Method and equipment for preparing porous gelatin microspheres

Abstract

The invention relates to the technical field of biomedical materials, in particular to a method and equipment for preparing porous gelatin microspheres. The method for preparing the porous gelatin microspheres is realized by utilizing a micro-fluidic chip with a micro-mixing area and a micro-dispersion area and a curing system, and comprises the following steps: injecting gelatin derivative fluid and polymer fluid into the micro-mixing area at 30-60 DEG C, so that the gelatin derivative fluid and the polymer fluid flow and are mixed in the micro-mixing area, and the micro-mixing area is formed; and carrying out time sequence phase separation and self-assembly under the periodic influence of the microstructure of the micro-mixing region to form a micro-mixing fluid, and carrying out micro-dispersion and curing to obtain the porous gelatin microspheres. Loading a load into the fluid solution to prepare the porous gelatin microspheres containing the load. According to the method, the microsphere industrialization process of the porous gelatin material is simplified, the nano-structure characteristics of the porous gelatin microspheres can be efficiently reserved, the quality of the nano-structure porous gelatin microspheres is improved, and efficient development, batch production and application of porous gelatin products can be accelerated and promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a method and equipment for preparing porous gelatin microspheres. Background Art

[0002] Gelatin microspheres, due to their excellent biocompatibility, biodegradability, small size, and high specific surface area, have shown significant application potential in fields such as tissue engineering, regenerative medicine, and drug delivery. Furthermore, the unique structural characteristics of porous gelatin microspheres enhance their water absorption and permeability, expanding their application prospects and offering advantages in drug and cell delivery. Recent research has shown that a porous material based on a PEO-PPO-PEO block copolymer and methacrylated gelatin, characterized by its unique nanostructure, has demonstrated significant enhancement effects in tissue engineering and has broad application prospects. However, the current production capacity of porous gelatin biomaterials is insufficient, making clinical application of these products difficult. Therefore, accelerating the industrialization of nanostructured porous gelatin microspheres is of great significance.

[0003] However, the industrialization process of this type of nanostructured porous gelatin microspheres has long been limited by the complexity of the preparation process, which has seriously restricted the development, mass production and clinical application of them or their loaded products; currently, the main method for small-scale production of the above-mentioned nanostructured porous gelatin microspheres is to use physical methods such as stirring and ultrasound, but the preparation process is complicated, the operation is difficult, and the stability is insufficient; in addition, due to the inherent thermosensitivity and rheological properties of gelatin materials, traditional microfluidic technology is difficult to achieve precise control, and the conventional emulsification cross-linking method has bottlenecks such as poor process stability and insufficient cell compatibility. Therefore, the currently prepared nanostructured porous gelatin microspheres still have quality shortcomings such as uneven size, insufficient stability, poor uniformity, low batch reproducibility, and high temperature or organic solvents that easily damage cell activity.

[0004] Microfluidics utilizes microchannels (tens to hundreds of microns in size) to regulate fluid interfacial tension and shear force to process or manipulate tiny fluids (with volumes ranging from picoliters to nanoliters). Microfluidic chips are the core of microfluidic technology for preparing microspheres. By introducing curved or spiral channels, adding microcolumn arrays or obstacles, the geometric structure of microchannels can be optimized to enhance fluid mixing and shear effects. However, regardless of whether several spatially deflected and curved serpentine configurations are introduced into the microchannels of the microfluidic chip, or whether obstacles are continued to be added, it is still difficult to obtain a uniform and stable internal nanostructure in the porous gelatin microspheres prepared through these two types of microchannels, which still restricts the efficient development, large-scale quantitative production and clinical application of porous gelatin microsphere products.

[0005] In the prior art, CN112275336A and CN109622078A both disclose microfluidic chips with serpentine or wavy spatial deflection channels. However, CN112275336A uses microfluidic chips to prepare gel microspheres, while CN109622078A uses microfluidic chips for non-Newtonian fluids.

[0006] CN111286456A discloses setting a first obstacle and a second obstacle in the screening area of ​​a microfluidic channel. The technical purpose is to achieve cell screening and prevent cell clusters from blocking the crushing area and affecting the quality of nanovesicles.

[0007] The above existing technologies are difficult to simultaneously solve the problems of self-assembly and phase separation sequentiality and realize the preparation of nanostructured porous gelatin microspheres. Therefore, the efficient development, large-scale quantitative production and clinical application of nanostructured porous gelatin microspheres still face technical challenges. Summary of the Invention

[0008] The present invention provides a method and apparatus for preparing porous gelatin microspheres. The method and apparatus utilize a process flow of micro-mixing, micro-dispersion, and light-curing based on microfluidic technology, in conjunction with temperature control, to achieve periodic sequential phase separation and self-assembly during flow mixing. This simplifies the industrialization process for microspheres of porous gelatin materials, retains the nanostructure characteristics of the porous gelatin microspheres, and improves the quality of the nanostructured porous gelatin microspheres. This can accelerate the efficient development, mass production, and application of porous gelatin products.

[0009] To solve the above technical problems, the first object of the present invention is to provide a method for preparing porous gelatin microspheres, which is achieved by using a microfluidic chip with micro-mixing and micro-dispersing areas and a curing system, comprising the following steps: 1) Micromixing: injecting a gelatin derivative fluid and a polymer into the micromixing region at 30-60°C, causing the two to flow and mix within the micromixing region, and undergoing sequential phase separation and self-assembly under the periodic influence of the microstructure of the micromixing region to form a micromixing product; 2) Microdispersion: injecting the continuous phase into the microdispersion area to microdisperse the micromixed product to form microdroplets; 3) Solidification: The microdroplets are solidified using a solidification system to prepare porous gelatin microspheres.

[0010] In the present invention, phase separation refers to a two-phase separation process in which the gelatin derivative fluid and the polymer are contacted and flow-mixed after being injected into the micro-mixing region, flow through the microstructure, and under the influence of the microstructure, the separation strength of the two phases of the gelatin derivative fluid and the polymer is greater than the self-assembly of the two phases; self-assembly refers to a two-phase self-assembly process in which the self-assembly strength of the two phases of the gelatin derivative fluid and the polymer is greater than the self-assembly of the two phases; phase separation and self-assembly processes always occur during the flow mixing process of the gelatin derivative fluid and the polymer, especially during the flow through the periodic microstructure, but there is a difference in the strength of the two, thus manifesting as a periodic and temporal process of "first phase separation, then self-assembly"; as the flow mixing progresses, the gelatin derivative fluid and the polymer are fully and evenly mixed, and the strength of the phase separation and self-assembly caused by the influence of the microstructure in the later stage tends to balance; micro-dispersion occurs under the action of the continuous phase in the micro-dispersion region, and micro-droplets with controllable particle size that are uniformly mixed with the gelatin derivative fluid and the polymer can be obtained.

[0011] In the preparation method of the present invention, temperature also affects the flow mixing in the micro-mixing and thus affects the efficiency of the flow mixing; therefore, in addition to setting the microstructure on the inner wall surface of the micro-mixing area, it is also necessary to control the flow mixing temperature to 30-60°C. The two work together to achieve efficient, sufficient and uniform flow mixing of gelatin and polymer, and achieve the purpose of micro-dispersion and solidification in the continuous phase to form porous gelatin microspheres with controllable particle size; of course, based on the mechanism that the above microstructure can enhance the flow mixing effect of raw materials, the load is uniformly mixed in the porous gelatin microspheres, and the preparation process of the porous gelatin microspheres is applied to the preparation of drug-loaded drugs, cell-loaded preparations, etc., and the preparation process of the microspheres is optimized by applying to process steps such as detection, recovery, washing, filtration, automated operation, and post-processing.

[0012] In a specific embodiment of the present invention, in step 1) micro-mixing, the volume ratio of the gelatin derivative fluid to the polymer fluid is 0.25-4.

[0013] In a specific embodiment of the present invention, in step 3) curing, a photoinitiator is added to the gelatin derivative fluid to subject the microdroplets to a photocuring treatment.

[0014] In one embodiment of the present invention, the photocuring process utilizes visible light, such as 405 nm blue light or ultraviolet light.

[0015] In a specific embodiment of the present invention, a load is loaded into the gelatin derivative fluid or the polymer, and porous gelatin microspheres containing the load are prepared using the microfluidic chip and the curing system.

[0016] The second object of the present invention is to provide a microsphere making device for implementing the above-mentioned method, comprising a constant temperature system, a microfluidic system and a curing system, wherein the microfluidic system is arranged in the constant temperature system, and the microfluidic system includes at least one microfluidic chip; the microfluidic chip has at least one micro-mixing area and at least one micro-dispersing area, and the outlet of the micro-mixing area is connected to the inlet of the micro-dispersing area; the inner wall surface of the micro-mixing area is provided with a microstructure; the microfluidic chip is also provided with a first inlet for injecting gelatin derivative fluid, a second inlet for injecting polymer, a third inlet for injecting continuous phase and a droplet outlet for connecting the micro-dispersing area with the curing system.

[0017] In a specific embodiment of the present invention, the channel configuration of the micro-dispersed region is a T-shaped configuration, a flow focusing configuration or a capillary focusing configuration.

[0018] In a specific embodiment of the present invention, the microstructure is composed of spaced-apart raised microstripes on the inner wall surface, and the channel axis of the micro-mixing region is spatially deflected to form at least one spatially deflected configuration.

[0019] In a specific embodiment of the present invention, the distance between adjacent micro stripes is 30 to 80 μm, and the height of the protrusions is 3 to 10 μm.

[0020] In a specific embodiment of the present invention, the spatial deflection configuration is a serpentine configuration, a wave configuration or a sawtooth configuration.

[0021] Nanostructured porous gelatin microspheres are prepared based on the above method or microsphere making equipment.

[0022] The nanostructured porous gelatin microspheres are used for at least one application selected from the following (a) to (f): (a) As a three-dimensional cell culture scaffold, used in cell preparations to promote cell adhesion, proliferation or differentiation; (b) as drug delivery vehicles, loading therapeutic agents, diagnostic agents, or immunomodulators to achieve targeted or sustained release; (c) as injectable or implantable tissue filling materials for repairing soft tissue defects or regenerating bone tissue; (d) As a hemostatic material, used in wound dressings or surgical hemostasis to promote hemostasis by physical adsorption and activation of coagulation factors; (e) in the field of medical aesthetics as a soft tissue filler or to improve skin structure by stimulating fibroblasts to secrete collagen; (f) In tumor treatment, they can be used as drug-loaded microspheres to locally release chemotherapy drugs or as embolic agents to block the blood supply to tumor vessels.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention is simple and convenient. The mixing uniformity of raw materials is synergistically improved by temperature control and the design of the microstructure of the microfluidic chip. The porous gelatin microspheres can retain the nanostructure of the porous gelatin material after curing, and have good application prospects in the fields of cell preparation, optimized cell culture, tissue filling, drug delivery, hemostatic products, medical aesthetics, tumor treatment, etc. The present invention also improves the production efficiency of nanostructured porous gelatin microspheres, improves the quality of nanostructured porous gelatin microspheres, and makes the particle size of nanostructured porous gelatin microspheres uniform and controllable, which is conducive to accelerating the product development, large-scale quantitative production and clinical application of nanostructured porous gelatin microspheres. 2. The microsphere making equipment of the present invention can be used not only to prepare nanostructured porous gelatin microspheres, but also to prepare porous gelatin microspheres containing loads; the constant temperature system provides a temperature environment for the preparation of porous gelatin microsphere products. By utilizing the gain effect of the microstructure of the micro-mixing area, the micro-mixing, micro-dispersion, and solidification processes can be continuously completed in the equipment, and the automated preparation of porous gelatin microspheres can be realized.

[0024] 3. The porous gelatin microspheres with nanostructures of the present invention can be used as carriers of drugs and cell preparations, as well as carriers of other loads such as viruses, non-viral liposome nanoparticles, nucleic acids, proteins, metal ions, polysaccharides, polypeptides, etc., to prepare porous gelatin microspheres containing loads. At the same time, it can also provide a basis for the development of new porous gelatin products, and has good application prospects in the fields of cell preparations, optimized cell culture, tissue filling, drug delivery, hemostatic products, medical aesthetics, tumor treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A flowchart of the process for preparing porous gelatin microspheres provided in a specific embodiment of the present invention; Figure 2 A schematic structural diagram of a first microsphere manufacturing device provided in a specific embodiment of the present invention; Figure 3 A schematic structural diagram of a second microsphere manufacturing device provided in a specific embodiment of the present invention; Figure 4 A schematic structural diagram of a microsphere manufacturing system provided in a specific embodiment of the present invention; Figure 5 A schematic structural diagram of a first microsphere chip provided in a specific embodiment of the present invention; Figure 6 A schematic structural diagram of a second microsphere chip provided in a specific embodiment of the present invention; Figure 7 A schematic structural diagram of a third microsphere chip provided in a specific embodiment of the present invention; Figure 8A schematic structural diagram of a fourth microsphere chip provided in a specific embodiment of the present invention; Figure 9 A front view of the structure of the micro-mixing region provided in a specific embodiment of the present invention; Figure 10 A right side view of the structure of the micro-mixing region provided in a specific embodiment of the present invention; Figure 11 A schematic structural diagram of a microstructure provided for a specific embodiment of the present invention; Figure 12 A schematic structural diagram of a third microsphere manufacturing device provided in a specific embodiment of the present invention; Figure 13 This is a photograph of the micro-mixing area of ​​the microfluidic chip A prepared in Experimental Example 1 of the present invention; Figure 14 This is a photograph of the microstructure of the microfluidic chip A prepared in Experimental Example 1 of the present invention; Figure 15 This is a morphology of the internal structure of the porous gelatin microspheres prepared in Experimental Example 1 of the present invention under a scanning electron microscope; Figure 16 This is a photograph of the micro-mixing area of ​​the microfluidic chip B prepared in Experimental Example 3 of the present invention; Figure 17 This is a photograph of the microfluidic chip B without microstructure prepared in Experimental Example 3 of the present invention; Figure 18 This is a photograph of the micro-mixing area of ​​the microfluidic chip C prepared in Experimental Example 3 of the present invention; Figure 19 This is a microstructure photo of the microfluidic chip C prepared in Experimental Example 3 of the present invention; Figure 20 This is a photograph of the external structure of porous gelatin microspheres A prepared in Experimental Example 3 of the present invention under an optical microscope; Figure 21 This is a photograph of the external structure of porous gelatin microspheres B prepared in Experimental Example 3 of the present invention under an optical microscope; Figure 22 This is a photograph of the external structure of porous gelatin microspheres C prepared in Experimental Example 3 of the present invention under an optical microscope; Figure 23 This is a photo of the external structure of the porous gelatin microspheres A1 prepared in Experimental Example 4 of the present invention under an optical microscope; Figure 24 This is a photograph of the external structure of the porous gelatin microspheres A2 prepared in Experimental Example 4 of the present invention under an optical microscope.

[0026] In the accompanying drawings, the components represented by the reference numerals are as follows: 1. Microfluidic chip; 11. Micromixing region; 12. Microdispersion region; 13. First inlet; 14. Second inlet; 15. Third inlet; 16. Droplet outlet; 17. Microstructure; 18. Fourth inlet; 19. Fifth inlet 10. Batching device; 20. Microfluidic system; 30. Curing system; 40. Constant temperature system; 50. Liquid injection system. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be described in detail below. Obviously, the experimental examples described are only some of the experimental examples of the present invention, rather than all of the experimental examples. Based on the experimental examples in the present invention, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present invention.

[0028] Based on the attached Figure 1 The method for preparing porous gelatin microspheres is implemented by using a microfluidic chip 1 having micro-mixing and micro-dispersing areas and a curing system, comprising the following steps: 1) Micromixing: The gelatin derivative fluid and the polymer are injected into the micromixing region 11 at 30-60°C, where they flow and mix in the micromixing region 11 and undergo sequential phase separation and self-assembly under the periodic influence of the microstructure 17 of the micromixing region 11, thereby forming a micromixing product. 2) Microdispersion: The continuous phase is injected into the microdispersion region 12 to microdisperse the micromixed product to form microdroplets; 3) Solidification: The microdroplets are solidified using a solidification system 30 to prepare porous gelatin microspheres.

[0029] In the present invention, the gelatin derivative fluid is a fluid obtained by dissolving methacryloylated gelatin (GelMA) having a methacrylate (MA) substitution degree ≥90% in a solvent; the methacryloylated gelatin (GelMA) is also a biomaterial that can be photocured, so the curing of step 3) can be preferentially based on photocuring, although the curing of step 3) can also be achieved by other methods; the concentration of GelMA in the gelatin derivative fluid is 0.1 to 0.3 g / mL, and it has better fluidity in the microchannel of the micromixing area; the solvent for dissolving GelMA can be selected from any one of deionized water, phosphate buffer, glucose solution, physiological saline solution, and cell culture medium.

[0030] In the present invention, the polymer in the polymer fluid is a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer; preferably, the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is 0.01 to 0.4 g / mL, which has better flow inside the microchannel of the micromixing region; the balance in the polymer fluid is a solvent selected from any one of deionized water, phosphate buffer, glucose solution, physiological saline solution and cell culture medium; wherein the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is derived from the trade name Poloxamer 188, also known as Pluronic F68, and the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer can also be derived from other polyether products.

[0031] In the present invention, the organic matter in the continuous phase fluid is one of fluorinated oil (HFE-7500), silicone oil, liquid paraffin, hydrocarbon oil, and vegetable oil to achieve micro-dispersion; a conventional surfactant can also be added to the continuous phase to enhance the micro-droplet generation effect.

[0032] Referring to CN115850729A, in the present invention, methacrylated gelatin and polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer are mixed by flow in a micro-mixing process, and periodic temporal phase separation and self-assembly process occurs along with the flow mixing, thereby improving the uniformity of micro-mixing of methacrylated gelatin and polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer in a microchannel; micro-dispersion under the action of a continuous phase fluid can form micro-droplets of uniform size; and then, after a curing treatment, the methacrylated gelatin and polyoxyethylene-polyoxypropylene block copolymer in the micro-droplets are "locked" The uniform distribution of propylene glycol-polyoxyethylene block copolymer preserves the nanostructure of the porous gelatin microspheres. However, if the micro-mixing uniformity of methacrylated gelatin and polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer in the microchannel is poor, the micro-dispersion process will form microdroplets of different sizes (including small-sized microdroplets formed by the dispersion of methacrylated gelatin or polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer alone), and the number of solidified "locked" nanostructures will be reduced, thereby affecting the quality of the porous gelatin microspheres per unit weight.

[0033] In some embodiments, in step 1) micromixing, the volume ratio of the gelatin derivative fluid to the polymer is 0.25-4.

[0034] In order to control the volume ratio of the gelatin derivative fluid and the polymer fluid flowing and mixing in the microfluidic chip 1, when the flow rates of the raw materials injected into the micro-mixing area 11 are the same, the ratio of the cross-sectional areas of the channels used to inject the gelatin derivative fluid and the polymer is changed respectively to control the volume ratio of the raw materials flowing and mixing; of course, when the cross-sectional area ratio of the square channels for injecting the gelatin derivative fluid and the polymer fluid is fixed (for example, the cross-sectional area ratio of the square channels is 1:1), the flow rates of the raw materials can be adjusted respectively to control the volume ratio of the micro-mixing.

[0035] In some instances, a photoinitiator is added to the gelatin derivative fluid, and in step 3), a photocuring treatment is performed based on visible light; the photoinitiator is a conventional photoinitiator, and its concentration is also a conventional concentration; the type of photoinitiator can be determined based on whether photocuring is achieved by irradiation with 405 nm visible light; the photoinitiator can also be any one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and 2-hydroxy-2-methyl-1-phenyl-1-propanone or 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide as disclosed in CN115850729A.

[0036] In other examples, the photoinitiator can also be injected separately into the micro-mixing area 11 for flow mixing with the gelatin derivative fluid and the polymer fluid, but the injection amount needs to be determined based on the flow rate of the aforementioned gelatin derivative fluid and the concentration of the photoinitiator in the gelatin derivative fluid; the photoinitiator can also be dissolved in the aforementioned solvent for dissolving GelMA and then injected into the micro-mixing area 11 for flow mixing.

[0037] It should be noted that when the curing in step 3) is light curing, the processes of micro-mixing, micro-dispersion and light curing are all carried out in the same environment at 30-60°C.

[0038] In some instances, the surface of porous gelatin microspheres (solid state) obtained by photoinitiator and photocuring is coated with a continuous phase fluid (flowing state). After the photocured product is recovered, it needs to be purified to remove the continuous phase fluid and the solvent required for micromixing and microdispersion of the gelatin derivative fluid and the polymer fluid; the purification process can be achieved through unit operations such as emulsification, centrifugation, filtration, washing, elution, and drying, and can also be achieved by using microbial culture decomposition and culture facilities; among them, elution is achieved using deionized water, phosphate buffer, glucose solution, physiological saline solution or cell culture medium.

[0039] In some examples, the solvent introduced into the gelatin derivative fluid and the polymer fluid due to micro-mixing and micro-dispersion can also be removed based on the above purification method, or spray-dried.

[0040] In some examples, uncrosslinked photocurable biomaterial monomers, residual photoinitiators, and free polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymers can also be removed based on the above purification treatment method, such as elution.

[0041] In some examples, after solidification or purification, the recovered porous gelatin microsphere product is freeze-dried or spray-dried for subsequent storage and application.

[0042] In some examples, micro-mixing and micro-dispersion temperature control methods: 1. Place the microfluidic chip 1 in an environment of 30-60°C; 2. A temperature control structure is provided inside the microfluidic chip 1 to control the temperature of the micro-mixing region 11 and the micro-dispersing region 12 to be 30-60° C.; In the above two temperature control methods, the microfluidic chip 1 may be placed in an environment of 30-60°C, which may cause heat transfer obstruction and temperature difference; the built-in temperature control structure is prone to cause local overheating; the two temperature control methods can be implemented separately or combined to make up for the defects when implemented separately; the above two temperature control methods can be selected from any one of resistance heating, thermoelectric heating, fluid circulation temperature control, phase change material temperature control, and magnetocaloric effect heating.

[0043] In some examples, a loading substance is loaded into a gelatin derivative fluid or a polymer fluid to prepare porous gelatin microspheres containing the loading substance.

[0044] In other examples, the load may be injected into the micro-mixing region 11 alone to achieve flow mixing with the gelatin derivative fluid and the polymer fluid.

[0045] In some examples, the cargo is a drug or a cell.

[0046] In other examples, the load may also be biologically active substances such as viruses, non-viral liposome nanoparticles, nucleic acids, proteins, metal ions, polysaccharides, and polypeptides.

[0047] In some examples, the prepared porous gelatin microspheres or porous gelatin microspheres containing a load can achieve particle collection and storage through structural design, and can achieve post-processing purification processes such as particle recovery and washing by adding corresponding facilities.

[0048] In some examples, the microdroplets obtained by microdispersion before solidification are spherical or prolate ellipsoidal.

[0049] Based on the attached Figure 2-12, a microsphere making device for realizing the method of preparing porous gelatin microspheres, includes a constant temperature system 40, a microfluidic system 20 and a curing system 30, the microfluidic system 20 is arranged in the constant temperature system 40; the microfluidic system 20 includes at least one microfluidic chip 1; the microfluidic chip 1 has at least one micro-mixing area 11 and at least one micro-dispersing area 12; the outlet of the micro-mixing area 11 is connected to the inlet of the micro-dispersing area 12; the inner wall surface of the micro-mixing area 11 is provided with a microstructure 17; the microfluidic chip 1 is also provided with a first inlet 13 for injecting gelatin derivative fluid, a second inlet 14 for injecting polymer fluid, a third inlet 15 for injecting continuous phase fluid and a droplet outlet 16 for connecting the micro-dispersing area 12 and the curing system 30.

[0050] In some instances, the constant temperature system 40 includes a constant temperature chamber and a constant temperature facility, and the constant temperature facility and the microfluidic system 20 are arranged in the constant temperature chamber; the constant temperature facility is based on one of resistance heating, thermoelectric heating, fluid circulation temperature control, phase change material temperature control, and magnetocaloric effect heating to achieve temperature control of the constant temperature chamber at 30 to 60°C.

[0051] In other examples, a temperature control structure may be implanted inside the microfluidic chip 1 to compensate for the defect of the microfluidic chip 1 being affected by heat transfer in a constant temperature chamber and causing temperature difference interference.

[0052] In other examples, a constant temperature platform may be provided in the constant temperature chamber, and the microfluidic chip 1 may be placed on the constant temperature platform to prepare porous gelatin microspheres.

[0053] As attached Figure 2 、 3 As shown, in some instances, in order to achieve the preparation of gelatin derivative fluid, polymer fluid and continuous phase fluid, the microfluidic system 20 also includes a dispensing device 11; the dispensing device 11 includes a first dispensing container for preparing gelatin derivative fluid, a second dispensing container for preparing polymer fluid and a third dispensing container for preparing continuous phase; the first dispensing container, the second dispensing container and the third dispensing container are all sterile containers, which can be sterile centrifuge tubes, sterile culture bottles, sterile reagent bottles, anaerobic tanks, sterile transport bottles, etc.

[0054] In some examples, in order to facilitate the control of the temperature of the gelatin derivative fluid and the polymer fluid, the batching device 11 is set in a constant temperature room to achieve preheating to prevent the cold mass brought in by the raw materials from affecting the quality of micro-mixing and micro-dispersion.

[0055] In some examples, the constant temperature chamber is a sterile constant temperature chamber; in order to keep the sterile constant temperature chamber sterile, an ultraviolet sterilization lamp can be set in the sterile constant temperature chamber, and the sterile constant temperature chamber can also be connected to the output end of the high-temperature steam device.

[0056] In some examples, the batching device 11 further includes a fourth batching container for storing a photoinitiator and / or a fifth batching container for storing a load; the fourth batching container and / or the fifth batching container are the same as the first batching container, the second batching container or the third batching container and can be sterile containers; the photoinitiator or the load must be preheated.

[0057] In some instances, in order to prevent photocuring from occurring prematurely during the micromixing and microdispersion process and thereby clogging the micromixing area 11 or the microdispersion area 12, the constant temperature chamber is placed in a lightless state, or the microfluidic chip 1 is made of a low-light-transmittance material, or the outer surface of the microfluidic chip 1 is covered with a light-shielding layer or a light-shielding device.

[0058] In some examples, the outlet of the batching device 11 is directly connected to the inlet of the microfluidic chip 1 so as to inject the raw materials for preparing the porous gelatin microspheres into the microfluidic chip 1 .

[0059] In other embodiments, the outlet of the dispensing device 11 is connected to the inlet of the microfluidic chip 1 through a pipe; the material of the pipe can be metal or plastic, or medical metal or medical plastic; the delivery method of the pipe includes: Gravity flow transport, for example, the outlet height of the batching device 11 is higher than the inlet height of the microfluidic chip 1, and the raw materials are injected into the micro-mixing area 11 by natural gravity; Siphon transport uses the pressure difference generated by the height difference between the raw material liquid level of the batching device 11 and the inlet of the microfluidic chip 1 to make the liquid continue to flow after passing the high point of the pipeline; Capillary transport relies on the surface tension of the liquid to spontaneously penetrate through a microchannel (such as a capillary with an inner diameter of 0.5-2.5 mm) and flow into the inlet of the microfluidic chip 1; Pressure delivery, for example, injecting sterile pressurized gas into the ingredient container to push the raw materials in the ingredient container into the inlet of the microfluidic chip 1; Pump delivery, set a pump on the pipeline, such as a syringe pump; establish a liquid injection system 50 composed of multiple syringe pumps between the batching device 11 and the microfluidic chip 1, as shown in the attached Figure 3 As shown; the injection system 50 is responsible for controlling the raw material ratio and flow rate of preparing porous gelatin microspheres by controlling the working process of the pump, providing a stable environment for the batch production of porous gelatin microspheres; preferably, the injection pump is a constant flow injection pump.

[0060] See attached Figure 4In some instances, when the number of micro-mixing areas 11 or micro-dispersing areas 12 in the microfluidic system 20 is ≥2, a liquid distributor may be further provided on the pipeline between the dosing device 11 and the microfluidic chip 1 or outside the microfluidic chip 1 to distribute the raw materials of the dosing device 11 and then inject them into each micro-mixing area 11 or micro-dispersing area 12; optionally, the liquid distributor is "one inlet and n outlets", where n is a natural number ≥2, and n is equal to the number of the same inlets of the microfluidic system 20, such as the number of the first inlet, the second inlet or the third inlet; optionally, the "one inlet and n outlet" liquid distributor can be composed of multiple "one inlet and a (<n) out" distributors connected in series / parallel in stages, and the number of effective outlets is n, which is equal to the number of the same inlets of the microfluidic system 20; where the same inlet refers to the first inlet 13, the second inlet 14, the third inlet 15, the fourth inlet 18 or the fifth inlet 19.

[0061] In other embodiments, when the microfluidic chip 1 has multiple microfluidic chips 1 or multiple micro-dispersing areas 12, a distribution channel that can achieve "one inlet and n outlets" is provided inside the microfluidic chip 1. The inlet of the distribution channel is connected to the inlet of the microfluidic chip 1, and the outlet of the distribution channel is respectively connected to the inlet of each micro-mixing area 11 or the inlet of each micro-dispersing area 12.

[0062] In some instances, the outlets of multiple micro-mixing regions 11 are connected to multiple micro-dispersing regions 12 through a converging and redistributing channel, and the converging and redistributing channel is "b in c out"; wherein b is equal to the number of micro-mixing regions 11 in each microfluidic chip 1, and c is equal to the number of micro-dispersing regions 12 in the same microfluidic chip 1.

[0063] See attached Figure 5 、 7 In some instances, in order to enable the photoinitiator to be directly injected into the micro-mixing region 11 to participate in the flow mixing process, the microfluidic chip 1 further has a fourth inlet 18 for injecting the photoinitiator, and the fourth inlet 18 is connected to the inlet of the micro-mixing region 11.

[0064] See attached Figure 6 、 8 In some instances, in order to enable the load to be directly injected into the micro-mixing region 11 to participate in the flow mixing process, the microfluidic chip 1 further has a fifth inlet 19 for injecting the load, and the fifth inlet 19 is connected to the inlet of the micro-mixing region 11; either the fourth inlet 18 or the fifth inlet 19 can be selected according to actual needs.

[0065] In some embodiments, the microstructure 17 is composed of spaced microstripes protruding from the inner wall surface, and the channel axis of the micro-mixing region 11 is spatially deflected to form at least one spatially deflected configuration. In some embodiments, the microfluidic chip 1 can be mounted horizontally, vertically, or tilted.

[0066] In some embodiments, the microstructure 17 is composed of micro-stripes that are raised on the inner wall surface and arranged at intervals; preferably, the spacing between adjacent micro-stripes is 30 to 80 μm (as shown in the attached figure). Figure 9 The height of the protrusion is 3 to 10 μm (see attached Figure 10 、 11 As shown); the channel cross section of the micro-mixing region 11 can also be square.

[0067] It should be noted that the adjacent spacing refers to the shortest spacing between adjacent planes based on the plane where each micro-stripe extends along the inner wall surface; the protrusion height refers to the height of the micro-stripe relative to the inner wall surface, or the length extending to the center of the micro-mixing area 11 on the plane where the micro-stripe is located based on the inner wall surface.

[0068] It should also be noted that the spatial deflection of the axis of the channel of the micro-mixing region 11 forms at least one spatial deflection configuration, which can enhance the sequential flow mixing effect of the gelatin solution and the polymer solution. The periodic sequential phase separation and self-assembly process under the influence of the microstructure 17 occurs along with the sequential flow mixing process. To achieve the periodic sequential phase separation and self-assembly process, the microstructure 17 needs to meet the following conditions: 1. The microstructure 17 is arranged on the inner wall surface of the micro-mixing region 11, as shown in the attached Figure 9-11 As shown, the microstructure 17 is arranged at the bottom of the channel of the micro-mixing region 11 having a spatial deflection configuration on the same horizontal plane; Second, the arrangement of the microstructures 17 changes periodically to affect the periodic sequential phase separation and self-assembly of the fluid flowing and mixing in the micro-mixing region 11; Figure 9-11 As shown, the microstructure 17 is a raised micro-stripes arranged at intervals at the bottom of the channel of the micro-mixing region 11, and the channel axis of the micro-mixing region 11 is spatially deflected to form at least one periodic serpentine configuration; as shown in the attached Figure 9 As shown, along the flow direction of the flow mixing, the angle of the micro stripes gradually changes from 90° to 0°, and then from 0° to 90°, thus forming a periodic change, and so on; Therefore, when the gelatin derivative fluid and the polymer fluid flow through the micro-mixing region 11, not only flow mixing occurs, but also temporal phase separation and self-assembly occur under the influence of the microstructure 17, thereby achieving the purpose of enhancing the flow mixing effect.

[0069] In the present invention, the microfluidic chip 1 is prepared based on 3D printing technology.

[0070] In some embodiments, the spatial deflection configuration is a serpentine configuration, a wave configuration, or a sawtooth configuration; Figure 9 、 12 As shown, the channel axis of the micro-mixing region 11 is deflected on a plane to form at least one spatially deflected serpentine configuration.

[0071] As attached Figure 5-9 As shown in Figures 13 and 14, there are 65 serpentine configurations in the micro-mixing region 11. The micro-stripes arranged at intervals at the bottom of each serpentine configuration have an angle with the channel axis of the micro-mixing region that changes periodically twice.

[0072] In some examples, the inner wall surface is any location, including but not limited to the bottom, top, or side of the channel of the micro-mixing zone 11 .

[0073] In some embodiments, the micro stripes extend in the channel of the micro mixing region 11 to form stripes, arcs or rings; Figure 9 As shown, from the channel orthographic projection direction of the micro-mixing region 11, although the micro stripes extend in the channel, adjacent micro stripes do not actually intersect with each other, and the planes or lines where adjacent micro stripes are located may have intersection points or intersection lines in space.

[0074] In some examples, to achieve fine dispersion, the channel configuration of the fine dispersion region 12 is a T-shaped configuration, a flow focusing configuration, or a capillary focusing configuration.

[0075] In some examples, the third inlet 15 can be connected to the inlet of the micro-dispersed region 12 through a plurality of continuous phase microchannels; the plurality of microchannels are arranged at intervals around the inlet of the micro-dispersed region 12; Figure 5-7 As shown, there are two microchannels, and both microchannels are connected to the inlet of the micro-dispersing area 12, but the two microchannels are arranged relative to each other so that the continuous phase can cover the micro-mixed fluid entering the micro-dispersing area 12 and form micro-droplets after micro-dispersing in the micro-dispersing area.

[0076] See attached Figure 12 In some embodiments, the curing system 30 is a light curing system; the light curing system is set in a constant temperature room to prevent the micro droplets from being affected by low temperature (below 30-60°C) and phase separation due to untimely light curing.

[0077] In some examples, the photocuring system includes a 405 nm visible light lamp to irradiate the microdroplets to cause photocuring.

[0078] In some instances, the outlet of the solidification system 30 is connected to a collection device or a purification device, such as a culture device, a collector, a freeze dryer, etc., to achieve one or more processes such as recovery and collection, storage, purification (removal of the continuous phase of the porous gelatin microspheres coated on the surface that has not been solidified), and application of the porous gelatin microspheres.

[0079] Nanostructured porous gelatin microspheres are prepared based on the above method or microsphere making equipment.

[0080] The nanostructured porous gelatin microspheres are used for at least one application selected from the following (a) to (f): (a) As a three-dimensional cell culture scaffold, used in cell preparations to promote cell adhesion, proliferation or differentiation; (b) as drug delivery vehicles, loading therapeutic agents, diagnostic agents, or immunomodulators to achieve targeted or sustained release; (c) as injectable or implantable tissue filling materials for repairing soft tissue defects or regenerating bone tissue; (d) As a hemostatic material, used in wound dressings or surgical hemostasis to promote hemostasis by physical adsorption and activation of coagulation factors; (e) in the field of medical aesthetics as a soft tissue filler or to improve skin structure by stimulating fibroblasts to secrete collagen; (f) In tumor treatment, they can be used as drug-loaded microspheres to locally release chemotherapy drugs or as embolic agents to block the blood supply to tumor vessels.

[0081] The present invention provides the following experimental examples: Experimental Example 1 Study on the Effect of Microfluidics on the Nanostructure of Porous Gelatin Microspheres This experimental example provides a method for preparing porous gelatin microspheres, comprising the following steps: 1. Microfluidic chip design: see attached Figure 5 A microfluidic chip is designed with a micro-mixing region 11, the microfluidic chip also being designed with a first inlet 13 for injecting a gelatin solution, a second inlet 14 for injecting a polymer solution, and a third inlet 15 for injecting a continuous phase fluid, and a droplet outlet 16 connected to the outlet of the micro-mixing region 11; the inner wall surface of the channel of the micro-mixing region 11 has micro-stripes with a spacing of 30 to 80 μm and a height of 3 to 10 μm; and 65 serpentine-shaped spatial deflection flow channels in the micro-mixing region; 2. Preparation of microfluidic chip: A microfluidic chip template with micro stripes was prepared based on photocuring 3D printing, including a substrate and a top plate. The substrate and the top plate were bonded, and bubbles were removed by vacuum. The plates were placed in an 80°C oven for heating and curing. The bonding surface was treated with plasma spraying. Finally, the substrate and the top plate were bonded. After heating at 80°C, the substrate and the top plate were irreversibly bonded to obtain a microfluidic chip. Photos of the micro-mixing area of ​​the microfluidic chip are shown in the attached figure. Figure 13 and 14 As shown; according to the attached Figure 13 and 14 It can be seen that a raised, spaced-apart micro-striped structure was successfully prepared on the inner wall surface of the micro-mixing region, and coordinated with the spatial deflection configuration of the micro-mixing region, it has a periodic change; 3. Preparation of porous gelatin material: GelMA solution: GelMA was dissolved in a 0.75 wt% LAP (water-soluble photoinitiator) solution to prepare a GelMA solution; F68 solution: Dissolve F68 in PBS (phosphate buffered saline) to prepare F68 solution; The GelMA solution, F68 solution, HFE7500 solution, and microfluidic chip were placed on a temperature-controlled platform at 37°C for insulation; Use a 1 mm inner diameter silicone tube to connect the first inlet of the microfluidic chip to the GelMA solution; use a 1 mm inner diameter silicone tube to connect the second inlet of the microfluidic chip to the F68 solution; use a 1 mm inner diameter silicone tube to connect the third inlet of the microfluidic chip to the HFE7500 solution; Using a syringe pump as the driving force, the GelMA solution and the F68 solution were perfused into the microfluidic chip at a volume ratio of 2:1 for flow mixing, and sequential phase separation and self-assembly occurred to form a micromixed fluid; Injecting the continuous phase through the third inlet into the inlet of the micro-dispersing region to micro-disperse the micro-mixed fluid to form micro-droplets; The microdroplets obtained from the microdroplet outlet were photocured using 405 nm blue light, frozen, and critical point dried to obtain porous gelatin microspheres.

[0082] The porous gelatin microspheres obtained in this experimental example were squeezed and broken, and the microstructure of the cross section was observed under an optical microscope. The results showed that there was no significant difference in the cross-sectional microstructure of the porous gelatin microspheres compared with those prepared by traditional hand-made methods.

[0083] The cross section (internal structure) of the porous gelatin microspheres in this experimental example was photographed using a scanning electron microscope, as shown in the attached figure. Figure 15 shown.

[0084] By the attached Figure 15 It can be seen that the microfluidic chip with microstripe can be used to prepare a porous gelatin material that forms a porous structure of connected nano-microspheres. The design of microstripe in the micro-mixing area will not change the nanoporous structure inside the nano-gelatin microspheres.

[0085] Experimental Example 2 Study on the Effect of Microstripes in the Micromixing Region on the Size of Porous Gelatin Microspheres This experimental example provides a method for preparing porous gelatin microspheres, comprising the following steps: 1. Design of microfluidic chips with different micro-stripe structures: Microfluidic chip A: The same microfluidic chip as in Experimental Example 1 was used; Microfluidic chip B: Based on Figure 5 Microfluidic chip B was prepared. The difference between microfluidic chip B and microfluidic chip A is that the inner wall of the micro-mixing area is a smooth surface without micro-stripes. Microfluidic chip C: Based on the Figure 5 Microfluidic chip C was prepared, which differed from microfluidic chip A in that the spacing of the micro-stripes was greater than 80 μm and the height was greater than 10 μm; 2. Microfluidic chips AC were prepared based on the same microfluidic chip preparation method as in Experimental Example 1. The photo of the micro-mixing area of ​​microfluidic chip A is shown in the attached figure. Figure 13 、 14 As shown; the photo of the mixing area of ​​microfluidic chip B is shown in the attached Figure 16 、 17 As shown; the photo of the micro-mixing area of ​​the microfluidic chip C is shown in the attached Figure 18 、 19 shown.

[0086] 3. Preparation of porous gelatin microspheres AC: The method for preparing porous gelatin microspheres AC in this experimental example is the same as that for preparing porous gelatin microspheres in Experimental Example 1; wherein, porous gelatin microspheres A are prepared based on microfluidic chip A; porous gelatin microspheres B are prepared based on microfluidic chip B; and porous gelatin microspheres C are prepared based on microfluidic chip C.

[0087] The porous gelatin microspheres AC were photographed using an optical microscope to observe the external dimensions of the porous gelatin microspheres. The photo of the porous gelatin microspheres A under an optical microscope is shown in the attached figure. Figure 20 The photo of porous gelatin microspheres B under an optical microscope is shown in the attached figure. Figure 21 The photo of porous gelatin microspheres C under optical fiber microscope is shown in the attached figure. Figure 22 shown.

[0088] By the attached Figure 20-22It can be seen that the porous gelatin microspheres B not only have different sizes, but also the gelatin derivatives and polymers are separated in a single porous gelatin microsphere B; the porous gelatin microspheres C have different sizes compared with the porous gelatin microspheres B. Although the uniformity of the gelatin derivatives and polymers in a single porous gelatin microsphere has been improved to a certain extent, the size and uniformity are still poor and fall short of expectations; while the porous gelatin microspheres A not only have uniform size, but also the gelatin derivatives and polymers are evenly mixed in each porous gelatin microsphere A, which is better than the porous gelatin microspheres B and C; this indicates that designing micro-stripes with a spacing of 30 to 80 μm and a height of 3 to 10 μm in the micro-mixing area has a significant gain effect on the size and uniformity of the porous gelatin microspheres.

[0089] Experimental Example 3 Study on the influence of ambient temperature on the formation process of porous gelatin microspheres This experimental example provides a method for preparing porous gelatin microspheres, comprising: The microfluidic chip A of Experimental Example 2 was placed at temperatures A1 (room temperature) and A2 (37°C temperature control platform) to prepare porous gelatin microspheres A1 and A2, respectively, according to the method for preparing porous gelatin microspheres of Experimental Example 2.

[0090] The porous gelatin microspheres A1 and A2 were photographed using an optical microscope to observe the external dimensions of the porous gelatin microspheres. The photo of the porous gelatin microspheres A1 under an optical microscope is shown in the attached figure. Figure 23 The photo of porous gelatin microspheres A2 under an optical microscope is shown in the attached figure. Figure 24 shown.

[0091] By the attached Figure 23 、 24 It can be seen that temperature also affects the micromixing and microdispersion process of the microfluidic chip; the porous gelatin microspheres A2 prepared at room temperature (25°C) have poor uniformity in the flow mixing of gelatin derivatives and polymers, which leads to size differences after microdispersion; while the porous gelatin microspheres A1 prepared at 37°C have excellent quality uniformity in the flow mixing of gelatin derivatives and polymers, and can form porous gelatin microspheres with uniform size after microdispersion; this shows that when porous gelatin microspheres are prepared based on micromixing and microdispersion of gelatin derivatives and polymers, the microstructure of the micromixing area has a synergistic effect with the ambient temperature of micromixing and microdispersion, which is beneficial to improving the uniformity of material mixing in the porous gelatin microspheres, ensuring dimensional stability and excellent product quality, solving the problems of efficient development, large-scale production and industrial application of porous gelatin materials and drug-loaded or cell-loaded products, and can accelerate the promotion of porous gelatin.

Claims

1. A method for preparing porous gelatin microspheres, characterized in that: The method is realized by using a microfluidic chip (1) having micro-mixing and micro-dispersing areas and a curing system (30), comprising the following steps: 1) Micromixing: injecting a gelatin derivative fluid and a polymer fluid into a micromixing region (11) at 30-60° C., causing the two to flow and mix in the micromixing region (11), and causing sequential phase separation and self-assembly under the periodic influence of the microstructure (17) of the micromixing region (11) to form a micromixed fluid; 2) Micro-dispersion: injecting the continuous phase fluid into the micro-dispersion area (12) to micro-disperse the micro-mixed fluid to form micro-droplets; 3) Solidification: The microdroplets are solidified using a solidification system (30) to prepare porous gelatin microspheres with nanostructures.

2. The method according to claim 1, wherein: Step 1) micro-mixing, wherein the volume ratio of the gelatin derivative fluid to the polymer fluid is 0.25-4.

3. The method according to claim 1, wherein: Step 3) curing: adding a photoinitiator to the gelatin derivative fluid to subject the microdroplets to a light-curing treatment; preferably, the light-curing treatment utilizes visible light.

4. The method according to claim 1, wherein: A load is loaded into the gelatin derivative fluid or the polymer fluid, and porous gelatin microspheres containing the load are prepared using the microfluidic chip (1) and the curing system (30).

5. The method according to claim 4, characterized in that: The load is a drug or a cell.

6. A microsphere manufacturing device for implementing the method according to any one of claims 1 to 5, characterized in that: The invention comprises a constant temperature system (40), a microfluidic system (20) and a curing system (30), wherein the microfluidic system (20) is arranged in the constant temperature system (40), and the microfluidic system (20) comprises at least one microfluidic chip (1); the microfluidic chip (1) has at least one micro-mixing area (11) and at least one micro-dispersing area (12), and the outlet of the micro-mixing area (11) is connected to the inlet of the micro-dispersing area (12); the inner wall surface of the micro-mixing area (11) is provided with a microstructure (17); the microfluidic chip (1) is also provided with a first inlet (13) for injecting a gelatin derivative fluid, a second inlet (14) for injecting a polymer fluid, a third inlet (15) for injecting a continuous phase fluid, and a droplet outlet (16) for connecting the micro-dispersing area (12) and the curing system (30).

7. The microsphere manufacturing device according to claim 6, characterized in that: The channel configuration of the micro-dispersed area (12) is a T-shaped configuration, a flow focusing configuration or a capillary focusing configuration.

8. The microsphere manufacturing device according to claim 6, characterized in that: The microstructure (17) is composed of spaced micro stripes raised on the inner wall surface, and the channel axis of the micro-mixing region (11) is spatially deflected to form at least one spatially deflected configuration.

9. The microsphere manufacturing device according to claim 8, characterized in that: The adjacent spacing between the micro stripes is 30 to 80 μm, and the height of the protrusions is 3 to 10 μm.

10. The microsphere manufacturing device according to claim 8, characterized in that: The spatial deflection configuration is a serpentine configuration, a wave configuration or a sawtooth configuration.

11. Nanostructured porous gelatin microspheres prepared by the method according to any one of claims 1 to 5 or the microsphere-making device according to any one of claims 6 to 10.

12. The use of the nanostructured porous gelatin microspheres according to claim 11, characterized in that: The nanostructured porous gelatin microspheres are used for at least one application selected from the following (a)-(f): (a) As a three-dimensional cell culture scaffold, used in cell preparations to promote cell adhesion, proliferation or differentiation; (b) as a drug delivery carrier, loading therapeutic agents, diagnostic agents or immunomodulators to achieve targeted release or sustained release; (c) as injectable or implantable tissue filling materials for repairing soft tissue defects or regenerating bone tissue; (d) As a hemostatic material, used in wound dressings or surgical hemostasis to promote hemostasis by physical adsorption and activation of coagulation factors; (e) in the field of medical aesthetics as a soft tissue filler or to improve skin structure by stimulating fibroblasts to secrete collagen; (f) In tumor treatment, drug-loaded microspheres can be used to block the blood supply to tumor vessels through local sustained drug release or embolization.

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