Preparation method and application of exosome-loaded microspheres based on microfluidic focusing technology
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-30
- Publication Date
- 2026-08-11
Smart Images
Figure CN122537327A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biopharmaceutical formulation technology, and in particular to a method for preparing and applying exosome-loaded microspheres based on microfluidic focusing technology. Background Technology
[0002] Stem cell exosomes, as natural nanoscale vesicles, possess low immunogenicity, good biocompatibility, and the potential to cross biological barriers, making them promising therapeutic agents or drug carriers for neurodegenerative diseases and tissue repair. However, direct administration of exosomes suffers from problems such as short in vivo half-life, easy clearance, and poor targeting. Encapsulating them in microsphere carriers can achieve sustained release, protection, and targeted delivery.
[0003] Currently, the preparation of microspheres loaded with bioactive ingredients mainly relies on traditional emulsification-solvent evaporation methods or emulsification-chemical crosslinking methods. These methods typically disperse a solution containing the material in an oil phase under vigorous stirring to form an emulsion, and then solidify the droplets by evaporating the solvent or adding a chemical crosslinking agent (such as glutaraldehyde). However, these methods still have the following inherent drawbacks: 1. Poor uniformity: Relying on shear force for fragmentation, the resulting emulsion droplets have a wide size distribution, leading to uneven microsphere size and large batch-to-batch variations. This directly affects the accuracy of drug dosage and the repeatability of release kinetics.
[0004] 2. Risk of activity damage: The mechanical force and local heat generated by high-speed shearing may damage the structure and activity of fragile biological macromolecules such as exosomes.
[0005] 3. Chemical residues and safety issues: Chemical cross-linking agents may remain in the product, causing cytotoxicity or immune responses, and post-processing is complicated.
[0006] 4. Low process controllability: The process is affected by many factors such as stirring speed, temperature, and container geometry, making it difficult to achieve precise control and stable scale-up. Summary of the Invention
[0007] In view of the above, the main objective of this invention is to propose a method for preparing exosome microspheres based on microfluidic focusing technology and its application, so as to solve the above-mentioned technical problems.
[0008] This invention proposes a method for preparing exosome-loaded microspheres based on microfluidic focusing technology, the method comprising the following steps: Step 1: Add gelatin to the buffer solution and stir in a water bath until completely dissolved to form a gel solution; after the gel solution cools, stir while adding stem cell exosome suspension drop by drop. After the addition is complete, continue stirring in the dark to form an exosome-loaded gel precursor solution; add surfactant to perfluorocarbon oil and stir to form a stabilized oil phase. Step 2: Inject the exosome-loaded gel precursor solution and the stabilized oil phase into two sterile syringes, respectively, to obtain an aqueous phase syringe and an oil phase syringe. Attach the aqueous phase syringe and oil phase syringe to the syringe pump. Connect the aqueous phase syringe to the central channel inlet of the flow-focusing microfluidic chip using a PTFE tube. Connect the oil phase syringe to the sheath flow channel inlets on both sides of the flow-focusing microfluidic chip using PTFE tubes. Start the syringe pump to form a monodisperse water-in-oil droplet emulsion and collect it. Step 3: Cool the collected monodisperse water-in-oil droplet emulsion. During the cooling process, when the temperature passes the sol-gel transition point of gelatin, the gelatin molecules inside the monodisperse water-in-oil droplet emulsion change from a random coil state to a triple helix structure. The network structure forms and locks in water and exosomes to obtain an oil phase emulsion containing solid gel microspheres. Step 4: Centrifuge the oil phase emulsion containing solid gel microspheres and remove the supernatant oil phase to obtain a precipitate; repeatedly wash the precipitate with pre-cooled anhydrous ethanol, and add a lyophilization protectant solution after washing to obtain a suspension; pre-freeze the suspension, and then perform main drying and desorption drying in sequence to obtain exosome-loaded microspheres.
[0009] This invention also proposes an application of exosome-carrying microspheres based on microfluidic focusing technology, wherein the exosome-carrying microspheres are prepared using any of the preparation methods for exosome-carrying microspheres based on microfluidic focusing technology described above, and the exosome-carrying microspheres are applied to lymphatic targeted delivery of tumor vaccines.
[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention achieves highly monodisperse preparation of exosome-loaded microspheres through the synergistic effect of microfluidic focusing technology and a specific flow rate ratio, resulting in a qualitative improvement in particle size uniformity. This precise and controllable microfluidic molding process completely solves the inherent defects of traditional methods, such as wide particle size distribution and large batch-to-batch differences caused by mechanical shearing, laying a solid foundation for the accuracy of subsequent drug delivery and the repeatability of release kinetics; 2. This invention fundamentally solves the core technical bottleneck of "difficulty in simultaneously achieving high encapsulation efficiency and high activity" in the preparation of exosome-loaded microspheres through a three-pronged process combination of "mild microfluidic molding - gradient cooling curing - trehalose freeze-drying protection". This invention does not simply achieve physical encapsulation of exosomes, but rather preserves the natural structure and biological function of exosomes entirely through a shear-free process, realizing a qualitative leap from "drug-loaded material" to "active formulation". 3. The exosome-loaded microspheres prepared in this invention exhibit significantly superior overall performance compared to existing technologies in terms of controllable release behavior, storage stability, and biological efficacy. The in vitro release behavior displays typical sustained-release characteristics, and the exosome activity remains excellent after long-term storage, with cell proliferation-promoting effects remarkably close to those of fresh exosomes. These combined advantages provide a reliable guarantee for the clinical translation and industrial production of exosome microspheres in regenerative medicine, tumor therapy, and other fields.
[0011] 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 means of embodiments of the invention. Attached Figure Description
[0012] Figure 1 This is a flowchart illustrating the steps of a method for preparing exosome-loaded microspheres based on microfluidic focusing technology proposed in this invention. Figure 2 This is a comparison chart of the encapsulation efficiency and exosome activity retention rate between the embodiments of the present invention and the prior art; Figure 3 This is a comparison diagram of the in vitro release behavior of embodiments of the present invention and prior art; Figure 4 This is a comparison chart of the storage stability of embodiments of the present invention and existing technologies; Figure 5 This is a comparison diagram of cell proliferation promotion experiments between embodiments of the present invention and existing technologies. Detailed Implementation
[0013] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0014] These and other aspects of the embodiments of the present invention will become clear from the following description and accompanying drawings. In these descriptions and drawings, some specific embodiments of the present invention are specifically disclosed to illustrate some ways of implementing the principles of the embodiments of the present invention; however, it should be understood that the scope of the embodiments of the present invention is not limited thereto.
[0015] Example 1 Please see Figure 1 This embodiment provides a method for preparing exosome-loaded microspheres based on microfluidic focusing technology, the method comprising the following steps: Step 1: Add 1g of gelatin to a buffer solution (PBS, pH=7, 10mL), and stir in a 37°C water bath until completely dissolved to form a 10% (w / v) gel solution. After the gel solution cooled to 25°C, 1 mL of stem cell exosome suspension with a protein concentration of 2 mg / mL was added dropwise while stirring at a rate of 200 rpm. After the addition was complete, the mixture was stirred at a rate of 200 rpm for 13 min in the dark to form an exosome-loaded gel precursor solution (aqueous phase). Add 0.5g of surfactant (Krytox 157FSH) to 50mL of perfluorocarbon oil and stir at 200rpm at 25°C until completely dissolved to form a stabilized oil phase with a concentration of 1% (w / v).
[0016] Step 2: Inject the exosome-carrying gel precursor solution and the stabilized oil phase into two sterile syringes respectively to obtain an aqueous phase syringe and an oil phase syringe, and then install the aqueous phase syringe and the oil phase syringe onto the injection pump. A PTFE tube is used to connect the aqueous phase injector to the central channel inlet of the flow-focusing microfluidic chip (PDMS material, central channel width 100μm, height 100μm, sheath flow channel of the same size); a PTFE tube is used to connect the oil phase injector to the sheath flow channel inlets on both sides of the flow-focusing microfluidic chip. Start the syringe pump, set the aqueous phase flow rate to 20 μL / min and the total oil phase flow rate to 200 μL / min (i.e., the flow rate of each sheath to 100 μL / min), and control the ratio of the total oil phase flow rate to the aqueous phase flow rate to 10:1 to form a monodisperse water-in-oil (W / O) droplet emulsion and collect it.
[0017] It should be noted that the aqueous phase and oil phase converge in the flow focusing region of the flow focusing microfluidic chip. The oil phase exerts hydrodynamic compression and focusing on the central aqueous phase flow from both sides, forming a stable "cone-jet" structure. Under the combined action of interfacial tension and viscous shear, the jet undergoes periodic fracture downstream.
[0018] Step 3: After cooling the collected monodisperse water-in-oil droplet emulsion to 35°C, the temperature is then reduced to 4°C at a rate of 2°C / min. (During the cooling process, when the temperature passes the sol-gel transition point of gelatin (approximately 25~28°C), the gelatin molecules inside the monodisperse water-in-oil droplet emulsion change from a random coil state to a triple helix structure, and a network structure is formed and locks in water and exosomes) to obtain an oil phase emulsion containing solid gel microspheres.
[0019] Step 4: Centrifuge the oil phase emulsion containing solid gel microspheres at 3°C and 2800 rpm for 10 min, and remove the supernatant oil phase to obtain the precipitate; The precipitate was repeatedly washed with pre-cooled anhydrous ethanol (4°C, 50 mL). After washing, a lyophilization protectant solution (PBS containing 5% trehalose, 10 mL) was added to obtain a suspension. The suspension was pre-frozen at -80℃ for 4 hours, then dried at -50℃ and pressure below 10 Pa for 24 hours, followed by desorption drying for 6 hours to obtain exosome-loaded microspheres, denoted as A1.
[0020] Example 2 This embodiment provides a method for preparing exosome-loaded microspheres based on microfluidic focusing technology, the method comprising the following steps: Step 1: Add 1g of gelatin to the buffer solution (PBS, pH=7.2, 10mL), and stir in a 38℃ water bath until completely dissolved to form a 10% (w / v) gel solution; After the gel solution cooled to 27°C, 1 mL of stem cell exosome suspension with a protein concentration of 2 mg / mL was added dropwise while stirring at a rate of 220 rpm. After the addition was complete, the mixture was stirred at a rate of 220 rpm for 14 min in the dark to form an exosome-loaded gel precursor solution (aqueous phase). Add 0.5g of surfactant (Krytox 157FSH) to 50mL of perfluorocarbon oil and stir at 225rpm at 25°C until completely dissolved to form a stabilized oil phase with a concentration of 1% (w / v).
[0021] Step 2: Inject the exosome-carrying gel precursor solution and the stabilized oil phase into two sterile syringes respectively to obtain an aqueous phase syringe and an oil phase syringe, and then install the aqueous phase syringe and the oil phase syringe onto the injection pump. A PTFE tube is used to connect the aqueous phase injector to the central channel inlet of the flow-focusing microfluidic chip (PDMS material, central channel width 100μm, height 100μm, sheath flow channel of the same size); a PTFE tube is used to connect the oil phase injector to the sheath flow channel inlets on both sides of the flow-focusing microfluidic chip. Start the syringe pump, set the aqueous phase flow rate to 20 μL / min and the total oil phase flow rate to 200 μL / min (i.e., the flow rate of each sheath to 100 μL / min), and control the ratio of the total oil phase flow rate to the aqueous phase flow rate to 10:1 to form a monodisperse water-in-oil droplet emulsion and collect it.
[0022] It should be noted that the aqueous phase and oil phase converge in the flow focusing region of the flow focusing microfluidic chip. The oil phase exerts hydrodynamic compression and focusing on the central aqueous phase flow from both sides, forming a stable "cone-jet" structure. Under the combined action of interfacial tension and viscous shear, the jet undergoes periodic fracture downstream.
[0023] Step 3: After cooling the collected monodisperse water-in-oil droplet emulsion to 35°C, the temperature is then reduced to 4°C at a rate of 2°C / min. (During the cooling process, when the temperature passes the sol-gel transition point of gelatin (approximately 25~28°C), the gelatin molecules inside the monodisperse water-in-oil droplet emulsion change from a random coil state to a triple helix structure, and a network structure is formed and locks in water and exosomes) to obtain an oil phase emulsion containing solid gel microspheres.
[0024] Step 4: Centrifuge the oil phase emulsion containing solid gel microspheres at 4°C and 2900 rpm for 11 min, and remove the supernatant oil phase to obtain the precipitate; The precipitate was repeatedly washed with pre-cooled anhydrous ethanol (4°C, 50 mL). After washing, a lyophilization protectant solution (PBS containing 5% trehalose, 10 mL) was added to obtain a suspension. The suspension was pre-frozen at -80℃ for 4 hours, then dried at -50℃ and pressure below 10 Pa for 24 hours, followed by desorption drying for 6 hours to obtain exosome-loaded microspheres, denoted as A2.
[0025] Example 3 This embodiment provides a method for preparing exosome-loaded microspheres based on microfluidic focusing technology, the method comprising the following steps: Step 1: Add 1g of gelatin to the buffer solution (PBS, pH=7.4, 10mL), and stir in a 40℃ water bath until completely dissolved to form a 10% (w / v) gel solution; After the gel solution cools to 30°C, 1 mL of stem cell exosome suspension with a protein concentration of 2 mg / mL is added dropwise while stirring at a rate of 240 rpm. After the addition is complete, continue stirring at a rate of 240 rpm for 15 min in the dark to form an exosome-loaded gel precursor solution (aqueous phase). Add 0.5g of surfactant (Krytox 157FSH) to 50mL of perfluorocarbon oil and stir at 250rpm at 25°C until completely dissolved to form a stabilized oil phase with a concentration of 1% (w / v).
[0026] Step 2: Inject the exosome-carrying gel precursor solution and the stabilized oil phase into two sterile syringes respectively to obtain an aqueous phase syringe and an oil phase syringe, and then install the aqueous phase syringe and the oil phase syringe onto the injection pump. A PTFE tube is used to connect the aqueous phase injector to the central channel inlet of the flow-focusing microfluidic chip (PDMS material, central channel width 100μm, height 100μm, sheath flow channel of the same size); a PTFE tube is used to connect the oil phase injector to the sheath flow channel inlets on both sides of the flow-focusing microfluidic chip. Start the syringe pump, set the aqueous phase flow rate to 20 μL / min and the total oil phase flow rate to 200 μL / min (i.e., the flow rate of each sheath to 100 μL / min), and control the ratio of the total oil phase flow rate to the aqueous phase flow rate to 10:1 to form a monodisperse water-in-oil droplet emulsion and collect it.
[0027] It should be noted that the aqueous phase and oil phase converge in the flow focusing region of the flow focusing microfluidic chip. The oil phase exerts hydrodynamic compression and focusing on the central aqueous phase flow from both sides, forming a stable "cone-jet" structure. Under the combined action of interfacial tension and viscous shear, the jet undergoes periodic fracture downstream.
[0028] Step 3: After cooling the collected monodisperse water-in-oil droplet emulsion to 35°C, the temperature is then reduced to 4°C at a rate of 2°C / min. (During the cooling process, when the temperature passes the sol-gel transition point of gelatin (approximately 25~28°C), the gelatin molecules inside the monodisperse water-in-oil droplet emulsion change from a random coil state to a triple helix structure, and a network structure is formed and locks in water and exosomes) to obtain an oil phase emulsion containing solid gel microspheres.
[0029] Step 4: Centrifuge the oil phase emulsion containing solid gel microspheres at 5°C and 3000 rpm for 12 min, and remove the supernatant oil phase to obtain the precipitate; The precipitate was repeatedly washed with pre-cooled anhydrous ethanol (4°C, 50 mL). After washing, a lyophilization protectant solution (PBS containing 5% trehalose, 10 mL) was added to obtain a suspension. The suspension was pre-frozen at -80℃ for 4 hours, then dried at -50℃ and pressure below 10 Pa for 24 hours, followed by desorption drying for 6 hours to obtain exosome-loaded microspheres, denoted as A3.
[0030] Example 4 This embodiment provides an application of exosome-carrying microspheres based on microfluidic focusing technology. The exosome-carrying microspheres are prepared using the preparation method of exosome-carrying microspheres based on microfluidic focusing technology as described above. The exosome-carrying microspheres are used for lymphatic targeted delivery of tumor vaccines.
[0031] To verify the effectiveness of the present invention, the encapsulation efficiency and exosome activity retention rate of the exosome-loaded microspheres prepared by the traditional emulsification method (control group 1), the non-optimal flow rate ratio group (control group 2, which was prepared using a microfluidic chip but the ratio of the total flow rate of the oil phase to the flow rate of the water phase was adjusted to 5:1 (water phase 20 μL / min, oil phase 100 μL / min, the rest is the same as in Example 1) and Examples 1-3 of the present invention were evaluated.
[0032] As shown in Table 1 and Figure 2 As shown, traditional emulsification methods, due to severe shear forces, damage the exosome membrane structure, resulting in an encapsulation efficiency of only 64.8% and a sharp drop in activity retention to 51.6%, meaning that nearly half of the exosomes are inactivated during the preparation process. Control group 2, although employing microfluidic technology, suffered from an inappropriate flow rate ratio, achieving only 77.5% encapsulation efficiency and 71.3% activity retention, respectively. In contrast, the three embodiments of this invention exhibit superior performance: the encapsulation efficiency remained stable above 92% (reaching 93.0% in embodiment 3), and the activity retention rate approached 90% (reaching 90.1% in embodiment 3). This demonstrates that the synergistic effect of the specific "10:1 flow rate ratio" and microfluidic focusing technology of this invention is not simply a process optimization, but rather a fundamental solution to the technical challenge of easily inactivating active vesicles like exosomes during encapsulation by avoiding eddy current impact through laminar flow shearing, achieving a dual breakthrough in high loading capacity and high activity.
[0033] Table 1 Comparison of exosome encapsulation efficiency and activity retention rate
[0034] It should be noted that, in Figure 2 In the examples, experimental groups 1-3 correspond to the exosome-carrying microspheres prepared in Examples 1-3 of this invention, respectively.
[0035] Please refer to Table 2 and Figure 3The in vitro release kinetics of the microspheres prepared by the traditional emulsification method (control group 1), the non-gradient cooling group (control group 3, using the microspheres of Example 1 of this invention, but without gradient cooling in step 3, instead the collected droplet emulsion was directly placed in a 4°C refrigerator for rapid cooling and solidification), and the microspheres prepared by Examples 1-3 of this invention were studied for 72 hours. As shown in Table 2 and the corresponding release curves, the microspheres prepared by the traditional emulsification method showed a severe burst release within 2 hours, with a cumulative release rate of 38.4%, indicating that their internal structure was loose and could not effectively encapsulate exosomes. Although control group 3 (rapid cooling and solidification) was better than the traditional emulsification method, the burst release at 2 hours still reached 28.5%. In contrast, the release curves of the three examples of this invention were flat and robust: the cumulative release rate at 2 hours was only about 12%, at 24 hours about 48%, and at 72 hours about 78%, showing typical sustained release characteristics. The 26% initial release difference (12.1% in Example 3 vs. 38.4% in Control Group 1) demonstrates that the "2℃ / min gradient cooling" process employed in this invention provides sufficient time for molecular chain rearrangement when passing the gelatin sol-gel transition point, resulting in a more uniform and dense three-dimensional network structure than the quenching method. This is not simply an adjustment of the cooling rate, but a precise intervention in the kinetics of gel network formation, fundamentally transforming the release behavior of microspheres from "uncontrollable burst release" to "controllable sustained release."
[0036] It should be noted that the 72-hour in vitro release kinetics study was conducted as follows: 30 mg of each microsphere was placed in a dialysis bag (MWCO 100 kDa) and immersed in a release medium containing PBS with a pH of 7.4, and the mixture was incubated at 37°C with shaking. Release solutions were collected at predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours), and an equal volume of fresh medium was added. The cumulative release of exosomal proteins in the release solution was determined using the BCA method, and the cumulative release rate (%) was calculated.
[0037] Table 2 Experimental Results (Cumulative Release Rate / %)
[0038] Please refer to Table 3 and Figure 4The stability of traditional emulsification method (control group 1), wet microspheres (control group 4, using the method of Example 1 of this invention but without freeze-drying, stored in wet microsphere suspension at 4°C), and freeze-dried microspheres prepared in Examples 1-3 of this invention were investigated after storage at 4°C for 4 weeks. As shown in Table 3, the exosome protein residue rate of wet microspheres dropped to 85.3% after 1 week of storage, and only 51.2% remained after 4 weeks, indicating that exosomes are easily degraded and inactivated in a liquid environment. Although freeze-dried microspheres prepared by traditional emulsification method are superior to wet microspheres, the residue rate is only 72.5% after 4 weeks. However, the three examples of this invention showed excellent performance: the exosome protein residue rate remained stable at over 93% after 4 weeks of storage (reaching 93.8% in Example 3), with almost no loss compared to the initial content. The 21.3% difference in residual rate (93.8% in Example 3 vs. 72.5% in Control Group 1) demonstrates that the present invention, through the three-in-one process combination of "microfluidic precision molding - gradient gel solidification - trehalose freeze-drying protection", does not simply extend the shelf life of microspheres, but fundamentally constructs a long-term stable microenvironment for exosomes, providing a fundamental guarantee for their commercial clinical application.
[0039] Table 3 Experimental Results (Exosomal Protein Residue Rate / %)
[0040] Please refer to Table 4 and Figure 5 The proliferation promotion experiments of L929 fibroblasts were conducted using the traditional emulsification method (control group 1), fresh exosomes (control group 5, untreated, with protein concentration adjusted to be comparable to the release solution), a blank control group (culture medium without exosomes), and the microsphere release solutions prepared in Examples 1-3 of this invention, to assess the degree of preservation of exosome biological function. As shown in Table 4, the microsphere release solution prepared by the traditional emulsification method could only promote cell proliferation to 132.5%, which was a limited improvement compared to the blank control, indicating that most of the released exosomes had lost their biological activity. In contrast, the microsphere release solutions of the three examples of this invention could significantly promote cell proliferation to about 180% (185.2% in Example 3), which was very close to the proliferation-promoting effect of fresh exosomes (195.7%). This 52.7% difference in proliferation rate (185.2% in Example 3 vs. 132.5% in Control Group 1) fully demonstrates that the present invention does not simply physically encapsulate exosomes within microspheres, but rather preserves the natural structure and biological function of exosomes to the greatest extent possible through a gentle, shear-free preparation process and precise freeze-drying protection technology. This complete preservation of biological efficacy signifies that the exosome-loaded microspheres provided by the present invention have evolved from "materials with drug-loaded forms" to "active formulations with therapeutic functions."
[0041] Table 4. Experimental Results (Relative Cell Proliferation Rate / %)
[0042] In summary, this invention, through innovative microfluidic focusing technology combined with a gradient cooling curing process and a trehalose freeze-drying protection system, successfully achieved the precise and controllable construction and complete preservation of exosome-loaded gelatin microspheres, significantly improving the microsphere particle size uniformity, exosome encapsulation efficiency, bioactivity retention rate, controllable release behavior, and storage stability. Experimental results show that, compared with the traditional emulsification method, the encapsulation efficiency of the exosome-loaded microspheres prepared by this invention increased from 64.8% to over 93.0%, and the bioactivity retention rate increased from 51.6% to over 90.1%, solving the core problem of significant exosome inactivation due to severe shear stress in traditional processes. In in vitro release studies, the burst release rate of the microspheres of this invention decreased from 38.4% to below 12.1% after 2 hours, and the release curve exhibited typical sustained-release characteristics, demonstrating the precise control effect of the gradient cooling process on the gel network structure. Storage stability experiments showed that the exosome retention rate of the microspheres of this invention remained above 93% after storage at 4°C for 4 weeks, while that of traditional emulsified microspheres was only 72.5%, and that of wet microspheres was even lower at 51.2%. This indicates that the freeze-drying protection system constructed in this invention provides a long-term stable microenvironment for exosomes. More importantly, cell proliferation experiments confirmed that the exosomes released by the microspheres of this invention promoted cell proliferation at a rate as high as 185.2%, extremely close to that of fresh exosomes (195.7%), while that of traditional emulsified microspheres was only 132.5%. This 52.7% efficiency difference fully demonstrates that this invention does not simply achieve physical encapsulation of exosomes, but rather completely preserves the natural structure and biological function of exosomes through a gentle, shear-free process throughout.
[0043] This invention not only solves the key technical bottleneck of "difficulty in achieving both high encapsulation efficiency and high activity" in the preparation of exosome-loaded microspheres, but also ensures batch-to-batch consistency and reliability through the precise controllability of microfluidic technology. This lays a solid technical foundation for its clinical translation and industrial production in the fields of regenerative medicine, tissue engineering, tumor treatment and drug delivery, and has significant scientific research value and broad prospects for industrial application.
[0044] It should be understood that although the steps in the flowcharts of the various embodiments of the present invention are shown sequentially according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the various embodiments may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be performed alternately or in turn with other steps or at least a portion of the sub-steps or stages of other steps.
[0045] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0046] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.
Claims
1. A method for preparing exosome-loaded microspheres based on microfluidic focusing technology, characterized in that, The method includes the following steps: Step 1: Add gelatin to the buffer solution and stir in a water bath until completely dissolved to form a gel solution; after the gel solution cools, stir while adding stem cell exosome suspension drop by drop. After the addition is complete, continue stirring in the dark to form an exosome-loaded gel precursor solution; add surfactant to perfluorocarbon oil and stir to form a stabilized oil phase. Step 2: Inject the exosome-loaded gel precursor solution and the stabilized oil phase into two sterile syringes, respectively, to obtain an aqueous phase syringe and an oil phase syringe. Attach the aqueous phase syringe and oil phase syringe to the syringe pump. Connect the aqueous phase syringe to the central channel inlet of the flow-focusing microfluidic chip using a PTFE tube. Connect the oil phase syringe to the sheath flow channel inlets on both sides of the flow-focusing microfluidic chip using PTFE tubes. Start the syringe pump to form a monodisperse water-in-oil droplet emulsion and collect it. Step 3: Cool the collected monodisperse water-in-oil droplet emulsion. During the cooling process, when the temperature passes the sol-gel transition point of gelatin, the gelatin molecules inside the monodisperse water-in-oil droplet emulsion change from a random coil state to a triple helix structure. The network structure forms and locks in water and exosomes to obtain an oil phase emulsion containing solid gel microspheres. Step 4: Centrifuge the oil phase emulsion containing solid gel microspheres and remove the supernatant oil phase to obtain a precipitate; repeatedly wash the precipitate with pre-cooled anhydrous ethanol, and add a lyophilization protectant solution after washing to obtain a suspension; The suspension was pre-frozen, and then subjected to main drying and analytical drying in sequence to obtain exosome-loaded microspheres.
2. The method for preparing exosome-loaded microspheres based on microfluidic focusing technology according to claim 1, characterized in that, In step 1, during the process of obtaining the gel solution, the mass of gelatin added is 1g, the buffer solution is PBS, the pH of the buffer solution is 7~7.4, the volume of the buffer solution is 10mL, the water bath temperature is 37~40℃, and the concentration of the gel solution is 10%. In the process of obtaining the exosome-loaded gel precursor solution, the gel solution was cooled to 25~30℃, the stirring rate was 200~240rpm, the protein concentration of the stem cell exosome suspension was 2mg / mL, the volume of the stem cell exosome suspension was 1mL, the stirring rate was 200~240rpm and the stirring time was 13~15min in the dark. In the process of obtaining the stabilized oil phase, the surfactant used was Krytox 157FSH, with a mass of 0.5g and a volume of 50mL of perfluorocarbon oil. The stirring temperature was 25℃, the stirring speed was 200~250rpm, and the concentration of the stabilized oil phase was 1%.
3. The method for preparing exosome-loaded microspheres based on microfluidic focusing technology according to claim 2, characterized in that, In step 2, the flow-focusing microfluidic chip is made of PDMS material. The central channel of the flow-focusing microfluidic chip has a width of 100μm and a height of 100μm. Both sides of the flow-focusing microfluidic chip are provided with sheath flow channels of the same size.
4. The method for preparing exosome-loaded microspheres based on microfluidic focusing technology according to claim 3, characterized in that, In step 2, during the process of obtaining the monodisperse water-in-oil droplet emulsion, the flow rate of the aqueous phase is set to 20 μL / min, the total flow rate of the oil phase is set to 200 μL / min, and the ratio of the total flow rate of the oil phase to the flow rate of the aqueous phase is controlled to be 10:
1.
5. The method for preparing exosome-loaded microspheres based on microfluidic focusing technology according to claim 4, characterized in that, In step 3, during the process of obtaining the oil phase emulsion containing solid gel microspheres, the collected monodisperse water-in-oil droplet emulsion is cooled to 35°C and then the temperature is reduced to 4°C at a rate of 2°C / min.
6. The method for preparing exosome-loaded microspheres based on microfluidic focusing technology according to claim 5, characterized in that, In step 4, during the process of obtaining the precipitate, the centrifugation temperature is 3~5℃, the centrifugation rate is 2800~3000rpm, and the centrifugation time is 10~12min. During the process of obtaining the suspension, the temperature of anhydrous ethanol was 4℃ and the volume of anhydrous ethanol was 50mL. The lyophilization protectant solution used was PBS containing 5% trehalose and the volume of the lyophilization protectant solution was 10mL. In the process of obtaining exosome-loaded microspheres, the pre-freezing temperature was -80℃ and the pre-freezing time was 4h, the main drying temperature was -50℃ and the main drying time was 24h, and the desorption drying time was 6h.
7. An application of exosome-carrying microspheres based on microfluidic focusing technology, characterized in that, The exosome-loaded microspheres are prepared using the method for preparing exosome-loaded microspheres based on microfluidic focusing technology as described in any one of claims 1 to 6, and the exosome-loaded microspheres are used for lymphatic targeted delivery of tumor vaccines.