Long-acting, non-embolism filling monodisperse chitosan microspheres and preparation method thereof

Abstract

The application provides a long-acting, non-embolism filling monodisperse chitosan microsphere, the base material of the microsphere is cross-linked chitosan, the cross-linked chitosan is formed by cross-linking reaction of component A and a cross-linking agent, or formed by cross-linking reaction of component A, component B and the cross-linking agent; the component A is at least one of chitosan and modified chitosan, and the component B is at least one of hyaluronic acid, collagen and gelatin; the particle size of the microsphere is 20-120 mu m, and the variation coefficient of the microsphere particle size is not more than 5%. The application also provides a preparation method of the microsphere. The monodispersity of the microsphere is excellent, the degradation time is relatively longer, the water absorption performance is good, the embolism risk can be reduced, the filling effect maintaining time can be prolonged, and the effect of improving wrinkles can be increased, and the problems of short filling effect maintaining time and easy to cause blood vessel embolism of the filling hyaluronic acid product used in the existing medical and beauty field can be solved.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetic materials and relates to a long-lasting, non-embolizing filled monodisperse chitosan microsphere and its preparation method. Background Technology

[0002] In recent years, injectable cosmetic procedures have gained widespread acceptance and are commonly used to fill and improve wrinkles caused by aging. Currently, hyaluronic acid is the primary material used in injectable cosmetic procedures. However, hyaluronic acid degrades relatively quickly, resulting in a short-lived filling effect. Furthermore, high-molecular-weight hyaluronic acid typically requires grinding or sieving before injection. The irregular lumps formed during grinding and sieving can easily cause vascular embolism during injection, potentially leading to serious medical accidents. Therefore, developing microsphere products with good biocompatibility and excellent monodispersity for injectable filling would significantly improve the safety of injectable fillings by addressing the risk of vascular embolism caused by the irregular shape of existing hyaluronic acid fillers. If the degradation rate of injectable microsphere products can be appropriately slowed down, further enhancing their water absorption and moisturizing properties, it is hoped that the problem of short-lived hyaluronic acid filling effects can be solved while achieving superior wrinkle improvement results compared to hyaluronic acid. Summary of the Invention

[0003] To address the issues of short-lasting filling effects and the risk of vascular embolism caused by irregular shapes in existing hyaluronic acid filler products used in the medical aesthetics field, this invention provides a long-lasting, non-embolizing monodisperse chitosan microsphere filler and its preparation method, in order to reduce the risk of embolism and prolong the duration of the filling effect.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] A long-lasting, non-embolizing filled monodisperse chitosan microsphere is disclosed. The matrix material of the microsphere is cross-linked chitosan, which is formed by cross-linking component A and a cross-linking agent, or by cross-linking components A and B and a cross-linking agent. Component A is at least one of chitosan and modified chitosan, and component B is at least one of hyaluronic acid, collagen, and gelatin. The particle size of the microsphere is 20–120 μm, and the coefficient of variation of the microsphere particle size does not exceed 5%.

[0006] In the above-mentioned long-lasting, non-embolizing filled monodisperse chitosan microsphere technical solution, when the cross-linked chitosan is formed by the cross-linking reaction of component A and component B with a cross-linking agent, the microsphere uses the polymer network formed by the cross-linking reaction of component A and the cross-linking agent as the backbone, and the polymer formed by the cross-linking reaction of component B and the cross-linking agent is distributed in the backbone, with the mass ratio of component A to component B being (50-95):(50-5). Further, when the cross-linked chitosan is formed by the cross-linking reaction of component A and component B with a cross-linking agent, the preferred mass ratio of component A to component B is (70-95):(30-5).

[0007] In the above-mentioned long-lasting, non-embolizing filled monodisperse chitosan microspheres, the modified chitosan includes carboxylated chitosan. Carboxylated chitosan contains a large number of carboxyl, hydroxyl, and amino groups, which have excellent water absorption capacity, thus giving the chitosan microspheres good water absorption properties and making the chitosan microspheres have better wrinkle improvement capabilities.

[0008] In the aforementioned long-lasting, non-embolizing filled monodisperse chitosan microspheres, the degradation time of the microspheres is 0.5–2 years. Based on the actual application requirements for degradation, the molecular weight and molecular chain composition of the cross-linked chitosan can be adjusted by selecting the types and molecular weights of components A and B, thereby regulating the degradation time of the microspheres.

[0009] In the above-mentioned long-lasting, non-embolizing filled monodisperse chitosan microspheres, the crosslinking agent includes any one of genipin, glutaraldehyde, terephthalaldehyde, and proanthocyanidins.

[0010] In the aforementioned long-lasting, non-clogging filled monodisperse chitosan microspheres, the microspheres may or may not have a porous structure. When the microspheres have a porous structure, they have stronger water absorption capacity and faster water absorption speed, achieving a water absorption volume several times their own volume, resulting in better wrinkle improvement.

[0011] This invention also provides a method for preparing the above-mentioned long-lasting, non-embolizing filled monodisperse chitosan microspheres, the method comprising the following steps:

[0012] (1) Preparation of internal phase fluid: The polymer is dissolved or fully dispersed in an acidic aqueous solvent to obtain an internal phase fluid; the concentration of the polymer in the internal phase fluid is 0.001-0.2 g / mL; the polymer is component A, or component A and component B;

[0013] Preparation of external phase fluid: Dissolve oil-soluble surfactant in oil phase solvent to obtain external phase fluid; the concentration of oil-soluble surfactant in external phase fluid is 0.05-10 g / mL;

[0014] Preparation of the collection solution: Dissolve the crosslinking agent in an oil phase solvent to obtain the collection solution; the concentration of the crosslinking agent in the collection solution is 0.1 wt.% to 5 wt.%.

[0015] (2) The inner phase fluid is introduced into the injection tube of the microfluidic device, and the outer phase fluid is introduced into the collection tube of the microfluidic device. A monodisperse water-in-oil emulsion is formed at the outlet of the injection tube of the microfluidic device. The water-in-oil emulsion is collected in a container filled with the collection liquid. After the polymer and the crosslinking agent have fully crosslinked, monodisperse chitosan microspheres are obtained.

[0016] (3) Washing removes the collected liquid from the surface of the monodisperse chitosan microspheres.

[0017] In the above-mentioned method for preparing long-lasting, non-embolizing filled monodisperse chitosan microspheres, the internal phase fluid prepared in step (1) also contains a substance that easily generates gas. When the internal phase fluid used in step (2) contains a substance that easily generates gas, during the preparation process in step (2), the substance that easily generates gas in the internal phase fluid reacts with the acidic aqueous solvent to generate gas, thus forming monodisperse chitosan microspheres with a porous structure. The substance that easily generates gas includes ammonium bicarbonate.

[0018] In the above-mentioned method for preparing long-lasting, non-embolizing filled monodisperse chitosan microspheres, the external phase fluid prepared in step (1) also contains a dehydrating substance. When the external phase fluid used in step (2) contains a dehydrating substance, step (2) prepares monodisperse chitosan microspheres with a porous structure. The dehydrating substance includes ethanol.

[0019] In the above-mentioned method for preparing long-lasting, non-embolizing filled monodisperse chitosan microspheres, the acidic aqueous solvent used to prepare the internal phase fluid is an aqueous acetic acid solution, typically with a concentration of 1 wt.% to 5 wt.%.

[0020] In the above-mentioned method for preparing long-lasting, non-embolizing filled monodisperse chitosan microspheres, the oil phase solvent is soybean oil, n-octanol, or liquid paraffin, and the oil-soluble surfactant is Span80, Span85, Twen80, or polyglycerol condensed castor oil.

[0021] In the above-mentioned method for preparing long-lasting, non-embolizing filled monodisperse chitosan microspheres, the injection tube and the collection tube of the microfluidic device used in step (2) are coaxially arranged. The inner diameter of the outlet of the injection tube is 20-60 μm, and the inner diameter of the receiving tube is 100-300 μm. A feasible structure of the microfluidic device is as follows: Figure 1As shown, it includes an injection tube, a connecting tube, and a collection tube, and is used in conjunction with an injection pump; the injection tube is made of a cylindrical glass capillary tube, with its tail drawn into a conical shape; the collection tube is a cylindrical glass capillary tube; the connecting tube is a square glass tube with a square through hole in its center; the tail of the injection tube is inserted into the head of the collection tube and connected through the connecting tube; the injection tube, connecting tube, and collection tube are arranged coaxially.

[0022] In the above-described method for preparing long-lasting, non-clogging filled monodisperse chitosan microspheres, the size of the water-in-oil emulsion can be adjusted by regulating the channel size of the microfluidic device, the flow rates of the internal and external phase fluids, and the polymer concentration in the internal phase fluid. The size of the chitosan microspheres can then be adjusted by regulating the size of the water-in-oil emulsion. The specific values ​​of these factors can be flexibly adjusted according to the actual application requirements for the chitosan microsphere size.

[0023] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0024] 1. This invention provides a long-lasting, non-embolic filling monodisperse chitosan microsphere. The matrix material of the microsphere is cross-linked chitosan, which is formed by a cross-linking reaction of component A, or component A and component B with a cross-linking agent. Component A is at least one of chitosan and modified chitosan, and component B is at least one of hyaluronic acid and collagen. The coefficient of variation of the microsphere particle size does not exceed 5%. Because the matrix material of the microsphere is formed by a cross-linking reaction of component A and a cross-linking agent, the chitosan or modified chitosan used as component A has excellent biocompatibility and biodegradability. Its degradation time is adjustable and controllable. By selecting component A, the degradation time of the microsphere can be controlled within 0.5 to 2 years, thus maintaining its filling effect for a longer period, solving the problem of rapid degradation and short retention time of hyaluronic acid filling effect. Simultaneously, the excellent monodispersity of the microsphere effectively avoids the vascular embolism problem caused by the irregular shape and uneven size of existing hyaluronic acid injection filler materials, resulting in higher safety.

[0025] 2. The long-lasting, non-embolizing filled monodisperse chitosan microspheres provided by this invention have excellent absorption properties because the matrix material of the microspheres is formed by the cross-linking reaction of component A and a cross-linking agent. The polymer network formed by the reaction of chitosan or modified chitosan as component A with the cross-linking agent has excellent water absorption properties. Therefore, the microspheres have good absorption performance. Furthermore, by introducing a porous structure into the microspheres, the water absorption capacity and water absorption rate of the microspheres can be improved, and the water absorption volume can reach several times the volume of the microspheres themselves. Compared with existing hyaluronic acid injection filling products, it can produce a better wrinkle improvement effect.

[0026] 3. The long-lasting, non-embolizing filled monodisperse chitosan microspheres provided by the present invention have uniform particle size, ranging from 20 to 120 μm, and excellent monodispersity, which is beneficial for using smaller diameter injection needles and reducing skin trauma.

[0027] 4. The matrix material of the long-lasting, non-embolizing monodisperse chitosan microspheres provided by this invention is mainly chitosan or modified chitosan, formed by adding at least one of collagen, hyaluronic acid, and gelatin through a cross-linking reaction. Collagen enhances the filling effect, gelatin promotes collagen regeneration, and hyaluronic acid strengthens water retention. The matrix material is a polymeric network framework formed by the cross-linking reaction of chitosan or cross-linked chitosan with a cross-linking agent. The polymers formed by the reaction of collagen, hyaluronic acid, and gelatin with the cross-linking agent are distributed in the framework. The degradation time of the framework is long, while the reaction of collagen, hyaluronic acid, and gelatin with the cross-linking agent is slow, resulting in a relatively lower degree of cross-linking and a relatively faster degradation rate. Therefore, the remaining porous framework after degradation will remain in vivo for a relatively longer time, which is beneficial for cell adhesion and growth. All of these factors can improve the filling effect. Therefore, the chitosan microspheres provided by this invention have broad application prospects in the field of medical aesthetic filling.

[0028] 5. This invention also provides a method for preparing the above-mentioned long-lasting, non-embolizing filled monodisperse chitosan microspheres. This method has a simple production process, low production cost, and can achieve continuous production of low-dose, high-yield products. Furthermore, the microspheres prepared by this method have uniform and flexibly controllable sizes, allowing for precise control of microsphere size according to actual application requirements. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of the primary capillary microfluidic device used in this invention. In the diagram, 1-injection tube, 2-connecting tube, and 3-collection tube.

[0030] Figure 2 This is an optical image of the W / O emulsion prepared in Example 1.

[0031] Figure 3 These are optical images of the chitosan microspheres prepared in Example 1.

[0032] Figure 4 Figures (A) and (B) show the diameter (Diamiter) and coefficient of variation (CV) of the W / O emulsion and chitosan microspheres prepared in Example 1 as a function of the flow rate ratio (V0) of the external and internal phase fluids. o / V i The changing curve.

[0033] Figure 5 This is a scanning electron microscope image of the chitosan microspheres prepared in the second group of experiments in Example 1 after air drying.

[0034] Figure 6 This is a scanning electron microscope image of the cross-section of the chitosan microspheres prepared in the second group of experiments in Example 1 after air drying.

[0035] Figure 7 These are optical images of the W / O emulsion prepared in Example 2.

[0036] Figure 8 These are optical images of the chitosan microspheres prepared in Example 2.

[0037] Figure 9 Figures (A) and (B) show the diameter (Diamiter) and coefficient of variation (CV) of the W / O emulsion and chitosan microspheres prepared in Example 2 as a function of the flow rate ratio (VL) of the external and internal phase fluids. o / V i The changing curve.

[0038] Figure 10 This is a scanning electron microscope image of the chitosan microspheres prepared in the fourth group of experiments in Example 2 after air drying. Detailed Implementation

[0039] The following examples further illustrate the long-lasting, non-embolizing filled monodisperse chitosan microspheres and their preparation method provided by the present invention. It should be noted that the following examples are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0040] In the following embodiments, the microfluidic device used is a single-stage capillary microfluidic device, the structural schematic of which is shown in the figure. Figure 1As shown, the assembly includes an injection tube 1, a connecting tube 2, and a collection tube 3, and is used in conjunction with an injection pump. The injection tube 1 is made of a cylindrical glass capillary tube. The tail end of the cylindrical glass capillary tube is drawn into a conical shape using a needle puller, and then polished on sandpaper until the inner diameter of the conical opening is approximately 20–60 μm. The outer diameter of its cylindrical section is 960 μm, and the inner diameter is 550 μm. The collection tube 3 is also made of a cylindrical glass capillary tube, with both ends polished smooth. The outer diameter of the collection tube is 960 μm, and the inner diameter is 150–300 μm. The connecting tube 2 is a square glass tube, with both ends polished smooth. A square through-hole with a size of 1.0 × 1.0 mm is located in the center. After fabrication, the injection tube 1, connecting tube 2, and collection tube 3 are ultrasonically cleaned in anhydrous ethanol and then dried. The tail end of the injection tube 1 is inserted into the head end of the collection tube 2 and connected via the connecting tube 3. Injection tube 1, connecting tube 2, and collection tube 3 are coaxially arranged and fixed to the glass slide with AB glue. A steel tube can be fitted onto injection tube 1, and the non-inlet end is fixed with AB glue to facilitate connection to the injection pump. The inlet end of connecting tube 2 is fixed with a flat-tipped needle with AB glue, and the non-inlet end of connecting tube 2 is sealed with AB glue. Each flat-tipped needle is connected to the injection pump through a fitting.

[0041] Example 1

[0042] In this embodiment, monodisperse chitosan microspheres of different particle sizes were prepared by changing the experimental conditions. The steps are as follows:

[0043] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0044] Preparation of the inner phase fluid: Chitosan with a molecular weight of 30-200 kDa, a degree of deacetylation of 75%-95%, and a viscosity of 100-500 mPa·s was dissolved in 2 wt.% glacial acetic acid, filtered, and allowed to stand to defoam, thus obtaining the inner phase fluid; the concentration of chitosan in the inner phase fluid was 0.02 g / mL.

[0045] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in the external phase fluid was 0.05 g / mL.

[0046] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 0.5 wt.%.

[0047] (2) Preparation of monodisperse chitosan microspheres

[0048] The structure adopted is as follows Figure 1The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan and the crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0049] In this step, a total of eight sets of experiments were conducted:

[0050] The microfluidic apparatus used in the first through sixth groups of experiments was the same: the inner diameter of the conical nozzle of the injection tube was 40 μm, and the inner diameter of the collection tube was 200 μm. The flow rate control conditions were as follows: in the first group of experiments, the internal phase fluid flow rate was 200 μL / h, and the external phase fluid flow rate was 500 μL / h; in the second through sixth groups of experiments, the internal phase fluid flow rate was 100 μL / h, and the external phase fluid flow rates were 500, 750, 1000, 2000, and 2500 μL / h, respectively.

[0051] The microfluidic apparatus used in the first to sixth groups of experiments was the same, with the inner diameter of the conical nozzle of the injection tube being 20 μm and the inner diameter of the collection tube being 150 μm. The flow rate control conditions were as follows: in the seventh to eighth groups of experiments, the flow rate of the inner phase fluid was 40 μL / h, and the flow rate of the outer phase fluid was 2250 and 3200 μL / h, respectively.

[0052] (3) Washing

[0053] The collected liquid from washing the surface of the chitosan microspheres prepared in step (2) with deionized water was used, and the resulting chitosan microspheres were stored in pure water. For subsequent scanning electron microscopy testing, the chitosan microspheres were air-dried before testing.

[0054] Figure 2 These are optical images of the W / O emulsions prepared in this embodiment, where images a to h are optical images of the W / O emulsions prepared in the first to eighth groups of experiments, respectively. Figure 3 These are optical images of the chitosan microspheres prepared in this embodiment, where images a through h are optical images of the chitosan microspheres prepared in the first through eighth groups of experiments, respectively. Figures 2-3 It can be seen that the W / O emulsion and chitosan microspheres have good sphericity and monodispersity.

[0055] Figure 4 Figures (A) and (B) show the diameter (Diamiter) and coefficient of variation (CV) of the W / O emulsion and chitosan microspheres prepared in this embodiment as a function of the flow rate ratio (VL) of the external and internal phase fluids. o / V i The changing curve, from Figure 4It can be seen that the diameter of the chitosan microspheres prepared in this embodiment is 20-90 μm, and the CV value is no more than 3%, indicating that the chitosan microspheres prepared by the method of the present invention have uniform and precisely adjustable size and good monodispersity.

[0056] Figure 5 This is a scanning electron microscope image of the chitosan microspheres prepared in the second group of experiments in this embodiment after air drying. Figure 6 This is a scanning electron microscope (SEM) image of the cross-section of the chitosan microspheres prepared in the second group of experiments in this embodiment after air drying. Figures 5-6 It can be seen that the chitosan microspheres prepared in this embodiment have good sphericity, dense cross-linking, and a small number of pore structures on the surface of the chitosan microspheres.

[0057] Example 2

[0058] In this embodiment, monodisperse chitosan microspheres of different particle sizes were prepared by changing the experimental conditions. The steps are as follows:

[0059] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0060] Preparation of the inner phase fluid: Chitosan with a molecular weight of 30-200 kDa, a degree of deacetylation of 75%-95%, and a viscosity of 100-500 mPa·s was dissolved in 2 wt.% glacial acetic acid, filtered, and allowed to stand to defoam, thus obtaining the inner phase fluid; the concentration of chitosan in the inner phase fluid was 0.04 g / mL.

[0061] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in the external phase fluid was 0.05 g / mL.

[0062] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 0.5 wt.%.

[0063] (2) Preparation of monodisperse chitosan microspheres

[0064] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan and the crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0065] In this step, a total of four sets of experiments were conducted:

[0066] The microfluidic apparatus used in the first to sixth groups of experiments was the same, with the inner diameter of the conical nozzle of the injection tube being 60 μm and the inner diameter of the collection tube being 300 μm. The flow rate control conditions were as follows: in the first to fourth groups of experiments, the flow rates of the inner phase fluid were 600, 400, 300, and 200 μL / h, respectively, and the flow rate of the outer phase fluid was 2000 μL / h.

[0067] (3) Washing and drying

[0068] The collected liquid from washing the surface of the chitosan microspheres prepared in step (2) with deionized water was used, and the resulting chitosan microspheres were stored in pure water. For subsequent scanning electron microscopy testing, the chitosan microspheres were air-dried before testing.

[0069] Figure 7 These are optical images of the W / O emulsions prepared in this embodiment, where images a to d are optical images of the W / O emulsions prepared in the first to fourth groups of experiments, respectively. Figure 8 These are optical images of the chitosan microspheres prepared in this embodiment, where images a through d are optical images of the chitosan microspheres prepared in the first through fourth groups of experiments, respectively. Figures 7-8 It can be seen that the W / O emulsion and chitosan microspheres have good sphericity and monodispersity.

[0070] Figure 9 Figures (A) and (B) show the diameter (Diamiter) and coefficient of variation (CV) of the W / O emulsion and chitosan microspheres prepared in this embodiment as a function of the flow rate ratio (VL) of the external and internal phase fluids. o / V i The changing curve, from Figure 9 It can be seen that the chitosan microspheres prepared in this embodiment have a diameter of 60-120 μm, and the CV value does not exceed 2.5%. The microspheres are uniform in size and have good monodispersity.

[0071] Figure 10 This is a scanning electron microscope image of the chitosan microspheres prepared in the fourth group of experiments in this embodiment after air drying. As can be seen from the image, the chitosan microspheres have good sphericity and a small number of pore structures on their surface.

[0072] Example 3

[0073] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0074] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0075] Preparation of the inner phase fluid: Chitosan with a molecular weight of 30-200 kDa, a degree of deacetylation of 75%-95%, and a viscosity of 100-500 mPa·s was dissolved in 2 wt.% glacial acetic acid, filtered, and allowed to stand to defoam, thus obtaining the inner phase fluid; the concentration of chitosan in the inner phase fluid was 0.04 g / mL.

[0076] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in external phase fluid was 0.1 g / mL.

[0077] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 0.5 wt.%.

[0078] (2) Preparation of monodisperse chitosan microspheres

[0079] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan and the crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0080] In this step, a total of four sets of experiments were conducted:

[0081] The microfluidic apparatus used in the first to fourth groups of experiments was the same, with the inner diameter of the conical nozzle of the injection tube being 60 μm and the inner diameter of the collection tube being 300 μm. The flow rate control conditions were as follows: in the first to fourth groups of experiments, the flow rates of the inner phase fluid were 600, 400, 300, and 200 μL / h, respectively, and the flow rate of the outer phase fluid was 2000 μL / h.

[0082] (3) Washing and drying

[0083] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0084] Example 4

[0085] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0086] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0087] Preparation of the inner phase fluid: Chitosan with a molecular weight of 200-500 kDa, a degree of deacetylation of 75%-95%, and a viscosity of 300-600 mPa·s was dissolved in 2 wt.% glacial acetic acid, filtered, and allowed to stand to defoam, thus obtaining the inner phase fluid; the concentration of chitosan in the inner phase fluid was 0.02 g / mL.

[0088] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in the external phase fluid was 0.05 g / mL.

[0089] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 0.5 wt.%.

[0090] (2) Preparation of monodisperse chitosan microspheres

[0091] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan and the crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0092] In this step, a total of four sets of experiments were conducted:

[0093] The microfluidic apparatus used in the first to fourth groups of experiments was the same, with the inner diameter of the conical nozzle of the injection tube being 60 μm and the inner diameter of the collection tube being 300 μm. The flow rate control conditions were as follows: in the first to fourth groups of experiments, the flow rates of the inner phase fluid were 600, 400, 300, and 200 μL / h, respectively, and the flow rate of the outer phase fluid was 2000 μL / h.

[0094] (3) Washing and drying

[0095] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0096] Example 5

[0097] In this embodiment, monodisperse chitosan microspheres with a porous structure are prepared by the following steps:

[0098] Preparation of the inner phase fluid: Chitosan with a molecular weight of 200–500 kDa, a degree of deacetylation of 75%–95%, and a viscosity of 300–600 mPa·s was dissolved in 2 wt.% glacial acetic acid. After filtration, the solution was allowed to stand to defoam, and then ammonium bicarbonate solution was added to obtain the inner phase fluid. In the inner phase fluid, the concentration of chitosan was 0.02 g / mL, and the concentration of ammonium bicarbonate was 10 wt.%.

[0099] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in the external phase fluid was 0.05 g / mL.

[0100] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 0.5 wt.%.

[0101] (2) Preparation of monodisperse chitosan microspheres with porous structure

[0102] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the inner phase fluid into the injection tube 1 and the outer phase fluid into the collection tube 3. At the outlet of the injection tube, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan and crosslinking agent have fully crosslinked. Simultaneously, during this preparation step, ammonium bicarbonate in the inner phase fluid reacts with acetic acid to generate gas, resulting in monodisperse chitosan microspheres with a porous structure.

[0103] The microfluidic device used in this step has an injection tube with a conical inner diameter of 20 μm and a collection tube with an inner diameter of 150 μm; the internal phase flow rate is controlled at 40 μL / h, and the external phase fluid flow rate is controlled at 2000 μL / h.

[0104] (3) Washing and drying

[0105] The collected liquid on the surface of the chitosan microspheres with porous structure prepared by washing with deionized water (2) was used to store the obtained chitosan microspheres with porous structure in pure water.

[0106] Example 6

[0107] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0108] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0109] Preparation of the inner phase fluid: Chitosan with a molecular weight of 200-500 kDa, a degree of deacetylation of 75%-95%, and a viscosity of 300-600 mPa·s was dissolved in 2 wt.% glacial acetic acid. Then, collagen was added and fully dissolved. After filtration, the mixture was allowed to stand to defoam, and the inner phase fluid was obtained. The concentration of chitosan in the inner phase fluid was 0.04 g / mL, and the concentration of collagen was 0.01 g / mL.

[0110] Preparation of external phase fluid: Span85 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span85 in the external phase fluid was 0.01 g / mL.

[0111] Preparation of the collecting solution: Dissolve the cross-linking agent genipin in n-octanol to obtain the collecting solution; the concentration of the cross-linking agent in the collecting solution is 1 wt.%.

[0112] (2) Preparation of monodisperse chitosan microspheres

[0113] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the chitosan, collagen and crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0114] The microfluidic device used in this step has an injection tube with a conical inner diameter of 40 μm and a collection tube with an inner diameter of 200 μm; the internal phase flow rate is controlled at 100 μL / h, and the external phase fluid flow rate is controlled at 1000 μL / h.

[0115] (3) Washing and drying

[0116] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0117] Example 7

[0118] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0119] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0120] Preparation of the inner phase fluid: Carboxylated chitosan with a molecular weight of 100-200 kDa was dissolved in 2 wt.% glacial acetic acid, then hyaluronic acid was added and fully dissolved. After filtration, the mixture was allowed to stand to defoam, and the inner phase fluid was obtained. The concentration of carboxylated chitosan in the inner phase fluid was 0.07 g / mL, and the concentration of hyaluronic acid was 0.03 g / mL.

[0121] Preparation of external phase fluid: Span85 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span85 in the external phase fluid was 0.005 g / mL.

[0122] Preparation of the collection solution: Dissolve the cross-linking agent proanthocyanidins in n-octanol to obtain the collection solution; the concentration of the cross-linking agent in the collection solution is 0.1 wt.%.

[0123] (2) Preparation of monodisperse chitosan microspheres

[0124] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the carboxylated chitosan, hyaluronic acid and crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0125] The microfluidic device used in this step has an injection tube with a conical inner diameter of 40 μm and a collection tube with an inner diameter of 200 μm; the internal phase flow rate is controlled at 200 μL / h, and the external phase fluid flow rate is controlled at 1500 μL / h.

[0126] (3) Washing and drying

[0127] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0128] Example 8

[0129] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0130] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0131] Preparation of the inner phase fluid: Chitosan with a molecular weight of 30-200 kDa was dissolved in 2 wt.% glacial acetic acid, then type I collagen was added and fully dissolved. After filtration, the mixture was allowed to stand to defoam, and the inner phase fluid was obtained. The concentration of chitosan in the inner phase fluid was 0.19 g / mL, and the concentration of collagen was 0.01 g / mL.

[0132] Preparation of external phase fluid: Span80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Span80 in the external phase fluid was 0.005 g / mL.

[0133] Preparation of the collection solution: Dissolve the cross-linking agent proanthocyanidins in n-octanol to obtain the collection solution; the concentration of the cross-linking agent in the collection solution is 5 wt.%.

[0134] (2) Preparation of monodisperse chitosan microspheres

[0135] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 of the microfluidic device and the external phase fluid into the collection tube 3 of the microfluidic device. At the outlet of the injection tube of the microfluidic device, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until chitosan, type I collagen and crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0136] The microfluidic device used in this step has an injection tube with a conical inner diameter of 40 μm and a collection tube with an inner diameter of 200 μm; the internal phase flow rate is controlled at 200 μL / h, and the external phase fluid flow rate is controlled at 1600 μL / h.

[0137] (3) Washing and drying

[0138] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0139] Example 9

[0140] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0141] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0142] Preparation of the inner phase fluid: Carboxylated chitosan with a molecular weight of 100-200 kDa was dissolved in 2 wt.% glacial acetic acid, then hyaluronic acid and type I collagen were added and fully dissolved. After filtration, the mixture was allowed to stand to defoam, and the inner phase fluid was obtained. The concentration of carboxylated chitosan in the inner phase fluid was 0.07 g / mL, the concentration of hyaluronic acid was 0.02 g / mL, and the concentration of type I collagen was 0.01 g / mL.

[0143] Preparation of external phase fluid: Twen80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Twen80 in external phase fluid was 0.01 g / mL.

[0144] Preparation of the collection solution: Dissolve the cross-linking agent proanthocyanidins in n-octanol to obtain the collection solution; the concentration of the cross-linking agent in the collection solution is 2 wt.%.

[0145] (2) Preparation of monodisperse chitosan microspheres

[0146] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 and the external phase fluid into the collection tube 3. At the outlet of the injection tube, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until the carboxylated chitosan, hyaluronic acid, type I collagen and crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0147] The microfluidic device used in this step has an injection tube with a conical inner diameter of 60 μm and a collection tube with an inner diameter of 200 μm; the internal phase flow rate is controlled at 500 μL / h, and the external phase fluid flow rate is controlled at 2000 μL / h.

[0148] (3) Washing and drying

[0149] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

[0150] Example 10

[0151] In this embodiment, the preparation of monodisperse chitosan microspheres is carried out through the following steps:

[0152] (1) Prepare the internal phase, external phase fluid and the collection liquid.

[0153] Preparation of the inner phase fluid: Chitosan with a molecular weight of 100-300 kDa was dissolved in 5 wt.% glacial acetic acid. Hyaluronic acid, type I collagen, and gelatin were then added and fully dissolved. After filtration, the mixture was allowed to stand to defoam, yielding the inner phase fluid. The concentration of chitosan in the inner phase fluid was 0.014 g / mL, the concentration of hyaluronic acid was 0.03 g / mL, the concentration of type I collagen was 0.03 g / mL, and the concentration of gelatin was [not specified].

[0154] Preparation of external phase fluid: Twen80 was dissolved in n-octanol to obtain external phase fluid; the concentration of Twen80 in external phase fluid was 0.01 g / mL.

[0155] Preparation of the collection solution: Dissolve the cross-linking agent proanthocyanidins in n-octanol to obtain the collection solution; the concentration of the cross-linking agent in the collection solution is 5 wt.%.

[0156] (2) Preparation of monodisperse chitosan microspheres

[0157] The structure adopted is as follows Figure 1 The microfluidic device shown uses an injection pump to input the internal phase fluid into the injection tube 1 and the external phase fluid into the collection tube 3. At the outlet of the injection tube, a monodisperse W / O emulsion is formed by shearing. The monodisperse W / O emulsion is collected in a container filled with the collection liquid and allowed to stand until chitosan, hyaluronic acid, type I collagen, gelatin and crosslinking agent are fully crosslinked to obtain monodisperse chitosan microspheres.

[0158] The microfluidic device used in this step has an injection tube with a conical inner diameter of 60 μm and a collection tube with an inner diameter of 200 μm; the internal phase flow rate is controlled at 500 μL / h, and the external phase fluid flow rate is controlled at 2000 μL / h.

[0159] (3) Washing and drying

[0160] The collected liquid on the surface of the chitosan microspheres prepared by (2) was washed with deionized water, and the resulting chitosan microspheres were stored in pure water.

Claims

1. A long-acting, non-embolic, filling monodisperse chitosan microspheres, characterized in that, The microspheres have a base material of cross-linked chitosan formed by cross-linking reaction of component A and component B with a cross-linking agent; the component A is at least one of chitosan and modified chitosan, the modified chitosan includes carboxymethyl chitosan, and the component B is at least one of hyaluronic acid, collagen and gelatin; the microspheres have a particle size of 20-120 μm, and a coefficient of variation of the particle size of the microspheres is not more than 5%; the microspheres have a high molecular network formed by cross-linking reaction of the component A and the cross-linking agent as a skeleton, and a high molecule formed by cross-linking reaction of the component B and the cross-linking agent is distributed in the skeleton, and a mass ratio of the component A to the component B is (50-95):(50-5); The microspheres are prepared by the following method: (1) preparing an internal phase fluid: dissolving or sufficiently dispersing a polymer in an acidic aqueous phase solvent to obtain the internal phase fluid; in the internal phase fluid, the concentration of the polymer is 0.001-0.2 g / mL; the polymer is the component A and the component B; preparing an external phase fluid: dissolving an oil-soluble surfactant in an oil phase solvent to obtain the external phase fluid; in the external phase fluid, the concentration of the oil-soluble surfactant is 0.05-10 g / mL; preparing a collection liquid: dissolving a cross-linking agent in the oil phase solvent to obtain the collection liquid; in the collection liquid, the concentration of the cross-linking agent is 0.1 wt.%-5 wt.%; the oil phase solvent is n-octanol, and the cross-linking agent is genipin or procyanidin; (2) inputting the internal phase fluid into a syringe of a microfluidic device, inputting the external phase fluid into a collection tube of the microfluidic device, shearing at an outlet of the syringe of the microfluidic device to form monodisperse water-in-oil emulsions, collecting the water-in-oil emulsions by using a container containing the collection liquid, and obtaining monodisperse chitosan microspheres after sufficient cross-linking reaction of the polymer and the cross-linking agent; (3) washing to remove the collection liquid on the surface of the monodisperse chitosan microspheres; In the internal phase fluid prepared in step (1), the internal phase fluid contains a gas-producing substance when the internal phase fluid is used in step (2), and the gas-producing substance in the internal phase fluid reacts with the acidic aqueous phase solvent to produce gas during the preparation in step (2), that is, monodisperse chitosan microspheres with a porous structure are formed; or, the external phase fluid prepared in step (1) also contains a dehydrating substance, and the external phase fluid contains the dehydrating substance when the external phase fluid is used in step (2), and monodisperse chitosan microspheres with a porous structure are prepared in step (2).

2. The long-acting, non-embolic, filling monodisperse chitosan microspheres according to claim 1, characterized by, The microspheres have a degradation time of 0.5-2 years, and the degradation time of the microspheres can be adjusted by adjusting the molecular weight and molecular chain structure of the cross-linked chitosan.

3. The long-acting, non-embolic, filling monodisperse chitosan microspheres according to claim 1, wherein, The gas-producing substance includes ammonium bicarbonate, and the dehydrating substance includes ethanol.

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