Method of making uniform microspheres and apparatus used

Abstract

This application provides a method and apparatus for preparing uniform microspheres, and particularly relates to a microfluidic chip that can be used to prepare microspheres. The chip and apparatus of this application can be easily switched between various production modes, including small-scale research, pilot-scale production, and industrial-scale production. They are easily standardized and mass-produced to meet different application scenarios, and such equipment can easily achieve long-term uninterrupted production. The microspheres prepared by this application have excellent dimensional uniformity and sphericity, and are biodegradable in vivo for use in medical aesthetics or clinical treatment.

Description

Technical Field

[0001] This application belongs to the field of microsphere manufacturing technology and relates to a method for preparing microspheres on a pilot-scale or industrial scale. In particular, it relates to a method that can easily switch between multiple production modes such as small-scale research, pilot-scale production and industrial production to prepare microspheres with excellent size uniformity and spherical shape. This application also relates to the equipment and components used in this method. Background Technology

[0002] Microspheres prepared using polymers as the main material have been widely used in the health product industry, especially in human health products. For example, these microspheres may be used for medical aesthetic purposes or clinical treatment purposes.

[0003] Microspheres for medical aesthetic purposes are well-known. For example, some microsphere formulations sold in China as Class III medical devices are mainly used for facial injection filling: Polycaprolactone microsphere facial filler for injection (composition: polycaprolactone microspheres, glycerin, carboxymethyl cellulose, PBS), Sodium hyaluronate-hydroxypropyl methylcellulose gel containing polyvinyl alcohol gel microspheres for medical use (composition: sodium hyaluronate, hydroxypropyl methylcellulose, polyvinyl alcohol microspheres, balanced saline), polylactic acid facial filler (lyophilized powder composed of L-polylactic acid microspheres, sodium carboxymethyl cellulose, and mannitol), etc. The polymer microsphere materials involved in these commercially available products include polycaprolactone, polylactic acid (especially poly-L-lactic acid), polyvinyl alcohol, etc.

[0004] Microsphere formulations for clinical treatment have been marketed for many years. For example, Novartis's SANDOSTATIN® LAR DEPOT (octreotide acetate) for intraluteal injection consists of one vial of microspheres and one vial of diluent. The two vials are mixed immediately before use to suspend the microspheres. Each 20mg vial of microspheres contains: 22.4mg octreotide acetate, 377.6mg D,L-lactide-glycolic acid copolymer (also known as polylactic-co-glycolic acid copolymer, polylactic-co-glycolic acid copolymer, polylactide-glycolic acid, poly(lactic-co-glycolic acid), PLGA, etc.), and 81.9mg mannitol; each vial of diluent contains: 12mg mannitol, 14mg sodium carboxymethyl cellulose, 4mg poloxamer 188, and water for injection to a final volume of 2ml. Another example of a similar microsphere formulation is Recordati Rare's intramuscular injection long-release bis(hydroxynaphthyl)paretide microsphere suspension (SIGNIFOR). ®LAR (pasireotide) comprises one vial of microsphere formulation and one vial of diluent. The two vials are mixed immediately before use to suspend the microspheres. Each 30mg vial of microsphere formulation contains: 41.13mg of pasireotide, 39.44mg of PLGA (50-60:40-50 type), and 39.44mg of PLGA (50:50 type); each vial of diluent contains: 90mg of mannitol, 14mg of sodium carboxymethyl cellulose, 4mg of poloxamer 188, and water for injection to a final volume of 2ml. For example, the Chinese patent CN103458895B (Chinese patent application number 201180049447.2) discloses a rotigotine microsphere formulation approved for marketing in China, composed of rotigotine, poly(lactic-co-glycolic acid) (PLGA), and stearic acid. All of the above products use PLGA as the microsphere matrix.

[0005] In addition, Takeda / AbbVie's long-acting gonadotropin-releasing hormone microspheres (LUPRON DEPOT) for intramuscular injection are packaged in a pre-filled dual-chamber syringe. The microsphere chamber contains 11.25 mg of leuprolide acetate, 99.3 mg of polylactic acid, and 19.45 mg of mannitol. The dilution chamber contains 7.5 mg of sodium carboxymethyl cellulose, 75 mg of mannitol, 1.5 mg of polysorbate 80, an appropriate amount of pH adjuster glacial acetic acid, and water for injection to a final volume of 1.5 ml.

[0006] As is well known in this field, the polymeric materials used to prepare microspheres, i.e., microsphere-forming materials, mainly fall into two categories: natural polymers and synthetic polymers. Natural polymeric materials include polysaccharides (such as chitosan, alginate, starch, cellulose, etc.), proteins (such as gelatin, albumin, collagen, etc.), and so on. Synthetic polymeric materials include polyesters (such as polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA, such as poly-L-lactic acid (PLLA)), polycaprolactone (PCL), etc.), polyethylene glycol (PEG), polyvinyl alcohol (PVA), etc. Among synthetic polymers, polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), and polycaprolactone (PCL) are currently the most widely used and most extensively studied classic microsphere forming materials.

[0007] Microspheres for medical aesthetics or clinical treatment are typically prepared using spray drying, spray antisolvent extraction, and solvent evaporation methods. The classic method is solvent evaporation, which involves dispersing a dispersed phase (internal phase) containing microsphere material / organic solvent into an aqueous system through stirring / emulsification, and then evaporating the solvent to obtain microspheres. For example, a preferred preparation method in CN103458895B is as follows: Weigh 0.4491g of rotigotine, 0.5060g of PLGA75254A (Mr42-58kDa), 0.5055g of PLGA50502.5A (Mr15-35kDa), and 0.0371g of stearic acid. Add 7.5ml of dichloromethane and stir to dissolve. Add the solution to 750ml of 0.5% PVA aqueous solution with a 100rpm peristaltic pump while stirring at 1200-2000rpm. After emulsification for 2min, reduce the stirring speed to evaporate the solvent for 5h. Filter the resulting solution through a 1200-mesh sieve to collect the microspheres. Rinse the microspheres on the 1200-mesh sieve with purified water 3-5 times, freeze-dry, and then sieve through a 100-mesh sieve to obtain the final microspheres. However, microspheres prepared using these classic methods have insurmountable problems, such as poor surface sphericity and particle size uniformity. Sphericity issues affect the permeability of microspheres within the injection needle during injection, especially for facial injections where smaller needles (e.g., 27g-32g) are typically used to reduce recipient discomfort. Furthermore, microsphere size uniformity is a widespread problem. Wide particle size distributions and significant differences in degradation or drug release rates between large and small microspheres can easily lead to burst release or delayed release. Sieving microspheres using methods such as those described in CN103458895B significantly reduces product yield.

[0008] To overcome the aforementioned problems related to surface sphericity and particle size uniformity, microfluidics has been proposed for the fabrication of microspheres. Microfluidics is a novel approach to microsphere fabrication, a highly interdisciplinary technology integrating chemistry, physics, life sciences, microelectronics, materials science, and computer science, currently primarily applied in the fields of chemistry and life sciences. It is a technique for manipulating or controlling nanoliter or picoliter-level liquids within microchannels at the micrometer scale, and allows for seamless switching between small-scale research, pilot-scale production, and industrial-scale production through parallel and / or parallel replication of microchannel units.

[0009] The core component of microfluidic technology is the microfluidic chip, which is fabricated using microfabrication techniques on materials such as silicon wafers, quartz, glass, polymethylmethacrylate (PMMA), and polydimethylsiloxane (PDMS) to create channels of various micrometer-scale structures and sizes according to specific needs. Because of its extremely small size and micro-manipulation capabilities, the microfluidic chip not only requires minimal sample volume but also enables continuous, automated production.

[0010] Currently, there are mature methods for fabricating microfluidic chips, such as soft photolithography and molding, thermoforming and injection molding, laser processing technology, chip bonding and packaging, etc. For example, Wu Yuanqing et al. (Microfluidic Chip Technology, Chemical Industry Press, 2022) and Dy, Aaron J. et al. (Fabricating microfluidic valve mastermolds in SU-8 photoresist, Journal of Micromechanics and Microengineering, 2014, DOI:10.1088 / 0960-1317 / 24 / 5 / 057001) described a method of fabricating a mold on a silicon wafer using SU-8 photoresist, then casting polydimethylsiloxane for curing, and finally bonding the chips using plasma or thermal diffusion methods to obtain microfluidic chips.

[0011] Numerous studies have documented the fabrication of microspheres, particularly for medical aesthetics or clinical treatment, using microfluidic chips. For example, CN104588139B (201510025274.0) discloses a microfluidic chip for fabricating microspheres, composed of a chip body, a cooling chip, and a volatile exhaust chip. It includes a U-shaped double V-shaped mixing channel I and a double V-shaped mixing channel II, a circular cooling double U-shaped channel, and a shaping collection pool and outlet on one side of the cooling double U-shaped channel. CN113797986B (202111168539.4) discloses a microfluidic chip with coaxially arranged capillaries, comprising an integrated glass chip substrate, a continuous phase inlet, an intermediate phase inlet, a dispersed phase inlet, a sample inlet capillary, a collection capillary, a collection port, a capillary nesting assembly, and a capillary coaxial fine-tuning assembly. Its design primarily addresses the issue of three-dimensional coaxial arrangement between components. CN116139790A (202310276830.6) discloses a microfluidic chip for preparing polylactic acid microspheres, comprising a continuous phase inlet, a continuous phase microchannel, a dispersed phase inlet, a dispersed phase microchannel, and an outlet microchannel. CN116651525B (202310669820.9) discloses a microfluidic chip comprising first, second, and third syringe needles and the channels they define. CN220531647U (202322163010.4) discloses a capillary microfluidic chip comprising a substrate, a capillary, and a capillary nesting assembly; a mounting groove is formed within the substrate, a fixing groove is formed on the inner wall of the mounting groove, and a liquid outlet is formed on the bottom wall of the fixing groove. CN119897038A (202411801484.X) discloses a 3D-printed microfluidic chip for preparing leuprolide sustained-release microspheres. The chip contains a dispersed phase, a continuous phase, and microsphere channels. The dispersed phase and continuous phase channels intersect to form the microsphere formation area, and the surface of the chip has a dispersed phase inlet, a continuous phase inlet, and a microsphere outlet.

[0012] Existing microfluidic chips may still have some shortcomings. For example, a single chip channel makes it difficult to effectively increase microsphere yield; heating and curing or cooling after sphere formation makes it difficult to increase microsphere sphericity; installation requires on-site installation and adjustment, and chip failure can affect production efficiency; a single channel for venting during production can seriously affect the large-scale integration of chips and the large-scale production of microspheres; the cost of 3D-printed multi-channel chips is high; and the inclusion of a microsphere curing stage in some chips leads to complex structures, making industrial production difficult and significantly reducing production efficiency.

[0013] Therefore, there is an urgent need in the field for new methods and equipment to prepare microspheres, especially methods that can easily switch between various production modes such as small-scale research, pilot-scale production and industrial production to prepare microspheres with excellent size uniformity. Summary of the Invention

[0014] The purpose of this application is to provide a method for preparing microspheres with uniform size and spherical shape, and another purpose is to provide the equipment used to prepare the microspheres and the microfluidic chip configured therewith.

[0015] In this application, the term "microsphere" should be understood as a group of individual microspheres, which can be a group of microspheres ranging from a few to tens of thousands to billions, or even an unlimited number of microspheres. For example, the aforementioned "product for generating collagen in mammals" contains microspheres in its smallest packaging for single or multiple use, with each use containing a group of microspheres of a certain number, the number of which varies depending on the composition of the microspheres, the occasion of use, the target user, etc.

[0016] Therefore, the first aspect of this application provides a microfluidic chip for preparing microspheres, the microfluidic chip being substantially circular in shape, comprising three circular sheets sequentially and tightly stacked together from top to bottom: a dispersed phase flow sheet, a microsphere forming sheet, and a continuous phase flow sheet, characterized in that: A circular dispersed phase inlet is penetrating the center of the dispersed phase flow plate. 4 to 24 or 6 to 18 groove-shaped dispersed phase channels are uniformly radiated from the dispersed phase inlet on the lower surface of the dispersed phase flow plate. The length of the channel from the center of the flow plate is 1 / 6 to 1 / 2 of the radius of the dispersed phase flow plate, for example, preferably 1 / 4 to 1 / 2. The microsphere forming sheet has a small through hole at the end of each dispersed phase flow channel. The lower edge of the through hole extends downward to form a capillary nozzle, which allows the dispersed phase entering from the dispersed phase inlet to flow along each dispersed phase flow channel to the through hole, and then through the through hole to the capillary nozzle to drip out. A circular continuous phase inlet passes through the center of the continuous phase flow plate. The upper surface of the continuous phase flow plate has a number of groove-shaped continuous phase channels that are uniformly radiated outward from the continuous phase inlet. The length of the channel extends from the continuous phase inlet to the outlet at the outer edge of the flow plate.

[0017] According to the microfluidic chip of the first aspect of this application, a dispersed phase liquid flow of a microsphere forming material (usually a biodegradable polymer, such as polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, etc., in which physiologically active ingredients, such as chemical components for medical aesthetics or clinical treatment purposes, can be added according to the requirements of use) enters from the dispersed phase flow sheet, reaches the microsphere forming sheet, and forms tiny droplets that are injected into the continuous phase liquid flow in the continuous phase flow sheet. The tiny droplets then form a spherical shape during the liquid flow process, and the spherical droplets can then be solidified, separated, and dried in subsequent processes to form microspheres.

[0018] According to the microfluidic chip of the first aspect of this application, the outlet is located at the edge outside the circular continuous phase flow sheet.

[0019] According to the microfluidic chip of the first aspect of this application, when the dispersed phase flow sheet, the microsphere forming sheet, and the continuous phase flow sheet are tightly stacked and assembled... The dispersed phase inlet of the dispersed phase flow plate is coaxially arranged with the continuous phase inlet of the continuous phase flow plate; The dispersed phase flow channel of the dispersed phase flow plate corresponds in position to the continuous phase flow channel of the continuous phase flow plate, and is separated by a microsphere forming plate, thereby enabling: After entering from the dispersed phase inlet, the dispersed phase liquid flows outward to the end of the disc along the dispersed phase flow channel, passes through the through holes of the microsphere forming plate, and drips into the continuous phase liquid flow of the continuous phase flow channel of the continuous phase flow plate in the form of small droplets through the capillary nozzle. After entering from the continuous phase inlet, the continuous phase liquid flows along the continuous phase flow channel to the outer edge of the disc, and at the capillary nozzle, it carries the tiny droplets of dispersed phase away from the microfluidic chip.

[0020] According to the microfluidic chip of the first aspect of this application, a guide nozzle is provided at the outlet of the continuous phase channel at the end of the continuous phase flow plate to the bottom of the flow plate, so that the continuous phase liquid carrying the dispersed phase micro-droplets can flow down.

[0021] The microfluidic chip according to the first aspect of this application has a radius of 2 to 20 cm, for example, a radius of 3 to 15 cm, preferably a radius of 3 to 10 cm.

[0022] According to the microfluidic chip of the first aspect of this application, the width of the groove of the dispersed phase channel is 1 to 10 mm, for example 1 to 8 mm, preferably 1 to 5 mm; and / or, the depth of the groove of the dispersed phase channel is 0.5 to 5 mm, for example 0.5 to 4 mm, preferably 0.5 to 3 mm.

[0023] According to the microfluidic chip of the first aspect of this application, the inlet radius of the dispersed phase is 0.5~5mm, preferably 0.5~4mm, and more preferably 0.5~3mm.

[0024] According to the microfluidic chip of the first aspect of this application, the cross-sectional area of ​​the dispersed phase inlet is 5% to 60% of the total cross-sectional area through which the dispersed phase flow passes, preferably 10% to 50%, and more preferably 20% to 40%.

[0025] According to the microfluidic chip of the first aspect of this application, the width of the groove of the continuous phase channel is 1 to 10 mm, for example 1 to 8 mm, preferably 2 to 5 mm; and / or, the depth of the groove of the continuous phase channel is 0.5 to 5 mm, for example 0.5 to 4 mm, preferably 1 to 3 mm.

[0026] According to the microfluidic chip of the first aspect of this application, the total cross-sectional area through which the liquid flows through the continuous phase channel is 60% to 130% of the cross-sectional area of ​​the continuous phase inlet, preferably 70% to 110%, and more preferably 80% to 90%.

[0027] According to the microfluidic chip of the first aspect of this application, the continuous phase inlet radius can be 4~10mm, preferably 4.5~8mm.

[0028] According to the microfluidic chip of the first aspect of this application, the diameter of the capillary pore of the capillary protrusion on the microsphere forming sheet is 10~1000μm, preferably 10~500μm, more preferably 10~250μm, and more preferably 10~200μm.

[0029] According to the microfluidic chip of the first aspect of this application, the cross-sectional area of ​​the dispersed phase inlet is 10-60% of the total cross-sectional area of ​​the dispersed phase channel, preferably 15-50%, and more preferably 20-40%.

[0030] According to the microfluidic chip of the first aspect of this application, the "capillary correction flow rate" is calculated using the following formula: Capillary corrected flow rate (ml / min) = (Dispersed phase inlet cross-sectional area ÷ Total capillary orifice cross-sectional area) × Dispersed phase inlet flow rate. Within the permissible inlet flow rate range of the dispersed phase pumped in by the constant flow pump, the capillary-corrected flow rate of the microfluidic chip is greater than 10 ml / min, greater than 20 ml / min, greater than 30 ml / min, greater than 40 ml / min, greater than 50 ml / min, greater than 60 ml / min, or greater than 70 ml / min; and / or, the capillary-corrected flow rate of the microfluidic chip is less than 200 ml / min, less than 190 ml / min, less than 180 ml / min, less than 170 ml / min, less than 160 ml / min, less than 150 ml / min, less than 140 ml / min, less than 130 ml / min, less than 120 ml / min, less than 110 ml / min, or less than 100 ml / min. Preferably, the capillary-corrected flow rate is in the range of 30~150 ml / min, or 40~140 ml / min, 50~130 ml / min, or 50~120 ml / min.

[0031] According to the microfluidic chip of the first aspect of this application, the capillary protrusion extends from the surface of the microsphere forming sheet, and the protrusion height is 1 / 5 to 1 / 2, preferably 1 / 4 to 1 / 2, of the depth of the continuous phase flow channel groove.

[0032] According to the microfluidic chip of the first aspect of this application, when the dispersed phase flow sheet, the microsphere forming sheet, and the continuous phase flow sheet are tightly stacked and assembled, the dispersed phase liquid flows unidirectionally in the direction of the dispersed phase inlet, the dispersed phase channel, the through hole, the capillary nozzle, the continuous phase channel, and the outlet, and will not seep into other positions between the dispersed phase flow sheet and the microsphere forming sheet.

[0033] According to the microfluidic chip of the first aspect of this application, when the dispersed phase flow sheet, the microsphere forming sheet, and the continuous phase flow sheet are tightly stacked and assembled, the continuous phase liquid flows unidirectionally in the direction of the continuous phase inlet, the continuous phase flow channel, and the outlet, and will not seep into other positions between the continuous phase flow sheet and the microsphere forming sheet.

[0034] According to the microfluidic chip of the first aspect of this application, a guide nozzle is provided at the end of the continuous phase channel of the continuous phase flow sheet, through the outlet of the continuous phase flow sheet, and downward to the flow sheet, so that the continuous phase liquid carrying the dispersed phase micro-droplets can be dripped downward.

[0035] According to the microfluidic chip of the first aspect of this application, the outlet at the end of the continuous phase channel is located inside the continuous phase sheet or at the outer edge of the continuous phase sheet.

[0036] The microfluidic chip according to the first aspect of this application is made of silicon wafer, glass, quartz, polymethyl methacrylate, or polydimethylsiloxane.

[0037] Furthermore, the second aspect of this application provides a method for preparing the microfluidic chip described in any one of the first aspects of this application. The microfluidic chip is made of a glass substrate and includes the following steps: using glass as a substrate, using photoresist as a patterning protective layer, transferring a preset microchannel pattern to the photoresist layer by photolithography, forming a microchannel structure on the glass substrate by chemical etching, and finally sealing the two glass sheets by bonding to obtain a closed glass microfluidic chip.

[0038] According to the method of the second aspect of this application, it includes the following steps: (1) Cleaning the glass substrate: Select three circular glass substrates, thoroughly clean the glass substrates with a cleaning solution (e.g., Piranha solution), and then dry them with nitrogen gas; (2) Spin coating of photoresist: Take two glass substrates used to prepare dispersed phase flow sheets and continuous phase flow sheets, drop positive photoresist onto the center of the cleaned and dried glass substrates, and use a spin coater to spread the photoresist evenly on the surface of the glass substrates; (3) Pre-baking treatment: Place the glass substrates with the spin-coated photoresist film on a hot plate for pre-baking treatment to allow the solvent in the photoresist to fully evaporate and the photoresist film to solidify; (4) Ultraviolet exposure: According to the liquid flow channel arrangement of the dispersed phase sheet and the continuous phase sheet, the mask with the preset microchannel pattern is tightly attached to the surface of the photoresist film after pre-baking treatment. The attached glass substrate is irradiated with ultraviolet light to achieve the initial transfer of the microchannel pattern; (5) Development treatment: The glass substrate after ultraviolet exposure is placed in the developing solution for development treatment, so that the surface of the glass substrate forms a pattern consistent with the mask. The glass substrate is then cleaned and dried; (6) Hardening and chemical etching: The developed glass substrate is placed on the hot plate again for hardening treatment. Then the glass substrate is placed in the hydrofluoric acid etching solution for chemical etching to form a dispersed phase. (7) Removing adhesive and drilling: Immerse the etched dispersed phase flow sheet and continuous phase flow sheet in acetone solvent for adhesive removal, then clean and dry; then use an ultrasonic drilling machine to drill holes at the corresponding positions of the dispersed phase inlet of the dispersed phase flow sheet and the continuous phase inlet of the continuous phase flow sheet, and then use photosensitive resin to bond a hollow pipe opening protruding from the flow sheet surface on the side away from the liquid flow channel to form the dispersed phase inlet and the continuous phase inlet, thus obtaining the dispersed phase flow sheet and the continuous phase flow sheet; (8) Fabricating microsphere forming sheets: Set the microsphere forming sheet glass substrate with a groove structure, and clean the glass substrate; (9) Removing adhesive and drilling: Immerse the etched dispersed phase flow sheet and the continuous phase flow sheet in acetone solvent solvent for adhesive removal, then clean and dry; then use an ultrasonic drilling machine to drill holes at the corresponding positions of the dispersed phase inlet of the dispersed phase flow sheet and the continuous phase inlet of the continuous phase flow sheet, and then use photosensitive resin to bond a hollow pipe opening protruding from the flow sheet surface to form the dispersed phase inlet and the continuous phase inlet, thus obtaining the dispersed phase flow sheet and the continuous phase flow sheet; (10) Fabricating microsphere forming sheets: Set the microsphere forming sheet glass substrate with a groove structure, and clean the glass substrate; (11) Removing adhesive and drilling: Immerse the etched dispersed phase flow sheet and the continuous phase flow sheet in acetone solvent solvent, then clean and dry the glass substrate; (12) Removing adhesive and drilling: Immerse the etched dispersed phase flow sheet and the continuous phase flow sheet in acetone solvent solvent, then use photosensitive resin to bond a hollow pipe opening on the side away from the liquid flow channel to form the dispersed phase inlet and the continuous phase inlet, thus obtaining the dispersed phase flow sheet and the continuous phase flow sheet; (13) Fabricating microsphere forming sheets: Set the microsphere forming sheet glass substrate with a groove structure, and clean the glass substrate; (14) Removing adhesive and drilling: Immerse the etched disperse The capillary nozzle is connected to a capillary tube with a predetermined aperture by high-temperature bonding. Then, a through hole corresponding to the capillary hole is drilled on the corresponding position on the back side of the capillary tube of the microsphere forming sheet using a computer numerical control milling machine to obtain the microsphere forming sheet; (9) Bonding and packaging: The dispersed phase flow sheet, microsphere forming sheet and continuous phase flow sheet are precisely aligned and stacked from top to bottom, placed in a high-temperature furnace and pressure is applied to make the three glass sheets permanently fuse under high temperature and high pressure conditions to achieve bonding and sealing, and obtain a microfluidic chip with a glass substrate having a closed microchannel; Optionally, the flow nozzle is bonded to each liquid outlet of the continuous phase flow sheet by photosensitive resin bonding.

[0039] Furthermore, a third aspect of this application provides an apparatus unit for preparing uniform microspheres, which basically includes: a microfluidic chip (as described in the first aspect of this application); a dispersed phase solution storage bottle for storing the dispersed phase solution during microsphere preparation; a continuous phase solution storage bottle for storing the continuous phase solution during microsphere preparation; a constant flow pump for introducing the dispersed phase solution and the continuous phase solution into the microfluidic chip; a liquid guide tube for connecting the dispersed phase solution storage bottle, the constant flow pump, and the dispersed phase inlet of the microfluidic chip, and for connecting the continuous phase solution storage bottle, the constant flow pump, and the continuous phase inlet of the microfluidic chip; a receiving tray disposed below the microfluidic chip for receiving liquid flowing out from the outlet at the end of the continuous phase flow channel of the microfluidic chip; and a collection bottle connected to the outlet at the bottom of the receiving tray via the liquid guide tube for collecting the continuous phase solution containing dispersed phase microdroplets.

[0040] According to the device unit of the third aspect of this application, the preparation of uniform microspheres is basically carried out in the following steps: (i) the continuous phase inlet is connected to the continuous phase solution storage bottle through a liquid guide tube, and the continuous phase solution is pumped into the continuous phase flow plate of the microfluidic chip through a constant flow pump, flowing through the continuous phase flow channel until it flows out from the outlet of the flow plate; (ii) the dispersed phase inlet is connected to the dispersed phase solution storage bottle through a liquid guide tube, and the dispersed phase solution is pumped into the dispersed phase flow plate of the microfluidic chip through a constant flow pump, and then passes through the through holes and capillary nozzles of the microsphere forming plate, entering in the form of tiny droplets. (iii) The dispersed micro-droplets are introduced into and dispersed in the continuous phase solution in the continuous phase channel, and carried away by the continuous phase solution to the outlet of the microfluidic chip; (iv) The continuous phase solution with dispersed micro-droplets suspended in it is dripped from the outlet into the receiving tray, and then discharged into the collection bottle through the outlet of the receiving tray; (iv) The dispersed micro-droplets formed at the capillary nozzle undergo spheroidization and preliminary solidification in the continuous phase solution, and then liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle.

[0041] Additionally, the liquid microspheres obtained in step (iv) above can be further processed as follows: (v) After the liquid obtained in step (iv) has been left to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. Optionally, the solid microspheres are washed and dried (e.g., under reduced pressure) with water to obtain microspheres in the form of fine powder.

[0042] Furthermore, the fourth aspect of this application provides an apparatus for preparing uniform microspheres to accommodate different production scales, which is composed of any number of the apparatus units described in any of the third aspects of this application connected in parallel.

[0043] According to the fourth aspect of this application, all components of all equipment units operate independently and in parallel, or several equipment units form a group and each group operates independently and in parallel.

[0044] According to the equipment of the fourth aspect of this application, 2 to 100 (e.g., 2 to 50, 2 to 20, 2 to 10) dispersed phase solution storage bottles, continuous phase solution storage bottles, and collection bottles of equipment units are shared to form a group, forming several groups that operate independently and in parallel. In this application, unless otherwise stated, "parallel operation" generally refers to the parallel assembly of equipment units or groups with independent spatial structures, followed by the merging of subsequent production processes. For example, liquids containing dispersed phase droplets from collection bottles of different equipment units or groups can be combined and then concentrated together in subsequent processes for solidification, cleaning, drying, etc.

[0045] Furthermore, the fifth aspect of this application provides a method for preparing microspheres, which uses the equipment unit described in the third aspect or the equipment described in the fourth aspect of this application. The method first prepares a continuous phase solution and a dispersed phase solution, and then operates according to the following steps: (a) adding the continuous phase solution and the dispersed phase solution to the continuous phase solution storage bottle and the dispersed phase solution storage bottle, respectively; starting the constant flow pump; and adjusting and controlling the flow rates of the two pumps for the continuous phase solution and the dispersed phase solution by monitoring the microsphere particle size in the suspension collected by the receiving tray, so that the microsphere particle size reaches the desired value or range; (b) (b) The dispersed phase microdroplets formed at the capillary nozzle undergo spheroidization and preliminary solidification in the continuous phase solution, resulting in liquid microspheres that are loosely deposited at the bottom of the continuous phase solution in the collection bottle. (c) After the liquid obtained in step (b) is allowed to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. The solid microspheres are then rinsed with water and dried (under reduced pressure) to obtain microspheres in the form of fine powder.

[0046] According to the method of the fifth aspect of this application, the device unit or device operates substantially according to the following steps: (i) a continuous phase inlet is connected to a continuous phase solution storage bottle via a liquid guide tube, wherein a constant flow pump pumps the continuous phase solution into the continuous phase flow plate of the microfluidic chip, flowing through the continuous phase flow channel until it flows out from the outlet of the flow plate; (ii) a dispersed phase inlet is connected to a dispersed phase solution storage bottle via a liquid guide tube, wherein a constant flow pump pumps the dispersed phase solution into the dispersed phase flow plate of the microfluidic chip, which then passes through the through-holes and capillary nozzles of the microsphere forming plate in the form of tiny droplets. (iii) The continuous phase solution, which is dispersed into the continuous phase channel, is carried away by the continuous phase solution to the outlet of the microfluidic chip; (iv) The continuous phase solution, which is suspended with dispersed phase microdroplets, is dripped from the outlet into the receiving tray, and then discharged into the collection bottle through the outlet of the receiving tray; (iv) The dispersed phase microdroplets formed at the capillary nozzle undergo spheroidization and preliminary solidification in the continuous phase solution to obtain liquid microspheres that are loosely deposited at the bottom of the continuous phase solution in the collection bottle.

[0047] In this application, terms indicating direction, such as front, back, up, down, left, right, etc., are often used for descriptive convenience rather than as absolute directional indications, unless they have a specific meaning in the corresponding context. In this application, unless otherwise specified, the term "particle size" used to characterize microsphere size refers to the diameter of the microsphere.

[0048] The microfluidic chips, their device units, devices, and methods for preparing microspheres provided in this application have numerous advantages that can be recognized from the context of this application, including but not limited to: 1) It can easily switch freely between different output requirements of research-oriented small-scale production, pilot production, and large-scale commercial production; 2) The equipment is easy to standardize and assemble on a large scale to meet different usage scenarios; 3) The prepared microspheres have a round and uniform structure and size, avoiding the waste caused by the usual sieving process; 4) It has multiple applications. It can be used to adjust the microsphere size to prepare drug-containing microspheres with different drug release requirements for clinical treatment purposes, or to prepare drug-free microspheres that can be biodegraded in vivo for medical aesthetic purposes. 5) The equipment unit or equipment can easily achieve long-term uninterrupted production by simply replenishing the continuous or dispersed phase solution to the continuous or dispersed phase solution storage bottle regularly, and by periodically processing the fluid in the collection bottle for subsequent processes. Therefore, the production capacity can be unlimited. Attached Figure Description

[0049] Figure 1This application presents a schematic diagram of a microfluidic chip assembly state.

[0050] Figure 2 This application presents an axial schematic diagram of a circular microfluidic chip in its assembled state. View a is taken from the direction of the dispersed phase inlet, and view b is taken from the direction of the continuous phase inlet.

[0051] Figure 3 This application relates to a microfluidic chip in... Figure 1 A schematic diagram showing the separation of three circular thin plates from different perspectives.

[0052] Figure 4 This application describes a microfluidic chip that is basically in... Figure 1 The diagram shows the separation of three circular thin plates from each other, with the dispersed phase plate and the microsphere forming plate viewed from bottom to top, and the continuous phase plate viewed from top to bottom.

[0053] Figure 5 This application presents a schematic diagram showing the three circular thin films of a microfluidic chip in a separated state.

[0054] Figure 6 This application presents a schematic diagram showing the three circular thin films of a microfluidic chip in a separated state.

[0055] Figure 7 This application presents schematic diagrams of a dispersed phase flow sheet of a microfluidic chip viewed from both above and below.

[0056] Figure 8 This application presents schematic diagrams of a microsphere-forming sheet of a microfluidic chip viewed from both above and below.

[0057] Figure 9 This application presents schematic diagrams of a continuous phase flow sheet of a microfluidic chip viewed from three perspectives: directly above, diagonally above, and diagonally below.

[0058] Figure 10 Schematic diagram of the equipment unit for preparing microspheres.

[0059] Figure 11 : Optical microscope image (monitor photograph) of the drug-containing microsphere T01a sample prepared on a research scale taken from the receiving tray.

[0060] Figure 12 : Optical microscope image (monitor photograph) of the drug-containing microsphere T01a sample prepared on a research scale after curing and drying.

[0061] Figure 13 Scanning electron microscope image of the drug-containing microsphere T01a sample prepared on a research scale after curing and drying.

[0062] Figure 14 Scanning electron microscope image of the T04 microsphere sample prepared on a research scale after curing and drying.

[0063] Figure 15 A typical photograph (partial field of view) of a microsphere sample with particle size measured by an optical microscope. In the figure, for example, microsphere C1, D=60.77um represents the diameter of the microsphere, and C=190.92um represents the projected perimeter of the microsphere.

[0064] Figure 16 Scanning electron microscope image of microspheres prepared by classical methods. Detailed Implementation

[0065] The present application can be further described through the following embodiments; however, the scope of the present application is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present application without departing from its spirit and scope. The present application provides a general and / or specific description of the materials and test methods used in the experiments. Although many materials and operating methods used to achieve the purposes of this application are well known in the art, they are still described in as much detail as possible herein. The following embodiments further illustrate the present application, but are not intended to limit it.

[0066] Example 1: Microfluidic Chip This embodiment provides an implementation scheme for the design and fabrication of the microfluidic chip of this application. The microfluidic chip is essentially in the shape of a disc, such as... Figures 1-4 As shown, this microfluidic chip can form microspheres by dripping tiny dispersed phase droplets into a continuous phase liquid flow.

[0067] Figure 1 This is a schematic diagram of the assembled state of the microfluidic chip, shown as a side view of the circular plane of the chip. The microfluidic chip includes (for ease of description, from top to bottom) three (preferably with the same radius) circular sheets that are tightly stacked together in sequence: a dispersed phase flow sheet 11, a microsphere forming sheet 12, and a continuous phase flow sheet 13.

[0068] The basic principle of microsphere fabrication using microfluidic chips is as follows: A dispersed phase liquid stream of a microsphere-forming material (usually a biodegradable polymer, such as polycaprolactone, polylactic acid, or polylactic-glycolic acid copolymer, etc., in which physiologically active ingredients, such as chemical components for medical aesthetics or clinical treatment purposes, can be added according to the application requirements) enters from the dispersed phase flow plate 11 and reaches the microsphere-forming plate 12, forming tiny droplets that are then introduced into the continuous phase liquid stream in the continuous phase flow plate 13. These tiny droplets of the dispersed phase then form spherical shapes during the liquid flow process. These spherical droplets are subsequently solidified, separated, and dried to form microspheres.

[0069] The microfluidic chip is designed to allow a dispersed phase liquid containing microsphere-forming material to enter from the dispersed phase flow plate 11, reach the microsphere-forming plate 12, and form tiny droplets that fall into the continuous phase liquid flow in the continuous phase flow plate 13. These dispersed phase droplets then gradually form spherical shapes (i.e., spheroidization) during the liquid flow process. These spherical droplets are then solidified, separated, and dried in subsequent processes to form microspheres.

[0070] Figure 2 This is an axial schematic diagram of the assembled disc-shaped microfluidic chip. In the figure, a is a view viewed from the dispersed phase inlet 112, and b is a view viewed from the continuous phase inlet 132.

[0071] Figure 3 In order to be in Figure 1 A schematic diagram showing the separation of three circular thin plates from different perspectives.

[0072] Figure 4 For basically in Figure 1 A schematic diagram showing the separation of three circular thin plates from each other in terms of viewing angle. The dispersed phase flow plate 11 and the microsphere forming plate 12 are viewed from the bottom upwards, while the continuous phase flow plate 13 is viewed from the top downwards.

[0073] According to the microfluidic chip of this embodiment, a circular dispersed phase inlet 112 penetrates the center of the dispersed phase flow sheet 11. The lower surface of the dispersed phase flow sheet 11 has 4 to 24 (preferably 6 to 18, 12 in this embodiment) groove-shaped dispersed phase flow channels 111 that are uniformly diverged from the dispersed phase inlet 112 outward from the flow sheet. The length of the flow channel from the center of the flow sheet is 1 / 6 to 1 / 2 (preferably 1 / 4 to 1 / 2, about 1 / 3 in this embodiment) of the radius of the dispersed phase flow sheet 11.

[0074] When the microfluidic chip according to this embodiment is tightly stacked and assembled with the dispersed phase flow sheet 11, the microsphere forming sheet 12 is provided with a small through hole 122 at the end of each dispersed phase flow channel 111. The lower edge of the through hole 122 extends downward to form a capillary nozzle 121, which allows the dispersed phase entering from the dispersed phase inlet 112 to flow along each dispersed phase flow channel 111 to the through hole 122, and then through the through hole 122 to reach the capillary nozzle 121 and drip out.

[0075] The three components—dispersed phase flow plate 11, microsphere forming plate 12, and continuous phase flow plate 13—are made of different materials. Figure 1 The diagram schematically shows the obscured through-hole 122 and capillary nozzle 121 in the microsphere forming sheet 12. Figure 3 The diagram schematically shows the blocked through-hole 122 in the microsphere forming sheet 12.

[0076] According to the microfluidic chip of this embodiment, a circular continuous phase inlet 132 penetrates the center of the continuous phase flow plate 13. The upper surface of the continuous phase flow plate 13 has groove-shaped continuous phase channels 131, the same number as the dispersed phase channels 111, evenly radiating outwards from the continuous phase inlet 132. The length of each channel extends from the continuous phase inlet 132 to the outlet 134 at the outer edge of the flow plate. Thus, the number of dispersed phase channels 111, continuous phase channels 131, and capillary nozzles 121 are equal.

[0077] According to the microfluidic chip of this embodiment, the outlet 134 is located at the edge outside the circular continuous phase flow plate 13.

[0078] According to the microfluidic chip of this embodiment, when the dispersed phase flow sheet 11, the microsphere forming sheet 12, and the continuous phase flow sheet 13 are tightly stacked and assembled... The dispersed phase inlet 112 of the dispersed phase flow plate 11 is coaxially arranged with the continuous phase inlet 132 of the continuous phase flow plate 13; The dispersed phase flow channel 111 of the dispersed phase flow plate 11 corresponds to the continuous phase flow channel 131 of the continuous phase flow plate 13, and is separated by a microsphere forming plate 12, thereby enabling: After entering from the dispersed phase inlet 112, the dispersed phase liquid flows outward to the end of the disc along the dispersed phase channel 111, passes through the through hole 122 of the microsphere forming plate 12, and drips into the continuous phase liquid flow of the continuous phase channel 131 of the continuous phase flow plate 13 in the form of small droplets through the capillary nozzle 121. After entering from the continuous phase inlet 132, the continuous phase liquid flows along the continuous phase channel 131 to the outer edge of the disc, and at the capillary nozzle 121, it carries the dispersed phase micro-droplets away from the microfluidic chip.

[0079] Generally, in this embodiment, the disc-shaped microfluidic chip is mounted vertically with the disc axis in operation. Figure 1 The installation scheme is shown. Based on this, as follows: Figures 1-4 As shown, a guide nozzle 133 is provided at the outlet 134 (i.e., the edge outside the circular flow plate) at the end of the continuous phase flow channel 131 of the continuous phase flow plate 13, so that the continuous phase liquid flow carrying the dispersed phase micro-droplets can flow down (to a separately configured collector).

[0080] The microfluidic chip according to this embodiment has a radius of 2~20cm, for example, a radius of 3~15cm, preferably a radius of 3~10cm, and the chip in this embodiment has a radius of 5cm.

[0081] According to the microfluidic chip of this embodiment, the width of the groove in the dispersed phase channel 111 is 1~10mm, for example 1~8mm, preferably 1~5mm, and in this embodiment it is designed to be 2mm. The depth of the groove in the dispersed phase channel 111 is 0.5~5mm, for example 0.5~4mm, preferably 0.5~3mm, and in this embodiment it is designed to be 1mm. Based on the liquid flow capacity of the dispersed phase channel 111, the size of the dispersed phase inlet 112 can be easily determined. Generally speaking, the cross-sectional area of ​​the dispersed phase inlet 112 is 5%~60% of the total cross-sectional area through which the liquid flows in the dispersed phase channel 111, preferably 10%~50%, and more preferably 20%~40%. For example, the size of the dispersed phase flow channel 111 groove designed in this embodiment is 2mm×1mm, with a total of 12 channels; the radius of the dispersed phase inlet 112 designed can be 0.5~5mm, preferably 0.5~4mm, preferably 0.5~3mm, and the actual radius set in this embodiment is 1.5mm. Thus, the cross-sectional area of ​​the dispersed phase inlet is 29.4% of the total cross-sectional area of ​​the dispersed phase flow channel.

[0082] According to the microfluidic chip of this embodiment, the width of the groove in the continuous phase flow channel 131 is 1~10mm, for example 1~8mm, preferably 2~5mm, and in this embodiment it is designed to be 3mm. The depth of the groove in the continuous phase flow channel 131 is 0.5~5mm, for example 0.5~4mm, preferably 1~3mm, and in this embodiment it is designed to be 2mm. Based on the liquid flow capacity of the continuous phase flow channel 131, the size of the continuous phase inlet 132 can be easily determined. Generally speaking, the total cross-sectional area through which the liquid flows in the continuous phase flow channel 131 is 60%~130% of the cross-sectional area of ​​the continuous phase inlet 132, preferably 70%~110%, preferably 80%~90%, for example about 85%. For example, the size of the groove in the continuous phase flow channel 131 designed in this embodiment is 3mm×2mm, with a total of 12 channels, and the radius of the designed continuous phase inlet 132 can be 4~10mm, preferably 4.5~8mm, and the actual set radius is 5.2mm.

[0083] According to the microfluidic chip of this embodiment, the diameter of the capillary pore of the capillary protrusion 121 on the microsphere forming sheet 12 is 10~1000μm, preferably 10~500μm, more preferably 10~250μm, and more preferably 10~200μm. The actual diameter of the capillary pore designed in this embodiment is 45μm. It should be noted that the capillary aperture of the capillary nozzle 121 is not equal to the diameter of the prepared microspheres. Generally speaking, the particle size of the dispersed phase droplets exiting the capillary aperture may be larger than the particle size of the final solid microspheres. This depends on many factors, such as the flow rate or volume, viscosity, and temperature of the dispersion and continuous phase. The desired droplet size can be obtained by comprehensive adjustment. However, the particle size of the droplets is not constant. After subsequent spheroidization, solidification, separation, and drying, the diameter of the formed microspheres will usually shrink. This size change and the proportion of change will vary depending on the microsphere forming material and composition, the combination of the dispersed phase and the continuous phase, and other factors.

[0084] Furthermore, the cross-sectional area of ​​the dispersed phase inlet is smaller than the total cross-sectional area of ​​the 12 dispersed phase channels, approximately 20-40% of the latter. However, the cross-sectional area of ​​the dispersed phase inlet is much larger than the total cross-sectional area of ​​the 12 capillary nozzle orifices. For example, for the dispersed phase flow sheet and microsphere forming sheet in this embodiment, the dispersed phase inlet r = 1.5 mm, with a cross-sectional area of ​​7.0650 square millimeters, and the total cross-sectional area of ​​the 12 dispersed phase channels is 24 square millimeters. The former is approximately 29.4% (7.0650 / 24) of the latter, meaning the inlet is small and the outlet is large. However, the total cross-sectional area of ​​the 12 capillary nozzle orifices on the microsphere forming sheet is 0.0191 square millimeters, much smaller than the cross-sectional area of ​​the dispersed phase inlet. Therefore, in terms of flow velocity, the flow velocity in the dispersed phase channels is smaller than the flow velocity at the dispersed phase inlet, but the flow velocity at the capillary orifices is very large. In this way, the huge flow rate difference between the dispersed phase inlet and the capillary nozzle can be buffered through the dispersed phase flow channel in the middle. This buffering will help avoid the uniformity of microsphere particle size caused by fluctuations in the inlet flow rate due to external factors such as constant flow pump.

[0085] Furthermore, since the dispersed phase flow rate at the dispersed phase inlet can be recorded by a constant flow pump, this flow rate is equal to the total liquid volume flowing out of the entire capillary. However, the flow velocity at the capillary (which should be understood as the velocity of the dispersed phase liquid flowing through the capillary) is affected by the cross-sectional area of ​​the dispersed phase inlet and the total cross-sectional area of ​​the capillary. The size of the microspheres is more directly affected by the liquid flow velocity at the capillary outlet. To more conveniently describe the liquid flow velocity at the capillary outlet, this paper introduces the concept of "capillary correction flow rate" for the microfluidic chip of this application, which is calculated by the following formula: Capillary corrected flow rate (ml / min) = (Cross-sectional area of ​​dispersed phase inlet ÷ Total cross-sectional area of ​​capillary orifice) × Dispersed phase inlet flow rate.

[0086] For example, in Embodiment 5 of this application, when using the chip of Embodiment 1 to prepare microspheres, the flow rates used in the small-scale and pilot-scale tests were 0.25 ml / min and 0.3 ml / min, respectively, and their capillary-corrected flow rates were 92.5 ml / min and 111.0 ml / min, respectively; in Embodiments 6 and 7 of this application, when using the chip of Embodiment 1 to prepare microspheres, the flow rates used were 0.20 ml / min and 0.15 ml / min, respectively, and their capillary-corrected flow rates were 74.0 ml / min and 55.5 ml / min, respectively; in Embodiment 8 of this application, when using the chip of Embodiment 3 to prepare microspheres, the flow rate used was 0.35 ml / min, and its capillary-corrected flow rate was 124.5 ml / min.

[0087] The applicant has unexpectedly discovered that the aforementioned capillary corrected flow rate, within an appropriate range, is beneficial for the control of microsphere size, as will be further described below.

[0088] It should be noted that the amount (volume) of dispersed phase solution delivered to the microfluidic chip by the constant flow pump per unit time is called the flow rate (measured in ml / min or other equivalent units). This flow rate can be easily read by the constant flow pump. Based on the principle of material balance, the amount (volume) of dispersed phase solution flowing through all the capillaries of the microsphere forming sheet into the continuous phase channel per unit time is equal to the flow rate read by the constant flow pump. However, the velocity of the liquid passing through the dispersed phase inlet and the capillary is different because the cross-sectional area of ​​the dispersed phase inlet is different from the total cross-sectional area of ​​the capillary. The latter has a smaller total cross-sectional area, and the liquid passes through faster, but this velocity is not easy to monitor. Based on the above parameters such as flow rate and cross-sectional area, which can be read or measured, the "capillary-corrected flow rate" can reflect the velocity of the liquid passing through the capillary, and this velocity is directly related to the size of the formed microspheres. In particular, the "capillary-corrected flow rate" is not a reflection of the actual flow rate, but should be understood as the velocity of the liquid passing through the capillary.

[0089] In addition, for continuous phase flow sheets, the total cross-sectional area through which the liquid flows through the continuous phase channel is basically the same as the cross-sectional area of ​​the continuous phase inlet. There are no velocity limiting points at the inlet and outlet of this type, and the difference in flow velocity between the two does not affect the size of the microspheres.

[0090] According to the microfluidic chip of this embodiment, the capillary protrusion 121 provided above protrudes from the surface of the microsphere forming sheet 12. The protrusion height can be 1 / 5 to 1 / 2 of the groove depth of the continuous phase flow channel 131, preferably 1 / 4 to 1 / 2. In this embodiment, the protrusion height is designed to be 1 / 3.

[0091] According to the microfluidic chip of this embodiment, when the dispersed phase flow sheet 11, the microsphere forming sheet 12, and the continuous phase flow sheet 13 are tightly stacked and assembled, the dispersed phase liquid flows unidirectionally in the direction of the dispersed phase inlet 112, the dispersed phase channel 111, the through hole 122, the capillary nozzle 121, the continuous phase channel 131, and the outlet 134, and will not seep into other positions between the dispersed phase flow sheet 11 and the microsphere forming sheet 12.

[0092] According to the microfluidic chip of this embodiment, when the dispersed phase flow sheet 11, the microsphere forming sheet 12, and the continuous phase flow sheet 13 are tightly stacked and assembled, the continuous phase liquid flows unidirectionally in the direction of the continuous phase inlet 132, the continuous phase flow channel 131, and the outlet 134, and will not seep into other positions between the continuous phase flow sheet 13 and the microsphere forming sheet 12.

[0093] According to another embodiment of the microfluidic chip in this example, a plurality of through holes 122 arranged longitudinally (i.e., in the flow channel direction) or laterally can be provided on the microsphere forming sheet 12 corresponding to the dispersed phase flow channel 111, as well as capillary protrusions 121 at corresponding positions and in corresponding numbers. The arrangement of such a plurality of through holes can improve production efficiency.

[0094] Example 2: Method for fabricating microfluidic chips This embodiment provides a general method for fabricating the microfluidic chip designed in Example 1 using glass as the material. The basic principle of this fabrication method is as follows: using glass as a substrate, photoresist as a patterning protective layer, a pre-defined microchannel pattern is transferred to the photoresist layer using photolithography, then a microchannel structure is formed on the glass substrate using chemical etching, and finally, the two glass substrates are sealed using a bonding process to obtain a closed glass microfluidic chip. The specific steps are as follows: Step 1) Glass substrate cleaning: Select 3 circular glass substrates with no scratches or impurities on the surface. Use a cleaning solution (i.e., Piranha solution, with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 7:3, purchased from Merck Group) to thoroughly clean the glass substrates, removing organic matter, oil, and impurities adhering to the surface of the glass substrates. After cleaning, use nitrogen to blow dry the substrates to ensure that the surface of the glass substrates is clean and free of impurities, laying the foundation for subsequent photoresist coating.

[0095] Step 2) Photoresist spin coating: Take two glass substrates used to prepare dispersed phase flow sheets and continuous phase flow sheets, and drop positive photoresist (AZ6130 positive photoresist, purchased from Merck Performance Materials) onto the center of the cleaned and dried glass substrates. Start the spin coater and control the spin coater speed to 3000 rpm to make the photoresist spread evenly on the surface of the glass substrates to form a photoresist film of uniform thickness (the thickness of the film can be adjusted according to the actual microchannel size requirements).

[0096] Step 3) Pre-baking treatment: Place the glass substrate with the spin-coated photoresist film on a hot plate for pre-baking treatment. Control the temperature of the hot plate to 95°C. The pre-baking time is set according to the type of photoresist and the thickness of the film. Pre-baking allows the solvent in the photoresist to fully evaporate, promotes the curing of the photoresist film, and enhances the adhesion between the photoresist and the glass substrate.

[0097] Step 4) Ultraviolet Exposure: Based on the liquid flow channel arrangement of the dispersed phase sheet and the continuous phase sheet, the mask with the preset microchannel pattern is tightly bonded to the surface of the photoresist film after pre-baking, ensuring that there are no gaps or offsets between the mask and the glass substrate; the bonded glass substrate is irradiated with ultraviolet light, causing irreversible chemical changes in the photoresist in the irradiated area, and realizing the initial transfer of the microchannel pattern.

[0098] Step 5) Development process: The glass substrate exposed to ultraviolet light is placed in a special developer for the corresponding type of photoresist for development. The photoresist in the ultraviolet-irradiated area is dissolved and removed by the developer, while the photoresist in the unirradiated area is retained. Finally, a microchannel pattern window that is consistent with the mask pattern is formed on the surface of the glass substrate for subsequent etching. After development is completed, the glass substrate is removed and cleaned and dried.

[0099] Step 6) Hardening and Chemical Etching: The developed glass substrate is placed on a hot plate again for hardening to further solidify the remaining photoresist, enhance its etching resistance, and prevent it from falling off during subsequent etching. After hardening, the glass substrate is placed in a hydrofluoric acid etching solution for chemical etching. The glass areas not protected by the photoresist are etched by the hydrofluoric acid etching solution to form a groove structure. This groove is the dispersed phase channel or continuous phase channel of the microfluidic chip described in Example 1. The etching depth of the channel is adjusted by controlling the etching time according to the actual application requirements. After etching, the glass substrate is removed and cleaned.

[0100] Step 7) Resin Removal and Drilling of the Fabrication: The etched dispersed phase and continuous phase wafers are immersed in acetone solvent until the remaining photoresist on the surface of the glass substrate is completely removed. After removal, the wafers are cleaned and dried. Then, an ultrasonic drilling machine is used to drill holes at the corresponding positions of the dispersed phase inlet of the dispersed phase wafer and the continuous phase inlet of the continuous phase wafer, so that the holes penetrate the glass substrate to form the liquid inlet of the microfluidic chip. During the drilling process, it is ensured that the holes are precisely connected to the microchannels and are not blocked. After drilling, a hollow tube port extending out of the wafer surface is bonded to the side of the dispersed phase and continuous phase wafers by photosensitive resin using an adhesive method, forming the dispersed phase inlet and the continuous phase inlet (the aperture of the two inlets can be designed and adjusted as needed). The two inlets are used to connect to the tubing to introduce the dispersed phase liquid or the continuous phase liquid into the microfluidic chip, thereby obtaining the dispersed phase wafer and the continuous phase wafer of the microfluidic chip described in Example 1.

[0101] Step 8) Fabrication of the microsphere forming sheet: At the location where the capillary protrusion is set on the glass substrate of the microsphere forming sheet, a capillary with a predetermined aperture (the aperture of the capillary can be designed and adjusted as needed) is connected by high-temperature bonding. Then, a through hole 122 corresponding to the capillary hole is drilled on the corresponding position on the back side of the capillary of the microsphere forming sheet using a computer numerical control (CNC) milling machine. The design of the capillary protrusion protruding from the microsphere forming sheet is as required in Example 1. Preferably, the surface of the protruding end of the capillary can be polished smooth. The microsphere forming sheet can thus be obtained.

[0102] Step 9) Bonding and Encapsulation: The dispersed phase flow sheet, microsphere forming sheet, and continuous phase flow sheet are precisely aligned and stacked from top to bottom according to the design in Example 1. They are placed in a high-temperature furnace, and the furnace temperature is raised to near the glass softening point (approximately 800°C). Simultaneously, a preset pressure is applied, causing the three glass sheets to permanently fuse under high temperature and high pressure conditions, achieving bonding and sealing. This ultimately yields a microfluidic chip with a glass substrate and closed microchannels. Additionally, if necessary, nozzles can be bonded to the liquid outlets of the continuous phase flow sheet using photosensitive resin bonding.

[0103] The microfluidic chip described in Example 1 can be obtained using the above method.

[0104] Of course, materials such as silicon wafers, quartz, polymethyl methacrylate, and polydimethylsiloxane can also be used to fabricate the microfluidic chip designed in this paper using conventional methods in the field.

[0105] Example 3: Microfluidic Chip This embodiment provides another design scheme for the microfluidic chip of this application. This scheme is essentially the same as that of Embodiment 1, the main difference being that the outlet of the liquid flow is not located at the edge of the continuous phase flow plate. Figures 5-9As shown, the microfluidic chip is basically disc-shaped and can form microspheres by dripping tiny dispersed phase droplets into a continuous liquid flow.

[0106] Figure 5 and Figure 6 This is a schematic diagram of the three circular sheets of the microfluidic chip in a separated state, including (for ease of description, in the direction from top to bottom) three circular sheets (preferably with the same radius) that are tightly stacked together in sequence: dispersed phase flow sheet 21, microsphere forming sheet 22, and continuous phase flow sheet 23.

[0107] The basic principle of fabricating microspheres using the microfluidic chip in this embodiment is essentially the same as in Embodiment 1: the dispersed phase liquid of the microsphere forming material enters from the dispersed phase flow plate 21, reaches the microsphere forming plate 22, and forms tiny droplets that fall into the continuous phase liquid flow in the continuous phase flow plate 23. These tiny droplets of the dispersed phase then form spherical shapes during the liquid flow process. These spherical droplets are then solidified, separated, and dried in subsequent processes to form microspheres.

[0108] The microfluidic chip is designed to allow a dispersed phase liquid containing microsphere-forming material to enter from the dispersed phase flow plate 21, reach the microsphere-forming plate 22, and form tiny droplets that fall into the continuous phase liquid flow in the continuous phase flow plate 23. These dispersed phase droplets then gradually form spherical shapes (i.e., spheroidization) during the liquid flow process. These spherical droplets are then solidified, separated, and dried in subsequent processes to form microspheres.

[0109] Figure 7 This is a schematic diagram of the dispersed phase flow sheet 21 of the microfluidic chip in this embodiment, viewed from both above and below. Figure 8 This is a schematic diagram of the microsphere forming sheet 22 of the microfluidic chip in this embodiment, viewed from both above and below. Figure 9 This is a schematic diagram of the continuous phase flow plate 23 of the microfluidic chip in this embodiment, viewed from three angles: directly above, diagonally above, and diagonally below.

[0110] According to the microfluidic chip of this embodiment, a circular dispersed phase inlet 212 penetrates the center of the dispersed phase flow sheet 21. The lower surface of the dispersed phase flow sheet 21 has 4 to 24 (preferably 6 to 18, 8 in this embodiment) groove-shaped dispersed phase flow channels 211 that are uniformly radiated outward from the dispersed phase inlet 212. The length of the flow channel from the center of the flow sheet is 1 / 6 to 1 / 2 (preferably 1 / 4 to 1 / 2, about 1 / 3 in this embodiment) of the radius of the dispersed phase flow sheet 21.

[0111] When the microfluidic chip according to this embodiment is tightly stacked and assembled with the dispersed phase flow sheet 21, the microsphere forming sheet 22 is provided with a small through hole 222 at or near the end of each dispersed phase flow channel 211. The lower edge of the through hole 222 extends downward to form a capillary protrusion 221, which allows the dispersed phase entering from the dispersed phase inlet 212 to flow along each dispersed phase flow channel 211 to the through hole 222, and then through the through hole 222 to reach the capillary protrusion 221 and drip out.

[0112] According to the microfluidic chip of this embodiment, a circular continuous phase inlet 232 penetrates the center of the continuous phase flow plate 23. The upper surface of the continuous phase flow plate 23 has groove-shaped continuous phase channels 231, the same number as the dispersed phase channels 211, evenly radiating outwards from the continuous phase inlet 232. The length of each channel extends outwards from the continuous phase inlet 232 to near the outer edge of the flow plate, and an outlet 234 is formed at the end of the channel, penetrating the continuous phase flow plate 23. Thus, the number of dispersed phase channels 211, continuous phase channels 231, and capillary nozzles 221 are equal.

[0113] According to the microfluidic chip of this embodiment, when the dispersed phase flow plate 21, the microsphere forming plate 22, and the continuous phase flow plate 23 are tightly stacked and assembled, the dispersed phase inlet 212 of the dispersed phase flow plate 21 and the continuous phase inlet 232 of the continuous phase flow plate 23 are coaxially arranged; the dispersed phase flow channel 211 of the dispersed phase flow plate 21 and the continuous phase flow channel 231 of the continuous phase flow plate 23 are correspondingly positioned and separated by the microsphere forming plate 22, thereby enabling the dispersed phase liquid to flow from... After entering through the dispersed phase inlet 212, the dispersed phase flows outward from the disc along the dispersed phase channel 211 to the end, passes through the through hole 222 of the microsphere forming plate 22, and drips into the continuous phase liquid flow of the continuous phase channel 231 of the continuous phase flow plate 23 in the form of small droplets through the capillary nozzle 221; after entering through the continuous phase inlet 232, the continuous phase liquid flows outward from the disc along the continuous phase channel 231, and carries the small dispersed phase droplets away from the microfluidic chip at the capillary nozzle 221.

[0114] Generally, in this embodiment, the disc-shaped microfluidic chip is mounted with the disc axis in a vertical position during operation, i.e., referring to... Figure 1 The installation scheme is shown. Based on this, as follows: Figures 5-9 As shown, a guide nozzle 233 is provided at the end of the continuous phase flow channel 231 of the continuous phase flow plate 23, through the outlet 234 of the continuous phase flow plate 23, so that the continuous phase liquid carrying the dispersed phase micro-droplets can drip down (to a separately configured collector); the outlet 234 is located inside the continuous phase flow plate instead of at the outer edge of the continuous phase flow plate as in Embodiment 1.

[0115] The microfluidic chip according to this embodiment has a radius of 2~20cm, for example, a radius of 3~15cm, preferably a radius of 3~10cm, and the chip in this embodiment has a radius of 5cm.

[0116] According to the microfluidic chip of this embodiment, the width of the groove in the dispersed phase channel 211 is 1~10mm, for example 1~8mm, preferably 1~5mm, and in this embodiment it is designed to be 2mm. The depth of the groove in the dispersed phase channel 211 is 0.5~5mm, for example 0.5~4mm, preferably 0.5~3mm, and in this embodiment it is designed to be 1.5mm. Based on the liquid flow capacity of the dispersed phase channel 211, the size of the dispersed phase inlet 212 can be easily determined. Generally speaking, the cross-sectional area of ​​the dispersed phase inlet 212 is 5%~60% of the total cross-sectional area through which the liquid flows in the dispersed phase channel 211, preferably 10%~50%, and more preferably 10%~40%. For example, the size of the dispersed phase flow channel 211 groove designed in this embodiment is 2mm×1.5mm, with a total of 8 channels; the radius of the dispersed phase inlet 212 can be 0.5~5mm, preferably 0.5~4mm, preferably 0.5~3mm, and the actual radius set in this embodiment is 1.6mm. Thus, the cross-sectional area of ​​the dispersed phase inlet is 33.5% of the total cross-sectional area of ​​the dispersed phase flow channel.

[0117] In this application, it is preferred that both the dispersed phase inlet and the continuous phase inlet be circular, which facilitates the connection of such inlets to the dispersed phase and continuous phase storage bottles via hoses.

[0118] According to the microfluidic chip of this embodiment, the width of the groove in the continuous phase flow channel 231 is 1~10mm, for example 1~8mm, preferably 2~5mm, and in this embodiment it is designed to be 3.5mm. The depth of the groove in the continuous phase flow channel 231 is 0.5~6mm, for example 0.5~6mm, preferably 1~4mm, and in this embodiment it is designed to be 2.6mm. Based on the liquid flow capacity of this continuous phase flow channel 231, the size of the continuous phase inlet 212 can be easily determined. Generally speaking, the total cross-sectional area through which the liquid flows in the continuous phase flow channel 231 is 60%~130% of the cross-sectional area of ​​the continuous phase inlet 232, preferably 70%~110%, preferably 80%~90%, for example about 85%. For example, the size of the groove in the continuous phase flow channel 231 designed in this embodiment is 3.5mm×2.6mm, with a total of 8 channels, and the radius of the designed continuous phase inlet 232 can be 4~10mm, preferably 4.5~8mm, and the actual set radius is 5.2mm.

[0119] According to the microfluidic chip of this embodiment, the diameter of the capillary pore of the capillary protrusion 221 on the microsphere forming sheet 22 is 10~1000μm, preferably 10~500μm, more preferably 10~250μm, and even more preferably 10~200μm. The actual diameter of the capillary pore designed in this embodiment is 60μm.

[0120] According to the microfluidic chip of this embodiment, the capillary protrusion 221 provided above protrudes from the surface of the microsphere forming sheet 22. The protrusion height can be 1 / 5 to 1 / 2 of the groove depth of the continuous phase flow channel 231, preferably 1 / 4 to 1 / 2. In this embodiment, the protrusion height is designed to be 1 / 4.

[0121] According to the microfluidic chip of this embodiment, when the dispersed phase flow sheet 21, the microsphere forming sheet 22, and the continuous phase flow sheet 23 are tightly stacked and assembled, the dispersed phase liquid flows unidirectionally in the direction of the dispersed phase inlet 212, the dispersed phase channel 211, the through hole 222, the capillary nozzle 221, the continuous phase channel 231, and the outlet 234, and will not seep into other positions between the dispersed phase flow sheet 21 and the microsphere forming sheet 22.

[0122] According to the microfluidic chip of this embodiment, when the dispersed phase flow sheet 21, the microsphere forming sheet 22, and the continuous phase flow sheet 23 are tightly stacked and assembled, the continuous phase liquid flows unidirectionally in the direction of the continuous phase inlet 232, the continuous phase flow channel 231, and the outlet 234, and will not seep into other positions between the continuous phase flow sheet 21 and the microsphere forming sheet 22.

[0123] According to another embodiment of the microfluidic chip in this example, a plurality of through holes 222 arranged longitudinally (i.e., in the flow channel direction) or laterally can be provided on the microsphere forming sheet 22 corresponding to the dispersed phase flow channel 211, for example, 1 to 5 or 1 to 3, and capillary protrusions 221 at corresponding positions and numbers. The arrangement of such a plurality of through holes can improve production efficiency.

[0124] Furthermore, the microfluidic chip provided in Embodiment 3 is fabricated according to the method described in Embodiment 2. Of course, it can also be fabricated using other methods commonly used in the art, such as those described in the literature by Wu Yuanqing et al. (Microfluidic Chip Technology, Chemical Industry Press, 2022) and Dy, Aaron J. et al. (Fabricating microfluidic valve master molds in SU-8photoresist, Journal of Micromechanics and Microengineering, 2014, DOI:10.1088 / 0960-1317 / 24 / 5 / 057001). Given the convenience and cost advantages of glass fabrication, this application preferably uses glass as the material for fabricating the microfluidic chip. Of course, using other materials such as silicon wafers is also feasible.

[0125] Example 4: Equipment and apparatus for preparing uniform microspheres The key component used in the fabrication of microspheres in this application is the microfluidic chip described herein. Of course, other auxiliary components are also necessary. This embodiment uses the microfluidic chip provided herein to design a device for fabricating uniform microspheres. This device consists of an unlimited number of illustrative examples, such as... Figure 10 The device unit shown comprises: a microfluidic chip (which is in an assembled state, for example, the structure described in Embodiment 1 or Embodiment 3, or an equivalent structure within the scope of this application); a dispersed phase solution storage bottle for storing the dispersed phase solution during microsphere preparation; a continuous phase solution storage bottle for storing the continuous phase solution during microsphere preparation; a constant flow pump for introducing the dispersed phase solution and the continuous phase solution into the microfluidic chip; a liquid guide tube for connecting the dispersed phase solution storage bottle, the constant flow pump, and the dispersed phase inlet of the microfluidic chip, and for connecting the continuous phase solution storage bottle, the constant flow pump, and the continuous phase inlet of the microfluidic chip; a receiving tray disposed below the microfluidic chip for receiving liquid flowing out from the outlet at the end of the continuous phase flow channel of the microfluidic chip; and a collection bottle connected to the outlet at the bottom of the receiving tray via the liquid guide tube for collecting the continuous phase solution containing dispersed phase droplets.

[0126] It should be noted that, Figure 10 One unit of the device is schematic, and the size ratio between the components is not limited to that shown in the illustration. For example, the device can continuously produce microspheres without interruption (as long as there is no desire to stop), and the size of the solution storage bottle is actually much larger than the size of the microfluidic chip.

[0127] Furthermore, based on the desired production scale, for laboratory-scale production, the equipment for preparing microspheres can consist of a limited number of the aforementioned equipment units, such as five such equipment units, where all components of each equipment unit are independent and operate in parallel. Figure 10 After the equipment unit completes its operation, the liquids in the five collection bottles are combined; alternatively, the dispersed phase solution storage bottle, continuous phase solution storage bottle, and collection bottle of the five equipment units can be shared, thus simplifying the operation. For pilot-scale or production scale, the desired number of the above equipment units can be arranged to jointly constitute the equipment for preparing microspheres according to this application. All components between all equipment units can be independent and operate in parallel, or several equipment units, such as 5, 8, 10, 20, or other quantities, can be formed into a group. The solution storage bottles and collection bottles within the group are shared, while the groups operate in parallel. In addition, since the microfluidic chip, equipment units, and equipment of this application can achieve uninterrupted continuous production, and the facility scale can be expanded by adding equipment units and / or their working groups, the efficiency of preparing microspheres according to this application will be quite high, and the output can meet the desired small-scale or large-scale requirements.

[0128] by Figure 10 Taking the device unit for preparing microspheres shown in Example 1 and using the microfluidic chip described in Example 1 as an example, the device unit for preparing uniform microspheres operates basically according to the following steps when the microfluidic chip is assembled: (i) The continuous phase inlet is connected to the continuous phase solution storage bottle through a liquid guide tube. During this process, the continuous phase solution is pumped into the continuous phase flow plate of the microfluidic chip by a constant flow pump, and flows through the continuous phase flow channel until it flows out from the outlet of the flow plate. (ii) The dispersed phase inlet is connected to the dispersed phase solution storage bottle through a liquid guide tube. During this process, the dispersed phase solution is pumped into the dispersed phase flow plate of the microfluidic chip through a constant flow pump. Then, it passes through the through holes and capillary protrusions of the microsphere forming plate and enters and disperses into the continuous phase solution in the continuous phase flow channel in the form of tiny droplets. It is then carried away by the continuous phase solution to the outlet of the microfluidic chip. (iii) A continuous phase solution containing tiny droplets of dispersed phase is dripped from the outlet into the receiving tray, and then discharged into the collection bottle through the outlet of the receiving tray. (iv) The dispersed phase microdroplets formed at the capillary nozzle undergo spheroidization and preliminary solidification in the continuous phase solution, and then the liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle.

[0129] In the preparation of uniform microspheres in this application, the liquid microspheres obtained in step (iv) above can be further processed as follows: (v) After the liquid obtained in step (iv) has been left to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. Optionally, the solid microspheres are washed and dried (e.g., under reduced pressure) with water to obtain microspheres in the form of fine powder.

[0130] Example 5: Preparation of microspheres (PLGA, drug-containing) As described herein, the commercially available injectable octreotide acetate microsphere suspension (SANDOSTATIN® LAR DEPOT, octreotide acetate) uses D,L-lactide-glycolic acid copolymer (PLGA) as the microsphere material. This embodiment attempts to utilize the microfluidic chip of this application and its assembled device unit or device octreotide acetate microspheres. Octreotide acetate is readily available commercially and can also be prepared according to existing literature methods, such as those described in EP29579 and US4395403. The octreotide acetate used in this embodiment was prepared according to the method described in US4395403. This embodiment uses PLGA (model B6001-1, 65:35 polylactic acid-glycolic acid copolymer) from EVONIK Corporation and a Fluigent LU-FEZ-0345 constant flow pump. The dispersed phase solution composition is a solution containing 0.4% octreotide acetate and 6.0% PLGA, prepared using dichloromethane as a solvent. The continuous phase solution consists of a 3% aqueous solution of polyvinyl alcohol (PVA 17-88).

[0131] In this embodiment, microfluidic chips designed and manufactured in Examples 1 and 2 were used, and a device unit (research scale) was installed as described in Example 4. A group consisting of 5 device units (a small-scale production device can be constructed by having 5 such groups working in parallel) was used to prepare drug-containing microspheres. The preparation environment, the temperature of the dispersed phase solution, and the continuous phase solution were all at room temperature (24±1°C), and the humidity was RH 58~65%.

[0132] 1. Basic process for microsphere preparation: (a) Add the continuous phase solution and the dispersed phase solution to the continuous phase solution storage bottle and the dispersed phase solution storage bottle respectively, start the constant flow pump, and adjust and control the flow rate of the two pumps for the continuous phase solution and the dispersed phase solution by monitoring the microsphere particle size in the suspension collected by the receiving tray, so that the microsphere particle size reaches the desired value or range. (Step (a) above describes the method for adjusting a single device unit. The same adjustment is applied to a group consisting of multiple device units, or to a larger manufacturing group formed by parallel setups of these units. It should be noted that by controlling the uniformity of the capillary orifice diameter of each capillary nozzle in the same microfluidic chip, it is easy to achieve uniformity of the microsphere diameter flowing out from different capillary nozzles of the same microfluidic chip. Under this premise, it is also easy to achieve uniformity of the microsphere diameter flowing out from different microfluidic chips. Therefore, if the flow rate of the constant flow pump between different chips is adjusted to be basically consistent, it is easy to achieve uniformity of the microsphere diameter flowing out from different microfluidic chips.) (b) The process from the moment the dispersed phase microdroplets formed at the capillary nozzle enter the continuous phase solution until they are in the collection bottle, wherein the dispersed phase microdroplets undergo spheroidization and preliminary solidification in the continuous phase solution, and then the liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle. (c) After the liquid obtained in step (b) has been left to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. The solid microspheres are then rinsed with water and dried (under reduced pressure) to obtain microspheres in the form of fine powder.

[0133] 2. Scale-up preparation of microspheres Using the microfluidic chip (12 capillary channels) designed and manufactured in Examples 1 and 2, and installed as a device unit as described in Example 4, octreotide acetate microspheres were prepared on a small-scale research basis. The dispersed phase solution, the continuous phase solution, and the temperature of the preparation environment were all room temperature (24 ± 1 °C).

[0134] The flow rate of the dispersed phase liquid at the dispersed phase inlet (hereinafter the same) is set to 0.25 ml / min, and the flow rate of the continuous phase liquid at the continuous phase inlet (hereinafter the same) is set to 5.0 ml / min. Based on the aforementioned dispersed phase solution composition, the theoretical yield of solid microspheres is 0.96 g / hour / chip.

[0135] The above steps (a) to (c) are performed continuously for 48 hours. Step (b) involves collecting samples every 12 hours, followed by step (c), resulting in four batches of finely powdered microspheres. These four batches are designated T01a, T01b, T01c, and T01d according to the order of collection. The mixed microspheres, with equal weight proportions of the four batches, are designated T01. The phrase "collecting samples every 12 hours" or other similar expressions in this application refer to collecting samples for approximately 30 minutes at 12-hour intervals during continuous production and microsphere collection. These samples are distinct from the final total production sample obtained in step (c) (i.e., the final mixed product obtained throughout the entire production process). The samples collected at these intervals reflect the state of the microspheres at a specific point in time during the production process. If the state of the microspheres at all points is essentially the same, they will be compared with the total production sample obtained in step (c).

[0136] The particle size of each batch of microsphere samples was determined using an industrial optical microscope. For each sample, the diameter of 60-70 microspheres was recorded, and the average diameter and standard deviation were calculated. The particle sizes of the five types of microspheres (T01a, T01b, T01c, T01d, and T01), expressed as mean ± SD, were 57.4 ± 1.3 μm, 56.3 ± 1.1 μm, 57.9 ± 1.4 μm, 55.5 ± 1.4 μm, and 56.8 ± 1.2 μm, respectively. The average particle size of the five types of microspheres was 56.8 μm.

[0137] The specific operating steps for determining the particle size of microsphere samples using an industrial optical microscope are as follows: First, the optical microscope is pre-calibrated, and the accuracy of the scale is verified and adjusted on the monitoring display using a standard micrometer scale. Then, the microsphere sample is taken, uniformly dispersed on a clean glass slide, and placed on the microscope stage. By adjusting the microscope's focal length and illumination system, the microsphere image is clearly displayed on the monitoring display. Finally, using the measuring software integrated with the microscope, the microsphere image is automatically captured directly on the display, the outline of a specified number of microspheres is automatically selected, and their diameter values ​​are automatically read and recorded. Furthermore, the mean and standard deviation of the microsphere sample's particle size can be calculated. The particle size determination of microsphere samples using an industrial optical microscope in this application was performed using a BM19A-UV-G type particle size statistical analysis microscope (Shanghai Optical Instrument Factory).

[0138] In addition, the particle size of the above five microsphere samples was determined using a scanning electron microscope (ZEISS GeminiSEM 300, Carl Zeiss AG, Germany) in the same manner. The results were 56.9±1.1μm, 56.7±1.4μm, 57.1±1.2μm, 55.2±1.0μm, and 57.5±1.4μm, respectively. The average particle size of the five microspheres was 56.7μm.

[0139] In addition, with 48 hours of continuous production, 44.1g of dried solid microspheres were obtained, which means that the actual yield of solid microspheres is 0.92g / hour / chip, which is close to the theoretical value.

[0140] Figures 11-13 The images show an optical microscope image (monitor photo) of the T01a sample taken from the liquid receiving tray, an optical microscope image (monitor photo) of the T01a sample after curing and drying, and a scanning electron microscope image of the T01a sample after curing and drying.

[0141] The above results show that the measurements obtained using optical microscopy and scanning electron microscopy are consistent, thus verifying the reliability of the optical microscopy results. These results also indicate that there was no significant difference in the diameter of the microspheres harvested at different times during the 48-hour preparation period, demonstrating that the microfluidic chip and device unit for preparing microspheres in this application exhibit excellent production stability.

[0142] 3. Pilot-scale preparation of microspheres As described in this embodiment, a group consisting of 6 equipment units is formed, and 5 such groups working in parallel constitute a pilot-scale production equipment, totaling 30 equipment units (i.e., 30 microfluidic chips, 5 groups with 5 collection bottles) to prepare the above-mentioned drug-containing octreotide acetate microspheres. The temperature of the dispersed phase solution, the continuous phase solution, and the preparation environment are all room temperature 24±1°C.

[0143] Under optical microscope monitoring conditions, the flow rate of the dispersed phase liquid flow in each device unit was adjusted to within the range of 0.30±0.02 ml / min, and the flow rate of the continuous phase liquid flow was adjusted to within the range of 6.5±0.5 ml / min, so that the particle size of the micro-droplet suspension collected from the receiving trays of all device units was basically the same. At this continuous small-scale production, a daily production scale of over 500 g can be easily achieved, and a daily production scale of over 1 kg can be achieved by arranging a group of 50 microfluidic chip device units.

[0144] The above steps (a) to (c) were used for continuous production for 48 hours. Step (b) was collected every 12 hours, and step (c) was performed. The collection bottles of the five groups were collected, solidified, rinsed and dried at regular intervals to obtain 20 batches of solid microspheres. In addition, the microspheres of these batches were mixed according to the sampling time or the unit group to obtain 10 mixed batches. Their average particle size was measured and the results are shown in Table 1.

[0145] Table 1: Average particle size determination results of microspheres obtained by the five equipment unit groups at different time points.

[0146] The mean and sd values ​​of the results for the 30 sampling scenarios were calculated to be 65.43 ± 1.55 μm.

[0147] The results show that a pilot-scale production facility (30 units in total) consisting of multiple equipment units operating in parallel exhibits remarkably uniform microsphere size across groups composed of different microfluidic chips, between groups composed of different units, and at different times during continuous production. The study also demonstrated that using different flow rates of dispersed and continuous phases in both the small-scale and pilot-scale trials yielded microspheres of varying sizes, indicating that the desired microsphere size can be easily obtained by adjusting the flow rates of the two solutions.

[0148] Furthermore, the uniformity of the results from the five equipment unit groups shown in Table 1 above allows for the parallel arrangement of such equipment unit groups, such as 20, 50, 100, or 200 groups. This easily meets the equipment requirements for larger pilot-scale and commercial-scale production. Since each equipment unit group occupies little space, the overall space required for such a large-scale parallel arrangement of equipment unit groups is small, making it easy to achieve large-scale production of microspheres within a limited space. The drug-containing microspheres prepared above can be used for clinical treatment.

[0149] Example 6: Preparation of polycaprolactone microspheres This embodiment refers to the method of Example 5, and prepares microspheres using polycaprolactone (model P398622, Aladdin) as the material. Given that this microsphere material is biodegradable in the human body, these microspheres can be used as a facial filler in medical aesthetics.

[0150] As described in this embodiment, a group consisting of five device units (i.e., using five microfluidic chips as described in Example 1, sharing a collection bottle, a dispersed phase storage bottle, and a continuous phase storage bottle) was prepared. The dispersed phase solution consisted of a 5.0% polycaprolactone solution in dichloromethane, and the continuous phase solution consisted of a 2.5% polyvinyl alcohol (PVA 17-88) aqueous solution. The temperatures of the dispersed phase solution, the continuous phase solution, and the preparation environment were all room temperature (24 ± 1 °C). Under optical microscopy monitoring, the flow rate of the dispersed phase liquid flow in each device unit was adjusted to within the range of 0.20 ± 0.02 ml / min, and the flow rate of the continuous phase liquid flow was adjusted to within the range of 6.0 ± 0.5 ml / min, so that the particle size of the microdroplet suspension collected from the receiving trays of all device units was substantially the same. The operation of steps (a) to (c) described in Example 5 was used for continuous production for 48 hours. Step (b) was collected every 12 hours. Step (c) was then performed to solidify, rinse, and dry the microspheres in the collection bottle, resulting in 4 batches of solid microspheres. The 4 batches of samples were recorded as T02a, T02b, T02c, and T02d in the order of collection. The mixed microspheres of the four batches of microspheres were mixed in equal weight proportions and recorded as T02.

[0151] The particle size of each batch of microsphere samples was determined using an optical microscope according to the method described in this paper. The particle sizes of the five types of microspheres, T02a, T02b, T02c, T02d and T02, were 43.8±1.6μm, 46.1±1.2μm, 42.8±1.7μm, 45.2±1.3μm and 44.9±1.6μm, respectively, expressed as mean ± SD. The mean of the average particle size of the five types of microspheres was 44.6μm.

[0152] In addition, the statistical particle size results of the microspheres obtained in step (c) of this embodiment were determined to be: mean 44.28 μm, standard deviation 1.78 μm, and relative standard deviation 4.02%. These results are basically consistent with the microspheres collected during the production process described above.

[0153] Example 7: Preparation of polylactic acid microspheres This embodiment refers to the method of Example 5, using poly-L-lactic acid (PLLA, LACTEL®, intrinsic viscosity 0.90-1.20 dL / g, DURECT) as the material to prepare microspheres. Given that this microsphere material is biodegradable in the human body, these microspheres can be used as a facial filler in medical aesthetics.

[0154] As described in this embodiment, a group consisting of five device units (i.e., using five microfluidic chips as described in Embodiment 1, sharing a collection bottle, a dispersed phase storage bottle, and a continuous phase storage bottle) was prepared. The dispersed phase solution consisted of a 6.5% poly(L-lactic acid) solution in dichloromethane, and the continuous phase solution consisted of a 3.0% polyvinyl alcohol (PVA 17-88) aqueous solution. The temperature of the dispersed phase solution, the continuous phase solution, and the preparation environment were all room temperature (24 ± 1 °C). Under optical microscope monitoring conditions, the flow rate of the dispersed phase liquid flow in each device unit was adjusted to within the range of 0.15 ± 0.02 ml / min, and the flow rate of the continuous phase liquid flow was adjusted to within the range of 5.5 ± 0.5 ml / min, so that the particle size of the microdroplet suspension collected from the receiving trays of all device units was basically the same. The operation of steps (a) to (c) described in Example 5 was used for continuous production for 48 hours. Step (b) was collected every 12 hours. Step (c) was then performed to solidify, rinse, and dry the microspheres in the collection bottle, resulting in 4 batches of solid microspheres. The 4 batches of samples were recorded as T03a, T03b, T03c, and T03d in the order of collection. The mixed microspheres of the four batches of microspheres were mixed in equal weight proportions and recorded as T03.

[0155] The particle size of each batch of microsphere samples was determined using an optical microscope according to the method described in this paper. The particle sizes of the five types of microspheres, T03a, T03b, T03c, T03d and T03, were 31.2±1.2μm, 29.3±1.5μm, 30.6±1.4μm, 31.8±1.6μm and 29.7±1.8μm, respectively. The average particle size of the five types of microspheres was 30.5μm.

[0156] In addition, the statistical particle size results of the microspheres obtained in step (c) of this embodiment were determined to be: mean 30.27 μm, standard deviation 1.71 μm, and relative standard deviation 5.65%. These results are basically consistent with the microspheres collected during the production process described above.

[0157] Example 8: Preparation of polylactic acid microspheres This embodiment refers to the method of Embodiment 5, using the microfluidic chip designed in Embodiment 3 and manufactured in Embodiment 2, and assembling a device unit (research scale) as described in Embodiment 4, to prepare microspheres using poly-L-lactic acid (LACTEL®, intrinsic viscosity 0.90-1.20 dL / g, DURECT). Given that this microsphere material is biodegradable in the human body, these microspheres can be used as a facial filler in medical aesthetics.

[0158] This embodiment consists of a group of 5 device units (i.e., using 5 microfluidic chips, which share a collection bottle, a dispersed phase storage bottle, and a continuous phase storage bottle). The dispersed phase solution consists of a 5.0% poly(L-lactic acid) solution in dichloromethane, and the continuous phase solution consists of a 2.0% polyvinyl alcohol (PVA 17-88) aqueous solution. The temperature of the dispersed phase solution, the continuous phase solution, and the preparation environment are all room temperature (24±1°C). Under optical microscope monitoring conditions, the flow rate of the dispersed phase liquid in each device unit is adjusted to within the range of 0.35±0.02 ml / min, and the flow rate of the continuous phase liquid is adjusted to within the range of 7.0±0.5 ml / min, so that the particle size of the microdroplet suspension collected from the receiving trays of all device units is basically the same. The operation of steps (a) to (c) described in Example 5 was used for continuous production for 48 hours. Step (b) was collected every 12 hours. Step (c) was then performed to solidify, rinse, and dry the microspheres in the collection bottle, resulting in 4 batches of solid microspheres. The 4 batches of samples were recorded as T04a, T04b, T04c, and T04d in the order of collection. The mixed microspheres of the four batches of microspheres were mixed in equal weight proportions and recorded as T04.

[0159] The particle size of each batch of microsphere samples was determined using an optical microscope according to the method described in this paper. The particle sizes of the five types of microspheres, T04a, T04b, T04c, T04d and T04, were 73.8±1.9μm, 75.1±1.2μm, 71.4±1.7μm, 73.6±2.2μm and 74.2±1.6μm, respectively, expressed as mean ± SD. The mean of the average particle size of the five types of microspheres was 73.6μm.

[0160] In addition, samples from batch T04 were cured and dried before being measured using a scanning electron microscope. The results are as follows: Figure 14 As shown, the microspheres exhibiting uniform size are consistent with the results obtained using optical microscopy.

[0161] It has been unexpectedly discovered that capillary-corrected flow rate design within the range of 30–150 ml / min, particularly 40–140 ml / min, and especially 50–130 ml / min, is beneficial. Below this range, the microsphere particle size tends to gradually increase over long-term production, while above this range, the variation in microsphere particle size becomes greater. This finding is illustrated by the following examples.

[0162] Example 9: Preparation of polycaprolactone microspheres Polycaprolactone microspheres were prepared according to Example 6.

[0163] Dispersed phase solution: 5.0% polycaprolactone in dichloromethane solution; continuous phase solution: 2.5% polyvinyl alcohol (PVA 17-88) aqueous solution, flow rate 6.0±0.5 ml / min. A microfluidic chip was designed according to Example 1 and prepared according to the method of Example 2, and the device unit was configured according to Example 4.

[0164] The microspheres were prepared using a device unit consisting of a microfluidic chip and produced continuously for 48 hours. In step (b), the microspheres were collected every 8 hours (the droplets collected during the 5-minute period were collected; unless otherwise specified, the average value of the microspheres collected at a certain time point is the microspheres obtained after post-processing such as solidification and drying of the droplets collected at that time point). Then, the operation in step (c) was performed to solidify, rinse, and dry the microspheres in the collection bottle, resulting in 6 batches of solid microspheres. The particle size of the microsphere samples was measured using an optical microscope, and the diameter of 60-70 microspheres was recorded for each sample. The average diameter and standard deviation were calculated.

[0165] result: (1) With dispersed phase inlet r=1.5mm (cross-sectional area 7.0650) and capillary d=45μm (12-hole total cross-sectional area 0.0191), different dispersed phase inlet flow rates were set, and the average diameter and standard deviation of microsphere particle size (μm) at the 6 sampling points are shown in Table 2 below.

[0166] Table 2:

[0167] Note: *Dispersed phase inlet flow rate (±0.01 ml / min), **Capillary corrected flow rate (ml / min).

[0168] The results in the table above show that: for the four chips with calibration flow rates in the range of 55-129 mL / min, the microsphere particle size differences at their respective six sampling points are small, and the particle size differences read by the optical microscope at each sampling point are very small (small SD value); for the two chips with calibration flow rates of 18.5 and 37.0 mL / min, the microsphere particle size at their respective six sampling points shows a significant decreasing trend over time, although the microsphere particle size at each sampling point is basically consistent (small SD value); for the two chips with calibration flow rates of 148.0 and 185.0 mL / min, the particle size differences at each sampling point read by the optical microscope are large (large SD value), although the overall microsphere particle size at the six sampling points remains stable. This indicates that, with the pore sizes of both the dispersed phase inlet and the capillary fixed, adjusting the dispersed phase inlet flow rate to maintain a calibration flow rate of 50-130 mL / min can not only maintain the consistency of microsphere particle size at a certain time point, but also maintain the consistency of microsphere particle size before and after a long period of production.

[0169] (2) With a dispersed phase inlet flow rate of 0.30±0.01 ml / min and a capillary d=45 μm (total cross-sectional area of ​​12 wells 0.0191), different dispersed phase inlet r (mm) were set. The average diameter and standard deviation of the microsphere particle size (μm) obtained at the 6 sampling points are shown in Table 3 below.

[0170] Table 3:

[0171] Note: *Dispersed phase inlet flow rate (±0.01 ml / min), **Capillary corrected flow rate (ml / min).

[0172] The results in the table above show that: for the two chips with corrected flow rates of 59.7 and 118.5 ml / min, the microsphere particle size differences at their respective six sampling points are small, and the particle size differences read by the optical microscope at each sampling point are very small (small SD value); for the two chips with corrected flow rates of 12.3 and 31.6 ml / min, the microsphere particle size at their respective six sampling points shows a significant decreasing trend over time, although the particle size at each sampling point is basically consistent (small SD value); for the two chips with corrected flow rates of 159.8 and 207.3 ml / min, the particle size differences at each sampling point read by the optical microscope are large (large SD value), although the overall particle size at the six sampling points remains stable. This indicates that, with a fixed dispersed phase inlet flow rate and capillary pore size, adjusting the dispersed phase inlet radius to maintain a corrected flow rate of 50–120 ml / min can not only maintain the consistency of microsphere particle size at a certain time point, but also maintain the consistency of microsphere particle size before and after a long period of production.

[0173] (3) With a dispersed phase inlet flow rate of 0.25±0.01 ml / min and a dispersed phase inlet r=1.3 mm (cross-sectional area of ​​5.3066 square millimeters), different capillary inner diameters (d, unit μm) were set. The average diameter and standard deviation of the microsphere particle size (μm) obtained at the 6 sampling points are shown in Table 4 below.

[0174] Table 4:

[0175] Note: *Dispersed phase inlet flow rate (±0.01 ml / min), **Capillary corrected flow rate (ml / min).

[0176] The results in the table above show that: for the chip with a calibration flow rate of 88.0, the microsphere particle size difference at the six sampling points is small, and the particle size difference read by the optical microscope at each sampling point is very small (small SD value); for the chip with a calibration flow rate of 28.7, the microsphere particle size at the six sampling points shows a significant decreasing trend over time, although the particle size at each sampling point is basically the same (small SD value); for the chip with a calibration flow rate of 156.5, the particle size difference read by the optical microscope at each sampling point is large (large SD value), although the overall particle size of the microspheres at the six sampling points remains stable. In some additional tests conducted according to the table above, the parameters were adjusted to make the calibration flow rate approximately 15 ml / min, approximately 50 ml / min, approximately 125 ml / min, and 195 ml / min, respectively. The results were consistent with the trend in Table 2, namely, at a calibration flow rate of 15 ml / min, the microsphere diameter at the six sampling points gradually decreased; at a calibration flow rate of 195 ml / min, the relative standard deviation of the microsphere diameter at the six sampling points was greater than 9.5%; while at calibration flow rates of 50 ml / min and 125 ml / min, the microsphere diameter at the six sampling points remained basically unchanged, and the relative standard deviation was less than 3.2%. This indicates that, with the dispersed phase inlet flow rate and dispersed phase inlet r fixed, adjusting the capillary pore size to make the calibration flow rate between 50 and 130 ml / min can not only maintain the consistency of microsphere particle size at a certain time point, but also maintain the consistency of microsphere particle size before and after long-term production.

[0177] Example 10: Preparation of poly-L-lactic acid microspheres Poly-L-lactic acid microspheres were prepared according to Example 8.

[0178] Dispersed phase solution: 5.0% poly(L-lactic acid) in dichloromethane solution; continuous phase solution: 2.5% polyvinyl alcohol (PVA17-88) aqueous solution, flow rate 7.0 ± 0.5 ml / min. A microfluidic chip was designed according to Example 3 and prepared according to the method of Example 2, and the device unit was configured according to Example 4.

[0179] The microspheres were prepared using a device unit consisting of a microfluidic chip and produced continuously for 48 hours. In step (b), the microspheres were collected every 8 hours (the droplets collected during the 5-minute period were collected; unless otherwise specified, the average value of the microspheres collected at a certain time point is the microspheres obtained after post-processing such as solidification and drying of the droplets collected at that time point). Then, the operation in step (c) was performed to solidify, rinse, and dry the microspheres in the collection bottle, resulting in 6 batches of solid microspheres. The particle size of the microsphere samples was measured using an optical microscope, and the diameter of 60-70 microspheres was recorded for each sample. The average diameter and standard deviation were calculated.

[0180] result: (1) With dispersed phase inlet r=1.25mm (cross-sectional area 4.90625) and capillary d=50μm (total cross-sectional area of ​​8 holes 0.0157), different dispersed phase inlet flow rates were set, and the average diameter and standard deviation of microsphere particle size (μm) at the 6 sampling points are shown in Table 5 below.

[0181] Table 5:

[0182] Note: *Dispersed phase inlet flow rate (±0.01 ml / min), **Capillary corrected flow rate (ml / min).

[0183] The results in the table above show that: for the chip with a calibration flow rate of 93.8, the microsphere particle size difference at the six sampling points is small, and the particle size difference read by the optical microscope at each sampling point is very small (small SD value); for the chip with a calibration flow rate of 31.2, the microsphere particle size at the six sampling points shows a significant decreasing trend with the sampling time, although the microsphere particle size at each sampling point is basically consistent (small SD value); for the chip with a calibration flow rate of 187.5, the particle size difference read by the optical microscope at each sampling point is large (large SD value), although the overall microsphere particle size at the six sampling points remains stable. Furthermore, referring to Example 9, variations in the dispersed phase inlet radius, capillary inner diameter, and dispersed phase inlet flow rate were applied to keep the calibration flow rate within the range of 20~250 ml / min. It was also found that when the calibration flow rate is less than 50 ml / min, the generated microsphere particle size tends to decrease with the extension of working time, while when the calibration flow rate is greater than 130 ml / min, the microsphere particle size fluctuation at each sampling time point is significantly larger. Specifically, in some additional tests conducted according to the table above, the parameters were adjusted so that the calibration flow rates were approximately 15 ml / min, 53 ml / min, 128 ml / min, and 215 ml / min, respectively. The results were consistent with the trend in Table 2, namely, when the calibration flow rate was 15 ml / min, the microsphere diameters at the six sampling points gradually decreased; when the calibration flow rate was 215 ml / min, the relative standard deviations of the microsphere diameters at the six sampling points were all greater than 11.2%; while at the calibration flow rates of 53 ml / min and 128 ml / min, the microsphere diameters at the six sampling points remained basically unchanged and the relative standard deviations were all less than 2.4%.

[0184] In this embodiment, the microspheres (microsphere 1) obtained at the first sampling point using the 93.8 flow rate correction scheme are typically shown in the photographs obtained using optical microscopy with particle size determination. Figure 15 As shown in the figure, the diameters of several typical microspheres read are all in the range of 60-63 μm.

[0185] Example 11: Preparation of microspheres using classical methods The classic method for preparing microspheres is the emulsification method, which typically involves preparing an O / W emulsion by adjusting the shear of a dichloromethane solution containing a polymer material (optionally and a drug), followed by solidification of the microspheres during the removal of the dichloromethane, and separation of the microspheres. For example, the methods described in CN103536537B (Chinese application number 201310494030.8) and CN103458895B (Chinese application number 201180049447.2) are used in this embodiment. This embodiment refers to the process of Example 1 of CN103458895B and uses poly-L-lactic acid (PLLA, LACTEL®) as the polymer material to prepare microspheres without octreotide acetate.

[0186] A dispersed phase solution (a solution containing 15.0% PLLA (LACTEL®, with a higher PLLA concentration than in Example 7 due to a different preparation process) prepared using dichloromethane as a solvent) and a continuous phase solution (1% PVA 17-88 aqueous solution) were prepared. 20 ml of the dispersed phase was slowly injected into 1500 ml of the continuous phase while stirring at 1800 rpm at room temperature. Emulsification was continued for 5 minutes, followed by stirring at 250 rpm for approximately 5 hours to evaporate the solvent. After standing for 1-2 hours, the supernatant (mainly aqueous phase) was poured off. The remaining liquid microsphere suspension at the bottom of the flask was further solidified by removing the solvent under reduced pressure (unless otherwise specified, organic solvent removal in this application is achieved using a rotary evaporator) to obtain solid microspheres. These solid microspheres were then rinsed with water and dried (under reduced pressure) to obtain fine powder microspheres. A typical scanning electron microscope image of these microspheres is shown below. Figure 16 The figure shows a situation where the microsphere particle size varies greatly (3~50μm). As described in CN103458895B, a 1200-mesh sieve and a 100-mesh sieve are required to obtain microspheres, resulting in a wide distribution range of microsphere particle size from 6.5μm to 150μm. As microspheres for controlling drug release, a "sieving" process is needed to obtain microspheres with a narrow particle size distribution to ensure uniform drug release. This "sieving" process significantly reduces production efficiency, and due to the small size of the microspheres (below 100μm), the "sieving" work is extremely difficult. However, using the method of this application, extremely uniform microspheres can be obtained without any "sieving" process. The statistical particle size of the microspheres obtained by the classical method in this embodiment was determined using the method of this application. The results were: mean 29.84μm, standard deviation 18.76μm, relative standard deviation 62.87%, indicating a large degree of dispersion. As can be seen, the statistical average particle size of the microspheres prepared by the classical method in this embodiment, which is 29.84 μm, is basically the same as that of the microspheres obtained in Example 7, which is 30.27 μm. The main difference between the two is that the particle size uniformity is huge, that is, the dispersion of the microsphere particle size is different.

[0187] This application illustrates the detailed method of this application through the above embodiments, but this application is not limited to the above detailed method, that is, it does not mean that this application must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of the product of this application, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

Claims

1. A microfluidic chip for fabricating microspheres, the microfluidic chip being substantially circular in shape, comprising three circular thin sheets sequentially and tightly stacked together from top to bottom: a dispersed phase flow sheet, a microsphere forming sheet, and a continuous phase flow sheet, characterized in that: A circular dispersed phase inlet passes through the center of the dispersed phase flow plate. 4 to 24 groove-shaped dispersed phase channels radiate uniformly from the dispersed phase inlet outwards from the lower surface of the dispersed phase flow plate. The length of each channel from the center of the flow plate is 1 / 6 to 1 / 2 of the radius of the dispersed phase flow plate. The microsphere forming sheet has a small through hole at the end of each dispersed phase flow channel. The lower edge of the through hole extends downward to form a capillary nozzle, which allows the dispersed phase entering from the dispersed phase inlet to flow along each dispersed phase flow channel to the through hole, and then through the through hole to the capillary nozzle to drip out. A circular continuous phase inlet passes through the center of the continuous phase flow plate. The upper surface of the continuous phase flow plate has a number of groove-shaped continuous phase channels that are uniformly radiated outward from the continuous phase inlet. The length of the channel extends from the continuous phase inlet to the outlet at the outer edge of the flow plate.

2. The microfluidic chip according to claim 1, characterized in that: It allows the dispersed phase liquid flow of the microsphere forming material to enter from the dispersed phase flow plate, reach the microsphere forming plate, and form tiny droplets that fall into the continuous phase liquid flow in the continuous phase flow plate. These tiny droplets then form spherical shapes during the liquid flow process. These spherical droplets can then be solidified, separated, and dried in subsequent processes to form microspheres. The outlet is located at the edge of the circular continuous phase flow plate. A guide nozzle is provided at the outlet at the end of the continuous phase flow channel of the continuous phase flow plate, allowing the continuous phase liquid flow carrying the dispersed phase tiny droplets to drip down. The radius of the chip is 2~20cm; the width of the groove in the dispersed phase flow channel is 1~10mm. The depth of the groove in the dispersed phase flow channel is 0.5~5mm; the radius of the dispersed phase inlet is 0.5~5mm; the cross-sectional area of ​​the dispersed phase inlet is 5%~60% of the total cross-sectional area through which the liquid flows in the dispersed phase flow channel; the width of the groove in the continuous phase flow channel is 1~10mm; the depth of the groove in the continuous phase flow channel is 0.5~5mm; the total cross-sectional area through which the liquid flows in the continuous phase flow channel is 60%~130% of the cross-sectional area of ​​the continuous phase inlet; the radius of the continuous phase inlet is 4~10mm; the diameter of the capillary orifice of the capillary protrusion on the microsphere forming sheet is 10~1000μm; and / or, the cross-sectional area of ​​the dispersed phase inlet is 10~60% of the total cross-sectional area of ​​the dispersed phase flow channel.

3. The microfluidic chip according to any one of claims 1 to 2, characterized in that When the dispersed phase flow plate, the microsphere forming plate, and the continuous phase flow plate are tightly stacked and assembled... The dispersed phase inlet of the dispersed phase flow plate is coaxially arranged with the continuous phase inlet of the continuous phase flow plate; The dispersed phase flow channel of the dispersed phase flow plate corresponds in position to the continuous phase flow channel of the continuous phase flow plate, and is separated by a microsphere forming plate, thereby enabling: After entering from the dispersed phase inlet, the dispersed phase liquid flows outward to the end of the disc along the dispersed phase flow channel, passes through the through holes of the microsphere forming plate, and drips into the continuous phase liquid flow of the continuous phase flow channel of the continuous phase flow plate in the form of small droplets through the capillary nozzle. After entering from the continuous phase inlet, the continuous phase liquid flows along the continuous phase flow channel to the outer edge of the disc, and at the capillary nozzle, it carries the tiny droplets of dispersed phase away from the microfluidic chip.

4. The microfluidic chip according to any one of claims 1 to 3, characterized in that: Its capillary corrected flow rate is in the range of 30~150ml / min or 50~120ml / min; the capillary nozzle protrudes from the surface of the microsphere forming plate, and the protrusion height is 1 / 5 to 1 / 2 of the depth of the continuous phase flow channel groove; when the dispersed phase flow plate, microsphere forming plate, and continuous phase flow plate are tightly stacked and assembled, the dispersed phase liquid flows unidirectionally in the direction of dispersed phase inlet, dispersed phase flow channel, through hole, capillary nozzle, continuous phase flow channel, and outlet, and will not seep into other positions between the dispersed phase flow plate and the microsphere forming plate; the dispersed phase flow plate, microsphere forming plate, and continuous phase flow plate are tightly stacked and assembled. During close-layer stacking, the continuous phase liquid flows unidirectionally in the direction of continuous phase inlet, continuous phase channel, and outlet, and will not seep into other positions between the continuous phase flow sheet and the microsphere forming sheet; a guide nozzle is provided at the end of the continuous phase channel of the continuous phase flow sheet, penetrating the outlet of the continuous phase flow sheet and extending downwards from the flow sheet, so that the continuous phase liquid carrying the dispersed phase micro-droplets can drip down; the outlet at the end of the continuous phase channel is located inside the continuous phase flow sheet or at the outer edge of the continuous phase flow sheet; and / or, it is made of silicon wafer, glass, quartz, polymethyl methacrylate, or polydimethylsiloxane.

5. A method for preparing a microfluidic chip according to any one of claims 1 to 4, wherein the microfluidic chip is made of a glass substrate, comprising the following steps: using glass as a substrate, using photoresist as a patterning protective layer, transferring a preset microchannel pattern to the photoresist layer by photolithography, forming a microchannel structure on the glass substrate by chemical etching, and finally sealing the two glass substrates by bonding to obtain a closed glass microfluidic chip.

6. The method of claim 5, comprising the following steps: (1) Cleaning of glass substrates: Select three circular glass substrates, thoroughly clean the glass substrates with cleaning solution, and then dry them with nitrogen gas; (2) Photoresist spin coating: Take two glass substrates used to prepare dispersed phase flow sheets and continuous phase flow sheets, drop positive photoresist onto the center of the cleaned and dried glass substrates, and use a spin coater to spread the photoresist evenly on the surface of the glass substrates. (3) Pre-baking treatment: The glass substrate with the photoresist film spin-coated is placed on a hot plate for pre-baking treatment to allow the solvent in the photoresist to fully evaporate and the photoresist film to solidify. (4) Ultraviolet exposure: According to the liquid flow channel arrangement of the dispersed phase sheet and the continuous phase sheet, the mask with the preset microchannel pattern is tightly attached to the surface of the photoresist film after pre-baking treatment, and the attached glass substrate is irradiated with ultraviolet light to achieve the initial transfer of the microchannel pattern. (5) Development treatment: The glass substrate exposed to ultraviolet light is placed in the developing solution for development treatment, so that the surface of the glass substrate forms a pattern consistent with the mask. The glass substrate is then cleaned and dried. (6) Hardening and chemical etching: The developed glass substrate is placed on a hot plate again for hardening treatment, and then the glass substrate is placed in hydrofluoric acid etching solution for chemical etching to form a groove structure of dispersed phase flow channel or continuous phase flow channel. The glass substrate is then cleaned. (7) Removal and drilling: The glass substrates of the etched dispersed phase sheet and the continuous phase sheet are immersed in acetone solvent for removal of adhesive, and then cleaned and dried. Then, an ultrasonic drilling machine is used to drill holes at the corresponding positions of the dispersed phase inlet of the dispersed phase sheet and the continuous phase inlet of the continuous phase sheet. Then, a hollow pipe opening protruding from the sheet surface is bonded to the side away from the liquid flow channel by photosensitive resin to form the dispersed phase inlet and the continuous phase inlet, thus obtaining the dispersed phase sheet and the continuous phase sheet. (8) Fabrication of microsphere forming sheet: At the position where the capillary protrusion is set on the glass substrate of the microsphere forming sheet, a capillary with a predetermined aperture is connected by high temperature bonding. Then, a through hole corresponding to the capillary hole is drilled on the corresponding position on the back side of the capillary of the microsphere forming sheet using a computer numerical control milling machine to obtain the microsphere forming sheet. (9) Bonding and encapsulation: The dispersed phase flow sheet, microsphere forming sheet and continuous phase flow sheet are precisely aligned and stacked from top to bottom, placed in a high-temperature furnace and pressure is applied to make the three glass sheets permanently fuse under high temperature and high pressure conditions to achieve bonding and sealing, and obtain a microfluidic chip with a glass substrate having closed microchannels; optionally, the flow outlets of each liquid outlet of the continuous phase flow sheet are bonded by photosensitive resin bonding.

7. The equipment unit for preparing uniform microspheres basically includes: A microfluidic chip as described in any one of claims 1 to 4; Dispersed phase solution storage bottle, used to store the dispersed phase solution during the preparation of microspheres; Continuous phase solution storage bottle, used to store continuous phase solution during microsphere preparation; A constant flow pump is used to introduce dispersed phase solutions and continuous phase solutions into the microfluidic chip; The liquid guide tube is used to connect the dispersed phase solution storage bottle, the constant flow pump, and the dispersed phase inlet of the microfluidic chip, and to connect the continuous phase solution storage bottle, the constant flow pump, and the continuous phase inlet of the microfluidic chip. A liquid receiving tray is disposed below the microfluidic chip and is used to receive liquid flowing out from the outlet at the end of the continuous phase flow channel of the microfluidic chip; A collection bottle, connected to the outlet at the bottom of the receiving tray via a guide tube, is used to collect a continuous phase solution containing tiny droplets of the dispersed phase. Furthermore, this device unit operates essentially as follows when preparing uniform microspheres: (i) The continuous phase inlet is connected to the continuous phase solution storage bottle through a liquid guide tube. During this process, the continuous phase solution is pumped into the continuous phase flow plate of the microfluidic chip by a constant flow pump, and flows through the continuous phase flow channel until it flows out from the outlet of the flow plate. (ii) The dispersed phase inlet is connected to the dispersed phase solution storage bottle through a liquid guide tube. During this process, the dispersed phase solution is pumped into the dispersed phase flow plate of the microfluidic chip through a constant flow pump. Then, it passes through the through holes and capillary protrusions of the microsphere forming plate and enters and disperses into the continuous phase solution in the continuous phase flow channel in the form of tiny droplets. It is then carried away by the continuous phase solution to the outlet of the microfluidic chip. (iii) A continuous phase solution containing tiny droplets of dispersed phase is dripped from the outlet into the receiving tray, and then discharged into the collection bottle through the outlet of the receiving tray. (iv) The dispersed phase microdroplets formed at the capillary nozzle, from the moment they enter the continuous phase solution until they are in the collection bottle, undergo spheroidization and preliminary solidification in the continuous phase solution, and then the liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle. Optional (v) After the liquid obtained in step (iv) has been left to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. Optionally, the solid microspheres are washed and dried (e.g., under reduced pressure) with water to obtain microspheres in the form of fine powder.

8. An apparatus for preparing uniform microspheres to accommodate different production scales, comprising any number of the apparatus units described in claim 7 connected in parallel; all components of all apparatus units are independent and operate in parallel, or several apparatus units form a group and each group operates independently and in parallel; and / or, wherein 2 to 100 apparatus units of dispersed phase solution storage bottles, continuous phase solution storage bottles, and collection bottles are shared to form a group, forming several groups that operate independently and in parallel.

9. A method for preparing microspheres, wherein the preparation is carried out using the equipment unit of claim 7 or the equipment of claim 8, the method comprising first preparing a continuous phase solution and a dispersed phase solution respectively, and then operating according to the following steps: (a) Add the continuous phase solution and the dispersed phase solution to the continuous phase solution storage bottle and the dispersed phase solution storage bottle respectively, start the constant flow pump, and adjust and control the flow rate of the two pumps for the continuous phase solution and the dispersed phase solution by monitoring the microsphere particle size in the suspension collected by the receiving tray, so that the microsphere particle size reaches the desired value or range. (b) The process from the moment the dispersed phase microdroplets formed at the capillary nozzle enter the continuous phase solution until they are in the collection bottle, wherein the dispersed phase microdroplets undergo spheroidization and preliminary solidification in the continuous phase solution, and then the liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle. (c) After the liquid obtained in step (b) has been left to stand for an appropriate time, the upper layer of liquid is poured out. The remaining liquid microsphere suspension at the bottom of the bottle is solidified by removing the solvent under reduced pressure to obtain solid microspheres. The solid microspheres are then rinsed with water and dried to obtain microspheres in the form of fine powder.

10. The method of claim 9, wherein the device unit or device operates substantially according to the following steps: (i) The continuous phase inlet is connected to the continuous phase solution storage bottle through a liquid guide tube. During this process, the continuous phase solution is pumped into the continuous phase flow plate of the microfluidic chip by a constant flow pump, and flows through the continuous phase flow channel until it flows out from the outlet of the flow plate. (ii) The dispersed phase inlet is connected to the dispersed phase solution storage bottle through a liquid guide tube. During this process, the dispersed phase solution is pumped into the dispersed phase flow plate of the microfluidic chip through a constant flow pump. Then, it passes through the through holes and capillary protrusions of the microsphere forming plate and enters and disperses into the continuous phase solution in the continuous phase flow channel in the form of tiny droplets. It is then carried away by the continuous phase solution to the outlet of the microfluidic chip. (iii) A continuous phase solution containing tiny droplets of dispersed phase is dripped from the outlet into the receiving tray, and then discharged into the collection bottle through the outlet of the receiving tray. (iv) The dispersed phase microdroplets formed at the capillary nozzle undergo spheroidization and preliminary solidification in the continuous phase solution, and then the liquid microspheres are deposited in a loose state at the bottom of the continuous phase solution in the collection bottle.

Images