CN118903551A - Modularized preparation method of sodium hyaluronate composite gel loaded with degradable microspheres for injection - Google Patents

Abstract

The invention discloses a modularized preparation method of sodium hyaluronate composite gel loaded with degradable microspheres for injection, which introduces a freeze-drying process, through the chemical crosslinking modularized treatment of the sodium hyaluronate in the environment of-50 to-80 ℃, the solid storage of the crosslinked gel is realized. The preparation process can modularly treat the crosslinked gel through single mass production, can be used for subsequent multiple times, and can directly load the microspheres after re-dissolution according to the requirement. Compared with the traditional gel preparation process, the preparation process can directly compound the crosslinked gel and the microspheres, increases the flexibility and convenience of process operation, and after the crosslinked gel is freeze-dried and modularized, the crosslinked sodium hyaluronate gel is stored in a solid state, so that the crosslinked sodium hyaluronate gel can be taken and used at any time, and the gel system can still keep stable physical and chemical properties after being redissolved, thereby ensuring the effectiveness of the product.

Description

Modular preparation method of a sodium hyaluronate composite gel loaded with degradable microspheres for injection

Technical Field

The invention relates to a modular preparation method of a sodium hyaluronate composite gel loaded with degradable microspheres for injection.

Background Art

In recent years, injectable gel systems have played a cosmetic role in filling and regenerating the injection site and achieved minimally invasive treatments such as nasolabial folds, and restoration and/or correction of fat loss in human immunodeficiency virus (HIV) patients. Currently, most of the filling and regeneration products on the market are cross-linked or non-cross-linked sodium hyaluronate gel composite regeneration microspheres, such as polylactic acid (PLA), poly-L-lactic acid (PLLA), polycaprolactone (PCL), hydroxyapatite (CaHA), etc.

Patent CN201510593332.X discloses a method for preparing a mixed gel of microspheres and cross-linked sodium hyaluronate. In the patent, the microsphere material is mixed with the cross-linked sodium hyaluronate gel to prepare a gel-microsphere complex. In the method described in the patent, the gel is directly compounded with the microsphere material after cross-linking. The preparation process is continuous and cannot be used as needed. Therefore, it is necessary to further adjust the preparation process of the cross-linked sodium hyaluronate gel composite microspheres.

Summary of the invention

The purpose of the present invention is to provide a modular preparation method for a sodium hyaluronate composite gel loaded with degradable microspheres for injection. This technology introduces a freeze-drying process, freeze-drying the cross-linked sodium hyaluronate in a modular manner, produces a single batch, and stores it in a solid state, so that the cross-linked hyaluronic acid can be used immediately and the process flexibility is improved. The present invention is achieved through the following technical solutions;

The present invention discloses a modular preparation method of a sodium hyaluronate composite gel loaded with degradable microspheres for injection, comprising the following steps:

1) Sodium hyaluronate is dissolved in an alkaline solution and then cross-linked, and the cross-linked sodium hyaluronate gel is dialyzed for 4 to 8 days and then freeze-dried to obtain a freeze-dried gel;

2) crushing the freeze-dried gel into gel particles, fully re-dissolving the gel particles at a certain concentration to obtain a cross-linked gel, adding microspheres and sodium hyaluronate solution in a specific ratio to the cross-linked gel and mixing them evenly to obtain a mixed gel;

3) Filling the mixed gel into a pre-filled syringe and sterilizing it with high-pressure steam to obtain a sodium hyaluronate composite gel loaded with degradable microspheres for injection.

As a further improvement, the freeze-drying temperature of the cross-linked gel of the present invention is -50 to -80°C, the storage temperature is 0 to -80°C, the reconstitution solution of the freeze-dried gel is phosphate buffered saline or physiological saline, the pH is 6.8 to 7.5, and the osmotic pressure is 280 to 340 mOsm/L.

As a further improvement, the mass fraction of the microsphere material of the present invention in the mixed gel is 5 to 30%; the average particle size of the microsphere material is 10 to 100 μm.

As a further improvement, the alkaline solution described in the present invention is any one of sodium hydroxide and potassium hydroxide, and the solution concentration is 0.1-5.0 mol/L.

As a further improvement, the molecular weight of the sodium hyaluronate added in step 2) of the present invention is 500,000 to 3,000,000 Da, the concentration of sodium hyaluronate in the cross-linked gel is 1 to 15%, and the mass fraction of sodium hyaluronate in the mixed gel is 1 to 50%.

As a further improvement, the cross-linking agent used in the cross-linking reaction of the sodium hyaluronate gel in step 1) of the present invention is one or more of divinyl sulfone, 1,4-butanediol diglycidyl ether, oxalic acid dihydrazide, carbodiimide, and glycidyl methacrylate, with a concentration of 0.1 to 10 mg/mL.

As a further improvement, in step 2) of the present invention, the microspheres are added to the gel system in batches and multiple times, stirred at 100-800 rpm/min for 4-16 hours, and manually stirred every 20-50 minutes to uniformly mix the composite gel system.

As a further improvement, the sodium hyaluronate gel component described in the present invention includes sodium hyaluronate and cross-linked sodium hyaluronate. Sodium hyaluronate can be replaced by collagen, chitosan, gelatin, sodium alginate, fibrin, silk fibroin and artificial synthetic gel. The artificial synthetic gel is any one of polyethylene glycol, polyvinyl alcohol, polyethylene oxide and polyacrylamide.

As a further improvement, the material of the microspheres described in the present invention is one or a composite of several of polylactic acid, poly-L-lactic acid, polycaprolactone and hydroxyapatite.

As a further improvement, in step 2) of the present invention, one or more active ingredients such as liposomes, exosomes, amino acids, polypeptides, proteins, vitamins and local anesthetics are also added to the cross-linked gel.

The present invention relates to a sodium hyaluronate composite gel loaded with degradable microspheres for injection and a modular preparation method thereof, which is composed of degradable microspheres, a salt solution of a cross-linked sodium hyaluronate gel, and a sodium hyaluronate mobile phase. The present invention introduces freeze-drying technology in the form of modular assembly, freeze-drying the cross-linked sodium hyaluronate gel after dialysis, crushing it and fully re-dissolving it at a certain concentration, loading microspheres, making it uniformly distributed in the sodium hyaluronate gel, and preparing a microsphere gel composite system. The present invention realizes the modular preparation of the three processes of microspheres, cross-linked sodium hyaluronate gel, and the composite of the two. The cross-linked gel is stored in a solid state, so that the cross-linked gel can be taken and used at any time, thereby improving the process flexibility. After the composite gel for injection prepared by the present invention is injected into the local tissue of the human body, it has the effect of improving and modifying the defects of facial soft tissues and tissue contours. With the gradual degradation of the microspheres, the collagen growth is continuously stimulated to ensure its long-term filling effect.

Compared with the prior art, the innovation and beneficial effects of the present invention are mainly reflected in:

(1) In the composite gel preparation method, a freeze-drying process is introduced. By placing the cross-linked sodium hyaluronate gel in an environment of -50 to -80°C, the chemical cross-linking of sodium hyaluronate is modularized, thereby achieving solid-state storage of the cross-linked gel. This preparation process can modularize the cross-linked gel through a single large-scale production, so that it can be taken multiple times in the future and directly loaded with microspheres after re-dissolving as needed. Compared with the traditional gel preparation process, this preparation process can directly compound the cross-linked gel with the microspheres, increasing the flexibility and convenience of the process operation.

(2) After the cross-linked gel is freeze-dried and modularized, the cross-linked sodium hyaluronate gel is stored in a solid state, which allows the cross-linked gel to be taken and used at any time, and the gel system can still maintain stable physical and chemical properties after re-dissolution, thereby ensuring the effectiveness of the product. In addition, this preparation process also provides a more convenient and reliable method for the subsequent freeze-drying and re-dissolution operations of the cross-linked gel.

(3) The freeze-dried gel particles are re-dissolved in a phosphate buffer solution or physiological saline with a pH of 6.8 to 7.5 and an osmotic pressure of 280 to 340 mOsm/L, so that the composite gel can better meet the physiological adaptation conditions of the human body, maintain filling stability in the in vivo environment, improve its biocompatibility, and effectively reduce the inflammatory response caused by the implant material.

The present invention uniformly mixes cross-linked sodium hyaluronate gel and regenerative microspheres, and after the formed mixed gel is injected into the skin, it can replace the lost collagen and stimulate the regeneration of collagen in the human body, with significant and lasting effects. The sodium hyaluronate composite gel product loaded with degradable microspheres for injection provided by the present invention has uniform and fine particles, is easy to inject, has a long local action time, good plasticity, and obvious filling effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an electron microscope image of microspheres prepared in Example 1 of the present invention;

Figure 2 (a) and (b) are comparison diagrams of the viscous modulus and elastic modulus of the products prepared in Example 1 and Comparative Examples 1 and 2 of the present invention;

FIG3 is a comparison chart of the dynamic viscosity of the products prepared in Example 1 and Comparative Examples 1 and 2 of the present invention;

FIG4 is a comparison chart of the maximum pushing force and the average pushing force of the products prepared in Example 1 of the present invention and Comparative Examples 1 and 2.

DETAILED DESCRIPTION

The technical solution of the present invention is further described below through specific embodiments in conjunction with the accompanying drawings of the specification, but the present invention is by no means limited to the embodiments.

Example 1

(1) Preparation of poly (L-lactic acid) microspheres: 50 g of poly (L-lactic acid) with a molecular weight of 90,000 Da was added to 500 mL of dichloromethane to dissolve as the oil phase, and then slowly added to 3000 mL of a 2.0% polyvinyl alcohol aqueous solution. After the addition was completed, the mixture was emulsified at room temperature at a speed of 2500 rpm/min for 30 min, and then the dichloromethane solution was evaporated by continuous stirring. After washing, the filter cake was obtained by suction filtration, and dried and sieved to obtain poly (L-lactic acid) microspheres with a particle size of 10 to 50 μm. The surface morphology of the poly (L-lactic acid) microspheres was observed by electron microscopy, as shown in FIG1 .

(2) Preparation of cross-linked sodium hyaluronate gel: 3.6 g of sodium hyaluronate dry powder with a molecular weight of 2,000,000 Da was dissolved in 50 mL of 0.2 mol/L sodium hydroxide solution. After the sodium hyaluronate was completely dissolved, 110 μL of divinyl sulfone was added. After stirring for 12 min, the mixture was transferred to an environment of 2 to 8 °C and allowed to stand for about 16 h to obtain a cross-linked sodium hyaluronate gel.

(3) Modular preparation of cross-linked gel: The cross-linked sodium hyaluronate gel obtained in step (2) was purified and dialyzed in ultrapure water for 4 days. After the dialysis, the cross-linked gel was taken out and firstly placed in a -80°C freezer for quick freezing, and then freeze-dried using a freeze dryer at a freeze-drying temperature of -40°C. The obtained white solid was sealed and stored in a -80°C freezer.

(4) Preparation of sodium hyaluronate composite gel loaded with degradable microspheres for injection: Take 1.0g of freeze-dried gel particles, add 55mL of phosphate buffer, and stir at a speed of 140rpm/min for 6-8h to fully swell the cross-linked gel. Shear and disperse the re-dissolved gel at a speed of 6000rpm/min and a pressure of 0.08MPa. Take 50g of cross-linked sodium hyaluronate gel after shearing and homogenization, add 10g of poly-L-lactic acid microspheres, 5.5g of 2% sodium hyaluronate mobile phase, 0.8mL of 30% lidocaine hydrochloride solution, and then add 0.4mL of 1% sodium hydroxide solution to adjust the pH to 6.0-7.5, add 2mL of phosphate buffer to adjust the osmotic pressure to 260-350mOsm/L, and stir at a speed of 120rpm/min for 8h-9h until mixed evenly. Then, it was filled into a prefilled syringe and centrifuged to eliminate bubbles. It was sterilized with high-pressure steam at a temperature of 121° C. for 15 minutes to obtain a sodium hyaluronate composite gel loaded with degradable microspheres for injection. The preparation process took 3 days.

Example 2

(1) The preparation steps of poly-L-lactic acid microspheres and cross-linked sodium hyaluronate gel are the same as those in Example 1.

(2) Preparation of cross-linked sodium hyaluronate gel: Dissolve 1.6 g of sodium hyaluronate dry powder with a molecular weight of 2,000,000 Da in 50 mL of 0.4 mol/L sodium hydroxide solution. After the sodium hyaluronate is completely dissolved, add 130 μL of divinyl sulfone. After stirring for 12 min, transfer the mixture to an environment of 2 to 8 °C and let it stand for about 16 h to obtain a cross-linked sodium hyaluronate gel.

(3) Modular preparation of cross-linked gel: The cross-linked sodium hyaluronate gel obtained in step (2) was purified and dialyzed in ultrapure water for 4 days. After the dialysis, the cross-linked gel was taken out and firstly placed in a -80°C freezer for quick freezing, and then freeze-dried using a freeze dryer at a freeze-drying temperature of -50°C. The obtained white solid was sealed and stored in a -20°C freezer.

(4) Preparation of sodium hyaluronate composite gel loaded with degradable microspheres for injection: Take 1.0g of freeze-dried gel particles (freeze-dried particles storage time is 0d), add 55mL of normal saline, stir at a speed of 140rpm/min for 6-8h to fully swell the cross-linked gel. Shear and disperse the reconstituted gel at a speed of 6000rpm/min and a pressure of 0.08MPa. Take 50g of cross-linked sodium hyaluronate gel after shearing and homogenization, add 16g of poly-L-lactic acid microspheres, 5.5g of 2% sodium hyaluronate mobile phase, 0.8mL of 30% lidocaine hydrochloride solution, and then add 0.4mL of 1% sodium hydroxide solution to adjust the pH to 6.0-7.5, add 2mL of phosphate buffer to adjust the osmotic pressure to 260-350mOsm/L, and stir at a speed of 120rpm/min for 8h-9h until mixed evenly. Then, it was filled into a prefilled syringe and centrifuged to eliminate bubbles. It was sterilized with high-pressure steam at a temperature of 121° C. for 15 minutes to obtain a sodium hyaluronate composite gel loaded with degradable microspheres for injection. The preparation process took 3 days.

Example 3

(1) The preparation steps of poly-L-lactic acid microspheres and cross-linked sodium hyaluronate gel are the same as those in Example 1.

(2) Preparation of cross-linked sodium hyaluronate gel: 3.6 g of sodium hyaluronate dry powder with a molecular weight of 2,000,000 Da was dissolved in 50 mL of 0.2 mol/L sodium hydroxide solution. After the sodium hyaluronate was completely dissolved, 110 μL of divinyl sulfone was added. After stirring for 12 min, the mixture was transferred to an environment of 2 to 8 °C and allowed to stand for about 16 h to obtain a cross-linked sodium hyaluronate gel.

(3) Modular preparation of cross-linked gel: The cross-linked sodium hyaluronate gel obtained in step (2) was purified and dialyzed in ultrapure water for 4 days. After the dialysis, the cross-linked gel was taken out and firstly placed in a -80°C freezer for quick freezing, and then freeze-dried using a freeze dryer at a freeze-drying temperature of -80°C. The obtained white solid was sealed and stored in a -20°C freezer.

(4) Preparation of sodium hyaluronate composite gel loaded with degradable microspheres for injection: 1.0 g of freeze-dried gel particles were added to 75 mL of phosphate buffer and stirred at a speed of 140 rpm/min for 6 to 8 hours to fully swell the cross-linked gel. The reconstituted gel was sheared and dispersed at a speed of 6000 rpm/min and a pressure of 0.08 MPa. 70 g of cross-linked sodium hyaluronate gel after shearing and homogenization was added to 9 g of poly-L-lactic acid microspheres, 5.5 g of 2% sodium hyaluronate mobile phase, 0.8 mL of 30% lidocaine hydrochloride solution, and then 0.4 mL of 1% sodium hydroxide solution was added to adjust the pH to 6.0 to 7.5, and 2 mL of phosphate buffer was added to adjust the osmotic pressure to 260 to 350 mOsm/L, and stirred at a speed of 120 rpm/min for 8 to 9 hours until the mixture was uniformly mixed. Then, it was filled into a prefilled syringe and centrifuged to eliminate bubbles. It was sterilized with high-pressure steam at a temperature of 121° C. for 15 minutes to obtain a sodium hyaluronate composite gel loaded with degradable microspheres for injection. The preparation process took 3 days.

Comparative Example 1

(1) The preparation steps of poly-L-lactic acid microspheres and cross-linked sodium hyaluronate gel are the same as those in Example 1.

(2) Modular preparation of sodium hyaluronate composite gel loaded with degradable microspheres: The preparation steps are different from those in Example 1 only in the storage time of the freeze-dried gel particles. The storage time of the freeze-dried gel particles used is 30 days, and the rest of the preparation process is the same as that in Example 1.

Comparative Example 2

(1) The preparation steps of poly-L-lactic acid microspheres are the same as those in Example 1.

(2) The preparation of the cross-linked sodium hyaluronate gel and the preparation of the sodium hyaluronate composite gel loaded with degradable microspheres for injection were not significantly different from those in Example 1. The difference between the preparation process of this comparative example and that of Example 1 was mainly reflected in that the freeze-drying process of step (3) of Example 1 was not introduced into this comparative example. The time required for the preparation of the entire composite gel was 7 days. The remaining technical features were the same as those in Example 1.

SEM test: The sample was treated with gold spraying, and the surface morphology of the PLLA microspheres was characterized by scanning electron microscopy, with an acceleration voltage of 5 kV. The results are shown in Figure 1. As can be seen from the figure, the surface of the PLLA microspheres is smooth, the particles are uniform, and the particle size is in the range of 10 to 50 μm.

Rheological test: The viscous modulus and elastic modulus of Example 1 and Comparative Examples 1 and 2 were measured by a rheometer at 25°C and in the range of 0.05-10 Hz; the dynamic viscosity of Example 1 and Comparative Examples 1 and 2 was measured at a shear rate of 0.1-10s ^-1 (25±0.1)°C, and the results are shown in Figures 2 and 3. As can be seen from the figure, compared with Example 1 and Comparative Example 1, the cross-linked sodium hyaluronate gel was immediately re-dissolved after freeze-drying and was placed for 30 days after freeze-drying and then re-dissolved. The two groups of composite gels have a high degree of overlap in the viscous modulus, elastic modulus and dynamic viscosity curves, and there is no obvious difference, indicating that the modular preparation of cross-linked sodium hyaluronate gel has no effect on the physical and chemical properties of the composite gel.

Pushing force test: After the samples were placed in a room temperature environment for equilibrium using a universal material testing machine, the pushing forces of Example 1 and Comparative Examples 1 and 2 were measured at a pushing speed of 30 mm/min, and the results are shown in Figure 4. As can be seen from the figure, by comparing the maximum and average pushing force values of Example 1 with those of Comparative Examples 1 and 2, the pushing force deviations of Example 1 and Comparative Example 1 are both less than 0.5 N; in addition, the pushing forces of Example 1 and Comparative Example 1 are both lower than those of Comparative Example 2, ranging from 15 to 20 N, indicating that the composite gel prepared by chemical cross-linking modular treatment has stable pushing force and is easier to inject.

In addition, after the composite gel treated with chemical cross-linking modularization was placed at room temperature for 30 days, it still appeared as a uniform milky white gel in appearance without stratification, and there was no significant difference compared with the composite gel stored at 2-8°C. As can be seen from Table 1, the dynamic viscosity index of Comparative Example 1 only decreased by 4.46% before and after being placed at room temperature for 30 days, which is within the error range, indicating that the composite gel has not been degraded and is in a stable state.

Table 1

Composite gel Evaluation Metrics 0d 30d Rate of change Duration/d Comparative Example 1 Dynamic viscosity 232,212 221,841 -4.46% 30

The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. A modular preparation method for a sodium hyaluronate composite gel loaded with degradable microspheres for injection, characterized in that it comprises the following steps: 1) Sodium hyaluronate is dissolved in an alkaline solution and then cross-linked, and the cross-linked sodium hyaluronate gel is dialyzed for 4 to 8 days and then freeze-dried to obtain a freeze-dried gel; 2) crushing the freeze-dried gel into gel particles, fully re-dissolving the gel particles at a certain concentration to obtain a cross-linked gel, adding microspheres and sodium hyaluronate solution in a specific ratio to the cross-linked gel and mixing them evenly to obtain a mixed gel; 3) Filling the mixed gel into a pre-filled syringe and sterilizing it with high-pressure steam to obtain a sodium hyaluronate composite gel loaded with degradable microspheres for injection. 2. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1 is characterized in that the freeze-drying temperature of the cross-linked gel is -50 to -80°C, the storage temperature is 0 to -80°C, the reconstitution solution of the freeze-dried gel is phosphate buffered saline or physiological saline, the pH is 6.8 to 7.5, and the osmotic pressure is 280 to 340 mOsm/L. 3. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1 is characterized in that the mass fraction of the microsphere material in the mixed gel is 5 to 30%; and the average particle size of the microsphere material is 10 to 100 μm. 4. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that the alkaline solution is any one of sodium hydroxide and potassium hydroxide, and the solution concentration is 0.1 to 5.0 mol/L. 5. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that the molecular weight of the sodium hyaluronate added in step 2) is 500,000 to 3,000,000 Da, the concentration of sodium hyaluronate in the cross-linked gel is 1 to 15%, and the mass fraction of sodium hyaluronate in the mixed gel is 1 to 50%. 6. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that the cross-linking agent used in the cross-linking reaction of the sodium hyaluronate gel in the step 1) is one or more of divinyl sulfone, 1,4-butanediol diglycidyl ether, oxalic acid dihydrazide, carbodiimide, and glycidyl methacrylate, and the concentration is 0.1 to 10 mg/mL. 7. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1 is characterized in that in the step 2), the microspheres are added to the gel system in batches and multiple times, stirred at 100-800 rpm/min for 4-16 hours, and manually stirred every 20-50 minutes to make the composite gel system evenly mixed. 8. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that the sodium hyaluronate gel component includes sodium hyaluronate and cross-linked sodium hyaluronate, and sodium hyaluronate can be replaced by collagen, chitosan, gelatin, sodium alginate, fibrin, silk fibroin and artificial synthetic gel, and the artificial synthetic gel is any one of polyethylene glycol, polyvinyl alcohol, polyethylene oxide and polyacrylamide. 9. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that the material of the microspheres is a composite of one or more of polylactic acid, poly-L-lactic acid, polycaprolactone, and hydroxyapatite. 10. The modular preparation method of the sodium hyaluronate composite gel loaded with degradable microspheres for injection according to claim 1, characterized in that in the step 2), one or more active ingredients such as liposomes, exosomes, amino acids, polypeptides, proteins, vitamins and local anesthetics are also added to the cross-linked gel.