Preparation method of non-spherical calcium alginate hydrogel particles based on microemulsion

Abstract

The invention relates to a preparation method of non-spherical calcium alginate hydrogel particles based on microemulsion. According to the method, ETPTA is used as an oily dispersion phase of a proportioning pre-gel phase, liquid paraffin is used as an external oily continuous phase, and a water-in-oil (W / O) emulsion of water-based micro-droplets containing calcium ions is used as a reaction phase; a three-phase system of ETPTA-sodium alginate aqueous solution / liquid paraffin (O / W / O) is adopted, and the ETPTA-sodium alginate Janus-shaped micro-droplets with controllable morphology and size are stably prepared by adjusting the three-phase flow in a micro-channel of a double-hole focused flow capillary tube. A micro-emulsion formed by mixing a calcium chloride aqueous solution containing a surfactant and liquid paraffin is used in a lower flow path of the chip to cure a sodium alginate phase in situ. According to the method, the problem of biotoxicity caused by the fact that ultraviolet light or an acid solution must be introduced in a traditional non-spherical hydrogel microsphere preparation method based on Janus liquid drops is effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of materials synthesis technology, specifically relating to a method for preparing non-spherical calcium alginate hydrogel microparticles based on microemulsions. Background Technology

[0002] Hydrogels, as green materials with excellent biocompatibility and biodegradability, have wide applications in biology, chemistry, and medicine. In recent years, microfluidics, thanks to its precise control over micron-sized fluids, has become an important method for preparing hydrogel microparticles with good monodispersity and controllable morphology. Furthermore, hydrogel microparticles prepared using microfluidics have been widely applied in cell encapsulation, targeted therapy, tumor treatment, drug sustained release, and biomedical diagnostics. Compared to traditional spherical hydrogels, non-spherical hydrogels possess advantages such as morphological anisotropy, better mass transfer surface area, and better tissue and cell adhesion. Studies have shown that the morphology of microparticles has a crucial impact on the release kinetics and in vivo circulation mechanisms of the encapsulated drugs. Therefore, researching an efficient method for preparing non-spherical hydrogel microparticles with excellent monodispersity and controllable size and morphology is of great significance.

[0003] Traditional methods for preparing non-spherical hydrogel microparticles (such as photolithography, electrospraying, and emulsion crosslinking) are simple to operate and widely used, but they suffer from inherent limitations such as poor controllability, low preparation efficiency, and low product monodispersity. Microfluidic technology, with its advantages of high throughput, high stability, and good controllability, offers a new solution to these challenges in preparing non-spherical hydrogel microparticles. Currently, microfluidic preparation strategies for non-spherical hydrogel microparticles mainly include hydrolithography and droplet microfluidics. Hydrolithography, due to its reliance on precise mask fabrication and the excessively long exposure polymerization process, severely limits its production efficiency, making it difficult to meet the demands of large-scale preparation. In contrast, droplet microfluidics, with its stable dispersion process and ease of achieving high-throughput production through parallel scale-up, exhibits a more significant overall advantage in terms of preparation efficiency and controllability.

[0004] Droplet microfluidics, with its advantages of stability, efficiency, and simple control, has gradually become an important method for preparing non-spherical hydrogel microparticles. However, current methods for preparing non-spherical hydrogel microparticles mainly rely on photopolymerization curing or ion crosslinking curing with calcium ion chelates and acidic solutions. Photopolymerization curing requires ultraviolet light and cytotoxic photoinitiators, which significantly weaken the biocompatibility of the hydrogel material. Secondly, photopolymerization curing mostly uses synthetic materials, which often have higher production costs compared to natural hydrogel materials. On the other hand, ion crosslinking curing strategies based on natural polysaccharides such as alginate and pectin offer an alternative approach to address the biotoxicity issues of ultraviolet light and photoinitiators. However, ion crosslinking curing strategies for hydrogel microparticles based on droplet microfluidics generally use acidic solutions incorporating calcium ion chelates as the reaction phase. This strategy, due to the introduction of acidic solutions, also significantly weakens the biocompatibility of the prepared system, thus limiting its application. Without acidic solutions, solidified microspheres cannot be formed, and hydrogel microparticles cannot be successfully prepared.

[0005] In summary, existing techniques for preparing non-spherical hydrogels based on Janus droplets, if ionic crosslinking curing is used, rely on acidic solutions to trigger the release of calcium ions from calcium ion chelates (such as disodium calcium ethylenediaminetetraacetate) (e.g., CN118122231A). The acidic environment can damage the stability of bioactive substances (such as cells and protein drugs). If photopolymerization curing is used, ultraviolet light and photoinitiators can lead to biotoxicity, and the synthetic materials are expensive. Furthermore, existing microemulsion reaction phases often use a corn oil-calcium chloride system (see Microfluidic generation of monodispersed Janusalginate hydrogel microparticles using water-in-oil emulsion reactant, doi:10.1063 / 5.0077916), but this is not suitable for Janus droplet generation in the ETPTA-sodium alginate-liquid paraffin three-phase system, making it difficult to prepare non-spherical structures. Therefore, developing a microemulsion reaction phase preparation method that does not require ultraviolet light or acidic solutions and is compatible with three-phase systems is key to solving the problems of biocompatibility and morphology controllability of non-spherical calcium alginate hydrogel particles. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a method for preparing non-spherical calcium alginate hydrogel microparticles based on microemulsions, thereby reducing the biotoxicity issues of existing preparation methods, improving the preparation efficiency and biocompatibility of the product, and enabling the product to be used as a drug or cell microcarrier for biological injection therapy.

[0007] This invention discloses an apparatus and method for the continuous preparation of non-spherical calcium alginate hydrogel microparticles based on a microemulsion reaction phase. A two-stage microfluidic chip is designed, using a mixed microemulsion of calcium chloride aqueous solution and liquid paraffin as the reaction phase. In the microchannel, the in-situ synthesis of non-spherical calcium alginate hydrogel microparticles under acid-free and UV-free conditions is realized.

[0008] The core concept of this invention is as follows: 1. To address the step-by-step requirements of Janus droplet generation and solidification, a two-stage cross-focusing chip structure is designed—the upper flow path utilizes the interfacial tension balance of a three-phase system (dispersed phase I / II + continuous phase III), and by adjusting the flow ratio and surfactant concentration, morphology-controllable Janus droplet generation is achieved; the lower flow path introduces a microemulsion reaction phase VI (liquid paraffin-calcium chloride), utilizing the highly dispersed calcium ions in the microemulsion to achieve in-situ rapid solidification of Janus droplets, avoiding the uneven solidification caused by traditional blocky reaction phases; 2. To address biotoxicity issues, ultraviolet light and acidic solutions are abandoned, and a mechanism of direct cross-linking of sodium alginate with calcium ions is adopted. Simultaneously, highly biocompatible liquid paraffin and Span 80 / FS-30 surfactants are selected to ensure the product's applicability in biomedical applications; 3. To address system compatibility issues, the surfactant concentrations (Span 80 0.2~0.4wt%, FS-30) are screened. Compared to the reaction phase (10-30wt%), the microemulsion reaction phase VI (0-1wt%) makes it compatible with the three-phase system, avoiding droplet aggregation and morphological damage.

[0009] Specifically, the technical solution adopted in this invention includes the following steps: A method for preparing non-spherical calcium alginate hydrogel microparticles based on microemulsions, comprising: (1) Dissolve sodium alginate in water and use it as pregel dispersion phase I. Use ethoxylated trimethylolpropane triacrylate as oily dispersion phase II. Dissolve sorbitan oleate in liquid paraffin as continuous phase III and reaction phase oil phase IV. The reaction phase aqueous phase V is an aqueous solution of calcium chloride. Mix reaction phase oil phase IV and reaction phase aqueous phase V evenly, wherein the mass fraction of reaction phase aqueous phase V is 10~30%, to obtain a W / O emulsion containing calcium ions in aqueous microdroplets as reaction phase VI; wherein the concentration of sorbitan oleate in continuous phase III and reaction phase oil phase IV is 0.2~0.4wt%. (2) The pregelated dispersion phase I, the oily dispersion phase II, the continuous phase III, and the reactive phase VI are respectively introduced into a microfluidic chip with a composite microchannel using an injection pump. The chip has two cross-focusing structures. From the inlet to the outlet of the dispersion phase, the first cross-focusing structure is called the upper flow path, which is used for the generation of Janus-type droplets. The second cross-focusing structure is called the lower flow path, which is used for the generated Janus droplets to contact the reactive phase and solidify to form hydrogel microparticles. The pregelated dispersion phase I, the oily dispersion phase II, and the continuous phase III enter the upper flow path in a perpendicular flow direction. The generated Janus-type droplets and the reactive phase VI flow through the lower flow path in a perpendicular flow direction, and an ionic cross-linking and solidification reaction occurs. The flow rate of the reactive phase VI is controlled to be 20 ~ 40 mL / h to form non-spherical hydrogel microspheres. (3) Collect the reaction products flowing out of the microchannel of the microfluidic chip after the reaction, separate and purify them by filtration, filter out the oily dispersed phase II, continuous phase III and reaction phase VI, and wash the hydrogel particles thoroughly with n-hexane and pure water to obtain non-spherical calcium alginate hydrogel particles.

[0010] Furthermore, the pregel dispersion phase I mentioned in step (1) also contains a water-soluble surfactant, which is fluorinated polyoxyethylene ether FS-30, with a concentration of 0~1 wt%. When the concentration of FS-30 is 0.05~1 wt%, it forms non-spherical hydrogel particles with a single concave structure; when the concentration of FS-30 is 0, it forms non-spherical hydrogel particles with a biconvex elliptical structure.

[0011] Furthermore, the mass content of calcium chloride in the aqueous phase V of the reaction phase in step (1) is 20 wt%.

[0012] Furthermore, the mass ratio of aqueous phase V in reaction phase VI in step (1) is 20-30 wt%.

[0013] Furthermore, the microfluidic chip mentioned in step (2) is at least one of quartz glass chip, PDMS chip, glass capillary, and acrylic composite chip; the inner diameter of the droplet generator in the microchannel is 70μm to 160μm.

[0014] Furthermore, the flow rate of the pregel dispersion I in step (2) is controlled at 0.125 ~ 0.500 mL / h.

[0015] Furthermore, the flow rate of the oily dispersed phase II in step (2) is controlled at 0.125 ~ 0.500 mL / h.

[0016] Furthermore, the flow rate of continuous phase III in step (2) is controlled at 2 to 9 mL / h.

[0017] Furthermore, in step (2), the flow ratio of the pregelated dispersion phase I to the oily dispersion phase II is between 1.00 and 3.00.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: 1. The microfluidic preparation method for non-spherical calcium alginate hydrogel microparticles based on microemulsion proposed in this invention can precisely control the size, proportion, and morphology of pre-gelled Janus microdroplets, and can generate non-spherical calcium alginate hydrogel microparticles with controllable size and height, including biconvex and single concave surfaces.

[0019] 2. The structure of the Janus microdroplets described in this invention can be adjusted by the type and concentration of water-soluble and oil-soluble surfactants in the liquid phase without damaging the structure of the hydrogel particles. The structure and size of the Janus microdroplets described in this invention can be achieved by adjusting the volume ratio of the two dispersed phases, thereby obtaining Janus microdroplets with specific morphology and size.

[0020] 3. The hydrogel microparticles prepared by the method of the present invention have stable quality, uniform morphology, high monodispersity, and high biocompatibility.

[0021] 4. This invention is based on ion crosslinking curing of microemulsions, which effectively avoids the biotoxicity problems caused by the use of acidic reaction phases or ultraviolet light in existing microfluidic curing reaction methods. It does not involve organic reagents with biotoxicity and has the advantages of being green, environmentally friendly and having excellent biocompatibility.

[0022] 5. The preparation method of the present invention has certain versatility and can realize the continuous preparation of non-spherical hydrogel particles based on ion crosslinking reaction curing, except for calcium alginate materials, by following the ion crosslinking curing method designed according to the concept of the present invention.

[0023] 6. This invention is based on droplet microfluidics technology, which can easily achieve large-scale synthesis of microparticles through multiple parallel microchannels, thus making it suitable for industrial production.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0026] Figure 1a This is a schematic diagram of a microfluidic chip with a two-stage microchannel.

[0027] Figure 1b This is a schematic diagram of the channel dimensions of a microfluidic chip.

[0028] Figure 2 This is a schematic diagram of the feeding reaction of the microfluidic chip of the present invention.

[0029] Figure 3 These are calcium alginate hydrogel particles with a single concave structure prepared in Example 2 of this invention. The scale bar in the figure is 100 micrometers in length.

[0030] Figure 4a This is a phase diagram of the fine emulsion state formed by high-speed mixing of liquid paraffin and calcium chloride aqueous solution at different phase volume ratios and surfactant (Span 80) concentrations used in this invention.

[0031] Figure 4b This is a photograph of the fine emulsion reaction phase formed by high-speed mixing of liquid paraffin and calcium chloride aqueous solution in different proportions used in this invention.

[0032] Figure 4c This invention investigates the effect of the surfactant (Span 80) concentration in the liquid paraffin and calcium chloride aqueous solution on the size and monodispersity of the calcium chloride aqueous solution droplets in the resulting fine emulsion. The phase volume ratio of the calcium chloride aqueous solution is 30 wt%.

[0033] Figure 4d This invention illustrates the effect of the volume ratio of liquid paraffin to calcium chloride aqueous solution in the mixture on the size and monodispersity of the calcium chloride aqueous solution droplets in the resulting fine emulsion. The concentration of Span 80 was 0.2 wt%.

[0034] Figure 5 This diagram illustrates the actual flow process of the microemulsion reaction phase used in this invention within the flow path channel of the microfluidic chip shown in Figure 1 under different flow rate conditions. The letters represent the following meanings: Qw represents the aqueous dispersion phase (sodium alginate aqueous solution); Qo represents the oil dispersion phase (ETPTA); Qc represents the continuous phase (liquid paraffin); and Qe represents the emulsion reaction phase (a microemulsion formed by high-speed mixing of liquid paraffin and calcium chloride aqueous solution). Figure 6a This is the preparation process of Janus-shaped microdroplets with controllable morphology and size under a high-speed camera in Embodiment 2 of the present invention. The sodium alginate part has a single concave structure, and the time required for the generation of a single droplet is about 230 milliseconds. The scale bar length in the figure is 250 micrometers.

[0035] Figure 6b The image shows Janus-shaped ETPTA-sodium alginate microdroplets dispersed in a liquid paraffin solution, wherein the sodium alginate portion has a single concave structure. The scale bar in the image is 250 micrometers long.

[0036] Figure 6c for Figure 6b The diameter distribution of the Janus-shaped microdroplets prepared in the study is statistically shown. The characteristic diameter of the EPTA phase in the Janus droplets is 180.2 μm with a coefficient of variation of 2.4%, while the characteristic diameter of the sodium alginate phase is 170.3 μm with a coefficient of variation of 2.5%.

[0037] Figure 7 This is a biocompatibility test of the calcium alginate hydrogel microparticles with a single concave structure prepared in Example 2 of the present invention.

[0038] Figure 8 This study tested the adhesion properties of the calcium alginate hydrogel microparticles with a single concave structure prepared in Example 2 of the present invention to the mouse intestine.

[0039] Figure 9 The figure shows the test results provided for Comparative Example 2 of this invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] This invention provides a method for preparing non-spherical calcium alginate hydrogel particles using a microfluidic chip with a two-segment microchannel structure. The two-segment microchannel structure in the embodiment is as follows: Figure 1a , Figure 1b As shown, the chip has two cross-focusing structures from the dispersed phase inlet to the outlet. The first cross-focusing structure, called the upper flow path, is used for the generation of Janus-type droplets, and the second cross-focusing structure, called the lower flow path, is used for the generated Janus droplets to contact the reaction phase and solidify to form hydrogel microparticles.

[0042] The manufacturing method is as follows: First, a groove with a width and depth between 1.0 and 1.7 mm is machined on the acrylic substrate using a CNC machine tool. Specific dimensions are as follows: Figure 1bAs shown, the acrylic substrate is 3 mm thick. After processing, the recessed acrylic substrate is joined to another acrylic substrate of the same size using a thermoforming machine, sealing the recesses to form microchannels. Glass capillaries are drawn to a point using a capillary puller (P-97; Sutter Instruments Co., USA), polished, and then embedded into the microchannels; the specific dimensions are shown in Figure 1. The interface between the glass capillary and the acrylic substrate is sealed and fixed with epoxy resin.

[0043] Based on the equilibrium relationship of emulsions in a multiphase system within a microchannel, this invention prepares structurally controllable multiphase Janus-type microdroplets in an upper flow path microchannel, and then performs an ionic crosslinking and curing reaction with a fine emulsion reaction phase formed by liquid paraffin and calcium chloride aqueous solution in a lower flow path microchannel, thereby in-situ curing the Janus-type microdroplets. Figure 2 This method enables the preparation and control of non-spherical calcium alginate hydrogel microparticles. The upper flow path microchannel refers to the first cross-shaped focusing channel from the inlet direction, and the lower flow path microchannel refers to the second cross-shaped focusing channel.

[0044] This invention utilizes a three-phase system with sodium alginate aqueous solution / ethoxylated trimethylolpropane triacrylate (ETPTA) as the dispersed phase and liquid paraffin as the continuous phase to prepare monodisperse and uniformly morphologically uniform Janus-shaped microdroplets primarily composed of sodium alginate / ETPTA. By adjusting the proportions of each phase and the concentrations of the oil-soluble surfactant sorbitan oleate (Span 80) and the water-soluble surfactant fluorinated polyoxyethylene ether (FS-30), a multi-structured microemulsion is stably generated within microchannels. Specifically, controlling the concentration of FS-30 to be greater than 0.05 wt% and the concentration of Span 80 to be greater than 0.08 wt% results in the formation of calcium alginate hydrogel microparticles with a single concave structure; controlling the FS-30 concentration to 0 and only adjusting the Span 80 concentration to be greater than 0.08 wt% results in calcium alginate hydrogel microparticles with a biconvex ellipsoidal structure; and controlling the flow ratio of the dispersed and continuous phases can alter the size of the calcium alginate hydrogel microparticles.

[0045] This invention utilizes a fine emulsion formed by high-speed mixing of liquid paraffin and calcium chloride aqueous solution as the reaction phase to prepare non-spherical calcium alginate hydrogel microparticles by in-situ ionic crosslinking and solidification of Janus-shaped droplets within microchannels. The reaction process of the pre-gelled Janus-shaped microdroplets within the microchannels is controlled by adjusting the physical properties of the reaction phase.

[0046] Example 1 (1) Dissolve 0.75 g of sodium alginate in water to prepare a 50 mL solution as the pregel dispersion phase I; use ethoxylated trimethylolpropane triacrylate as the oily dispersion phase II; dissolve 0.2 g of sorbitan oleate in liquid paraffin to prepare a 100 g solution as the continuous phase III; dissolve 0.2 g of sorbitan oleate in liquid paraffin to prepare a 100 g solution as the reaction phase oil phase IV; dissolve 20 g of calcium chloride in water to prepare a 100 g solution as the reaction phase aqueous phase V; mix reaction phase oil phase IV and reaction phase aqueous phase V at a mass ratio of 7:3, and stir at 30,000 rpm for 5 minutes using a high-speed homogenizer to prepare a 200 g fine emulsion as the reaction phase VI.

[0047] (2) Load the prepared dispersed phase I, dispersed phase II, and continuous phase III into syringes and place them on a microinjection pump. Connect the injection pump to the microfluidic chip using a PTFE tubing to input the target solution into the microfluidic chip, such as... Figure 3 As shown, with the flow rates of dispersed phases I and II both at 0.25 mL / h and continuous phase III at 6 mL / h, monodisperse Janus microdroplets with characteristic diameters between 170-180 mm can be obtained. Furthermore, by adjusting the concentration of dehydrated sorbitan oleate and its relationship to the flow rates of phases I to III, Janus microdroplets with different sizes and structures can be obtained.

[0048] (3) Inject reaction phase VI into the lower flow path of the microfluidic chip at a flow rate of 40 mL / h to trigger the ionic crosslinking and solidification reaction of Janus microdroplets generated in the upper flow path.

[0049] (4) Collect the reaction products flowing out of the microchannel after the reaction, separate and purify them by filtration, filter the oily dispersed phase, continuous phase and reaction phase, and wash the hydrogel particles thoroughly with n-hexane and pure water to obtain calcium alginate hydrogel particles with a biconvex ellipsoidal structure.

[0050] Example 2 (1) Dissolve 0.75 g sodium alginate and 0.125 g fluorinated polyoxyethylene ether (FS-30) in water to prepare a 50 mL solution as the pregel dispersion phase I; take ethoxylated trimethylolpropane triacrylate as the oily dispersion phase II; dissolve 0.2 g sorbitan oleate in liquid paraffin to prepare a 100 g solution as the continuous phase III; dissolve 0.2 g sorbitan oleate in liquid paraffin to prepare a 100 g solution as the reaction phase oil phase IV; dissolve 20 g calcium chloride in water to prepare a 100 g solution as the reaction phase aqueous phase V; mix reaction phase oil phase IV and reaction phase aqueous phase V at a mass ratio of 7:3, and stir at 30,000 rpm for 5 minutes using a high-speed homogenizer to prepare a 200 g fine emulsion as the reaction phase VI.

[0051] (2) The prepared solutions were loaded into syringes and placed on microinjection pumps. The syringe pumps were connected to the microfluidic chip using PTFE tubing. The target solutions were then fed into the microfluidic chip. The flow rates of dispersed phase I and dispersed phase II were both 0.25 mL / h, and the flow rate of continuous phase III was 6 mL / h. Monodisperse Janus microdroplets with characteristic diameters between 170-180 mm could be obtained. Furthermore, by adjusting the concentration of dehydrated sorbitan oleate and its relationship with the flow rates of phases I to III, Janus microdroplets with different sizes and structures could be obtained.

[0052] (3) Inject reaction phase VI into the lower flow path of the microfluidic chip at a flow rate of 40 mL / h to trigger the ionic crosslinking and solidification reaction of Janus microdroplets generated in the upper flow path.

[0053] (4) Collect the reaction products flowing out of the microchannel after the reaction, separate and purify them by filtration, filter the oily dispersed phase, continuous phase and reaction phase, and wash the hydrogel particles thoroughly with n-hexane and pure water to obtain calcium alginate hydrogel particles with a single concave structure.

[0054] Example 3: Surfactant Concentration Exploration Experiment (1) First, prepare a 20% calcium chloride aqueous solution as the aqueous phase V of the reaction phase; mix different masses of Span 80 with liquid paraffin to prepare solutions with mass fractions of 0, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, 0.3%, 0.4%, and 0.5% as the oil phase IV of the reaction phase; mix the aqueous phase V and the oil phase IV of the reaction phase in different mass ratios to prepare a mixed solution with a total mass of 40g. Stir the prepared mixed solution for 5 minutes at 30,000 rpm using a high-speed homogenizer, observe the condition of the prepared emulsion, and draw a phase diagram (see [link]). Figure 4a ).

[0055] (2) Use a camera to observe the state of the prepared microemulsion, such as... Figure 4b As shown. Where (i) the mass fraction of the aqueous phase V in the emulsion is 30% and the Span 80 concentration is 0; (ii) the mass fraction of the aqueous phase V in the emulsion is 30% and the mass fraction of Span 80 in the oil phase IV in the emulsion is 0.2%; (iii) the mass fraction of the aqueous phase V in the emulsion is 10% and the mass fraction of Span 80 in the oil phase IV in the emulsion is 0.2%; (iv) the mass fraction of the aqueous phase V in the emulsion is 40% and the mass fraction of Span 80 in the oil phase IV in the emulsion is 0.2%.

[0056] from Figure 4a , 4b It can be seen that different concentrations of Span 80 result in different emulsion states. Without the addition of Span 80, the emulsion is clear; with the addition of Span 80, a stable emulsion can only be obtained when the mass fraction of Span 80 is 0.2-0.5% and the mass fraction of the aqueous phase V in the reaction phase is 10-30%.

[0057] (3) Take 200 mL of the prepared reaction phase emulsion VI and disperse it in 1 mL of liquid paraffin. Use a microscope to observe the size and dispersion of the calcium chloride aqueous solution droplets in the emulsion. Calculate the average diameter and coefficient of variation of the droplets. Figure 4c and Figure 4d As shown.

[0058] from Figure 4c , Figure 4d It can be seen that the average droplet diameter and coefficient of variation in the prepared reactive emulsion VI decrease with increasing Span concentration in the reactive phase (e.g., Figure 4c As shown); the average droplet diameter and coefficient of variation in the prepared reactive emulsion VI increased with the increase of the proportion of the aqueous reactive phase V (e.g. Figure 4d (As shown).

[0059] Example 4: Flow Exploration Experiment (1) The preparation process of calcium alginate hydrogel microparticles was carried out according to Example 2. In step (3), the flow rate of reaction phase VI was adjusted to 15 mL / h, 30 mL / h, and 45 mL / h. The flow conditions of the flow path under the microfluidic chip were observed by taking pictures using a high-speed camera.

[0060] like Figure 5As shown, the dense reactive phase VI emulsion appears black under a microscope. It forms a stable laminar turbulent flow path in the microfluidic chip, with a clear boundary between it and the continuous phase III liquid paraffin. Furthermore, as the flow rate of reactive phase VI increases, the width of the intermediate paraffin phase is gradually compressed, and the contact area between the microdroplets generated in the downstream flow path and reactive phase VI gradually increases.

[0061] from Figure 5 It can be seen that when the flow rate of reaction phase VI is too low (ca. 15 mL / h), the generated droplets do not come into complete contact with the reaction phase, and the reaction is insufficient. Therefore, the droplets are prone to coalescence, generating large-sized and non-uniform calcium alginate microparticles. When the flow rate of reaction phase VI is too high (ca. 45 mL / h), a large shear force will be generated on the generated droplets in the downstream flow path. Under the shear force, the droplets are prone to deformation or have difficulty maintaining the Janus morphology. The generated hydrogel microparticles cannot maintain a perfect hemispherical morphology.

[0062] Therefore, controlling the flow rate of reaction phase VI to 20-40 mL / h is necessary to obtain hydrogel microparticles with a better hemispherical morphology.

[0063] Example 5 (1) Dissolve 0.75 g sodium alginate in water to prepare a 50 mL solution as the pregel dispersion phase I; use ethoxylated trimethylolpropane triacrylate as the oily dispersion phase II; dissolve 0.2 g sorbitan oleate in liquid paraffin to prepare a 100 g solution as the continuous phase III.

[0064] (2) The prepared dispersed phase I, dispersed phase II, and continuous phase III were respectively loaded into syringes and placed on a micro-injection pump. The injection pump was connected to the microfluidic chip using a PTFE tube. The target solution was then introduced into the microfluidic chip, with the flow rates of dispersed phase I and dispersed phase II both being 0.25 mL / h, and the flow rate of continuous phase III being 6 mL / h. The formation process of Janus-type droplets in the flow path of the microfluidic chip was observed using a high-speed camera and a microscope. Figure 6a As shown, the time required to generate a single Janus-type droplet is approximately 230 ms.

[0065] (3) Collect the product flowing out of the microchannel and observe it under a microscope, then count the characteristic diameter of the generated Janus-type droplets. For example... Figure 6b and Figure 6c As shown, the generated EPTA-sodium alginate microdroplets exhibit a monodisperse Janus-type structure, with the sodium alginate phase having a characteristic diameter of approximately 170 micrometers and the EPTA phase having a characteristic diameter of approximately 180 micrometers.

[0066] Example 6 (1) The calcium alginate hydrogel microparticles collected in Example 2 were co-cultured with mouse fibroblast embryonic cells (NIH3T3) in a 24-well cell culture plate. Meanwhile, mouse fibroblast embryonic cells (NIH3T3) were cultured separately in another 24-well cell culture plate as a control group.

[0067] (2) Carefully aspirate the old culture medium from the culture plate, add preheated PBS solution (approximately 2-3 times the volume of the original culture medium), and gently shake to wash the microspheres. After the microspheres have settled, carefully aspirate the PBS. Repeat this process 3 times to remove any culture medium components and metabolic waste that may interfere with staining. Perform the same procedure on the control group.

[0068] (3) Culture for 1-3 days and stain to observe cell growth.

[0069] (4) The safe concentration and biocompatibility of the hydrogel microspheres were verified, and the results are shown in [the table below]. Figure 7 The results showed that hydrogel microspheres at concentrations of 0-100 mg / mL had no significant toxic effect on mouse fibroblast embryonic cells (NIH3T3). Furthermore, live / dead staining results indicated that, compared to the control group, it had no significant effect on the generation of either cell type within 1-3 days. Figure 7 ).

[0070] Example 7 (1) Calcium alginate hydrogel microparticles were prepared according to Example 2. Specifically, 0.2 g of CY5.5 monomer was dissolved in 100 mL of dimethyl sulfoxide to prepare a 2 mg / mL stock solution; 0.75 g of sodium alginate, 0.125 g of fluorinated polyoxyethylene ether (FS-30) and 0.05 mL of the prepared CY5.5 stock solution were dissolved in water to prepare a 50 mL solution as the pregel dispersion phase I; the remaining steps were completely consistent with those in Example 2 to obtain CY5.5 labeled non-spherical hydrogel microparticles.

[0071] Calcium alginate hydrogel microparticles were prepared according to Example 2. Specifically, 0.2 g of CY5.5 monomer was dissolved in 100 mL of dimethyl sulfoxide to prepare a 2 mg / mL stock solution. 0.75 g of sodium alginate, 0.125 g of fluorinated polyoxyethylene ether (FS-30), and 0.05 mL of the prepared CY5.5 stock solution were dissolved in water to prepare a 50 mL solution, which was used as pre-gel dispersion phase I. Dispersion phase II was replaced with dispersion phase I and injected into the microchannels together with the original dispersion phase. The remaining steps were exactly the same as in Example 2, resulting in CY5.5-labeled spherical hydrogel microparticles.

[0072] The prepared CY5.5-labeled sucker-shaped and spherical hydrogel particles were resuspended in 1 mL of physiological saline and vortexed to prepare two homogeneous suspensions suitable for enema. A 2 mg / mL free dye solution prepared with physiological saline was used as a control.

[0073] (2) Mice were fasted (with free access to water) for 16 hours prior to the experiment to reduce interference from gastric contents and feces on fluorescence signals and the effect on the gavage procedure. Mice were randomly divided into three groups: one group received an enema of CY5.5-labeled hydrogel microsphere dispersion, one group received an enema of CY5.5-labeled suction cup-shaped hydrogel microsphere dispersion, and one group received an enema of free CY5.5 dye solution. Mice were immobilized, their tails were lifted to expose the anus, and the soft, thin catheter connected to the syringe was lubricated and gently inserted approximately 3 cm into the colon. 200 mL of sample was slowly injected per mouse.

[0074] (3) Thoroughly shave the fur off the mouse's abdomen using an electric shaver, taking care to avoid scratching the skin. Place the mouse in the induction box and anesthetize it with isoflurane. Once fully anesthetized, move it to the nasal cone of the imaging platform and maintain anesthesia. Fix the mouse in a supine position on the imaging plate, ensuring the abdomen is fully extended and facing the lens. Select an excitation filter wavelength of 640 nm and an emission filter wavelength of 700 nm. Acquire images at the following key time points: immediately after drug administration (0 hours); 6 hours, 12 hours; and 24 hours.

[0075] (4) Twenty-four hours after administration, mice were euthanized by cervical dislocation or other humane methods. The mice were quickly dissected, and the intact stomach, small intestine, cecum, and colon were removed. These organs were then laid flat in a culture dish, and fluorescence imaging was performed again to accurately locate the distribution of the microspheres. A comparison of the adhesion of hydrogel microspheres with different morphologies in the mouse intestine was obtained, as shown in the attached figure. Figure 8 As shown in the fluorescence images taken 6, 12, and 24 hours after enema, the mouse intestinal tissue injected with non-spherical hydrogel microparticles exhibited the highest fluorescence intensity and area. The results indicate that, compared to spherical hydrogel microparticles, non-spherical hydrogel microparticles can remain in intestinal tissue for a longer period.

[0076] Comparative Example 1 The preparation of calcium alginate hydrogel microparticles was carried out according to CN118122231A, as follows: (1) Dissolve 0.75g sodium alginate, 2.80g disodium calcium ethylenediaminetetraacetate, and 0-0.5g fluorinated polyoxyethylene ether (FS-30) in water to prepare a 50mL solution as the pregel dispersion phase I; use ethoxylated trimethylolpropane triacrylate as the oily dispersion phase II; dissolve 0-1g dehydrated sorbitan oleate in liquid paraffin to prepare a 100g solution as the continuous phase III; mix soybean oil and liquid paraffin at a mass ratio of 2:97 to prepare a 200g solution as the reaction phase IV.

[0077] (2) The prepared solutions were loaded into syringes and placed on microinjection pumps. The syringe pumps were connected to the microfluidic chip using PTFE tubing. The target solutions were then fed into the microfluidic chip, with the flow rates of dispersed phase I and dispersed phase II ranging from 5 to 30 mL / h, and the flow rate ratio of dispersed phase I to dispersed phase II ranging from 0.25 to 4.00. It was found that the pregelated dispersed phase I did not react with the reaction phase in the downstream flow path, but maintained its droplet shape until it flowed into the collection dish.

[0078] This is because disodium calcium ethylenediaminetetraacetate (EDTA) was added to pregel dispersion phase I. Under acidic conditions, disodium calcium ethylenediaminetetraacetate decomposes into EDTA ions, sodium ions, and calcium ions. Therefore, only by adding acetic acid to the reaction phase in the downstream flow path to create acidic conditions will the disodium calcium ethylenediaminetetraacetate in pregel dispersion phase I release calcium ions and undergo an ionic cross-linking reaction with the sodium alginate molecules in pregel dispersion phase I to generate a calcium alginate hydrogel network, thus preparing calcium alginate hydrogel microparticles. In Comparative Example 1, reaction phase IV did not contain acetic acid, and there were no acidic conditions or divalent metal ions in the reaction phase in the downstream flow path. Therefore, pregel dispersion phase I will not react with the reaction phase in the downstream flow path.

[0079] Comparative Example 1 shows that traditional ionic crosslinking requires an acidic solution to trigger calcium release and cannot be cured under acid-free conditions.

[0080] Comparative Example 2 The preparation process of calcium alginate hydrogel microparticles in Example 2 is the same as that in step (1), the reaction phase VI is replaced by: a. the mass fraction of aqueous phase V in the emulsion is 30%, and the mass fraction of Span 80 in oil phase IV is 0.5%; b. the mass fraction of aqueous phase V in the emulsion is 30%, and the mass fraction of Span 80 in oil phase IV is 0.2%; c. the mass fraction of aqueous phase V in the emulsion is 32%, and the mass fraction of Span 80 in oil phase IV is 0.2%.

[0081] The prepared calcium alginate hydrogel particles are as follows Figure 9As shown in the figure, when the concentration of Span 80 is too high (0.5wt%), the interfacial tension of the reactive phase VI is too low, causing Janus droplets to fuse when they come into contact with the microemulsion in the downstream flow path, easily generating large hydrogel particles. The overall hydrogel particles are not uniform in size and have poor monodispersity. When the proportion of the aqueous phase V in the reactive phase is too high (32wt%), the calcium ion concentration in the microemulsion is too high, causing sodium alginate to solidify rapidly, and the generated hydrogel particles cannot maintain a perfect hemispherical morphology.

[0082] Comparative Example 2 shows that the concentration of surfactant Span 80 must be matched with the reaction phase to achieve morphology control.

Claims

1. A process for the preparation of non-spherical calcium alginate hydrogel microparticles based on microemulsions, characterized in that, The method comprises the following steps: (1) dissolving sodium alginate in water to obtain a pre-gel dispersed phase I, using ethoxylated trimethylolpropane triacrylate as an oily dispersed phase II, dissolving sorbitan oleate in liquid paraffin to obtain a continuous phase III and a reaction phase oil phase IV, and mixing the reaction phase water phase V and the reaction phase oil phase IV uniformly, wherein the mass fraction of the reaction phase water phase V is 10-30%, to obtain a W / O emulsion containing calcium ion-containing aqueous microdroplets as a reaction phase VI; wherein the concentration of sorbitan oleate in the continuous phase III and the reaction phase oil phase IV is 0.2-0.4 wt%, (2) respectively feeding the pre-gel dispersed phase I, the oily dispersed phase II, the continuous phase III and the reaction phase VI into a composite microchannel microfluidic chip with two-stage cross-focusing structures by using injection pumps, wherein the first cross-focusing structure from the dispersed phase inlet to the outlet in the chip is an upper flow path for generating Janus droplets, and the second cross-focusing structure is a lower flow path for the generated Janus droplets to contact the reaction phase VI for solidification; the pre-gel dispersed phase I, the oily dispersed phase II and the continuous phase III enter the upper flow path in a vertically intersecting flow direction, the generated Janus droplets flow through the lower flow path in a vertically intersecting flow direction, and an ionic cross-linking solidification reaction occurs, the flow rate of the reaction phase VI is controlled to be 20-40 mL / h, and non-spherical hydrogel microspheres are formed; (3) collecting the reaction product flowing out of the microchannel of the microfluidic chip after the reaction, separating and purifying the reaction product by suction filtration, removing the oily dispersed phase II, the continuous phase III and the reaction phase VI, and washing the hydrogel microparticles with n-hexane and pure water to obtain non-spherical calcium alginate hydrogel microparticles.

2. The production method according to claim 1, wherein The pre-gel dispersed phase I in step (1) further comprises a water-soluble surfactant, which is fluorine-containing polyoxyethylene ether FS-30, wherein when the concentration of FS-30 is 0.05-1 wt%, non-spherical hydrogel microparticles with a single concave structure are formed; and when the concentration of FS-30 is 0, non-spherical hydrogel microparticles with a double-convex elliptical structure are formed.

3. The production method according to claim 1, wherein The mass content of calcium chloride in the reaction phase water phase V in step (1) is 20 wt%.

4. The production method according to claim 3, wherein The mass ratio of the water phase V in the reaction phase VI in step (1) is 20-30 wt%.

5. The production method according to claim 1, wherein The microfluidic chip in step (2) is at least one of a quartz glass chip, a PDMS chip, a glass capillary and a composite chip, and the inner diameter of the droplet generator in the microchannel is 70-160 μm to obtain Janus droplets with a diameter of 50-500 μm.

6. The production method according to claim 5, wherein The flow rate of the pre-gel dispersed phase I in step (2) is controlled to be 0.125-0.500 mL / h.

7. The production method according to claim 5 or 6, characterized by, The flow rate of the oily dispersed phase II in step (2) is controlled to be 0.125-0.500 mL / h.

8. The production method according to claim 5 or 6, wherein The flow rate of the continuous phase III in step (2) is controlled to be 2-9 mL / h.

9. The production method according to claim 8, wherein The flow rate ratio of the pre-gel dispersed phase I to the oily dispersed phase II in step (2) is between 1.00 and 3.

00. The flow rate ratio of the pre-gel dispersed phase I to the oily dispersed phase II in step (2) is between 1.00 and 3.00.

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