Hydroxyapatite composite microsphere, injection filling preparation and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a hydroxyapatite composite microsphere, an injection filling preparation and a preparation method and application of the hydroxyapatite composite microsphere and the injection filling preparation. The hydroxyapatite composite microsphere comprises the following components in final concentration: a water phase: 1wt%-3wt% of hydroxyapatite, 5wt%-10wt% of recombinant collagen, 1wt%-5wt% of reduced glutathione, 3wt%-3.5 wt% of a cross-linking agent EDC, and 1.5 wt%-1.75 wt% of NHS; the oil phase is prepared by dissolving span 80 and Tween 80 in liquid paraffin, so that the final concentrations of span 80 and Tween 80 are respectively 3wt%-5wt% and 1wt%. The invention also discloses a preparation method of the hydroxyapatite composite microsphere, and an injection filler containing the hydroxyapatite composite microsphere. According to the multi-heavy-component modified hydroxyapatite, the problems of poor hydrophilicity, irregular shape and the like of the existing hydroxyapatite microsphere material are solved, so that the product is uniform in sphere shape and controllable in particle size specification, and has good biocompatibility.

Description

Technical Field

[0001] This application belongs to the field of biomedical materials technology, and in particular relates to a hydroxyapatite composite microsphere, an injection filling formulation, and its preparation method and application. Background Technology

[0002] The use of soft tissue fillers has received increasing attention, with short-acting fillers including hyaluronic acid and collagen. Hyaluronic acid, a first-generation facial filler, degrades rapidly, making it difficult to maintain its clinical efficacy after injection and requiring repeated injections, increasing safety risks. Collagen, a large protein molecule and an important component of the extracellular matrix, is gradually broken down by collagenase after injection, also requiring repeated injections to see better results. These short-acting fillers generally degrade too quickly in the body, requiring multiple touch-ups, increasing the burden on consumers. Permanent implantable products (such as Artefill) have drawbacks: the collagen solution component gradually degrades, non-degradable PMMA microspheres pose a risk of foreign body granulomas, and they can form crystals deep within the skin that cannot degrade, resulting in foreign body irritation and poor biocompatibility.

[0003] Currently, hydroxyapatite calcium filler products have been approved for marketing. These products generally contain hydroxyapatite calcium microspheres, matrix components, and selectively added anesthetic components. As the matrix components degrade, the hydroxyapatite calcium microspheres can stimulate the production of endogenous collagen. Chinese patents CN 111249189 A and CN 111544656 A disclose injectable fillers formed by mixing hydroxyapatite with a collagen-containing gel matrix; Chinese patent CN102492032 A discloses a soft tissue filler material formed by mixing hydroxyapatite with sodium hyaluronate and recombinant collagen. The above-mentioned patented technologies use hydroxyapatite as the single microsphere component, which has poor hydrophilicity, irregular shape, and is prone to adverse reactions. Furthermore, it is difficult to distribute evenly, and the hydroxyapatite particles are prone to displacement and accumulation during injection. In comparison, regularly shaped microsphere materials, due to their unique advantages such as good flowability, show better application prospects.

[0004] Currently, traditional methods for preparing microsphere fillers mostly produce single-component biodegradable polymers. There are no reports on the preparation of three-component composite microsphere fillers formed by combining collagen with hydroxyapatite and reduced glutathione. Summary of the Invention

[0005] Purpose of the invention: In view of the above-mentioned prior art, this application provides a hydroxyapatite composite microsphere, an injection filling formulation, its preparation method and application.

[0006] Technical solution: The hydroxyapatite composite microspheres described in this application comprise the following components at the following final concentrations: Aqueous phase: hydroxyapatite 1wt%-3wt%, recombinant collagen 5wt%-10wt%, reduced glutathione 1wt%-5wt%, crosslinking agent EDC 3wt%-3.5wt%, NHS 1.5wt%-1.75wt%; Oil phase: Span 80 and Tween 80 dissolved in liquid paraffin to final concentrations of 3wt%-5wt% and 1wt%, respectively.

[0007] As a technical solution, the volume ratio of the aqueous phase to the oil phase is 1:4-5, preferably 1:5.

[0008] In some embodiments, the volume ratio of the aqueous phase to the oil phase is 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, or 1:5.

[0009] As a technical solution, the mass ratio of EDC to NHS is 2:1.

[0010] As one technical solution, the final concentration of hydroxyapatite is 3wt%, the final concentration of recombinant collagen is 10wt%, and the final concentration of reduced glutathione is 1wt%-5wt%, preferably 2.5wt%-5wt%.

[0011] In some embodiments, the final concentration of the reduced glutathione is 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3 wt%, 3.1 wt%, 3.2 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, and 5 wt%.

[0012] As a technical solution, the recombinant collagen is recombinant type III collagen, and its amino acid sequence is shown in SEQ ID NO: 1.

[0013] The method for preparing hydroxyapatite composite microspheres described in this application involves crosslinking an aqueous phase and an oil phase, specifically including the following steps: (1) Preparation of aqueous phase: Prepare the prescribed amount of hydroxyapatite aqueous solution, sonicate in water bath, add the prescribed amount of recombinant collagen and reduced glutathione and shake to mix; then add the prescribed amount of EDC and NHS respectively and mix to obtain the aqueous phase; (2) Preparation of oil phase: Dissolve the prescribed amounts of Span 80 and Tween 80 in liquid paraffin, stir and mix well to obtain the oil phase; (3) Slowly drop the above-mentioned mixed aqueous phase into the oil phase, and emulsify and crosslink at room temperature while stirring. (4) Centrifuge to remove supernatant, transfer the sample to a dialysis bag, dialyze in distilled water for 2-3 days, concentrate, pre-freeze at -80°C overnight, and freeze-dry to obtain hydroxyapatite composite microspheres.

[0014] As a technical solution, in step (1), the water bath is ultrasonic for 20 minutes.

[0015] As a technical solution, in step (2), the mixing speed is 500-1000 rpm.

[0016] As a technical solution, in step (3), the emulsification crosslinking is carried out for 6 hours and the stirring speed is set to 500-1000 rpm.

[0017] As a technical solution, in step (4), the sample is transferred to a 14kD dialysis bag, dialyzed in distilled water for 2-3 days (changing the water 4-5 times a day), concentrated, pre-frozen at -80°C overnight, and freeze-dried for 1-2 days.

[0018] As a technical solution, in step (4), centrifugation includes three steps: 3000g rotation speed for 10min; washing with 4-5 times the volume of petroleum ether three times and centrifuging (1000g rotation speed for 5min); washing with 4-5 times the volume of 50% ethanol three times and centrifuging (1000g rotation speed for 5min).

[0019] This application also discloses the application of the hydroxyapatite composite microspheres in medical aesthetics, plastic surgery, filling, and tissue defect repair. In particular, its application in filler materials, such as injectable fillers for medical aesthetics.

[0020] This application discloses an injectable filler formulation comprising the hydroxyapatite composite microspheres.

[0021] As one technical solution, the injectable filling formulation comprises hydroxyapatite composite microspheres and sodium hyaluronate gel, wherein the mass ratio of the hydroxyapatite composite microspheres to the sodium hyaluronate gel is 1:6.

[0022] The method for preparing the injectable filler formulation described in this application includes the following steps: (1) Preparation of sodium hyaluronate gel: Sodium hyaluronate was dissolved in sodium hydroxide and stirred until it was completely dissolved to obtain a sodium hyaluronate solution; BDDE was added to the mixed solution for cross-linking reaction; after the reaction was completed, the solution was dialyzed, and then homogenized after adding PBS buffer to obtain sodium hyaluronate gel. (2) After mixing hydroxyapatite composite microspheres with sodium hyaluronate gel, the mixture is degassed and sterilized to obtain an injectable filler containing composite microspheres.

[0023] The injectable filler formulation described in this application can be used for filling and repairing skin tissue or soft tissue, such as for filling and repairing the face, trunk or limbs, and can also be used for filling one or more of the following: tear troughs, crow's feet, stretch marks, temples, nose, chin, nasolabial folds, corners of the mouth, forehead and temples, neck, etc.

[0024] Beneficial effects: Compared with the prior art, the hydroxyapatite composite microspheres of this application have the following advantages: (1) By modifying hydroxyapatite with multiple components, the problems of poor hydrophilicity and irregular shape of existing hydroxyapatite microsphere materials are solved, so that the product spheres have uniform shape and controllable particle size, and have good biocompatibility; (2) At the same time, in response to the problem of single microsphere components, recombinant collagen and reduced glutathione are added simultaneously. Through cross-linking reaction, the molecules of reduced glutathione and recombinant collagen are extended (such as the -COOH of GSH and the -NH2 of collagen forming amide bonds (-CONH-)). While taking into account the advantages of reduced glutathione and recombinant collagen, the problem of poor stability of reduced glutathione is solved, and the dual effect of "anti-wrinkle + brightening" is achieved; For example, the macromolecular network of collagen can form a "physical barrier" to isolate oxygen, ROS and GSH from direct contact, and reduce GSH (2) The oxidation of the reduced glutathione enhances its antioxidant properties, achieving a 1+1>2 effect; (3) The hydroxyapatite composite microspheres can be better dispersed in the gel, reducing the hardening caused by microsphere accumulation and the ineffective filling due to insufficient microsphere quantity, ensuring the smoothness and continuity of the injection process, and facilitating clinical injection operations.

[0025] The hydroxyapatite composite microspheres of this application have a triple effect: First, the gel carrier provides immediate filling; second, the cross-linked microspheres work continuously to stimulate collagen regeneration, achieving a long-lasting filling effect, while the antioxidant properties of GSH can reduce inflammatory reactions during injection and have whitening and brightening effects; third, after the cross-linked microspheres are gradually absorbed, the regenerated collagen will gradually fill the space of the original microspheres, exerting a continuous filling effect. Attached Figure Description

[0026] Figure 1 This is an optical microscope image (10X) of the hydroxyapatite composite microspheres prepared in Example 1. Figure 2 This is an optical microscope image (40X) of hydroxyapatite in Comparative Example 1. Figure 3 This is the result of DPPH free radical scavenging rate; Figure 4 This is the result of tyrosinase inhibition rate; Figure 5 This is the result of subcutaneous implantation in a rabbit. Detailed Implementation

[0027] The technical solution of this application will be described in detail below with reference to specific embodiments.

[0028] In the examples, hydroxyapatite was purchased from Shaanxi Haibo Biotechnology Co., Ltd., product number HB-QJLHS02; sodium hyaluronate was purchased from Bloomage Biotechnology Co., Ltd., product number HA-EP-N2.5; reduced glutathione, liquid paraffin, Tween 80, Span 80, EDC, and NHS were all commercially available products.

[0029] Preparation of recombinant type III collagen (1) Obtain the amino acid sequence of recombinant collagen. The sequence was obtained by splicing the active fragment from human type III collagen (UniProt database number: P02461). The amino acid sequence is shown in SEQ ID NO: 1. The amino acid sequence of recombinant type III collagen (SEQ ID NO: 1): MHHHHHHENLYFQGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGI PGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGE KGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGP AGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAAGERGAPGFRGPAGPNGIPGEKGPAGER (2) The pET28a-rCol plasmid with the target protein amino acid sequence was transformed into E. coli BL21 (DE3) competent cells (C504-02, purchased from Nanjing Novizan Biotechnology Co., Ltd.) to prepare the strain seed liquid. The fermentation broth was obtained by shaking flask inoculation and induction. The cells were collected, ultrasonically disrupted, and centrifuged to obtain the cell supernatant. The fusion protein (His tag-TEV restriction site-recombinant collagen) was obtained by nickel column purification. After TEV protease digestion (JE1006-00, purchased from Nanjing Novizan Biotechnology Co., Ltd.), the His tag, TEV protease and other impurities at the front end were removed by gel filtration chromatography to obtain the purified recombinant type III collagen.

[0030] Examples 1 to 13, Comparative Examples 1 to 5 Hydroxyapatite composite microspheres were prepared according to the components and ratios shown in Tables 1 and 2.

[0031] Table 1. Microsphere components and ratios from Examples 1 to 13

[0032] Table 2. Microsphere components and ratios of Comparative Examples 1 to 5

[0033] Note: 0% means no addition.

[0034] The specific preparation method of hydroxyapatite composite microspheres includes the following steps: (1) Aqueous phase preparation: Prepare aqueous solutions of hydroxyapatite with the concentrations (mass fractions) shown in Tables 1 and 2, sonicate in a water bath for 20 min, add recombinant type III collagen and reduced glutathione and shake to mix, then add EDC and NHS respectively and mix. (2) Preparation of oil phase: Prepare oil phase according to the volume ratio of water phase to oil phase, dissolve Span 80 and Tween 80 in liquid paraffin, stir and mix well, stirring at 500-1000 rpm; (3) The above-mentioned mixed aqueous phase was slowly dripped into the oil phase, and emulsified and crosslinked at room temperature with stirring for 6 hours. The rotation speed was set as shown in Table 1 and Table 2. (4) Centrifuge to remove supernatant (3000g, 10min); wash with 4-5 times volume of petroleum ether and centrifuge three times (1000g, 5min); wash with 4-5 times volume of 50% ethanol and centrifuge three times (1000g, 5min). (5) The sample was transferred to a 14kD dialysis bag and dialyzed in distilled water for 2-3 days (changing the water 4-5 times a day). After concentration, it was pre-frozen at -80°C overnight and freeze-dried for 1-2 days to obtain microspheres.

[0035] Example 8 Microsphere Performance Testing (1) Optical microscope photographs of microspheres The composite microspheres prepared in Example 1 and the hydroxyapatite in Comparative Example 1 were observed under an optical microscope. The results are as follows: Figure 1 and Figure 2 As shown in the figure, the composite microspheres prepared in Example 1 have a regular morphology and uniform particle size, ranging from 20 to 100 μm; while the hydroxyapatite has an irregular morphology and is agglomerated.

[0036] (2) Microsphere pushing force test ①Preparation of sodium hyaluronate gel 150 mg / ml sodium hyaluronate was prepared by dissolving sodium hyaluronate in 0.25 M sodium hydroxide solution and stirring until the sodium hyaluronate was completely dissolved. 10% BDDE was added to the mixed solution, and a cross-linking reaction was carried out at 30 °C for 20 h. After the reaction was completed, the solution was dialyzed, and after adding PBS buffer, it was homogenized to obtain 15 mg / ml sodium hyaluronate gel. ② The microspheres prepared in Examples 1-13 and Comparative Example 1 were mixed with HA hydrogel at a mass ratio of 1:6, and the pushing force was measured using a 27G needle.

[0037] The results are shown in Table 3. The average pushing force of the prepared composite microsphere gel (Examples 1-13) is smaller, indicating that the composite microspheres are uniformly dispersed in the gel without clumping. Compared with hydroxyapatite alone, the composite microspheres improve hydrophilicity and reduce pushing force.

[0038] Table 3 Propellant Force Test Results

[0039] Example 9: Determination of antioxidant properties The antioxidant properties of the samples prepared in Examples 1, 2, 4, Comparative Example 2, and Comparative Example 3 were determined. The determination method was the DPPH free radical scavenging test. 3 mg of DPPH powder was weighed, dissolved in anhydrous ethanol, and the volume was adjusted to 50 mL. The reaction solutions from each group in the table below were accurately transferred to 5 mL EP tubes, mixed thoroughly, and reacted in the dark for 30 min. The absorbance was then measured at 517 nm. The experiment was repeated in triplicate, and the scavenging rate was calculated. The reaction solutions for the blank group, test group, and control group are shown in Table 4.

[0040]

[0041] Table 4. DPPH Free Radical Scavenging Rate Test Reaction Solution

[0042] The results are as follows Figure 3 As shown, the DPPH scavenging rate test results indicate that the DPPH scavenging rate of the samples gradually increased with the increase of the reduced glutathione concentration in Examples 1, 2, and 4, with Example 1 showing the highest scavenging rate at 80.67%. When the amount of reduced glutathione added to the microspheres was 0, there was no difference in scavenging rate between the sample and the blank control group. At the same amount of reduced glutathione added, the scavenging rate of Example 3 was higher than that of Comparative Example 3, which had a scavenging rate of only 14%. Therefore, the preparation of composite microspheres improves the antioxidant properties of reduced glutathione.

[0043] Example 10 Tyrosinase Inhibition Rate The tyrosinase inhibition rate of the samples prepared in Examples 1, 2, 4, Comparative Example 2, and Comparative Example 3 was determined. The determination methods are shown in Table 5: 9.85 mg of L-levodopa was weighed and dissolved in 10 mL of PB. For the tyrosinase solution, 1.72 mg of tyrosinase was weighed and dissolved in 10 mL of PB. The reaction solutions from each group in the table below were accurately transferred to 96-well plates, and the absorbance of the samples was measured. Each experiment was performed in triplicate, and the average value was taken.

[0044]

[0045] In the formula: A0 is the absorbance of the control group minus the absorbance of the blank group. A1 represents the absorbance of the test sample group minus the absorbance of the blank sample group. Table 5. Procedure for determining tyrosinase inhibition rate

[0046] The results are as follows Figure 4 As shown, the tyrosinase inhibition rate test results indicate that the tyrosinase inhibition rate of the samples increased with the increase of reduced glutathione concentration in Examples 1, 2, and 4, with Example 1 showing the highest inhibition rate at 86%. When the amount of reduced glutathione added to the microspheres was 0, the sample showed no inhibitory effect. At the same amount of reduced glutathione added, the inhibition rate of Example 3 was higher than that of Comparative Example 3, which had an inhibition rate of only 6%. Therefore, the preparation of composite microspheres improved the tyrosinase inhibition rate of reduced glutathione.

[0047] Example 11 An injectable filler formulation is prepared by the following method: (1) Preparation of sodium hyaluronate gel: 150 mg / ml sodium hyaluronate was prepared by dissolving sodium hyaluronate in 0.25 M sodium hydroxide, stirring and mixing until the sodium hyaluronate was completely dissolved to obtain a sodium hyaluronate solution; 10% BDDE was added to the mixed solution, and cross-linking reaction was carried out at 30℃ for 12 h. After the reaction was completed, the solution was dialyzed, and then homogenized after adding PBS buffer to obtain 15 mg / ml sodium hyaluronate gel. (2) The hydroxyapatite composite microspheres prepared in Example 1, Comparative Example 4 and Comparative Example 5 were mixed with sodium hyaluronate gel at a mass ratio of 1:6 and then degassed and sterilized to obtain an injection filler containing composite microspheres.

[0048] Example 12 Subcutaneous Implantation Experiment in Rabbits Experimental methods: The samples prepared in Example 1, Comparative Example 4 and Comparative Example 5 were mixed with sodium hyaluronate gel at a mass ratio of 1:6, and the gel was used as a blank control. The samples were injected subcutaneously into rabbits (about 200 μL / rabbit). The degradation of the implants was observed at 1 week, 4 weeks, 13 weeks, 26 weeks, 52 weeks, 78 weeks and 104 weeks after injection.

[0049] Experimental results are as follows Figure 5 As shown: Test results indicate that the filling effect of Example 1 can be maintained for 24 months. The microspheres combined with gel without hydroxyapatite have a slightly weaker supporting effect and completely degrade after 24 months. Comparative Example 4, due to the lack of cross-linking of components, experienced rapid volume degradation in the early stages, but later, the hydroxyapatite stimulated the body to generate collagen, which had a certain effect on increasing the volume of the implant. In summary, the composite microspheres prepared in this application can stimulate collagen regeneration and achieve a long-lasting filling effect without the need for continuous injections to maintain tissue structure and performance.

Claims

1. A hydroxyapatite composite microsphere, characterized in that, Includes the following components at their final concentrations: Aqueous phase: hydroxyapatite 1wt%-3wt%, recombinant collagen 5wt%-10wt%, reduced glutathione 1wt%-5wt%, cross-linking agent EDC 3wt%-3.5wt%, NHS 1.5wt%-1.75wt%; Oil phase: Span 80 and Tween 80 were dissolved in liquid paraffin to achieve final concentrations of 3wt%-5wt% and 1wt%, respectively.

2. The hydroxyapatite composite microspheres according to claim 1, characterized in that, The volume ratio of the aqueous phase to the oil phase is 1:4-5, preferably 1:

5.

3. The hydroxyapatite composite microspheres according to claim 1, characterized in that, The mass ratio of EDC to NHS is 2:

1.

4. The hydroxyapatite composite microspheres according to claim 1, characterized in that, The final concentration of hydroxyapatite is 3 wt%, the final concentration of recombinant collagen is 10 wt%, and the final concentration of reduced glutathione is 1 wt%-5 wt%, preferably 2.5 wt%-5 wt%.

5. The hydroxyapatite composite microspheres according to claim 1, characterized in that, The recombinant collagen is recombinant type III collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

6. A method for preparing hydroxyapatite composite microspheres according to any one of claims 1-5, characterized in that, The preparation involves crosslinking the aqueous and oil phases, specifically including the following steps: (1) Preparation of aqueous phase: Prepare the prescribed amount of hydroxyapatite aqueous solution, sonicate in water bath, add the prescribed amount of recombinant collagen and reduced glutathione and shake to mix; then add the prescribed amount of EDC and NHS respectively and mix to obtain the aqueous phase; (2) Preparation of oil phase: Dissolve the prescribed amounts of Span 80 and Tween 80 in liquid paraffin, stir and mix well to obtain the oil phase; (3) Slowly drop the above-mentioned mixed aqueous phase into the oil phase, and emulsify and crosslink at room temperature while stirring. (4) Centrifuge to remove supernatant, transfer sample to dialysis bag, dialyze in distilled water for 2-3 days, concentrate, pre-freeze overnight, freeze dry to obtain hydroxyapatite composite microspheres.

7. The application of the hydroxyapatite composite microspheres according to any one of claims 1-5 in medical aesthetics, plastic surgery, filling and tissue defect repair.

8. An injectable filler formulation, characterized in that, It comprises the hydroxyapatite composite microspheres described in any one of claims 1-5.

9. The injectable filler formulation according to claim 8, characterized in that, It comprises hydroxyapatite composite microspheres and sodium hyaluronate gel, wherein the mass ratio of the hydroxyapatite composite microspheres to the sodium hyaluronate gel is 1:

6.

10. The method for preparing the injectable filling formulation according to claim 8 or 9, characterized in that, Includes the following steps: (1) Preparation of sodium hyaluronate gel: Sodium hyaluronate was dissolved in sodium hydroxide and stirred until it was completely dissolved to obtain a sodium hyaluronate solution; BDDE was added to the mixed solution for cross-linking reaction; after the reaction was completed, the solution was dialyzed, and then homogenized after adding PBS buffer to obtain sodium hyaluronate gel. (2) After mixing hydroxyapatite composite microspheres with sodium hyaluronate gel, the mixture is degassed and sterilized to obtain an injectable filler containing composite microspheres.

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