Thermo-sensitive physical hydrogel lyophilized formulation, preparation method therefor and use thereof

Abstract

The present invention provides a thermo-sensitive physical hydrogel lyophilized formulation, a preparation method therefor and a use thereof. Raw materials for preparing the thermo-sensitive physical hydrogel lyophilized formulation comprise a thermo-sensitive polymer, sodium hyaluronate and a phosphate buffer solution; the phase-transition temperature of the thermo-sensitive polymer is 35±2℃; the molecular weight of the sodium hyaluronate is 2,200,000-2,800,000, and the mass-to-volume ratio of the sodium hyaluronate to the phosphate buffer solution is 3-12 mg:1 ml. According to the present invention, the sodium hyaluronate and the thermo-sensitive polymer of specific molecular weights and dosages of are compounded to achieve rapid gelation transition under the condition of physiological temperature, forming an interpenetrating three-dimensional network structure, which has better structural stability, facilitates cell growth and repair, has long degradation time, and can reduce the inconvenience of repeated injection. Moreover, the prepared lyophilized formulation can solve the problems in microsphere products of proneness to needle clogging, precipitation and degradation, and poor stability.

Description

A thermosensitive physical hydrogel freeze-dried preparation and its preparation method and application

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2023107913768, filed on June 30, 2023, entitled “A Thermosensitive Physical Hydrogel Freeze-Dried Preparation, Its Preparation Method and Application,” the entire disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the technical field of pharmaceutical and chemical engineering, and in particular to a temperature-sensitive physical hydrogel freeze-dried preparation and a preparation method and application thereof. Background Art

[0004] In recent years, with the improvement of people's living standards, the demand for medical aesthetic products has continued to increase, driving the continuous upgrading of plastic surgery technologies and products. In the field of minimally invasive cosmetic procedures, those seeking beauty are increasingly favoring non-invasive, immediate-repair fillers, which have been widely accepted by consumers since their launch. These non-invasive, immediate-repair injectable fillers are generally the preferred choice for rhinoplasty, chin augmentation, lip augmentation, and wrinkle reduction. Directly injected into the dermis, they provide moisturizing, lubricating, and sculpting benefits.

[0005] Currently popular instant-filling injectables consist of 20-50μm microparticles made from materials such as polycaprolactone (PCL) and polylactic acid (PL), mixed in a carboxymethylcellulose solution. After subcutaneous injection, the carboxymethylcellulose solution forms a gel that provides an immediate fill. As the gel degrades, the microspheres within the formulation begin to function. By adjusting the molecular weight of the polymer microspheres to control their degradation time in the body, the formulation achieves a long-lasting effect of stimulating collagen regeneration.

[0006] This type of preparation is generally a pre-filled injection. In order to prevent the microspheres from settling and agglomerating, resulting in uneven distribution and tissue inflammatory reactions, the gel preparation concentration of this preparation is often higher, and a thinner needle is generally used to reduce pain. At the same time, it is prevented that the microspheres in the body are phagocytosed to achieve a lasting stimulation effect. The microsphere particle size is often above 40μm. This preparation makes the procedure inconvenient and is very easy to clog the needle. On the one hand, the microsphere preparation process generally adopts an emulsification-solidification-screening process. This process requires the introduction of reagents such as dichloromethane and polyvinyl alcohol. These reagents have low limit requirements in implantable preparations and are difficult to remove. In order to achieve a suitable microsphere particle size distribution, screening will remove some non-target particle size microspheres, resulting in complex processes and high raw material costs. On the other hand, as the preparation degrades after implantation into the body, the carboxymethyl cellulose gel tends to loosen its structure, the gel viscosity decreases, and the cell adhesion is weak, making it difficult to support the long-term stable suspension of microspheres, and there is a risk of microsphere sedimentation, agglomeration, and injection displacement. The weakening of the microsphere dispersion support function will lead to the microspheres not being evenly dispersed. In addition, PCL and PLLA materials have poor affinity with tissues, making it impossible to repair tissues evenly.

[0007] Summary of the Invention

[0008] The present invention provides a temperature-sensitive physical hydrogel freeze-dried preparation and its preparation method and application, which are used to solve the defects of microsphere products in the prior art, such as easy needle clogging, easy precipitation, nodules and vascular embolism, easy degradation and poor stability of pre-filled preparations, etc., to achieve needle-free, painless and safe injection with good filling effect.

[0009] In a first aspect, the present invention provides a thermosensitive physical hydrogel freeze-dried preparation, the preparation raw materials including a thermosensitive polymer, sodium hyaluronate and phosphate buffer;

[0010] The phase transition temperature of the thermosensitive polymer is 35±2°C;

[0011] The molecular weight of the sodium hyaluronate is 2.2 million to 2.8 million, and the mass-to-volume ratio of the sodium hyaluronate to the phosphate buffer is 3-12 mg:1 ml.

[0012] In an embodiment of the present invention, the temperature-sensitive physical hydrogel freeze-dried preparation is prepared by mixing the preparation raw materials and then freeze-drying.

[0013] Degradable amphiphilic block polymers are a typical type of temperature-sensitive material, commonly used in injectable gel drug delivery systems, cartilage repair, tissue engineering scaffolds, etc., and are a relatively mature implant material. However, thermosensitive polymers that meet the requirements of undergoing a sol-gel phase transition at physiological temperature (i.e., a phase transition temperature of 35±2°C) have low molecular weights and short degradation cycles, making it difficult to meet the requirements of long-term medical aesthetic filling. The present invention has found that by compounding sodium hyaluronate with a specific molecular weight and dosage with the thermosensitive polymer, in-situ solidification filling can be achieved, and an interpenetrating three-dimensional gel network structure can be formed, which has better structural stability, is conducive to cell attachment and growth, and prolongs the effective repair time, i.e., a long degradation cycle of more than 6 months, thereby reducing the inconvenience caused by repeated injections. On the other hand, the present invention abandons pre-filled products in which microspheres are dissolved in gel and makes freeze-dried preparations, which can effectively prevent the preparation from oxidative decomposition and prolong the storage time. After reconstitution, the freeze-dried preparation is an aqueous solution, which is easy to inject, convenient for doctors to operate, and solves the problem of easy needle clogging.

[0014] The phosphate buffer described herein is prepared by dissolving disodium hydrogen phosphate and potassium dihydrogen phosphate in water. Reference is made to the preparation method for phosphate buffer (pH 7.3) in 8004 Buffer, Part III of the Chinese Pharmacopoeia 2020. The phosphate buffer provides salt balance and an adjustable pH buffer, alleviating discomfort in patients after injection.

[0015] In some embodiments of the present invention, the thermosensitive polymer is an amphiphilic block copolymer having an AB diblock, ABA triblock, or BAB triblock structure, wherein A represents a hydrophilic segment and B represents a hydrophobic segment. The mass-to-volume ratio of the thermosensitive polymer to the phosphate buffer is 100-300 mg:1 ml. The polymer concentration in the phosphate buffer affects the phase transition temperature. Under the same conditions, a higher concentration results in a lower phase transition temperature. If the concentration, i.e., the mass-to-volume ratio, of the thermosensitive polymer to the phosphate buffer exceeds this range, a phase transition temperature of 35±2°C cannot be achieved.

[0016] Among them, the triblock structure is superior to the diblock structure.

[0017] In a preferred embodiment of the present invention, the weight average molecular weight of segment A is in the range of 800-4000, the weight average molecular weight of segment B is in the range of 3000-7000, and the molecular weight ratio of segments A to B is 1:(2-3), preferably 1:2.5.

[0018] The present invention controls the weight-average molecular weight and ratio of the hydrophilic segment and the hydrophobic segment, can achieve rapid dissolution into a sol under low temperature conditions, reduces the difficulty of sol preparation, and is conducive to the rapid reconstitution of the freeze-dried preparation.

[0019] In an embodiment of the present invention, the A segment is a hydrophilic segment such as PEG, preferably PEG, and the B segment is one or more hydrophobic segments such as PCL, PLA, PLLA, PLCA, and PLGA. It is understood that PLA can be in the form of PLLA, PDLA, and PDLLA. For polymers such as PLCA and PLGA, which are composed of two or more monomers, the ratio of the monomers can be arbitrary.

[0020] In some embodiments of the present invention, the thermosensitive polymer is PCL-PEG-PCL, PLCA-PEG-PLCA, PLGA-PEG-PLGA, or PLLA-PEG-PLLA.

[0021] In a preferred embodiment of the present invention, the thermosensitive polymer is PLGA-PEG-PLGA.

[0022] It is understood that the preferred conditions of the present invention may be combined arbitrarily, provided they do not conflict. For example, a preferred thermosensitive polymer of the present invention is PLGA-PEG-PLGA. Further preferably, the weight-average molecular weight of PEG is in the range of 800-4000, the weight-average molecular weight of PLGA is in the range of 3000-7000, and the molecular weight ratio of PEG to PLGA is 1:(2-3), more preferably 1:2.5.

[0023] In a second aspect, the present invention provides a method for preparing the above-mentioned temperature-sensitive physical hydrogel freeze-dried preparation.

[0024] The preparation method provided by the present invention comprises mixing the preparation raw materials and then freeze-drying them.

[0025] In a specific embodiment of the present invention, the preparation method comprises: dissolving sodium hyaluronate in a phosphate buffer at 4-10°C, then dissolving a thermosensitive polymer in the phosphate buffer in which the sodium hyaluronate has been dissolved, filling, and freeze-drying at a pre-freezing temperature of -45 to -35°C.

[0026] The present invention selects a pre-freezing temperature of -45 to -35°C for freeze-drying. Under these freeze-drying conditions, the interaction between the thermosensitive polymer of the present invention and the hydrophilic groups of sodium hyaluronate results in uniform reconstitution and a short reconstitution time. The thermosensitive polymer must have the aforementioned preferred weight-average molecular weight and ratio of hydrophilic and hydrophobic segments.

[0027] Furthermore, the filling can be filled into vials at a volume of 1 ml / bottle. Vials are generally 5 ml in volume. It is understood that it can also be used for pre-filled injections, but the shelf life is relatively shorter.

[0028] In a preferred embodiment of the present invention, the preparation method of the thermosensitive physical hydrogel freeze-dried preparation comprises: adding HA at a mass-to-volume ratio of 8 mg / ml to phosphate buffer at 4-10°C, stirring for 0.5-1 hour until completely dissolved, then adding 200 mg / ml of thermosensitive polymer, continuing to stir for 1-2 hours until completely dissolved, and then filling into a vial at a volume of 1 ml / bottle, and freeze-drying at a pre-freezing temperature of -40°C.

[0029] In a third aspect, the present invention provides an injectable tissue filler comprising the above-mentioned temperature-sensitive physical hydrogel lyophilized preparation.

[0030] In an embodiment of the present invention, the thermosensitive physical hydrogel freeze-dried preparation is reconstituted and then administered to the target tissue of the subject by skin or subcutaneous administration, including but not limited to injection.

[0031] The present invention applies the prepared thermosensitive physical hydrogel freeze-dried preparation to the subcutaneous tissue or the skin after redissolving it. It can undergo a sol-gel phase transition at physiological temperature, achieve an in-situ solidification and immediate filling and repair effect on the application site, and can form an interpenetrating three-dimensional network structure with higher stability. It has good cell adhesion and is conducive to cell proliferation, reducing the risk of poor filling effect caused by subcutaneous displacement of the gel, and achieving the effect of targeted positioning and long-lasting filling. The gel components all have good biocompatibility and controllable biodegradability, which can effectively avoid the risk of vascular embolism and improve the operability of injection. Therefore, it can be used as an injectable tissue filler in the fields of medical beauty and tissue repair.

[0032] Furthermore, the reconstitution solution used for the reconstitution is water, and the amount thereof is equal to the sol volume of the temperature-sensitive physical hydrogel freeze-dried preparation before freeze-drying.

[0033] For example, if a phosphate buffer solution containing a thermosensitive polymer and sodium hyaluronate is filled into a vial at a volume of 1 ml per vial and lyophilized, 1 ml of water is used for reconstitution. It is understood that the amount of the reconstitution solution can vary without affecting the effectiveness of the solution.

[0034] In some embodiments of the present invention, the injectable tissue filler is suitable for injection through a 27-29G needle.

[0035] The present invention adopts a relatively thin needle of 27-29G, which can ensure the convenience of operation for doctors and prevent needle blockage, and can also relieve the pain of patients.

[0036] It should be noted that, in order to further promote the filling effect and prevent adverse reactions after filling such as inflammation, the injectable tissue filler may also include cell growth factors, anti-inflammatory drugs, etc., which can be added and mixed before the hydrogel is freeze-dried.

[0037] It should be noted that, unless otherwise defined, the scientific and technical terms used in this specification have the same meanings as those commonly understood by those skilled in the art. Furthermore, unless otherwise inconsistent with the context, singular nouns used in this specification include the plural form of the noun, and plural nouns also include the singular form of the noun.

[0038] The present invention provides a thermosensitive physical hydrogel freeze-dried preparation and its preparation method and application. Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] The present invention adopts a compound of sodium hyaluronate and a thermosensitive polymer with a specific molecular weight and dosage to achieve a rapid gelation transition under physiological temperature conditions, forming an interpenetrating three-dimensional network structure with better structural stability, which is conducive to cell growth and repair, and has a long degradation time, which can reduce the inconvenience of repeated injections.

[0040] Furthermore, the optimized freeze-drying conditions shorten the reconstitution time of the freeze-dried preparation, facilitate clinical operation, and solve the problems of short storage time, uneven dispersion, easy oxidation and decomposition, and sedimentation and needle blockage of microsphere-filled preparations. When injected, it is in liquid form, so a thinner needle can be used to reduce patient pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a schematic diagram of the freeze-dried gel structure provided in Example 1 of the present invention;

[0042] FIG2 is a physical picture of the lyophilized gel in Example 1 of the present invention, which is a transparent liquid after dissolution (left) and a translucent gel after the transparent liquid is bathed in 37° C. water (right);

[0043] FIG3 is a 400× Masson staining image of collagen fiber proliferation in the subcutaneous tissue of an animal after the lyophilized hydrogel preparation provided in Example 1 of the present invention was reconstituted and implanted subcutaneously. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0046] The English and Chinese abbreviations of some chemical substances involved in this invention are as follows:

[0047] PCL: Polycaprolactone

[0048] PLA: Polylactic acid

[0049] PLLA: poly (L-lactic acid)

[0050] PDLA: dextrorotatory polylactic acid

[0051] PDLLA: racemic polylactic acid

[0052] PLCA: polylactic acid-citric acid

[0053] PLGA: polylactic-co-glycolic acid

[0054] PBS: Phosphate buffered saline

[0055] HA: Sodium hyaluronate.

[0056] Example 1

[0057] This embodiment provides a thermosensitive physical hydrogel freeze-dried preparation, the preparation method of which is as follows:

[0058] Phosphate buffer was prepared by referring to the preparation method of phosphate buffer (pH 7.3) in 8004 buffer solution in the General Rules of the Chinese Pharmacopoeia 2020 Part III.

[0059] HA with a molecular weight of 2.8 million was added to phosphate buffer at a mass-to-volume ratio of 8 mg / ml and stirred at 500 rpm for 0.5-1 h until dissolved.

[0060] Thermosensitive polymer: PLGA(5000)-PEG(2000)-PLGA(5000) with an A:B segment ratio of 1:2.5 was added to the phosphate buffer solution containing HA at a concentration of 200 mg / ml (still based on the volume of pure phosphate buffer solution, the same below), and stirred at 500 rpm for 1-2 hours until dissolved.

[0061] Then, the mixture was filled into 5 ml vials at a volume of 1 ml / vial, the pre-freezing temperature was set to -40°C, and freeze-dried according to the freeze-drying parameters shown in Table 1 below.

[0062] Table 1

[0063] After freeze-drying is completed, the lid is pressed and sealed to obtain a freeze-dried hydrogel preparation.

[0064] The schematic diagram of the gel structure of the freeze-dried hydrogel preparation obtained in this example after freeze-drying is shown in Figure 1. As can be seen from the figure, an interpenetrating three-dimensional gel network structure is formed, which is conducive to cell attachment and growth.

[0065] Example 2

[0066] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0067] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 100 mg / ml.

[0068] Example 3

[0069] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0070] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 300 mg / ml.

[0071] Example 4

[0072] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0073] Sodium hyaluronate (HA) was added to the phosphate buffer at a mass volume ratio of 3 mg / ml.

[0074] Example 5

[0075] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 4 in that:

[0076] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 100 mg / ml.

[0077] Example 6

[0078] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 4 in that:

[0079] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 300 mg / ml.

[0080] Example 7

[0081] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0082] Sodium hyaluronate HA was added into phosphate buffer at a mass volume ratio of 12 mg / ml.

[0083] Example 8

[0084] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 7 in that:

[0085] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 100 mg / ml.

[0086] Example 9

[0087] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 7 in that:

[0088] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 300 mg / ml.

[0089] Example 10

[0090] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0091] The pre-freezing temperature was set at -45°C, and freeze-drying was performed according to the freeze-drying parameters shown in Table 2 below.

[0092] Table 2

[0093] Example 11

[0094] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0095] The pre-freezing temperature was set to -35°C, and freeze-drying was performed according to the freeze-drying parameters shown in Table 3 below.

[0096] Table 3

[0097] Example 12

[0098] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0099] The molecular weight of sodium hyaluronate HA is 2.2 million.

[0100] Example 13

[0101] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0102] Thermosensitive polymer: PCL(5000)-PEG(2000)-PCL(5000) with an A:B segment ratio of 1:2.5.

[0103] Example 14

[0104] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0105] Thermosensitive polymer: PLCA(4000)-PEG(2000)-PLCA(4000) with an A:B segment ratio of 1:2.5.

[0106] Example 15

[0107] This example provides a thermosensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0108] Thermosensitive polymer: PLLA(6000)-PEG(2000)-PLLA(6000) with an A:B segment ratio of 1:3.

[0109] Comparative Example 1

[0110] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0111] Sodium hyaluronate HA was added into phosphate buffer at a mass volume ratio of 2 mg / ml.

[0112] Comparative Example 2

[0113] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from comparative example 1 in that:

[0114] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 100 mg / ml.

[0115] Comparative Example 3

[0116] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from comparative example 1 in that:

[0117] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 300 mg / ml.

[0118] Comparative Example 4

[0119] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0120] Sodium hyaluronate HA was added into phosphate buffer at a mass volume ratio of 13 mg / ml.

[0121] Comparative Example 5

[0122] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from comparative example 4 in that:

[0123] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 100 mg / ml.

[0124] Comparative Example 6

[0125] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from comparative example 4 in that:

[0126] The thermosensitive polymer was added to the phosphate buffer solution containing the dissolved HA at a concentration of 300 mg / ml.

[0127] Comparative Example 7

[0128] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0129] The molecular weight of sodium hyaluronate HA is 2 million.

[0130] Comparative Example 8

[0131] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0132] The molecular weight of sodium hyaluronate HA is 3 million.

[0133] Comparative Example 9

[0134] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0135] Thermosensitive polymer: PLGA(2000)-PEG(2000)-PLGA(2000) with an A:B segment ratio of 1:1.

[0136] Comparative Example 10

[0137] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0138] Thermosensitive polymer: PLGA(7000)-PEG(2000)-PLGA(7000) with an A:B segment ratio of 1:3.5.

[0139] Comparative Example 11

[0140] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0141] The pre-freezing temperature was set at -50°C, and freeze-drying was performed according to the freeze-drying parameters shown in Table 4 below.

[0142] Table 4

[0143] Comparative Example 12

[0144] This comparative example provides a temperature-sensitive physical hydrogel freeze-dried preparation, which differs from Example 1 in that:

[0145] The pre-freezing temperature was set at -30°C, and freeze-drying was performed according to the freeze-drying parameters shown in Table 5 below.

[0146] Table 5

[0147] Experimental example

[0148] The following tests were performed on the freeze-dried hydrogel preparations prepared in the examples and comparative examples:

[0149] 1. Take one vial of hydrogel freeze-dried preparation, inject 1 ml of pure water, vortex for 5 minutes, and pour it onto a filter membrane with a pore size of 0.2 μm. Turn on the vacuum pump for filtration and observe whether there is any unmelted film on the filter membrane to determine whether the freeze-dried gel is completely dissolved.

[0150] 2. Return the solution gel dissolved according to Test 1 to room temperature (25°C) to a liquid state. Place the gel in a syringe, expel the air, and test the extrusion force of the solution gel using a 29G needle in a pressure testing machine. If the sustained force of the piston is ≤15N, the composition has a moderate extrusion force and is well suited for surgical procedures.

[0151] 3. Pour the solution gel dissolved according to Test 1 into a glass test tube and place it in a water bath. Start heating at 33°C in a water bath, increasing the temperature by 1°C at a time until it reaches 38°C. Incubate at each temperature for 10 minutes until the gel reaches the water bath temperature. Use the test tube inversion method to determine whether the current water bath temperature has reached the sol-gel transition temperature. If the sol has no fluidity after the phase transition and does not flow after inverting the test tube, this method can be used to test the phase transition temperature of the gel formulation.

[0152] 4. Insulate the gel obtained by Test 3 at 37°C. Place approximately 0.5 ml of the gel into the sample holder of the rheometer and test the loss modulus and storage modulus of the gel at different frequencies. When the storage modulus is greater than the loss modulus, the sample is in a gel state at that frequency; otherwise, it is in a solution state. This method tests the stability of the gel in different application scenarios. 1-10 Hz is the daily vibration frequency of the gel, and the scanning range is 0.1-100 Hz (refer to YYT0308-2015 Medical Sodium Hyaluronate Gel 5.8 Shear Viscosity).

[0153] The test results of each group are shown in Table 6.

[0154] Table 6

[0155] The lyophilized hydrogel preparations obtained in Examples 1-15 were in a transparent liquid state after dissolution, which then transformed into a translucent gel state after incubation in water at 37°C. Figure 2 shows a photo of the sample in Example 1, which was in a transparent liquid state after dissolution (left) and a translucent gel state after incubation in water at 37°C (right).

[0156] 5. Fifty-four healthy New Zealand white rabbits were screened and divided into 27 groups of two rabbits each. The samples prepared in the Examples and Comparative Examples were reconstituted and injected subcutaneously into the left and right backs of each rabbit. Each rabbit received three injections on each side of the back, with 0.1 ml injected at each site, separated by 2 cm. Skin redness and swelling reactions were observed for 7 days. Two rabbits in each group were sacrificed at 1 month and 3 months, respectively. Skin papules of the injected material were removed and microscopically observed for collagen proliferation. The results are shown in Table 7.

[0157] Table 7

[0158] In addition, FIG3 is a 400×Masson staining image of collagen fiber proliferation in the subcutaneous tissue of the animal 3 months after the reconstituted sample of Example 1 was implanted subcutaneously. As can be seen from the figure, collagen fiber proliferation is obvious in the tissue injection site.

[0159] 6. Take the 24 vials of hydrogel freeze-dried preparation prepared in each group of Examples 1, 13, 14, and 15, add 7.28 ml of Sorensen buffer to each vial (reference method: YY-0473-2004 Surgical Implant Polylactide Copolymer and Blend In Vitro Degradation Test 4.2.1), vortex for 5 minutes until dissolved, and place the container in an oscillating water bath at 37±1°C. Take two degradation samples from each group every month and test the content of the degradation product lactic acid. When the lactic acid content reaches the maximum value and the concentration no longer changes, it indicates that it has been completely degraded (reference method: YYT 1806.1-2021 In Vitro Degradation of Biopharmaceutical Materials In Vitro Degradation Performance Evaluation Method for Degradable Polyesters 5.4). Degradation tests are performed on the samples of the examples.

[0160] Lyophilized preparations with the same formulations as those in Example 1, Example 13, Example 14, and Example 15, except that they did not contain HA, were prepared and named Example 1', Example 13', Example 14', and Example 15'. Degradation tests were performed using the same method as above. The results are shown in Table 8.

[0161] Table 8

[0162] According to the above experimental results, the present invention adopts high molecular weight sodium hyaluronate and thermosensitive polymer to be dissolved in phosphate buffer, and makes lyophilized preparation, can effectively prevent oxidation, makes polymer and sodium hyaluronate stable and difficult to decompose, and the hydrogel lyophilized preparation is aqueous solution after adding water and redissolving, is easy to inject, solves the problems such as microsphere preparation instability, easy blocking needle, and the hydrophilic group of compounded sodium hyaluronate interacts with the hydrophilic group of thermosensitive polymer, improves the speed of redissolving. After lyophilized preparation is redissolved and injected, when tending to physiological temperature, the micelle aggregation degree formed by polymer in solution increases and gel state phase transition occurs, forms interpenetrating three-dimensional network structure with sodium hyaluronate, and structural stability is good, strengthens tissue filling effect. In addition, compounding high molecular weight sodium hyaluronate, not only can accelerate the thermal gelation of amphiphilic block copolymer, and makes up for the fast disadvantage of block copolymer degradation rate, prolongs degradation cycle, reduces the number of supplementary injections, and provides cell attachment scaffold structure simultaneously, is easy to tissue repair.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention. Industrial Applicability

[0164] The present invention provides a thermosensitive physical hydrogel freeze-dried formulation, its preparation method, and application. The raw materials for preparing the thermosensitive physical hydrogel freeze-dried formulation include a thermosensitive polymer, sodium hyaluronate, and phosphate buffer. The thermosensitive polymer has a phase transition temperature of 35±2°C. The molecular weight of the sodium hyaluronate is 2.2 million to 2.8 million, and the mass-to-volume ratio of the sodium hyaluronate to the phosphate buffer is 3-12 mg:1 ml. The present invention utilizes a combination of sodium hyaluronate and a thermosensitive polymer of specific molecular weight and dosage to achieve a rapid gelation transition at physiological temperatures, forming an interpenetrating three-dimensional network structure. This provides improved structural stability, facilitates cell growth and repair, and has a long degradation time, reducing the inconvenience of repeated injections. Furthermore, the freeze-dried formulation can address the problems of microsphere products such as needle clogging, precipitation, degradation, and poor stability, demonstrating excellent economic value and application prospects.

Claims

1. A thermosensitive physical hydrogel freeze-dried preparation, characterized in that: The preparation raw materials include thermosensitive polymer, sodium hyaluronate and phosphate buffer; The phase transition temperature of the thermosensitive polymer is 35±2°C; The molecular weight of the sodium hyaluronate is 2.2 million to 2.8 million, and the mass-to-volume ratio of the sodium hyaluronate to the phosphate buffer is 3-12 mg:1 ml.

2. The thermosensitive physical hydrogel freeze-dried preparation according to claim 1, characterized in that: The thermosensitive polymer is an amphiphilic block copolymer having an AB type diblock, ABA type triblock or BAB type triblock structure, wherein A represents a hydrophilic segment and B represents a hydrophobic segment. The mass volume ratio of the thermosensitive polymer to the phosphate buffer is 100-300 mg:1 ml.

3. The thermosensitive physical hydrogel freeze-dried preparation according to claim 2, characterized in that: The weight average molecular weight of the A segment is in the range of 800-4000, the weight average molecular weight of the B segment is in the range of 3000-7000, and the molecular weight ratio of the A and B segments is 1:(2-3), preferably 1:2.

5.

4. The thermosensitive physical hydrogel freeze-dried preparation according to claim 3, characterized in that: The A segment is PEG, and the B segment is one or more of PCL, PLA, PLLA, PLCA, and PLGA; Preferably, the thermosensitive polymer is PLGA-PEG-PLGA.

5. The method for preparing the thermosensitive physical hydrogel freeze-dried preparation according to any one of claims 1 to 4, characterized in that: The raw materials are mixed and then freeze-dried.

6. The method for preparing the thermosensitive physical hydrogel freeze-dried preparation according to claim 5, characterized in that: include: At 4-10°C, sodium hyaluronate is dissolved in phosphate buffer, and then the thermosensitive polymer is dissolved in the phosphate buffer in which the sodium hyaluronate has been dissolved. The mixture is filled and freeze-dried at a pre-freezing temperature of -45 to -35°C.

7. An injectable tissue filler, characterized in that: The invention comprises the thermosensitive physical hydrogel freeze-dried preparation according to any one of claims 1 to 4.

8. The injectable tissue filler according to claim 7, characterized in that: The thermosensitive physical hydrogel lyophilized preparation is reconstituted and then administered to the target tissue of the subject by the skin or subcutaneous administration.

9. The injectable tissue filler according to claim 8, characterized in that: The reconstitution solution used in the reconstitution is water, and the amount used is equal to the sol volume of the temperature-sensitive physical hydrogel freeze-dried preparation before freeze-drying.

10. The injectable tissue filler according to any one of claims 7 to 9, characterized in that: The injectable tissue filler is suitable for injection through a 27-29G needle.

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