A temperature-sensitive physical hydrogel lyophilized preparation, and a preparation method and application thereof

Abstract

This invention provides a thermosensitive physical hydrogel lyophilized formulation, its preparation method, and its application. The raw materials for preparing the thermosensitive physical hydrogel lyophilized formulation include a thermosensitive polymer, sodium hyaluronate, and phosphate buffer. The phase transition temperature of the thermosensitive polymer is 35±2℃. The molecular weight of the sodium hyaluronate is 2.2 million to 2.8 million, and its mass-to-volume ratio with the phosphate buffer is 3-12 mg:1 ml. This invention uses a specific molecular weight and dosage of sodium hyaluronate and a thermosensitive polymer to achieve rapid gelation transformation under physiological temperature conditions, forming an interpenetrating three-dimensional network structure with better structural stability, facilitating cell growth and repair, and extending degradation time, thus reducing the inconvenience of repeated injections. Furthermore, the lyophilized formulation can solve problems such as needle clogging, precipitation, degradation, and poor stability associated with microsphere products.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and chemical technology, and in particular to a thermosensitive physical hydrogel freeze-dried formulation, its preparation method, and its application. Background Technology

[0002] In recent years, with the improvement of people's living standards, the demand for medical aesthetic products has continued to increase, promoting the continuous upgrading of plastic surgery and cosmetic techniques and products. In the field of minimally invasive cosmetic procedures, beauty seekers are increasingly favoring non-invasive, immediate-repairing filler products, which have been widely recognized by consumers since their launch. These non-invasive, immediate-repairing injectable fillers are generally the first choice for rhinoplasty, chin augmentation, lip augmentation, and anti-wrinkle fillers. They are injected directly into the dermis, providing moisturizing, lubrication, and shaping effects.

[0003] Currently popular injectable fillers are mostly formulations made from materials such as polycaprolactone (PCL) and polylactic acid (PLA), containing microparticles of 20-50 μm in size, mixed with an aqueous solution of carboxymethyl cellulose. After subcutaneous injection, the gel formed by the carboxymethyl cellulose aqueous solution provides an immediate filling effect. Then, as the gel degrades, the microspheres in the formulation begin to function. By adjusting the molecular weight of the polymer microspheres, their degradation time in the body can be controlled, achieving a long-lasting effect of stimulating collagen regeneration.

[0004] These types of formulations are generally pre-filled injectable solutions. To prevent microspheres from settling and agglomerating, leading to uneven distribution and tissue inflammation, the gel concentration of these formulations is often high. Furthermore, to reduce pain, finer needles are typically used. To avoid microspheres being phagocytosed by the body and to achieve a sustained stimulating effect, the microsphere particle size is often above 40 μm. This makes the process inconvenient and prone to needle clogging. On one hand, the microsphere preparation process generally employs emulsification-solidification-sieving, which requires the introduction of reagents such as dichloromethane and polyvinyl alcohol. These reagents have low tolerance requirements in implanted formulations and are difficult to remove. Moreover, to achieve a suitable microsphere particle size distribution, sieving removes some non-target microspheres, resulting in a complex process and high raw material costs. On the other hand, as the carboxymethyl cellulose gel degrades after implantation, its structure becomes loose, gel viscosity decreases, and cell adhesion weakens, making it difficult to support long-term stable suspension of the microspheres. This poses a risk of microsphere settling, agglomeration, and injection displacement. The weakening of the microsphere dispersion and support effect leads to the microspheres not being uniformly dispersed. In addition, the poor affinity between PCL and PLLA materials and tissues results in the inability to uniformly repair tissues. Summary of the Invention

[0005] This invention provides a thermosensitive physical hydrogel freeze-dried formulation, its preparation method, and its application, to solve the defects of existing technologies such as microsphere products being prone to needle clogging, precipitation, nodules, and vascular embolism, and pre-filled formulations being prone to degradation and having poor stability, thereby achieving needle-free, painless, safe injection with good filling effect.

[0006] In a first aspect, the present invention provides a thermosensitive physical hydrogel freeze-dried formulation, the raw materials for which include a thermosensitive polymer, sodium hyaluronate and phosphate buffer.

[0007] The phase transition temperature of the thermosensitive polymer is 35±2℃;

[0008] The sodium hyaluronate has a molecular weight of 2.2 million to 2.8 million, and its mass-to-volume ratio with the phosphate buffer is 3-12 mg: 1 ml.

[0009] In an embodiment of the present invention, the temperature-sensitive physical hydrogel freeze-dried formulation is prepared by freeze-drying the raw materials.

[0010] Biodegradable amphiphilic block polymers are a typical class of temperature-sensitive materials, commonly used in injectable gel drug delivery systems, cartilage repair, and tissue engineering scaffolds. They are a relatively mature implant material. However, temperature-sensitive polymers that undergo a sol-gel phase transition at physiological temperatures (i.e., a phase transition temperature of 35±2℃) have low molecular weights and short degradation cycles, making them unsuitable for long-lasting cosmetic filling. This invention has discovered that by combining sodium hyaluronate with the aforementioned temperature-sensitive polymer at specific molecular weights and dosages, in-situ curing and filling can be achieved, forming an interpenetrating three-dimensional gel network structure with better structural stability, facilitating cell adhesion and growth, and extending the effective repair time—that is, a longer degradation cycle of over 6 months—reducing the inconvenience of repeated injections. Furthermore, this invention abandons pre-filled products where microspheres are dissolved in gel, instead producing a lyophilized formulation, which effectively prevents oxidative decomposition and extends storage time. The lyophilized formulation, after reconstitution, is an aqueous solution, easy to inject, convenient for doctors, and solves the problem of needle clogging.

[0011] The phosphate buffer solution described in this invention is prepared by dissolving disodium hydrogen phosphate and potassium dihydrogen phosphate in water, referring to the preparation method of phosphate buffer solution (pH 7.3) in Buffer Solution 8004 of the General Chapter 3 of the Chinese Pharmacopoeia 2020. The function of the phosphate buffer solution is to maintain salt balance and provide an adjustable pH buffering effect, reducing patient discomfort after injection.

[0012] In some embodiments of the present invention, the thermosensitive polymer is an amphiphilic block copolymer with 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-to-volume ratio of the thermosensitive polymer to the phosphate buffer is 100-300 mg: 1 ml. The concentration of the polymer 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 the above range, a phase transition temperature of 35 ± 2 °C cannot be obtained.

[0013] Among them, the triblock structure is superior to the diblock structure.

[0014] 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 and B is 1:(2-3), preferably 1:2.5.

[0015] This invention controls the weight-average molecular weight and ratio of hydrophilic and hydrophobic segments, enabling rapid dissolution into a sol under low-temperature conditions, reducing the difficulty of sol preparation, and facilitating the rapid reconstitution of freeze-dried formulations.

[0016] In embodiments of the present invention, segment A is a hydrophilic segment such as PEG, preferably PEG, and segment B 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, as well as PDLA and PDLLA. For polymers such as PLCA and PLGA, which are polymerized from two or more monomers, the proportion of monomers can be arbitrary.

[0017] In some embodiments of the present invention, the thermosensitive polymer is PCL-PEG-PCL, PLCA-PEG-PLCA, PLGA-PEG-PLGA, or PLLA-PEG-PLLA.

[0018] In a preferred embodiment of the present invention, the thermosensitive polymer is PLGA-PEG-PLGA.

[0019] It is understood that the preferred conditions of the present invention can be combined arbitrarily without conflict. For example, the preferred thermosensitive polymer of the present invention is PLGA-PEG-PLGA. More 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.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned thermosensitive physical hydrogel lyophilized formulation.

[0021] The preparation method provided by the present invention includes mixing the raw materials and then freeze-drying them.

[0022] In a specific embodiment of the present invention, the preparation method includes: dissolving sodium hyaluronate in phosphate buffer at 4-10°C, then dissolving the thermosensitive polymer in the aforementioned phosphate buffer containing dissolved sodium hyaluronate, filling the container, and freeze-drying at a pre-freezing temperature of -45 to -35°C.

[0023] This invention selects a pre-freezing temperature of -45 to -35°C for freeze-drying. Under these freeze-drying conditions, combined with the interaction between the thermosensitive polymer of this invention and the hydrophilic groups of sodium hyaluronate, the reconstitution effect is uniform and the reconstitution time is short. The thermosensitive polymer must be selected based on the aforementioned preferred weight-average molecular weight and ratio of hydrophilic and hydrophobic segments.

[0024] Furthermore, the filling can be done at a rate of 1 ml per vial. Vials are typically selected in 5 ml volumes. It is understood that pre-filled injectable solutions can also be used, but the shelf life will be shorter.

[0025] In a preferred embodiment of the present invention, the preparation method of the thermosensitive physical hydrogel lyophilized formulation includes: adding HA to phosphate buffer at a mass-volume ratio of 8 mg / ml at 4-10°C, stirring for 0.5-1 h until completely dissolved, then adding 200 mg / ml of thermosensitive polymer, continuing to stir for 1-2 h until completely dissolved, then filling into vials at a volume of 1 ml / bottle, and lyophilizing at a pre-freezing temperature of -40°C.

[0026] Thirdly, the present invention provides an injectable tissue filler comprising the above-mentioned thermosensitive physical hydrogel lyophilized formulation.

[0027] In embodiments of the present invention, the thermosensitive physical hydrogel lyophilized formulation is reconstituted and then administered to the target tissue of the subject via skin or subcutaneous application. Administration methods include, but are not limited to, injection.

[0028] This invention involves reconstituted a freeze-dried thermosensitive physical hydrogel formulation and applying it subcutaneously or intradermally. It undergoes a sol-gel phase transition at physiological temperatures, achieving in-situ solidification and immediate filling and repair effects at the application site. Furthermore, it forms a more stable interpenetrating three-dimensional network structure with good cell adhesion, promoting cell proliferation and reducing the risk of poor filling effect due to gel displacement under the skin, thus achieving targeted and long-lasting filling. All gel components exhibit good biocompatibility and controllable biodegradability, effectively avoiding the risk of vascular embolism and improving injection operability. Therefore, it can be used as an injectable tissue filler in fields such as medical aesthetics and tissue repair.

[0029] Furthermore, the resolution solution used is water, and its amount is equal to the sol volume of the temperature-sensitive physical hydrogel lyophilized formulation before lyophilization.

[0030] For example, if a phosphate buffer solution containing a thermosensitive polymer and sodium hyaluronate is dispensed into vials at a volume of 1 ml / vial and then lyophilized, 1 ml of water should be used for reconstitution. It is understandable that an error in the amount of reconstitution solution used is permissible and does not affect the effectiveness of the solution.

[0031] In some embodiments of the invention, the injectable tissue filler is adapted to be injected using a 27-29G needle.

[0032] This invention uses a relatively fine needle of 27-29G, which can ensure convenient operation for doctors, prevent needle blockage, and reduce patient pain.

[0033] It should be noted that, in order to further promote the filling effect and prevent adverse reactions such as inflammation after filling, 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.

[0034] It should be noted that, unless otherwise defined, the scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. Furthermore, unless conflicting with the context, singular nouns used in this specification include their plural forms, and vice versa.

[0035] This invention provides a thermosensitive physical hydrogel freeze-dried formulation, its preparation method, and its application. Compared with the prior art, this invention has at least the following beneficial effects:

[0036] This invention uses sodium hyaluronate with a specific molecular weight and dosage, combined with a thermosensitive polymer, to achieve rapid gelation transformation under physiological temperature conditions, forming an interpenetrating three-dimensional network structure with better structural stability, which is conducive to cell growth and repair. The degradation time is long, which can reduce the inconvenience of repeated injections.

[0037] Furthermore, optimized freeze-drying conditions shorten the reconstitution time of freeze-dried formulations, facilitating clinical operation and solving problems such as short storage time, uneven dispersion, easy oxidation and decomposition, sedimentation and needle clogging of microsphere-filled formulations. The formulations are in liquid form during injection, allowing the use of finer needles and reducing patient discomfort. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the freeze-dried gel structure provided in Embodiment 1 of the present invention;

[0039] Figure 2 These are actual images of the gel after freeze-drying in Example 1 of the present invention, which is in a transparent liquid state (left) and a translucent gel state after being bathed in a water bath at 37°C (right).

[0040] Figure 3 The image shows collagen fiber proliferation in an animal after the hydrogel freeze-dried formulation provided in Example 1 of this invention was reconstituted and implanted subcutaneously. The image was stained with 400× Masson staining. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] The English abbreviations and Chinese translations of some of the chemical substances involved in this invention are as follows:

[0044] PCL: Polycaprolactone

[0045] PLA: Polylactic acid

[0046] PLLA: Polylactic Acid

[0047] PDLA: dextrorotatory polylactic acid

[0048] PDLLA: Racemic polylactic acid

[0049] PLCA: Polylactic acid-citric acid

[0050] PLGA: Polylactic-Co-glycolic Acid

[0051] PBS: Phosphate Buffer

[0052] HA: Sodium hyaluronate.

[0053] Example 1

[0054] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, the preparation method of which is as follows:

[0055] Phosphate buffer was prepared according to the preparation method of phosphate buffer (pH 7.3) in 8004 buffer solution, as per the General Chapter of Part III of the Chinese Pharmacopoeia 2020.

[0056] Add HA with a molecular weight of 2.8 million to phosphate buffer at a mass-to-volume ratio of 8 mg / ml, and stir at 500 rpm for 0.5-1 h until dissolved.

[0057] Add PLGA(5000)-PEG(2000)-PLGA(5000), a thermosensitive polymer with an A / B segment ratio of 1:2.5, to the phosphate buffer solution containing dissolved HA at a concentration of 200 mg / ml (based on the volume of the plain phosphate buffer solution, the same below). Stir at 500 rpm for 1-2 hours until dissolved.

[0058] Then, fill 1ml / bottle into 5ml vials, set the pre-freezing temperature to -40℃, and freeze-dry according to the freeze-drying parameters shown in Table 1 below.

[0059] Table 1

[0060]

[0061] After freeze-drying, the product is capped and sealed to obtain a hydrogel freeze-dried formulation.

[0062] A schematic diagram of the gel structure of the lyophilized hydrogel formulation obtained in this embodiment is shown below. Figure 1 As shown in the figure, an interpenetrating three-dimensional gel network structure is formed, which facilitates cell adhesion and growth.

[0063] Example 2

[0064] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0065] The thermosensitive polymer was added at a concentration of 100 mg / ml to the phosphate buffer solution used to dissolve HA.

[0066] Example 3

[0067] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0068] The thermosensitive polymer was added at a concentration of 300 mg / ml to the phosphate buffer solution used to dissolve HA.

[0069] Example 4

[0070] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0071] Sodium hyaluronate (HA) was added to phosphate buffer at a mass-volume ratio of 3 mg / ml.

[0072] Example 5

[0073] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 4 in that:

[0074] The thermosensitive polymer was added at a concentration of 100 mg / ml to the phosphate buffer solution used to dissolve HA.

[0075] Example 6

[0076] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 4 in that:

[0077] The thermosensitive polymer was added at a concentration of 300 mg / ml to the phosphate buffer solution used to dissolve HA.

[0078] Example 7

[0079] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0080] Sodium hyaluronate (HA) was added to phosphate buffer at a mass-volume ratio of 12 mg / ml.

[0081] Example 8

[0082] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 7 in that:

[0083] The thermosensitive polymer was added at a concentration of 100 mg / ml to the phosphate buffer solution used to dissolve HA.

[0084] Example 9

[0085] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 7 in that:

[0086] The thermosensitive polymer was added at a concentration of 300 mg / ml to the phosphate buffer solution used to dissolve HA.

[0087] Example 10

[0088] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0089] Set the pre-freezing temperature to -45℃ and perform freeze-drying according to the freeze-drying parameters shown in Table 2 below.

[0090] Table 2

[0091]

[0092]

[0093] Example 11

[0094] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0095] Set the pre-freezing temperature to -35℃ and perform freeze-drying according to the freeze-drying parameters shown in Table 3 below.

[0096] Table 3

[0097]

[0098] Example 12

[0099] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0100] Sodium hyaluronate (HA) has a molecular weight of 2.2 million.

[0101] Example 13

[0102] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0103] Thermosensitive polymer: PCL(5000)-PEG(2000)-PCL(5000) with an A:B segment ratio of 1:2.5.

[0104] Example 14

[0105] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0106] Thermosensitive polymer: PLCA(4000)-PEG(2000)-PLCA(4000) with an A:B segment ratio of 1:2.5.

[0107] Example 15

[0108] This embodiment provides a thermosensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0109] Thermosensitive polymer: PLLA(6000)-PEG(2000)-PLLA(6000) with an A:B segment ratio of 1:3.

[0110] Comparative Example 1

[0111] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0112] Sodium hyaluronate (HA) was added to phosphate buffer at a mass-volume ratio of 2 mg / ml.

[0113] Comparative Example 2

[0114] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Comparative Example 1 in that:

[0115] The thermosensitive polymer was added at a concentration of 100 mg / ml to the phosphate buffer solution used to dissolve HA.

[0116] Comparative Example 3

[0117] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Comparative Example 1 in that:

[0118] The thermosensitive polymer was added at a concentration of 300 mg / ml to the phosphate buffer solution used to dissolve HA.

[0119] Comparative Example 4

[0120] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0121] Sodium hyaluronate (HA) was added to phosphate buffer at a mass-volume ratio of 13 mg / ml.

[0122] Comparative Example 5

[0123] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Comparative Example 4 in that:

[0124] The thermosensitive polymer was added at a concentration of 100 mg / ml to the phosphate buffer solution used to dissolve HA.

[0125] Comparative Example 6

[0126] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Comparative Example 4 in that:

[0127] The thermosensitive polymer was added at a concentration of 300 mg / ml to the phosphate buffer solution used to dissolve HA.

[0128] Comparative Example 7

[0129] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0130] Sodium hyaluronate (HA) has a molecular weight of 2 million.

[0131] Comparative Example 8

[0132] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0133] Sodium hyaluronate (HA) has a molecular weight of 3 million.

[0134] Comparative Example 9

[0135] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0136] Thermosensitive polymer: PLGA(2000)-PEG(2000)-PLGA(2000) with an A and B segment ratio of 1:1.

[0137] Comparative Example 10

[0138] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0139] Thermosensitive polymer: PLGA(7000)-PEG(2000)-PLGA(7000) with an A:B segment ratio of 1:3.5.

[0140] Comparative Example 11

[0141] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0142] Set the pre-freezing temperature to -50℃ and perform freeze-drying according to the freeze-drying parameters shown in Table 4 below.

[0143] Table 4

[0144]

[0145] Comparative Example 12

[0146] This comparative example provides a temperature-sensitive physical hydrogel lyophilized formulation, which differs from Example 1 in that:

[0147] Set the pre-freezing temperature to -30℃ and perform freeze-drying according to the freeze-drying parameters shown in Table 5 below.

[0148] Table 5

[0149]

[0150] Experimental Example

[0151] The hydrogel lyophilized formulations prepared in the examples and comparative examples were subjected to the following tests:

[0152] 1. Take one vial of hydrogel lyophilized 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 to filter and observe whether there are unmelted films on the filter membrane to determine whether the lyophilized gel has completely dissolved.

[0153] 2. After the solution gelled according to test method 1 is restored to room temperature (25°C) and becomes liquid, it is loaded into a syringe, air is expelled, and the extrusion force of the solution gel is tested under a pressure testing machine using a 29G needle. If the continuous force of the piston sliding is ≤15N, it indicates that the corresponding composition has a moderate extrusion force and good adaptability for the surgeon's operation.

[0154] 3. Pour the dissolved gel solution from test method 1 into a glass test tube, place it in a water bath, and heat it from 33°C, increasing the temperature by 1°C each time, until it reaches 38°C. Heat the tube for 10 minutes at each temperature to allow the gel to reach the water bath temperature. Use the inverted test tube method to determine if the current water bath temperature has reached the sol-gel transition temperature. After the sol undergoes a phase transition, it becomes non-flowable and does not flow after inverting the test tube. This method is used to test the phase transition temperature of the gel formulation.

[0155] 4. Incubate the gel obtained according to method 3 at 37℃. Take approximately 0.5 ml of gel and place it in the sample stage of the rheometer. Test the loss modulus and storage modulus of the gel at different frequencies. When the storage modulus is greater than the loss modulus, it indicates that 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 under different applicable scenarios. 1-10 Hz is the frequency at which the gel is normally subjected to vibration, and the scanning range is 0.1-100 Hz (refer to YYT0308-2015 Medical Sodium Hyaluronate Gel 5.8 Shear Viscosity).

[0156] The test results for each group are shown in Table 6.

[0157] Table 6

[0158]

[0159]

[0160]

[0161]

[0162] Among them, the hydrogel freeze-dried formulations obtained in Examples 1-15 are transparent liquids after dissolution, and the transparent liquids are translucent gels after being bathed in a water bath at 37°C. Figure 2 The images show the samples from Example 1 after dissolution, which are in a transparent liquid state (left) and a translucent gel state after being bathed in a water bath at 37°C (right).

[0163] 5. Fifty-four healthy New Zealand white rabbits were selected 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. Three injection points were made on each side of the back of each rabbit, with 0.1 ml injected at each point and 2 cm apart. Skin redness and swelling were observed for 7 days. Two rabbits from each group were sacrificed at 1 month and 3 months, respectively. Skin wheals of the injected material were collected and observed under a microscope for collagen hyperplasia. The results are shown in Table 7.

[0164] Table 7

[0165]

[0166]

[0167] in addition, Figure 3 Three months after the sample from Example 1 was reconstituted and implanted subcutaneously into the animal, a 400× Masson staining image was obtained to show collagen fiber proliferation. As can be seen from the image, collagen fiber proliferation was significant at the tissue injection site.

[0168] 6. Take the hydrogel lyophilized formulations from each of the 24 vials prepared in Examples 1, 13, 14, and 15, and add 7.28 ml of Sorensen buffer to each vial (preparation method refers to: YY-0473-2004 In Vitro Degradation Test of Polylactide Copolymers and Blends for Surgical Implants 4.2.1). Vortex for 5 minutes until dissolved, and place the container in a shaking water bath at 37±1℃. Take 2 degradation samples from each group every month to test the content of lactic acid, the degradation product. When the lactic acid content reaches its maximum value and the concentration no longer changes, it indicates that it has been completely degraded (refer to: YYT 1806.1-2021 In Vitro Degradation of Biomedical Materials, Evaluation Method for In Vitro Degradation Performance, Degradable Polyesters 5.4). Perform degradation tests on the samples from the examples.

[0169] Another lyophilized formulation was prepared with the same formulation as Examples 1, 13, 14, and 15 except that it did not contain HA. These formulations were named Examples 1', 13', 14', and 15', and were subjected to degradation tests using the same methods as described above. The results are shown in Table 8.

[0170] Table 8

[0171] Group Degradation completion time Example 1 8 months Example 13 6 months Example 14 5 months Example 15 6 months Example 1' 6 months Example 13' 5 months Example 14' 4 months Example 15' 5 months

[0172] Based on the above experimental results, this invention utilizes high-molecular-weight sodium hyaluronate and a thermosensitive polymer dissolved in phosphate buffer to prepare a lyophilized formulation. This effectively prevents oxidation, stabilizing the polymer and sodium hyaluronate and preventing decomposition. The hydrogel lyophilized formulation, after reconstitution with water, forms an aqueous solution, facilitating injection and solving problems such as instability and needle clogging associated with microsphere formulations. Furthermore, the interaction between the hydrophilic groups of the compounded sodium hyaluronate and the hydrophilic groups of the thermosensitive polymer enhances the reconstitution rate. After reconstitution and injection, as the temperature approaches physiological temperature, the increased micelle aggregation of the polymer in the solution leads to a gel phase transition, forming an interpenetrating three-dimensional network structure with sodium hyaluronate. This structure exhibits good stability and enhances tissue filling. Additionally, the compounded high-molecular-weight sodium hyaluronate not only accelerates the thermal gelation of the amphiphilic block copolymer but also compensates for the rapid degradation rate of block copolymers, extending the degradation cycle, reducing the number of re-injections, and providing a scaffold structure for cell adhesion, facilitating tissue repair.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A freeze-dried formulation of a temperature-sensitive physical hydrogel, characterized in that, The preparation raw materials include a temperature-sensitive polymer, sodium hyaluronate and a phosphate buffer; The phase transition temperature of the temperature-sensitive polymer is 35±2℃; The molecular weight of the sodium hyaluronate is 2.2-2.8 million, and the mass-volume ratio of the sodium hyaluronate to the phosphate buffer is 3-12 mg:1 ml; The temperature-sensitive polymer is an amphiphilic block copolymer with a two-block A-B, a three-block A-B-A or a three-block B-A-B structure, wherein A represents a hydrophilic segment, and B represents a hydrophobic segment, and the mass-volume ratio of the temperature-sensitive polymer to the phosphate buffer is 100-300 mg:1 ml; The weight average molecular weight of the A segment is in the range of 800-4000, and 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); The A segment is PEG, and the B segment is one or more of PCL, PLA, PLLA, PLCA and PLGA; The temperature-sensitive physical hydrogel lyophilized preparation is prepared by lyophilizing the preparation raw materials after mixing at a pre-freezing temperature of-45 to-35℃.

2. The temperature-sensitive physical hydrogel lyophilized formulation according to claim 1, characterized in that, The molecular weight ratio of the A and B segments is 1:2.

5.

3. The temperature-sensitive physical hydrogel lyophilizate according to claim 1 or 2, characterized in that The temperature-sensitive polymer is PLGA-PEG-PLGA.

4. Process for the preparation of a lyophilized formulation of a temperature- sensitive physical hydrogel according to any one of claims 1 to 3, characterized in that, The preparation raw materials are mixed and then lyophilized.

5. The method of claim 4, wherein the temperature-sensitive physical hydrogel lyophilizate is prepared by, Comprising: The sodium hyaluronate is dissolved in the phosphate buffer at 4-10℃, then the temperature-sensitive polymer is dissolved in the aforementioned phosphate buffer in which the sodium hyaluronate has been dissolved, filled, and lyophilized at a pre-freezing temperature of-45 to-35℃.

6. An injectable tissue filler, characterized in that, The temperature-sensitive physical hydrogel lyophilized preparation according to any one of claims 1-3.

7. The injectable tissue filler of claim 6, wherein, The temperature-sensitive physical hydrogel lyophilized preparation is reconstituted and then applied to the target tissue of a subject by skin or subcutaneous administration.

8. The injectable tissue filler of claim 7, wherein, The reconstitution solution used for the reconstitution is water, and the amount of the reconstitution solution is equal to the volume of the sol of the temperature-sensitive physical hydrogel lyophilized preparation before lyophilization.

9. The injectable tissue filler of any one of claims 6-8, wherein, The injectable tissue filler is suitable for injection through a needle with a gauge of 27-29G.

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