A cross-linking agent, a hyaluronic acid soft tissue filler, and a preparation method

Abstract

The application provides a crosslinking agent, the general formula of the crosslinking agent is shown in formula (I) or formula (II): in the formula, m is greater than or equal to 1, n is greater than or equal to 1, and l is greater than or equal to 1. The crosslinking agent described in the application can realize effective crosslinking of hyaluronic acid by simply adjusting pH, can limit excessive swelling of hyaluronic acid, and can make the hyaluronic acid filler have better mechanical properties, supporting properties and stability, and can reduce the occurrence of complications. The crosslinking agent introduces PCL or PLA in the form of a molecular chain into the filler, not only retains the advantages of PCL / PLA in stimulating the generation of collagen and elastic fibers in the skin, but also can be degraded by hyaluronidase to quickly dispose the risks such as adverse embolism, and improves the safety of treatment. This scheme provides a safe, reliable and high-effective option for the medical and cosmetic industry.

Description

Technical Field

[0001] This invention relates to the field of hyaluronic acid technology, specifically to a crosslinking agent, a hyaluronic acid soft tissue filler, and a preparation method thereof. Background Technology

[0002] Hyaluronic acid fillers hold a significant share of the medical aesthetics market and maintain a continuous growth trend, thanks to the excellent biocompatibility and biodegradability of hyaluronic acid, an endogenous component. Hyaluronic acid is mainly composed of repeating units of N-acetylglucosamine and D-glucuronic acid, rich in carboxyl and hydroxyl groups, making it easy to cross-link and modify. Cross-linking can form a more stable three-dimensional network structure, and the increased mechanical strength provides better support for soft tissue filling, while also prolonging the degradation cycle in vivo, improving and extending the filling and shaping effect. Hyaluronic acid can also be rapidly degraded by hyaluronidase into low molecular weight hyaluronic acid or oligosaccharides, thus better managing adverse reactions or reversing suboptimal treatment results.

[0003] However, as the market pursues higher levels of filling effects, hyaluronic acid fillers have several limitations. For example, hyaluronic acid fillers are prone to swelling and have weak mechanical properties, which may cause adverse reactions such as overfilling, vascular compression, deformation, and displacement; their biological inertness prevents them from stimulating collagen regeneration. In contrast, PCL or PLA microsphere fillers have the advantage of effectively stimulating the production of collagen and elastin fibers in the skin, thereby significantly improving skin texture and elasticity. New filler materials formed by combining PCL / PLA microspheres with hyaluronic acid fillers can combine immediate filling function with long-term collagen regeneration stimulation. However, the size and shape of PCL / PLA microspheres affect their collagen regeneration effect in vivo, therefore, the control of their size and shape is very strict. More concerning is that adverse reactions such as excessive inflammation, vascular embolism, tissue hardening, and infection that occur in different individuals after PCL / PLA microsphere filling are difficult to correct and treat in a timely manner. This is mainly because there is currently no effective method to rapidly degrade PCL / PLA microspheres as quickly as hyaluronic acid, which brings huge potential safety risks to the application of this type of material.

[0004] Therefore, there is an urgent need for a hyaluronic acid-based filler that can provide immediate filling and stimulate collagen regeneration, while also being resistant to excessive water absorption and swelling, having better mechanical elasticity, and being rapidly degraded by hyaluronidase for quick treatment of adverse reactions. Summary of the Invention

[0005] To address the problems existing in the prior art, this application introduces PCL / PLA into the hyaluronic acid cross-linking network in the form of molecular chains to prepare a hyaluronic acid-based filler that can achieve immediate filling and stimulate collagen regeneration. It has resistance to excessive water absorption and swelling, better mechanical elasticity, and can be rapidly degraded by hyaluronidase for rapid treatment of adverse reactions.

[0006] This application provides a crosslinking agent, the general formula of which is shown in formula (I) or formula (II):

[0007] Formula (I)

[0008]

[0009] Equation (II)

[0010] In the formula, m≥1, n≥1, l≥1.

[0011] Further, in formula (I), 1≤l≤68, 1≤m≤26, 1≤n≤68, preferably 4≤l≤45, 2≤m≤18, 4≤n≤45; in formula (II), 1≤l≤68, 1≤m≤42, 1≤n≤68, preferably 4≤l≤45, 2≤m≤28, 4≤n≤45.

[0012] Further, the weight-average molecular weight of the crosslinking agent shown in formula (I) is 750-9600, and the weight-average molecular weight of the crosslinking agent shown in formula (II) is 700-9500; more preferably, the weight-average molecular weight of the crosslinking agent shown in formula (I) is 1100-6500, and the weight-average molecular weight of the crosslinking agent shown in formula (II) is 1000-6500.

[0013] This application also provides a method for preparing the crosslinking agent, comprising:

[0014] Diblock polymers were prepared using allyl polyethylene glycol and lactone compounds as raw materials;

[0015] Carboxylated allyl polyethylene glycol was prepared using allyl polyethylene glycol and acid anhydrides as raw materials;

[0016] The carboxylated allyl polyethylene glycol and the diblock polymer are reacted to prepare a triblock polymer;

[0017] Hydroxytriblock polymers were prepared by reacting mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone, and the aforementioned triblock polymer.

[0018] An epoxy-based triblock polymer, i.e., the crosslinking agent, is prepared by reacting epichlorohydrin, TBAB, and an alkali with the hydroxyl triblock polymer.

[0019] Furthermore, the weight-average molecular weight of the allyl polyethylene glycol is 160-3000; more preferably, the weight-average molecular weight is 200-2000.

[0020] Furthermore, when preparing the diblock polymer, the preferred molar ratio of allyl polyethylene glycol to lactone compound is 1:(1-50).

[0021] Furthermore, when preparing carboxylated allyl polyethylene glycol, the molar ratio of allyl polyethylene glycol to acid anhydride is preferably 1:(1-3); and the acid anhydride is even more preferably succinic anhydride.

[0022] Furthermore, when preparing the triblock polymer, a catalyst is added to carry out the reaction; more preferably, the molar ratio of the carboxylated allyl polyethylene glycol, the catalyst and the diblock polymer is (1-2):(1-2):(2-1).

[0023] Furthermore, when preparing the hydroxyl triblock polymer, the preferred molar ratio of mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone and the triblock polymer is (1-3):(1-3):1.

[0024] Furthermore, when preparing the epoxy triblock polymer, the molar ratio of epichlorohydrin, TABA, base and the triblock polymer is (3-6):(0.1-0.3):(2-5):1.

[0025] This application provides a method for preparing a hyaluronic acid soft tissue filler, comprising: performing a crosslinking reaction between hyaluronic acid and any of the aforementioned crosslinking agents to obtain a hyaluronic acid soft tissue filler.

[0026] Further, the mass ratio of hyaluronic acid to the crosslinking agent is 1:(0.1-2); more preferably, the mass ratio is 1:(0.5-1).

[0027] Furthermore, the crosslinking reaction is carried out under alkaline conditions; more preferably, the crosslinking reaction is carried out under conditions of 9 ≤ pH ≤ 11.

[0028] The cross-linking agent provided in this application can effectively cross-link hyaluronic acid by simply adjusting the pH, limiting excessive swelling of hyaluronic acid and giving the hyaluronic acid filler better mechanical properties, support, and stability, while reducing the occurrence of complications. Notably, this cross-linking agent introduces PCL or PLA into the filler in the form of molecular chains, which not only retains the advantages of PCL / PLA in stimulating collagen and elastin production in the skin, but also allows for rapid resolution of risks such as adverse embolisms through hyaluronidase degradation, thus improving treatment safety. This approach provides the medical aesthetics industry with a safe, reliable, and highly effective option. Attached Figure Description

[0029] Figure 1 The 1H NMR spectrum of the epoxy crosslinking agent described in formula (I) is shown. Detailed Implementation

[0030] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0031] This application provides a crosslinking agent, the general formula of which is shown in formula (I) or formula (II):

[0032] Formula (I)

[0033]

[0034] Equation (II)

[0035] In the formula, m≥1, n≥1, l≥1.

[0036] In some implementations, 1 ≤ l ≤ 68, for example, the value of l can be selected from 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68. In some preferred implementations, it is further preferred that the value of l is 4 ≤ l ≤ 45.

[0037] In some implementations, 1 ≤ n ≤ 68, for example, the value of n can be selected from 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68. In some preferred implementations, it is further preferred that the value of n is 4 ≤ n ≤ 45.

[0038] In some embodiments, in the crosslinking agent described in formula (I), 1 ≤ m ≤ 26, for example, the value of m can be selected from 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26. In some preferred embodiments, it is further preferred that the value of m is 2 ≤ m ≤ 18.

[0039] In some embodiments, in the crosslinking agent described in formula (II), 1 ≤ m ≤ 42, for example, the value of m can be selected from 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42. In some preferred embodiments, it is further preferred that the value of m is 2 ≤ m ≤ 28.

[0040] In some embodiments, the weight-average molecular weight of the crosslinking agent shown in formula (I) is 750-9600, for example, 750, 800, 900, 1000, 1100, 1300, 1500, 1800, 2000, 2200, 2500, 2800, 3000, 3200, 3500, 3800, 4000, 4200, 4500, 4800, 5000, 5200, 5500, 5800, 6000, 6200, 6500, 6800, 7000, 7200, 7500, 7800, 8000, 8200, 8500, 8800, 9000, 9200, 9500, 9600. In some preferred embodiments, the weight-average molecular weight of the crosslinking agent shown in formula (I) is 1100-6500.

[0041] In some embodiments, the crosslinking agent shown in formula (II) has a weight-average molecular weight of 700-9500, for example, 700, 1000, 1200, 1500, 1800, 2000, 2200, 2500, 2800, 3200, 3500, 3800, 4000, 4200, 4500, 4800, 5000, 5200, 5500, 5800, 6000, 6200, 6500, 6800, 7000, 7200, 7500, 7800, 8000, 8200, 8500, 8800, 9000, 9200, 9500. In some preferred embodiments, the crosslinking agent shown in formula (II) has a weight-average molecular weight of 1000-6500.

[0042] This application also provides a method for preparing the crosslinking agent, comprising:

[0043] Step 1: Prepare a diblock polymer using allyl polyethylene glycol and lactone compounds as raw materials;

[0044] Step 2: Prepare carboxylated allyl polyethylene glycol using allyl polyethylene glycol and acid anhydride as raw materials;

[0045] Step 3: The carboxylated allyl polyethylene glycol and the diblock polymer are reacted to prepare a triblock polymer;

[0046] Step 4: Reaction of mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone with the triblock polymer to prepare a hydroxyl triblock polymer;

[0047] Step 5: Epichlorohydrin, TBAB, and an alkali are reacted with the hydroxyl triblock polymer to prepare an epoxy triblock polymer, i.e., the crosslinking agent.

[0048] In step one, when preparing the diblock polymer using allyl polyethylene glycol and lactone compounds as raw materials, allyl polyethylene glycol and lactone compounds are mixed under a protective atmosphere, and an initiator is added to carry out the reaction. The reaction time is 2-12 hours, and the reaction temperature is 100-160℃. After the reaction is completed, precipitation is carried out, and the diblock copolymer is obtained after drying for 12-36 hours.

[0049] When preparing the crosslinking agent of formula (I), the above steps involve the following reaction.

[0050] When preparing the crosslinking agent of formula (II), the following reaction occurs in the above steps.

[0051]

[0052] In some embodiments, the allyl polyethylene glycol is used as a raw material, and its weight-average molecular weight is 160-3000, for example, 160, 200, 400, 700, 1000, 1200, 1500, 1700, 2000, 2200, 2500, 2700, 3000; in some preferred embodiments, its weight-average molecular weight is 200-2000.

[0053] In some preferred embodiments, the lactone compound is ε-caprolactone or lactide.

[0054] In some preferred embodiments, the molar ratio of allyl polyethylene glycol to ε-caprolactone or lactide is 1:(1-50), for example 1:1, 1:3, 1:5, 1:8, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:38, 1:40, 1:42, 1:45, 1:48, 1:50.

[0055] In some preferred embodiments, the molar ratio of allyl polyethylene glycol to ε-caprolactone or lactide is 1:(14-40).

[0056] In some preferred embodiments, the initiator is stannous 2-ethylhexanoate.

[0057] In step two, when preparing carboxylated allyl polyethylene glycol using allyl polyethylene glycol and acid anhydride as raw materials, allyl polyethylene glycol is dissolved in an organic solvent, and acid anhydride and initiator are added to carry out the reaction. The reaction temperature is room temperature, and the reaction time is 12-36 hours. After the reaction is completed, precipitation is carried out, and after drying for 12-36 hours, carboxylated allyl polyethylene glycol is obtained.

[0058] When preparing the crosslinking agent of formula (I) or formula (II), the above steps involve the following reaction.

[0059] In some preferred embodiments, the anhydride is succinic anhydride.

[0060] In some preferred embodiments, the molar ratio of allyl polyethylene glycol to succinic anhydride is 1:(1-3), for example, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3.

[0061] In step three, when the carboxylated allyl polyethylene glycol and the diblock polymer are reacted to prepare the triblock polymer, the carboxylated allyl polyethylene glycol is dissolved in an organic solvent, a catalyst and the diblock polymer are added, and the reaction is carried out under a protective atmosphere at room temperature for 12-36 hours. After the reaction is completed, the product is precipitated, washed, and dried for 12-36 hours to obtain the triblock polymer.

[0062] When preparing the crosslinking agent of formula (I), the above steps involve the following reaction.

[0063] When preparing the crosslinking agent of formula (II), the following reaction occurs in the above steps.

[0064]

[0065] In some preferred embodiments, the catalyst is 4-dimethylaminopyridine.

[0066] In some preferred embodiments, the molar ratio of the carboxylated allyl polyethylene glycol, the catalyst, and the diblock polymer is (1-2):(1-2):(2-1). For example, the molar ratio of the carboxylated allyl polyethylene glycol to the catalyst is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 2:1, 2:1.1, 2:1.2, 2:1.3, 2:1.4, or 2:1.5. , 2:1.6, 2:1.7, 2:1.8, 2:1.9, 2:2; for example, the molar ratio of the carboxylated allyl polyethylene glycol to the diblock polymer is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 2:1, 2:1.1, 2:1.2, 2:1.3, 2:1.4, 2:1.5, 2:1.6, 2:1.7, 2:1.8, 2:1.9, 2:2.

[0067] In some preferred embodiments, the catalyst is 4-dimethylaminopyridine.

[0068] In step four, when preparing the hydroxyl triblock polymer by reacting mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone, and the triblock polymer, mercaptoethanol and 2,2-dimethoxy-2-phenylacetophenone are dissolved, the triblock polymer is added, and the mixture is reacted under ultraviolet light for 10-60 minutes. After washing and drying, the hydroxyl triblock polymer is obtained.

[0069] When preparing the crosslinking agent of formula (I), the above steps involve the following reaction.

[0070] When preparing the crosslinking agent of formula (II), the following reaction occurs in the above steps.

[0071]

[0072] In some preferred embodiments, the molar ratio of mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone, and the triblock polymer is (1-3):(1-3):1. For example, the molar ratio of mercaptoethanol to the triblock polymer is 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:11:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1... The molar ratios of 2,2-dimethoxy-2-phenylacetophenone and the triblock polymer are 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2:1, 2:2, 2:3:1, 2:4:1, 2.5:1, 2:6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, and 3:1.

[0073] In some preferred embodiments, the molar ratio of mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone and the triblock polymer is 2:2:1.

[0074] In step five, when preparing the epoxy triblock polymer by reacting epichlorohydrin, TBAB, and an alkali with the hydroxyl triblock polymer, the hydroxyl triblock polymer is heated and melted, and then epichlorohydrin and TBAB are added to carry out the reaction at a temperature of 30-50°C. Then, an alkali is added, and the sodium hydroxide can be added all at once or in multiple batches. Subsequently, the epoxy triblock polymer is obtained by filtration, dissolution, precipitation, and drying, which is the crosslinking agent.

[0075] When preparing the crosslinking agent of formula (I), the above steps involve the following reaction.

[0076] When preparing the crosslinking agent of formula (II), the following reaction occurs in the above steps.

[0077]

[0078] In some preferred embodiments, the base is NaOH.

[0079] In some preferred embodiments, the molar ratio of epichlorohydrin, TBAB, NaOH, and the hydroxyl triblock polymer in the reaction to prepare the epoxy triblock polymer is 3-6:0.1-0.3:2-5:1, for example,

[0080] The molar ratio of epichlorohydrin to the hydroxyl triblock polymer is 3:1, 4:1, 5:1, or 6:1; the molar ratio of TABA to the hydroxyl triblock polymer is 0.1:1, 0.2:1, or 0.3:1; and the molar ratio of NaOH to the hydroxyl triblock polymer is 2:1, 3:1, 4:1, or 5:1.

[0081] In some preferred embodiments, the molar ratio of epichlorohydrin, TBAB, NaOH, and the hydroxyl triblock polymer in the reaction to prepare the epoxy triblock polymer is 5:0.2:3:1.

[0082] In some implementations, the organic solvent used in steps one through five is 1,4-dioxane and / or dichloromethane.

[0083] In some implementations, the protective atmosphere described in steps one through five is nitrogen.

[0084] In some implementations, the drying described in steps one through five can be vacuum drying or freeze drying.

[0085] This application provides a method for preparing a hyaluronic acid soft tissue filler, comprising: performing a crosslinking reaction between hyaluronic acid and the above-mentioned crosslinking agent to obtain a hyaluronic acid soft tissue filler.

[0086] Further, hyaluronic acid and the crosslinking agent are mixed and dissolved according to a mass ratio, and alkali is added to carry out a crosslinking reaction. The reaction temperature is 20-50℃ and the reaction time is 2-12h. After washing, a hydrogel product is obtained. The hydrogel product is then washed to remove unreacted BDDE, and the hyaluronic acid soft tissue filler is obtained.

[0087] When hyaluronic acid soft tissue fillers are prepared using the crosslinking agent described in formula (I), the following reaction occurs.

[0088] When hyaluronic acid soft tissue fillers are prepared using the crosslinking agent described in formula (II), the following reaction occurs.

[0089]

[0090] In some embodiments, the mass ratio of hyaluronic acid to the crosslinking agent is 1:(0.1-2), for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.

[0091] In some preferred embodiments, the mass ratio of hyaluronic acid to the crosslinking agent is 1:(0.5-1).

[0092] In some embodiments, the crosslinking reaction is carried out under alkaline conditions with pH > 7. In some preferred embodiments, the crosslinking reaction is carried out under conditions with pH ≤ 9 ≤ pH ≤ 11.

[0093] Example

[0094] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0095] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0096] Example 1

[0097] Preparation of APEG-PCL-OH diblock polymer: Under a nitrogen atmosphere and with mechanical stirring, 8 g of allyl polyethylene glycol (0.004 mol), 13.7 g of ε-caprolactone (0.12 mol), and 0.3 g of stannous 2-ethylhexanoate were added to a 250 mL three-necked flask and refluxed at 130 °C for 8 hours. After the reaction was complete, 100 mL of diethyl ether was used for precipitation. The product was filtered and vacuum dried for 24 hours to obtain the APEG-PCL-OH diblock copolymer.

[0098] Preparation of carboxylated APEG: 6 g of allyl polyethylene glycol (0.003 mol) was dissolved in 25 ml of 1,4-dioxane, and then 0.48 g of succinic anhydride (0.0048 mol) and 0.37 g of 4-dimethylaminopyridine (0.003 mol) were added to the solution. After stirring at room temperature for 24 hours, the mixture was precipitated with 100 ml of diethyl ether. The product was then vacuum dried for 24 hours to obtain carboxylated APEG.

[0099] Preparation of APEG-PCL-APEG: 3g of APEG-COOH (0.0014mol) was dissolved in 30ml of dichloromethane and stirred at room temperature for 30 minutes. Then, 1.2g of 4-dimethylaminopyridine (0.0014mol) was added, and 4.05g of APEG-PCL-OH (0.001mol) was added to the reaction mixture. The mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. After precipitation with 100ml of diethyl ether, the mixture was dialyzed with water to remove unreacted residual PEG-COOH. Finally, the mixture was lyophilized for 24 hours to obtain the APEG-PCL-APEG triblock copolymer.

[0100] Preparation of hydroxy PEG-PCL-PEG: 0.076 g mercaptoethanol (0.000978 mol) and 0.25 g 2,2-dimethoxy-2-phenylacetophenone (0.000978 mol) were completely dissolved in 30 ml of ethanol. Then, 3 g APEG-PCL-APEG (0.000489 mol) was added to the above solution. After mixing, the solution was stirred and reacted under ultraviolet light for 30 minutes. After dialysis with water for 3 days, unreacted residual reactants were removed. Finally, the hydroxy PEG-PCL-PEG triblock copolymer was obtained by lyophilization.

[0101] Preparation of epoxy-based PEG-PCL-PEG: 2g of hydroxyl PEG-PCL-PEG triblock copolymer (0.000318mol) was heated to melt, and 0.146g of epichlorohydrin (0.00159mol) and 0.02g of TBAB (0.000064mol) were added. The mixture was reacted at 40°C. After a period of time, 0.038g of sodium hydroxide (0.000954mol) was added in portions. The mixture was filtered while hot, and the dioxane was dissolved and ether precipitated. The mixture was then vacuum dried to obtain the epoxy-based triblock polymer, i.e., the crosslinking agent. Finally, the epoxy-based PEG-PCL-PEG triblock copolymer was obtained by freeze-drying.

[0102] Compared with Example 1, the only difference between Examples 2 and 3 is that the weight-average molecular weight of allyl polyethylene glycol is different when preparing the diblock polymer, thus preparing different epoxy-based PEG-PCL-PEG crosslinking agents. All other parameters are the same.

[0103] Compared with Example 1, the only difference between Examples 4 and 5 is that the weight-average molecular weight of allyl polyethylene glycol is different when preparing carboxylated APEG, thus preparing different epoxy-based PEG-PCL-PEG crosslinking agents. All other parameters are the same.

[0104] Compared with Example 1, the only difference between Examples 6 and 7 is that the molar ratio of allyl polyethylene glycol to ε-caprolactone is different when preparing the diblock polymer, thus preparing different epoxy-based PEG-PCL-PEG crosslinking agents. All other parameters are the same.

[0105] Compared with Example 1, the only difference in Example 8 is that ε-caprolactone was replaced with lactide when preparing the diblock polymer, and an epoxy-based PEG-PLA-PEG crosslinking agent was prepared. All other parameters were the same.

[0106] Compared with Example 8, the only difference between Examples 9 and 10 is that the molar ratio of allyl polyethylene glycol to lactide is different when preparing the diblock polymer, thus preparing different epoxy-based PEG-PLA-PEG crosslinking agents. All other parameters are the same.

[0107] The epoxy triblock copolymers obtained in Examples 1-10 were tested using the following specific methods.

[0108] 1. Chemical structure characterization

[0109] The chemical structure of the resulting copolymer was characterized by dissolving the raw material in deuterated chloroform and then using proton nuclear magnetic resonance spectroscopy.

[0110] 2. Calculate the epoxy value

[0111] The epoxy value of the epoxy triblock copolymer was determined using the hydrochloric acid-acetone method. Taking Example 1 as an example, the specific operation was as follows: 1 g of the lyophilized epoxy triblock copolymer was accurately weighed and placed in a 250 mL stoppered conical flask. 20 mL of hydrochloric acid-acetone solution was added, and the flask was sealed tightly. After shaking well, the flask was placed in the dark and allowed to stand for 30 min. Five drops of a mixed indicator solution (10 mL of cresol red, 30 mL of thymol blue, 0.01 mol / L sodium hydroxide, and 0.01 mol / L hydrochloric acid adjusted to neutral) were added, and the solution was titrated with 0.15 mol / L sodium hydroxide standard titrant until a purple-blue color was obtained. The volume of sodium hydroxide standard titrant consumed was recorded, and the epoxy value was calculated.

[0112] according to Figure 1 It can be seen that the peak with a chemical shift of approximately 4.05 corresponds to the hydrogen in the repeating unit of polyethylene glycol, the peaks with chemical shifts of approximately 2.25, 1.8, and 1.4 correspond to the hydrogen at positions d, e, and f in polycaprolactone, and the two peaks with a chemical shift of approximately 2.75 correspond to the hydrogen at positions a and b on the epoxy group. Combined with the epoxy value calculated by the hydrochloric acid-acetone titration method, the successful preparation of the epoxy triblock crosslinking agent can be confirmed.

[0113] 3. Detecting molecular weight

[0114] The weight-average molecular weight and degrees of polymerization (n, m, l) of the epoxy triblock copolymer were determined using gel permeation chromatography.

[0115] The parameters of Examples 1-10 and the test results of the prepared crosslinking agents are shown in Table 1.

[0116] In the preparation of the diblock polymer, the weight-average molecular weight of the allyl polyethylene glycol used is denoted as Mw1.

[0117] The weight-average molecular weight of the allyl polyethylene glycol used in the preparation of carboxylated APEG is denoted as Mw2.

[0118] When preparing diblock polymers, the molar ratio of allyl polyethylene glycol to lactone is denoted as "x:y";

[0119] The weight-average molecular weight of an epoxy triblock copolymer is denoted as Mw3;

[0120] "n:m:l" refers to the ratio of n, m, and l in an epoxy triblock copolymer.

[0121] Table 1

[0122]

[0123] Example 11

[0124] Preparation of hyaluronic acid filler: 0.1 g HA powder was dissolved in 10 ml of deionized water containing 0.1 g epoxy PEG-PCL-PEG and 2.5 g NaOH. The mixture was reacted at 40 °C for 6 h. The reaction mixture was washed three times with deionized water to obtain a hydrogel product. The hydrogel was dialyzed against phosphate buffer for 24 h, and then against deionized water for 24 h to completely remove unreacted BDDE.

[0125] In this embodiment, the crosslinking agent is an epoxy-based PEG-PCL-PEG triblock copolymer, which was prepared using the method described in Example 1.

[0126] The difference between Examples 12-20 and Example 11 is that the epoxy triblock copolymers prepared in Examples 2-10 were used as crosslinking agents to prepare hyaluronic acid fillers, while other parameters remained the same.

[0127] The difference between Examples 21 and 22 and Example 11 is that the mass ratio of hyaluronic acid to crosslinking agent is different, namely 1:0.5, 1:0.8 and 1:1.2, respectively, while other parameters are the same.

[0128] Comparative Example 1

[0129] Compared with Example 11, the only difference in Comparative Example 1 is that PCL microspheres were used as crosslinking agents to prepare hyaluronic acid fillers. The hyaluronic acid used in this example is the same as that in Example 11.

[0130] Comparative Example 2

[0131] The only difference between Comparative Example 2 and Comparative Example 1 is that Comparative Example 2 uses PLA microspheres as a crosslinking agent to prepare hyaluronic acid filler, while other parameters are the same.

[0132] The hyaluronic acid fillers in Examples 11-22 and Comparative Examples 1-2 were tested, as detailed below.

[0133] The viscoelasticity G of hyaluronic acid fillers, including the elastic modulus G, was tested using a rotational rheometer. ， and loss modulus G ，， The unit is Pa. Where G ， The elastic modulus reflects the elastic properties of a material, that is, its ability to return to its original shape after being subjected to external forces. The magnitude of the elastic modulus directly reflects the material's ability to store and release energy. Generally speaking, the larger the elastic modulus, the stronger the material's rigidity. ，， Viscoelasticity refers to the damping characteristics of a material, that is, the degree of energy loss when the material is subjected to external forces. The magnitude of the loss modulus directly affects the vibration damping ability of the material; generally speaking, the larger the loss modulus, the more pronounced the viscous behavior of the material. The testing conditions for viscoelasticity are: using a 12mm clamp, testing at 37℃, and the test mode is Oscillation Frequency Sweep Test.

[0134] The swelling coefficient of hyaluronic acid fillers was tested using a gravimetric method. The hydrogel sample was immersed in deionized water for at least 24 hours, then quickly removed from the water, the surface water was removed with filter paper, and the sample was weighed to obtain the equilibrium swelling mass Ws. The equilibrium swelling ratio (ESR) of the hydrogel was calculated using the following formula: ESR = Ws / Wd, where Wd is the dry weight of the gel.

[0135] The degradation cycle of hyaluronic acid fillers was tested using an in vitro simulated body fluid method, with PBS as the degradation medium and 37°C as the degradation temperature.

[0136] The test results of the hyaluronic acid fillers in Examples 11-25 and Comparative Examples 1-2 are shown in Table 2.

[0137] Table 2

[0138]

[0139]

[0140] Comparing the data in Tables 1 and 2, it can be seen that by adjusting the feed ratio and controlling the reaction conditions, epoxy crosslinking agents with specific molecular structures can be synthesized. The higher the PCL / PLA content in the epoxy crosslinking agent, the stronger its ability to limit swelling. This is because both PCL and PLA have hydrophobic properties, thus weakening the hydrophilicity of the gel and limiting the degree of swelling. Simultaneously, the degradation period also varies with the PCL / PLA content; both types of molecules can extend the degradation period of the filler.

[0141] The hyaluronic acid filler prepared in this application significantly limits the swelling of hyaluronic acid, and at the same time, the preparation process eliminates the use of traditional organic solvents, reducing the environmental burden and potential health risks in the production process.

[0142] The crosslinking agent prepared in this application retains the advantages of PCL / PLA in stimulating the production of collagen and elastin fibers in the skin, and also improves the safety of treatment. This approach provides the medical aesthetics industry with a safer, more reliable, and more effective option.

[0143] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A crosslinking agent, said crosslinking agent having the general formula shown in formula (I) or formula (II): ； Formula (I); ； Formula (II); In the formula (I), 4≤l≤68, 2≤m≤26, and 4≤n≤68; In the formula (II), 4≤l≤68, 2≤m≤42, and 4≤n≤68.

2. The crosslinking agent according to claim 1, wherein, In the formula (I), 4≤l≤45, 2≤m≤18, and 4≤n≤45.

3. The crosslinking agent according to claim 1, wherein, In the formula (II), 4≤l≤45, 2≤m≤28, and 4≤n≤45.

4. The crosslinking agent according to claim 1, wherein, The weight-average molecular weight of formula (I) is 750-9600.

5. The crosslinking agent according to claim 1, wherein, The weight-average molecular weight of formula (I) is 1100-6500.

6. The crosslinking agent according to claim 1, wherein, The weight-average molecular weight of formula (II) is 700-9500.

7. The crosslinking agent according to claim 1, wherein, The weight-average molecular weight of formula (II) is 1000-6500.

8. A method for preparing the crosslinking agent according to any one of claims 1-7, wherein, include: Diblock polymers were prepared using allyl polyethylene glycol and lactone compounds as raw materials; Carboxylated allyl polyethylene glycol was prepared using allyl polyethylene glycol and acid anhydrides as raw materials; The carboxylated allyl polyethylene glycol and the diblock polymer are reacted to prepare a triblock polymer; Hydroxytriblock polymers were prepared by reacting mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone, and the aforementioned triblock polymer. An epoxy-based triblock polymer was prepared by reacting epichlorohydrin, TBAB, and an alkali with the hydroxyl triblock polymer, thus obtaining a crosslinking agent.

9. The preparation method according to claim 8, wherein, The weight-average molecular weight of the allyl polyethylene glycol is 160-3000.

10. The preparation method according to claim 8, wherein, The weight-average molecular weight of the allyl polyethylene glycol is 200-2000.

11. The preparation method according to claim 8, wherein, In preparing the diblock polymer, the molar ratio of allyl polyethylene glycol to lactone compound is 1:(1-50).

12. The preparation method according to claim 8, wherein, In the preparation of carboxylated allyl polyethylene glycol, the molar ratio of allyl polyethylene glycol to acid anhydride is 1:(1-3).

13. The preparation method according to claim 8, wherein, In the preparation of carboxylated allyl polyethylene glycol, the acid anhydride is succinic anhydride.

14. The preparation method according to claim 8, wherein, In the preparation of the triblock polymer, the carboxylated allyl polyethylene glycol and the diblock polymer react in the presence of a catalyst.

15. The preparation method according to claim 14, wherein, The molar ratio of the carboxylated allyl polyethylene glycol, the catalyst, and the diblock polymer is (1-2):(1-2):(2-1).

16. The preparation method according to claim 8, wherein, In preparing the hydroxyl triblock polymer, the molar ratio of mercaptoethanol, 2,2-dimethoxy-2-phenylacetophenone and the triblock polymer is (1-3):(1-3):

1.

17. The preparation method according to claim 8, wherein, When preparing the epoxy triblock polymer, the molar ratio of epichlorohydrin, TBAB, base and the hydroxyl triblock polymer is (3-6):(0.1-0.3):(2-5):

1.

18. A hyaluronic acid soft tissue filler, wherein, The crosslinking agent includes any one of claims 1-7 or any crosslinking agent prepared by any one of the preparation methods of claims 8-17.

19. The filler according to claim 18, wherein, The filler also includes hyaluronic acid, which undergoes a cross-linking reaction with the cross-linking agent to obtain a hyaluronic acid soft tissue filler.

20. The filler according to claim 19, wherein, The mass ratio of the hyaluronic acid to the crosslinking agent is 1:(0.1-2).

21. The filler according to claim 19, wherein, The mass ratio of the hyaluronic acid to the crosslinking agent is 1:(0.5-1).

Images