Injectable microphase-separated hydrogel, preparation method therefor, and use thereof

Abstract

The present invention relates to an injectable microphase-separated hydrogel, a preparation method therefor, and use thereof. The hydrogel prepared by the process of the present invention uses collagen as a raw material. By controlling the steps of dissolution, room-temperature stirring, low-temperature stirring, and chemical crosslinking, a uniform bubble solution is preferentially formed in the process of room-temperature stirring; then, microphase separation occurs by triggering the temperature-sensitive characteristic of the collagen material at a low temperature; with the increase of the viscosity of the solution, the bubble morphology tends to be stable, and continuous stirring enables the collagen solution to reach a thermodynamic and kinetic steady state, forming a stable and uniform bubble solution; finally, by means of chemical crosslinking, a chemically crosslinked network is formed, in which three-dimensional networks and microsphere structures are interlaced. These two structures together endow the microphase-separated hydrogel with strong support, resistance to heat and humidity, resistance to enzymatic degradation, ability to be injected, and injection uniformity. Using this process, the hydrogel still maintains good performance after high-temperature and high-pressure treatment, and has a white appearance, thereby avoiding the Tyndall phenomenon after injection.

Description

An injectable microphase separation hydrogel, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an injectable microphase separation hydrogel, its preparation method, and its applications. Background Technology

[0002] Collagen is the most abundant protein in animals, accounting for more than 30% of the total protein in the body, and is also a major component of the extracellular matrix. Collagen possesses high tensile strength, biodegradability, low antigenic activity, low irritation, low cytotoxicity, and the ability to promote cell growth, cell adhesion, and synergistic wound repair with newly formed cells and tissues when used as artificial organ frameworks or wound dressings. It is an ideal skincare ingredient and biomedical material with wide applications in medical dressings, cosmetic skincare, and regenerative medicine. Collagen-based materials belong to thermosensitive polymers, a class of the most important smart polymer materials. They can undergo reversible phase transitions between hydrophilic and hydrophobic states under temperature control. Thermosensitive polymers are divided into two categories: one is polymers with a lower critical solution temperature (LCST), i.e., LCST-type polymers. When the temperature is below their LCST, the polymer dissolves in solution and forms a homogeneous phase; when the temperature is above the LCST, the polymer solubility decreases and phase separation occurs. Another type is polymers with a high critical solution temperature (UCST), also known as UCST-type polymers. When the temperature is higher than its UCST, the polymer completely dissolves and becomes homogeneous; otherwise, phase separation occurs.

[0003] Phase separation occurs when the solubility of polymer materials decreases and molecular aggregation occurs due to changes in the external environment, resulting in reduced transparency, turbidity, or even white precipitation. Microphase separation, on the other hand, occurs when polymer materials have not yet undergone a macroscopic phase transition, but have formed microscopic phase regions ranging from nanometers to micrometers in size, exhibiting a state between homogeneous and heterogeneous phases. For example, in collagen or polypeptide materials, the viscosity of the solution increases at low temperatures, reducing the intermolecular distance. Adjusting the pH or ionic strength of the collagen solution can then affect the charge distribution on the collagen molecular chains, enhancing the interactions between collagen molecules and causing further aggregation. Microscopically, this results in a regular "D-band" distribution, while macroscopically, it presents a white appearance.

[0004] Collagen-based materials are typical amphiphilic polymers, meeting the characteristics of temperature-sensitive polymers, meaning their mechanical properties can be adjusted by temperature. However, this type of temperature-controlled phase separation is reversible, and the resulting mechanical properties are relatively low, which does not meet the application requirements of biomedical materials, thus limiting their development and utilization. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an injectable microphase-separated hydrogel, its preparation method, and its applications. The present invention employs an Interfacial Crosslinking Technique for Microphase Separation (ICT-MS) technology. Through mechanical stirring and low-temperature processing, the collagen solution reaches thermodynamic and kinetic steady state, resulting in microphase separation and a stable state. Subsequently, a crosslinking agent is added to chemically crosslink the collagen molecules and conduct interfacial crosslinking with the bubble film within the structure, forming a stable and uniform chemical network. This ultimately yields an injectable microphase-separated hydrogel with high strength, resistance to damp heat and enzymatic hydrolysis, stable performance, and a white appearance.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides an injectable microphase separation hydrogel, the injectable microphase separation hydrogel comprising a three-dimensional network structure formed by collagen molecules and a microsphere structure formed by cross-linking of collagen molecules on the surface of a bubble liquid membrane, wherein the microsphere structure and the three-dimensional network structure are interspersed.

[0008] Furthermore, the preparation steps of the injectable microphase separation hydrogel include preparing a collagen solution, stirring at room temperature, stirring at low temperature, and interfacial chemical crosslinking.

[0009] The collagen concentration in the injectable microphase-separated hydrogel is 20 mg / mL to 60 mg / mL (e.g., 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 48 mg / mL, 50 mg / mL, 52 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 58 mg / mL, 60 mg / mL).

[0010] The particle size D50 of the injectable microphase separation hydrogel is 30–300 μm (e.g., 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm).

[0011] The particle size D90 of the injectable microphase separation hydrogel is 50–500 μm (e.g., 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm, 240 μm, 260 μm, 280 μm, 300 μm, 320 μm, 340 μm, 360 μm, 380 μm, 400 μm, 420 μm, 440 μm, 460 μm, 480 μm, 500 μm).

[0012] The collagen in the injectable microphase separation hydrogel can be recombinant collagen or natural collagen.

[0013] The molecular weight of the collagen is 3,000 to 500,000 Daltons.

[0014] The natural collagen is selected from one or more of the following: porcine collagen, bovine collagen, fish collagen, human collagen, hydrolysate of porcine collagen, hydrolysate of bovine collagen, and hydrolysate of fish collagen.

[0015] Furthermore, the recombinant collagen is recombinant human collagen.

[0016] Furthermore, the collagen is selected from one or more of type I, type II, type III, type V, type XI, type XII, and type VXII recombinant human collagen.

[0017] In the preparation process of the injectable microphase-separated hydrogel, collagen molecules are first fully dissolved in an aqueous solution. Then, mechanical stirring is used to reduce the binding force between collagen and water, allowing the collagen molecules to arrange themselves in an orderly manner. At the same time, air is introduced, and stirring is performed for a certain period of time to make the bubbles uniformly dispersed in the liquid solution. As the solution temperature decreases, the viscosity of the liquid phase increases, providing support for the stability of the bubbles. During this process, stirring is continued, and the system gradually reaches thermodynamic and kinetic stability, forming a stable foam solution. Finally, a cross-linking agent is added to form covalent bonds between collagen molecules and to perform interfacial cross-linking on the surface of the bubble liquid film, forming a dense, stable, and uniform chemical cross-linked network.

[0018] This invention achieves the preparation of injectable microphase-separated hydrogels through a four-stage process sequence and parameter control. The hydrogel network structure includes both a three-dimensional network formed by collagen molecular chains and microsphere structures formed by the cross-linking of collagen molecules on the surface of a bubble-like liquid film. The three-dimensional network provides the gel with water absorption and retention capabilities, achieving the gel's viscosity, injectability, and support; the microsphere structures do not easily absorb water, providing the gel with strong resistance to enzymatic hydrolysis and also enhancing the hydrogel's support. The two structures are intertwined and uniformly distributed, resulting in a gel with strong support, resistance to damp heat, resistance to enzymatic hydrolysis, injectability, and uniform injection.

[0019] Optionally, the preparation method of the injectable microphase separation hydrogel further includes the steps of washing, drying, resolution, and sterilization.

[0020] Secondly, the present invention provides a method for preparing the injectable microphase separation hydrogel described in the first aspect, the specific steps of which are as follows:

[0021] 1) Prepare a collagen solution and adjust the pH value;

[0022] 2) Stir the collagen solution from step 1) at a constant rate at room temperature;

[0023] 3) Lower the temperature of the collagen solution in step 2) and continue stirring at a constant rate to obtain a milky white foam solution;

[0024] 4) Add a cross-linking agent to the solution in step 3), continue stirring at low temperature, and carry out a cross-linking reaction to obtain a gel;

[0025] 5) Sterilize the gel obtained in step 4) to obtain the injectable microphase separation hydrogel.

[0026] Preferably, in step 1), the concentration of the collagen solution is 20–200 mg / mL (e.g., 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL), more preferably 20–150 mg / mL.

[0027] The higher the concentration of collagen solution, the easier it is for the system to reach thermodynamic and kinetic steady state. However, the uniformity of the cross-linking network deteriorates during the chemical cross-linking process, and the injectability of the final product decreases. Therefore, the appropriate collagen solution concentration is 20–200 mg / mL.

[0028] Preferably, in step 1), the pH range is 8.0–12.0 or the pH range is 5.0–7.0. The pH range is compatible with the type of crosslinking agent in step 4).

[0029] Step 2) During the room temperature stirring process, mechanical force is used to reduce the binding force between collagen and water and to arrange collagen molecular chains in an orderly manner. At the same time, air is introduced and stirred to make the bubbles evenly dispersed in the liquid solution, providing a basis for the subsequent hydrogel microsphere structure.

[0030] Preferably, in step 2), room temperature stirring refers to mechanical stirring at a temperature of 20–30°C.

[0031] The mechanical stirring is performed at a medium speed, with a stirring rate of 100–500 rpm (e.g., 100 rpm, 120 rpm, 140 rpm, 150 rpm, 160 rpm, 180 rpm, 200 rpm, 220 rpm, 240 rpm, 250 rpm, 260 rpm, 280 rpm, 300 rpm, 320 rpm, 340 rpm, 350 rpm, 360 rpm, 380 rpm, 400 rpm, 420 rpm, 440 rpm, 450 rpm, 460 rpm, 480 rpm, 500 rpm), preferably 100–300 rpm. The stirring rate should not be too high. If the stirring rate is too high (exceeding 500 rpm), the formed bubbles will be relatively large, resulting in larger microsphere structures and a higher risk of needle clogging, which would not meet the injection requirements for clinical use.

[0032] The stirring time is 2 to 30 minutes (e.g., 2 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 25 minutes, 26 minutes, 28 minutes, 30 minutes), preferably 5 to 15 minutes, so that the formed fine bubbles are evenly dispersed in the collagen solution.

[0033] Without a room-temperature stirring step, adequate air cannot be introduced into the collagen solution, preventing the formation of microsphere structures in the hydrogel. If the room-temperature stirring time is too short (less than 2 minutes), the amount of air introduced into the collagen solution is insufficient, and the dispersion uniformity decreases, resulting in a small proportion and uneven distribution of microsphere structures after cross-linking, thus reducing the hydrogel product's resistance to enzymatic hydrolysis. As the room-temperature stirring time increases, the amount of air introduced reaches saturation, and the number of microsphere structures in the hydrogel will not continue to increase, with little change in product performance. Therefore, the suitable stirring time is 2–30 minutes.

[0034] Preferably, in step 3), the temperature is 2–10°C (e.g., 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C). Under low-temperature conditions, the hydrophobic interactions on the temperature-sensitive collagen molecular chains are enhanced, microphase separation occurs in the system, and the viscosity of the collagen solution increases, the strength of the bubble film gradually improves, and a stable foam solution is formed.

[0035] At this point, the viscosity of the collagen solution increases, the intermolecular distance decreases, and the interaction between collagen molecules is enhanced, causing the collagen molecules to further aggregate, resulting in a macroscopic milky white appearance. Especially for recombinant collagen, collagen hydrolysates, gelatin, or peptide raw materials with shorter molecular fragments, milky white collagen hydrogel products are more conducive to reducing the risk of the Tyndall effect after injection, providing a better solution for the clinical application of recombinant collagen raw materials.

[0036] Meanwhile, the invention requires mechanical stirring during the low-temperature process to effectively retain the introduced bubbles as the solution viscosity increases. The kinetics and thermodynamics of the bubble solution reach equilibrium, resulting in a higher degree of bubble solidification. A cross-linking reaction of collagen occurs on the surface of the bubble liquid film, and the resulting injectable microphase separation hydrogel has a more uniform distribution of three-dimensional network and microspheres.

[0037] Step 3) Stirring is performed at a medium speed, with a stirring rate of 100–500 rpm, preferably 100–300 rpm. More preferably, the stirring rate is the same as that in step 2).

[0038] Step 3) The stirring time is 10 to 90 minutes (e.g., 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes), preferably 20 to 70 minutes.

[0039] Step 3) The low-temperature stirring time should not be too long. The longer the low-temperature stirring time (e.g., more than 100 minutes), the more air bubbles are introduced into the system. In high-viscosity solutions, the air bubbles are unevenly distributed and easily aggregate, affecting the uniformity of the subsequent cross-linking network and reducing the injectability of the final hydrogel. Moreover, excessive foam in the solution tends to become dry foam, which also reduces the efficiency of subsequent cross-linking reactions. At the same time, the low-temperature stirring time should not be too short to avoid affecting the air bubble fixation process, thereby limiting the formation of microspheres in the hydrogel material and affecting the hydrogel's resistance to enzymatic hydrolysis.

[0040] Preferably, in step 4), the reactive groups on the collagen molecular chain include one or more of carboxyl, amino, and hydroxyl groups, and the cross-linking agent is one or more of glutaraldehyde, polyamine, and BDDE.

[0041] The polyamine is selected from carbodiimide and endogenous polyamine.

[0042] The endogenous polyamines include spermidine, spermine, and their derivatives.

[0043] Furthermore, in step 4), when the crosslinking agent is glutaraldehyde and / or BDDE, step 1) adjusts the pH range of the collagen solution to 8.0–12.0.

[0044] Furthermore, in step 4), when the cross-linking agent is a polyamine, the pH range of the collagen solution is adjusted to 5.0–7.0 in step 1).

[0045] When the crosslinking agent is an endogenous polyamine, a catalyst also needs to be added.

[0046] The catalyst comprises one or more of the following: carbodiimide, triphenylphosphine, bromide, carbomonium salt, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0047] When the crosslinking agent or catalyst is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), an auxiliary agent needs to be added; the auxiliary agent includes any one or more of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt), with NHS being preferred.

[0048] Furthermore, in step 4), the stirring time is 40–60 min, and the temperature is the same as in step 3).

[0049] Furthermore, in step 4), the stirring rate is the same as that in step 3).

[0050] After adding the crosslinking agent and catalyst, it is still necessary to continue stirring for a period of time under the low temperature conditions in step 3). The low temperature stirring time is 40-60 min (e.g., 40 min, 42 min, 45 min, 47 min, 50 min, 52 min, 55 min, 57 min, 60 min). This not only ensures that the crosslinking agent and catalyst are evenly distributed, and that a crosslinking network is formed on the surface of the bubble liquid film as the subsequent chemical crosslinking reaction begins, but also facilitates the stabilization and solidification of the bubble morphology and quantity, maintains the state of collagen microphase separation, further reduces the probability of reversible collagen microphase separation reaction, and prevents the stable bubbles formed in the system from being destroyed by changes in the subsequent crosslinking reaction conditions, thereby facilitating the formation of a uniform and stable microsphere structure within the hydrogel.

[0051] The amount of crosslinking agent used is 3% to 20% of the collagen protein mass (e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%).

[0052] The catalyst is used in an amount of 10% to 80% of the collagen protein mass (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%). The adjuvant is used in an amount of 1% to 50% of the catalyst mass (e.g., 1%, 3%, 5%, 8%, 10%, 13%, 15%, 18%, 20%, 23%, 25%, 28%, 30%, 33%, 35%, 38%, 40%, 43%, 45%, 48%, 50%).

[0053] Appropriate amounts of cross-linking agents and catalysts can cause collagen to undergo cross-linking reactions to form a three-dimensional network structure. This can affect the density of the cross-linked network formed in the microphase separation hydrogel, the uniformity of the distribution of microcapsule structures, and the proportion of microcapsule structures, thereby affecting the injectability and enzyme resistance of the microphase separation hydrogel.

[0054] Excessive crosslinking agents and / or catalysts can lead to the formation of a relatively dense three-dimensional collagen network structure in the microphase-separated hydrogel. While this results in high mechanical strength, it can negatively impact the injectability of the hydrogel and increase the difficulty of subsequent cleaning steps, posing a risk to the safety of the final product. Conversely, insufficient crosslinking and / or catalyst dosages make it difficult to form a strong, supportive three-dimensional collagen network structure, thus reducing the mechanical strength of the product.

[0055] Preferably, the crosslinking reaction time in step 4) is 16-72 h; the reaction temperature is 5-30℃ (e.g., 5℃, 8℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 30℃).

[0056] Furthermore, before sterilization in step 5), the gel undergoes washing, drying, and reconstitution steps.

[0057] The cleaning time is at least 72 hours, and the cleaning agent is one or more of purified water, physiological saline, and buffer salt solution.

[0058] The drying process can be freeze-drying.

[0059] Preferably, the solvent used for reconstitution in step 5) is one or more of phosphate buffer, physiological saline, and purified water.

[0060] Preferably, in the injectable microphase-separated hydrogel described in step 5), the collagen concentration is 20 mg / mL to 60 mg / mL (e.g., 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 48 mg / mL, 50 mg / mL, 52 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 58 mg / mL, 60 mg / mL).

[0061] Furthermore, the injectable microphase separation hydrogel described in step 5) may also include excipients such as lidocaine hydrochloride and glycerin, wherein lidocaine hydrochloride is used to increase injection comfort, and glycerin is a protein protectant and stabilizer. These excipients can be added during the reconstitution process.

[0062] Furthermore, in step 5), sterilization is performed using moist heat sterilization, epoxy sterilization, or irradiation sterilization.

[0063] The conditions for moist heat sterilization are 105–121°C for 30–90 minutes.

[0064] Thirdly, the present invention provides the application of the injectable phase-separated hydrogel described in the first aspect or the injectable microphase-separated hydrogel obtained according to the preparation method described in the second aspect in the preparation of tissue filling and repair materials or drug carriers for non-therapeutic purposes.

[0065] Specifically, the tissue filler can be soft tissue filler, bone tissue filler, etc. The tissue filler can be used to eliminate wrinkles (such as periorbital wrinkles, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear troughs, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), anti-aging, scar elimination, wound repair, intraoperative and postoperative venous hemostasis, etc.

[0066] The tissue repair material can be bone tissue repair material, cartilage tissue repair material, corneal tissue repair material, cardiovascular tissue repair material, liver tissue repair material, etc.

[0067] The drug uses an injectable phase-separable hydrogel as a carrier to achieve sustained release, controlled release, and targeted drug delivery.

[0068] The beneficial effects of this invention are:

[0069] 1. This invention provides an effective microphase separation-interfacial crosslinking technique (ICT-MS) for preparing collagen materials, targeting their unique characteristics. First, room temperature stirring achieves the ordered arrangement of collagen molecules, and the introduced bubbles are uniformly dispersed between the collagen molecular chains. Then, by lowering the temperature, the thermosensitive properties of the collagen material are triggered, enhancing the hydrophobic interactions on the collagen molecular chains and leading to microphase separation. Simultaneously, the increased solution viscosity provides support for the stability of the bubble film in the liquid phase, gradually bringing the collagen solution to thermodynamic and kinetic steady state, forming a stable foam solution with a milky white appearance. Then, a crosslinking agent is added, and the collagen undergoes chemical crosslinking, forming an interwoven three-dimensional network and microsphere structure. These two structures are intertwined and uniformly distributed. The three-dimensional network endows the hydrogel with good water absorption and retention capabilities, while the microsphere structure is not easy to absorb water and can resist the penetration of collagenase solution, providing the hydrogel with strong resistance to enzymatic hydrolysis. It also enhances the support of the hydrogel. The two structures complement each other and together endow the microphase separation hydrogel with strong support, resistance to damp heat, resistance to enzymatic hydrolysis, injectability, and injection uniformity.

[0070] 2. This invention employs a sequence of dissolution, room temperature stirring, low temperature stirring, and finally chemical cross-linking. By controlling the collagen concentration during dissolution and the stirring rate and time during stirring, a fine and uniform bubble solution is first formed. As the temperature decreases, the viscosity of the collagen solution increases, and the bubble morphology is fixed. By controlling the low temperature stirring time, not only can the bubbles be retained to the maximum extent, but the aggregation and accumulation of bubbles can also be effectively prevented, thereby forming a uniform and stable foam solution. This provides favorable preconditions for the subsequent chemical cross-linking process, ultimately forming a three-dimensional network and a uniformly distributed microsphere structure of collagen chemical cross-linking network.

[0071] 3. This invention controls the density of the crosslinking network formed by the hydrogel and the distribution of the microsphere structure by adjusting the amount of crosslinking agent and / or catalyst, thereby regulating the properties of the prepared hydrogel to adapt to different application scenarios.

[0072] 4. The injectable microphase separation hydrogel prepared by this invention is white in appearance and has a watery texture. After injection, it can effectively avoid the Tyndall effect and is particularly suitable for recombinant collagen or polypeptide raw materials. The milky white hydrogel product has a wider application prospect and use value. Attached Figure Description

[0073] Figure 1 is a SEM image of the injectable microphase separation hydrogel prepared in Example 3.

[0074] Figure 2 is a SEM image of the injectable microphase separation hydrogel prepared in Example 7.

[0075] Figure 3 is a SEM image of the cross-linked collagen hydrogel prepared in Comparative Example 2.

[0076] Figure 4 shows a SEM image of the cross-linked collagen hydrogel prepared in Comparative Example 3.

[0077] Figure 5 shows a SEM image of the cross-linked collagen hydrogel prepared in Comparative Example 4.

[0078] Figure 6 is a schematic diagram of the crosslinking process of the injectable microphase separation hydrogel of the present invention, wherein a is the collagen dissolution stage, which is a clear and transparent solution; b is the room temperature stirring stage, which forms a bubble solution; c is the low temperature stirring (initial) stage, in which the bubbles gradually solidify; and d is the low temperature stirring (end point) stage, in which a white foam solution is formed.

[0079] Figure 7 is a schematic diagram of the white foam solution produced by low-temperature stirring (endpoint) in Figure 6.

[0080] Figure 8 shows a comparison of the final product appearances of Example 7 (left), Comparative Example 5 (middle), and Comparative Example 2 (right). Detailed Implementation

[0081] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0082] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are all conventional experimental methods and reagents in the art, or commercially available.

[0083] Example 1: Injectable microphase separation hydrogel

[0084] 1) Prepare a 20 mg / mL collagen (10,000–40,000 Daltons) solution with purified water, adjust the pH to 11.5 ± 0.1, and wait for the solid to completely dissolve;

[0085] 2) Stir at a medium speed of 300 rpm for 5 minutes at room temperature (25℃);

[0086] 3) Place the collagen solution in a 5°C environment and stir continuously at 300 rpm for 70 min to obtain a milky white foamy solution;

[0087] As shown in Figure 6, in step 1), collagen dissolves to form a clear and transparent solution (Figure 6a); in step 2), the collagen solution is stirred at room temperature to form a bubble solution (Figure 6b); in step 3), as the stirring proceeds at low temperature, the bubbles gradually solidify, eventually forming a milky white foam solution (Figure 6c); as shown in Figure 7, the milky white foam solution of collagen formed at the end of the low-temperature stirring has fine foam and good uniformity.

[0088] 4) Then add 20% of the amount of collagen protein used in BDDE, stir continuously at 300 rpm and 5℃ for 60 min, and then place at 20℃ for cross-linking for 72 h;

[0089] 5) After cross-linking, the gel was cut into small pieces, washed with purified water for 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain an injectable microphase separation hydrogel.

[0090] Based on the preparation process of Example 1, samples of Examples 2 to 14 were prepared according to the specific process parameters in Table 1 (collagen concentration, pH value, room temperature stirring time, low temperature stirring conditions, crosslinking agent dosage, catalyst dosage, continued stirring time, and crosslinking reaction conditions). Except for the changes in the parameters in Table 1, all other parameters were the same as those in Example 1.

[0091] Table 1. Summary of parameters for each example

[0092] Note: The amounts of cross-linking agent and catalyst are both percentages of the amount of collagen protein used.

[0093] Comparative Example 1

[0094] Prepare a 150 mg / mL collagen solution with purified water, adjust the pH to 11.5 ± 0.1, and after the solid is completely dissolved, place the collagen solution directly in a 5°C environment and stir at a medium speed of 300 rpm for 20 min. Then add 20% BDDE of the amount of collagen protein used, continue stirring for 60 min, and then place it at 20°C for cross-linking for 72 h.

[0095] The cross-linked gel was cut into small pieces, washed with purified water for 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain collagen hydrogel.

[0096] Comparative Example 2

[0097] Prepare a 150 mg / mL collagen solution with purified water, adjust the pH to 11.5 ± 0.1, and stir at a medium speed of 300 rpm at room temperature (25℃) for 15 min after the solid is completely dissolved. Then add 20% BDDE of the amount of collagen protein used, stir for 60 min, and then place at 20℃ for cross-linking for 72 h.

[0098] The cross-linked gel was cut into small pieces, washed with purified water for 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain collagen hydrogel.

[0099] Comparative Example 3

[0100] Prepare a 150 mg / mL collagen solution with purified water, adjust the pH to 11.5 ± 0.1, and stir at 300 rpm at room temperature (25℃) for 15 min after the solid is completely dissolved. Then place the collagen solution in a 5℃ environment and stir at 300 rpm for 5 min. Add 20% BDDE of the amount of collagen protein used, and continue stirring at 300 rpm for 60 min at 5℃. Then place it at 20℃ for cross-linking for 72 h.

[0101] The cross-linked gel was cut into small pieces, washed with purified water for at least 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain collagen hydrogel.

[0102] Comparative Example 4

[0103] Prepare a 150 mg / mL collagen solution with purified water, adjust the pH to 11.5 ± 0.1, and stir at 300 rpm at room temperature (25℃) for 15 min after the solid is completely dissolved. Then place the collagen solution in a 5℃ environment and stir at 300 rpm for 100 min. Add 20% BDDE of the amount of collagen protein used, and continue stirring at 5℃ and 300 rpm for 60 min. Then place it at 20℃ for cross-linking for 72 h.

[0104] The cross-linked gel was cut into small pieces, washed with purified water for at least 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain collagen hydrogel.

[0105] Comparative Example 5

[0106] Prepare a 150 mg / mL collagen solution with purified water, adjust the pH to 11.5 ± 0.1, and stir at 300 rpm at room temperature (25℃) for 15 min after the solid is completely dissolved. Then place the collagen solution in a 5℃ environment and let it stand for 20 min. Add 20% BDDE of the amount of collagen protein used, stir at 300 rpm at 5℃ for 60 min, and then place it at 20℃ for cross-linking for 72 h.

[0107] The cross-linked gel was cut into small pieces, washed with purified water for at least 72 hours, freeze-dried, and reconstituted in physiological saline containing lidocaine hydrochloride and glycerol to make the collagen concentration 35 mg / mL. Finally, it was sterilized at 121°C for 30 minutes to obtain collagen hydrogel.

[0108] Performance Testing 1: Push Force Test (Injectability and Push Force Data)

[0109] The extrusion force of the examples and comparative examples was tested using an electronic universal tensile testing machine, and the state of the extruded gel was observed.

[0110] Performance testing 2. Elastic modulus test

[0111] The elastic modulus of the hydrogel samples of the examples and comparative examples was detected using a rheometer. The rheometer parameters were: operating gap: 45000 μm, loading gap: 1000 μm, operating temperature: 37 °C, deformation: 0.1%, frequency: 1 Hz, and running time: 60 s.

[0112] Performance testing 3 Degradation performance

[0113] A 60 U / mL collagenase solution was prepared. 1 g of each of the hydrogel samples from the examples and comparative examples were added to 2 mL of the collagenase solution and degraded at 37°C and 150 rpm. Observations were made every 30 minutes to compare the enzymatic resistance of each sample. Complete degradation was defined as the sample being completely degraded by the collagenase solution, with no obvious lumps of insoluble matter remaining in the bottle. The results of performance tests 1-3 are summarized in Table 2.

[0114] Table 2 Summary of Performance Data

[0115] Data from Examples 1-3 show that with prolonged stirring time at room temperature, the dispersion uniformity of bubbles in the liquid solution is better, the distribution of microspheres and cross-linked networks in the microphase-separated hydrogel is more uniform, exhibiting better injectability and lower extrusion force. Data from Examples 3-5 show that the longer the low-temperature stirring time, the more stable bubbles are in the foam solution, and the more microsphere structures are formed after cross-linking, exhibiting better resistance to enzymatic hydrolysis and mechanical properties. Data from Examples 5-7 show that in the dissolution step, the higher the collagen concentration, the higher the cross-linking efficiency, and the denser the formed cross-linked network, exhibiting higher modulus and resistance to enzymatic hydrolysis. Products with higher modulus are more suitable for deep injection filling. Data from Examples 7-9 show that reducing the amount of cross-linking agent reduces the support and resistance to enzymatic hydrolysis of the final product; conversely, the higher the amount of cross-linking agent, the higher the modulus of the gel and the higher the resistance to enzymatic hydrolysis. Furthermore, as can be seen from Examples 5 and 9, the utilization rate of the crosslinking agent is higher at high collagen concentrations. Even with a 3% crosslinking agent dosage, the final gel product in Example 9 (150 mg / mL) exhibits stronger support and resistance to enzymatic hydrolysis than that of Example 5, which has a lower collagen concentration (20 mg / mL) and a higher crosslinking agent dosage (20%). However, the injectability of Example 9 is weaker than that of the gel with lower collagen concentrations. Therefore, the sample prepared with lower collagen concentrations is more hydrated and softer, making it more suitable for shallow injection. Data from Examples 10–14 show that using different types of crosslinking agents and / or catalysts to react with the carboxyl, amino, or hydroxyl groups on collagen can all produce injectable microphase separation hydrogels with strong support and high resistance to enzymatic hydrolysis, indicating that the process described in this invention has high applicability.

[0116] Compared to Example 7, in Comparative Example 1, the room temperature stirring step was omitted, and low-temperature stirring was performed directly. Collagen molecules rapidly aggregated, increasing viscosity and making it difficult to introduce air bubbles. The number of air bubbles was small, and it was difficult to evenly disperse them in the collagen solution. Therefore, fewer microsphere structures were formed after cross-linking, reducing the product's resistance to enzymatic hydrolysis. The uneven distribution of the three-dimensional network and microsphere structures formed by collagen molecules reduced injection uniformity. The gel extruded from the injection needle was discontinuous, exhibiting particle aggregation and high extrusion force. In Comparative Example 2, only room temperature stirring was performed, without a low-temperature stirring step. The viscosity of the collagen solution did not increase, and the air bubbles lacked stable support. Therefore, the air bubbles broke rapidly as they formed. The cross-linking process only formed a three-dimensional network between collagen molecules, failing to form microsphere structures. Therefore, the final product had the worst resistance to enzymatic hydrolysis and appeared as a transparent gel (as shown in Figure 8, right). In Comparative Example 3, the low-temperature stirring time was too short (5 min). Insufficient low-temperature stirring time affected the number of stable air bubbles in the foam solution, thus affecting the final product's resistance to enzymatic hydrolysis. In Comparative Example 4, the excessively long low-temperature stirring time (100 min) caused the bubble films in the foam solution to come into contact with each other, adhere to each other, and agglomerate, resulting in large particles in the cross-linked gel and a gel pushing force as high as 29.05 N. Although the enzymatic hydrolysis was improved, it seriously affected its use. In the preparation process of Comparative Example 5, the collagen solution was not stirred when it was allowed to stand at low temperature, which disrupted the kinetic and thermodynamic equilibrium of the bubble solution. As a result, some of the bubbles introduced at room temperature broke down during the temperature drop. As the solution viscosity increased, only a small number of bubbles were retained. Therefore, the number of microsphere structures in the cross-linked network of the hydrogel sample was less (less than that in Comparative Example 3). Although stirring was started after the catalyst was added, the cross-linking reaction had already begun, and the system viscosity increased, making it difficult to introduce more bubbles. Therefore, it was impossible to increase the number of microsphere structures. This led to a significant reduction in enzymatic hydrolysis resistance and a decrease in the "whiteness" of the final hydrogel product, resulting in a semi-transparent state (as shown in Figure 8). This is not conducive to solving the "Tyndall effect" that occurs after injection.

[0117] Performance testing 4. Microscopic morphology testing

[0118] After lyophilization, the samples were examined using scanning electron microscopy (SEM). As seen in the SEM images of Examples 3 and 7 (Figures 1 and 2), the injectable microphase hydrogel exhibits an interwoven distribution of microsphere structures and a three-dimensional network, thus demonstrating good injectability, strength, and resistance to enzymatic degradation. Even after high-temperature sterilization, both structures remained stable, indicating the stability of the formed microsphere and three-dimensional network structures, and the hydrogel product maintained good performance even after high-temperature treatment.

[0119] Observation of the electron microscope (SEM) micrographs (Figures 2-5) of Example 7 and Comparative Examples 2-4 shows that Example 7 has significantly more microsphere structures, thus exhibiting better resistance to enzymatic hydrolysis (Figure 2). The SEM image (Figure 3) of Comparative Example 2 shows an ordered cross-linked network structure, but no microsphere structures are observed. This is because the low-temperature stirring step was not performed, preventing the formation of microsphere structures, resulting in the worst resistance to enzymatic hydrolysis. The SEM image (Figure 4) of Comparative Example 3 shows that the short low-temperature stirring time significantly reduces the microsphere structure, affecting the hydrogel's resistance to enzymatic hydrolysis. The SEM image (Figure 5) of Comparative Example 4 shows numerous obvious microsphere agglomerations, caused by excessively long low-temperature stirring time leading to bubble aggregation and accumulation. Consequently, needle blockage is significant during hydrogel extrusion, which is detrimental to clinical use.

[0120] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An injectable microphase separation hydrogel, characterized in that: The injectable microphase separation hydrogel includes a three-dimensional network structure formed by collagen molecules and a microsphere structure formed by cross-linking of collagen molecules on the surface of a bubble liquid membrane. The microsphere structure and the three-dimensional network structure are interspersed. The concentration of collagen in the injectable microphase separation hydrogel is 20 mg / mL to 60 mg / mL. The molecular weight of the collagen is 3,000 to 500,000 Daltons.

2. The injectable microphase separation hydrogel according to claim 1, characterized in that: The preparation steps of the injectable microphase separation hydrogel include preparing a collagen solution, stirring at room temperature, stirring at low temperature, and interfacial chemical crosslinking. Preferably, the room temperature is 20–30°C, and the low temperature is 2–10°C.

3. The injectable microphase separation hydrogel according to claim 1, characterized in that: The particle size D50 of the injectable microphase separation hydrogel is 30–300 μm; the particle size D90 of the injectable microphase separation hydrogel is 50–500 μm. Preferably, the collagen is recombinant collagen or natural collagen; More preferably, the collagen is recombinant human collagen.

4. A method for preparing an injectable microphase separation hydrogel, characterized in that, The preparation method includes the following steps: 1) Prepare a collagen solution and adjust the pH value; 2) Stir the collagen solution from step 1) at a constant rate at room temperature; 3) Lower the temperature of the collagen solution in step 2) and continue stirring at a constant rate to obtain a milky white foam solution; 4) Add a cross-linking agent to the solution obtained in step 3), continue stirring at low temperature, and carry out a cross-linking reaction to obtain a gel; 5) Sterilize the gel obtained in step 4) to obtain the injectable microphase separation hydrogel.

5. The preparation method according to claim 4, characterized in that: The concentration of the collagen solution obtained in step 1) is 20–200 mg / mL, preferably 20–150 mg / mL; the pH value range is 8.0–12.0 or 5.0–7.

0.

6. The preparation method according to claim 4, characterized in that: In step 2), the room temperature stirring temperature is 20-30℃, the stirring speed is 100-500 rpm, and the stirring time is 2-30 min, preferably 5-15 min.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The temperature in step 3) is 2 to 10°C; the stirring rate is 100 to 500 rpm; the stirring time is 10 to 90 min, preferably 20 to 70 min; preferably, the stirring rate in step 3) is the same as the stirring rate in step 2).

8. The preparation method according to claim 4 or 5, characterized in that: The crosslinking agent is selected from one or more of glutaraldehyde, polyamines, and BDDE; Preferably, when the crosslinking agent is glutaraldehyde and / or BDDE, step 1) adjusts the pH range of the collagen solution to 8.0–12.0; when the crosslinking agent is a polyamine, step 1) adjusts the pH range of the collagen solution to 5.0–7.

0. Preferably, the stirring time in step 4) is 40 to 60 minutes.

9. The preparation method according to claim 4, characterized in that: Step 4) The polyamine is selected from carbodiimide or endogenous polyamine; Preferably, the endogenous polyamine includes spermidine, spermine, and their derivatives; The amount of the cross-linking agent is 3% to 20% of the collagen protein content; The cross-linking reaction temperature is 5–30°C, and the cross-linking reaction time is 16–72 h.

10. The use of the injectable microphase-separated hydrogel according to any one of claims 1 to 3 or the injectable microphase-separated hydrogel prepared by any one of claims 4 to 9 in the preparation of tissue filling and repair materials or drug carriers.

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