Preparation method of collagen microspheres and collagen microspheres
By using a three-channel microfluidic device and pressure control with an inert macromolecular crowding agent, the problems of cross-linking curing and solvent residue in the preparation of collagen microspheres were solved, achieving collagen microspheres with uniform particle size and mass production, and possessing excellent biocompatibility and injectability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INSTITUTE OF PROCESS ENGINEERING CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-01
AI Technical Summary
In existing methods for preparing collagen microspheres, cross-linking and curing lead to denaturation, the process can easily damage the structure, and there are solvent residue issues, making it difficult to achieve uniform particle size and mass production.
A three-channel microfluidic device was used to prepare collagen microspheres without cross-linking by using an inert macromolecular crowding agent such as polyethylene glycol and controlling the pressure in different channels, resulting in microspheres with uniform particle size.
This method enables the efficient and stable preparation of collagen microspheres with uniform particle size, avoids the use of cross-linking agents, is simple to operate, and has no toxic or harmful components added, making it suitable for mass production.
Smart Images
Figure CN121944940A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of polymer microspheres, specifically to a method for preparing collagen microspheres and collagen microspheres. Background Technology
[0002] Collagen is a major component of connective tissue, accounting for about one-third of the protein content in animal bodies. It functions to support organs and maintain mechanical stability, elasticity, and strength. As a natural high-molecular-weight biomaterial, collagen has a unique triple helix structure and exhibits low immunogenicity, good biocompatibility, and biodegradability. Collagen can be molded into sponges, gels, membrane dressings, microspheres, etc., and clinically it is mainly used for wound repair, bone repair, surgical sutures, and tissue engineering.
[0003] Collagen-based microspheres possess characteristics such as large specific surface area and good cell adhesion, making them excellent biomaterials for cell culture and drug delivery. Currently, there are several methods for preparing collagen microspheres, mainly including emulsification-solvent evaporation, spray drying, and phase separation. As the most commonly used method, solvent evaporation involves creating oil / water, water / oil, oil / water / oil, or water / oil / water emulsions by mechanical stirring or ultrasonic treatment. Then, cross-linking agents or other cross-linking methods are added to cross-link and solidify the collagen phase to obtain collagen microspheres. However, collagen denaturation caused by cross-linking and solidification often limits its applications. Spray drying uses an atomizer to spray a collagen solution in a hot gas stream formed by inert gas. During spraying, the solution evaporates, and the collagen shrinks into a shell to obtain microspheres. This method is simple and convenient, but it involves high temperatures, which can easily damage the collagen structure. Phase separation reduces the solubility of collagen by adding inorganic salts and non-solvent substances to the collagen solution environment, thereby preparing collagen microspheres. However, the difficulty in separating and removing added ingredients can also lead to problems such as solvent residue. Summary of the Invention
[0004] To at least partially solve the above problems, according to one aspect of this application, embodiments of this application provide a method for preparing collagen microspheres, comprising:
[0005] Step S1: Dissolve collagen in an acidic solution to obtain the first solution;
[0006] Step S2: Dissolve the inert macromolecular crowding agent in water to obtain a second solution;
[0007] Step S3: Obtain the oil phase;
[0008] Step S4: Inject the first solution, the second solution, and the oil phase into the first channel, the second channel, and the third channel of the three-channel microfluidic device, respectively. The first channel intersects with the second and third channels, and the second channel is isolated from the third channel.
[0009] Step S5: Apply pressure to the first channel, the second channel and the third channel respectively, so that the first solution flows sequentially through the first junction of the first channel and the second channel and the second junction of the first channel and the third channel in the first channel;
[0010] Step S6: Collect the effluent at the outlet of the first channel to obtain collagen microspheres.
[0011] Preferably, the pressure applied to the first channel is 8-20 kPa; the pressure applied to the second channel is 15-30 kPa.
[0012] Preferably, when the pressure applied to the first channel is increased, the pressure applied to the second channel is decreased; or, when the pressure applied to the first channel is decreased, the pressure applied to the second channel is increased.
[0013] Preferably, the pressure applied to the third channel is 8-15 kPa.
[0014] Preferably, the acidic solution is an acetic acid solution, a citric acid solution, or a lactic acid solution. An acetic acid solution is preferred, and when preparing the acetic acid solution, the volume ratio of acetic acid to water is 1:180 to 1:220.
[0015] Preferably, the collagen is animal-derived collagen and / or recombinant collagen; the inert macromolecular crowding agent is polyethylene glycol, dextran, dextran, or polyethylene oxide.
[0016] Preferably, the molecular weight of the inert macromolecular crowding agent is between 2000 and 8000.
[0017] Preferably, the oil phase contains 0.5%-2.0% surfactant.
[0018] According to another aspect of this application, this application provides collagen microspheres prepared by any of the methods described above, the collagen microspheres comprising: collagen, an inert macromolecular crowding agent, and an oil phase.
[0019] Preferably, the collagen microspheres contain 1%-2% collagen and 10%-30% inert macromolecular crowding agent.
[0020] The method for preparing collagen microspheres provided in this application involves a three-channel microfluidic device. In the first channel, an aqueous solution containing dissolved collagen serves as the dispersed phase. Under pressure, this dispersed phase is encapsulated by an aqueous solution containing an inert macromolecular crowding agent in the second channel. Due to the macromolecular crowding effect, the collagen rapidly gels to form microspheres. Subsequently, an oil phase in the third channel acts as the continuous phase, encapsulating the composite liquid in the second channel to obtain collagen microspheres with uniform particle size. The method provided in this application is simple in design and highly operable. Through a water / water / oil approach, it achieves efficient and stable preparation of collagen microspheres with uniform particle size in a microfluidic device. In a preferred embodiment, polyethylene glycol is introduced as an inert macromolecular crowding agent and used to encapsulate the collagen solution at the micrometer scale. During contact with the collagen, the polyethylene glycol solution significantly accelerates the liquid-gel transition of collagen molecules, enabling instantaneous assembly and solidification of the collagen. Furthermore, the above-mentioned method for preparing collagen microspheres with uniform particle size involves placing collagen, an inert macromolecular crowding agent, and an oil phase in the first, second, and third channels of a microfluidic device, respectively, and preparing collagen microspheres with uniform particle size under different pressure conditions. This preparation process ensures the uniformity of the microsphere particle size; it does not involve the addition of toxic or harmful components; and the preparation method enables the mass production of microspheres with a high degree of industrialization.
[0021] The collagen microspheres provided in this application have a uniform particle size. Both the collagen and polyethylene glycol in these microspheres have excellent biocompatibility and low immune rejection in vivo, making them safe biological implants. The uniformly sized collagen microspheres of this application are spherical with a regular morphology and have excellent injectability during use.
[0022] Compared with the prior art, this application has the following technical effects:
[0023] (1) The preparation of collagen microspheres without modification is achieved by using the macromolecular crowding effect and without relying on cross-linking agents or photocuring treatment;
[0024] (2) The method for generating high-throughput droplets of uniform-size collagen microspheres provided in this application is simple to operate and does not involve the addition of any toxic or harmful components;
[0025] (3) The collagen microspheres with uniform particle size prepared in this application have controllable particle size and high uniformity, and can be mass-produced. Attached Figure Description
[0026] Figure 1 This is a schematic diagram illustrating the preparation process of uniform-sized collagen microspheres in the embodiments of this application;
[0027] Figure 2 This is a light micrograph of the collagen microspheres prepared in Example 1 of this application;
[0028] Figure 3 This is a light micrograph of the collagen microspheres prepared in Example 2 of this application;
[0029] Figure 4 This is a light micrograph of the collagen microspheres prepared in Example 3 of this application;
[0030] Figure 5 This is a light micrograph of the collagen microspheres prepared in Example 4 of this application;
[0031] Figure 6 This is a light micrograph of the collagen microspheres prepared in Example 5 of this application;
[0032] Figure 7 This is a light microscopic image of the collagen microspheres prepared in Example 6 of this application. Detailed Implementation
[0033] The preferred embodiments of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection claimed in this application.
[0034] The terms "first," "second," and similar words used in this application do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Words such as "including" or "contains" mean that the element preceding the word encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as "above," "below," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.
[0035] Example 1
[0036] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0037] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0038] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0039] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 20 kPa for the first channel, 20 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0040] Example 2
[0041] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0042] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0043] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0044] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 15 kPa for the first channel, 20 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0045] Example 3
[0046] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0047] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0048] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0049] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 10 kPa for the first channel, 20 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0050] Example 4
[0051] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0052] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0053] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0054] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 8 kPa for the first channel, 20 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0055] Example 5
[0056] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0057] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0058] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0059] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 15 kPa for the first channel, 15 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0060] Example 6
[0061] A method for preparing collagen microspheres with uniform particle size includes the following steps:
[0062] (1) Dissolve the extracted tissue collagen in acetic acid solution (20.0 mL water containing 0.1 mL pure acetic acid) to prepare a first solution with a mass concentration of 20 mg / mL;
[0063] (2) Dissolve 1.0 g of polyethylene glycol with a molecular weight of 8000 in 5 mL of ultrapure water and stir until completely dissolved to make a second solution;
[0064] (3) The first solution, the second solution, and the oil phase (containing 1% surfactant) were injected into the first channel (channel 1), the second channel (channel 2), and the third channel (channel 3) of the three-channel microfluidic device, respectively. The pressure conditions applied to each channel were: 15 kPa for the first channel, 30 kPa for the second channel, and 8 kPa for the third channel. After applying pressure, the collagen microspheres were collected at the outlet of the first channel.
[0065] The particle size and sphericity of the collagen microspheres in Examples 1-6 were tested, and the results are as follows: Figures 2-7 As shown, Table 1 summarizes the test results.
[0066] Table 1
[0067] Water / water / oil microsphere particle size (μm) Collagen microsphere sphericity Example 1 34 Incomplete Example 2 33 Incomplete Example 3 36 Incomplete Example 4 36 whole Example 5 33 whole Example 6 34 Incomplete
[0068] As shown in Table 1, the overall particle size of the collagen microspheres prepared in Examples 1 to 6 did not change much, indicating that the target product can be stably prepared based on the high-throughput microfluidic device.
[0069] The results from Examples 1 to 6 also show that the pressure of the first and second channels can effectively control the sphericity of the collagen microspheres. When the pressure parameter of the second channel is fixed, the sphericity of the collagen microspheres gradually increases as the pressure parameter of the first channel decreases. When the pressure parameter of the first channel is fixed, the sphericity of the collagen microspheres gradually decreases as the pressure parameter of the second channel increases. Therefore, when preparing collagen microspheres, collagen microspheres with uniform particle size and complete sphericity can be prepared by reasonably controlling the pressure of the first and second channels. Combining Examples 4 and 5, it can be seen that in order to obtain collagen microspheres with complete sphericity, when the pressure applied to the first channel is increased, the pressure applied to the second channel is decreased; or, when the pressure applied to the first channel is decreased, the pressure applied to the second channel is increased.
[0070] Based on the various embodiments of this application described above, in the absence of explicit denial or conflict, the technical features of one embodiment may be advantageously combined with one or more other embodiments.
[0071] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.
Claims
1. A method for preparing collagen microspheres, characterized in that, include: Step S1: Dissolve collagen in an acidic solution to obtain the first solution; Step S2: Dissolve the inert macromolecular crowding agent in water to obtain a second solution; Step S3: Obtain the oil phase; Step S4: Inject the first solution, the second solution, and the oil phase into the first channel, the second channel, and the third channel of the three-channel microfluidic device, respectively. The first channel intersects with the second and third channels, and the second channel is isolated from the third channel. Step S5: Apply pressure to the first channel, the second channel and the third channel respectively, so that the first solution flows sequentially through the first junction of the first channel and the second channel and the second junction of the first channel and the third channel in the first channel; Step S6: Collect the effluent at the outlet of the first channel to obtain collagen microspheres.
2. The method according to claim 1, characterized in that, The pressure applied to the first channel is 8-20 kPa; The pressure applied to the second channel is 15-30 kPa.
3. The method according to claim 2, characterized in that, When the pressure applied to the first channel is increased, the pressure applied to the second channel is decreased; or, When the pressure applied to the first channel is reduced, the pressure applied to the second channel is increased.
4. The method according to claim 1, characterized in that, The pressure applied to the third channel is 8-15 kPa.
5. The method according to claim 1, characterized in that, The acidic solution is an acetic acid solution, a citric acid solution, or a lactic acid solution.
6. The method according to claim 1, characterized in that, Collagen is animal-derived collagen and / or recombinant collagen; The inert macromolecular crowding agent is polyethylene glycol, dextran, dextran, or polyethylene oxide.
7. The method according to claim 6, characterized in that, The molecular weight of inert macromolecular crowding agents is between 2000 and 8000.
8. The method according to claim 1, characterized in that, The oil phase contains 0.5%-2.0% surfactant.
9. A collagen microsphere, characterized in that, Prepared by the method according to any one of claims 1-8, comprising: collagen, an inert macromolecular crowding agent, and an oil phase.
10. The collagen microspheres according to claim 9, characterized in that, The collagen microspheres contain 1%-2% collagen and 10%-30% inert macromolecular crowding agents.