Collagen gel microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to collagen gel microspheres as well as a preparation method and application thereof. The preparation method of the collagen gel microspheres comprises the following steps: forming atomized liquid drops from a first solution under the action of airflow, collecting the atomized liquid drops by adopting a second solution, and enabling the atomized liquid drops to be self-assembled to form the microspheres, the first solution comprises an acidic aqueous solution of collagen; the flow velocity V1 of the first solution is 0.1-100 mL / h, and the speed V1 of the air flow is 0.5-100 L / min. The method comprises the following steps: enabling an acidic aqueous solution of collagen to form atomized liquid drops by utilizing airflow, and collecting the atomized liquid drops to promote rapid self-assembly of collagen in the liquid drops in a salt solution to form collagen gel microspheres; the method is simple and efficient, and the prepared microspheres are good in uniformity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to collagen gel microspheres and a preparation method and application thereof. Background Art

[0002] Collagen has been widely used in food, cosmetics and medical fields due to its good biocompatibility, biodegradability and wide source channels. Especially in the biomedical field, products of different forms and with different physical properties can be prepared to meet the requirements of different functions, parts, indications and methods of use.

[0003] At present, in the field of biomedicine, the main application forms of collagen materials include collagen diaphragms, collagen gels, collagen sponges and collagen coatings. Among them, collagen diaphragms and collagen sponges are mainly used for filling and hemostasis of skin, oral cavity and surgery; collagen coatings mainly realize the functions of improving the biocompatibility of the material surface, promoting cell adhesion and growth, and preventing material bleeding; collagen gels are mainly used for injection filling and in vitro cell culture. For collagen gel, when it is used as an injection filling material, if the collagen concentration or cross-linking degree is too high, the injectability becomes worse; and if the collagen concentration or cross-linking degree is reduced, its degradation time in the body will be significantly shortened, affecting the use effect. When collagen gel is used for in vitro cell culture, the surface area of ​​collagen gel is small, and the number of cells that can be cultured is small, resulting in low cell culture efficiency.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide collagen gel microspheres and a preparation method and application thereof. Collagen gel microspheres are prepared in a simple and easy manner, which can have high cross-linking degree, good injectability, and large specific surface area, improve the operability and use effect of injection filling, and can also improve the efficiency of in vitro cell culture.

[0006] The first aspect of the present invention provides a method for preparing collagen gel microspheres, comprising the following steps: a first solution forms atomized droplets under the action of an airflow, a second solution is used to collect the atomized droplets and the atomized droplets are self-assembled to form microspheres; the first solution comprises an acidic aqueous solution of collagen;

[0007] The flow rate V1 of the first solution is 0.1 to 100 mL / h, and the speed V1 of the air flow is 0.5 to 100 L / min.

[0008] In a specific embodiment of the present invention, the flow rate V1 of the first solution is 5 to 50 mL / h; the velocity V2 of the air flow is 1 to 15 L / min.

[0009] In a specific embodiment of the present invention, the first solution and the airflow are introduced through a coaxial needle, and the first solution and the airflow contact at the outlet of the coaxial needle, so that the first solution forms the atomized droplets under the action of the airflow; the coaxial needle is composed of two inner needles with different inner diameters and an outer needle in a coaxial sleeve manner.

[0010] In a specific embodiment of the present invention, the G number X1 of the inner needle is 14G to 34G, and the G number X2 of the outer needle is 7G to 27G. Furthermore, the difference between the G numbers of the inner needle and the outer needle is X1-X2=5-8.

[0011] In a specific embodiment of the present invention, the distance d between the second solution and the outlet of the coaxial needle is 3 to 30 cm. Furthermore, the liquid surface of the second solution is perpendicular to the axial direction of the coaxial needle.

[0012] In a specific embodiment of the present invention, the flow rate V1 (mL / min) of the first solution, the velocity V2 (L / min) of the airflow, the inner diameter D1 (mm) of the inner needle, the inner diameter D2 (mm) of the outer needle, and the distance d (cm) between the second solution and the outlet of the coaxial needle satisfy:

[0013]

[0014] In a specific embodiment of the present invention, the pH of the first solution is 0.5 to 4. Furthermore, in the first solution, the concentration of collagen is 0.1 wt% to 10 wt%.

[0015] In a specific embodiment of the present invention, the first solution further comprises at least one of metal ions and photocrosslinking agents.

[0016] In a specific embodiment of the present invention, in the first solution, the concentration of the photocrosslinking agent is 0.005 wt % to 5 wt %.

[0017] In a specific embodiment of the present invention, the photocrosslinking agent includes at least one of riboflavin, riboflavin sodium phosphate, camphorquinone, curcumin and eosin Y.

[0018] In a specific embodiment of the present invention, in the first solution, the concentration of the metal ions is 0.0001 wt % to 2 wt %.

[0019] In a specific embodiment of the present invention, the metal ions include Ca 2+ Mg 2+ , Fe 2+ , Fe3+ 、Zn 2+ , Cu 2+ 、Al 3+ 、Zr 2+ and Ti 3+ At least one of .

[0020] In a specific embodiment of the present invention, the second solution comprises at least one of PBS buffer, DPBS buffer, TBE buffer, Tris-HCl buffer, HEPES buffer, PIPEs buffer, MOPS buffer, TEA buffer, Tricine buffer, sodium carbonate-sodium bicarbonate buffer and NaCl solution.

[0021] In a specific embodiment of the present invention, the pH of the second solution is 5 to 8. When the second solution is a buffer solution, the concentration is 1X to 10X; when the second solution is a NaCl solution, the concentration is 2 to 4M.

[0022] In a specific embodiment of the present invention, the second solution is in a flowing state. Further, the second solution flows at a rotation speed of 50 to 5000 rpm.

[0023] In a specific embodiment of the present invention, the temperature of the second solution is 20-40°C.

[0024] In a specific embodiment of the present invention, the second solution further comprises sodium alginate, sodium bicarbonate or a chemical cross-linking agent.

[0025] In a specific embodiment of the present invention, in the second solution, the concentration of sodium alginate is 0.001 wt % to 0.5 wt %.

[0026] In a specific embodiment of the present invention, in the second solution, the concentration of sodium bicarbonate is 0.1 wt% to 5 wt%.

[0027] In a specific embodiment of the present invention, in the second solution, the concentration of the chemical crosslinking agent is 0.02 wt% to 3 wt%. Further, the chemical crosslinking agent includes at least one of glutaraldehyde, formaldehyde, EDC-NHS, genipin and maleimide crosslinking agents.

[0028] In a specific embodiment of the present invention, the method further comprises: collecting the microspheres, and then washing and drying them. Further, the collection method comprises: centrifugation and / or filtration.

[0029] In a specific embodiment of the present invention, before collecting the microspheres, the method further comprises: subjecting the second solution containing the atomized droplets to light treatment.

[0030] The second aspect of the present invention provides collagen gel microspheres prepared by the preparation method of the first aspect.

[0031] The third aspect of the present invention provides a use of the collagen gel microspheres of the second aspect in preparing a preparation for injection filling or in preparing a hemostatic spray.

[0032] The fourth aspect of the present invention provides a use of the collagen gel microspheres of the second aspect in in vitro cell culture.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention utilizes airflow to form atomized droplets from an acidic aqueous solution of collagen, and uses a second solution to collect the atomized droplets. The pH and ionic strength of the collagen droplets change rapidly, so that the procollagen molecules in the droplets quickly self-assemble to form collagen gel microspheres. The method of the present invention is simple and efficient, and the obtained microspheres have good uniformity.

[0035] (2) The present invention can further regulate the composition of the acidic aqueous solution and / or the collected solution of collagen, introduce metal ions, chemical crosslinking agents and / or photocrosslinking agents, and simultaneously achieve self-assembly and chemical crosslinking and / or photocrosslinking, thereby further improving the stability of the collagen gel microspheres and extending the degradation time;

[0036] (3) The collagen gel microspheres of the present invention not only have good stability and long degradation time, but also have good fluidity, and can be used to prepare preparations for tissue injection filling; and the collagen gel microspheres of the present invention have a large specific surface area, and when used for in vitro cell culture, can significantly improve the cell culture efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 A schematic diagram of the structure of a device for preparing collagen gel microspheres provided in an embodiment of the present invention;

[0039] Figure 2 This is a particle size distribution diagram of the collagen gel microspheres prepared in Example 1 of the present invention;

[0040] Figure 3This is a particle size distribution diagram of the collagen gel microspheres prepared in Example 2 of the present invention;

[0041] Figure 4 This is a particle size distribution diagram of the collagen gel microspheres prepared in Example 3 of the present invention;

[0042] Figure 5 This is a particle size distribution diagram of the collagen gel microspheres prepared in Example 4 of the present invention.

[0043] Reference numerals:

[0044] 1-first solution channel; 2-air flow channel; 3-coaxial needle;

[0045] 4- atomized droplets; 5- collection container; 6- lighting equipment. DETAILED DESCRIPTION

[0046] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0047] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0048] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0049] At present, collagen gel has the following problems when used for injection filling or in vitro cell culture: collagen gel used for injection filling cannot take into account both injectability and degradation time; collagen culture efficiency for in vitro cell culture is low. In order to solve these problems, the present invention makes collagen into hydrogel microspheres. Since there is no connection between the microspheres, the microspheres have good fluidity and ensure their injection performance; at the same time, the cross-linking degree between the collagen in the microspheres is increased to extend the degradation time. In addition, the gaps between the microspheres are similar to the macroporous structure in the scaffold material, which facilitates the migration of cells and can be injected into the implanted material. When it is used as a carrier for in vitro cell culture, its ultra-large specific surface area can adhere to more cells, greatly improving the efficiency of cell culture. Moreover, collagen microspheres covered with cells can be directly injected during cell therapy, which can greatly improve the survival efficiency of cells in the body and enhance the therapeutic effect.

[0050] Based on this, the first aspect of the present invention provides a method for preparing collagen gel microspheres, comprising the following steps: a first solution forms atomized droplets under the action of an airflow, a second solution is used to collect the atomized droplets and the atomized droplets are self-assembled to form microspheres; the first solution comprises an acidic aqueous solution of collagen;

[0051] The flow rate V1 of the first solution is 0.1-100 mL / h, and the velocity V2 of the air flow is 0.5-100 L / min.

[0052] The preparation method of the present invention utilizes the force of airflow to make the acidic aqueous solution of collagen enter the second solution in the form of atomized droplets, and the atomized droplets can quickly self-assemble in the second solution to form collagen gel microspheres, which simplifies the process flow of microspheres on the one hand and ensures the uniformity of microspheres on the other hand. In addition, the preparation method of the present invention does not need to introduce substances such as organic solvents and emulsifiers, thereby improving the biological safety of microspheres.

[0053] The flow rate of the first solution of the present invention and the speed of the airflow are regulated to be within the above ranges, respectively. On the one hand, the formation of atomized droplets is ensured, and on the other hand, the flow rate of the first solution and the speed of the airflow can be adjusted to obtain collagen gel microspheres of different particle sizes to meet the needs of different scenarios. For example, in different embodiments, the flow rate of the first solution can be 0.1mL / h, 1mL / h, 3mL / h, 5mL / h, 8mL / h, 10mL / h, 30mL / h, 50mL / h, 80mL / h, 100mL / h or a range consisting of any two of them; the speed of the airflow can be 0.5L / min, 1L / min, 3L / min, 5L / min, 8L / min, 10L / min, 30L / min, 50L / min, 80L / min, 100L / min or a range consisting of any two of them.

[0054] In a specific embodiment of the present invention, the gas used for the gas flow may include but is not limited to air, nitrogen, argon, carbon dioxide, etc.

[0055] In a specific embodiment of the present invention, the flow direction of the first solution is the same as the flow direction of the gas flow.

[0056] In a specific embodiment of the present invention, the flow rate V1 of the first solution is 5 to 50 mL / h; the velocity V2 of the air flow is 1 to 15 L / min.

[0057] In a specific embodiment of the present invention, the first solution and the airflow are introduced through a coaxial needle, and the first solution and the airflow contact at the outlet of the coaxial needle, so that the first solution forms atomized droplets under the action of the airflow; the coaxial needle is composed of two inner needles with different inner diameters and an outer needle in a coaxial sleeve manner.

[0058] In a specific embodiment of the present invention, the G number X1 of the inner needle is 14 to 34G, and the corresponding inner diameter size is from 1.600mm to 0.083mm. The G number of the inner needle can be, for example, 14G, 15G, 16G, 17G, 18G, 19G, 20G, 22G, 25G, 28G, 30G, 34G or a range consisting of any two of them; the G number X2 of the outer needle is 7G to 27G, and the corresponding inner diameter size is from 3.810mm to 0.210mm. The G number of the outer needle can be, for example, 7G, 8G, 9G, 10G, 11G, 12G, 13G, 14G, 15G, 16G, 17G, 18G, 19G, 20G, 21G, 23G, 24G, 25G, 26G, 27G or a range consisting of any two of them. This helps to make the first solution form evenly dispersed atomized droplets under the action of the airflow, further improving the uniformity of the formed microspheres.

[0059] In a specific embodiment of the present invention, the difference between the G numbers of the inner needle and the outer needle is X1-X2=5-8, wherein the difference in G number refers to the value of the G number of the inner needle minus the value of the G number of the outer needle. For example, the difference in G number between the inner needle and the outer needle can be 5, 6, 7, 8 or a range consisting of any two of them, thereby ensuring the sufficiency and uniformity of the first solution forming atomized droplets under the action of the airflow. It can be understood that the inner diameter of the outer needle is larger than the outer diameter of the inner needle to ensure that the outer needle can be sleeved outside the inner needle.

[0060] In a specific embodiment of the present invention, the distance d between the second solution and the outlet of the coaxial needle is 3 to 30 cm, for example, 3 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm or any two thereof. Furthermore, the liquid surface of the second solution is perpendicular to the axial direction of the coaxial needle.

[0061] In a specific embodiment of the present invention, the flow rate V1 of the first solution, the velocity V2 of the air flow, the inner diameter D1 of the inner needle, the inner diameter D2 of the outer needle, and the distance d between the second solution and the outlet of the coaxial needle satisfy:

[0062]

[0063] For example, It can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5 or any two of them. By regulating the first solution and the air flow velocity, the needle size and the liquid surface distance between the coaxial needle and the second solution, the uniformity and sphericity of the collagen gel microspheres can be further improved and the microspheres can be regulated to have a suitable particle size. It should be noted that when calculating the results corresponding to the above relationship, the units represented by V1, V2, D1, D2 and d are not considered, and only the corresponding values ​​when V1 is in mL / h, V2 is in L / min, D1 is in mm, D2 is in mm, and d is in cm are used to calculate the results corresponding to the corresponding relationship.

[0064] In a specific embodiment of the present invention, the pH of the first solution is 0.5 to 4, for example, it can be 0.5, 1, 1.5, 2, 3, 4 or any two thereof. Further, in the first solution, the concentration of collagen is 0.1 wt% to 10 wt%, for example, it can be 0.1 wt%, 1 wt%, 3 wt%, 5 wt%, 8 wt%, 10 wt% or any two thereof.

[0065] In a specific embodiment of the present invention, the acid used to prepare the first solution includes but is not limited to at least one of hydrochloric acid, phosphoric acid, sulfuric acid, citric acid and acetic acid. Furthermore, the pH of the acid used to prepare the first solution can be 0.5-4.

[0066] In a specific embodiment of the present invention, the first solution includes an optional photocrosslinker. Further, in the first solution, the concentration of the photocrosslinker is 0.005wt% to 5wt%, for example, it can be 0.005wt%, 0.01wt%, 0.1wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt% or any two of them. The photocrosslinker is further added to the first solution, and after the microspheres are formed, they can be further subjected to light treatment to increase the crosslinking degree of the microspheres, ensure the stability of the microspheres and prolong the degradation time.

[0067] In a specific embodiment of the present invention, the photocrosslinking agent includes at least one of riboflavin, riboflavin sodium phosphate, camphorquinone, curcumin and eosin Y.

[0068] In a specific embodiment of the present invention, the first solution includes optional metal ions. Further, in the first solution, the concentration of the metal ions is 0.0001wt% to 2wt%, for example, it can be 0.0001wt%, 0.001wt%, 0.01wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt% or any two thereof. Thus, the speed of self-assembly to form microspheres can be accelerated by the interaction between the collagen fibers and the metal ions in the reaction system, and the stability of the microspheres can be further improved.

[0069] In a specific embodiment of the present invention, the metal ions include Ca 2+ Mg 2+ , Fe 2+ , Fe 3+ 、Zn 2+ , Cu 2+ 、Al 3+ 、Zr 2+ and Ti 3+ At least one of .

[0070] In a specific embodiment of the present invention, the second solution includes at least one of PBS buffer (phosphate buffer), DPBS buffer (phosphate buffer without calcium and magnesium ions), TBE buffer (tris(hydroxymethyl)aminomethane-borate buffer), Tris-HCl buffer (tris(hydroxymethyl)aminomethane-hydrochloric acid buffer), HEPES buffer (hydroxyethylpiperazineethanesulfonic acid buffer), PIPEs buffer (piperazine-1,4-bis(2-ethylsulfonic acid) buffer), MOPS buffer (3-(N-morpholino)propanesulfonic acid buffer), TEA buffer (triethanolamine buffer), Tricine buffer (tri(hydroxymethyl)methylglycine buffer), sodium carbonate-sodium bicarbonate buffer and NaCl solution.

[0071] The concentration of each buffer solution can be 1 to 10 times that of the buffer solution used in biological experiments, i.e., 1X to 10X, the concentration of the NaCl solution can be 2 to 4M, and the pH of each buffer solution and the NaCl solution is 5 to 8. The microenvironment of the atomized droplets is acidic. After being collected by the second solution, the pH and ionic strength of the second solution are significantly different from the microenvironment of the atomized droplets, so that the atomized droplets can self-assemble in the second solution to form collagen gel microspheres.

[0072] In a specific embodiment of the present invention, the second solution is in a flowing state. Further, the second solution flows at a speed of 50 to 5000 rpm. In actual operation, mechanical stirring, magnetic stirring, etc. can be used to make the second solution flow. When the atomized droplets contact the liquid surface of the second solution, they are carried away by the flowing second solution to prevent the atomized droplets from merging at the same position and affecting the uniformity of the microsphere particle size.

[0073] In a specific embodiment of the present invention, the temperature of the second solution is 20-40° C., for example, 20° C., 25° C., 30° C., 35° C., 40° C., or any two thereof. Properly increasing the temperature of the second solution helps to accelerate the self-assembly speed of the atomized droplets in the second solution.

[0074] In a specific embodiment of the present invention, the second solution optionally includes sodium alginate. Further, in the second solution, the concentration of sodium alginate is 0.001wt% to 0.5wt%, for example, it can be 0.001wt%, 0.01wt%, 0.05wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt% or any two thereof, thereby further improving the speed of self-assembly of the atomized droplets in the second solution to form microspheres.

[0075] In a specific embodiment of the present invention, the second solution includes sodium bicarbonate. Further, the concentration of sodium bicarbonate is 0.1wt% to 5wt%, for example, it can be 0.1wt%, 0.5wt%, 1wt%, 3wt%, 5wt% or any two of them. The sodium bicarbonate in the second solution will react with the acidic environment of the atomized droplets to generate CO2, forming bubbles and occupying space in the formed gel, and when the CO2 is released, a porous structure will be formed in the gel microspheres.

[0076] In a specific embodiment of the present invention, the second solution includes an optional chemical crosslinking agent. Further, in the second solution, the concentration of the chemical crosslinking agent is 0.02wt% to 3wt%, for example, it can be 0.02wt%, 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt% or any two thereof. The use of a relatively low concentration of the chemical crosslinking agent ensures biological safety while further chemically crosslinking the microspheres in the second solution to increase the degree of crosslinking.

[0077] In a specific embodiment of the present invention, the chemical crosslinking agent includes at least one of glutaraldehyde, formaldehyde, EDC-NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide), genipin and maleimide crosslinking agents. Among them, maleimide crosslinking agents include but are not limited to 4-maleimidobutyric acid.

[0078] In a specific embodiment of the present invention, the method of the present invention further comprises: collecting the microspheres, and then washing and drying them. Furthermore, the collection method includes but is not limited to centrifugation and / or filtration.

[0079] In actual operation, pure water can be used for multiple washing to remove possible excess salt and residual chemical cross-linking agents. Drying methods include but are not limited to freeze drying, supercritical carbon dioxide drying, etc. For subsequent applications, conventional sterilization treatments can also be performed after drying, including but not limited to irradiation, ethylene oxide, supercritical methods, etc.

[0080] In a specific embodiment of the present invention, when a photocrosslinking agent is added to the first solution, before collecting the microspheres, the second solution containing the collected atomized droplets is subjected to light treatment. Furthermore, during the light treatment, the wavelength of the light is 300 to 500 nm, and the light intensity is 100 to 10000 mW / cm 2 The irradiation time is 3 to 300 minutes. Through the light treatment, the collagen gel microspheres self-assembled in the second solution can be further photo-crosslinked.

[0081] The second aspect of the present invention provides collagen gel microspheres prepared by the preparation method of the first aspect.

[0082] The collagen gel microspheres of the present invention have high cross-linking degree, good injectability, and large specific surface area, improve the operability and use effect of injection filling, and can also improve the efficiency of in vitro cell culture.

[0083] In a specific embodiment of the present invention, the D50 particle size of the collagen gel microspheres is preferably 40 to 300 μm, for example, it can be 40 μm, 50 μm, 60 μm, 70 μm, 90 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 220 μm, 250 μm, 280 μm, 300 μm or a range consisting of any two of them.

[0084] The third aspect of the present invention provides a use of the collagen gel microspheres of the second aspect in preparing a preparation for injection filling or in preparing a hemostatic spray.

[0085] After sterilization, the collagen gel microspheres of the present invention can be mixed with physiological saline in different proportions to prepare an injectable preparation for injection filling of tissues. They can also be placed in a pressure container and added with high-pressure gas to prepare a hemostatic spray.

[0086] The fourth aspect of the present invention provides a use of the collagen gel microspheres of the second aspect in in vitro cell culture.

[0087] After sterilization, the collagen gel microspheres of the present invention can also be added to a cell culture medium as a cell microcarrier for 3D culture and expansion of cells.

[0088] Figure 1 A schematic diagram of the structure of the device for preparing collagen gel microspheres provided in an embodiment of the present invention. The device for preparing collagen gel microspheres in an embodiment of the present invention may include: a first solution channel 1 for conveying a first solution, an air flow channel 2 for conveying a gas, a coaxial needle 3, and a collection container 5 for containing a second solution. Among them, the first solution channel 1 is connected to the inner needle of the coaxial needle 3, and the air flow channel 2 is connected to the outer needle of the coaxial needle 3. The other end of the first solution channel 1 can be connected to a container for storing and supplying the first solution, and the other end of the air flow channel 2 can be connected to a container for storing and supplying an air flow.

[0089] Furthermore, the liquid surface of the second solution in the collecting container 5 and the axial direction of the coaxial needle 3 are perpendicular to each other.

[0090] Furthermore, the distance d between the liquid surface of the second solution in the collection container 5 and the outlet of the coaxial needle 3 is 3 to 30 cm.

[0091] Furthermore, the device for preparing collagen gel microspheres further comprises an illumination device 6 . The illumination device 6 can perform illumination treatment on the solution in the collection container 5 .

[0092] When the device of the present invention is used to prepare collagen gel microspheres, the first solution and the high-speed gas enter the coaxial needle 3 through the first solution channel 1 and the air flow channel 2 respectively, the first solution introduced by the inner needle of the coaxial needle 3 and the gas introduced by the outer needle of the coaxial needle 3 converge at the outlet of the coaxial needle, and the first solution forms atomized droplets 4 under the action of the air flow. The atomized droplets 4 are collected by the collection container 5, and when the atomized droplets 4 enter the second solution contained in the collection container 5, they are quickly self-assembled to form collagen gel microspheres.

[0093] Example 1

[0094] This embodiment provides a method for preparing collagen gel microspheres, comprising the following steps:

[0095] (1) 3 g of freeze-dried collagen with an intact triple helix structure was prepared into a solution with a collagen concentration of 3 wt % using a phosphoric acid aqueous solution at pH 2, and CaCl 2 was added to the solution to make its concentration in the solution 1 wt %, thereby obtaining an acidic aqueous solution of collagen.

[0096] (2) Weigh a certain amount of sodium alginate and dissolve it in 5X DPBS buffer. The ratio of sodium alginate to DPBS buffer is 5:1000 (m / V). After it is fully dissolved, centrifuge at high speed to remove the undissolved sodium alginate and take the supernatant as the collection liquid.

[0097] (3) According to Figure 1 Assemble the structure schematic diagram shown, load the acidic aqueous solution of collagen into a syringe, connect the syringe to the inner needle of the coaxial needle 3 (inner needle model 29G), and use a microinjection pump for injection; the outer needle of the coaxial needle 3 (outer needle model 23G) is connected to high-purity nitrogen; place the coaxial needle 3 above the collection container 5 containing the collection liquid, so that the outlet of the coaxial needle 3 is 10 cm away from the liquid surface of the collection liquid, use a heater to heat the collection liquid to keep the temperature of the collection liquid at 37°C, and use a magnetic stirrer to make the collection liquid flow at a rotation speed of 300rpm.

[0098] (4) Start the microinjection pump and high-purity nitrogen gas, adjust the injection speed of the acidic aqueous solution of collagen to 5 mL / h, and the nitrogen flow rate to 1.5 L / min, so that atomized droplets are sprayed out from the coaxial needle 3, and the atomized droplets are collected by the collection liquid; since the atomized droplets contain Ca 2+ When encountering sodium alginate in the collection liquid, ionic cross-linking can be achieved quickly. At the same time, the collagen in the atomized droplets is in an acidic microenvironment. When entering the collection liquid, the pH and ionic strength of the solution change rapidly, causing the procollagen in the droplets to quickly self-assemble into collagen fibers to obtain collagen gel microspheres.

[0099] (5) Centrifuging the collected liquid obtained in step (4) at a centrifugal force of 4000G for 10 minutes, then removing the supernatant and collecting the lower layer of collagen gel microspheres; washing the collagen gel microspheres with pure water and centrifuging to remove the supernatant, repeating this process three times, and then adding a conventional collagen protein lyophilization protective agent to the collagen gel microspheres, freeze-drying, and obtaining dried collagen gel microspheres.

[0100] Example 2

[0101] This embodiment provides a method for preparing collagen gel microspheres, comprising the following steps:

[0102] (1) 3 g of freeze-dried collagen with an intact triple helix structure was taken and a solution with a collagen concentration of 3 wt% was prepared using a phosphoric acid aqueous solution with a pH of 2. At the same time, riboflavin was added to the solution to a concentration of 0.1 wt% to obtain an acidic aqueous solution of collagen.

[0103] (2) According to Figure 1Assemble as shown in the structural schematic diagram, load the acidic aqueous solution of collagen into a syringe, connect the syringe to the inner needle of the coaxial needle 3 (inner needle model 23G), and use a microinjection pump for injection; the outer needle of the coaxial needle 3 (outer needle model 18G) is connected to high-purity nitrogen; use 10X PBS buffer as the collecting liquid, place the coaxial needle 3 above the collecting container 5 containing the collecting liquid, make the outlet of the coaxial needle 3 20 cm away from the liquid surface of the collecting liquid, use a heater to heat the collecting liquid, keep the collecting liquid temperature at 37°C, and use a magnetic stirrer to make the collecting liquid flow at a speed of 500rpm.

[0104] (3) Start the microinjection pump and high-purity argon gas, adjust the injection speed of the acidic aqueous solution of collagen to 15 mL / h, and the nitrogen flow rate to 2 L / min, so that atomized droplets are sprayed out from the coaxial needle 3, and the atomized droplets are collected by the collecting liquid; and use a 200 W blue light lamp to irradiate the collecting liquid from directly above, the blue light lamp is 25 cm away from the surface of the collecting liquid, and the irradiation time is 2 h; the collagen in the atomized droplets is in an acidic microenvironment. When entering the collecting liquid, the pH and ionic strength of the solution change rapidly, so that the procollagen in the droplets quickly self-assembles to form collagen fibers, thereby obtaining collagen gel microspheres, which are further cross-linked by light treatment.

[0105] (4) Centrifuging the collected liquid obtained in step (3) at a centrifugal force of 4000G for 10 minutes, then removing the supernatant and collecting the lower layer of collagen gel microspheres; washing the collagen gel microspheres with pure water and centrifuging to remove the supernatant, repeating this process three times, and then adding a conventional collagen protein lyophilization protective agent to the collagen gel microspheres, freeze-drying, and obtaining dried collagen gel microspheres.

[0106] Example 3

[0107] This embodiment provides a method for preparing collagen gel microspheres, comprising the following steps:

[0108] (1) Take 5 g of freeze-dried collagen with an intact triple helix structure, use a hydrochloric acid aqueous solution with a pH of 2 to prepare a solution with a collagen concentration of 5 wt%, and add CaCl2 to the solution to make its concentration in the solution 1 wt%, to obtain an acidic aqueous solution of collagen.

[0109] (2) Weigh a certain amount of sodium alginate and dissolve it in 10X DPBS buffer at a ratio of 5:1000 (m / V). After it is fully dissolved, centrifuge at high speed to remove the undissolved sodium alginate, take the supernatant, add glutaraldehyde to the supernatant to a concentration of 2 wt%, and obtain a collection solution.

[0110] (3) According to Figure 1Assemble as shown in the structural schematic diagram, load the acidic aqueous solution of collagen into the syringe, connect the syringe to the inner needle of the coaxial needle 3 (inner needle model 27G), and use a microinjection pump for injection; the outer needle of the coaxial needle 3 (outer needle model 20G) is connected to high-purity nitrogen; place the coaxial needle 3 above the collection container 5 containing the collection liquid, so that the outlet of the coaxial needle 3 is 15 cm away from the liquid surface of the collection liquid, use a heater to heat the collection liquid to keep the temperature of the collection liquid at 37°C, and use a magnetic stirrer to make the collection liquid flow at a rotation speed of 600rpm.

[0111] (4) Start the microinjection pump and high-purity argon gas, adjust the injection speed of the acidic aqueous solution of collagen to 10 mL / h, and the nitrogen flow rate to 3 L / min, so that atomized droplets are sprayed out from the coaxial needle 3, and the atomized droplets are collected by the collection liquid; since the atomized droplets contain Ca 2+ When it encounters sodium alginate in the collecting liquid, it can quickly achieve ionic cross-linking. At the same time, the collagen in the atomized droplets is in an acidic microenvironment. When it enters the collecting liquid, the pH and ionic strength of the solution change rapidly, causing the procollagen in the droplets to quickly self-assemble into collagen fibers; at the same time, the glutaraldehyde in the collecting liquid reacts with the amino groups in the collagen molecules to form chemical covalent bonds to obtain collagen gel microspheres.

[0112] (5) Centrifuging the collected liquid obtained in step (4) at a centrifugal force of 4000G for 10 minutes, then removing the supernatant and collecting the lower layer of collagen gel microspheres; washing the collagen gel microspheres with pure water and centrifuging to remove the supernatant, repeating this process three times, and then adding a conventional collagen lyophilization protective agent to the collagen gel microspheres, freeze-drying, and obtaining dried collagen gel microspheres.

[0113] (6) In order to further remove the residual glutaraldehyde in the collagen gel microspheres, the microspheres were treated with supercritical carbon dioxide (SCCO2). The solid collagen gel microspheres were placed in a SCCO2 extraction kettle, and SCCO2 was introduced. The pressure of the extraction kettle was controlled to be 30 MPa, the SCCO2 flow rate was 1 L / min, and the extraction time was maintained for 1 h.

[0114] Example 4

[0115] This embodiment provides a method for preparing collagen gel microspheres, comprising the following steps:

[0116] (1) Take 5 g of freeze-dried collagen with an intact triple helix structure, use a hydrochloric acid aqueous solution with a pH of 2 to prepare a collagen solution with a concentration of 5 wt%, add CaCl2 to the solution to make its concentration in the solution 2 wt%, and add riboflavin sodium phosphate to make its concentration 0.05 wt%, to obtain an acidic aqueous solution of collagen.

[0117] (2) Weigh a certain amount of sodium alginate and dissolve it in 10X PBS buffer. The ratio of sodium alginate to PBS buffer is 3:1000 (m / V). After it is fully dissolved, centrifuge at high speed to remove the undissolved sodium alginate, take the supernatant, and then add sodium bicarbonate to the supernatant to make the concentration of sodium bicarbonate 2wt%, to obtain a collection solution.

[0118] (3) According to Figure 1 Assemble the structure schematic diagram shown, load the acidic aqueous solution of collagen into the syringe, connect the syringe to the inner needle of the coaxial needle 3 (inner needle model 25G), and use a microinjection pump for injection; the outer needle of the coaxial needle 3 (outer needle model 18G) is connected to high-purity nitrogen; place the coaxial needle 3 above the collection container 5 containing the collection liquid, so that the outlet of the coaxial needle 3 is 5 cm away from the liquid surface of the collection liquid, use a heater to heat the collection liquid to keep the temperature of the collection liquid at 37°C, and use a magnetic stirrer to make the collection liquid flow at a rotation speed of 400rpm.

[0119] (4) Start the microinjection pump and high-purity argon gas, adjust the injection speed of the acidic aqueous solution of collagen to 10 mL / h, and the nitrogen flow rate to 2 L / min, so that atomized droplets are sprayed out of the coaxial needle 3, and the atomized droplets are collected by the collecting liquid; start the coaxial needle so that the atomized droplets sprayed out of the coaxial needle are irradiated from the collecting liquid with a 200 W blue light lamp from above, the blue light lamp is 30 cm away from the liquid surface of the collecting liquid, and the irradiation time is 2 h; because the atomized droplets contain Ca 2+ When encountering sodium alginate in the collection liquid, ion cross-linking can be achieved quickly. At the same time, the collagen in the atomized droplets is in an acidic microenvironment. When entering the collection liquid, the pH and ionic strength of the solution change rapidly, and the acid reacts with the sodium bicarbonate in the collection liquid to generate CO2, which causes the procollagen in the droplets to quickly self-assemble into collagen fibers. The generated CO2 forms bubbles and occupies space in the gel. When it is released, gel microspheres with a porous structure are formed. During light treatment, riboflavin sodium phosphate in the collagen gel microspheres is activated as a photochemical cross-linking agent and undergoes photochemical cross-linking with collagen.

[0120] (5) filtering the collected liquid obtained in step (4) using a 300-mesh sieve, adding a conventional collagen lyophilization protective agent to the collected collagen gel microspheres, and freeze-drying to obtain dried collagen gel microspheres.

[0121] (6) In order to further remove the residual riboflavin sodium phosphate in the collagen gel microspheres, the microspheres were treated with supercritical carbon dioxide (SCCO2). The solid collagen gel microspheres were placed in a SCCO2 extraction kettle, and SCCO2 was introduced. The pressure of the extraction kettle was controlled at 35 MPa, the SCCO2 flow rate was 3 L / min, and the extraction time was maintained at 1.5 h.

[0122] Example 5

[0123] This example is based on the preparation method of Example 1, with the only difference being that in step (4), the flow rate of nitrogen is different.

[0124] In this embodiment, the flow rate of nitrogen is 2 L / min.

[0125] Example 6

[0126] This example is based on the preparation method of Example 2, with the only difference being that in step (4), the flow rate of nitrogen is different.

[0127] In this embodiment, the flow rate of nitrogen is 2.5 L / min.

[0128] Example 7

[0129] This example refers to the preparation method of Example 3, with the only difference being that in step (4), the flow rate of nitrogen is different.

[0130] In this embodiment, the flow rate of nitrogen is 4.5 L / min.

[0131] Example 8

[0132] This example refers to the preparation method of Example 3, except that the distance between the outlet of the coaxial needle and the liquid surface of the collected liquid in step (3) and the flow rate of the nitrogen in step (4) are different. In this example, the distance between the outlet of the coaxial needle and the liquid surface of the collected liquid is 30 cm, and the flow rate of the nitrogen is 5 L / min.

[0133] Example 9

[0134] This example refers to the preparation method of Example 4, with the only difference being that in step (4), the injection speed of the acidic aqueous solution of collagen is different.

[0135] In this embodiment, the injection rate of the acidic aqueous solution of collagen is 20 mL / h.

[0136] Experimental example

[0137] The performance of the collagen gel microspheres of different embodiments was tested as follows, and the test results are shown in Table 1. Figure 2 to Figure 5 The particle size distribution diagrams of the collagen gel microspheres prepared in Examples 1 to 4 of the present invention are respectively shown.

[0138] In vitro degradation test: 50 mg of the freeze-dried collagen gel microsphere product prepared in different embodiments was placed in 15 mL of 2u / mL collagenase solution, and the container was placed in a 37°C constant temperature shaking box to observe the degradation and record the time corresponding to complete degradation.

[0139] Table 1 Performance test results of different collagen gel microspheres

[0140]

[0141] Note: The sphericity is mainly determined by taking SEM photos of microspheres, performing image processing on the SEM photos, and randomly selecting 50 microspheres. If the proportion of microspheres with a sphericity (the ratio of the short diameter to the long diameter of the microspheres) above 0.9 is above 90%, the sphericity test result is considered regular; teardrop-shaped means that its sphericity does not meet the above-mentioned regularity requirements, and the microspheres are mainly teardrop-shaped particles.

[0142] From the above test results, it can be seen that the present invention uses airflow to form atomized droplets from the acidic aqueous solution of collagen, and collects the atomized droplets to promote the rapid self-assembly of collagen in the droplets in the salt solution to form collagen gel microspheres; the method of the present invention is simple and efficient, and the obtained microspheres have good uniformity. In addition, the introduction of metal ions, chemical crosslinkers and / or photocrosslinkers into the acidic aqueous solution of collagen or the collected solution, and the simultaneous realization of self-assembly and chemical crosslinking and / or photocrosslinking can further improve the stability of the collagen gel microspheres and prolong the degradation time. Further, by regulating the acidic aqueous solution of collagen and the airflow velocity, the needle size, and the distance between the coaxial needle and the liquid surface of the second solution, the uniformity, sphericity, etc. of the collagen gel microspheres can be further improved.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing collagen gel microspheres, characterized in that: The method comprises the following steps: a first solution forms atomized droplets under the action of airflow, a second solution is used to collect the atomized droplets and the atomized droplets are self-assembled to form microspheres; the first solution comprises an acidic aqueous solution of collagen; The flow rate V1 of the first solution is 0.1 to 100 mL / h, and the speed V1 of the air flow is 0.5 to 100 L / min.

2. The preparation method according to claim 1, characterized in that: The flow rate V1 of the first solution is 5 to 50 mL / h; the velocity V2 of the air flow is 1 to 15 L / min.

3. The preparation method according to claim 1, characterized in that: The first solution and the airflow are introduced through a coaxial needle, and the first solution and the airflow are in contact at the outlet of the coaxial needle, so that the first solution forms the atomized droplets under the action of the airflow; the coaxial needle is composed of two inner needles with different inner diameters and an outer needle in a coaxial sleeve manner; Preferably, the G number X1 of the inner needle is 14G to 34G; the G number X2 of the outer needle is 7G to 27G; Preferably, the difference between the G numbers of the inner needle and the outer needle is X1-X2=5-8; Preferably, the distance d between the second solution and the outlet of the coaxial needle is 3 to 30 cm; Preferably, the liquid surface of the second solution is perpendicular to the axial direction of the coaxial needle.

4. The preparation method according to claim 3, characterized in that: The flow rate V1 of the first solution, the velocity V2 of the air flow, the inner diameter D1 of the inner needle, the inner diameter D2 of the outer needle, and the distance d between the second solution and the outlet of the coaxial needle satisfy: Among them, the unit of V1 is mL / min, the unit of V2 is L / min, the unit of D1 is mm, the unit of D2 is mm, and the unit of d is cm.

5. The preparation method according to claim 1, characterized in that: The first solution has at least one of the following characteristics: (1) The pH of the first solution is 0.5 to 4; (2) In the first solution, the concentration of collagen is 0.1wt% to 10wt%; (3) the first solution includes at least one of metal ions and a photocrosslinking agent; (4) the first solution further comprises a photocrosslinking agent, wherein the concentration of the photocrosslinking agent is 0.005 wt % to 5 wt %; the photocrosslinking agent comprises at least one of riboflavin, riboflavin sodium phosphate, camphorquinone, curcumin and eosin Y; (5) The first solution further comprises optional metal ions, wherein the concentration of the metal ions is 0.0001 wt% to 2 wt%; the metal ions include Ca 2+ Mg 2+ , Fe 2+ , Fe 3+ 、Zn 2+ , Cu 2+ 、Al 3+ 、Zr 2+ and Ti 3+ At least one of .

6. The preparation method according to claim 1, characterized in that: The second solution has at least one of the following characteristics: (1) the second solution comprises at least one of PBS buffer, DPBS buffer, TBE buffer, Tris-HCl buffer, HEPES buffer, PIPEs buffer, MOPS buffer, TEA buffer, Tricine buffer, sodium carbonate-sodium bicarbonate buffer and NaCl solution; (2) the second solution is in a flowing state; (3) the second solution flows at a speed of 50 to 5000 rpm; (4) The temperature of the second solution is 20 to 40° C.; (5) the second solution further comprises optionally sodium alginate, sodium bicarbonate or a chemical cross-linking agent; (6) the second solution optionally includes sodium alginate, and the concentration of the sodium alginate is 0.001 wt% to 0.5 wt%; (7) the second solution optionally includes sodium bicarbonate, wherein the concentration of the sodium bicarbonate is 0.1 wt% to 5 wt%; (8) The second solution optionally includes a chemical crosslinking agent, wherein the concentration of the chemical crosslinking agent is 0.02wt% to 3wt%; the chemical crosslinking agent includes at least one of glutaraldehyde, formaldehyde, EDC-NHS, genipin and maleimide crosslinking agents.

7. The preparation method according to claim 1, characterized in that: The method further comprises: collecting the microspheres, and then washing and drying them; Preferably, before collecting the microspheres, the method further comprises: subjecting the second solution containing the atomized droplets to light treatment.

8. A collagen gel microsphere, characterized in that: The preparation method is described in any one of claims 1 to 7.

9. Use of the collagen gel microspheres according to claim 8 in preparing a preparation for injection filling or in preparing a hemostatic spray.

10. Use of the collagen gel microspheres according to claim 8 in in vitro cell culture.

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