CN107930542B

CN107930542B - Micro-fluidic technology for continuously preparing calcium alginate microgel by one-step method

Info

Publication number: CN107930542B
Application number: CN201711115297.6A
Authority: CN
Inventors: 王华楠; 张丽媛
Original assignee: Shenzhen Hua-Nova Biotechnology Ltd
Current assignee: Shenzhen Sino Biological Technology Co., Ltd.
Priority date: 2017-11-13
Filing date: 2017-11-13
Publication date: 2020-11-20
Anticipated expiration: 2037-11-13
Also published as: WO2019091145A1; CN107930542A

Abstract

The invention realizes the one-step method continuous preparation of the multi-cavity calcium alginate microgel capable of immobilizing bioactive substances based on the microfluidic technology, and can realize high-throughput and continuous production of the microgel material. Firstly, different hydrogel prepolymer solutions in a microfluidic channel can form a parallel and stable flow field, so that the hydrogel prepolymer solutions forming different chambers of the microgel are injected into a microfluidic chip to form a water phase solution of a multiphase parallel fluid, then the water phase solution and a non-blended fluid (oil phase) are mixed through a T-shaped channel or a microfluidic channel designed by fluid focusing to form a water-in-oil emulsion liquid drop, and the cross-linking of alginic acid in the liquid drop is initiated immediately after the liquid drop is formed, so that the preparation of the multi-chamber microgel is realized; and then, the emulsion liquid drops are cleaned on the microfluidic chip, so that the microgel is rapidly conveyed to a water phase, the calcium alginate microgel immobilized with bioactive substances is prepared by a one-step method, and the method is particularly suitable for industrial application.

Description

Micro-fluidic technology for continuously preparing calcium alginate microgel by one-step method

Technical Field

The invention belongs to the field of material micromachining, the technical field of biological material preparation, the field of tissue engineering and cell therapy, and particularly relates to a method for preparing microgel embedded and immobilized living cells or bioactive molecular drugs by using a microfluidic device.

Background

In recent years, hydrogels have been widely used in many biomedical fields, especially in the field of tissue engineering, where hydrogels have become very important biomaterials. The hydrogel is a cross-linked network consisting of water-insoluble but water-swellable macromolecules, is similar to human tissues and organs, and consists of a water-rich porous network, and the porous structure is similar to the human tissues and organs and is very beneficial to effective exchange of substances. Meanwhile, some hydrogels composed of natural polymers have good biocompatibility and biodegradability and mechanical properties similar to those of human tissues, so that the hydrogels can be used as tissue engineering scaffold materials to support cell growth and tissue regeneration. One branch of the hydrogel material is composed of a biomacromolecule prepolymer which has good biocompatibility and mild gelation conditions, so that the material is widely used for three-dimensional encapsulation of cells. Among them, many natural polymer materials including collagen, fibrin, or alginate are used in a large amount for embedding cells. Although hydrogel is extensively studied and used in the field of tissue engineering as a biomedical material, the conventional hydrogel cell-embedding technology still has many problems to be solved in the application of tissue engineering: 1) the block hydrogel has larger size (>1cm), the pore size of a polymer network is in a nanometer level, and the diffusion rate and distance of biological macromolecules are low, so that the survival rate of living cells embedded in the block hydrogel is reduced due to low exchange efficiency of nutrient substances and metabolites; 2) the hydrogel as a carrier for bioactive substance delivery has no injectability, can only be implanted in a block, and cannot be implanted in a minimally invasive intervention manner; 3) when the block hydrogel is used in a macromolecule or cell embedding process, the uniform distribution of a load substance in a gel system is difficult to ensure. In contrast, the use of micro-gels on a micron scale as carriers for biologically active substances provides an effective solution to the above problems. Because the microgel has small size, the microgel is beneficial to rapid diffusion of substances, and cells encapsulated in the microgel can effectively exchange with external nutrient components, signal factors, excrement and the like. Meanwhile, the microgel particles can be directly injected, and an effective way is provided for the direct intravenous injection transplantation of active macromolecular drugs and living cells. Therefore, the realization of high-throughput preparation of the cell-loaded microgel is beneficial to promoting the development and clinical application of tissue engineering and cell therapy technology.

Emulsion technology is widely used to prepare microgels encapsulating biologically active substances, such as biologically active macromolecular drugs or living cells. The method generally utilizes water-oil two-phase unblended fluid to form a dispersed phase and a continuous phase under the action of shearing force. The typical preparation method is as follows: first, a bioactive substance (e.g., bioactive macromolecules or living cells) is dispersed in an aqueous solution of a hydrogel prepolymer, which is blended with a continuous oil to form a water-in-oil single emulsion, and then a polymerization or crosslinking reaction is induced to gel the hydrogel prepolymer within the droplets to obtain a solidified microgel. These conventional emulsion methods have the following problems: 1) living cells need to be dispersed in the dispersed microemulsion for a long time, and the cells cannot exchange nutrients and gas with the outside of liquid drops, so that the metabolic activity of the cells is gradually weakened, and the activity of the cells is reduced when the cells are in the emulsion for a long time; 2) in order to form a stable emulsion, a surfactant is required to be added into an oil phase or an aqueous phase, and the surfactant can damage cell membranes to generate cytotoxicity, so that the potential cytotoxicity problem exists when the cells are contacted with the surfactant for a long time; 3) the use of cytotoxic cross-linking agents or initiators of cross-linking reactions is often required to initiate polymerization or cross-linking of the hydrogels, and the long term exposure of cells to these components is also associated with cytotoxicity. Therefore, the conventional emulsion method for preparing cell-immobilized microgel has difficulty in maintaining high cell survival rate and metabolic activity of cells, and requires additional time-consuming and labor-consuming oil phase cleaning after immobilization, thus being not applicable to industrial continuous processing.

Recently, a batch of micro-processing technologies for cell immobilization, including photolithography, micro-template technology, emulsion technology, etc., have been emerging, and the problem of how to maintain the survival rate of immobilized cells exists, so that it is difficult to be really applied to the industrial scale production of cell-immobilized microgel. Moreover, these conventional techniques are based on batch production processes, which cannot achieve high-throughput continuous preparation, and product performance varies from batch to batch.

The micro-fluidic droplet technology is a micro-processing technology for accurately controlling immiscible multiphase fluid based on a micro-fluidic chip, and can realize continuous sample introduction and fast produce microgel or microcapsules with monodispersity and accurately controllable size. Compared with the traditional water-in-oil (W/O) or oil-in-water (O/W) single emulsion droplet technology, single emulsion droplets with uniform size can be prepared by a micro-fluidic device with a T-shaped flow channel or a fluid focusing structure. And the monodisperse microgel can be prepared by different polymerization modes by using the template. However, as with conventional emulsion methods, microfluidic single emulsion droplet technology is not suitable for continuous processing of cell-loaded microgels. This is due to cytotoxicity caused by prolonged exposure of the immobilized cells to oil phase, surfactants, cross-linking agents, etc. during the preparation of the sample. Therefore, it is necessary to intermittently collect the product and rapidly break the emulsion to transfer the cells into a biocompatible aqueous solution to maintain the cell viability of the microgel prepared, and the process is time consuming and laborious. Therefore, how to realize that the cells are immobilized into the micro gel and quickly separated from the oil phase greatly ensures the activity of the cells, can realize continuous preparation and improves the production efficiency.

In addition, the water-in-water (W/W) emulsion technology can also realize the preparation of the microgel. The method uses a two-phase fluid containing water, can form liquid drops by utilizing no mixing between the two phases, and uses the liquid drops as a template to realize the production of the microgel particles in the aqueous solution. The method avoids additional step of breaking emulsion, and can realize one-step cell embedding. However, this system is limited to a combination of specific unblended aqueous solutes, such as dextran and polyethylene glycol, and the aqueous solute concentration is high, 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 the purpose of embedding cells in microgel in a solid carrier and maintain the survival rate of cells is still a technical problem to be solved in the field. This prompts researchers to develop new technologies that are simpler, more efficient, and can realize industrial production. Although the microgel material has important application potential in the field of biomedicine, the function and the structure of the microgel material are relatively single, and more functional expansion is difficult to perform. Therefore, multi-chamber microgel materials have recently become a hot spot for biomedical applications and research. The multi-cavity microgel particles are microgel materials consisting of more than two different cavities, and the different cavities can be the same or different materials. Compared with the traditional single-cavity microgel, the multi-cavity microgel has multiple structures and can be endowed with different functions. Due to the complex structure and function, the multi-cavity microgel has important application potential in the field of biomedicine, particularly in the field of immobilization and delivery of bioactive substances. The conventional method for preparing multi-cavity microgel materials is generally to emulsify aqueous solutions of several different hydrogel monomers or prepolymers and rapidly initiate polymerization or crosslinking, so as to obtain stable microgel particles with a multi-cavity structure. The commonly used materials for preparing the multi-cavity microgel comprise hydrogel prepolymer which can be crosslinked by light initiation, alginate which can be rapidly crosslinked by ions and the like. However, these rapid and vigorous crosslinking reactions usually have strong biological toxicity, which affects the biological activity of the immobilized material, especially when embedding cells, these methods generally have strong cytotoxicity, thus causing the cell survival rate to be significantly reduced, and limiting the development and application in this field. Due to the complex structure and function, the multi-cavity microgel has important application potential in the field of biomedicine, particularly in the field of immobilization and delivery of bioactive substances.

Existing techniques for preparing multiwell microgels include microfluidic chip technology [ y.du, e.lo, s.ali, a.khademhosseini, p.natl acad.sci.2008,105, 9522-9527; s.seiffert, angelw.chem.int.ed 2013,52, 11462-; b) d.dendikuri, s.s.gu, d.c.pregibon, t.a.hatton, p.s.doyle, lab.chip 2007,7, 818-.

Fluid imprint techniques [ d.dendiukuri, d.c.pregibon, j.collins, t.a.hatton, p.s.doyle, nat.mater.2006,5, 365-; b) D.Dendikuri, S.S.Gu, D.C.Pregibon, T.A.Hatton, P.S.Doyle, Lab.Chip 2007,7, 818-; and the loss of prepolymer solution, encapsulated medicine or cells is large in the preparation process, and the encapsulation is usually less than 50%.

The centrifugal preparation process [ K.Maeda, H.Onoe, M.Takinoue, S.Takeuchi, adv.Mater.2012,24,1340-1346 ] is to add an alginate aqueous solution as a hydrogel prepolymer into a multi-channel capillary, and inject the liquid in the capillary into a solution containing calcium ions by using a centrifugal technology. However, the method has high cost due to high structural design and processing cost of the multi-channel capillary; moreover, because the addition amount of the prepolymer in the capillary is limited, continuous sample addition can not be carried out, continuous processing can not be realized, and the method is not suitable for industrial production; also, this technique is difficult to use for cell encapsulation, since cross-linked alginic acid requires the use of high concentration calcium ion solutions, which is detrimental to cell survival.

Microfluidic chip technology [ y.du, e.lo, s.ali, a.khademhosseini, p.natl acad.sci.2008,105, 9522-9527; s. seiffert, angelw.chem.int.ed 2013,52, 11462-. The method usually requires a violent gelation reaction so as to rapidly fix the multi-cavity structure, and when the method is applied to embedding of bioactive substances, protein molecules are easy to lose bioactivity, and the survival rate of cells is reduced; meanwhile, in the prior art, firstly, micro-fluidic chips are used for preparing microgel particles, and after the microgel particles are collected, a second step of cleaning is needed to elute an oil phase, so that the microgel is re-dispersed in an aqueous solution, and therefore, continuous sample injection preparation cannot be realized, and the method is not suitable for industrial application.

Disclosure of Invention

The invention realizes the one-step method continuous preparation of the multi-cavity calcium alginate microgel capable of immobilizing bioactive substances based on the microfluidic technology, and can realize high-throughput and continuous production of the microgel material. Firstly, different hydrogel prepolymer solutions in a microfluidic channel can form a parallel and stable flow field, so that the hydrogel prepolymer solutions forming different chambers of the microgel are injected into a microfluidic chip to form a water phase solution of a multiphase parallel fluid, then the water phase solution and a non-blended fluid (oil phase) are mixed through a T-shaped channel or a microfluidic channel designed by fluid focusing to form a water-in-oil emulsion liquid drop, and the cross-linking of alginic acid in the liquid drop is initiated immediately after the liquid drop is formed, so that the preparation of the multi-chamber microgel is realized; and then, the emulsion liquid drops are cleaned on the microfluidic chip, so that the microgel is rapidly conveyed to a water phase, and the calcium alginate microgel immobilized with bioactive substances is prepared by a one-step method. The preparation method has good biocompatibility, can well maintain the biological activity of the embedded active substances (such as bioactive macromolecules or living cells), and is an effective method for continuously preparing the microgel product for the biological medicine in high flux.

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

(1) preparing solution

Dissolving water-soluble alginate serving as a raw material in water to prepare an alginic acid aqueous solution, then adding a crosslinking reaction initiator, and dispersing bioactive substances and/or nanoparticles in the aqueous solution to obtain a hydrogel prepolymer solution serving as an aqueous phase solution for preparing a water-in-oil emulsion system, wherein:

the cross-linking reaction initiator is selected from one or more of a chelate aqueous solution of calcium-ethylene diamine tetraacetic acid, a chelate aqueous solution of calcium-nitrilotriacetic acid, calcium carbonate nanoparticles, calcium sulfate nanoparticles and calcium phosphate nanoparticles; the final concentration of the crosslinking reaction initiator is recorded as 10-1000mM in terms of calcium content; preferably 20-500mM, more preferably 25-100 mM;

mixing fluorinated oil, fluorinated surfactant and acidic substance to obtain a first heavy oil phase of the water-in-oil emulsion system;

mixing fluorinated oil and perfluoroalcol or perfluoroacid to obtain a mixed solution as a second heavy oil phase;

(2) injecting the water phase solution obtained in the step I into the microfluidic chip at a first flow rate, injecting the first heavy oil phase solution into the microfluidic chip from a second input port at a second flow rate, blending the water phase solution and the first heavy oil phase through an emulsification channel, and blending the two unblended phases through the emulsification channel to form a water-in-oil emulsion droplet; at the downstream of the emulsification channel, alginic acid in the single emulsion droplets undergoes a gelation reaction and is rapidly solidified (within 0.00001-1 second) to form alginic acid microgel;

(3) injecting a second heavy oil phase into the microfluidic chip from a third input port at a third flow rate, fully blending the second heavy oil phase and the emulsion obtained in the step (2) in a mixing channel arranged at the downstream of the emulsifying channel, and enabling the oil-water mixture dispersed with the alginic acid microgel to flow out of the chip from an output channel; inputting the output solution into a collection phase aqueous solution through a conduit, dispersing the calcium alginate microgel loaded with bioactive substances and/or nanoparticles in the collection phase aqueous solution, automatically separating an oil phase from a water phase, and sinking to the bottom of the aqueous solution, wherein the alginic acid microgel is automatically dispersed in the aqueous solution; collecting the aqueous solution to obtain the final product.

In the above-mentioned technical solution, specifically, the microfluidic chip has a microfluidic channel with a fluid focusing structure, a T-shaped mixing structure, a cocurrent flow type or cross structure, and further has at least 3-phase liquid input ports, an emulsification channel and an output channel. The 3-phase liquid input port comprises: an aqueous phase solution input, a first heavy oil phase input, and a second heavy oil phase input; and the inner wall surface of the micro-channel is subjected to hydrophobic treatment.

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

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

In the technical solution described above, specifically, the embedded bioactive substance and/or nanoparticles in step (1) are selected from one or more of the following combinations: living cells, water-soluble active protein drug molecules, nanoparticles; the skilled person can choose the purpose according to the implementation, for example:

the living cells are one or more of primary culture cells, subculture cells, cell strain culture cells and heterozygotes generally;

the water-soluble active protein drug molecules are generally one or more of protein drugs, polypeptide drugs, enzyme drugs and cell growth factors.

Proteinaceous drugs, for example: serum albumin, gamma globulin, insulin;

polypeptide drugs, for example: oxytocin, glucagon;

enzyme-like drugs, for example: digestive enzymes (pepsin, pancreatin, and maltogenic amylase), antiinflammatory enzymes (lysozyme and trypsin), and cardiovascular disease therapeutic enzymes (kallikrein for dilating blood vessel and lowering blood pressure);

cell growth factors, for example: interferon, interleukin, tumor necrosis factor, osteogenesis protein-2, osteogenesis protein-7, fibroblast growth factor, angiopoietic growth factor, etc

The nanoparticles are typically: metal or metal oxide nanoparticles such as nano-gold, nano-silver and nano-iron oxide, polymer nanoparticles such as polyethylene, polypropylene, polystyrene, polymethyl methacrylate and polylactic acid, fat-soluble vitamin, quinolone non-water-soluble drug nanoparticles, and one or more of hydroxyapatite, silicon dioxide, calcium phosphate, carbon nano-tubes and graphene inorganic non-metallic material nanoparticles. The size of the nano particles is 5-1000nm in diameter.

In the above-mentioned embodiments, specifically, the fluorinated oil is generally selected from one or more of the following combinations: perfluoropentane, perfluorohexane, perfluoroheptane, perfluorobutyl-methyl ether, perfluorooctane, perfluorononane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane, perfluorotetradecane, perfluoropentadecane, perfluorohexadecane, perfluoroheptadecane, perfluorodecalin.

In the technical solutions described above, in particular, the fluorinated surfactant is generally selected from one or a combination of several of the following: perfluoro Ethers (PFPE), perfluoro alkyl acid perfluoro ether-polyvinyl alcohol block copolymers, perfluoro ether-polyvinyl alcohol-perfluoro ether block copolymer surfactants; wherein the concentration of the fluorinated surfactant is 0.1 to 10 wt%, preferably 0.5 to 5 wt%.

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

in the above technical solution, specifically, the perfluoroalcohol in the second heavy oil phase is generally selected from one or a combination of several of the following: 22,33,444-heptafluoro-1-butanol, perfluoroundecanol, 1H-perfluoro-1-dodecanol, 1H-perfluoro-1-heptanol, 1H, 2H-perfluorooctanol, 1H-perfluorooctyl-1-ol, 1H-perfluoro-1-nonanol, 1H, 2H-perfluoro-1-dodecanol, 1H, 2H-perfluoro-1-decanol, 1H-perfluoro-1-tetradecanol, perfluoropentanol, perfluorohexanol, perfluoroheptanol, perfluorooctanol, perfluorononanol, perfluorodecanol;

the perfluoro acid is generally selected from one or a combination of more of the following: perfluorododecanoic acid, n-perfluoropentanoic acid, 5H-perfluoropentanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroheptanoic acid, perfluorohexanoic acid, perfluorobutanoic acid, perfluoroundecanoic acid; the concentration of the perfluoroalcohol or the perfluoroacid in the fluorinated oil in the second heavy oil phase is 1 to 100 v/v%, preferably 10 to 50 v/v%.

In the above-mentioned technical solutions, specifically, the aqueous solution of the collected phase in step (4) is a buffer solution which is non-cytotoxic and suitable for cell culture and has a pH buffer range of 6-8, and a solution of a cross-linking agent and a surfactant in the aqueous phase can be diluted and neutralized, and those skilled in the art can specifically select the solution according to the sample, and generally, the solution is selected from one or more of the following combinations: HEPES buffer solution, cell culture medium buffer solution, Phosphate Buffer Solution (PBS), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, PBS buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, citric acid-sodium citrate buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution, barbital sodium-hydrochloric acid buffer solution, Tris-hydrochloric acid buffer solution, and boric acid-borax buffer solution; wherein the buffer has an ion concentration of 10-2000mM, preferably 100-200 mM.

In the above-mentioned technical solution, specifically, the internal phase aqueous solution, the first heavy oil phase and the second heavy oil phase composed of the multiple fluids are respectively delivered to the corresponding micro-channels of the micro-fluidic device by the micro-pump or the micro-syringe at the first flow rate of 5-2000 μ L/hr, the second flow rate of 200-.

Preferably the first flow rate is from 10 to 500. mu.L/hr, more preferably from 20 to 100. mu.L/hr;

the preferred second flow rate is 500-;

the preferred third flow rate is 500-.

In the above technical solution, specifically, the time of the formed aqueous phase droplets from the upstream water-oil two-phase intersection channel to the second heavy oil-phase blending channel is 0.1-30s, preferably 0.1-10 s; so as to ensure that the alginic acid prepolymer is gelatinized in the chip channel to obtain the alginic acid microgel. The time from the blending of the microgel particles and the second heavy oil phase to the injection and collection of the aqueous solution phase is 0.1 to 30s, preferably 1 to 10 s; thereby ensuring that the two phases are fully mixed and simultaneously avoiding the cell toxicity caused by too long residence time of the microgel particles under the crosslinking condition.

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

Another objective of the present invention is to disclose the microgel product prepared by the microfluidic technology for continuously preparing calcium alginate microgel by the one-step method described above; the diameter of the microgel is 5-1000 mu m; the dispersion coefficient of the particle size distribution is 1-6%, the microgel prepared by the method is prepared into a water-in-oil emulsion from a microfluidic chip, the water-in-oil emulsion is dispersed in an oil phase, an aqueous solution is injected and collected through an output port and is spontaneously dispersed in the aqueous solution, and the time for dispersing the microgel in the oil phase is 1-60 seconds; when living cells are embedded in the microgel, the residence time of the cells in the emulsion is 1-60 seconds, and the survival rate of the cells is more than 85%.

Another objective of the present invention is to disclose a microfluidic technology for continuously preparing multi-cavity calcium alginate microgel particles by a one-step method, which is different from the microfluidic technology for continuously preparing calcium alginate microgel particles by a one-step method described above in that: increasing the variety of the hydrogel prepolymer solution and correspondingly increasing the number of the water phase input ports on the microfluidic chip;

typically, single chamber microgel preparation requires 3 inputs (1 aqueous phase input, 2 oil phase inputs); multi-chamber microgel preparation requires more than 2 input ports for aqueous solution, and others are unchanged. Specifically, the aqueous phase solution input port is used for inputting the aqueous gel prepolymer solution, so that when a person skilled in the art intends to prepare the multi-cavity calcium alginate microgel according to needs, the types of the aqueous gel prepolymer solution need to be increased, and correspondingly, the number of the aqueous phase solution input ports also needs to be increased.

When multi-cavity calcium alginate microgel particles are prepared, according to the method of the step I, various bioactive substances and/or nanoparticles are selected to prepare hydrogel prepolymer solutions for forming different cavities of the microgel, different hydrogel prepolymer solutions are respectively used as two-dimensional, three-dimensional or multi-dimensional parallel fluids to be injected from a plurality of water phase input ports on a microfluidic chip and are converged into water phase solutions, and then the multi-cavity microgel particles are prepared according to the methods of the step II, the step III, (2) and the step 3.

In the above-mentioned technical solution for preparing multi-cavity microgel particles, specifically, the sum of the flow rates of the aqueous phase fluids of the multiple input alginic acid hydrogel prepolymers is 5 to 2000 μ L/hr; the difference multiple of the input flow rates of the prepolymer aqueous solutions of all phases is 1-100 times, and is related to the size of the obtained multi-cavity microgel cavity.

In the technical scheme for preparing the multi-cavity microgel particles, in the specific cross-linking process, the alginic acid solution containing a plurality of bioactive substances and/or nanoparticles in the liquid drops simultaneously undergoes a gelation reaction, and is rapidly solidified to form the alginic acid microgel with a multi-cavity structure; the concentration of alginic acid in the hydrogel prepolymer solution of each phase forming different chambers of the microgel is 1-10 times (namely the concentration difference of alginic acid raw materials in each chamber).

Advantageous effects

1) The microgel immobilized with living cells and bioactive substances can be prepared by a one-step method, and the microgel with multiple cavities can be continuously prepared, so that the microgel immobilized with living cells and bioactive substances is suitable for industrial application.

2) The method is based on the alginic acid microgel material, has good biocompatibility and low cytotoxicity, and the microgel stays in the emulsion for less than 30 seconds, so that the microgel can be rapidly collected in an aqueous solution, the maintenance of the bioactivity of the encapsulated substance is facilitated, and the survival rate of the immobilized cells is high.

3) The preparation of the microgel with a complex three-dimensional structure can be realized through the design of a microfluidic chip channel, and the number of the microgel chambers can reach more than 6; can realize continuous and high-throughput preparation of the cell-loaded or drug-loaded microgel particles.

4) Through the design of the microfluidic chip, the size range of the microgel is regulated and controlled within 10-800 mu m, the particle size distribution is narrow, and the dispersion is less than 5%. The size of the liquid drop is determined by the size of the channel, and the larger the size of the channel is, the larger the liquid drop is; meanwhile, the ratio of the two flow rates of water and oil also influences the size of the liquid drops, and the larger the flow rate of the internal phase is, the larger the liquid drops are.

5) Can realize the preparation of the microgel of the hydrogel material in different crosslinking modes.

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

7) The microgel can be used as an extracellular matrix to support the survival and the function of cells in the microgel.

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

Drawings

FIG. 1 is a schematic diagram of one-step method for continuously preparing alginic acid microgel by microfluidic chip. In fig. 1, (+) means that alginic acid in the droplets starts to be crosslinked to form microgel particles.

Fig. 2 is a schematic diagram of a typical T-shaped mixing channel as described in example 1. 1 is water phase containing bioactive substances, and 2 is oil phase of continuous phase.

Fig. 3 is a schematic structural diagram of a mixing channel of an exemplary fluid focusing mechanism as described in example 1. 1 is water phase containing bioactive substances, and 2 is oil phase of continuous phase.

FIG. 4 is a diagram illustrating a one-step preparation method of microgel particles dispersed in an aqueous solution by a two-layer fluid focusing microfluidic chip. (a) The micro-gel is a phase-separated water phase formed after the emulsion is destroyed, wherein the phase-separated water phase contains a large number of micro-gel spheres (c), and green fluorescence labeled glucan (with the molecular weight of 5kDa) is encapsulated in the micro-gel, and can quickly permeate out of the micro-gel due to the small molecular weight; (b) is the oil phase of the continuous phase.

FIG. 5 is a size distribution of microgel particles prepared by the method described in example 1.

FIG. 6 is a schematic diagram of a design of a microfluidic chip for preparing water-in-oil emulsion droplets in comparative example 1. In fig. 6, the expression "(r) indicates that alginic acid begins to be cross-linked in the droplets to form microgel particles.

Fig. 7 is a microgel droplets dispersed in an oil phase prepared by a layer of a fluid focusing microfluidic chip as described in comparative example 1. (a) Are emulsion droplets prepared using microfluidic chips (stacked on top because the density of the aqueous phase is less than that of the oil phase), (c) contain crosslinked microgel particles in which green fluorescently labeled dextran (molecular weight 5kDa) is encapsulated; (b) is the oil phase of the continuous phase.

FIG. 8 is a graph showing the size of microgel particles formed at different flow rate ratios.

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

FIG. 10 shows the viability of cells loaded with microgel by different microfluidic chip methods.

FIG. 11 shows alginic acid gels prepared using different cross-linking agents. Wherein A is alginic acid microgel prepared by using the method of comparative example 1 and Ca-NTA as a crosslinking reaction initiator, and B is alginic acid microgel prepared by using the method of comparative example 2 and CaCO₃The nano particles are used as a crosslinking reaction initiator to prepare the alginic acid gel.

FIG. 12 is a schematic diagram of a one-step method for continuously preparing multi-chamber alginic acid microgel by microfluidic chip, wherein 1, 2,3, 4, n-hydrogel prepolymer solutions constituting different chambers of the microgel, A-water phase, B-fluorinated oil, C-oil phase containing perfluoroalcol, 4D output channel, and E-alginic acid in the droplets starts to be crosslinked to form microgel particles; the hydrogel prepolymer is excited and crosslinked, a multiple internal phase blending region M1 is formed, water-in-oil emulsion is formed, and droplets are blended to remove M2 and a third heavy oil phase blending region M3.

Fig. 13 shows two-chamber microgel particles prepared by the preparation method described in example 4.

Fig. 14 is a three-chamber microgel particles prepared by the preparation method described in example 5.

Fig. 15 is a four-chamber microgel particles prepared by the preparation method described in example 6.

FIG. 16 shows a one-step method for preparing microgel particles dispersed in an aqueous solution by a two-layer fluid focusing microfluidic chip in the method of example 7. FIG. A shows that the size of a cavity for preparing the dual-cavity microgel is controlled by adjusting the flow rate ratio of two input phases by using different two-phase alginic acid prepolymer aqueous solutions as input items. In the graph B, a, B, c and d are fluorescence micrographs of microgel particles obtained according to different preparation parameters corresponding to the curve in the graph A.

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

Fig. 18 is a graph showing the controlled, single cell level three-dimensional immobilization and assembly of two different cells using dual-chamber microgel as a template according to the method described in example 9. FIG. A, C is a fluorescence micrograph at different magnifications and FIG. B, D is a micrograph of the visible light field. The scale in the figure is 50 μm.

FIG. 19 is a diagram illustrating the micro gel prepared to have a two-chamber structure using the micro fluidic chip and the preparation method and parameters described in example 4.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.

The perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer surfactant is purchased from Beijing An core micro-nano technology, Inc.; all chemicals were purchased from sigma, inc, unless otherwise specified.

Example 1

As shown in fig. 1, a microfluidic device includes a first input channel, a second input channel, a third input channel, an output channel, a hydrogel prepolymer cross-linking channel, and M1 is a water-oil blending region, and two unblended phases pass through a microfluidic channel having a "T" structure (as shown in fig. 2) or a fluid focusing structure (as shown in fig. 3). M2 is a microfluidic channel of an emulsion prepared upstream blended with a second heavy oil. And carrying out hydrophobic treatment on the surface of the inner wall of the micro-channel.

Dissolving sodium alginate and fluorescein-labeled dextran (molecular weight is 10kDa) in deionized water to prepare an aqueous solution with the sodium alginate content of 1 w/v% and the fluorescein-labeled dextran content of 0.01 w/v%, and then adding a chelate aqueous solution with the final concentration of 100mM calcium ion-nitrilotriacetic acid (Ca-NTA); the water solution prepared by the method is used as the water phase of the water-in-oil emulsion to enter the first input channel. Adding perfluoroether (PFPE) surfactant and acetic acid into perfluorooctane oil solution to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of a water-in-oil emulsion system to enter a second input channel. The concentration of the surfactant in the oil phase was 2 v/v%, and the concentration of acetic acid was 1 v/v%. And blending the perfluoropentanol and the perfluorooctane oil to obtain a second heavy oil phase with the perfluoropentanol content of 10 v/v%, and entering a third input channel.

The aqueous phase solution and the first heavy oil phase are respectively injected into a first layer of 'fluid focusing' micro-channel of the micro-fluidic device from a first input port and a second input port by a constant flow pump through an injector (the chip structure is designed as shown in figure 1), and are blended with the first heavy oil phase through a micro-flow channel with a fluid focusing structure (as shown in figure 3), and the oil phase shears the aqueous phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rate of the inner phase is 100ul/hr and the flow rate of the first heavy oil phase is 1000 ul/hr. As the acetic acid in the oil phase enters the aqueous phase droplets, resulting in dropletsThe decrease of the pH value causes the Ca originally chelated with the NTA molecules to be stable²⁺The ions are no longer stable and form free Ca²⁺And ions which further form ionic bonds with the alginic acid high molecular chain initiate alginic acid to crosslink with calcium ions to form hydrogel. Thus, microgel particles are formed in the emulsion droplets and stably dispersed in the oil phase due to the presence of the surfactant.

Subsequently, the above-mentioned microgel dispersed water-in-oil single emulsion was passed through a downstream second layer of microchannels having a "fluid focus" structure (see FIG. 3), and blended with a second heavy oil composed of perfluorooctane having a perfluoropentanol content of 10 v/v%. Wherein the flow rate of the second heavy oil phase is 1000 ul/hr. After the mixed fluid is gradually blended through the U-shaped mixing micro-channel, 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 not stable any more, and the aqueous phase droplets (namely the microgel particles) are difficult to continue to be stably dispersed in the oil phase, so that the microgel and the oil phase are separated. Thus obtaining the dispersion liquid of the cell-loaded microgel particles in water. As shown in fig. 4, the prepared microgel particles were dispersed in water and separated from the oil phase, as seen by fluorescence micrographs, the microgel was dispersed in water, and fluorescein-labeled dextran in the initial aqueous solution rapidly permeated through the porous network of the microgel into the aqueous solution due to the smaller molecular weight. The size distribution of the resulting microgel particles was uniform, as shown in fig. 5. The distance between the first heavy fluid focusing micro-channel and the second heavy fluid focusing micro-channel is 2 cm, so that after alginic acid microgel is completely solidified, emulsion destruction is realized in a downstream channel, so that water and oil phases can be separated in a microfluidic chip channel, and one-step cell embedding is realized.

Comparative example 1 alginic acid two-step Process

In the microfluidic device shown in fig. 6, sodium alginate grafted with green fluorescent molecules is dissolved in deionized water, and then a chelate aqueous solution of calcium-nitrilotriacetic acid (Ca-NTA) is added, wherein the concentration of the fluorescently labeled sodium alginate is 1 wt%, and the concentration of the Ca-NTA is 100 mM; the aqueous solution thus prepared was used as the aqueous phase of the water-in-oil emulsion. Adding a perfluoro ether-polyvinyl alcohol-perfluoro ether (Krytox-PEG-Krytox) block copolymer serving as a surfactant into perfluorobutyl-methyl ether to obtain a mixed oil phase solution serving as an oil phase of the water-in-oil emulsion system.

The aqueous phase solution and the oil phase solution are respectively injected into the micro-channel of the microfluidic device from the first input port and the second input port by a constant flow pump through a syringe (as shown in figure 6), and the oil phase shears the aqueous phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rate of the inner phase is 100ul/hr and the flow rate of the oil phase is 1000 ul/hr. As shown in fig. 7, since the aqueous phase droplets are less dense than the oil phase, they float on top of the oil phase, and the fluorescence micrographs show that the resulting aqueous phase droplets are uniform in size and that the fluorescein-labeled dextran in the initial aqueous phase is still entrapped in the water-in-oil emulsion droplets.

Subsequently, acetic acid (final concentration 0.05 vol%) was added to the emulsion prepared in the above-described manner. The diffusion of acetic acid from the oil phase into the aqueous phase droplets causes a decrease in the pH of the droplets, resulting in Ca being stably chelated to the NTA molecules²⁺The ions are no longer stable and form free Ca²⁺Ions, which further form ionic bonds with the alginic acid polymer chains, form hydrogels. In this way, 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 solution, the microgel particles dispersed in the emulsion need to be washed. 30 seconds after the acetic acid is added into the emulsion, filtering the emulsion dispersed with the microgel to remove an oil phase, and then washing with a large amount of deionized water, thereby removing the oil phase and the surfactant and realizing the dispersion of the microgel in an aqueous solution.

Comparative example 2

Dissolving sodium alginate grafted with green fluorescent molecules in deionized water, and then adding CaCO₃Calcium carbonate nano particles are fully and uniformly mixed, wherein the concentration of the fluorescence labeled sodium alginate is 1 wt%, and the concentration of the calcium carbonate particles is 10 mg/ml; the aqueous solution thus prepared was used as the aqueous phase of the water-in-oil emulsion. Adding perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer as surfactant into perfluorooctane oil phase solution to obtain mixed solution, and using the mixed solution as surfactantIs the oil phase of a water-in-oil emulsion system. The surfactant concentration in the oil phase was 2 v/v%. The water-in-oil emulsion droplets were prepared using the method described in comparative example 1, followed by adding acetic acid (final concentration of 0.5 v/v%) to the emulsion, and after soaking for 120 seconds, the microgel dispersed emulsion was filtered to remove the oil phase, and then rinsed with a large amount of deionized water, thereby removing the oil phase and the surfactant, and achieving dispersion of the microgel in an aqueous solution. FIG. 11 alginic acid gels prepared using different cross-linking agents. Wherein A is alginic acid microgel prepared by using the method of comparative example 1 and Ca-NTA as a crosslinking reaction initiator, and B is alginic acid microgel prepared by using the method of comparative example 2 and CaCO₃The nano particles are used as a crosslinking reaction initiator to prepare the alginic acid gel. Micrographs of microgels show that the fluorescent-labeled alginic acid macromolecules in the microgels are not uniformly distributed, which is caused by the non-uniform distribution of calcium carbonate nanoparticles in the sodium alginate prepolymer solution.

Comparative example 3 different crosslinking reaction initiators

Dissolving the sodium alginate grafted with the green fluorescent molecules in deionized water, and then adding different crosslinking reaction initiators: calcium-nitrilotriacetic acid (Ca-NTA), calcium-ethylenediaminetetraacetic acid (Ca-EDTA), CaCO₃Calcium carbonate nanoparticles (average particle size about 300nm) were mixed well, wherein the concentration of fluorescently labeled sodium alginate was 1 wt%, the concentration of Ca-NTA and Ca-EDTA was 100mM, and the concentration of calcium carbonate particles was 10mg/ml (calcium ion concentration was 100 mM); the aqueous solution thus prepared was used as the aqueous phase of the water-in-oil emulsion. Adding a perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer serving as a surfactant into a perfluorooctane oil phase solution to obtain a mixed solution, and taking the mixed solution as an oil phase of a water-in-oil emulsion system. The surfactant concentration in the oil phase was 2 v/v%. The dispersion of the microgel in the aqueous solution was achieved by preparing water-in-oil emulsion droplets using the method described in comparative example 1, followed by adding acetic acid (final concentration of 0.1 v/v%) to the emulsion, filtering the microgel dispersed emulsion to remove the oil phase immediately after soaking for various times, and then rinsing with a large amount of deionized water to remove the oil phase and surfactant. Followed by fluorescence microscopy for microgel formation and no coagulationGel-ball formation is considered that the preparation parameters are not able to form microgels. As shown in Table 1, Ca-NTA and Ca-EDTA as the initiator of the crosslinking reaction can crosslink the alginate in a very short time to obtain microgel particles, which is mainly due to the fact that both are water-soluble chelates of calcium ions, can be uniformly dispersed in an aqueous solution, and the dissociation reaction of the calcium ions under the action of acid takes place instantaneously, thus being beneficial to the quick crosslinking of alginic acid. Relative ratio of CaCO₃The speed of calcium ion dissociation of the nano particles under the action of acid is obviously slower, microgel crosslinking is difficult to realize within 60 seconds after acetic acid is added into the emulsion phase, and when the soaking time in acid is more than 120 seconds, the microgel is formed. The speed of the crosslinking speed is directly related to two very important applications: 1. when used for the preparation of cell-loaded microgel particles, in order to ensure microgel formation, the experimental group using calcium carbonate nanoparticles as a crosslinking reaction initiator had a low cell survival rate because cells needed to stay in an acidic environment for a long time (fig. 10). 2. When Ca-NTA and Ca-EDTA are used as the cross-linking reaction initiator, the alginic acid prepolymer is quickly gelled in the emulsion droplets, which makes it possible to realize the cleaning of the emulsion droplets on a chip, which is one of the keys of the present invention for realizing the continuous preparation of microgel particles.

Table 1 effect of using different alginic acid crosslinking initiators and different crosslinking times on microgel formation as described in comparative example 3.

Example 2 control of dimensions

Dissolving sodium alginate in deionized water to obtain an aqueous solution with the sodium alginate content of 2 w/v%, and dissolving calcium-nitrilotriacetic acid (Ca-NTA) in deionized water to obtain an aqueous solution with the concentration of 100 mM; and blending the two solutions to obtain an aqueous solution, wherein the final concentration of the sodium alginate solution is 1 w/v%, the concentration of the Ca-NTA is 80mM, and the aqueous solution is used as an internal phase aqueous solution of the water-in-oil emulsion. The method comprises the steps of blending perfluorobutyl-methyl ether, a fluorinated surfactant of a perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer and acetic acid to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of a water-in-oil emulsion system. The concentration of the surfactant in the oil phase was 1 v/v%, and the concentration of acetic acid was 0.1 v/v%. And finally blending the perfluorooctanol and the perfluorobutyl-methyl ether to obtain a second heavy oil phase with the perfluorooctanol content of 10 v/v%.

The aqueous phase solution and the first heavy oil phase are respectively injected into a first layer of 'fluid focusing' micro-channel of the micro-fluidic device from a first input port and a second input port by a constant flow pump through an injector, and are mixed with the first heavy oil phase through a micro-fluidic channel (as shown in figure 1) with a 'T' -shaped structure or a fluid focusing structure, and the oil phase cuts the aqueous phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rate of the internal phase is 100ul/hr, and the flow rate ratio Qc/Qd (oil: water) of the internal phase aqueous solution is changed<And 20, 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 in the oil phase enters the aqueous phase droplets, the pH in the droplets decreases, and Ca which is originally stably chelated with NTA molecules²⁺The ions are no longer stable and form free Ca²⁺And ions which further form ionic bonds with the alginic acid high molecular chains are subjected to ionic crosslinking to form the hydrogel. In this way, microgel particles are formed in the emulsion droplets and are stably dispersed in the oil phase due to the presence of the surfactant. The results are shown in FIG. 8. The microgel particles are reduced in size in response to a reduction in the flow ratio of the aqueous phase to the first heavy oil phase.

EXAMPLE 3 embedding of Living cells

Cell culture: with NIH3T3(

CRL-1658^TM) Fibroblast cultures are exemplified by a proliferation medium consisting of DMEM containing 10% fetal bovine serum (FBS, Gibco). The culture conditions were 37 ℃, 95% relative humidity and 5% CO₂. Cell culture medium was changed after every three days. Before use, cells were detached with Phosphate Buffered Saline (PBS) using a trypsin/EDTA solution (0.25% trypsin/0.02% EDTA) for 5 minutes and suspended in medium for use.

And dissolving the green fluorescence labeled sodium alginate in a cell culture medium DMEM solution to prepare a 2 w/v% sodium alginate aqueous solution. Mixing the cell suspension, sodium alginate aqueous solution and calcium-ethylene diamine tetraacetic acid (Ca-EDTA) chelate aqueous solution to obtain mixed solution containing sodium alginate 1 w/v%, Ca-EDTA 100mM at final concentration, and cell 10%⁶One per ml. The above aqueous solution was used as the internal phase aqueous solution of the water-in-oil emulsion. The method comprises the steps of blending perfluorobutyl-methyl ether, a fluorinated surfactant of a perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer and acetic acid to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of a water-in-oil emulsion system. Wherein the concentration of the surfactant is 1 w/v%, and the concentration of the acetic acid is 0.1 v/v%. And blending perfluorooctanol and perfluorobutyl-methyl ether to obtain a second heavy oil phase with the perfluorooctanol content of 10 v/v%.

The aqueous phase solution and the first heavy oil phase are respectively injected into a first layer of 'fluid focusing' micro-channel of the micro-fluidic device from a first input port and a second input port by a constant flow pump through an injector, and are mixed with the first heavy oil phase through a micro-fluidic channel (as shown in figure 1) with a 'T' -shaped structure (figure 2), and the oil phase cuts the aqueous phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rate of the inner phase is 100ul/hr and the flow rate of the first heavy oil phase is 1000 ul/hr. Subsequently, the acetic acid in the oil phase enters the aqueous phase droplets, which causes the pH in the droplets to decrease, and the Ca originally stably chelated with the EDTA molecules²⁺The ions are no longer stable and form free Ca²⁺And ions which further form ionic bonds with the alginic acid high molecular chains, so that the alginic acid is subjected to ionic crosslinking to form the hydrogel. In this way, 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 microgel dispersed water-in-oil single emulsion flows through a downstream second layer having "fluid focusing" microchannels (see fig. 3) to form a blend with the second heavy oil. Wherein the flow rate of the second heavy oil phase is 2000 ul/hr. After the mixed fluid is gradually blended through the U-shaped mixing microchannel (fig. 1), the perfluorooctanol 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, and the aqueous phase droplets (i.e. microgel particles) are difficult to continue to be stably dispersed in the oil phase, so that the microgel and the oil phase are separated.

DMEM cell culture medium was blended with buffer HEPES at a concentration of 10mM, as an aqueous harvest solution. The output channel is blended with the collected aqueous solution through the conduit, the blended aqueous-oil mixture output from the chip is phase-separated in the collected aqueous solution, the microgel is directly phase-separated into the aqueous solution, and the acid in the buffer liquid drops is the damage to cells during the preparation process is minimized.

The cytotoxicity of the gel material was examined by using LIVE/DEAD fluorescence staining (LIVE/DEAD assay). First, the gel was washed with sterile PBS for 30 minutes before staining, 2mM calcein (green fluorescent dye labeled live cells) and 4mM ethidium bromide dimer (red fluorescent dye labeled dead cells) were added at room temperature, and examined using confocal laser scanning microscopy. As a result, as shown in the fluorescence micrograph of fig. 9, it can be seen that the cell survival rate was about 85%; the survival rate of the control experiment group is equivalent to that of the cells cultured on the cell culture plate directly, and the method of the invention is proved to have good biocompatibility (the result is shown in figure 10).

COMPARATIVE EXAMPLE 4 traditional emulsion method (cell embedding)

The cell-loaded microgel was prepared using the two-step preparation process of preparing microgel of comparative example 2. Dissolving green fluorescence labeled sodium alginate in cell culture medium DMEM solution, and adding chelate aqueous solution of calcium-ethylene diamine tetraacetic acid (Ca-EDTA) and cell dispersion solution, wherein the concentration of fluorescence labeled sodium alginate is 1 wt%, the concentration of Ca-NTA is 100mM, and the concentration of cell is 10⁶Per ml; the aqueous solution thus prepared was used as the aqueous phase of the water-in-oil emulsion. Adding a perfluoro ether-polyvinyl alcohol-perfluoro ether (Krytox-PEG-Krytox) block copolymer serving as a surfactant into perfluorobutyl-methyl ether to obtain a mixed oil phase solution serving as an oil phase of the water-in-oil emulsion system.

The aqueous phase solution and the oil phase solution are respectively injected into the micro-channel of the microfluidic device from the first input port and the second input port by a constant flow pump through a syringe (as shown in figure 6), and the oil phase shears the aqueous phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rate of the inner phase is 100ul/hr and the flow rate of the oil phase is 1000 ul/hr. As shown in fig. 7, the aqueous phase droplets floated on top of the oil phase due to their lower density relative to the oil phase, and the fluorescence micrographs showed that the resulting aqueous phase droplets were uniform in size and the fluorescein-labeled dextran in the initial aqueous phase was still entrapped in the water-in-oil emulsion droplets.

Subsequently, acetic acid (final concentration 0.1 vol%) was added to the emulsion prepared in the above-described manner. The diffusion of acetic acid from the oil phase into the aqueous phase droplets causes a decrease in the pH of the droplets, resulting in Ca being stably chelated to the NTA molecules²⁺The ions are no longer stable and form free Ca²⁺Ions, which further form ionic bonds with the alginic acid polymer chains, form hydrogels. In this way, 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 solution, the microgel particles dispersed in the emulsion need to be washed. 300 seconds after acetic acid is added into the emulsion, the emulsion dispersed with the microgel is filtered to remove an oil phase, and then a large amount of cell culture medium (DMEM) is used for washing, so that the oil phase and the surfactant are removed, and the cell-loaded microgel is finally dispersed in a DMEM aqueous solution.

The two-step microgel preparation technology can be used for embedding cells or bioactive substances such as protein drug molecules and the like, but the survival rate of the cells is seriously influenced because the cells are in contact with an acidic solution for too long time in the embedding and preparation processes. As shown in fig. 10, the cell survival rate of the microgel particles prepared in the two-step method according to the comparative example was extremely low, and was significantly reduced compared to the normal positive control (cells cultured adherently on two-dimensional plates).

EXAMPLE 4 Multi-Chamber microgel preparation

As shown in fig. 12, a microfluidic device for preparing multi-chamber microgel includes 1 st, 2 nd, 3 th, 4 th, 6 th input channels, which are merged together to form an internal phase input channel a, B is a first heavy oil phase input channel, C is a second heavy oil phase input channel, D is an output channel, E is a hydrogel prepolymer cross-linking channel, M1 is a mixing region of hydrogel prepolymer solutions forming different chambers of microgel, stable parallel flow is formed after the prepolymer solutions of different phases are input to a chip by using the characteristic that fluids in the microfluidic channels form stable parallel flow, and diffusion and exchange of substances between fluids are limited. M2 is a region of two blends of water and oil, the two phases that do not blend passing through a microfluidic channel with a "T" shaped structure (as shown in fig. 2) or a fluid focusing structure (as shown in fig. 3). M2 is a microfluidic channel of an emulsion prepared upstream blended with a second heavy oil. And carrying out hydrophobic treatment on the surface of the inner wall of the micro-channel.

Dissolving sodium alginate in deionized water to prepare a alginic acid hydrogel prepolymer solution; an aqueous chelate solution of calcium-nitrilotriacetic acid (Ca-NTA) was co-blended with nitrilotriacetic acid and calcium hydroxide in deionized water. And blending the two aqueous solutions, and then adding red or green fluorescein-labeled polystyrene nanoparticles (the particle diameter is 100nM) to uniformly disperse the polystyrene nanoparticles to obtain the alginic acid hydrogel prepolymer solution for preparing the microgel. Preparing with deionized water to obtain a chelate aqueous solution with sodium alginate content of 1 w/v% and fluorescein labeled nanoparticle content of 0.01 w/v%, and then adding 100mM calcium ion-nitrilotriacetic acid (Ca-NTA) final concentration; the aqueous solution thus prepared was used as the aqueous phase of the water-in-oil emulsion. Adding perfluoroether (PFPE) surfactant and acetic acid into the perfluorooctane oil phase solution to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the oil phase was 2 v/v%, and the concentration of acetic acid was 1 v/v%. And blending the perfluoropentanol and the perfluorooctane oil phase to obtain a second heavy oil phase with the perfluoropentanol content of 10 v/v%.

Inputting the prepared sodium alginate prepolymer dispersed with red fluorescent nanoparticles and green fluorescent nanoparticles into a chip from the 1 st and 2 nd input channels through a microfluidic channel structure as shown in fig. 13 to form parallel flow with stable flow rate (as shown in fig. 13), injecting the aqueous phase solution and the first heavy oil phase into a first layer of 'fluid focusing' microchannel of the microfluidic device from the first and second input ports respectively through a constant flow pump by using an injector (the chip structure is designed as shown in fig. 12), and carrying out fluid polymerizationThe microfluidic channels of the coke structure (see fig. 2) were blended with the first heavy oil phase and the oil phase sheared the aqueous phase into water-in-oil single emulsion droplets of uniform size distribution. Wherein the flow rates of the two internal phases are respectively 100ul/hr, and the flow rate of the first heavy oil phase is 1000 ul/hr. As acetic acid in the oil phase enters the aqueous phase droplets, the pH in the droplets is reduced, and Ca originally chelated with NTA molecules is stably generated²⁺The ions are no longer stable and form free Ca²⁺And ions which further form ionic bonds with the alginic acid high molecular chain initiate alginic acid to crosslink with calcium ions to form 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 microgel dispersed water-in-oil single emulsion flows through a downstream second layer having "fluid focusing" microchannels (see FIG. 2), blended with a second heavy oil consisting of perfluorooctane having a perfluoropentanol content of 10 v/v%. Wherein the flow rate of the second heavy oil phase is 1000 ul/hr. After the mixed fluid is gradually blended through the U-shaped mixing micro-channel (as shown in 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 is no longer stable, and the aqueous phase droplets (i.e., the microgel particles) are difficult to continue to be stably dispersed in the oil phase, so that the microgel and the oil phase are separated. The mixed output liquid is output to a collecting aqueous solution through a conduit connection, so that microgel particles with a two-chamber structure are obtained (see fig. 13B).

Example 5

The method described in example 4 was used to prepare aqueous alginate prepolymer solutions in which red fluorescent and green fluorescent labeled nanoparticles were dispersed, and three different aqueous internal phase solutions were injected into the dispersion using the input structure of aqueous internal phase solution as shown in fig. 14A, to form parallel flows of three different aqueous internal phase solutions. Wherein the flow rates of the three internal phases are respectively 100ul/hr, and the flow rate of the first heavy oil phase is 1500 ul/hr. Microgel was further prepared by the method described in example 4, and microgel particles having three different chambers were finally obtained (fig. 14B).

Example 6

An alginic acid prepolymer aqueous solution in which red fluorescent and green fluorescent labeled nanoparticles were dispersed was prepared by the method described in example 4, and four different internal phase aqueous solutions were injected into the alginate prepolymer aqueous solution using the internal phase aqueous solution input structure shown in fig. 15A, respectively, to form parallel flows of the four different internal phase aqueous solutions, wherein the flow rates of the four internal phases were 50ul/hr, respectively, and the flow rate of the first heavy oil phase was 1000 ul/hr. Microgel was further prepared by the method described in example 4, and microgel particles having four different chambers were finally obtained (fig. 15B).

Example 7

An aqueous solution of alginic acid prepolymer in which red fluorescent and green fluorescent labeled nanoparticles were dispersed, respectively, was prepared by the method described in example 4, and two different aqueous solutions of the internal phases were injected, respectively, using an input structure of the aqueous solutions of the internal phases as in fig. 13A to form parallel flows of the two different aqueous solutions of the internal phases, wherein the flow rates of the two aqueous solutions of the internal phases were adjusted such that the flow rate ratio (Q1/Q2) therebetween was from 1 to 9, the sum of the flow rates of the two phases was 100ul/hr, and the flow rate of the first heavy oil phase was 1000 ul/hr. Microgel was further prepared by the method described in example 4, and microgel particles having two different chambers with different chamber volume ratios were finally obtained (fig. 16).

Dissolving sodium alginate grafted with green fluorescent molecules in deionized water, and then adding a chelate aqueous solution of calcium-nitrilotriacetic acid (Ca-NTA), wherein the concentration of the fluorescence labeled sodium alginate is 1 wt%, and the concentration of the Ca-NTA is 100 mM; non-fluorescently labeled sodium alginate is dissolved in deionized water, and then an aqueous chelate solution of calcium ion-nitrilotriacetic acid (Ca-NTA) is added, wherein the concentration of the fluorescently labeled sodium alginate is 1 wt% and the concentration of the Ca-NTA is 100 mM. Two different aqueous internal phase solutions were injected separately using the aqueous internal phase input structure shown in fig. 13A to form parallel flows of the two different aqueous internal phase solutions, using the two aqueous hydrogel prepolymer solutions as different internal phases, in the same manner as described in example 4. Wherein the flow rates of the two-phase internal phase aqueous solution are both regulated to be 50ul/hr, the sum of the flow rates of the two phases is 100ul/hr, and the flow rate of the first heavy oil phase is 1000 ul/hr. Microgel was further prepared by the method described in example 4, and microgel particles having two chambers were finally obtained (fig. 17).

Example 8

NIH3T3 (NIH 3T 3) cultured by the method described in example 3

CRL-1658^TM) Fibroblasts, which were labeled with green and red live cell-tracing fluorescent dyes, respectively (ThermoFisher Scientific, USA, CellTracker, green and red live cell-tracing dyes)^TM). Alginic acid (Sigma, moderate viscosity, USA) was grafted with red and green fluorescent dyes (Sigma, USA), respectively, to obtain fluorescently labeled alginic acid dissolved in DMEM solution of cell culture medium.

Firstly, preparing a hydrogel prepolymer dispersed with living cells as a water phase: and dissolving the fluorescence labeled sodium alginate in a cell culture medium DMEM solution to prepare a 2 w/v% sodium alginate aqueous solution. And mixing the cell suspension, the sodium alginate aqueous solution and the calcium-ethylene diamine tetraacetic acid (Ca-EDTA) chelate aqueous solution to obtain a mixed solution, wherein the concentration of the sodium alginate is 1 w/v%, and the final concentration of the Ca-EDTA is 50 mM. Wherein, the green fluorescence labeling NIH3T3 living cells are dispersed in the red fluorescence labeling alginic acid water solution, the red fluorescence labeling NIH3T3 living cells are dispersed in the green fluorescence labeling alginic acid water solution, the cell concentration is 10 respectively⁶One per ml. The two cell dispersions are used as two internal phase aqueous solutions for preparing the microgel with two cavities. The method comprises the steps of blending perfluorobutyl-methyl ether, a fluorinated surfactant of a perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer and acetic acid to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of a water-in-oil emulsion system. Wherein the concentration of the surfactant is 1 w/v%, and the concentration of the acetic acid is 0.1 v/v%. Perfluoropentanol is blended with perfluorobutyl-methyl ether to give 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 as described above were introduced into the chip from the 1 st and 2 nd input channels through the microfluidic channel structure as shown in FIG. 13, respectively, to form parallel flows with stable flow rates (see FIG. 13), and the aqueous phase solution and the first heavy oil phase were introduced from the first heavy oil phase through the constant flow pump by means of syringes, respectivelyThe first and second input ports are injected into a first layer of "fluid focusing" microchannel of the microfluidic device (chip structure design shown in fig. 12), and through the microfluidic channel with fluid focusing structure (fig. 2), the microfluidic channel is blended with a first heavy oil phase, and the oil phase shears the water phase into water-in-oil single emulsion droplets with uniform size distribution. Wherein the flow rates of the two internal phases are respectively 50ul/hr, and the flow rate of the first heavy oil phase is 1000 ul/hr. The pH value in the liquid drops is reduced along with the entry of acetic acid in the oil phase into the liquid drops of the water phase, so that Ca originally chelated with EDTA molecules is stably generated²⁺The ions are no longer stable and form free Ca²⁺And ions which further form ionic bonds with the alginic acid high molecular chain initiate alginic acid to crosslink with calcium ions to form 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 microgel dispersed water-in-oil single emulsion was passed downstream through a second layer having "fluid focusing" microchannels (see FIG. 2), blended with a second heavy oil consisting of perfluorobutyl-methyl ether having a perfluoropentanol content of 10 v/v%. Wherein the flow rate of the second heavy oil phase is 1000 ul/hr. After the mixed fluid is gradually blended through the U-shaped mixing micro-channel (as shown in 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 is no longer stable, and the aqueous phase droplets (i.e., the microgel particles) are difficult to continue to be stably dispersed in the oil phase, so that the microgel and the oil phase are separated. DMEM cell culture medium was blended with buffer HEPES (pH7.2-7.4) at a concentration of 10mM, as a collecting aqueous solution. The output channel is blended with the collected aqueous solution through the conduit, the blended aqueous-oil mixture output from the chip is phase-separated in the collected aqueous solution, the microgel is directly phase-separated into the aqueous solution, and the acid in the buffer liquid drops is the damage to cells during the preparation process is minimized. The mixed solution of the dispersed cell-loaded dual-chamber microgel prepared by the method is connected and output to a collecting aqueous solution through a conduit, microgel particles with two chamber structures are collected, and different kinds of single living cells are loaded in different chambers. The results are shown in the fluorescence micrograph of FIG. 18.

EXAMPLE 9 Multi-Chamber microgel preparation

Preparing an alginic acid hydrogel prepolymer solution (the sodium alginate content is 1 w/v%, the concentration of calcium-ethylene diamine tetraacetic acid chelate is 100mM) by adopting the method described in example 4, and adding red fluorescent dye-labeled polystyrene nanoparticles (the average diameter is 100nm) and magnetic iron oxide nanoparticles (the average diameter is 100nm) into the hydrogel prepolymer solution respectively to obtain a two-phase aqueous solution for preparing the microgel with two cavities, wherein the content of the nanoparticles is 0.01 w/v%; the prepared alginic acid hydrogel prepolymer aqueous solution dispersed with different nano particles is used as two aqueous phase solutions for preparing microgel in two chambers. Adding perfluoroether (PFPE) surfactant and acetic acid into the perfluorooctane oil phase solution to obtain a mixed solution, and taking the mixed solution as a first heavy oil phase of the water-in-oil emulsion system. The concentration of the surfactant in the oil phase was 1 v/v%, and the concentration of acetic acid was 1 v/v%. And blending the perfluoropentanol and the perfluorooctane oil phase to obtain a second heavy oil phase with the perfluoropentanol content of 10 v/v%. By using the microfluidic chip and the preparation method and parameters described in example 4, a microgel with a two-chamber structure was prepared, as shown in fig. 19, in which two chambers were respectively loaded with red fluorescent dye-labeled polystyrene nanoparticles (average diameter 100nm) and magnetic iron oxide nanoparticles (black, average diameter 100 nm). The obtained microgel with two cavities has magnetic response, and the microgel particles with two cavities dispersed in the liquid can move directionally under the attraction of a magnet and can be orderly arranged in a magnetic field.

Claims

1. The microfluidic method for continuously preparing the calcium alginate microgel by the one-step method is characterized by comprising the following steps of: the method comprises the following steps:

(1) preparing solution

Dissolving water soluble alginate in water to prepare alginic acid water solution, adding cross-linking reaction initiator, dispersing bioactive substance and/or nano-particles in the water solution to obtain hydrogel prepolymer solution as water phase solution,

wherein:

the cross-linking reaction initiator is selected from one or more of a chelate aqueous solution of calcium-ethylene diamine tetraacetic acid, a chelate aqueous solution of calcium-nitrilotriacetic acid, calcium carbonate nanoparticles, calcium sulfate nanoparticles and calcium phosphate nanoparticles; the final concentration of the crosslinking reaction initiator is recorded as 10-1000mM in terms of calcium content;

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

mixing fluorinated oil and perfluoroalcohol or mixing fluorinated oil and perfluoroacid to obtain a second heavy oil phase;

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

(3) injecting a second heavy oil phase into the microfluidic chip from a third input port at a third flow rate, and after the second heavy oil phase and the emulsion obtained in the step (2) are fully blended in a mixing channel arranged at the downstream of the emulsifying channel, enabling the second heavy oil phase to flow out of the chip from an output channel; inputting the output solution into a collection phase aqueous solution, dispersing the calcium alginate microgel loaded with bioactive substances and/or nanoparticles in the collection phase aqueous solution, and collecting the aqueous solution to obtain a final product;

the acidic substance is selected from one or a combination of several of the following substances: 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 0.001-20 v/v%;

the time from the upstream water-oil two-phase intersection channel to the second heavy oil-phase blending channel of the formed water-phase droplets is 0.1-30s, and the time from the blending of the microgel particles and the second heavy oil phase to the injection of the collected water-phase droplets is 0.1-30 s;

the flow rate ratio of the water phase to the first heavy oil phase is 0.01-1; the ratio of the flow rate of the first heavy oil phase to the flow rate of the second heavy oil phase is 1: 0.5-50.

2. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the microfluidic chip is provided with a fluid focusing structure, a T-shaped mixing structure, a microfluidic channel with a cocurrent flow type or cross structure, at least 3-phase liquid input ports, an emulsifying channel and an output channel.

3. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the alginic acid raw material is alginate aqueous solution.

4. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the alginic acid raw material is a mixture of alginic acid and one or more of collagen, gelatin, hyaluronic acid, polyethylene glycol, polyvinyl alcohol, polyacrylamide, glucan, chitosan and agarose.

5. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: wherein the total concentration of alginic acid raw materials in the prepolymer solution is 0.1-8 w/v%.

6. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the embedded bioactive substance and/or the nanoparticles in the step (1) are selected from one or more of the following combinations: living cells, water-soluble active protein drug molecules, nanoparticles;

the living cells are primary culture cells, subculture cells, cell strain culture cells and heterozygotes;

the water-soluble active protein drug molecules are protein drugs, polypeptide drugs, enzyme drugs and cell growth factors;

the nano-particles are nano-particles of nano-gold, nano-silver, nano-iron oxide, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polylactic acid, fat-soluble vitamins, quinolone water-insoluble drugs, hydroxyapatite, silicon dioxide, calcium phosphate, carbon nano-tubes and graphene; the size of the nano particles is 5-1000nm in diameter.

7. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the fluorinated oil is selected from one or a combination of several of the following: perfluoropentane, perfluorohexane, perfluoroheptane, perfluorobutyl-methyl ether, perfluorooctane, perfluorononane, perfluorodecane, perfluoroundecane, perfluorododecane, perfluorotridecane, perfluorotetradecane, perfluoropentadecane, perfluorohexadecane, perfluoroheptadecane, perfluorodecalin.

8. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the fluorinated surfactant is selected from one or a combination of several of the following components: perfluoro ether, perfluoro alkyl acid perfluoro ether-polyvinyl alcohol block copolymer, perfluoro ether-polyvinyl alcohol-perfluoro ether block copolymer surfactant; wherein the concentration of the fluorinated surfactant is 0.1-10 wt%.

9. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the perfluorinated alcohol in the second heavy oil phase is selected from one or a combination of more of the following components: 22,33,444-heptafluoro-1-butanol, perfluoroundecanol, 1H-perfluoro-1-dodecanol, 1H-perfluoro-1-heptanol, 1H, 2H-perfluorooctanol, 1H-perfluorooctyl-1-ol, 1H-perfluoro-1-nonanol, 1H, 2H-perfluoro-1-dodecanol, 1H, 2H-perfluoro-1-decanol, 1H-perfluoro-1-tetradecanol, perfluoropentanol, perfluorohexanol, perfluoroheptanol, perfluorooctanol, perfluorononanol, perfluorodecanol; the perfluoro acid is selected from one or a combination of more of the following: perfluorododecanoic acid, n-perfluoropentanoic acid, 5H-perfluoropentanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroheptanoic acid, perfluorohexanoic acid, perfluorobutanoic acid, perfluoroundecanoic acid; the concentration of the perfluorinated alcohol or perfluorinated acid in the second heavy oil phase in the fluorinated oil is 1-100 v/v%.

10. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the aqueous solution of the collecting phase in the step (3) is a buffer solution which has no cytotoxicity and has a pH buffering range of 6-8, and the ionic concentration of the buffer solution is 10-2000 mM.

11. The microfluidic method for continuously preparing calcium alginate microgel by one-step method according to claim 1, wherein: the first flow rate is 5-2000 μ L/hr, the second flow rate is 200-.

12. The micro-fluidic method for continuously preparing the calcium alginate micro-gel by the one-step method according to claim 1, wherein the micro-gel is prepared by the following steps: the diameter of the microgel is 5-1000 mu m; the dispersion coefficient of the particle size distribution is 1-6%, and the time for dispersing the microgel in the oil phase is 1-60 seconds; when living cells are embedded in the microgel, the residence time of the cells in the emulsion is 1-60 seconds, and the survival rate of the cells is more than 80%.

13. The microfluidic method for continuously preparing the multi-cavity calcium alginate microgel particles by the one-step method is characterized by comprising the following steps of: the preparation method of the microgel particles comprises the steps of preparing hydrogel prepolymer solutions by using different bioactive substances and/or nanoparticles according to the method of the step I, injecting the hydrogel prepolymer solutions from a plurality of water phase input ports on a microfluidic chip, and preparing the multi-cavity microgel particles according to the methods of the step II, the step III, (2) and the step 3.

14. The microfluidic method for continuously preparing multi-cavity calcium alginate microgel particles in one step according to claim 13, wherein: the sum of the flow rates of the aqueous phase fluids of the plurality of hydrogel prepolymers is 5 to 2000. mu.L/hr.

15. The microfluidic method for continuously preparing multi-cavity calcium alginate microgel particles in one step according to claim 14, wherein: the flow rate difference multiple of the water phase fluids of the hydrogel prepolymers is 1 to 100 times.

16. The microfluidic method for continuously preparing multi-cavity calcium alginate microgel particles in one step according to claim 13, wherein: when the bioactive substances and/or the nano-particles in the step I are various, the concentration of alginic acid in the hydrogel prepolymer solution is 1-10 times different.