescence micrographs of the microgel particles obtained by the corresponding different preparation parameters in the graph of Fig. A, respectively.

Figure 17 is a microgel particle having a two-chamber structure prepared by the method described in Example 8.

Figure 18 is a diagram of the method of Example 9 using a dual chamber microgel as a template to achieve controlled, single cell level three dimensional immobilization and assembly of two different cells. Figures A and C are fluorescence micrographs of different magnifications, and Figures B and D are photomicrographs of the visible field. The scale in the figure is 50 μm.

Detailed ways

The following non-limiting examples are intended to provide a more complete understanding of the invention, but not in any way. Further, in the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and materials, reagents and the like used can be purchased from a biological or chemical reagent company.

Perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer surfactant was purchased from Beijing Anxin Micro-Nano Technology Co., Ltd.; all chemical reagents were purchased from sigma unless otherwise specified.

Example 1

As shown in FIG. 1 , a microfluidic device includes a first input channel, 2 second input channels, 3 third input channels, 4 output channels, 5 hydrogel prepolymer cross-link channels, and M1 is water. The two blended regions of the oil, the two phases that are not blended pass through a microfluidic channel having a "T" shaped structure (as shown in Figure 2) or a fluid focusing structure (shown in Figure 3). M2 is a microfluidic channel in which an emulsion prepared upstream is blended with a second heavy oil. The inner surface of the microchannel is subjected to a hydrophobic treatment.

Sodium alginate and fluorescein-labeled dextran (molecular weight: 10 kDa) were dissolved in deionized water to obtain an aqueous solution having a sodium alginate content of 1 w/v% and a fluorescein-labeled dextran content of 0.01 w/v%. Then, an aqueous solution of a chelate solution having a final concentration of 100 mM calcium ion-aminotriacetic acid (Ca-NTA) was added; the aqueous solution having the above configuration was used as the aqueous phase of the water-in-oil emulsion to enter the first input channel. A mixed solution of perfluorinated ethers (PFPE) surfactant and acetic acid added to the perfluorooctane oil solution is introduced into the second input channel as the first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the above oil phase was 2 v/v%, and the concentration of acetic acid was 1 v/v%. The perfluoropentanol was blended with perfluorooctane oil to obtain a second heavy oil phase having a perfluoropentanol content of 10 v/v% entering the third third input channel.

The aqueous phase solution and the first heavy oil phase are injected into the first layer of the "fluid focusing" microchannel of the microfluidic device from the first and second input ports by a constant current pump through a syringe (the chip structure design is as shown in FIG. 1). ), by mixing with the first heavy oil through a microfluidic channel having a fluid focusing structure (Fig. 3), the oil phase shears the aqueous phase into a water-in-oil single emulsion droplet of uniform size distribution. Among them, the flow rate of the internal phase was 100 ul / hr, and the flow rate of the first heavy oil phase was 1000 ul / hr. As the acetic acid enters the aqueous phase droplets in the oil phase, the pH in the droplets decreases, and the Ca ²⁺ ions that are originally chelated with the NTA molecules are no longer stable, forming free Ca ²⁺ ions, which are further combined with algae. The acid polymer chain forms an ionic bond, which initiates cross-linking of alginic acid with calcium ions to form a hydrogel. Therefore, microgel particles are formed in the emulsion droplets, and are stably dispersed in the oil phase due to the presence of the surfactant.

Subsequently, the above-mentioned microgel-dispersed water-in-oil single emulsion flows through the downstream second layer of microchannels having a "fluid focusing" structure (Fig. 3), and perfluoropentanol having a perfluoropentanol content of 10 v/v%. The second heavy oil composed of octane is blended. The flow rate of the second heavy oil phase was 1000 ul/hr. After the above mixed fluid is gradually blended through the U-shaped mixed microchannel, the perfluoropentanol in the second heavy oil phase elutes the perfluoroether surfactant which originally stabilizes the water-oil interface, so that the water-oil interface is no longer stable, and the water The phase droplets (i.e., microgel particles) are difficult to continue to be stably dispersed in the oil phase, and thus the microgels are phase separated from the oil phase. That is, a dispersion of the cell-loaded microgel particles in water is obtained. As shown in Figure 4, the prepared microgel particles were dispersed in water and separated from the oil phase. It was observed by fluorescence micrographs that the microgel was dispersed in water and the fluorescein-labeled dextran in the initial aqueous phase solution. Due to the small molecular weight, it quickly penetrates into the aqueous solution through the porous network of microgels. The size distribution of the obtained microgel particles was uniform as shown in FIG. Wherein, the distance between the first heavy fluid focusing microchannel and the second heavy fluid focusing microchannel is 2 cm to ensure that the alginic acid microgel is completely cured, and the emulsion is destroyed in the downstream channel, so that the water and oil are two The phase can be separated in the microfluidic chip channel to achieve one-step cell embedding.

Comparative Example 1 Alginic Acid Two-Step Method

The microfluidic device shown in FIG. 6 dissolves sodium alginate grafted with green fluorescent molecules in deionized water, followed by an aqueous solution of a calcium-ammonia triacetic acid (Ca-NTA) chelate, wherein the fluorescently labeled seaweed The sodium concentration was 1% by weight and the Ca-NTA concentration was 100 mM; the aqueous solution having the above configuration was used as the aqueous phase of the water-in-oil emulsion. The perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer was added as a surfactant to perfluorobutyl-methyl ether to obtain a mixed oil phase solution as the oil of the water-in-oil emulsion system. phase.

The aqueous phase solution and the oil phase solution are injected into the microchannel of the microfluidic device through the first and second input ports through a constant current pump (Fig. 6), and the oil phase shears the aqueous phase into a uniform size distribution. The water-in-oil single emulsion droplets. Among them, the internal phase flow rate was 100 ul / hr, and the oil phase flow rate was 1000 ul / hr. As shown in Fig. 7, since the density of the aqueous phase droplets is smaller than that of the oil phase, it floats on the upper layer of the oil phase, and the fluorescence micrograph shows that the size of the aqueous phase droplets is uniform, and the fluorescein-labeled in the initial aqueous phase The dextran is still encapsulated in the water-in-oil emulsion droplets.

Subsequently, acetic acid (final concentration: 0.05 vol%) was added to the emulsion prepared by the above method. The diffusion of acetic acid from the oil phase into the droplets of the aqueous phase causes a drop in the pH of the droplet, so that the Ca ²⁺ ion which is originally chelated with the NTA molecule is no longer stable, forming a free Ca ²⁺ ion, which is further combined with alginic acid. The polymer chain forms an ionic bond to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant. In order to convert the microgel particles into an aqueous phase solution, it is necessary to clean the dispersed microgel particles in the emulsion. 30 seconds after the acetic acid was added to the above emulsion, the microgel-dispersed emulsion was filtered to remove the oil phase, and then washed with a large amount of deionized water to remove the oil phase and the surfactant to realize dispersion of the microgel in the aqueous solution. in.

Comparative example 2

The sodium alginate grafted with the green fluorescent molecule is dissolved in deionized water, then the CaCO ₃ calcium carbonate nanoparticle is added and thoroughly mixed, wherein the concentration of the fluorescently labeled sodium alginate is 1 wt%, and the concentration of the calcium carbonate particle is 10 mg/ml. The aqueous solution in the above configuration was used as the aqueous phase of the water-in-oil emulsion. A perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer is added as a surfactant to a mixed solution obtained by adding a perfluorooctane oil phase solution as a water-in-oil emulsion system. Oil phase. The surfactant concentration in the above oil phase was 2 v/v%. The water-in-oil emulsion droplets were prepared by the method described in Comparative Example 1, and then acetic acid was added to the emulsion (final concentration was 0.5 v/v%). After soaking for 120 seconds, the microgel-dispersed emulsion was filtered to remove the oil phase. It is then rinsed with a large amount of deionized water to remove the oil phase and surfactant to effect dispersion of the microgel in the aqueous solution. Figure 11 is an alginate gel prepared using different crosslinkers. Wherein, A is an alginate microgel prepared by using the method described in Comparative Example 1, using Ca-NTA as a crosslinking reaction initiator, and B is the method described in Comparative Example 2, using CaCO ₃ nanoparticles as a crosslinking reaction. Alginate gel prepared by the agent. The micrograph of the microgel shows that the distribution of the fluorescently labeled alginic acid polymer in the microgel is not uniform due to the uneven distribution of the calcium carbonate nanoparticles in the sodium alginate prepolymer solution.

Comparative Example 3 Different Crosslinking Reaction Initiators

Sodium alginate grafted with green fluorescent molecules is dissolved in deionized water, followed by different crosslinking initiators: calcium-aminotriacetic acid (Ca-NTA), calcium-ethylenediaminetetraacetic acid (Ca-EDTA) CaCO ₃ calcium carbonate nanoparticles (average particle size about 300 nm) and well mixed, wherein the fluorescently labeled sodium alginate concentration is 1 wt%, the Ca-NTA and Ca-EDTA concentrations are 100 mM, and the calcium carbonate particle concentration is 10 mg/ml. (The calcium ion concentration was 100 mM); the aqueous solution having the above configuration was used as the aqueous phase of the water-in-oil emulsion. A perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer is added as a surfactant to a mixed solution obtained by adding a perfluorooctane oil phase solution as a water-in-oil emulsion system. Oil phase. The surfactant concentration in the above oil phase was 2 v/v%. The water-in-oil emulsion droplets were prepared by the method described in Comparative Example 1, and then acetic acid was added to the emulsion (final concentration was 0.1 v/v%). After soaking for different time, the microgel-dispersed emulsion was immediately filtered to remove the oil phase. Then, it is rinsed with a large amount of deionized water to remove the oil phase and the surfactant, and the microgel is dispersed in the aqueous solution. Subsequently, microgel formation was carried out by a fluorescence microscope, and if no gel spheres were formed, it was considered that the preparation parameters could not form a microgel. The results are shown in Table 1. Ca-NTA and Ca-EDTA act as cross-linking initiators to cross-link alginate in a very short time to obtain microgel particles, which are mainly due to the fact that both are calcium ions. The water-soluble chelate compound can be uniformly dispersed in an aqueous solution, and the dissociation reaction of calcium ions under the action of acid occurs instantaneously, which is favorable for rapid cross-linking of alginic acid. In contrast, CaCO ₃ nanoparticles dissociate calcium ions at a significantly slower rate under acid action, and it is difficult to achieve microgel cross-linking within 60 seconds of adding acetic acid to the emulsion phase, when the soaking time in acid is greater than 120 seconds. When the microgel is formed. The speed of cross-linking is directly related to two very important applications: 1. Experimental group for the formation of microgels for the preparation of microgel particles, using calcium carbonate nanoparticles as cross-linking initiators. Since the cells need to stay in an acidic environment for a long time, the cell survival rate is low (Fig. 10). 2. When Ca-NTA and Ca-EDTA are used as cross-linking initiators, the alginic acid prepolymer gels rapidly in the emulsion droplets, which makes it possible to clean the emulsion droplets on the chip. The present invention achieves one of the keys to the continuous preparation of microgel particles.

Table 1 shows the effect of different alginic acid crosslinking initiators and different crosslinking times on the formation of microgels as described in Comparative Example 3.

Example 2 for size control

Dissolving sodium alginate in deionized water to obtain an aqueous solution having a sodium alginate content of 2 w/v%, and dissolving calcium-ammonia triacetic acid (Ca-NTA) in deionized water to obtain an aqueous solution having a concentration of 100 mM; The blending configuration gave an aqueous solution in which the final solution concentration of sodium alginate was 1 w/v% and the concentration of Ca-NTA was 80 mM, and this aqueous solution was used as an aqueous solution of the internal phase of the water-in-oil emulsion. a mixed solution of perfluorobutyl-methyl ether, perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer fluorinated surfactant, and acetic acid, As the first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the above oil phase was 1 v/v%, and the concentration of acetic acid was 0.1 v/v%. Finally, perfluorooctyl alcohol was blended with perfluorobutyl-methyl ether to obtain a second heavy oil phase having a perfluorooctyl alcohol content of 10 v/v%.

Passing the above aqueous phase solution and the first heavy oil phase through a constant current pump into the first layer of "fluid focusing" microchannels of the microfluidic device from the first and second input ports, respectively, through a "T" shape structure or The microfluidic channel of the fluid focusing structure (Fig. 1) is blended with the first heavy oil, and the oil phase shears the aqueous phase into a water-in-oil single emulsion droplet of uniform size distribution. Wherein, the flow rate of the internal phase is 100 ul / hr, the flow rate ratio of the internal phase of the first heavy oil phase is changed by Qc / Qd (oil: water) < 20, and the flow rate of the second heavy oil phase is the same as the flow rate of the first heavy oil phase. As the acetic acid enters the aqueous phase droplets in the oil phase, the pH in the droplets decreases, and the Ca ²⁺ ions that are originally chelated with the NTA molecules are no longer stable, forming free Ca ²⁺ ions, which are further combined with algae. The acid polymer chain forms an ionic bond, and ion crosslinking occurs to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant. The result is shown in Figure 8. The size of the microgel particles decreases as the flow rate ratio of the aqueous phase to the first heavy oil phase decreases.

Example 3 Living Cell Embedding

Cell culture: with NIH3T3

For example, in fibroblast culture, the proliferation medium consists of DMEM containing 10% fetal bovine serum (FBS, Gibco). The culture conditions were 37 ° C, 95% relative humidity and 5% CO ₂ . Cell culture medium was changed every three days. Prior to use, cells were separated in phosphate buffered saline (PBS) using trypsin/EDTA solution (0.25% trypsin/0.02% EDTA) for 5 minutes and suspended in medium for use.

The green fluorescently labeled sodium alginate was dissolved in a cell culture medium DMEM solution to obtain a 2 w/v% aqueous sodium alginate solution. The cell suspension, the sodium alginate aqueous solution and the calcium-ethylenediaminetetraacetic acid (Ca-EDTA) chelate aqueous solution are blended to obtain a sodium alginate concentration of 1 w/v% in the mixed solution, and the final concentration of Ca-EDTA is 100 mM. The cell concentration was 10 ⁶ /ml. The above aqueous solution was used as an internal phase aqueous solution of the water-in-oil emulsion. a mixed solution of perfluorobutyl-methyl ether, perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer fluorinated surfactant, and acetic acid, As the first heavy oil phase of the water-in-oil emulsion system. The surfactant concentration was 1 w/v%, and the acetic acid concentration was 0.1 v/v%. The perfluorooctyl alcohol was blended with perfluorobutyl-methyl ether to obtain a second heavy oil phase having a perfluorooctyl alcohol content of 10 v/v%.

The aqueous phase solution and the first heavy oil phase are injected into the first layer of the "fluid focusing" microchannel of the microfluidic device from the first and second input ports respectively through a constant current pump through a syringe, and have a "T" shape structure ( Figure 2) Structure of the microfluidic channel (Figure 1), blended with the first heavy oil, the oil phase shears the aqueous phase into a water-in-oil single emulsion droplet of uniform size distribution. Among them, the flow rate of the internal phase was 100 ul / hr, and the flow rate of the first heavy oil phase was 1000 ul / hr. Subsequently, the acetic acid in the oil phase enters the droplets of the aqueous phase, resulting in a decrease in the pH of the droplets. The Ca ²⁺ ions which are originally chelating with the EDTA molecules are no longer stable, forming free Ca ²⁺ ions, which are further combined with algae. The acid polymer chain forms an ionic bond, which causes ion exchange of alginic acid to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant.

Subsequently, the above-described microgel-dispersed water-in-oil single emulsion flows through the second layer downstream to have a "fluid-focused" microchannel (Fig. 3), which is blended with the second heavy oil phase. The flow rate of the second heavy oil phase was 2000 ul/hr. After the above mixed fluid is gradually blended through the U-shaped mixed microchannel (Fig. 1), the perfluorooctyl alcohol in the second heavy oil phase elutes the surfactant which originally stabilizes the water-oil interface, so that the water-oil interface is no longer stable. The aqueous phase droplets (i.e., microgel particles) are difficult to continue to be stably dispersed in the oil phase, and thus the microgels are phase separated from the oil phase.

The DMEM cell culture medium was blended with a buffer HEPES having a buffer HEPES concentration of 10 mM, and the above solution was used as a collection aqueous solution. The output channel is blended with the collected aqueous solution through a conduit, and the blended water-oil mixture outputted from the chip is phase-separated in the collected aqueous solution, and the microgel is directly phase-separated into the aqueous phase solution to buffer the acid in the droplet, which is a preparation process Cell damage is minimized.

The cytotoxicity of the gel material was examined by using live-live fluorescent staining (LIVE/DEAD assay). First, the gel was washed with sterile PBS for 30 minutes before staining, and 2 mM calcein (green fluorescent dye-labeled living cells) and 4 mM ethidium bromide dimer (red fluorescent dye-labeled dead cells) were added at room temperature, and Use confocal laser scanning microscopy. As a result, as shown in the fluorescence micrograph of Fig. 9, the cell survival rate was found to be about 85%; the survival rate of the control experimental group which was directly cultured on the cell culture plate was comparable, demonstrating that the method of the present invention has a good biological phase. Capacity (results shown in Figure 10).

Comparative Example 4 Traditional Emulsion Method (Cell Embedding)

A cell-loaded microgel was prepared using a two-step preparation process for preparing a microgel in Comparative Example 2. The green fluorescently labeled sodium alginate was dissolved in a cell culture medium DMEM solution, followed by an aqueous solution of a chelate solution of calcium-ethylenediaminetetraacetic acid (Ca-EDTA) and a cell dispersion in which the concentration of the fluorescently labeled sodium alginate was 1% by weight. The Ca-NTA concentration was 100 mM, and the cell concentration was 10 ⁶ /ml; the aqueous solution having the above configuration was used as the aqueous phase of the water-in-oil emulsion. The perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer was added as a surfactant to perfluorobutyl-methyl ether to obtain a mixed oil phase solution as the oil of the water-in-oil emulsion system. phase.

The aqueous phase solution and the oil phase solution are injected into the microchannel of the microfluidic device through the first and second input ports through a constant current pump (Fig. 6), and the oil phase shears the aqueous phase into a uniform size distribution. The water-in-oil single emulsion droplets. Among them, the internal phase flow rate was 100 ul / hr, and the oil phase flow rate was 1000 ul / hr. As shown in Figure 7, since the density of the aqueous phase droplets is smaller relative to the oil phase, it floats on the upper layer of the oil phase, and the fluorescence micrograph shows that the size of the aqueous phase droplets is uniform and the fluorescence in the initial aqueous phase is obtained. The dextran-labeled dextran is still encapsulated in a water-in-oil emulsion droplet.

Subsequently, acetic acid (final concentration: 0.1 vol%) was added to the emulsion prepared by the above method. The diffusion of acetic acid from the oil phase into the droplets of the aqueous phase causes a drop in the pH of the droplet, so that the Ca ²⁺ ion which is originally chelated with the NTA molecule is no longer stable, forming a free Ca ²⁺ ion, which is further combined with alginic acid. The polymer chain forms an ionic bond to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant. In order to convert the microgel particles into an aqueous phase solution, it is necessary to clean the dispersed microgel particles in the emulsion. 300 seconds after the addition of acetic acid to the above emulsion, the microgel-dispersed emulsion was filtered to remove the oil phase, and then washed with a large amount of cell culture medium DMEM to remove the oil phase and the surfactant, and the cell-loaded microgel was finally Disperse in aqueous DMEM solution.

Although the above two-step preparation microgel technology can be used for embedding biological active substances such as cells or protein drug molecules, since the cells are in contact with the acidic solution for too long during embedding and preparation, the cell survival rate is seriously affected. As shown in Figure 10, the cell microgel particles were prepared using the two-step method of the comparative example, and the viability of the cells in the obtained microgel was extremely low, and the normal positive control (the cells were adherently cultured on a two-dimensional culture plate). ) Compared to the significant drop.

Example 4 Preparation of Multi-chamber Microgels

As shown in FIG. 12, a microfluidic device for preparing a multi-chamber microgel includes first, second, third, fourth, ..., n input channels, which together form an internal phase input channel. A, B is the first heavy oil phase input channel, C is the second heavy oil phase input channel, D is the output channel, E is the hydrogel prepolymer cross-linking channel, and M1 is the microgel different chamber hydrogel pre-form The mixed region of the polymer solution utilizes the characteristics of the fluid in the microfluidic channel to form a stable parallel fluid. The prepolymer solution of different phases forms a stable parallel flow after being input into the chip, and the material diffusion and exchange between the fluids are limited. M2 is a blending zone of water and oil, and the two phases that are not blended pass through a microfluidic channel having a "T" shape (as shown in Figure 2) or a fluid focusing structure (as shown in Figure 3). M2 is a microfluidic channel in which an emulsion prepared upstream is blended with a second heavy oil. The inner surface of the microchannel is subjected to a hydrophobic treatment.

Dissolving sodium alginate in deionized water to obtain alginate hydrogel prepolymer solution; blending ammonia triacetic acid and calcium hydroxide in deionized water to form a chelate of calcium-aminotriacetic acid (Ca-NTA) Aqueous solution. The above two aqueous solutions were blended, followed by red or green fluorescein-labeled polystyrene nanoparticles (particle diameter 100 nM), which were uniformly dispersed to obtain a microgel-formed alginic acid hydrogel prepolymer solution. The content of sodium alginate was 1 w/v% with deionized water, and the content of fluorescein-labeled nanoparticles was 0.01 w/v%, followed by the addition of a final concentration of 100 mM calcium ion-aminotriacetic acid (Ca-NTA). An aqueous solution; the aqueous solution having the above configuration is used as the aqueous phase of the water-in-oil emulsion. A mixed solution of perfluorinated ethers (PFPE) surfactant and acetic acid added to the perfluorooctane oil phase solution is used as the first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the above oil phase was 2 v/v%, and the concentration of acetic acid was 1 v/v%. The perfluoropentanol was blended with the perfluorooctane oil to obtain a second heavy oil phase having a perfluoropentanol content of 10 v/v%.

The sodium alginate prepolymer prepared by dispersing the red fluorescent and green fluorescent nanoparticles, respectively, is input into the chip from the first and second input channels through the microfluidic channel structure as shown in FIG. 13 to form a parallel flow with stable flow velocity ( As shown in Fig. 13), the aqueous phase solution and the first heavy oil phase are injected into the first layer of the "fluid focusing" microchannel of the microfluidic device from the first and second input ports by a constant current pump, respectively (the chip structure design is as shown in Fig. 12), blended with the first heavy oil through a microfluidic channel having a fluid focusing structure (Fig. 2), the oil phase shearing the aqueous phase into a water-in-oil single emulsion droplet of uniform size distribution. Among them, the flow rates of the two internal phases were 100 ul/hr, respectively, and the flow rate of the first heavy oil phase was 1000 ul/hr. As the acetic acid enters the aqueous phase droplets in the oil phase, the pH in the droplets decreases, and the Ca ²⁺ ions that are originally chelated with the NTA molecules are no longer stable, forming free Ca ²⁺ ions, which are further combined with algae. The acid polymer chain forms an ionic bond, which initiates cross-linking of alginic acid with calcium ions to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant.

Subsequently, the above-mentioned microgel-dispersed water-in-oil single emulsion flows through the second layer downstream to have a "fluid focusing" microchannel (Fig. 2), and perfluorooctane having a perfluoropentanol content of 10 v/v%. The second heavy oil of the composition is blended. The flow rate of the second heavy oil phase was 1000 ul/hr. After the above mixed fluid is gradually blended through the U-shaped mixed microchannel (Fig. 12), the perfluoropentanol in the second heavy oil phase elutes the perfluoroether surfactant which originally stabilizes the water-oil interface, so that the water-oil interface No longer stable, the aqueous phase droplets (i.e., microgel particles) are difficult to continue to be stably dispersed in the oil phase, and thus the microgels are phase separated from the oil phase. The microgel particles having a two-chamber structure were obtained by outputting the above mixed output liquid through a catheter connection to the collected aqueous solution (Fig. 13B).

Example 5

An alginic acid prepolymer aqueous solution in which red fluorescent and green fluorescent labeled nanoparticles were respectively dispersed was prepared by the method described in Example 4, and three different internal phase aqueous solutions were separately injected using the internal phase aqueous solution input structure as shown in FIG. 14A. Parallel fluids of three different internal phase aqueous solutions are formed. Among them, the flow rates of the three internal phases were 100 ul/hr, respectively, and the flow rate of the first heavy oil phase was 1500 ul/hr. The microgel was further prepared by the method described in Example 4 to finally obtain microgel particles having three different chambers (Fig. 14B).

Example 6

An alginate prepolymer aqueous solution in which red fluorescent and green fluorescent labeled nanoparticles were respectively dispersed was prepared by the method described in Example 4, and four different internal phase aqueous solutions were separately injected using the internal phase aqueous solution input structure as shown in FIG. 15A. Parallel fluids of four different internal phase aqueous solutions were formed, wherein the flow rates of the four internal phases were 50 ul/hr and the first heavy oil phase flow rate was 1000 ul/hr. The microgel was further prepared by the method described in Example 4 to finally obtain microgel particles having four different chambers (Fig. 15B).

Example 7

An alginate prepolymer aqueous solution in which red fluorescent and green fluorescent labeled nanoparticles were respectively dispersed was prepared by the method described in Example 4, and two different internal phase aqueous solutions were separately injected using the internal phase aqueous solution input structure as shown in FIG. 13A. Forming parallel fluids of two different internal phase aqueous solutions, wherein adjusting the flow rate of the aqueous phase of the two phase internal phase is such that the flow rate ratio (Q1/Q2) is from 1 to 9, and the sum of the two phase flow rates is 100 ul/hr, first The heavy oil phase flow rate was 1000 ul/hr. The microgels were further prepared by the method described in Example 4 to finally obtain microgel particles of two different chambers having different chamber volume ratios (Fig. 16).

The sodium alginate grafted with the green fluorescent molecule is dissolved in deionized water, followed by the aqueous solution of the calcium-aminotriacetic acid (Ca-NTA) chelate, wherein the concentration of the fluorescently labeled sodium alginate is 1 wt%, and the concentration of Ca-NTA 100 mM; non-fluorescently labeled sodium alginate dissolved in deionized water, followed by a calcium ion-ammonia triacetate (Ca-NTA) chelate aqueous solution with a fluorescently labeled sodium alginate concentration of 1 wt%, Ca-NTA The concentration is 100 mM. Using the above two aqueous hydrogel prepolymer aqueous solutions as different internal phases, two different internal phase aqueous solutions were separately injected according to the method described in Example 4 using the internal phase aqueous solution input structure as shown in FIG. 13A to form two different Parallel fluid of the internal phase aqueous solution. Wherein, the flow rate of the two-phase internal phase aqueous solution was adjusted to be 50 ul/hr, the sum of the two-phase flow rates was 100 ul/hr, and the first heavy oil phase flow rate was 1000 ul/hr. The microgel was further prepared by the method described in Example 4 to finally obtain microgel particles having two chambers (Fig. 17).

Example 8

The NIH3T3 was cultured using the method described in Example 3.

Fibroblasts, the cells were live cells green and red fluorescent dye labeled tracer (U.S. ThermoFisher Scientific Corporation, live cells green and red tracking dye CellTracker ^TM). Alginic acid (American Sigma, medium viscosity) was grafted with red and green fluorescent dyes (Sigma, USA) to obtain fluorescently labeled alginic acid dissolved in cell culture medium DMEM solution.

First, a hydrogel prepolymer in which living cells were dispersed was placed as an aqueous phase: fluorescently labeled sodium alginate was dissolved in a cell culture medium DMEM solution to obtain a 2 w/v% aqueous sodium alginate solution. The cell suspension, the aqueous sodium alginate solution and the calcium-ethylenediaminetetraacetic acid (Ca-EDTA) chelate aqueous solution are blended to obtain a sodium alginate concentration of 1 w/v% in the mixed solution, and the final concentration of Ca-EDTA is 50 mM. . Among them, the green fluorescent labeled NIH3T3 living cells were dispersed in a red fluorescently labeled aqueous alginic acid solution, and the red fluorescent labeled NIH3T3 living cells were dispersed in a green fluorescently labeled aqueous alginic acid solution at a cell concentration of 10 ⁶ /ml. The above two cell dispersions were used as two internal phase aqueous solutions for preparing two chamber microgels. a mixed solution of perfluorobutyl-methyl ether, perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer fluorinated surfactant, and acetic acid, As the first heavy oil phase of the water-in-oil emulsion system. The surfactant concentration was 1 w/v%, and the acetic acid concentration was 0.1 v/v%. The perfluoropentanol was blended with perfluorobutyl-methyl ether to obtain a second heavy oil phase having a perfluoropentanol content of 10 v/v%.

According to the method described in Example 4, the two cell dispersions prepared above were respectively input into the chip from the first and second input channels through the microfluidic channel structure as shown in FIG. 13, thereby forming a parallel flow with stable flow velocity (Fig. 13). The aqueous phase solution and the first heavy oil phase are injected into the first layer of the "fluid focusing" microchannel of the microfluidic device from the first and second input ports by a constant current pump, respectively (the chip structure design is as shown in FIG. 12). By blending with the first heavy oil through a microfluidic channel having a fluid focusing structure (Fig. 2), the oil phase shears the aqueous phase into a water-in-oil single emulsion droplet of uniform size distribution. The flow rates of the two internal phases were respectively 50 ul/hr, and the flow rate of the first heavy oil phase was 1000 ul/hr. As the acetic acid enters the aqueous phase droplets in the oil phase, the pH in the droplets decreases, and the Ca ²⁺ ions that are originally chelating with the EDTA molecules are no longer stable, forming free Ca ²⁺ ions, which are further combined with algae. The acid polymer chain forms an ionic bond, which initiates cross-linking of alginic acid with calcium ions to form a hydrogel. Thus, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant.

Subsequently, the above-mentioned microgel-dispersed water-in-oil single emulsion flows through the second layer downstream to have a "fluid focusing" microchannel (Fig. 2), and a perfluoropentanol having a perfluoropentanol content of 10 v/v%. - The second heavy oil phase consisting of methyl ether is blended. The flow rate of the second heavy oil phase was 1000 ul/hr. After the above mixed fluid is gradually blended through the U-shaped mixed microchannel (Fig. 12), the perfluoropentanol in the second heavy oil phase elutes the perfluoroether surfactant which originally stabilizes the water-oil interface, so that the water-oil interface No longer stable, the aqueous phase droplets (i.e., microgel particles) are difficult to continue to be stably dispersed in the oil phase, and thus the microgels are phase separated from the oil phase. The DMEM cell culture medium was blended with a buffer HEPES (pH 7.2-7.4) in which the buffer HEPES concentration was 10 mM, and the above solution was used as a collection aqueous solution. The output channel is blended with the collected aqueous solution through a conduit, and the blended water-oil mixture outputted from the chip is phase-separated in the collected aqueous solution, and the microgel is directly phase-separated into the aqueous phase solution to buffer the acid in the droplet, which is a preparation process Cell damage is minimized. The mixed solution of the dispersed cell-loaded double-chamber microgel prepared by the above method was connected to the collected aqueous solution through a catheter connection, and microgel particles having two chamber structures were collected, and the different chambers were immobilized. Different kinds of individual living cells. The results are shown in the fluorescence micrograph of Figure 18.

Example 9 Preparation of Multi-chamber Microgels

The alginic acid hydrogel prepolymer solution (the sodium alginate content was 1 w/v% and the calcium-ethylenediamine tetraacetic acid chelate concentration was 100 mM) was prepared by the method described in Example 4, and the hydrogel was pretreated. Red fluorescent dye-labeled polystyrene nanoparticles (average diameter 100 nm) and magnetic iron oxide nanoparticles (average diameter 100 nm) were added to the solution to obtain a two-phase aqueous solution for preparing two chamber microgels, wherein the nanoparticles were prepared. The content was 0.01 w/v%; the aqueous solution of the alginic acid hydrogel prepolymer in which the different nanoparticles were dispersed in the above configuration was used as the two aqueous phase solutions for preparing the two chamber microgels. A mixed solution of perfluorinated ethers (PFPE) surfactant and acetic acid added to the perfluorooctane oil phase solution is used as the first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the above oil phase was 1 v/v%, and the concentration of acetic acid was 1 v/v%. The perfluoropentanol was blended with the perfluorooctane oil to obtain a second heavy oil phase having a perfluoropentanol content of 10 v/v%.

Using the microfluidic chip described in Example 4 and the preparation method and parameters, a microgel having a two-chamber structure was prepared, as shown in FIG. 19, in which two chambers were respectively loaded with red fluorescent dye-labeled poly Styrene nanoparticles (average diameter 100 nm) and magnetic iron oxide nanoparticles (black, average diameter 100 nm). And the resulting two-chamber microgel has a magnetic response, and the two-chamber microgel particles dispersed in the liquid are oriented to move under the attraction of the magnet and can be arranged in an order in the magnetic field.

Claims

The microfluidic technology for continuously preparing calcium alginate microgel by one-step method is characterized in that it comprises the following steps:

(1) Preparation solution

1 using water-soluble alginate as raw material to prepare an aqueous solution of alginic acid in water, adding a crosslinking reaction initiator, and dispersing the biologically active substance and/or the nanoparticle in the aqueous solution to obtain a hydrogel prepolymer solution as water Phase solution, where:

The crosslinking reaction initiator is selected from the group consisting of a chelate aqueous solution of calcium-ethylenediaminetetraacetic acid, a chelate aqueous solution of calcium-aminotriacetic acid, calcium carbonate nanoparticles, calcium sulfate nanoparticles, and calcium phosphate nanoparticles. Or a combination of several; the final concentration of the crosslinking reaction initiator is recorded as a calcium content of 10-1000 mM;

2 blending a fluorinated oil, a fluorinated surfactant and an acidic substance to obtain a first heavy oil phase;

3 blending fluorinated oil with perfluoro alcohol or perfluoric acid to obtain a second heavy oil phase;

(2) injecting the aqueous phase solution obtained in step 1 into the microfluidic chip at a first flow rate, injecting the first heavy oil phase solution from the second input port into the microfluidic chip at a second flow rate, and emulsification of the aqueous phase solution and the first heavy oil phase Channel blending to form a water-in-oil emulsion droplet;

(3) injecting the second heavy oil phase into the microfluidic chip from the third input port at a third flow rate, and the second heavy oil phase and the emulsion obtained in the step (2) are thoroughly blended in the mixing channel disposed downstream of the emulsification channel, and then outputted from the output The channel flows out of the chip; the output solution is input into the collected phase aqueous solution, and the calcium alginate microgel carrying the biologically active substance and/or the nanoparticle is dispersed in the aqueous phase of the collecting phase, and the aqueous phase solution is collected to obtain the final product.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the microfluidic chip has a fluid focusing structure, a T-shaped hybrid structure, a co-flowing type or a cross structure. The microfluidic channel has at least a 3-phase liquid inlet, as well as an emulsification channel and an output channel.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the alginic acid raw material is alginic acid, alginate, aqueous alginate solution or alginic acid, and has high water solubility. a mixture of molecules.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the total concentration of the alginic acid raw material in the prepolymer solution is 0.1-8 w/v%.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the embedded biologically active substance and/or nanoparticle described in the step (1) is selected from the following a combination of one or more of: living cells, water-soluble active protein drug molecules, nanoparticles;

The living cells are primary cultured cells, subcultured cells, cell culture cells, and hybrids;

The water-soluble active protein drug molecule is a protein drug, a polypeptide drug, an enzyme drug, and a cell growth factor;

The nanoparticles are metal or metal oxide nanoparticles such as nano gold, nano silver, nano iron oxide, high molecular polymer nanoparticles such as polyethylene, polypropylene, polystyrene, polymethyl methacrylate and polylactic acid. a fat-soluble vitamin, a quinolone-based water-insoluble drug nanoparticle, a hydroxyapatite, a silica, a calcium phosphate, a carbon nanotube, a graphene inorganic non-metal material nanoparticle; the nanoparticle size diameter is greater than 5-1000 nm.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the fluorinated oil is selected from the group consisting of one or a combination of the following: perfluoropentane, all Fluorohexane, perfluoroheptane, perfluorobutyl-methyl ether, perfluorooctane, perfluorodecane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane , perfluorotetradecane, perfluoropentadecane, perfluorohexadecane, perfluoroheptadecane, perfluorodecalin.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the fluorinated surfactant is selected from the group consisting of one or a combination of the following: perfluoroether, Perfluoroalkyl acid perfluoroether-polyvinyl alcohol block copolymer, perfluoroether-polyvinyl alcohol-perfluoroether block copolymer surfactant; wherein the concentration of the fluorinated surfactant is from 0.1 to 10% by weight.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1 is characterized in that the acidic substance is selected from one or a combination of the following: sulfuric acid, nitric acid, hydrochloric acid, carbonic acid, phosphoric acid. , acetic acid or citric acid; the concentration of the acidic substance in the mixed solution is 0.001-20v / v%;
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the perfluoroalcohol in the second heavy oil phase is selected from one or a combination of the following: 22,33,444-heptafluoro-1-butanol, perfluoroundecyl alcohol, 1H, 1H-perfluoro-1-dodecanol, 1H, 1H-perfluoro-1-heptanol, 1H, 1H, 2H, 2H-perfluorooctyl alcohol, 1H, 1H-perfluorooctyl-1-ol, 1H, 1H-perfluoro-1-nonanol, 1H, 1H, 2H, 2H-perfluoro-1-dodecanol, 1H ,1H,2H,2H-perfluoro-1-nonanol, 1H, 1H-perfluoro-1-tetradecyl alcohol, perfluoropentanol, perfluorohexanol, perfluoroheptanol, perfluorooctyl alcohol , perfluorodecyl alcohol, perfluorononanol; said perfluoro acid is selected from one or a combination of the following: perfluorododecanoic acid, n-perfluoropentanoic acid, 5H-perfluoropentanoic acid, perfluorooctanoic acid, Perfluorodecanoic acid, perfluorodecanoic acid, perfluoroheptanoic acid, perfluorohexanoic acid, perfluorobutyric acid, perfluoroundecanoic acid; in the second heavy oil phase, perfluoroalcohol or perfluoric acid in fluorinated oil The concentration is 1-100 v/v%.
The microfluidic technology for continuously preparing a calcium alginate microgel according to claim 1 according to claim 1, wherein the aqueous phase collecting solution described in the step (4) is non-cytotoxic and has a pH buffering range of 6- 8 buffer solution, the buffer ion concentration of 10-2000 mM.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the first flow rate is 5-2000 μL/hr, the second flow rate is 200-20000 μL/hr, and the third flow rate It is 200-20000 μL/hr.
The microfluidic technology for continuously preparing calcium alginate microgel by the one-step method according to claim 1, characterized in that the formed aqueous phase droplets are from the upstream water-oil two-phase intersection channel to the second heavy oil phase blending channel time. For 0.1-30 s, the time from the blending of the microgel particles to the second heavy oil to the phase of collecting the aqueous solution is 0.1-30 s.
The microfluidic technology for continuously preparing a calcium alginate microgel by the one-step method according to claim 1, wherein the ratio of the aqueous phase flow rate to the flow rate of the first heavy oil phase is 0.01 to 1; the first heavy oil phase flow rate The flow rate ratio of the second heavy oil phase is 1:0.5 to 50.
The microgel prepared by the microfluidic technology for continuously preparing the calcium alginate microgel by the one-step method according to claim 1, wherein the microgel has a diameter of 5 to 1000 μm; the dispersion of the particle size distribution The coefficient is in the range of 1 to 6%, and the time during which the microgel is dispersed in the oil phase is 1 to 60 seconds; when the living cells are embedded in the microgel, the cells stay in the emulsion for 1 to 60 seconds, and the cells are Survival rate > 80%.
Microfluidic technology for continuously preparing multi-chamber calcium alginate microgel particles by one-step method, characterized in that when the bioactive substances and/or nanoparticles in step 1 are various, they are different according to the method of step 1 respectively. Bioactive substance and/or nanoparticle preparation hydrogel prepolymer solution, respectively injected from multiple water phase input ports on the microfluidic chip, and then prepared according to steps 2, 3, (2), (3) Room microgel particles.
The microfluidic technology for continuously preparing multi-chamber calcium alginate microgel particles by the one-step method according to claim 15, wherein the total flow velocity of the aqueous phase fluid of the plurality of hydrogel prepolymers is 5-2000 μL /hr.
The microfluidic technology for continuously preparing multi-chamber calcium alginate microgel particles by the one-step method according to claim 16, wherein the flow rate difference between the aqueous fluids of the plurality of hydrogel prepolymers is 1 -100 times.
The microfluidic technology for continuously preparing multi-chamber calcium alginate microgel particles by the one-step method according to claim 15, wherein when the bioactive substance and/or the nanoparticles of the step 1 are various, the water is coagulated. The concentration of alginic acid in the gel prepolymer solution is 1-10 times different.