Injectable microsphere-based hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable microsphere-based hydrogel as well as a preparation method and application thereof. The injectable microsphere-based hydrogel is formed by hydration of GM-Lp-coated PDA microspheres and normal saline. The GM-Lap (at) PDA microsphere is in a spherical shape with the particle size within 100 microns, and a macroporous structure is arranged on the GM-Lap (at) PDA microsphere. The preparation method comprises the following steps: embedding dopamine into a limited nano space between sheet layers of a two-dimensional nano clay material to carry out in-situ oxidation, so as to generate a polydopamine intercalated clay nanosheet with a free catechol group; the preparation method comprises the following steps: carrying out free radical polymerization on polydopamine intercalated clay nanosheets and methacrylated gelatin in the presence of a photoinitiator under the condition of ultraviolet irradiation, and carrying out a W / O emulsification method and a gradient freezing method. The dry microspheres are convenient to store and carry outside, high in biological safety and degradable, can be hydrated with normal saline to form injectable microsphere-based hydrogel when needed, can be used as an incompressible wound emergency hemostatic, and is suitable for complex and changeable pre-hospital first-aid scenes.

Description

Technical Field

[0001] The invention relates to a method for preparing an injectable microsphere-based gel used for emergency hemostasis of incompressible wounds, and belongs to the technical field of biomedical materials. Background Art

[0002] Uncontrolled bleeding after trauma causes 500,000 deaths worldwide each year. In both civilian and battlefield settings, more than one-third of hemorrhage deaths are caused by non-compressible intra-abdominal hemorrhage (NCIAH). Currently, NCIAH can only be definitively treated surgically, and there are limited strategies in the pre-hospital setting that can prolong patient survival to allow patients to undergo surgical treatment. In recent years, an injectable gel hemostatic material with tissue adhesion has attracted widespread attention for its ability to treat hard-to-reach wounds and can be used in minimally invasive surgery. This fluid-shaped hemostatic agent can quickly and fully contact and seal the bleeding wound surface, and is particularly suitable for hemostasis of some irregular, non-compressible wounds such as deep, narrow and micro-cavities. However, pre-made liquid gel hemostatic agents are not as convenient to carry and store as solid hemostatic agents, and are not suitable for some complex pre-hospital treatment environments that have high requirements for the storage and application temperature range of hemostatic materials.

[0003] As a product of partial hydrolysis of the natural polymer collagen, gelatin not only has high biocompatibility, no skin irritation, high absorption rate of tissue exudates, ideal cell adhesion, ability to maintain a moist wound environment, etc., but also has the advantages of low immunogenicity and low cost. At present, absorbable gelatin sponge is a commonly used hemostatic material in operating rooms, but it is mostly suitable for external hemostasis of open wounds, and its application scenarios are relatively limited. Therefore, it is very necessary to develop an injectable rapid hemostatic composite material with good biocompatibility and suitable for multiple scenarios. Summary of the invention

[0004] Based on the existing technical problems, the purpose of the present invention is to provide an injectable microsphere-based hydrogel, a preparation method and its use. The microspheres with interconnected macroporous structures are prepared by gradient freezing method using gelatin-based materials (GelMA) as the main raw material. The injectable microsphere-based hydrogel formed by hydration of dry microspheres with physiological saline is suitable for hemostasis of deep, irregular and incompressible wounds in the cavity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: The injectable microsphere-based gel is formed by hydration of GM-Lap@PDA microspheres and physiological saline; the GM-Lap@PDA microspheres are spherical in shape with a particle size of less than 100 μm and have a macroporous structure; the GM-Lap@PDA microspheres are in situ oxidized by embedding dopamine in the limited nanospace between the sheets of two-dimensional nanoclay materials to produce polydopamine intercalated clay nanosheets with free catechol groups; the polydopamine intercalated clay nanosheets and methacryloyl gelatin are free radically polymerized in the presence of a photoinitiator and under ultraviolet light irradiation, and are prepared by W / O emulsification method and gradient freezing method.

[0006] The preparation method of the above-mentioned injectable microsphere-based gel is as follows: (1) Dopamine is embedded in the limited nanospace between the sheets of two-dimensional nanoclay materials and undergoes in situ oxidation, producing polydopamine (PDA) intercalated clay nanosheets with free catechol groups. This not only induces interfacial adhesion, but its physical interaction with the polymer network can also enhance the mechanical properties of the hydrogel.

[0007] (2) Polydopamine (PDA) intercalated clay nanosheets and methacrylated gelatin were free radical polymerized in the presence of a photoinitiator and under ultraviolet light irradiation, and GM-Lap@PDA microspheres were prepared by W / O emulsification and gradient freezing methods.

[0008] (3) Hydrate the microspheres with saline to form an injectable microsphere-based hydrogel.

[0009] The preparation process specifically comprises the following steps: Step 1: Gelatin reacts with methacrylic anhydride to synthesize methacrylated gelatin (GelMA) (Reference: Liang M, Wei D, Ren P, et al. A visible light cross-linked underwater hydrogel adhesive with biodegradation and hemostatic ability[J]. Adv HealthcMater, 2024, 13, 2302538.). GelMA, photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and Tween 60 are dissolved in ultrapure water at 50 °C to form a GelMA aqueous solution.

[0010] In the solution, the concentration of GelMA was 20 wt%; the concentration of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was 0.5 wt%.

[0011] Step 2: Disperse dopamine hydrochloride and nanoclay (Laponite® XLG-XR) in ultrapure water and stir vigorously. Dopamine is embedded in the limited space between the nanoclay sheets for in-situ oxidation to produce polydopamine (PDA) intercalated clay nanosheets with free catechol groups.

[0012] The weight ratio of nanoclay to methacryloyl gelatin GelMA is 15 wt%; the mass ratio of dopamine hydrochloride to methacryloyl gelatin is 0-0.8 wt% and greater than 0; and the stirring time is 5 hours.

[0013] Step 3: Mix the GelMA aqueous solution and the solution in step 2 in equal volumes, stir at 800 rpm and perform ultrasonic defoaming to obtain a uniform solution.

[0014] Step 4: Add the homogeneous solution in step 3 dropwise into liquid paraffin containing Span 80, and stir continuously at high speed to form a W / O type emulsion.

[0015] The volume ratio of the aqueous solution to the liquid paraffin containing Span 80 is 1:10; the rotation speed is 450 rpm; and the stirring time is 20 min.

[0016] Step 5: The emulsion in step 4 is subjected to gradient freezing. The gradient freezing temperature and duration are: 20 min at 4°C, 30 min at -20°C, and 1 min at -196°C.

[0017] Step 6: Expose the frozen emulsion in step 5 to ultraviolet light for photo-crosslinking. Wash the emulsion with acetone to separate the oil phase, dry at room temperature, and collect the microspheres. The ultraviolet irradiation time is 5 min.

[0018] The use of GM-Lap@PDA microspheres as a powder hemostatic agent is to be applied directly to open wounds.

[0019] The above-mentioned GM-Lap@PDA microspheres are used as injectable microsphere-based hydrogels for emergency hemostasis of incompressible wounds. The injectable microsphere-based hydrogel formed by hydration of the microspheres with normal saline is injected into the bleeding site, wherein the mixing ratio of the microspheres and sterile normal saline is 100 mg / 300 μL.

[0020] The principle of the present invention is: using methacrylated gelatin (GelMA), two-dimensional nanomaterials (Laponite® XLG-XR) and dopamine (DA) as the main components, organic-inorganic composite cryogel microspheres are prepared by W / O emulsification and gradient freezing. First, dopamine is embedded in the limited nanospace of the clay sheet for in-situ oxidation to produce polydopamine (PDA) intercalated clay nanosheets with free catechol groups. This can not only induce interfacial adhesion, but also enhance the mechanical properties of the hydrogel through its physical interaction with the polymer network. Secondly, methacrylated gelatin is added, and free radical polymerization is carried out in the presence of a photoinitiator and under ultraviolet light irradiation to finally form GM-Lap@PDA microspheres. The dry microspheres can quickly hydrate with saline to form injectable microsphere-based hydrogels. This gel can be directly injected into irregular, incompressible wounds such as deep and narrow wounds through a 20 G needle, fully contact the bleeding wound surface and can quickly stop bleeding. It has high biosafety and is degradable, and is suitable for complex and changeable pre-hospital emergency scenarios.

[0021] Beneficial effects: The present invention has the following outstanding features: The microspheres of the present invention have simple preparation process, short preparation cycle, easy to obtain raw materials, easy to control performance, and can realize large-scale industrial production. At the same time, the microspheres have a wide temperature range of environmental adaptability, low storage cost and are easy to carry.

[0022] The microspheres prepared by the invention have interconnected macroporous structures, can quickly absorb water and blood, and can be directly used as powder hemostatic agents for open wounds.

[0023] The injectable microsphere-based hydrogel prepared by the present invention, which is formed by hydration of microspheres with physiological saline, can be directly injected into deep, narrow and other irregular incompressible wounds through a 20 G needle, fully contacts the bleeding wound surface and can quickly stop bleeding. It has high biosafety and is degradable, and is suitable for complex and changeable pre-hospital emergency scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The morphology diagram (Figures ac) and particle size distribution diagram (Figures df) of the microspheres prepared according to Example 1 of the present invention; Figure 2 The hydrogel prepared according to Example 1 of the present invention is shown in Figure a), the shear thinning property test result diagram (Figure b), the strain sweep test result diagram (Figure c) and the temperature sweep test result diagram (Figure d); Figure 3 This is a diagram showing the in vivo hemostatic effect of the hydrogel prepared according to Example 1 of the present invention in a SD rat liver bleeding model. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0026] Embodiment 1: Organic-inorganic composite cryogel microspheres were prepared by W / O emulsification and gradient freezing methods. The steps are as follows: (1) Dissolve 1.5 g of GelMA, 37.5 mg of photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and 150 mg of Tween 60 in 7.5 mL of ultrapure water at 50 °C to form a GelMA aqueous solution.

[0027] (2) Disperse 0, 6, and 12 mg of dopamine hydrochloride (corresponding to samples 1, 2, and 3) and 225 mg of nanoclay (Laponite® XLG-XR) in 7.5 mL of ultrapure water, respectively, and stir vigorously at room temperature to mix evenly. The solution without dopamine hydrochloride only needs to be stirred for 1 h to form a clear and transparent aqueous solution, while the solution with dopamine hydrochloride needs to be stirred vigorously for 5 h to form a dark viscous solution.

[0028] (3) The GelMA aqueous solution of step (1) and the solution of step (2) were mixed in equal volumes, stirred at 800 rpm for 10 min and defoamed by ultrasonication.

[0029] (4) The mixed solution of step (3) was added dropwise into liquid paraffin (150 mL) containing 300 mg of Span 80, and stirred continuously at 450 rpm for 20 min to form a W / O emulsion.

[0030] (5) After stirring the emulsion at 4 °C for 20 min, transfer it to -20 °C and freeze it for 30 min. Then, quick freeze it at -196 °C for 1 min.

[0031] (6) The frozen emulsion was exposed to ultraviolet light (365 nm) for 5 min for photocrosslinking. The emulsion was then washed with acetone to separate the oil phase and dried under ventilation at room temperature for 24 h to obtain GM-Lap@PDA microspheres.

[0032] Figure 1 The SEM and optical images in ac show that the three microsphere samples 1 to 3 prepared in Example 1 are all spherical in shape and have a macroporous structure. Figure 1 df shows that the average diameters of the three microspheres of sample 1 to sample 3 increase with the increase of dopamine content, but the average diameters are all within 100 μm, and the particle size distribution is relatively concentrated.

[0033] Embodiment 2: 100 mg of microspheres (samples 1-3) obtained in Example 1 and 0.3 mL of sterile saline were divided into two syringes (2.5 mL) connected by a Luer connector and pushed back and forth until they were mixed into a uniform injectable hydrogel. The rheological properties of the hydrogel were tested using a rotational rheometer. Figure 2 a shows that the pregels prepared from samples 1 to 3 can be injected through a 20 G needle and quickly form hydrogels that follow the shape of the mold in situ. Figure 2 b It can be seen that the gels prepared from samples 1 to 3 all have shear thinning properties. The hydrogels were subjected to oscillatory strain scanning at a constant angular frequency. The results are shown in Figure 2 c. The critical strain of the hydrogel prepared by sample 3 is the highest, which indicates that the presence of polydopamine makes the gel more stable. The temperature stability test of the hydrogels prepared by samples 1 to 3 is shown in Figure 2 d. The storage modulus of the hydrogels prepared from samples 1 to 3 is greater than the loss modulus (G′>G′′) in the temperature range of 10 °C to 60 °C, indicating that the three hydrogels maintain a gel state in this temperature range.

[0034] Embodiment 3:

[0035] The in vivo hemostatic performance of the hydrogel prepared from sample 3 was evaluated using a rat liver injury model ( Figure 3 a). After anesthetizing SD rats (~200 g, 6-8 weeks, male, N = 4), about 15 mm of liver tissue was cut off with surgical scissors. After the wound surface was naturally bleeding for 3 s, the blood was absorbed with sterile gauze, a weighed filter paper was placed under the liver, and 200 μL of the hydrogel prepared by sample 3 was immediately injected into the bleeding site. The bleeding situation of the wound surface was recorded by taking pictures every 30 s, and the filter paper was removed and weighed at 2 minutes to calculate the blood loss. The blank group without material treatment and the commercial hemostatic gelatin sponge group were used as controls. The hydrogel prepared by Example 3 can quickly stop bleeding within 30 s ( Figure 3 b). Compared with the blank control group (0.166 ± 0.020 g) and the commercial hemostatic gelatin sponge (0.119 ± 0.004 g), the blood loss in the hydrogel group was reduced to 0.037 ± 0.003 g within 2 min. This further illustrates that compared with block hemostatic agents, the advantage of injectable gels is that they can fully contact irregularly shaped wounds for rapid hemostasis.

[0036] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An injectable microsphere-based hydrogel, characterized in that GM-Lap@PDA microspheres are hydrated with physiological saline to form injectable microsphere-based hydrogels; GM-Lap@PDA microspheres are spherical in shape with a particle size of less than 100 μm and have interconnected macroporous structures on them; dopamine is embedded in the limited nanospace between the sheets of two-dimensional nanoclay materials for in-situ oxidation to produce polydopamine intercalated clay nanosheets with free catechol groups; polydopamine intercalated clay nanosheets and methacryloyl gelatin are subjected to free radical polymerization in the presence of a photoinitiator and under ultraviolet light irradiation, and GM-Lap@PDA dry microspheres are prepared by W / O emulsification and gradient freezing methods.

2. The method for preparing the injectable microsphere-based hydrogel according to claim 1, characterized in that: (1) Dopamine is embedded in the limited nanospace between the sheets of two-dimensional nanoclay materials and undergoes in situ oxidation, producing polydopamine (PDA) intercalated clay nanosheets with free catechol groups. This not only induces interfacial adhesion, but also enhances the mechanical properties of the hydrogel through its physical interaction with the polymer network. (2) Polydopamine intercalated clay nanosheets and methacrylated gelatin were subjected to free radical polymerization in the presence of a photoinitiator and under ultraviolet light irradiation, and GM-Lap@PDA microspheres were prepared by W / O emulsification and gradient freezing methods; (3) Hydrate the microspheres with saline to form an injectable microsphere-based hydrogel.

3. The method for preparing the injectable microsphere-based hydrogel according to claim 2, characterized in that: Here are the steps: Step 1: Gelatin reacts with methacrylic anhydride to synthesize methacrylic gelatin (GelMA), wherein methacrylic gelatin, a photoinitiator and Tween 60 are dissolved in ultrapure water at 50°C to form a GelMA aqueous solution; Step 2: Disperse dopamine hydrochloride and nanoclay in ultrapure water and stir vigorously. Dopamine is embedded in the limited nanospace of the nanoclay sheet for in-situ oxidation to produce polydopamine intercalated clay nanosheets with free catechol groups. Step 3: Mix the GelMA aqueous solution and the solution in step 2 in equal volumes, stir at high speed and remove bubbles by ultrasound to obtain a uniform solution, which is used as the aqueous phase for later use; Step 4: Add the aqueous phase obtained in step 3 dropwise into the oil phase, wherein the oil phase is liquid paraffin containing Span 80, and stir continuously at high speed to form a W / O type emulsion; Step 5: The emulsion obtained in step 4 is subjected to gradient freezing; the gradient freezing temperature and duration are: 20 min at 4 °C, 30 min at -20 °C, and 1 min at -196 °C; Step 6: exposing the frozen emulsion in step 5 to ultraviolet light for photo-crosslinking; washing the emulsion with acetone to separate the oil phase, and drying at room temperature to obtain GM-Lap@PDA microspheres.

4. The method for preparing the injectable microsphere-based hydrogel according to claim 3, characterized in that: In step 1, in the obtained solution, the concentration of GelMA is 20 wt %; the concentration of the photoinitiator is 0.5 wt %; and the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

5. The method for preparing the injectable microsphere-based hydrogel according to claim 3, characterized in that: In step 2, the weight ratio of nanoclay to methacryloyl gelatin is 15wt%; the mass ratio of dopamine hydrochloride to methacryloyl gelatin is 0-0.8wt% and greater than 0; and the stirring time is 5 hours.

6. The method for preparing the injectable microsphere-based hydrogel according to claim 3, characterized in that: In step 4, the volume ratio of the water phase to the oil phase is 1:10, the stirring speed is 450 rpm, and the stirring time is 20 min.

7. Use of the injectable microsphere-based hydrogel according to claim 1 as an emergency hemostatic agent for incompressible wounds.

8. The use according to claim 7, characterized in that The injectable microsphere-based hydrogel is injected directly into the bleeding site.

Images