A microfluidics-based multi-layer and multi-phase core-shell structured microsphere and a preparation method thereof

Abstract

The present invention provides a microfluidics-based multi-layered multi-phase core-shell structured microsphere and a preparation method therefor. The preparation method utilizes microfluidics technology. By selecting the types and feeding speeds of the core material, shell material, and truncation material, and designing the flow channel structure of the microfluidics device, the preparation of the multi-layered multi-phase core-shell structured microsphere is achieved. The core-shell structured microsphere prepared by this method has the advantages of highly uniform particle size, wide application range, customizable internal multi-phase materials, and gradual release of effective substances for different external environments. It can be applied to tissue engineering and biomedical applications such as cell culture, angiogenesis, drug delivery systems, synthesis of microparticles and microcapsules, biochemical analysis, targeted drug release, and disease treatment, and can be used to simulate complex physiological reactions in natural cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical and tissue engineering, and particularly relates to a multilayer and multiphase core-shell structure microsphere based on microfluidics and a preparation method thereof. Background Art

[0002] Microfluidic technology mainly realizes precise control of fluids by designing, fabricating, and operating different microchannel systems. The core of microfluidic technology lies in the microfluidic chip. The channel size of the microfluidic chip is very small, generally only a few micrometers to several hundred micrometers. It integrates a microchannel network and various functional units, and can achieve precise regulation of the microsphere preparation process. The high integration of this technology is conducive to the coexistence and interaction of multiple fluid phases. Compared with traditional microsphere preparation methods such as the emulsion method, the microspheres fabricated by microfluidic technology have uniform particle size, and have broad application prospects in the fields of physics, chemistry, biology, medicine, etc.

[0003] Existing preparation methods for core-shell structure microspheres include: emulsion method, template method, self-assembly method, deposition method, etc. Most of the core-shell structure microspheres prepared by these methods have poor monodispersity, which will affect the precise loading and release of active substances inside the core-shell structure microspheres. In the prior art, microfluidic technology is commonly used to solve the problem of poor monodispersity of core-shell structure microspheres, and microfluidic technology is also a commonly used technical means in the current technology for preparing core-shell structure microspheres. However, most of the microsphere materials fabricated by microfluidic technology have a single composition, and it is difficult to achieve targeted release for a specific environment, and cannot meet the usage requirements for microsphere materials with multiple functions. Summary of the Invention

[0004] To solve the problems existing in the prior art, the present invention provides a preparation method for a multilayer and multiphase core-shell structure microsphere based on microfluidics. The microspheres prepared by this method have the characteristics of a multilayer and multiphase structure, uniform sphere formation effect, and stable sphere formation efficiency. The multilayer and multiphase core-shell structure microspheres obtained by this method take into account various material properties, can achieve gradient release and targeted multiple release of loaded substances, take into account biocompatibility and material diversity, and are good media for multicellular co-culture and drug release.

[0005] The present invention also provides a multilayer and multiphase core-shell structure microsphere prepared by using the above preparation method. According to actual needs, the structure of the microsphere, the composition of each layer, and the number of layers can be adjusted by selecting microsphere materials, controlling the feeding speed of microsphere materials, setting the flow channel structure of the microfluidic device, etc., and at the same time, various functions such as drug loading and cell culture can be achieved.

[0006] A method for preparing multi-layer multi-phase core-shell structure microspheres based on microfluidics, comprising: a core material enters from the inlet of a main channel of a microfluidic device, is successively cut off by one or more layers of shell material and a cutting material entering the main channel, and is wrapped layer by layer, and then falls into a receiving solution for cross-linking and curing, thereby obtaining the multi-layer multi-phase core-shell structure microspheres;

[0007] The core material and each layer of shell material can be one or more materials respectively, and the core material and the shell materials of all layers are not the same material at the same time; multiple core materials, multiple shell materials of the same layer, core material and the adjacent shell material (the first layer shell material), and shell materials of adjacent layers are not miscible with each other;

[0008] The outermost shell material is a material that is oil / water reactive with the cut-off material; the cut-off material is isotropically compatible with the receiving solution.

[0009] In the above technical solution, the core material and the shell materials of all layers are not the same material at the same time, that is, at least one of the core material and the shell materials of all layers is a plurality of materials, so as to realize the preparation of multiphase core-shell structure microspheres. The core-shell structure continuous phase is cut off and wrapped by the cut-off material to form droplets and fall into the receiving solution. The cut-off material and the receiving solution are isotropic miscible materials. The cut-off material is dissolved by the receiving solution, and the core-shell structure droplets are solidified into microspheres to obtain multi-layer multiphase core-shell structure microspheres.

[0010] Preferably, when there are multiple core materials, each core material enters the main flow channel through an independent inlet flow channel;

[0011] The inlet flow channels of the adjacent layer shell materials, the inlet flow channel of the outermost layer shell materials and the inlet flow channel of the cut-off materials are arranged at intervals, and corresponding conveying sections and main flow channel outlet sections are formed on the main flow channel.

[0012] The outlet of the inlet flow channel of the core material is communicated with the inlet of the main flow channel, and the outlets of the inlet flow channel of the shell material and the inlet flow channel of the cut-off material are both communicated with the main flow channel.

[0013] It is particularly pointed out that the core material is cut and wrapped in shell materials in sequence as the first layer, the second layer, and the outermost layer from the inside to the outside.

[0014] As a further preference, the flow direction of each core material at the outlet of the corresponding inlet flow channel is the same as the flow direction of the material in the main flow channel, and the outlet end faces of the multiple core material inlet flow channels are flush on a plane perpendicular to the flow direction of the material in the main flow channel;

[0015] The first-layer shell material is a kind of material. There is one inlet channel for the first-layer shell material, and the outlet of this inlet channel is in communication with the inlet of the main channel; the flow direction of the first-layer shell material at the outlet of its inlet channel is perpendicular to the flow direction of the material in the main channel, and while multiple core materials flow out of the corresponding inlet channels, the multiple core materials are truncated and wrapped, forming a core with a dispersed phase of multiple materials.

[0016] In this technical solution, it is set that the outlet end faces of each core material are flush on the plane perpendicular to the plane where the flow direction of the material in the main channel is located. At the same time, it is set that the flow direction of the first-layer shell material at the outlet of its inlet channel is perpendicular to the flow direction of the material in the main channel, so as to ensure that the core materials are simultaneously vertically truncated and wrapped by the first-layer shell material the moment they flow out of the inlet channels, so that the multiple core materials are wrapped by the first-layer shell material before they come into contact with each other, forming a core with a dispersed phase of multiple materials, forming a structure similar to a "lotus pod shape"; that is, each core material is separately wrapped by the first-layer shell material (each core material does not contact each other), and after being truncated and wrapped by the second-layer shell material or the truncating material, a core with a dispersed phase of multiple materials is formed inside the first layer of shell.

[0017] As a further preference, when the number of wrapping layers outside the core material is two (the number of shell layers) and the number of core materials is four:

[0018] The feeding speed of the core material is 20 - 30 μL / min;

[0019] The feeding speed of the first-layer shell material is 400 - 600 μL / min;

[0020] The feeding speed of the second-layer shell material is 1000 - 1500 μL / min;

[0021] The feeding speed of the truncating material is 2000 - 4000 μL / min.

[0022] Even more preferably, the feeding speed of the core material is 25 μL / min;

[0023] The feeding speed of the first-layer shell material is 500 μL / min;

[0024] The feeding speed of the second-layer shell material is 1200 μL / min;

[0025] The feeding speed of the truncating material is 3000 μL / min.

[0026] As a further preference, when there are two kinds of shell materials for the layers other than the first-layer shell material, these two kinds of shell materials enter the main channel through two independent inlet channels, and the flow direction of each shell material at the outlet of the corresponding inlet channel is set at an angle of 45° with the flow direction of the material in the main channel; and the two inlet channels are symmetric with respect to the central axis of the main channel.

[0027] To better truncate the material in the main runner, as a further preference, when the shell materials of other layers except the first layer shell material are of one kind of material, this kind of material enters the main runner through two independent inlet runners respectively, and the flow direction of this kind of material at the outlet of the corresponding inlet runner is perpendicular to the flow direction of the material in the main runner, and the two inlet runners are symmetric with respect to the central axis of the main runner. Of course, this kind of material can also enter the main runner through one inlet runner.

[0028] As a further preference, the microfluidic device is a cascaded chip composed of a metal chip and a PDMS chip or a hydrogel chip;

[0029] The core material inlet runner and the first layer shell material inlet runner are arranged on the metal chip;

[0030] The main runner, the inlet runners of the shell materials of other layers except the first layer, and the truncating material inlet runner are all arranged on the PDMS chip or the hydrogel chip.

[0031] When the microfluidic chip is a cascaded chip composed of a metal chip and a PDMS chip or a hydrogel chip, the core material is the dispersed phase of multiple materials, and the multilayer multi-phase core-shell structure microspheres prepared therefrom are in a "lotus seed pod shape" structure, that is, each kind of core material is wrapped by the first layer shell material to form an integral structure, and each kind of core material does not contact each other.

[0032] The formation of the multilayer multi-phase core-shell structure microspheres in the "lotus seed pod shape" structure is, on the one hand, to truncate the core material through the feeding speed of the first layer shell material that is relatively fast in the process, and to control the position where the first layer shell material truncates the core material (i.e., the outlet of the core material inlet runner) so as to obtain the cores of multiple materials of the dispersed phase; on the other hand, it is to form a cascaded chip by combining the metal chip and the PDMS chip or the hydrogel chip, so as to wrap the cores of multiple materials of the dispersed phase flowing out of the metal chip.

[0033] As an even further preference, when the number of layers wrapped outside the core material is two:

[0034] The inner diameter of the core material inlet runner is 10 - 100 μm;

[0035] The inner diameter of the first layer shell material inlet runner is 10 - 100 μm;

[0036] The height of the conveying section (the first conveying section) between the inlet runners of the first layer shell material and the second layer shell material is 100 - 300 μm, and the width is 100 - 300 μm;

[0037] The height of the second layer shell material inlet runner is 10 - 100 μm, and the width is 10 - 100 μm;

[0038] The height of the conveying section (the second conveying section) between the second layer of shell material and the truncated material inlet channel is 600 - 800 μm, and the width is 600 - 800 μm;

[0039] The height of the truncated material inlet channel is 10 - 100 μm, and the width is 10 - 100 μm;

[0040] The height of the mainstream channel outlet section is 1000 μm, and the width is 1000 μm.

[0041] Preferably, the core material inlet channel and the inlet channel of the first layer of shell material are arranged at intervals, forming a core material conveying section on the mainstream channel; so that multiple core materials contact each other before being truncated by the first layer of shell material, and then are truncated and wrapped by the first layer of shell material to form the core of the continuous phase of multiple materials.

[0042] The purpose of setting the core material conveying section is to enable multiple core materials to contact each other (but not dissolve) after entering the mainstream channel, form the continuous phase of multiple materials, and then be truncated and wrapped by the first layer of shell material to form a core structure similar to a "mangosteen shape".

[0043] Further preferably, when there are two core materials, the flow direction of each core material at the outlet of the corresponding inlet channel forms a 45° angle with the flow direction of the material in the mainstream channel, and the two core material inlet channels are symmetrically arranged with respect to the central axis of the mainstream channel.

[0044] Further preferably, when the number of outer wrapping layers of the core material is two and the first layer of shell material is two:

[0045] The feeding speed of the core material is 5 - 15 μL / min;

[0046] The feeding speed of the first layer of shell material is 40 - 60 μL / min;

[0047] The feeding speed of the second layer of shell material is 260 - 330 μL / min;

[0048] The feeding speed of the truncated material is 800 - 1500 μL / min.

[0049] Even more preferably, the feeding speed of the core material is 10 μL / min;

[0050] The feeding speed of the first layer of shell material is 50 μL / min;

[0051] The feeding speed of the second layer of shell material is 300 μL / min;

[0052] The feeding speed of the truncated material is 1000 μL / min.

[0053] As a further preference, when the number of layers wrapping the core material is two and the first layer of shell material is of one type:

[0054] The feeding speed of the core material is 5 - 15 μL / min;

[0055] The feeding speed of the first layer of shell material is 80 - 120 μL / min;

[0056] The feeding speed of the second layer of shell material is 260 - 330 μL / min;

[0057] The feeding speed of the truncating material is 600 - 900 μL / min.

[0058] Even more preferably, the feeding speed of the core material is 10 μL / min;

[0059] The feeding speed of the first layer of shell material is 100 μL / min;

[0060] The feeding speed of the second layer of shell material is 300 μL / min;

[0061] The feeding speed of the truncating material is 850 μL / min.

[0062] As a further preference, when the first layer of shell material is of two types, the two types of shell materials enter the main flow channel through two independent inlet flow channels, and the flow direction of each shell material at the outlet of the corresponding inlet flow channel forms a 45° angle with the flow direction of the material in the main flow channel; and the two inlet flow channels are symmetric with respect to the central axis of the main flow channel.

[0063] To better truncate the material in the main flow channel, as a further preference, when the first layer of shell material is of one type, the material enters the main flow channel through two independent inlet flow channels respectively, and the flow direction of the material at the outlet of the corresponding inlet flow channel is perpendicular to the flow direction of the material in the main flow channel, and the two inlet flow channels are symmetric with respect to the central axis of the main flow channel. Of course, the one type of material can also enter the main flow channel through one inlet flow channel.

[0064] As a preference, when the core material is of one type, there is one inlet flow channel for the core material, and the flow direction of the core material at the outlet of its inlet flow channel is the same as the flow direction of the material in the main flow channel.

[0065] As a further preference, when the number of layers wrapping the core material is two and the first layer of shell material is of two types:

[0066] The feeding speed of the core material is 0.5 - 2 μL / min;

[0067] The feeding speed of the first layer of shell material is 1 - 6 μL / min;

[0068] The feeding speed of the second-layer shell material is 10 - 40 μL / min;

[0069] The feeding speed of the truncated material is 100 - 600 μL / min.

[0070] More preferably, the feeding speed of the core material is 1 μL / min;

[0071] The feeding speed of the first-layer shell material is 3 μL / min;

[0072] The feeding speed of the second-layer shell material is 20 μL / min;

[0073] The feeding speed of the truncated material is 200 μL / min.

[0074] It should be noted that in the relationship between the flow direction of the materials mentioned in this article at the outlet of their corresponding inlet channels and the flow direction of the materials in the main channel, the flow direction of the materials in the main channel is the flow direction of the materials at the connection of the inlet channel and the main channel, and is also the flow direction at the moment when the materials flow into the main channel. The central axis of the main channel is the central normal of the cross-section perpendicular to the flow direction of the materials in the main channel; if two inlet channels are symmetric with respect to the central axis of the main channel, it means that the two inlet channels are symmetric with respect to the central normal of the cross-section perpendicular to the flow direction of the materials at the connection of the two inlet channels and the main channel. If the main channel is a straight channel, then this central normal is the central axis of the main channel. In addition, the feeding speed of the materials mentioned in the article refers to the feeding speed of the materials entering the main channel through an inlet channel.

[0075] Preferably, the core material and the shell materials of other layers except the outermost layer shell material are each independently selected from one or more of hydrogels and polyester materials.

[0076] Preferably, the outermost layer shell material is selected from one of hydrogels and polyester materials.

[0077] More preferably, the hydrogels include photocurable hydrogels (such as gelatin methacrylate (GelMA), gelatin methacrylate hyaluronic acid, dextran methacrylate (DexMA), polyether F127 diacrylate (F127DA)) and non-photocurable hydrogels (such as sodium alginate, gelatin, chitosan (such as chitosan methacrylate CSMA), agarose, acrylamide-acrylic acid copolymer).

[0078] More preferably, the polyester materials include photocurable polyesters (such as photocurable polycaprolactone (such as polycaprolactone maleimide PCLMA), polyethylene glycol (diol) diacrylate (PEGDA 200)) and non-photocurable polyesters (such as polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA)).

[0079] Optionally, to make the material flow more smoothly in the microfluidic chip, a hydrophilic surfactant (Tween 20, Tween 40, Tween 60, Tween 80, PVA, polyether 188) or a lipophilic surfactant (such as sorbitan fatty acid ester, PFPE-PEG polymer) can be added to the core material and the shell material.

[0080] When the core material and the shell material are photocurable materials, a photoinitiator needs to be added to the photocurable materials. The photoinitiator can be LAP, TPO_L, etc., and the photoinitiator needs to be selected according to the specific components of the photocurable materials.

[0081] Preferably, the truncating material is one or more of edible oil, mineral oil, silicone oil, and hydrophilic surfactant.

[0082] Preferably, the receiving solution is one of edible oil, mineral oil, silicone oil, and hydrophilic surfactant.

[0083] As a further preference, the edible oil is selected from soybean oil, peanut oil, rapeseed oil, sunflower oil, and corn oil;

[0084] The mineral oil is liquid paraffin; the silicone oil is dimethyl silicone oil;

[0085] The hydrophilic surfactant is selected from Tween 20, Tween 40, Tween 60, Tween 80, PVA, gum arabic, and polyether 188.

[0086] When the core material has two layers of shell, the core material and the first layer of shell material, and the first layer of shell material and the second layer of shell material can be oil / water immiscible materials, that is, the multi-layer multi-phase core-shell structure microspheres are oil-water-oil or water-oil-water structures. Of course, the core material and the first layer of shell material, and the first layer of shell material and the second layer of shell material can also be materials with the same oiliness or the same wateriness. When they are materials with the same wateriness, the immiscibility of the adjacent two layers is achieved by the influence of intermolecular forces; when they are materials with the same oiliness, the immiscibility is achieved by the difference in osmotic pressure of the corresponding two layers of materials.

[0087] Preferably, a multi-channel plunger pump feeding device is used for supplying the core material, the shell material, and the truncating material.

[0088] Preferably, in the receiving solution, the crosslinking and curing method of the core-shell structure microspheres is one of photocuring, thermal curing, and pH curing (ionic crosslinking curing);

[0089] When photocuring is adopted, the concentration of the photoinitiator added to the photocurable material is 0.0005 - 0.005 g / mL;

[0090] When pH curing is adopted, the outermost shell material needs to be ion-modified.

[0091] In the receiving solution, the corresponding curing and forming method is selected according to the cross-linking mechanism of the spheroidizing material (core material, shell material) to fix the morphology of the microspheres. When the outermost shell material is a hydrogel, the receiving solution is an oily solution; when the outermost shell material is a polyester material, the receiving solution is an aqueous solution. After the microspheres wrapped by the outermost shell material fall into the receiving solution, photo-crosslinking, low-temperature crosslinking with the receiving solution, or ionic crosslinking with a solution for ionic modification of the outermost shell material needs to be used.

[0092] Preferably, the ambient temperature of the microfluidic device is 0 - 50 °C.

[0093] Preferably, the temperature control range of the receiving solution is 0 - 50 °C.

[0094] More preferably, the concentration of the photoinitiator in the photocurable material is 0.001 - 0.003 g / mL.

[0095] More preferably, when using photocuring, a visible light source with a wavelength of 405 nm or 365 nm and an intensity of 30 - 80 mW / cm 2 is used to perform photo-crosslinking on the microspheres, and the photo-irradiation time is 3 s - 3000 s. The photo-irradiation time is further preferably 120 s.

[0096] Preferably, if the outermost shell material is a hydrogel, after the multi-layer multi-phase core-shell structure microspheres are cross-linked in the receiving solution, the receiving solution needs to be washed and filtered to obtain the final microsphere product; if the outermost shell material is a polyester, the multi-layer multi-phase core-shell structure microspheres can be directly freeze-dried from the receiving solution to obtain the microsphere product after being cross-linked in the receiving solution.

[0097] More preferably, the microfluidic device is a PDMS chip or a hydrogel chip.

[0098] Taking the case where the core material is wrapped with two layers of shell materials as an example, when the microfluidic chip is a PDMS chip or a hydrogel chip, the multi-layer multi-phase core-shell structure microspheres prepared by it can be in a "mangosteen shape" structure, that is, different materials in the core material come into contact with each other to form a whole and then are truncated and wrapped by the first layer of shell material, and / or different materials in the first layer of shell material come into contact with each other and wrap the core material to form a whole and finally are wrapped by the second layer of shell material to form microspheres (taking the microspheres having two layers of shells as an example).

[0099] The formation of the multi-layer multi-phase core-shell structure microspheres in the "mangosteen shape" structure is achieved on the one hand by controlling the flow channel structure of the core material and the layer shell material in the microfluidic chip; on the other hand, by selecting appropriate core materials and shell materials to have a relatively fast cross-linking speed to ensure the morphological fixation of the spheroidizing materials.

[0100] As a further preference, when the number of layers wrapping the core material is two:

[0101] The height of the inlet channel of the core material is 10 - 100 μm, and the width is 10 - 100 μm;

[0102] The height of the core material conveying section is 10 - 100 μm, and the width is 10 - 100 μm;

[0103] The height of the inlet channel of the first layer of shell material is 10 - 100 μm, and the width is 10 - 100 μm;

[0104] The height of the conveying section (the first conveying section) between the inlet channels of the first layer of shell material and the second layer of shell material is 100 - 300 μm, and the width is 100 - 300 μm;

[0105] The height of the inlet channel of the second layer of shell material is 10 - 100 μm, and the width is 10 - 100 μm;

[0106] The height of the conveying section (the second conveying section) between the inlet channels of the second layer of shell material and the inlet channel of the truncating material is 600 - 800 μm, and the width is 600 - 800 μm;

[0107] The height of the inlet channel of the truncating material is 10 - 100 μm, and the width is 10 - 100 μm;

[0108] The height of the outlet section of the main channel is 1000 μm, and the width is 1000 μm.

[0109] As a further preference, when the PDMS chip is not subjected to surface hydrophilic modification, it supports the flow of lipophilic materials; after surface hydrophilic modification treatment, it can support the flow of hydrophilic materials; when manufacturing oil - water - oil or water - oil - water three - layer microspheres, the channels through which the aqueous materials flow need to be subjected to hydrophilic modification treatment; the hydrogel chip can support the flow of hydrophilic materials, and the channels through which the oily materials flow also need to be subjected to lipophilic treatment; the metal chip supports the flow of both hydrophilic and lipophilic materials.

[0110] As a further preference, the PDMS chip is made by lithography or 3D printing microfiber reverse molding; the hydrogel chip is made by 3D printing microfiber reverse molding or direct photocuring printing; the metal chip is made by welding multi - channel parallel metal micro - pipes.

[0111] As a specific preference, taking the core - structure microsphere with two layers of shell as an example, a preparation method of a microfluidics - based multi - layer and multi - phase core - shell structure microsphere includes the following steps:

[0112] Step1: (Feeding step) Use a multi - channel plunger pump feeding device to supply the sphere - forming materials (including the core material and the shell material) and the truncating material;

[0113] Step 2: (Preliminary forming step) Use a microfluidic device to prepare a multi-phase and multi-layer core-shell structure microsphere from the spherifying material, and the material is truncated into spherical droplets at the outlet of the microfluidic chip by the truncating material.

[0114] Step 3: (Morphology fixing step) The spherical droplets fall into the receiving solution, and the corresponding curing and forming method is selected according to the cross-linking mechanism of the spherifying material to fix the morphology of the multi-phase and multi-layer core-shell structure microsphere.

[0115] Step 4: (Post-treatment step) Post-treat the microspheres with fixed morphology according to the properties of the second-layer shell material of the microspheres (aqueous or oily).

[0116] Preferably, in the post-treatment step, if the second-layer shell material is a hydrogel, after the multi-layer and multi-phase core-shell structure microspheres are cross-linked in the receiving solution, the receiving solution needs to be washed and filtered to obtain the final microsphere product; if the second-layer shell material is polyester, the multi-layer and multi-phase core-shell structure microspheres can be directly freeze-dried after being cross-linked in the receiving solution to obtain the microsphere product.

[0117] A multi-layer and multi-phase core-shell structure microsphere is prepared by the preparation method of the microfluidic-based multi-layer and multi-phase core-shell structure microsphere described in any one of the above.

[0118] When the multi-layer and multi-phase core-shell structure microsphere includes a core, a first-layer shell, and a second-layer shell, the diameter of the core is preferably 10 - 100 μm;

[0119] The outer diameter of the first-layer shell is preferably 300 - 600 μm; the outer diameter of the second-layer shell is preferably 800 - 1000 μm.

[0120] As a further preference, when multiple core materials or multiple shell materials of the same layer are oily materials, the immiscibility between the multiple materials is achieved through different osmotic pressures; when they are aqueous materials, the immiscibility between the multiple materials is achieved through the influence of the intermolecular forces of the aqueous materials.

[0121] The preparation method of the microfluidic-based multi-layer and multi-phase core-shell structure microsphere of the present invention controls the composition and feeding speed of the core material, each layer of shell material, and the truncating material, and uses the flow channel design and cascade operation of the microfluidic device to generate multi-layer and multi-phase microsphere droplets; subsequently, the microsphere droplets are fixed and formed by using corresponding methods such as photo-crosslinking, temperature crosslinking (thermal crosslinking), pH crosslinking, etc., and the multi-layer and multi-phase core-shell structure microspheres are obtained by using post-treatment methods such as filtration or freeze-drying. At least one of the core and the multi-layers of shells in the obtained multi-layer and multi-phase core-shell structure microspheres has materials with multiple components, and can be customized into personalized multi-layer and multi-phase microspheres according to needs.

[0122] When loading cells into the multi-layer and multi-phase core-shell structure microspheres, the cell density added in the hydrogel material is 1 to 5×10 6 cells / mL.

[0123] When loading cells into the microspheres, after the cell microspheres are collected into the receiving solution, culture medium needs to be added dropwise to the receiving solution to maintain the cell viability, and the receiving solution is filtered by using an oil-loving and water-hating filter membrane to remove the oily solution in the culture medium.

[0124] Among them, the filter membrane material is one of polytetrafluoroethylene, polyvinylidene fluoride, hydrophobic polyethersulfone, and hydrophobic nylon.

[0125] When loading drugs, corresponding drugs can be loaded in the core and each layer of the shell as needed to achieve the step-by-step release of different drugs. Or corresponding drugs can be loaded in the core or a certain layer of the shell to achieve targeted drug release.

[0126] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0127] The preparation method of the microfluidics-based multi-layer and multi-phase core-shell structure microspheres of the present invention utilizes the microfluidics technology. By selecting the types and feeding speeds of the core material, shell material, and truncation material, and designing the flow channel structure in the microfluidics device, the preparation of the multi-layer and multi-phase core-shell structure microspheres is realized; the core-shell structure microspheres prepared by this method have the advantages of highly uniform particle size, wide application range, customizable internal multi-phase materials, and gradual release of effective substances for different external environments, etc. They can be applied to tissue engineering and biomedical applications such as multi-cell co-culture, angiogenesis, drug delivery systems, synthesis of microparticles and microcapsules, biochemical analysis, targeted drug release, and disease treatment, and can be used to simulate complex physiological reactions in natural cells. Description of the Drawings

[0128] Figure 1 is the preparation flow chart of the multi-layer and multi-phase core-shell structure microspheres;

[0129] Figure 2 is the schematic diagram of the microfluidics chip structure and the preliminary forming process of the multi-layer and multi-phase core-shell structure microspheres with a "mangosteen-shaped" core of one material;

[0130] Figure 3 is the schematic diagram of the microfluidics chip structure and the preliminary forming process of the multi-layer and multi-phase core-shell structure microspheres with two "mangosteen-shaped" cores of multiple materials as the continuous phase; among them, A is that the first layer of the shell is one material; B is that the first layer of the shell is a continuous phase of multiple materials;

[0131] Figure 4Schematic diagram of the microfluidic cascade chip structure and the preliminary forming process of microspheres with a "lotus pod shape" core, which is a multi-layer and multi-phase core-shell structure microsphere with a dispersed phase of multiple materials;

[0132] Figure 5 Physical diagram of a microsphere with a "lotus pod shape" core, which is a multi-layer and multi-phase core-shell structure microsphere with a dispersed phase of multiple materials. Specific implementation manners

[0133] The following embodiments will further illustrate the present invention, but do not limit the present invention thereby. The feeding speed of the materials mentioned in the following embodiments refers to the feeding speed of the materials entering the main flow channel through an inlet flow channel.

[0134] Embodiment 1

[0135] Schematic diagram of the structure of the microfluidic chip and the preliminary forming process of microspheres with a "mangosteen shape" core, which is a multi-layer and multi-phase core-shell structure microsphere with a single material in this embodiment, as Figure 2 shown. The microfluidic device is a PDMS chip, and the flow channels through which the aqueous materials flow are hydrophilically treated.

[0136] The microfluidic device has a main flow channel, a core material inlet flow channel, a first layer shell material inlet flow channel, a second layer shell material inlet flow channel, and a truncating material inlet flow channel, and the aforementioned flow channels are all straight flow channels. Among them, the outlet of the core material inlet flow channel is coaxially connected to the inlet of the main flow channel; there are two first layer shell material inlet flow channels, two second layer shell inlet flow channels, and two truncating material inlet flow channels respectively, and the outlets are all communicated with the main flow channel. The first layer shell material inlet flow channel, the second layer shell inlet flow channel, and the truncating material inlet flow channel are spaced apart on the main flow channel and divide the main flow channel into a first conveying section, a second conveying section, and a main flow channel outlet section in sequence.

[0137] The central axis of each first layer shell material inlet flow channel forms an angle of 45° with the central axis of the core material inlet flow channel; the central axis of each second layer shell material inlet flow channel forms an angle of 90° with the central axis of the first conveying section, and the central axis of each truncating material inlet flow channel forms an angle of 90° with the central axis of the second conveying section. And the two first layer shell material inlet flow channels, the two second layer shell material inlet flow channels, and the two truncating material inlet flow channels are all symmetrically arranged relative to the central axis of the main flow channel in pairs. The specific structure is shown in Figure 2 .

[0138] The inlet flow channels of the core material (height: 80 μm, width: 80 μm), the inlet flow channels of the first layer of shell material (height: 80 μm, width: 80 μm), the first conveying section (height: 120 μm, width: 120 μm), the inlet flow channels of the second layer of shell material (height: 80 μm, width: 80 μm), the second conveying section (height: 700 μm, width: 700 μm), the truncating material flow channel (height: 80 μm, width: 80 μm), and the mainstream channel outlet section (height: 1000 μm, width: 1000 μm) in the microfluidic device.

[0139] Preparation of the spherifying material and the truncating material:

[0140] The core material 1-1 is CSMA, with a concentration of 0.01 g / mL. It is prepared using 1×PBS as the solvent, and the concentration of the LAP initiator is 0.0025 g / mL;

[0141] The first layer of shell materials 1-2 and 1-3 are PEGDA200 (200 DA) and PCLMA (800 DA), with TPO_L as the initiator and a concentration of 0.0025 g / mL;

[0142] The second layer of shell material 1-4 is HAMA, with a concentration of 0.02 g / mL. It is prepared using 1×PBS as the solvent, and the concentration of the LAP initiator is 0.0025 g / mL;

[0143] The truncating material is corn oil, and the surfactant added is Span 80 with a concentration of 0.05 g / mL;

[0144] The receiving solution is corn oil.

[0145] A preparation method of a multi-layer and multi-phase core-shell structured microsphere with a "mangosteen-shaped" core of a single material, as Figure 1 and 2 shown, specifically includes the following steps:

[0146] (1) Preparation of microspheres: Control the environmental temperature of the microfluidic device to be 30 - 50 °C, and when the material is loaded with cells, the environmental temperature is 30 - 37 °C; Feed the core material into the core material inlet channel at a feed rate of 1 μL / min; Feed the first layer of shell materials 1 - 2 and 1 - 3 into the first layer of shell material inlet channels respectively, and the feed rates of both are 3 μL / min; Among them, the first layer of shell materials 1 - 2 and 1 - 3 are immiscible due to the different osmotic pressures between the oily materials; The core material 1 - 1 is intercepted, clamped, and wrapped by the first layer of shell materials 1 - 2 and 1 - 3, and flows through the first conveying section; Subsequently, it is intercepted by the second layer of shell material 1 - 4 flowing in from the second layer of shell material inlet channel to form a core - shell structure and is wrapped and conveyed into the second conveying section. The feed rate of the second layer of shell material 1 - 4 is 20 μL / min; Subsequently, it is intercepted by the intercepted material (feed rate of 200 μL / min) to form multi - layer and multi - phase core - shell structure droplets and fall into the receiving device from the mainstream channel outlet section. The receiving device is filled with corn oil to receive the droplets. In addition, a visible light source with a wavelength of 405 nm or 365 nm and an optical intensity of 30 - 80 mW / cm 2 irradiates the droplets for photocrosslinking. The photocrosslinking time is 120 s. After photocrosslinking, the droplets solidify into "mangosteen - shaped" microspheres with a core of a single material and a multi - layer and multi - phase core - shell structure.

[0147] (2) Post - treatment of microspheres: The microspheres obtained through the above steps have an oily solution attached to their surfaces and need to be filtered and washed. Use PBS as the filtering solution and wash them repeatedly three times.

[0148] When the hydrogel material is loaded with cells, since the microspheres in this embodiment have a water - oil - water structure, the cells are loaded into the outermost layer, that is, the second layer of the shell when loading cells in the microspheres. The cell density added to the second layer of shell material (hydrogel) is 1 - 5×10 6 cells / mL. Mix the obtained cell suspension with the hydrogel (the second layer of shell material), and use a pipette to repeatedly pipette the mixed solution for 20 - 30 s to finally obtain the cell - loaded hydrogel spherical material (the second layer of shell material); The steps of (1) are the same as the above steps; In step (2), it is necessary to add a culture medium to the oily receiving solution to maintain the activity of the cells, and filter the receiving solution through a polytetrafluoroethylene filter membrane to remove the oily solution in the culture medium, so that only the culture medium suitable for cell survival remains in the receiving solution, and finally obtain "mangosteen - shaped" microspheres with a core of a single material and a multi - layer and multi - phase core - shell structure loaded with cells.

[0149] Example 2

[0150] The schematic diagram of the microfluidic chip structure and the preliminary forming process of the "mangosteen - shaped" microspheres with a multi - layer and multi - phase core - shell structure with a core of multiple materials as a continuous phase in this embodiment is as Figure 3As shown. The microfluidic device is a PDMS chip, and the channels through which the aqueous material flows are hydrophilically treated.

[0151] The first case: When the core of the fabricated microspheres is a continuous phase of multiple materials and the first layer of shell is a single material:

[0152] The microfluidic device is provided with a main channel, a core material inlet channel, a first layer of shell material inlet channel, a second layer of shell material inlet channel, and a truncating material inlet channel. The outlet of the core material inlet channel is communicated with the inlet of the main channel, and the outlets of the first layer of shell material inlet channel, the second layer of shell material inlet channel, and the truncating material inlet channel are respectively communicated with the main channel; and the core material inlet channel, the first layer of shell material inlet channel, the second layer of shell material inlet channel, and the truncating material inlet channel are respectively arranged at intervals, successively separating the main channel into a core material conveying section, a first conveying section, a second conveying section, and a main channel outlet section. And, the main channel, the core material inlet channel, the first layer of shell material inlet channel, the second layer of shell material inlet channel, and the truncating material inlet channel are all straight channels.

[0153] Among them, there are two core material inlet channels, two first layer of shell material inlet channels, two second layer of shell material inlet channels, and two truncating material inlet channels respectively. The central axis of each core material inlet channel forms an angle of 45° with the central axis of the main channel; the central axis of the first layer of shell material inlet channel forms an angle of 90° with the central axis of the main channel; the central axis of the second layer of shell material inlet channel forms an angle of 90° with the central axis of the first conveying section; the central axis of the truncating material inlet channel forms an angle of 90° with the central axis of the second conveying section; and the two core material inlet channels, the two first layer of shell material inlet channels, the two second layer of shell material inlet channels, and the two truncating material inlet channels are all symmetrically arranged with respect to the central axis of the main channel in pairs. The specific structure is as Figure 3 shown in A.

[0154] The second case, when the core of the fabricated microspheres is a continuous phase of multiple materials and the first layer of shell is a continuous phase of multiple materials:

[0155] The number and structure of the channels in the microfluidic device are the same as those of the microfluidic device used in the first case. The only difference is that the central axis of the first layer of shell material inlet channel forms an angle of 45° with the central axis of the main channel. The specific structure is as Figure 3 shown in B.

[0156] When the first layer of shell material is a single material or a continuous phase of two materials, the sizes of the material inlet channels, each conveying section, and the main channel outlet section of the corresponding two microfluidic devices are the same. Specifically:

[0157] Core material inlet channel (height 80μm, width 80μm), core material conveying section (height 20μm, width 20μm), first layer shell material inlet channel (height 80μm, width 80μm), first conveying section (inner diameter height 200μm, width 200μm), second layer shell material inlet channel (height 80μm, width 80μm), second conveying section (height 700μm, width 700μm), truncation material inlet channel (height 80μm, width 80μm), main channel outlet section (height 1000μm, width 1000μm).

[0158] Preparation of spherifying material and truncation material:

[0159] When the first layer of shell material is a single material:

[0160] Core material 2-1 is DexMA, with a concentration of 0.05 g / mL, prepared using solvent 1xPBS;

[0161] Core material 2-2 is GelMA, with a concentration of 0.05 g / mL, prepared using 1xPBS as the solvent;

[0162] The first layer of shell material 2-3 is PLGA (33kDA, concentration 0.02 g / mL, solvent dichloromethane), and surfactant Span 80 with a concentration of 0.05 g / mL is added to it;

[0163] The second layer of shell material 2-4 is sodium alginate, with a concentration of 0.0025 g / mL, prepared using 1xPBS as the solvent, ion-modified with Ca-EDTA, and the pH of the second layer of shell material is adjusted to 7.2;

[0164] The truncation material is HFE-7500, and surfactant PFPE-PEG with a concentration of 0.02 g / mL is added simultaneously;

[0165] The receiving solution is HFE-7500.

[0166] When the first layer of shell material is multiple materials:

[0167] Core materials 3-1 and 3-2 are the same as 2-1 and 2-2 one by one;

[0168] The first layer of shell material 3-3 is PLGA (33kDA, concentration 0.02 g / mL, solvent dichloromethane), and surfactant Span 80 with a concentration of 0.05 g / mL is added to it;

[0169] The first layer of shell material 3-4 is PCL (80 kDa, concentration 0.02 g / mL, solvent is dichloromethane), and a surfactant Span 80 with a concentration of 0.05 g / mL is added to it;

[0170] The second layer of shell material 3-5 is the same as 2-4;

[0171] The truncating material is HFE-7500, and a surfactant PFPE-PEG with a concentration of 0.02 g / mL is added;

[0172] The receiving solution is HFE-7500.

[0173] A preparation method of a multi-layer and multi-phase core-shell structure microsphere with a "mangosteen-shaped" core as a continuous phase of multiple materials specifically includes the following steps:

[0174] (1) Preparation of microspheres: Control the environmental temperature of the microfluidic device to be 5-50 °C; when the first layer of shell material is a single material, the core materials 2-1 and 2-2 are respectively introduced into two core material inlet channels, and the feeding speed of the core materials is 10 μL / min; then the first layer of shell material is introduced into the first layer of shell material inlet channel:

[0175] When the first layer of shell material is a single material 2-3, the feeding speed of the first layer of shell material is 100 μL / min.

[0176] When the first layer of shell is a continuous phase of multiple materials, the core materials are 3-1 and 3-2, and the feeding speed of the core materials is 10 μL / min; then the first layer of shell material is introduced into the first layer of shell material inlet channel. When the first layer of shell material is materials 3-3 and 3-4, their feeding speeds are 50 μL / min and 50 μL / min respectively;

[0177] Subsequently, the core materials 2-1 and 2-2 (or 3-1 and 3-2) are truncated by the first layer of shell material 2-3 (or 3-3 and 3-4) and are wrapped and transported, flowing through the first transport section. Due to the influence of the intermolecular forces of the aqueous materials, the core materials 2-1 and 2-2 (or 3-1 and 3-2) after truncation are immiscible;

[0178] Subsequently, the core-shell structure is truncated by the second shell material 2-4 (or 3-5) with a feeding speed of 300 μL / min and is wrapped and transported; for the case where the first shell material is two materials, due to the different osmotic pressures between the oily materials, the truncated first shell materials 3-3 and 3-4 are immiscible and flow through the second transport section; subsequently, the truncated material (when the first shell material is two materials, the feeding speed of the truncated material is 1000 μL / min; when the first shell material is a single material, the feeding speed of the truncated material is 850 μL / min) truncates the multi-layer and multi-phase core-shell structure droplets and falls into the receiving device from the outlet channel. The receiving device is filled with an oil-soluble receiving solution HFE-7500 modified with an acid relative to the second shell material to receive the microspheres, thereby realizing the ionic cross-linking (pH cross-linking) of the microspheres. After ionic cross-linking, the droplets solidify into "mangosteen-shaped" core multi-material multi-layer and multi-phase core-shell structure microspheres.

[0179] (2) Post-treatment of microspheres: The microspheres obtained through the above steps have an oil-soluble solution attached to their surfaces and need to be filtered and washed. PBS is used as the filtering solution and washed repeatedly three times.

[0180] Example 3

[0181] The "lotus seed pod-shaped" core in this example is a schematic diagram of the microfluidic cascade chip structure and the preliminary forming process of multi-layer and multi-phase core-shell structure microspheres with a dispersed phase of multiple materials, as Figure 4 shown.

[0182] The microfluidic device is a cascade chip composed of a metal chip and a PDMS chip. The metal chip is provided with four core material inlet channels arranged side by side and a first shell material inlet channel; the PDMS chip is provided with a main channel, two second shell material inlet channels, and two truncated material inlet channels. All the inlet channels and the main channel are straight channels.

[0183] The outlet of each core material inlet channel is in communication with the inlet of the main channel, and the central axis of each core material inlet channel is parallel to or lies on the same straight line as the central axis of the main channel. The outlet end faces of all the core material inlet channels are located on the plane where the cross-section (perpendicular to the central axis) of the main channel is located. And the central axis of the first-layer shell material inlet channel is perpendicular to the central axis of the core material inlet channel, and the outlet of the first-layer shell material inlet channel is in communication with the inlet of the main channel. The outlets of the second-layer shell material inlet channel and the cutoff material inlet channel are respectively in communication with the main channel perpendicularly (the included angle between the central axes is 90°), and the first-layer shell material inlet channel, the second-layer shell material inlet channel, and the cutoff material inlet channel are arranged at intervals on the main channel, and the main channel is successively divided into a first conveying section, a second conveying section, and a main channel outlet section. And the two second-layer shell material inlet channels and the two cutoff material inlet channels are symmetrically arranged with respect to the central axis of the main channel in pairs. For the specific structure, see Figure 4 。

[0184] The channels through which the aqueous materials flow on the PDMS chip are subjected to hydrophilic treatment.

[0185] The core material inlet channels (inner diameter: 120 μm), the first-layer shell material inlet channels (inner diameter: 120 μm) in the above metal chip; the first conveying section (height: 800 μm, width: 800 μm), the second-layer shell material inlet channels (height: 120 μm, width: 120 μm), the second conveying section (height: 1000 μm, width: 1000 μm), the cutoff material channels (height: 700 μm, width: 700 μm), and the outlet channels (height: 1000 μm, width: 1000 μm) in the PDMS chip.

[0186] A preparation method of "lotus seed pod-shaped" core multi-material multi-layer multi-phase core-shell structure microspheres specifically includes the following steps:

[0187] Preparation of the sphering material and the cutoff material:

[0188] The core material 4-1 is acrylic resin (2000 DA), 4-2 is PEGDA 200 (200 DA), 4-3 is PCL (80 Kda, concentration: 0.02 g / mL, solvent: dichloromethane), 4-4 is PLGA (33 KDA, concentration: 0.02 g / mL, solvent: dichloromethane);

[0189] The first-layer shell material 4-5 is F127DA, with a concentration of 0.1 g / mL, prepared using 1xPBS as the solvent, and adding surfactant Span 80 with a concentration of 0.05 g / mL;

[0190] The second layer of shell material 4-6 is PLGA (33 kDa, concentration 0.02 g / mL, solvent dichloromethane), and surfactant Span 80 with a concentration of 0.05 g / mL is added;

[0191] The truncating material is an aqueous solution of Tween 20 with a concentration of 0.02 g / mL;

[0192] The receiving solution is an aqueous solution of Tween 20 with a concentration of 0.02 g / mL.

[0193] (1) Preparation of microspheres: Control the environmental temperature of the microfluidic device to be 37 - 50 °C; as Figure 4 shown, the core materials 4-1, 4-2, 4-3, 4-4 are respectively introduced into the four core material inlet channels of the metal chip, and the feeding rate of the core materials is 25 μL / min; among them, controlling the feeding rates of different core materials can achieve control of the proportion of different components in the core materials; the first layer of shell material 4-5 is introduced into the first layer of shell material inlet channel with a feeding rate of 500 μL / min; subsequently, the core materials 4-1 to 4-4 are truncated by the first layer of shell material 4-5 and are wrapped and transported to the first transport section in the PDMS chip. The truncated core materials 4-1, 4-2, 4-3, 4-4 are immiscible due to the different osmotic pressures between the oily materials and form a dispersed phase; the materials in the first transport section are truncated into a core-shell structure by the second layer of shell material 4-6 with a feeding rate of 1200 μL / min and are wrapped and transported, flowing through the second transport section; subsequently, they are truncated into multi-layer and multi-phase core-shell structure droplets by the truncating material with a feeding rate of 3000 μL / min and fall into the receiving device from the outlet channel. The receiving device is filled with an aqueous solution of Tween 20 with a concentration of 0.02 g / mL and a temperature of 4 °C to receive the droplets, and the droplets can be thermally cured. After the droplets are cured, multi-layer and multi-phase core-shell structure microspheres with a "lotus seed pod shape" core of a dispersed phase of multiple materials are formed.

[0194] (2) Post-treatment of microspheres: The microspheres soaked in the aqueous solution obtained through the above steps can be directly freeze-dried in a vacuum environment at -60 °C, and the aqueous solution will evaporate completely during the freeze-drying process, and finally microsphere products are obtained. As Figure 5 shown, it is a multi-layer and multi-phase core-shell structure microsphere with a core of a dispersed phase of four materials prepared in this embodiment.

Claims

1. A preparation method of a microfluidics-based multi-layer and multi-phase core-shell structure microsphere, characterized in that, include: The core material enters from the entrance of the main channel of the microfluidic device, is successively cut off and wrapped layer by layer by the two layers of shell material and the cutting material entering the main channel, and then falls into the receiving solution for cross-linking and curing, thereby obtaining the multi-layer multi-phase core-shell structure microspheres; There are four types of core materials, and the first layer shell material and the second layer shell material are both one type; the multiple core materials, the core materials and the adjacent shell materials, and the adjacent layers of shell materials are not miscible with each other; The outermost shell material and the cut-off material are oil / water reactive materials; the cut-off material and the receiving solution are isotropically compatible materials; Each core material enters the main flow channel through a separate inlet flow channel; The inlet flow channels of the adjacent layer shell materials, the inlet flow channels of the outermost layer shell materials and the inlet flow channels of the cut-off materials are arranged at intervals, and corresponding conveying sections and main flow channel outlet sections are formed on the main flow channel; The flow direction of each core material at the corresponding inlet flow channel outlet is the same as the flow direction of the material in the main flow channel, and the outlet end surfaces of the multiple core material inlet flow channels are flush on a plane perpendicular to the flow direction of the material in the main flow channel; The inlet flow channel of the first layer shell material is provided with an outlet of the inlet flow channel being connected to the inlet of the main flow channel; the flow direction of the first layer shell material at the outlet of the inlet flow channel is perpendicular to the flow direction of the material in the main flow channel, and the multiple core materials are cut off and wrapped when the multiple core materials flow out of their corresponding inlet flow channels, so as to form a core with dispersed phases of multiple materials; The second layer shell material enters the main channel through two independent inlet channels respectively, and the flow direction of the second layer shell material at the outlet of the corresponding inlet channel is perpendicular to the flow direction of the material in the main channel, and the two inlet channels are symmetrical with respect to the central axis of the main channel; The feed rate of core material is 20~30μL / min; The feed rate of the first shell material was 400-600 μL / min; The feed rate of the second shell material was 1000-1500 μL / min; The feed rate of cut-off material is 2000~4000μL / min.

2. The preparation method of the microfluidics-based multi-layer and multi-phase core-shell structure microspheres according to claim 1, wherein The microfluidic device is a cascade chip consisting of a metal chip and a PDMS chip or a hydrogel chip; The core material inlet channel and the first shell material inlet channel are arranged on the metal chip, and the main channel, the inlet channels of other shell materials and the cut-off material inlet channel are arranged on the PDMS chip or the hydrogel chip.

3. The preparation method of the microfluidics-based multi-layer and multi-phase core-shell structure microspheres according to claim 1, characterized in that, The core material and the shell materials of the other layers except the outermost shell material are independently selected from one or more of hydrogel and polyester materials; The outermost shell material is selected from one of hydrogel and polyester materials; The cut-off material is one or more of edible oil, mineral oil, silicone oil, and hydrophilic surfactant.

4. The preparation method of the microfluidics-based multi-layer and multi-phase core-shell structure microspheres according to claim 1, wherein, In the receiving solution, the cross-linking curing mode of the core-shell structure microspheres is one of light curing, heat curing and pH curing; When light curing is used, the concentration of the photoinitiator added to the light curing material is 0.0005~0.005 g / mL; When pH curing is used, the outermost shell material needs to be ion-modified.

5. A multi-layer and multi-phase core-shell structure microsphere, characterized in that, The microspheres are prepared by the method for preparing multi-layered multi-phase core-shell structured microspheres based on microfluidics according to any one of claims 1 to 4.

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