Polymer composition, soft tissue filler and preparation method and application thereof

Abstract

The invention discloses a polymer composition, a soft tissue filler and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The polymer composition comprises a biomacromolecule and a bioresponsive polymer, and the biomacromolecule and the bioresponsive polymer can form physical entanglement in the presence of active oxygen. As an effective component of the soft tissue filling agent, the polymer composition not only can significantly prolong the metabolism time and achieve a lasting filling effect, but also can endow the following properties: the process of network entanglement and tissue filling repair can be visually tracked; rOS generated in the aged skin can be removed for a long time, and the anti-inflammatory, anti-oxidation and anti-aging effects are achieved; long-term retention in skin tissues can be realized; the generation of collagen in tissues can be effectively promoted; water can be effectively locked and moisturized, and the dry state of the skin is improved; the skin repairing agent can repair damaged skin tissues and promote skin cell regeneration, and has good biocompatibility and biodegradability, and degradation products are non-toxic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a high molecular composition, a soft tissue filler and a preparation method and application thereof. BACKGROUND

[0002] Injection of tissue fillers is one of the effective means for treating tissue defects and deformation, repairing wrinkles and resisting aging, and is favored due to its simple operation, small trauma, short time, obvious repair effect and the like. At present, biological macromolecules and biodegradable polymer fillers are mainly used in clinical treatment. Although biological macromolecules have good biocompatibility, water retention, instant filling and rapid shaping effects, they generally have poor mechanical properties, fast metabolic speed, short retention time, no collagen stimulation regeneration, lack of biological responsiveness and functionality, and cannot track the tissue repair and filling process in real time.

[0003] To improve the short in-vivo retention time of biological macromolecule fillers, the related field proposes a strategy of chemical modification and / or chemical crosslinking of biological macromolecules, but faces problems such as the following: 1) both chemical modification and chemical crosslinking involve complex chemical reactions, which usually result in the residue of toxic chemicals and easily cause adverse reactions such as skin swelling and allergy; 2) the efficiency, controllability and batch stability of chemical modification are difficult to guarantee, which is not conducive to industrial production and transformation; 3) after introducing substituents on the biological macromolecule chain by chemical modification, the inherent physicochemical properties and biological activity of the biological macromolecule are affected, such as chemical structure, conformation, spatial structure, solubility, aggregation state and the like, which significantly affect its performance. SUMMARY

[0004] The present application provides a high molecular composition, a soft tissue filler and a preparation method and application thereof, which aims to solve the above-mentioned problems existing in the modification of biological macromolecule fillers by chemical modification and / or chemical crosslinking.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a high molecular composition, the main components of which include:

[0007] biological macromolecules, and biological responsive polymers;

[0008] The biological macromolecules and the biological responsive polymers can form a physical entanglement network in the presence of active oxygen;

[0009] The biological responsive polymers include any of the following chemical formulas:

[0010]

[0011] Among them, R1 is selected independently from each other. Either a mercapto group or a selenoyl group; R2 is independently selected from phenyl, C 7-20 Alkylphenyl, C 7-20 Heteroalkylphenyl, C 1-20 Alkyl or C 1-20 Any of the heteroalkyl groups; R3 is independently selected from C 1-20 Alkyl, C 1-20 Heteroalkyl or C 7-20 Any of the alkylphenyl groups; R4 is selected from thiol or selenool groups; n ≥ 5.

[0012] It should be noted that "alkylphenyl" refers to a phenyl group containing at least one alkyl chain; "heteroalkylphenyl" refers to an alkylphenyl group containing at least one heteroatom or a heteroatom functional group on its alkyl chain, including but not limited to etherylphenyl, thioetherylphenyl, carboxylphenyl, hydroxyphenyl, aminophenyl, esterylphenyl, ketylphenyl, aldehydeylphenyl, amamideylphenyl, thioamideylphenyl, ureophenyl, thioureaylphenyl, carbamate, etc.; "heteroalkyl" refers to an alkyl group containing at least one heteroatom or a heteroatom functional group on its main chain or side chain, including but not limited to esterylalkyl, aldehydeylalkyl, ketylalkyl, amamideylalkyl, thioamideylalkyl, ureoalkyl, thioureaylalkyl, carboxylalkyl, hydroxyalkyl, aminoalkyl, carbamateylalkyl, azalkyl, alkoxy, alkylthio, etc. The heteroatom can be nitrogen, oxygen, sulfur, phosphorus, etc.

[0013] In a preferred embodiment, R2 is independently selected from phenyl, C 7-20 Alkylphenyl, C 7-20 Ester-based phenyl, C 7-20 carbonyl phenyl, C 7-20 Amide phenyl, C 1-20 Alkyl, C 3-20 cycloalkyl, C 1-20 Nitrogen alkyl, C 1-20 Alkoxy, C 1-20 Alkylthio, C 2-20 Esteryl alkyl, C 2-20 Ketoalkyl, C 2-20 Aldehyde alkyl, C 2-20 Amide alkyl, C 2-20 Carboxyalkyl, C 2-20 Hydroxyalkyl, C 2-20 aminoalkyl, C 2-20 Thioamidoalkyl, C 2-20 Carbamate alkyl, C 2-20 Urea alkyl or C 2-20 Any of the thiourea alkyl groups; R3 is independently selected from C 1-20 Alkyl, C 1-20Nitrogen alkyl, C 1-20 Alkoxy, C 1-20 Alkylthio, C 2-20 Esteryl alkyl, C 2-20 Ketoalkyl, C 2-20 Aldehyde alkyl, C 2-20 Amide alkyl, C 2-20 Thioamidoalkyl, C 2-20 Ureaalkyl, C 2-20 Thiourea alkyl or C 2-20 Any of the urethane alkyl groups.

[0014] It should be noted that the embodiments of this application do not impose a special upper limit on the value of the degree of polymerization n, but in order to ensure that the polymer has good biocompatibility and suitable biodegradability, it is preferable that 5≤n≤500.

[0015] It should be noted that the embodiments of this application do not limit the synthesis method and process parameters of the polymer, and it can be prepared according to polymerization / condensation reactions known in the art. To facilitate implementation in the field, several exemplary synthesis methods are provided below, including:

[0016] Method 1: After modifying the side chains of an olefin using R1-R2, the modified product is used as a monomer for free radical polymerization to obtain a polymer with a degree of polymerization of 5-500. The initiator is selected from azobisisobutyronitrile, azobisisoheptanenitrile, dimethyl azobisisobutyrate, ammonium persulfate, potassium persulfate, or benzoyl peroxide, preferably azobisisobutyronitrile. The reaction temperature is 50-100℃, preferably 70℃; the reaction time is 12-72 h, preferably 24 h.

[0017] Method 2: After modifying the side chain of the diol using R1-R2, the modified product is used as a monomer for condensation polymerization to obtain the polymer, with a degree of polymerization of 5-500. The catalyst is selected from concentrated sulfuric acid, phosphoric acid, sulfonic acid, sodium hydroxide, potassium hydroxide, zinc chloride, or stannous chloride, preferably concentrated sulfuric acid; the reaction temperature is 110-150℃, preferably 130℃; the reaction time is 4-72 h, preferably 24 h.

[0018] Method 3: After modifying the side chains of a diol or / or a dicarboxylic acid using R1-R2, the modified product is used as a monomer for condensation polymerization to obtain a polymer with a degree of polymerization of 5-500. The condensation polymerization reaction can be carried out in the presence of a catalyst and a dehydrating agent. The catalyst is selected from sodium hydroxide, potassium hydroxide, potassium manganese acetate, potassium manganese acetate, or dicyclohexylcarbodiimide (DCC) / 4-dimethylaminepyridine (DMAP), preferably DCC / DMAP. The dehydrating agent is selected from aluminum bricks or molecular sieves, preferably molecular sieves. The reaction temperature is controlled at 20-100℃, preferably 25℃; the reaction time is controlled at 12-72h, preferably 72h.

[0019] Method 4: After side-chain modification of diols and / or diisocyanates using R1-R2, the modified products are used as monomers for addition polymerization to obtain polymers with a degree of polymerization of 5-500. The addition polymerization reaction can be carried out in the presence of a catalyst selected from triethylenediamine, triethylamine, trimethylbenzylamine, dimethylethanolamine, morpholine, organobismuth, dibutyltin dilaurate, or stannous octoate, preferably stannous octoate; the reaction temperature is 40-200℃, preferably 60℃; the reaction time is 12-72 h, preferably 24 h.

[0020] Method 5: After synthesizing N-carboxyl intracyclic anhydride (NCA) from amino acid derivative 1, the NCA ring-opening polymerization reaction initiated by the amino group is used to obtain the polymer (degree of polymerization 5-500). The structural formula of amino acid derivative 1 is as follows:

[0021] Method 6: After modifying the side chains of diamine and / or diisocyanate with R1-R2, the modified product is used as a monomer for addition polymerization to obtain a polymer. The polymerization reaction temperature is 10℃-120℃, preferably 25℃; the reaction time is controlled to be 2-48h, preferably 4-20h, and more preferably 12h.

[0022] Method 7: After side chain modification of the diamine and / or dicarboxylic acid of the R1-R2 pair, the modified product is used as a monomer and subjected to condensation polymerization in the presence of N,N-diisopropylethylamine (DIEA) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The polymerization reaction temperature is 25-60℃, preferably 25℃; the reaction time is controlled at 4-24h, preferably 6-20h, and more preferably 12h.

[0023] In a preferred embodiment, the bioresponsive polymer comprises:

[0024]

[0025]

[0026]

[0027] In a preferred embodiment, the biomacromolecule is at least one selected from hyaluronic acid, collagen, carboxymethyl cellulose, heparin sulfate, chondroitin sulfate, proteoglycan, chitosan, and sodium alginate. These biomacromolecules not only possess excellent biocompatibility, water-locking and moisturizing effects, instant filling, and rapid shaping, but also can physically entangle with the contained bioresponsive polymers under the action of reactive oxygen species to form a bioresponsive polymer-biomacromolecule complex, effectively enhancing the filling performance such as water-locking and moisturizing, instant filling, and rapid shaping.

[0028] In a preferred embodiment, the mass ratio of the bioresponsive polymer to the biomacromolecule is preferably 5-95:95-5, exemplarily 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, or any ratio within this range. In this application embodiment, by controlling the mass ratio, the network entanglement rate of the bioresponsive polymer and the biomacromolecule, the applicability of the filler, and the filling effect can be effectively regulated.

[0029] Secondly, embodiments of this application provide a soft tissue filler, wherein the soft tissue filler uses the polymer composition of this application as the active ingredient.

[0030] In a preferred embodiment, the soft tissue filler further comprises a solvent system for dispersing the polymeric composition, the solvent system comprising one or more of sterile water, physiological saline, phosphate buffer, acetate buffer, citrate buffer, tromethorphan buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, glycine buffer, and lactate buffer.

[0031] It should be noted that, in this embodiment of the application, a solvent system is added to disperse the polymer composition, so that the soft tissue filler is formed into an injectable filler product, which is convenient to use and has a good filling effect. Furthermore, the solvent composition is preferably compatible with the in vivo environment, which effectively avoids the occurrence of side effects during filling and repair.

[0032] In a preferred embodiment, the mass-to-volume concentration of the polymer composition in the solvent system is preferably 5-500 mg / mL, exemplarily 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, or any concentration within the range of the mass-to-volume concentration. In this embodiment, by controlling the mass-to-volume concentration, the filler can be effectively guaranteed to have excellent needle penetration and good filling effect.

[0033] Thirdly, the embodiments of this application also provide a method for preparing the soft tissue filler of this application, the steps of which include: dispersing the bioresponsive polymer and biomacromolecules in a solvent system, and then performing at least one of the following post-treatments in sequence: sterilization, canning, oxidation, defoaming, etc., to obtain the filler.

[0034] It should be noted that the embodiments of this application do not limit the order and specific method of dissolving and dispersing the bioresponsive polymer and biomacromolecule in the solvent system. It is preferred to disperse the bioresponsive polymer and biomacromolecule separately in their respective good medical solvents before mixing. The good solvent can be selected according to the properties of the substances. The embodiments of this application do not limit the specific post-treatment methods such as sterilization, oxidation, and defoaming.

[0035] Fourthly, embodiments of this application also provide applications in wound healing, tissue filling and / or repair, skin moisturizing and water retention, anti-aging, antioxidant therapy, and repair of the skin barrier.

[0036] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following:

[0037] This application's embodiments utilize a compounding of biomacromolecules and specifically structured bioresponsive polymers. The components can physically entangle under the influence of reactive oxygen species, forming a network entangled complex. This significantly prolongs the metabolic time, achieving a more lasting immediate filling effect. Furthermore, it endows the composite filler with at least the following biological properties: 1) excellent fluorescence imaging, enabling visualization and tracking of the network entanglement and tissue filling repair process; 2) long-lasting removal of ROS generated in aging skin, providing long-lasting anti-inflammatory, antioxidant, and anti-aging effects; 3) long-term retention in skin tissue, achieving a longer retention time than small-molecule antioxidants and biomacromolecule fillers; 4) effective promotion of collagen production in tissues; 5) effective water retention and moisturizing, improving dry skin; 6) rich nutritional components, repairing damaged skin tissue and promoting skin cell regeneration; 7) avoidance of toxic cross-linking agents and complex synthesis processes; 8) good biocompatibility and biodegradability, with non-toxic degradation products; and 9) simple structure, easy synthesis, and industrial transformation. Furthermore, this application involves simple compounding of biomacromolecules and bioresponsive polymers without any chemical reaction. It not only has advantages such as good operability and high batch stability, but also effectively avoids the residue and adverse reactions of toxic chemicals, and can completely maintain the inherent physicochemical properties and biological activity of biomacromolecules. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The images show the fluorescence of the soft tissue filler P1 / HA-inj injection solution at different time points after the addition of hydrogen peroxide, where a represents 0 min; b represents 1 min; c represents 5 min; and d represents 15 min.

[0040] Figure 2 The images show the fluorescence of the soft tissue filler P1 / HA-inj injection solution at different time points before and after intubation, where a is 0 min, b is 1 min, c is 5 min, and d is 15 min.

[0041] Figure 3 Cytotoxicity test diagrams of the soft tissue fillers provided in Examples 1-14;

[0042] Figure 4 Tissue sections of the heart, liver, spleen, lungs and kidneys of rats after injection of soft tissue filler P1 / HA-inj;

[0043] Figure 5 Cell closure rate diagrams for soft tissue fillers injected in Examples 1-14 and filling solutions injected in Comparative Examples 1-3;

[0044] Figure 6 The graph shows the changes in the content of the inflammatory factor TNF-α in the skin of rats after the soft tissue fillers of Examples 1, 5, 9, 12, and 14 and the filling injections of Comparative Examples 1-3 were injected into the skin of rats.

[0045] Figure 7 The graph shows the changes in superoxide dismutase (SOD) content in the back skin of rats after the soft tissue fillers of Examples 1, 5, 9, 12, and 14 and the filling injections of Comparative Examples 1-3 were injected into the skin of rats.

[0046] Figure 8 These are in vivo images of the soft tissue filler of Example 1 and the filling injection solutions of Comparative Examples 1-3 after injection into the skin of rats;

[0047] Figure 9 The image shows the collagen content of the back skin of rats after the soft tissue fillers of Examples 1, 5, 9, 12, and 14 and the filling injections of Comparative Examples 1-3 were injected into the skin of rats.

[0048] Figure 10 This is a diagram showing the changes in type I and type III collagen in the skin of the rat's back after the soft tissue filler was injected into the rat skin in Example 5.

[0049] Figure 11 The images show the changes in skin elasticity on the back of rats after the soft tissue fillers of Examples 1, 5, 9, 12, and 14 and the filling injections of Comparative Examples 1-3 were injected into the skin of rats. Detailed Implementation

[0050] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0051] Example 1

[0052] This embodiment provides polymer P1, and the preparation of a soft tissue filler P1 / HA-inj based on a composition of polymer P1 and hyaluronic acid. The synthesis steps of polymer P1 include:

[0053] 13.52 g of homocysteine ​​was weighed and added to a flask, and the mixture was subjected to three suction cycles and three openings. Then, 150 mL of deionized water and 14 mL of triethylamine were added to dissolve it. In an ice-water bath, 21.60 g of dinitrobenzyl bromide and 150 mL of methanol were slowly added. The mixture was stirred for 0.5 h, and then reacted at 25 °C for 24 h. The precipitate was collected in ice-cold diethyl ether, washed twice with acetone and ethanol respectively, and dried under vacuum for 48 h to obtain compound 1.

[0054] 3.00 g of compound 1 was dissolved in 70 mL of anhydrous tetrahydrofuran. Then, 2.56 g of triphosgene solution dissolved in 30 mL of THF was slowly added dropwise to the reaction flask while stirring. The reaction was carried out at 50 °C in the dark for 4 h. The product was recrystallized three times in a tetrahydrofuran / n-hexane mixed solvent and dried under vacuum to obtain compound 2.

[0055] 0.32 g of compound 2 was dissolved in 10 mL of DMF, and 0.5 g of polyethylene glycolamine with a molecular weight of 5000 was added. The mixture was reacted at 37 °C for 3 days. After the reaction, the mixture was precipitated three times with ice-cold diethyl ether, dried under vacuum at 37 °C for 48 h, dissolved in 20 mL of DMF, irradiated with 365 nm UV light for 2 h, purified three times with ice-cold diethyl ether precipitation, and dried under vacuum at 37 °C for 48 h to obtain polymer P1 with a degree of polymerization of 20. The structural formula of compound 1 is as follows: The structural formula of compound 2 is: The structural formula of polymer P1 is:

[0056] 1 H NMR (400MHz, DMSO-d6, 25℃): δ4.63ppm (SH-CH2-CH), 5.22ppm (SH-CH2-CH), 3.29ppm (CH3-O-CH2).

[0057] FT-IR: 2554cm -1 (-SH), 2879cm -1 (-CH2-), 3281cm -1 (-CONH-).

[0058] Preparation of soft tissue filler P1 / HA-inj:

[0059] Polymer P1 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P1. Hyaluronic acid was then mixed with the polymer P1 aqueous solution, and the mixture was filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P1 / HA-inj. The concentration of polymer P1 was 50 mg / mL, and the concentration of hyaluronic acid was 10 mg / mL.

[0060] Example 2

[0061] This embodiment provides polymer P2, and the preparation of a soft tissue filler P2 / COL-inj based on a polymer P2 and collagen composition. The synthesis steps of polymer P2 include:

[0062] 0.38 g of selenocysteine-intracyclic carboxylic anhydride was dissolved in 20 mL of DMF, and 0.5 g of a four-armed polyethylene glycolamine with a molecular weight of 10000 was added. The reaction was carried out at 37 °C for 3 days. After the reaction was completed, the product was precipitated three times with ice-cold ether and dried under vacuum at 37 °C for 48 h to obtain polymer P2 with a degree of polymerization of 10. Its structural formula is as follows:

[0063] 1 H NMR (400MHz, DMSO-d6, 25℃): δ4.61ppm (SeH-CH2-CH), 2.85ppm (SeH-CH2-CH), 3.29ppm (CH3-O-CH2).

[0064] FT-IR: 784cm -1 (-SeH), 1139cm -1 (COC), 3281cm -1 (-CONH-).

[0065] Preparation of soft tissue filler P2 / COL-inj:

[0066] Polymer P2 was dissolved in sterile water using a direct water-soluble method to obtain an aqueous solution of polymer P2. This polymer P2 aqueous solution was then mixed with an aqueous collagen solution, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P2 / COL-inj. The concentration of polymer P2 was 75 mg / mL, and the concentration of collagen was 10 mg / mL.

[0067] Example 3

[0068] This embodiment provides polymer P3, and the preparation of a soft tissue filler P3 / HS-inj based on a composition of polymer P3 and heparin sulfate. The synthesis steps of polymer P3 include:

[0069] 1.37 g of lysine-intracyclic carboxylic anhydride containing a carbamate bond and protected by a Boc thiol group was dissolved in 20 mL of DMF. 0.5 g of poly(tert-butoxycarbonyl-L-lysine) with a degree of polymerization of 30 was added, and the mixture was reacted at 37 °C for 3 days. After the reaction, the mixture was precipitated three times with ice-cold diethyl ether, dried under vacuum at 37 °C for 48 h, and then dissolved in 10 mL of trifluoroacetic acid. The mixture was stirred for 1 h, and the trifluoroacetic acid was removed by rotary evaporation. The mixture was then purified three times by precipitation with ice-cold diethyl ether to obtain polymer 3 with a degree of polymerization of 50. Its structural formula is as follows:

[0070]

[0071] 1 H NMR (400MHz, DMSO-d6, 25℃): δ4.51ppm(SH-CH2-CH), 5.12ppm(SH-CH2-CH), 1.21ppm(CH2-CH2-CH2).

[0072] FT-IR: 2556cm -1 (-SH), 2872cm -1 (-CH2-), 3279cm -1 (-CONH-), 1634cm -1 (-CONH-).

[0073] Preparation of soft tissue filler P3 / HS-inj:

[0074] Polymer P3 was dissolved in PBS buffer using a direct water-soluble method to obtain an aqueous solution of polymer P3. This polymer P3 aqueous solution was then mixed with an aqueous solution of heparin sulfate, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P3 / HS-inj. The concentration of polymer P3 was 200 mg / mL, and the concentration of heparin sulfate was 50 mg / mL.

[0075] Example 4

[0076] This embodiment provides polymer P4, and the preparation of a soft tissue filler P4 / CS-inj based on polymer P4 and chondroitin sulfate. The synthesis steps of polymer P4 include:

[0077] 2.00 g of chitosan was dissolved in an aqueous solution of NaHCO3. 2.07 g of a glutamic acid-cyclic intracyclic carboxylic anhydride with amide bonds and Cbz-protected thiol groups was added. The reaction was carried out in an ice-water bath for 24 h. The resulting aqueous solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 (MWCO 3500). Dialysis with deionized water was performed for 2 days. After freeze-drying, the solution was dissolved in 40 mL of methanol and degassed with high-purity nitrogen for 30 min. Then, 0.55 g of 10% Pd / C catalyst was added, hydrogen gas was introduced, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the mixture was filtered, washed three times with methanol, and the filtrates were combined and distilled under reduced pressure to obtain polymer P4 with a degree of polymerization of 50. Its structural formula is as follows:

[0078]

[0079] 1 H NMR (400MHz, DMSO-d6, 25℃): δ4.59ppm(SH-CH2-CH), 5.14ppm(SH-CH2-CH), 1.18ppm(CH3-CH2-CH2), 0.85ppm(CH3-CH2-CH2).

[0080] FT-IR: 2548cm -1 (-SH), 1420cm -1 (-CH2-), 3283cm -1 (-CONH-).

[0081] Preparation of soft tissue filler P4 / CS-inj:

[0082] Polymer P4 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P4. This polymer P4 aqueous solution was then mixed with an aqueous solution of chondroitin sulfate. Oxygen was then introduced for 30 minutes, followed by sterilization at 121℃ for 15 minutes, followed by canning and defoaming to obtain the soft tissue filler P4 / CS-inj. The concentration of polymer P4 and chondroitin sulfate was both 100 mg / mL.

[0083] Example 5

[0084] This embodiment provides polymer P5, and the preparation of a soft tissue filler P5 / COL-inj based on a polymer P5 and collagen composition. The synthesis steps of polymer P5 include:

[0085] 0.02 g of n-hexylamine was dissolved in an aqueous solution of NaHCO3. 4.66 g of γ-L-glutamyl-L-cysteine-glycine-N-carboxylic acid anhydride with Cbz-protected thiol groups was added. The reaction was carried out in an ice-water bath for 24 h. The resulting aqueous solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 (MWCO 3500). Dialysis with deionized water was performed for 2 days. After freeze-drying, the solution was dissolved in 40 mL of methanol and degassed with high-purity nitrogen for 30 min. 0.55 g of 10% Pd / C catalyst was added, and hydrogen gas was introduced. The reaction was stirred at room temperature for 24 h. After the reaction was complete, the mixture was filtered, washed three times with methanol, and the filtrates were combined and distilled under reduced pressure to obtain polymer 5 with a degree of polymerization of 50. Its structural formula is:

[0086]

[0087] 1 H NMR (400MHz, DMSO-d6, 25℃): δ2.94ppm(SH-CH2-CH), 2.32ppm(SH-CH2-CH), 1.79ppm(CH-CH2-CH2).

[0088] FT-IR: 2544cm -1 (-SH), 3286cm -1 (-CONH-), 1687cm -1 (-COOH).

[0089] Preparation of soft tissue filler P5 / COL-inj:

[0090] Polymer 5 was dissolved in acetate buffer using a direct water-soluble method to obtain an aqueous solution of polymer P5. This polymer P5 aqueous solution was then mixed with an aqueous collagen solution, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P5 / COL-inj. The concentration of polymer P5 was 50 mg / mL, and the concentration of heparin sulfate was 20 mg / mL.

[0091] Example 6

[0092] This embodiment provides polymer P6, and the preparation of a soft tissue filler P6 / PG-inj based on polymer P6 and proteoglycans. The synthesis steps of polymer P6 include:

[0093] 5.8 g of allyl alcohol was dissolved in 20 mL of THF, and 0.1 g of azobisisobutyronitrile was added. The mixture was reacted at 70 °C for 24 h. After the reaction was completed, the mixture was precipitated three times with ice-cold methanol and dried under vacuum at 50 °C for 48 h to obtain polyacrylol with a degree of polymerization of 300.

[0094] Under argon protection, 2.9 g of polyacrylamide was dissolved in 50 mL of N,N-dimethylformamide, followed by the addition of 0.78 g of DCC and 0.46 g of DMAP. After stirring for 30 min, 1.12 g of 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid was added, and the reaction was carried out at 25 °C for 24 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether and dried under vacuum for 48 h to obtain polymer P6 with a grafting rate of 20%, the structural formula of which is:

[0095]

[0096] 1 H NMR (400MHz, DMSO-d6, 25℃): 1.59ppm (CH3-CS), 3.34ppm (S-CH2-CO), 4.24-4.49ppm (O-CH2-CH), 1.89ppm (O-CH2-CH), 3.33-3.58ppm (CH-CH2-OH).

[0097] FT-IR: 3652cm -1 (-OH), 667cm -1 (-CS-), 1724cm -1 (-COO-).

[0098] Preparation of soft tissue filler P6 / PG-inj:

[0099] Polymer P6 was dissolved in citrate buffer using a direct water-soluble method to obtain an aqueous solution of polymer P6. This polymer P6 aqueous solution was then mixed with a proteoglycan aqueous solution, filtered through a 0.45 μm filter membrane, sterilized, bottled, and defoamed to obtain the soft tissue filler P6 / COL-inj. The concentration of polymer P6 was 20 mg / mL, and the concentration of proteoglycan was 30 mg / mL.

[0100] Example 7

[0101] This embodiment provides polymer P7, and the preparation of a soft tissue filler P7 / CMC-inj based on a composition of polymer P7 and carboxymethyl cellulose. The synthesis steps of polymer P7 include:

[0102] Under argon protection, 1.54 g of 3-(thiomercaptomethyl)-1,2-propanediol was added to a reaction flask. After cooling in an ice-water bath, 1.08 g of 1-chloro-2-methylthioethylene and 8 mL of dichloromethane were slowly added. The mixture was stirred for 1 h, then heated to 25 °C and reacted for 12 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether and dried under vacuum at room temperature for 48 h to obtain compound 3.

[0103] 1.50 g of thionyl diacetic acid was dissolved in 20 mL of DMSO, and 0.78 g of DCC and 0.46 g of DMAP were added. The mixture was stirred for 15 min, and then 2.26 g of compound 3 was added. The mixture was reacted at 25 °C for 72 h. After the reaction was completed, the mixture was precipitated three times with ice-cold methanol and dried under vacuum at 25 °C for 48 h to obtain polymer P7 with a degree of polymerization of 300.

[0104] The structural formula of compound 3 is:

[0105] The structural formula of polymer P7 is:

[0106] 1 H NMR (400MHz, DMSO-d6, 25℃): 3.34ppm (S-CH2-CO), 4.08-4.33ppm (O-CH2-CH), 2.44-2.69ppm (CH-CH2-S), 4.02ppm (S-CH2-S), 6.18ppm (S-CH-CH).

[0107] FT-IR: 1651cm -1 (C=C), 667cm -1 (-CS-), 1727cm -1 (-COO-).

[0108] Preparation of soft tissue filler P7 / CMC-inj:

[0109] Polymer P7 was dissolved in tromethamine buffer using a direct water-soluble method to obtain an aqueous solution of polymer P7. This aqueous solution was then mixed with carboxymethyl cellulose, and subsequently filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P7 / COL-inj. The concentration of polymer P7 was 80 mg / mL, and the concentration of carboxymethyl cellulose was 15 mg / mL.

[0110] Example 8

[0111] This embodiment provides polymer P8, and the preparation of a soft tissue filler P8 / HA-inj based on polymer P8 and hyaluronic acid. The synthesis steps of polymer P8 include:

[0112] 1.38 g of 3-(mercaptomethoxy)-1,2-propanediol was added to a reaction flask. After cooling in an ice-water bath, 1.08 g of 1-chloro-2-methylthioethylene and 8 mL of dichloromethane were slowly added. The mixture was stirred for 1 h, then heated to 25 °C and reacted for 12 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether and dried under vacuum at room temperature for 48 h to obtain compound 4.

[0113] 2.10 g of compound 4 was dissolved in 20 mL of xylene, and 40 μL of 98% concentrated sulfuric acid was added. The mixture was refluxed at 130 °C for 24 h. After the reaction was completed, the mixture was precipitated three times with ice-cold methanol, dried under vacuum at 70 °C for 48 h, and then dissolved in 10 mL of xylene. The mixture was purified three times by precipitation with ice-cold methanol to obtain polymer P8 with a degree of polymerization of 50.

[0114] The structural formula of compound 4 is:

[0115] The structural formula of polymer P8 is:

[0116] 1 H NMR (400MHz, DMSO, 25℃): δ6.18ppm(S-CH-CH), 2.34ppm(CH3-S-CH), 4.95ppm(O-CH2-S), 3.40ppm(O-CH-CH2).

[0117] FT-IR: 667cm -1 (-CS-)), 1651cm -1 (C=C), 1128ppm(COC).

[0118] Preparation of soft tissue filler P8 / HA-inj:

[0119] A direct water-soluble method was used, specifically dissolving polymer P8 in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to obtain an aqueous solution of polymer P8. This polymer P8 aqueous solution was then mixed with hyaluronic acid, and subsequently filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P8 / HA-inj. The concentration of polymer P8 was 45 mg / mL, and the concentration of hyaluronic acid was 25 mg / mL.

[0120] Example 9

[0121] This embodiment provides polymer P9, and the preparation of a soft tissue filler P9 / HA-inj based on polymer P9 and hyaluronic acid. The synthesis steps of polymer P9 include:

[0122] 3.47 g of 1,13-diaminotridecane-7-thiol with Boc-protected thiol groups was added to 20 mL of DMF, along with 1.68 g of hexamethylene diisocyanate (HDI). The reaction was carried out at 85 °C for 16 h. After the reaction was completed, the product was precipitated three times with ice-cold methanol, dried under vacuum at 80 °C for 48 h, and then dissolved in 15 mL of TFA. The mixture was stirred at room temperature for 1 h. The resulting mixture was then removed by rotary evaporation to remove the TFA, and purified three times by precipitation with ice-cold diethyl ether to obtain polymer P9, whose structural formula is:

[0123] 1 H NMR (400MHz, DMSO, 25℃): δ1.94ppm(SH-CH2-CH), 3.57ppm(SH-CH-CH), 1.25ppm(CH2-CH2-CH2), 3.97ppm(CH2-CH2-CH2).

[0124] FT-IR: 2556cm- 1 (-SH), 3478cm -1 (-NH-), 1693cm -1 (-CO-).

[0125] Preparation of soft tissue filler P9 / HA-inj:

[0126] Polymer P9 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P9. This solution was then mixed with hyaluronic acid, followed by oxygen purging for 30 minutes, sterilization at 121℃ for 15 minutes, canning, and defoaming to obtain the soft tissue filler P9 / HA-inj. The concentration of polymer P9 was 65 mg / mL, and the concentration of hyaluronic acid was 40 mg / mL.

[0127] Example 10

[0128] This embodiment provides polymer P10, and the preparation of a soft tissue filler P10 / HA-inj based on polymer P10 and hyaluronic acid. The synthesis steps of polymer P10 include:

[0129] Under argon protection, 2.19 g of 2-amino-3-thiol propamidohexylamine was added to a reaction flask, and the mixture was cooled in an ice-water bath. Then, 2.97 g of 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzyl chloroformate and 15 mL of diethyl ether were slowly added. After stirring for 1 h, the mixture was reacted at 25 °C for 12 h. After the reaction was complete, the mixture was precipitated three times with ice-cold diethyl ether and dried under vacuum at room temperature for 48 h to obtain compound 4.

[0130] 2.40 g of compound 4 was dissolved in 20 mL of DMF, and 0.84 g of hexamethylene diisocyanate (HDI) was added. The mixture was reacted at 85 °C for 16 h. After the reaction was completed, the mixture was precipitated three times with ice-cold methanol, dried under vacuum at 80 °C for 48 h, and then dissolved in 15 mL of TFA. The mixture was stirred at room temperature for 1 h. The TFA was removed by rotary evaporation of the resulting mixture, and the mixture was purified three times by precipitation with ice-cold diethyl ether to obtain polymer P9, whose structural formula is:

[0131] 1 H NMR (400MHz, DMSO, 25℃): δ8.01ppm(NH2-CH2-CH2), 3.18ppm(NH2-CH2-CH2), 1.23ppm(CH2-CH2-CH2), 3.94ppm(CH2-CH2-CH2), 7.28-7.78ppm(C6H4-CH2-O).

[0132] FT-IR: 3457cm -1 (-NH-), 1695cm -1 (-CO-).

[0133] Preparation of soft tissue filler P10 / HA-inj:

[0134] Polymer P10 was dissolved in glycine buffer using a direct water-soluble method to obtain an aqueous solution of polymer P10. This aqueous solution was then mixed with hyaluronic acid, filtered through a 0.45 μm filter membrane, sterilized, bottled, and defoamed to obtain the soft tissue filler P10 / HA-inj. The concentration of polymer P10 was 5 mg / mL, and the concentration of hyaluronic acid was 95 mg / mL.

[0135] Example 11

[0136] This embodiment provides polymer P11, and the preparation of a soft tissue filler P11 / HA-inj based on polymer P11 and hyaluronic acid. The synthesis steps of polymer P11 include:

[0137] 3.66 g of 2-amino-3-selenopropylammonohexylamine with a Boc-protected selenoyl group was dissolved in 20 mL of DMF, and 1.68 g of hexamethylene diisocyanate (HDI) was added. The reaction was carried out at 85 °C for 16 h. After the reaction was completed, the mixture was precipitated three times with ice-cold methanol, dried under vacuum at 80 °C for 48 h, and then dissolved in 12 mL of TFA. The mixture was stirred at room temperature for 1 h. The TFA was removed by rotary evaporation of the resulting mixture, and the mixture was purified three times by precipitation with ice-cold diethyl ether to obtain polymer P11, whose structural formula is:

[0138] 1 H NMR (400MHz, DMSO, 25℃): δ1.6-1.8ppm(SeH-CH2-CH), 3.6ppm(SeH-CH2-CH), 8.01ppm(NH2-CH2-CH2), 3.18ppm(NH2-CH2-CH2), 1.32ppm(NH2-CH2-CH2).

[0139] FT-IR: 2931cm -1 (-CH2-), 2247cm -1 (-NCO), 1695cm -1 (-CO-).

[0140] Preparation of soft tissue filler P11 / HA-inj:

[0141] Polymer P11 was dissolved in lactate buffer using a direct water-soluble method to obtain an aqueous solution of polymer P11. This aqueous solution was then mixed with hyaluronic acid, filtered through a 0.45 μm filter membrane, sterilized, bottled, and defoamed to obtain the soft tissue filler P11 / HA-inj. The concentration of polymer P11 was 150 mg / mL, and the concentration of hyaluronic acid was 60 mg / mL.

[0142] Example 12

[0143] This embodiment provides polymer P12, and the preparation of a soft tissue filler P12 / COL-inj based on polymer P12 and collagen. The synthesis steps of polymer P12 include:

[0144] Under argon protection, 1.54 g of dithiothreitol (DTT-ONB) was added to a reaction flask. After cooling in an ice-water bath, 4.28 g of 4-[[(chlorocarbonyl)oxy]methyl]phenylboronic acid and 15 mL of diethyl ether were slowly added. After stirring for 1 h, the mixture was reacted at 25 °C for 12 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether and dried under vacuum at room temperature for 48 h to obtain compound 5.

[0145] 2.55 g of compound 5 was dissolved in 20 mL of THF, and 0.84 g of hexamethylene diisocyanate and 0.01 g of stannous octoate were added. The mixture was reacted at 60 °C for 24 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether, dried under vacuum at 50 °C for 48 h, dissolved in 10 mL of THF, and irradiated with a 365 nm UV lamp at room temperature for 2 h. The mixture was then purified three times by precipitation with ice-cold diethyl ether to obtain polymer P12.

[0146] The structural formula of compound 5 is: The structural formula of polymer P12 is:

[0147] 1 H NMR (400MHz, DMSO, 25℃): δ4.2ppm (OH-B-C6H4), 7.28-7.78ppm (C6H4-CH2-O), 7.05ppm (CO-NH-CH2), 2.93ppm (CO-NH-CH2).

[0148] FT-IR: 3652cm -1 (-OH), 2252cm -1 (-NCO-), 1723cm -1 (-COO-), 667cm -1 (-CS-).

[0149] Preparation of soft tissue filler P12 / COL-inj:

[0150] Polymer P12 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P12. This aqueous solution was then mixed with collagen, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P12 / COL-inj. The concentration of polymer P12 was 80 mg / mL, and the concentration of collagen was 40 mg / mL.

[0151] Example 13

[0152] This embodiment provides polymer P13, and the preparation of a soft tissue filler P13 / HA-inj based on polymer P13 and hyaluronic acid. The synthesis steps of polymer P13 include:

[0153] 4.02 g of ONB-protected thiol-group-protected mercaptophenylalanine N-carboxylic acid anhydride was dissolved in 10 mL of DMF. 0.5 g of polyethylene glycol diamine with a molecular weight of 5000 was added, and the mixture was reacted at 37 °C for 3 days. After the reaction, the mixture was precipitated three times with ice-cold ether, dried under vacuum at 37 °C for 48 h, dissolved in 20 mL of DMF, irradiated with 365 nm UV light for 2 h, purified three times with ice-cold ether precipitation, and dried under vacuum at 37 °C for 48 h to obtain polymer P13 with a degree of polymerization of 50. The structural formula is as follows:

[0154] 1 H NMR (400MHz, DMSO, 25℃): δ3.52ppm (O-CH2-CH2), 5.52ppm (CH2-NH-CH), 7.05ppm (CO-NH-CH2), 7.04-7.36ppm (CH2-C6H4-SH).

[0155] FT-IR: 2566cm -1 (-SH), 1189cm -1 (COC), 3223cm -1 (-NH-).

[0156] Preparation of soft tissue filler P13 / HA-inj:

[0157] Polymer P13 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P13. This aqueous solution was then mixed with hyaluronic acid, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P13 / HA-inj. The concentration of polymer P13 was 95 mg / mL, and the concentration of hyaluronic acid was 5 mg / mL.

[0158] Example 14

[0159] This embodiment provides polymer P14, and the preparation of a soft tissue filler P14 / HA-inj based on polymer P14 and hyaluronic acid. The synthesis steps of polymer P14 include:

[0160] Under argon protection, 1.20 g of 3-amino-2-(aminomethyl)-1-propanethiol was added to a reaction flask. After cooling with ice water, 2.14 g of 4-[[(chlorocarbonyl)oxy]methyl]phenylboronic acid and 15 mL of diethyl ether were slowly added. The mixture was stirred for 1 h and then reacted at 25 °C for 12 h. After the reaction was completed, the precipitate was precipitated three times with ice-cold diethyl ether and dried under vacuum at room temperature for 48 h to obtain compound 6.

[0161] 2.98 g of compound 6 was dissolved in 20 mL of THF, and 1.04 g of malonic acid was added. The mixture was reacted at 60 °C for 24 h. After the reaction was completed, the mixture was precipitated three times with ice-cold diethyl ether. After vacuum drying at 50 °C for 48 h, the mixture was dissolved in 10 mL of THF and purified three times with ice-cold diethyl ether to obtain polymer P14.

[0162] The structural formula of compound 6 is: The structural formula of polymer P14 is:

[0163] 1 H NMR (400MHz, DMSO, 25℃): δ7.28-7-78ppm (B-C6H4-CH2), 2.67ppm (S-CH2-CH), 3.00-3.25ppm (CH-CH2-NH), 3.66ppm (CO-CH2-CO).

[0164] FT-IR: 671cm -1 (-CS-), 1677cm -1 (-CO), 1723cm -1 (-COO-).

[0165] Preparation of soft tissue filler P14 / HA-inj:

[0166] Polymer P14 was dissolved in physiological saline using a direct water-soluble method to obtain an aqueous solution of polymer P14. This polymer P14 aqueous solution was then mixed with hyaluronic acid, filtered through a 0.45 μm filter membrane for sterilization, canning, and defoaming to obtain the soft tissue filler P14 / HA-inj. The concentration of polymer P14 was 75 mg / mL, and the concentration of hyaluronic acid was 30 mg / mL.

[0167] To illustrate the actual effects of the soft tissue filler provided in this application, this application also provides filling injection solutions of Comparative Examples 1-3. Comparative Example 1 provides a 10 mg / mL hyaluronic acid injection solution; Comparative Example 2 provides a 10 mg / mL collagen injection solution; and Comparative Example 3 provides a 50 mg / mL glutathione injection solution. All solutions are in sterile water.

[0168] Test Example 1: Viscosity Test

[0169] The viscosities of the soft tissue fillers of Examples 1-14 and the filling injection solutions of Comparative Examples 1-3 before and after oxidation were tested using a Hacker torque rheometer. The test temperature was 37°C and the shear rate was 1001 / s. The test results are shown in Table 1.

[0170] Table 1: Viscosities of soft tissue fillers in Examples 1-14 and filling solutions in Comparative Examples 1-3 before and after oxidation

[0171]

[0172] According to the viscosity analysis in Table 1, the soft tissue fillers provided in Examples 1-14 have high viscosity and exhibit an increase in viscosity after oxidation. This is attributed to the physical entanglement between polymer P1-14 and biomolecules after oxidation. Physical entanglement can effectively slow down the metabolic rate of biomolecules, and the higher viscosity is beneficial for the soft tissue fillers to maintain their shape well within the skin tissue. In contrast, the filler injection solutions in Comparative Examples 1-3 did not form network entanglement, and their viscosity remained unchanged.

[0173] Test Example 2: Fluorescence Imaging Effect Test

[0174] Fluorescence tests were performed on small molecule cysteine ​​and polymer P1-14 using a fluorescence spectrophotometer, and the results are shown in Table 2.

[0175] Table 2: Fluorescence quantum yield of polymer P1-14

[0176]

[0177] As shown in Table 2, small molecule cysteine ​​has no fluorescence, while polymers 1-14 all have excellent fluorescence properties and high fluorescence quantum yields, with the highest fluorescence quantum yield reaching 52.5%.

[0178] The soft tissue filler P1 / HA-inj provided in Example 1 was subjected to fluorescence testing by adding hydrogen peroxide and oxygen. The results are as follows: Figure 1 and 2 As shown.

[0179] according to Figure 1 and 2 It is known that the addition of hydrogen peroxide and oxygen will quench the fluorescence of the soft tissue filler P1 / HA-inj, indicating that it forms physical entanglements.

[0180] Test Example 3: Biocompatibility and Biodegradability Test

[0181] Fibroblasts were selected for CCK-8 assays on polymer P1-14. Specifically, polymer P1-14 at a final concentration of 200 μg / mL was co-incubated with fibroblasts for 24 h, followed by the addition of CCK-8 solution. The absorbance at 450 nm was then measured using a microplate reader. The results are shown below. Figure 3 As shown, Figure 3 This is a graph showing the cytotoxicity test results for polymer P1-14.

[0182] according to Figure 3 It can be seen that polymers P1-14 all have good biocompatibility.

[0183] Four injection sites (50 μL each) of soft tissue fillers P1 / HA-inj, P5 / HA-inj, P9 / HA-inj, P12 / HA-inj, and P14 / HA-inj were injected into the back of SD rats. Two weeks later, the rats were sacrificed, and the heart, liver, spleen, lungs, and kidneys were collected for hematoxylin and eosin (H&E) staining. The H&E staining results of injection solution 1 are shown below. Figure 4 As shown.

[0184] according to Figure 4 It can be seen that none of the organs showed any damage or inflammatory response and were completely normal, indicating that the soft tissue filler has excellent biocompatibility.

[0185] Test Example 4: Cell Scratch Test

[0186] The ability of the soft tissue fillers provided in Examples 1-14 and the filler injections in Comparative Examples 1-3 to promote skin cell regeneration was verified using a cell scratch assay. The test results of the cell scratch assay were as follows: Figure 5 As shown.

[0187] according to Figure 5 It can be seen that after 24 hours of co-culture with cells, the closure area of ​​cell scratches treated with the soft tissue fillers provided in Examples 1-14 was over 40%, demonstrating that the soft tissue fillers provided in Examples 1-14 can repair damaged cells and skin tissue and promote skin cell regeneration. In contrast, the closure area of ​​the filling injections used in Comparative Examples 1-3 was only around 20%, significantly lower than that of the soft tissue fillers in Examples 1-14.

[0188] Test Example 5: In vitro scavenging of reactive oxygen species (ROS)

[0189] Animal experiments were conducted to verify the in vitro reactive oxygen species (ROS) scavenging effects of the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14 and the filling injection solutions provided in Comparative Examples 1-3. Six-week-old SD rats were used as experimental animals for model establishment. D-galactose was injected into the skin on the back of the rats once daily for four weeks. After model establishment, four injection points were made on the back of each rat, with 250 μL of the soft tissue filler provided in Examples 1, 5, 9, 12, and 14 or physiological saline injected at each point. The temperature was set at 24±2℃, the relative humidity at 50±10%, and the lighting time at 12 hours, allowing the animals free access to food. The experimental animals were sacrificed at 1, 2, 4, 6, and 12 weeks, and the skin tissue from the injected samples and the uninjected samples was harvested and fixed in 10% neutral buffered formalin solution.

[0190] The long-lasting anti-inflammatory and antioxidant capabilities of the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14 and the filling injections provided in Comparative Examples 1-3 were verified by testing the levels of inflammatory cytokines TNF-α and antioxidant cytokines, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and catalase (CAT) in the skin. Figure 6 and 7 The curves showing the changes in the levels of inflammatory factors TNF-α and SOD in the dorsal skin of rats at weeks 1, 2, 4, 6, and 12, respectively, after injection of the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14, the filling injection solutions of Comparative Examples 1 and 2, and physiological saline.

[0191] according to Figures 6 to 7It was found that, compared to the saline-treated rat dorsal skin, the content of the inflammatory factor TNF-α decreased over time, while the content of the four oxidative markers increased over time. Furthermore, the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14 remained effective after 12 weeks, indicating that the soft tissue fillers provided in these examples have anti-inflammatory and antioxidant capabilities for up to 12 weeks. In contrast, the filler injection in Comparative Example 3, after injection into the rat skin, rapidly increased antioxidant capacity in a short period, but its effect decreased rapidly from the fourth week onwards, failing to maintain its antioxidant effect long-term.

[0192] Test Example 6: Long-term retention effect

[0193] The retention time of the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14, and the filling injection solutions provided in Comparative Examples 1-3, in the dorsal skin of rats was tested using in vivo imaging. Fluorescently labeled injection solutions were prepared by conjugating the soft tissue fillers provided in Examples 1, 5, 9, 12, and 14, and the filling injection solutions provided in Comparative Examples 1-3, with FITC fluorescent molecules, and then injected into the dorsal skin of six-week-old SD rats. The diffusion of the fluorescently labeled injection solutions in the rat skin was tracked using in vivo imaging until the fluorescence completely disappeared. The results of in vivo imaging showed that the fluorescence of the soft tissue fillers injected in Examples 1, 5, 9, 12, and 14 completely disappeared after approximately 3 months, the fluorescence of Comparative Examples 1-2 completely disappeared after approximately 1 month, and the fluorescence of Comparative Example 3 completely disappeared after approximately 7 days. The in vivo imaging results for Examples 1 and Comparative Examples 1-3 are as follows: Figure 8 As shown. The results indicate that, compared to the filling injections of Comparative Examples 1-3, the soft tissue fillers of Examples 1, 5, 9, 12, and 14 exhibit superior long-term in vivo retention.

[0194] Test Example 7: Promoting Collagen Production

[0195] The soft tissue fillers of Examples 1, 5, 9, 12, and 14, and the small molecule injection solutions of Comparative Examples 1-3 were injected intradermally into the dermis of the dorsal skin of 8-week-old SD rats. Masson staining analysis was performed on the dorsal skin of the rats, and the results are as follows: Figure 9 As shown.

[0196] according to Figure 9 It can be seen that, compared with the normal control group, the soft tissue fillers in Examples 1, 5, 9, 12 and 14 can effectively increase the production of collagen in skin tissue, and the compound injection solution is significantly better than small molecule antioxidants and biological macromolecule fillers.

[0197] Furthermore, Sirius red staining was performed on the skin treated with the soft tissue filler of Example 5 to observe the changes in the content of type I and type III collagen. The results were as follows: Figure 10 As shown.

[0198] according to Figure 10 It can be seen that, compared with the PBS group, the content of type I collagen and type III collagen increased to some extent after treatment with the soft tissue filler in Example 5.

[0199] Test Example 8: Instant Fill Effect

[0200] The soft tissue fillers of Examples 1, 5, 9, 12, and 14, and the filling injection solutions of Comparative Examples 1-3, were injected intradermally into the dermis of the dorsal skin of 8-week-old SD rats. Skin elasticity tests were performed on the rats' dorsal skin at 0, 1, 2, 4, 6, and 12 weeks. The results are as follows: Figure 11 As shown.

[0201] according to Figure 11 It can be seen that, compared with the normal control group, the soft tissue fillers in Examples 1, 5, 9, 12 and 14 can effectively improve skin elasticity and still maintain good skin elasticity at 12 weeks, indicating that they have a long-lasting filling effect. Moreover, the soft tissue fillers in the examples are significantly better than the filling injections in the comparative examples.

[0202] Test Example 9: Skin Moisture Test

[0203] The soft tissue fillers provided in Examples 1, 5, 9, 12, and 14, and the filling injection solutions provided in Comparative Examples 1-3, were injected intradermally into the dermis of the dorsal skin of 8-week-old SD rats. The skin moisture content of the rats was measured using a skin moisture meter at 0, 1, 2, 4, 6, and 12 weeks, following relevant skin testing guidelines. The results are shown in Table 3.

[0204] Table 3: Water content of rat dorsal skin

[0205] Example 1 Example 5 Example 9 Example 12 Example 14 Comparative Example 1 Comparative Example 2 Comparative Example 3 1 week 20.51±0.49 22.19±0.53 20.40±0.36 21.68±0.63 20.89±0.51 19.20±0.62 19.19±0.43 19.43±0.41 2 weeks 20.74±0.55 22.40±0.48 20.67±0.51 21.93±0.45 21.02±0.59 19.32±0.56 19.33±0.52 19.64±0.46 4 weeks 20.89±0.41 22.77±0.79 20.87±0.48 22.14±0.55 21.24±0.62 19.47±0.67 18.70±0.63 19.81±0.57 6 weeks 21.12±0.63 23.05±0.57 20.75±0.65 22.29±0.69 21.39±0.48 19.62±0.43 18.49±0.51 19.61±0.47 12 weeks 21.03±0.57 22.81±0.42 20.63±0.54 21.97±0.44 21.10±0.52 19.31±0.31 18.31±0.38 19.55±0.43

[0206] According to the analysis in Table 3, the soft tissue fillers in Examples 1, 5, 9, 12, and 14 and the filling injection solutions provided in Comparative Examples 1-3 all have good water-locking and moisturizing effects, especially the soft tissue filler in Example 5, which has the best water-locking and moisturizing effect.

[0207] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0208] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application 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 or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A polymer composition, characterized in that, The components include: Biomacromolecules, and bioresponsive polymers; The biomacromolecules and the bioresponsive polymers can form a physically entangled network in the presence of reactive oxygen species; The bioresponsive polymer comprises any of the following chemical formulas: Among them, R1 is selected independently from each other. Either a mercapto group or a selenoyl group; R2 is independently selected from phenyl, C 7-20 Alkylphenyl, C 7-20 Heteroalkylphenyl, C 1-20 Alkyl or C 1-20 Any of the heteroalkyl groups; R3 is independently selected from C 1-20 Alkyl, C 1-20 Heteroalkyl or C 7-20 Any of the alkylphenyl groups; R4 is selected from thiol or selenool groups; n ≥ 5.

2. The polymer composition according to claim 1, characterized in that, R2 is independently selected from phenyl, C 7-20 Alkylphenyl, C 7-20 Ester-based phenyl, C 7-20 carbonyl phenyl, C 7-20 Amide phenyl, C 1-20 Alkyl, C 3-20 cycloalkyl, C 1-20 Nitrogen alkyl, C 1-20 Alkoxy, C 1-20 Alkylthio, C 2-20 Esteryl alkyl, C 2-20 Ketoalkyl, C 2-20 Aldehyde alkyl, C 2-20 Amide alkyl, C 2-20 Carboxyalkyl, C 2-20 Hydroxyalkyl, C 2-20 aminoalkyl, C 2-20 Thioamidoalkyl, C 2-20 Carbamate alkyl, C 2-20 Ureaalkyl, C 2-20 Thiourea alkyl or C 2-20 Any of the urethane alkyl groups; R3 is independently selected from C 1-20 Alkyl, C 1-20 Nitrogen alkyl, C 1-20 Alkoxy, C 1-20 Alkylthio, C 2-20 Esteryl alkyl, C 2-20 Ketoalkyl, C 2-20 Aldehyde alkyl, C 2-20 Amide alkyl, C 2-20 Thioamidoalkyl, C 2-20 Ureaalkyl, C 2-20 Thiourea alkyl or C 2-20 Any of the urethane alkyl groups.

3. The polymer composition according to claim 2, characterized in that, The bioresponsive polymer comprises:

4. The polymer composition according to any one of claims 1-3, characterized in that, The biomacromolecules are selected from at least one of hyaluronic acid, collagen, carboxymethyl cellulose, heparin sulfate, chondroitin sulfate, proteoglycan, chitosan, and sodium alginate.

5. The polymer composition according to claim 1, characterized in that, The mass ratio of the biomacromolecule to the bioresponsive polymer is 5-95:95-5.

6. A soft tissue filler, characterized in that, The active ingredient is the polymer composition described in any one of claims 1-5.

7. The soft tissue filler according to claim 6, characterized in that, It also includes solvent systems; The solvent system comprises any one of sterile water, physiological saline, phosphate buffer, acetate buffer, citrate buffer, tromethorphan buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, glycine buffer, and lactate buffer.

8. The soft tissue filler according to claim 7, characterized in that, The polymer composition has a mass-volume concentration of 5-500 mg / mL in the solvent system.

9. A method for preparing the soft tissue filler according to claim 8, characterized in that, The steps include: The bioresponsive polymer and biomacromolecules are dispersed in a solvent system and then post-processed to obtain the final product.

10. The use of the injectable soft tissue filler according to any one of claims 6 to 8 in wound healing, tissue filling and / or repair, skin moisturizing and water retention, anti-aging, antioxidant therapy, and skin barrier repair.

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