Encapsulated hyaluronic acid gel composition, method for preparing same, and use thereof

Abstract

Disclosed are an encapsulated hyaluronic acid gel composition, a method for preparing same, and use thereof. The encapsulated hyaluronic acid gel composition consists of a disulfide-cross-linked hyaluronic acid gel and a non-cross-linked hyaluronic acid solution droplet. The non-cross-linked hyaluronic acid solution droplet is encapsulated in the disulfide-cross-linked hyaluronic acid gel. The encapsulated hyaluronic acid gel composition provided herein features ease to extrude and inject and improved viscosity and stability of the gel. In addition, no additional cross-linking agents are required, thus reducing the relative proportion of modified hyaluronic acid in the gel and improving the biological safety of the gel.

Description

Encapsulated hyaluronic acid gel composition and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an encapsulated hyaluronic acid gel composition, a preparation method and an application thereof. Background Art

[0002] Hyaluronic acid is a non-sulfonated glycosaminoglycan composed of repeating disaccharide units (α-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine). It is present in all connective tissues and has excellent biocompatibility and unique physicochemical properties. However, its clinical application is limited due to its fluidity and rapid degradation by hyaluronidase in the body.

[0003] Cross-linking reactions can effectively reduce the fluidity of hyaluronic acid and delay its degradation and absorption in the body. However, improper cross-linking reactions may also impair the biocompatibility of hyaluronic acid. Currently, divinyl sulfoxide (DVS) or 1,4-butanediol diglycidyl ether (BDDE) are conventionally used for hyaluronic acid cross-linking reactions. A large number of unreacted single-end functional groups may remain in the reaction. These residual single-end functional groups are chemically bonded to the cross-linking material and cannot be removed through purification processes. Therefore, they may cause various toxic side effects during clinical use (Edsman et al., Dermatol Surg 2012, 38:1170-1179).

[0004] In the prior art, patent publication number CN101056891A provides a method for preparing a cross-linked hyaluronic acid gel with a low cross-linker content and excellent viscoelasticity. The method comprises stirring a mixture containing at least 10 w / v % hyaluronic acid, a cross-linker, and water under acidic (pH = 1-5) or alkaline (pH = 10-14) conditions. This method reduces the amount of cross-linker used by increasing the hyaluronic acid concentration. Patent publication number CN101107270A provides a method for preparing a homogeneous hydrogel comprising hyaluronic acid or a salt thereof cross-linked with divinyl sulfone (DVS), comprising the steps of: (a) providing an alkaline solution of hyaluronic acid or a salt thereof; (b) adding DVS to the solution of step (a), thereby cross-linking the hyaluronic acid or a salt thereof with the DVS to form a gel; and (c) treating the gel of step (b) with a buffer, wherein the gel swells and forms a hydrogel comprising hyaluronic acid or a salt thereof cross-linked with DVS. The gel prepared by this method exhibits enhanced homogeneity and enhanced softness.

[0005] The cross-linked hyaluronic acid gel prepared by the above-mentioned patented method through cross-linking reaction reduces the fluidity of hyaluronic acid and slows its degradation and absorption rate in the body, thereby reducing the amount of cross-linker used to a certain extent and improving the biosafety of the gel. However, due to the high gel strength of such cross-linked hyaluronic acid gel products, even after being crushed into granules, the pushing force is still large, which limits their clinical application. In addition, during injection, the mechanical extrusion of such gels destroys the non-cross-linked sites of the polymer, making it more susceptible to biochemical and enzymatic attacks in the body, resulting in rapid degradation of the gel, further affecting the clinical application of the product.

[0006] Therefore, to facilitate clinical injection, requirements are placed on the pushing force generated by the gel through the injection needle or extension tube. To reduce the product's pushing force, the hyaluronic acid gel needs to be crushed into fine particles. Furthermore, a certain proportion of non-cross-linked hyaluronic acid is usually added to the cross-linked hyaluronic acid to reduce the friction between the gel particles.

[0007] Patent publication number CN112334167 A provides a cross-linked polymer hydrogel composition comprising cross-linked hyaluronic acid, unmodified hyaluronic acid, and the like. One preparation method involves simultaneously dissolving thiol-modified hyaluronic acid, unmodified hyaluronic acid, and lidocaine hydrochloride in an aqueous solution. After adjusting the pH to approximately 6.8 to 7.6, the thiol groups are oxidized to disulfide bonds by oxygen, thereby cross-linking the thiol-modified hyaluronic acid. The resulting polymer hydrogel composition is an interpenetrating network gel. This method adds a certain proportion of non-cross-linked hyaluronic acid to produce a gel composition. However, the interpenetrating network gel does not reduce the pushing force of the gel.

[0008] For physical mixed two-phase gels that can reduce the gel extrusion force, such as These are made by homogenizing a mixture of cross-linked and non-cross-linked hyaluronic acid and are used for facial fillers or to increase viscosity in arthritis. These physically mixed two-phase gels are equivalent to non-cross-linked hyaluronic acid encapsulating / semi-encapsulating cross-linked hyaluronic acid gel particles. Due to the poor thermal stability of non-cross-linked hyaluronic acid, while these products can reduce the gel's extrusion force, the gel's viscosity stability is also reduced. Consequently, physically mixed two-phase gels generally exhibit poor viscosity stability.

[0009] Therefore, there is an urgent need in the art for a new hyaluronic acid gel composition that has the advantages of easy injection, good biosafety, and good gel viscosity stability, which has positive significance for the preparation and application of hyaluronic acid gel compositions and their derivatives.

[0010] Summary of the Invention

[0011] The technical problem to be solved by the present invention is that, although cross-linked hyaluronic acid gels prolong degradation time, they do not have the advantages of requiring little or no cross-linking agent, are not easy to inject, and have poor gel stability. Therefore, the present invention provides an encapsulated hyaluronic acid gel composition that is not only easier to push and inject, but also improves the viscosity stability of the gel. Furthermore, it does not require the addition of a cross-linking agent, reduces the relative proportion of modified hyaluronic acid in the gel, and improves the biosafety of the gel.

[0012] In order to solve the above technical problems, the present invention provides an encapsulated hyaluronic acid gel composition, which is composed of the following two parts:

[0013] 1) disulfide-crosslinked hyaluronic acid gel, and

[0014] 2) non-cross-linked hyaluronic acid solution mass;

[0015] Wherein, the non-cross-linked hyaluronic acid solution group is encapsulated in the disulfide bond cross-linked hyaluronic acid gel.

[0016] In clinical use, hyaluronic acid gel is typically injected locally into the target site via a needle or pushed into the affected area via an extension tube. After cross-linking, hyaluronic acid is often difficult to push due to its greater gel strength and poor flow and diffusion properties, and may even result in a dangerous needle burst. Furthermore, hyaluronic acid gel products must maintain the viscosity stability and elastic modulus (G') of the gel as much as possible during their shelf life. Therefore, cross-linked hyaluronic acid gels require special optimization for injection / push injection.

[0017] In the present invention, the encapsulated hyaluronic acid gel composition includes a disulfide-crosslinked hyaluronic acid gel and a non-crosslinked hyaluronic acid solution mass. The non-crosslinked hyaluronic acid solution mass is encapsulated in the disulfide-crosslinked hyaluronic acid gel. When the encapsulated hyaluronic acid gel is crushed and granulated and / or injected, some of the non-crosslinked hyaluronic acid solution masses are broken. These broken non-crosslinked hyaluronic acid solution masses can increase the lubricity of the gel particles and effectively reduce the pushing force of the gel, while the remaining intact non-crosslinked hyaluronic acid solution mass increases the viscosity stability and elastic modulus (G') of the gel.

[0018] Specifically, the particle size of the non-cross-linked hyaluronic acid solution mass is no more than 5000 μm.

[0019] Preferably, the particle size of the non-cross-linked hyaluronic acid solution mass is 50 to 2000 μm; more preferably, the particle size of the non-cross-linked hyaluronic acid solution mass is 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm or 2000 μm.

[0020] Specifically, the molecular weight of the non-cross-linked hyaluronic acid solution mass is 100 KDa to 2500 KDa.

[0021] Preferably, the molecular weight of the non-cross-linked hyaluronic acid solution mass is 1000KDa to 2200KDa; more preferably, the molecular weight of the non-cross-linked hyaluronic acid solution mass is 1100KDa, 1200KDa, 1300KDa, 1400KDa, 1500KDa, 1600KDa, 1700KDa, 1800KDa, 1900KDa, 2000KDa, 2100KDa or 2200KDa.

[0022] In the present invention, the encapsulated hyaluronic acid gel composition prepared from the large molecular weight non-cross-linked hyaluronic acid solution mass has a good lubricating effect and significantly reduces the pushing force of the gel.

[0023] Specifically, the content of the disulfide bond cross-linked hyaluronic acid gel is 4 to 50 mg / mL.

[0024] Preferably, the content of the disulfide-crosslinked hyaluronic acid gel is 10-25 mg / mL.

[0025] In the present invention, the content of the disulfide-crosslinked hyaluronic acid gel is expressed as the weight (mg / mL) of the disulfide-crosslinked hyaluronic acid per unit volume in the composition.

[0026] Specifically, the disulfide bond cross-linked hyaluronic acid gel is prepared by hyaluronic acid thiolation derivatives. The hyaluronic acid thiolation derivatives (HA-SH) refer to the hyaluronic acid derivatives containing sulfydryl, can be prepared by the thiolation modification of hyaluronic acid, also include various hyaluronic acid derivatives (such as carboxymethyl hyaluronic acid, acetylated hyaluronic acid and its sodium salt, potassium salt etc.) further by the thiolation derivatives prepared by thiolation modification, for example, using Shu etc., Biomacromolecules 2002, 3: 1304-1311 etc. The multiple hyaluronic acid thiolation derivative preparation method disclosed in previous documents can be used for preparing hyaluronic acid thiolation derivatives (HA-SH) of the present invention; Such hyaluronic acid thiolation derivatives (HA-SH) preferably maintain the initial structure, physiological function and biocompatibility of non-cross-linked hyaluronic acid, and can achieve effective disulfide bond cross-linking.

[0027] Specifically, the thiol content of the thiol derivative of hyaluronic acid is 5 to 250 μmol / g polymer.

[0028] Preferably, the thiol content of the thiol derivative of hyaluronic acid is 20 to 125 μmol / g polymer.

[0029] In the present invention, the thiol content of the thiol derivative of hyaluronic acid is expressed as the number of micromoles of thiol groups per gram of the thiol derivative of hyaluronic acid (μmol / g).

[0030] Specifically, the hyaluronic acid also includes salt forms thereof, and the salt forms include sodium salt, potassium salt, calcium salt and zinc salt.

[0031] Another aspect of the present invention provides a method for preparing an encapsulated hyaluronic acid gel composition, wherein the encapsulated hyaluronic acid gel composition is the aforementioned encapsulated hyaluronic acid gel composition, and the preparation method comprises the following steps:

[0032] Step 1: dissolving the thiolated hyaluronic acid derivative in a buffer solution, adding non-cross-linked hyaluronic acid solid particles, uniformly mixing, adjusting the pH, and forming a disulfide-cross-linked hyaluronic acid gel through an oxidation process;

[0033] Step 2, soaking the disulfide-crosslinked hyaluronic acid gel obtained in step 1 in a neutral buffer solution, and filtering to obtain an encapsulated hyaluronic acid gel composition;

[0034] In step 2, the non-cross-linked hyaluronic acid solid particles in the disulfide-cross-linked hyaluronic acid gel are restored to a dissolved state due to water absorption, forming a hyaluronic acid solution mass and being encapsulated in the disulfide-cross-linked hyaluronic acid gel. The disulfide-cross-linked hyaluronic acid gel is placed in an isotonic buffer solution. Due to osmotic action, the components of the neutral buffer penetrate the gel and contact the non-cross-linked hyaluronic acid solid particles. The non-cross-linked hyaluronic acid solid particles are restored to a dissolved state due to water absorption, thereby obtaining a hyaluronic acid solution mass.

[0035] Specifically, in step 1, the non-cross-linked hyaluronic acid solid particles are prepared by solvent precipitation and crushing and screening of a hyaluronic acid aqueous solution, and the precipitant used in the solvent precipitation is one of ethanol, acetone, ethyl acetate or dimethyl sulfoxide, or can be other precipitation solvents well known in the art.

[0036] In one embodiment, a 2% sodium chloride solution of hyaluronic acid is stirred while adding 2 volumes of anhydrous ethanol. The resulting flocculent precipitate is squeezed dry and shredded, and ethanol is added again to extract excess water, followed by filtration. The resulting solid precipitate is vacuum dried to remove residual ethanol and water. Further pulverization and screening are performed to obtain non-cross-linked hyaluronic acid solid particles of a predetermined particle size.

[0037] Specifically, the hyaluronic acid aqueous solution is one of sodium chloride, potassium chloride or magnesium chloride, and the concentration of the hyaluronic acid aqueous solution is 1% to 10%. When the concentration is lower than 1%, the solvent cannot precipitate the hyaluronic acid. When the concentration is higher than 10%, the salt will precipitate together with the hyaluronic acid, which is not conducive to the control of the particle size during subsequent crushing.

[0038] Preferably, the concentration of the hyaluronic acid aqueous solution is 2% to 4%.

[0039] Specifically, in step 1, the swelling degree of the non-cross-linked hyaluronic acid solid particles does not exceed 20%. After the non-cross-linked hyaluronic acid solid particles are added to the buffer solution, the non-cross-linked hyaluronic acid solid particles remain swollen but not dissolved by absorbing water for at least 30 minutes.

[0040] Non-crosslinked hyaluronic acid in an interpenetrating network structure cannot act as a lubricant, so the interpenetrating network gel is difficult to reduce the pushing force during injection. In the present invention, after the non-crosslinked hyaluronic acid solid particles are added to the hyaluronic acid thiolated derivative (HA-SH) buffer solution, the swelling degree of the non-crosslinked hyaluronic acid solid particles does not exceed 0.2, thereby limiting the mutual dissolution of the non-crosslinked hyaluronic acid and the hyaluronic acid thiolated derivative (HA-SH) to form an interpenetrating network structure.

[0041] Specifically, in step 2, the soaking time does not exceed 4 days.

[0042] Specifically, in step 1, the pH value of the buffer solution is 7-10.

[0043] Preferably, in step 1, the pH value of the buffer solution is 8-9.

[0044] Specifically, in step 1, the temperature is 10-50° C., and the mixing stirring speed is 100-2000 rpm.

[0045] Specifically, in step 1, the oxidation process is carried out in an aerobic environment, and the oxygen source of the aerobic environment is oxygen in the air or dissolved oxygen in the aqueous solution during the stirring process and / or pure oxygen introduced from the outside.

[0046] The physical mixing process and the introduction of pure oxygen can be carried out in a manner well known to those skilled in the art, such as mechanical stirring and mixing or the introduction of oxygen through a slender tube. This preparation method uses a buffer solution of thiolated hyaluronic acid derivatives (HA-SH) to achieve disulfide cross-linked gel under the oxidative action of oxygen, which has the advantage of not requiring the addition of a cross-linking agent. In order to accelerate the disulfide cross-linking rate, the dissolution pH can be adjusted to alkaline. When the pH exceeds 10, the cross-linking rate is too fast, resulting in uneven cross-linking and affecting the mechanical properties of the gel. The disulfide cross-linking reaction byproduct is water, which has many advantages such as a simple preparation process and the absence of impurities.

[0047] The present invention also provides an application of an encapsulated hyaluronic acid gel composition in medicine. The encapsulated hyaluronic acid gel composition is prepared by the above-mentioned preparation method of the encapsulated hyaluronic acid gel composition.

[0048] Specifically, the encapsulated hyaluronic acid gel composition is filled or crushed and then sterilized to prepare a final product;

[0049] The pulverization process refers to pulverizing the encapsulated hyaluronic acid gel composition into hyaluronic acid gel particles and then filling them into a container, and sterilizing to obtain the final product. The particle size of the hyaluronic acid gel particles is larger than the particle size of the non-cross-linked hyaluronic acid solid particles.

[0050] Specifically, the medical applications of the final product include prevention of postoperative tissue adhesion in the uterine cavity, pelvic and abdominal cavity, and ENT, treatment of osteoarthritis viscosity enhancement, soft tissue filling and enhancement, and water light injection.

[0051] Specifically, according to different clinical indications, the encapsulated hyaluronic acid gel composition can be directly filled into a container, or can be further crushed and sieved to obtain hyaluronic acid gel particles and then filled into a container, which can be a syringe of various specifications and / or a co-extruded film bag.

[0052] Gels for preventing postoperative tissue adhesions in uterine, pelvic, and ENT surgeries are typically dispensed into syringes or co-extruded film bags. Before use, the sterile container must be connected to a disposable extension tube with an inner diameter of 2.0 to 5 mm and a length of 60 to 350 mm. The non-cross-linked hyaluronic acid solid particles should be no larger than 2000 μm and no less than 500 μm. During filling and clinical push injection, some of the encapsulated hyaluronic acid solution clumps are crushed, allowing the hyaluronic acid solution to flow out, thus reducing the extrusion force of the encapsulated hyaluronic acid gel. When the non-cross-linked hyaluronic acid solid particles are smaller than 500 μm, only a very small amount of the hyaluronic acid solution clumps are crushed during filling and clinical push injection, failing to achieve the desired reduction in extrusion force.

[0053] In the treatment of osteoarthritis viscosity enhancement, soft tissue filling and enhancement injection, a 20-30G needle is generally used, and the particle size of non-cross-linked hyaluronic acid solid particles is controlled at 200-800μm.

[0054] For specialized applications, such as hydrating injections on the face, neck, hands, and private areas, which typically require dense, point-like injections, fine needles (such as 32G and 34G) can effectively reduce pain during injection. Therefore, it's important to control the particle size of the gel and the friction between the gel particles to ensure smooth passage of the gel through the needle. In this case, the particle size of the non-cross-linked hyaluronic acid solid particles should be less than 200μm and greater than 50μm.

[0055] The encapsulated hyaluronic acid gel composition of the present invention has the following beneficial effects:

[0056] 1. The encapsulated hyaluronic acid gel composition provided by the present invention has a smaller pushing force when passing through a thinner injection needle compared to an interpenetrating network gel, thereby solving the problem of difficult pushing; Compared to a mixed gel, the encapsulated hyaluronic acid gel composition provided by the present invention has better viscosity stability, particularly a composite gel with a higher non-crosslinking ratio. Therefore, the encapsulated hyaluronic acid gel composition of the present invention has the advantages of small pushing force and good viscosity stability. In addition, the encapsulated hyaluronic acid gel composition provided by the present invention does not need to be additionally added with a cross-linking agent during the preparation process, thereby reducing the relative proportion of modified hyaluronic acid in the gel, and the chemical modification degree is relatively lower, and the good biocompatibility also improves the biosafety of the gel.

[0057] 2. The encapsulated hyaluronic acid gel composition provided by the present invention has a simple preparation process and is easy to operate. It can be applied to various types of clinical symptoms, such as prevention of postoperative tissue adhesion in the uterine cavity, pelvic and abdominal cavity, and ENT, treatment of osteoarthritis viscosity enhancement, soft tissue filling and enhancement, and hyaluronic acid injection, etc., and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate a certain embodiment of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0059] FIG1 is a graph showing in vitro enzymatic hydrolysis curves of the hyaluronic acid gels prepared in Examples 10 to 12, Comparative Example 2, Comparative Example 4, and Comparative Example 6. DETAILED DESCRIPTION

[0060] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0061] Example 1 Preparation of non-cross-linked hyaluronic acid solid particles and testing of their anti-dissolution properties and swelling degree

[0062] Dissolve 2g of 1500KDa non-cross-linked hyaluronic acid in 200mL of 2% sodium chloride solution, add 400mL of ethanol while stirring, collect the flocculent precipitate, squeeze it dry, add 100mL of ethanol, let it stand for 12 hours, extract excess water, filter, and remove residual ethanol in the precipitate under reduced pressure. The precipitate is crushed and sieved to obtain particles with a particle size of 50 to 2000μm.

[0063] The non-cross-linked hyaluronic acid solid particles with a particle size of 50 to 2000 μm separated out were added to a 30°C, pH = 8.0 aqueous solution, stirred for 30 minutes, and filtered to obtain the non-cross-linked hyaluronic acid solid particles after water absorption and swelling. The filtrate was tested using the glucuronic acid method (YY / T 0962-2021, Appendix C, Determination of Sodium Hyaluronate Content). When the proportion of hyaluronic acid in the aqueous solution is less than 1%, it can be considered that the non-cross-linked hyaluronic acid solid particles do not dissolve under the test conditions; after filtration, the particles absorb surface moisture and the swelling degree of the solid particles is calculated.

[0064] Calculation method for solid particle swelling degree:

[0065] Q = (m2-m1) / m1; where Q is the swelling degree of non-cross-linked hyaluronic acid solid particles; m1 is the mass of non-cross-linked hyaluronic acid solid particles before the test (g); and m2 is the mass of non-cross-linked hyaluronic acid solid particles after the test (g).

[0066] Example 2 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0067] In Example 2, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the particle size of the non-cross-linked hyaluronic acid solid particles is 200-800 μm.

[0068] Example 3 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0069] In Example 3, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the particle size of the non-cross-linked hyaluronic acid solid particles is 50 to 200 μm.

[0070] Example 4 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0071] In Example 4, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the particle size of the non-cross-linked hyaluronic acid solid particles is 50-200 μm, and the sodium chloride used is 1% sodium chloride.

[0072] Example 5 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0073] In Example 5, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the molecular weight of the non-cross-linked hyaluronic acid is 1000 KDa.

[0074] Example 6 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0075] In Example 6, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the molecular weight of the non-cross-linked hyaluronic acid is 1000 KDa, and the particle size of the non-cross-linked hyaluronic acid solid particles is 50-200 μm.

[0076] Example 7 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0077] In Example 7, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the molecular weight of the non-cross-linked hyaluronic acid is 2200 KDa.

[0078] Example 8 Preparation of non-cross-linked hyaluronic acid solid particles and testing of anti-dissolution performance and swelling degree

[0079] In Example 8, the preparation method of non-cross-linked hyaluronic acid solid particles is the same as that of Example 1, except that the molecular weight of the non-cross-linked hyaluronic acid is 2200 KDa, and the particle size of the non-cross-linked hyaluronic acid solid particles is 50-200 μm.

[0080] The test results of Examples 1 to 8 are shown in Table 1 below:

[0081] Table 1

[0082] According to the contents of Table 1 above, it can be seen that under any combination of conditions of temperature range 10-50°C, pH value 7-9, and stirring speed 100-2000 rpm, the non-cross-linked hyaluronic acid solid particles can maintain water absorption and swelling but not dissolve for at least 30 minutes.

[0083] Example 9 Preparation of thiolated hyaluronic acid derivatives (HA-SH)

[0084] Hyaluronic acid thiolated derivatives (HA-SH) were prepared using 200 KDa sodium hyaluronate as raw material using the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3:1304-1311), with thiol contents of 25 μmol / g, 62 μmol / g, and 128 μmol / g, respectively.

[0085] Example 10 Preparation of an encapsulated hyaluronic acid gel composition for injection for viscosifying treatment of osteoarthritis, soft tissue filling and enhancement

[0086] 2.0 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 128 μmol / g was dissolved in 100 mL of buffer solution, and 0.2 g of non-cross-linked hyaluronic acid solid particles (1500 KDa, particle size 200-800 μm) was added. The mixture was evenly mixed, the pH was adjusted to 8.0, and the mixture was stirred. The mixture gradually lost fluidity and formed a disulfide-cross-linked hyaluronic acid gel after 5 minutes.

[0087] The disulfide-crosslinked hyaluronic acid gel was placed in 3 L of isotonic buffer solution four times, with the isotonic buffer solution replaced every two hours. The final step was to allow the encapsulated non-crosslinked hyaluronic acid solid particles to absorb water, swell, and eventually dissolve. The encapsulated hyaluronic acid gel was then filtered.

[0088] The encapsulated hyaluronic acid gel was crushed twice with a 300 μm mesh plate and then filled into a 1 mL syringe. After wet heat sterilization (121° C., 15 minutes), an encapsulated hyaluronic acid gel composition for osteoarthritis viscosity treatment, soft tissue filling and enhancement injection was obtained.

[0089] Example 11 Preparation of Encapsulated Hyaluronic Acid Gel Composition for Hyaluronic Acid Injection

[0090] 0.8 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 62 μmol / g was dissolved in 100 mL of buffer solution, and 0.4 g of non-cross-linked hyaluronic acid solid particles (1500 KDa, particle size 50-200 μm) was added. The mixture was evenly mixed, the pH was adjusted to 8.0, and the mixture was stirred. The mixture gradually lost fluidity and formed a disulfide-cross-linked hyaluronic acid gel after 15 minutes.

[0091] The disulfide-crosslinked hyaluronic acid gel was placed in 3 L of isotonic buffer solution four times, with the isotonic buffer solution being replaced every two hours. The final step was to allow the encapsulated non-crosslinked hyaluronic acid solid particles to absorb water, swell, and eventually dissolve, thereby obtaining an encapsulated hyaluronic acid gel.

[0092] The encapsulated hyaluronic acid gel was crushed twice with a 100 μm mesh plate and then filled into a 1 mL syringe. After wet heat sterilization (121° C., 15 minutes), the encapsulated hyaluronic acid gel composition for water light injection was obtained.

[0093] Example 12 Preparation of Encapsulated Hyaluronic Acid Gel Composition for Preventing Postoperative Tissue Adhesion

[0094] 0.5 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 25 μmol / g was dissolved in 100 mL of a buffer solution, 0.2 g of non-cross-linked hyaluronic acid solid particles (1500 KDa, particle size 500-2000 μm) was added, and the mixture was uniformly mixed. The pH was adjusted to 9.0, and pure oxygen was introduced while stirring. The mixture gradually lost fluidity, and a disulfide-cross-linked hyaluronic acid gel was formed after 25 minutes.

[0095] The disulfide-crosslinked hyaluronic acid gel was placed in 3 L of isotonic buffer solution four times, with the isotonic buffer solution replaced every two hours. The final step was to allow the encapsulated non-crosslinked hyaluronic acid solid particles to absorb water, swell, and eventually dissolve, thereby obtaining an encapsulated hyaluronic acid gel.

[0096] The encapsulated hyaluronic acid gel is filled into a 3 mL syringe or a 100 mL co-extruded film bag and sterilized by moist heat (121° C., 15 minutes) to obtain an encapsulated hyaluronic acid gel composition for preventing postoperative tissue adhesion.

[0097] Comparative Examples 1-6 were set up to prepare interpenetrating network gels and biphasic hyaluronic acid gels for different therapeutic uses, and were compared with the encapsulated hyaluronic acid gel provided by the present invention in terms of extrusion force, viscosity and in vitro anti-enzymatic ability.

[0098] Comparative Example 1 Preparation of Encapsulated Hyaluronic Acid Gel for Treatment of Osteoarthritis Viscosity, Soft Tissue Filling and Enhancement Injection (Interpenetrating Network Gel)

[0099] 2.0 g of thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 128 μmol / g was dissolved in 100 mL of buffer solution; 0.2 g of non-cross-linked hyaluronic acid (1500 KDa) was dissolved in 100 mL of buffer solution; the thiol-containing hyaluronic acid derivative (HA-SH) and non-cross-linked hyaluronic acid were mixed in a mass ratio of 9:1 and stirred until uniform, the pH was adjusted to 7.2, and the mixture was transferred into a sterile glass container after sterilization and filtration; the solution was sealed and stored at 50°C for 1 week, and the solution lost its fluidity and formed an interpenetrating network hyaluronic acid hydrogel.

[0100] The interpenetrating network hyaluronic acid gel is crushed twice with a 300 μm mesh plate and then filled into a 1 mL syringe. After moist heat sterilization (121° C., 15 minutes), the interpenetrating network hyaluronic acid gel for osteoarthritis viscosity treatment, soft tissue filling and enhancement injection is obtained.

[0101] Comparative Example 2 Preparation of Encapsulated Hyaluronic Acid Gel for Treatment of Osteoarthritis Viscosity Increase, Soft Tissue Filling and Enhancement Injection (Biphasic Hyaluronic Acid Gel)

[0102] Dissolve 2.0 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 128 μmol / g in 100 mL of buffer solution, adjust the pH to 7.2, and transfer to a sterile glass container after sterilization and filtration. Store in a sealed container at 50°C for 1 week until the solution loses fluidity and forms a disulfide-crosslinked hyaluronic acid gel.

[0103] 0.2 g of non-cross-linked hyaluronic acid (1500 KDa) was dissolved in 100 mL of buffer solution; thiolated hyaluronic acid derivative (HA-SH) and non-cross-linked hyaluronic acid were mixed in a mass ratio of 9:1 and stirred until uniform. The mixture was crushed twice through a 300 μm mesh plate and then filled into a 1 mL syringe. After moist heat sterilization (121°C, 15 minutes), a biphasic hyaluronic acid gel for osteoarthritis viscosity treatment, soft tissue filling and enhancement injection was obtained.

[0104] Comparative Example 3 Preparation of Encapsulated Hyaluronic Acid Gel for Water-Light Injection (Interpenetrating Network Gel)

[0105] 0.8 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 62 μmol / g was dissolved in 100 mL of buffer solution; 0.4 g of non-cross-linked hyaluronic acid (1500 KDa) was dissolved in 100 mL of buffer solution; the thiol-containing hyaluronic acid derivative (HA-SH) and non-cross-linked hyaluronic acid were mixed in a mass ratio of 2:1 and stirred until uniform, the pH was adjusted to 7.2, and the mixture was sterilized and filtered before being transferred into a sterile glass container; the mixture was sealed and stored at 50°C for 1 week, and the solution lost its fluidity and formed an interpenetrating network hyaluronic acid hydrogel.

[0106] The interpenetrating network hyaluronic acid gel was crushed twice with a 100 μm mesh plate and then filled into a 1 mL syringe. After wet heat sterilization (121° C., 15 minutes), the interpenetrating network hyaluronic acid gel for water light injection was obtained.

[0107] Comparative Example 4 Preparation of Encapsulated Hyaluronic Acid Gel for Water-Light Injection (Biphasic Hyaluronic Acid Gel)

[0108] 0.8 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 62 μmol / g was dissolved in 100 mL of buffer solution, the pH was adjusted to 7.2, and the solution was transferred into a sterile glass container after sterilization and filtration. The solution was sealed and stored at 50° C. for 1 week, during which time the solution lost its fluidity and formed a disulfide-crosslinked hyaluronic acid gel.

[0109] 0.4 g of non-cross-linked hyaluronic acid (1500 KDa) was dissolved in 100 mL of buffer solution; hyaluronic acid thiolate derivative (HA-SH) and non-cross-linked hyaluronic acid were mixed in a mass ratio of 2:1 and stirred until uniform. The mixture was crushed twice through a 100 μm mesh plate and then filled into a 1 mL syringe. After wet heat sterilization (121°C, 15 minutes), a dual-phase hyaluronic acid gel for hydro-light injection was obtained.

[0110] Comparative Example 5 Preparation of Encapsulated Hyaluronic Acid Gel for Preventing Postoperative Tissue Adhesion (Interpenetrating Network Gel)

[0111] 0.5 g of thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 25 μmol / g was dissolved in 100 mL of buffer solution; 0.2 g of non-cross-linked hyaluronic acid (1500 KDa) was dissolved in 100 mL of buffer solution; the thiol-containing hyaluronic acid derivative (HA-SH) and non-cross-linked hyaluronic acid were mixed in a mass ratio of 5:2 and stirred until uniform, the pH was adjusted to 7.2, and the mixture was transferred into a sterile glass container after sterilization and filtration; the solution was sealed and stored at 50°C for 2 weeks, and the solution lost its fluidity and formed an interpenetrating network hyaluronic acid hydrogel.

[0112] The interpenetrating network hyaluronic acid gel is filled into a 3 mL syringe or a 100 mL co-extruded film bag, and is sterilized by moist heat (121° C., 15 minutes) to obtain the interpenetrating network hyaluronic acid gel for preventing postoperative tissue adhesion.

[0113] Comparative Example 6 Preparation of Encapsulated Hyaluronic Acid Gel for Preventing Postoperative Tissue Adhesion (Biphasic Hyaluronic Acid Gel)

[0114] Dissolve 0.5 g of a thiol-containing hyaluronic acid derivative (HA-SH) with a thiol content of 25 μmol / g in 100 mL of buffer solution, adjust the pH to 7.2, and transfer into a sterile glass container after sterilization and filtration. Store in a sealed container at 50°C for 1 week until the solution loses fluidity and forms a disulfide-crosslinked hyaluronic acid gel.

[0115] Dissolve 0.2 g of non-cross-linked hyaluronic acid (1500 kDa) in 100 mL of buffer solution; mix the thiolated hyaluronic acid derivative (HA-SH) and non-cross-linked hyaluronic acid in a mass ratio of 5:2 and stir until uniform. Fill the biphasic hyaluronic acid gel into a 3 mL syringe or a 100 mL co-extruded film bag and sterilize it with moist heat (121°C, 15 minutes) to obtain the biphasic hyaluronic acid gel for preventing postoperative tissue adhesion.

[0116] Test Example 1 Hyaluronic Acid Gel Pushing Force and Viscosity Test

[0117] The gels prepared in Examples 10 to 12, Comparative Examples 1, 3, and 5 were tested for pushing force and viscosity.

[0118] Pushing force test method: Example 10 and Comparative Example 1 were equipped with a 27G needle; Example 11 and Comparative Example 3 were equipped with a 32G needle; Example 12 and Comparative Example 5 were equipped with an extension tube (with an inner diameter of 2.5 mm and a length of 150 mm), and the syringes were pushed at a speed of 30 mm / min, and the average pushing force of the syringe handle was recorded.

[0119] Viscosity test method: Take an appropriate amount of sample gel from each group and test it according to the third rotational viscometer method of 0633 Viscosity Determination Method in the 2020 edition of the Pharmacopoeia of the People's Republic of China. The test conditions are a shear rate of not less than 0.25 Hz and (25±0.1)°C.

[0120] Stability accelerated test conditions: 50°C, 75% humidity. The pushing force and viscosity of the samples were tested after 0 days, 137 days, and 274 days of acceleration. The test results are shown in Table 2 below.

[0121] Table 2

[0122] Examples 10-12 are encapsulated hyaluronic acid gels provided by the present invention, while Comparative Examples 1, 3, and 5 are interpenetrating network gels. The gels in Example 10 and Comparative Example 1 have identical component contents, cross-linked hyaluronic acid content, and gel particle size. Example 11 and Comparative Example 3, as well as Example 12 and Comparative Example 5, are consistent with those in Example 10 and Comparative Example 1.

[0123] According to Table 2, taking Example 10 and Comparative Example 1 as examples, Example 10 is an encapsulated hyaluronic acid gel, which has a smaller pushing force and a slightly higher viscosity than Comparative Example 1, but has comparable viscosity stability.

[0124] This shows that compared with interpenetrating network gel, under the premise of comparable viscosity stability, the encapsulated hyaluronic acid gel has smaller pushing force and is more convenient for clinical use.

[0125] Test Example 2 Hyaluronic Acid Gel Pushing Force and Viscosity Test

[0126] The gels prepared in Examples 10 to 12, Comparative Examples 2, 4, and 6 were tested for pushing force, viscosity, and elastic modulus G'.

[0127] Pushing force test method: Example 10 and Comparative Example 2 were equipped with a 27G needle; Example 11 and Comparative Example 4 were equipped with a 32G needle; Example 12 and Comparative Example 6 were equipped with an extension tube (with an inner diameter of 2.5 mm and a length of 150 mm), and the syringes were pushed at a speed of 30 mm / min, and the average pushing force of the syringe handle was recorded.

[0128] Viscosity test method: Take an appropriate amount of sample gel from each group and test it according to the third rotational viscometer method of 0633 Viscosity Determination Method in the 2020 edition of the Pharmacopoeia of the People's Republic of China. The test conditions are a shear rate of not less than 0.25 Hz and a temperature of (25±0.1)°C.

[0129] Stability accelerated test conditions: 50°C, 75% humidity, test the pushing force and viscosity of the sample after 0 days, 137 days, and 274 days of acceleration.

[0130] Elastic modulus G' test method: Take 1g sample and use a rheometer (Anton Paar, MCR 301) to test the sample. The test uses a flat plate with a diameter of 50mm, the plate spacing is set to 0.5mm, the test temperature is 25°C, and the shear stress is 0.001. The test results are shown in Table 3 below.

[0131] Table 3

[0132] Examples 10-12 are encapsulated hyaluronic acid gels, while Comparative Examples 2, 4, and 6 are biphasic hyaluronic acid gels. The gels in Example 10 and Comparative Example 2 have identical component contents, cross-linked hyaluronic acid content, and gel particle size. Example 11 and Comparative Example 4, as well as Example 12 and Comparative Example 5, are consistent with those in Example 10 and Comparative Example 2.

[0133] According to the data in Table 3, taking Example 10 and Comparative Example 2 as examples, Example 10 is an encapsulated hyaluronic acid gel. Compared with Comparative Example 2, its pushing force is equivalent, but its viscosity is greater and its viscosity stability is better.

[0134] This indicates that compared with the biphasic hyaluronic acid gel, the encapsulated hyaluronic acid gel has a higher viscosity and better viscosity stability under the premise of equivalent pushing force.

[0135] Test Example 3: Hyaluronic Acid Gel In Vitro Anti-enzymatic Performance Test

[0136] In vitro anti-enzymatic performance test method: Accurately weigh 0.5 g of the gel prepared in Examples 10 to 12, Comparative Example 2, Comparative Example 4, and Comparative Example 6, add 1.5 ml of hyaluronidase solution (10 U / mL), place the sample in a constant temperature shaker (37 ° C, 20 rpm), take out within the specified time, boil at 100 ° C for 10 minutes to inactivate, centrifuge the inactivated liquid, filter with a 0.22 um microporous filter membrane, take 1.0 mL of the filtrate, and use the glucuronic acid method (YY / T 0962-2021, Appendix C, Determination of sodium hyaluronate content) for content testing.

[0137] Calculation method for in vitro enzymatic hydrolysis of hyaluronic acid gel:

[0138] Enzyme hydrolysis rate / %=4a / b*100%; wherein, a is the sodium hyaluronate content in the inactivation solution after centrifugation; b is the sodium hyaluronate content in the sample.

[0139] The lower the enzymatic degradation rate, the better the gel's resistance to enzymatic degradation and the longer it can be stored in the body.

[0140] As shown in FIG1 , it is the in vitro enzymatic hydrolysis curves obtained for the hyaluronic acid gels prepared in Examples 10 to 12, Comparative Examples 2, 4, and 6.

[0141] Examples 10-12 are encapsulated hyaluronic acid gels, Comparative Examples 1, 3, and 5 are interpenetrating network gels, and Comparative Examples 2, 4, and 6 are biphasic hyaluronic acid gels. As shown in Figure 1, compared to the other comparative examples, Examples 10-12 exhibit lower enzymatic degradation rates at the same time point, exhibiting improved resistance to enzymatic degradation, and exhibit a longer in vivo storage duration.

[0142] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An encapsulated hyaluronic acid gel composition, characterized in that: The encapsulated hyaluronic acid gel composition consists of the following two parts: 1) disulfide-bonded cross-linked hyaluronic acid gel, and 2) non-cross-linked hyaluronic acid solution mass; Wherein, the non-cross-linked hyaluronic acid solution group is encapsulated in the disulfide bond cross-linked hyaluronic acid gel.

2. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The particle size of the non-cross-linked hyaluronic acid solution mass is no more than 5000 μm.

3. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The particle size of the non-cross-linked hyaluronic acid solution mass is 50 to 2000 μm.

4. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The molecular weight of the non-cross-linked hyaluronic acid solution mass is 100 KDa to 2500 KDa.

5. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The molecular weight of the non-cross-linked hyaluronic acid solution mass is 1000 KDa to 2200 KDa.

6. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The content of the disulfide bond cross-linked hyaluronic acid gel is 4-50 mg / mL.

7. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The content of the disulfide bond cross-linked hyaluronic acid gel is 10-25 mg / mL.

8. The encapsulated hyaluronic acid gel composition according to claim 1, characterized in that: The disulfide bond cross-linked hyaluronic acid gel is prepared from thiolated hyaluronic acid derivatives.

9. The encapsulated hyaluronic acid gel composition according to claim 8, characterized in that: The thiol content of the thiol derivative of hyaluronic acid is 5 to 250 μmol / g polymer.

10. The encapsulated hyaluronic acid gel composition according to claim 9, characterized in that: The thiol content of the thiol derivative of hyaluronic acid is 20 to 125 μmol / g polymer.

11. A method for preparing an encapsulated hyaluronic acid gel composition, characterized in that: The encapsulated hyaluronic acid gel composition is the encapsulated hyaluronic acid gel composition according to any one of claims 1 to 10, and the preparation method comprises the following steps: Step 1, dissolving the thiolated hyaluronic acid derivative in a buffer solution, adding non-cross-linked hyaluronic acid solid particles, mixing evenly, adjusting the pH, and forming a disulfide-bond cross-linked hyaluronic acid gel through an oxidation process; Step 2, soaking the disulfide-bond cross-linked hyaluronic acid gel obtained in step 1 in a neutral buffer solution, and filtering to obtain an encapsulated hyaluronic acid gel composition; Wherein, in step 2, the non-cross-linked hyaluronic acid solid particles in the disulfide-cross-linked hyaluronic acid gel recover the dissolved state due to water absorption, become hyaluronic acid solution clusters, and are wrapped in the disulfide-cross-linked hyaluronic acid gel.

12. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 1, the non-cross-linked hyaluronic acid solid particles are obtained by solvent precipitation and crushing and screening of a hyaluronic acid aqueous solution.

13. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 12, characterized in that: The hyaluronic acid aqueous solution is one of sodium chloride, potassium chloride or magnesium chloride, and the concentration of the hyaluronic acid aqueous solution is 1% to 10%.

14. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 13, characterized in that: The concentration of the hyaluronic acid aqueous solution is 2% to 4%.

15. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 1, the swelling degree of the non-cross-linked hyaluronic acid solid particles does not exceed 20%. After the non-cross-linked hyaluronic acid solid particles are added to the buffer solution, the non-cross-linked hyaluronic acid solid particles remain swollen but not dissolved for at least 30 minutes.

16. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 2, the soaking time is no more than 4 days.

17. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 1, the pH value of the buffer solution is 7-10.

18. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 1, the pH value of the buffer solution is 8-9.

19. The method for preparing the encapsulated hyaluronic acid gel composition according to claim 11, characterized in that: In step 1, the oxidation process is carried out in an aerobic environment, and the oxygen source of the aerobic environment is oxygen in the air or dissolved oxygen in the aqueous solution during the stirring process and / or pure oxygen introduced from the outside.

20. Use of an encapsulated hyaluronic acid gel composition in medicine, characterized in that: The encapsulated hyaluronic acid gel composition is prepared by the preparation method of the encapsulated hyaluronic acid gel composition according to claim 11.

21. Use of the encapsulated hyaluronic acid gel composition according to claim 20 in medicine, characterized in that: The encapsulated hyaluronic acid gel composition is filled or crushed and then sterilized to prepare a final product; The pulverizing process refers to pulverizing the encapsulated hyaluronic acid gel composition into hyaluronic acid gel particles and then filling them into a container, and obtaining the final product after sterilization. The particle size of the hyaluronic acid gel particles is larger than the particle size of the non-cross-linked hyaluronic acid solid particles.

22. Use of the encapsulated hyaluronic acid gel composition according to claim 21 in medicine, characterized in that: The medical applications of the final product include prevention of postoperative tissue adhesion in the uterine cavity, pelvic and abdominal cavity, and ENT, treatment of osteoarthritis adhesion, soft tissue filling and enhancement, and water light injection.

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