Biphase gel material formed based on hydrogen bond self-assembly as well as preparation method and application of biphase gel material

Abstract

The invention discloses a biphase gel material formed based on hydrogen bond self-assembly and a preparation method and application thereof.The biphase gel material is formed by a first polymer and a second polymer based on hydrogen bond self-assembly, the first polymer comprises a branched polymer, the second polymer comprises a long-chain linear multi-hydrogen-bond site polymer, and the long-chain linear multi-hydrogen-bond site polymer comprises a long-chain linear multi-hydrogen-bond site polymer. The branched polymer comprises at least one of the following substances: 1) a polyether polymer with hydroxyl at the tail end; and 2) a polymer obtained by carrying out a derivatization reaction on the hydroxyl of the polyether polymer in the step 1). A biological inert film can be formed in situ on the surface of the water-loss gel, so that invasion of exogenous bacteria is effectively blocked. A skeleton-vacuole structure is formed inside due to microscopic phase separation, so that a large number of bacteria or cells and the like can be loaded. The unique internal and external structures of the two-phase gel material can load wide and complex contents and provide a steady state for the contents, and meanwhile, the two-phase gel material can be widely applied to skin diseases, implant materials and the like due to excellent mechanical properties of the two-phase gel material.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a biphase gel material formed by hydrogen bond self-assembly, its preparation method, and its application. Background Technology

[0002] In the field of biomedical materials, gel materials are widely used in tissue engineering, drug delivery, wound dressings, and cell culture due to their unique three-dimensional network structure and excellent biocompatibility. However, the insufficient functionality of traditional gel materials in complex biological environments still restricts their clinical translation efficiency, specifically manifested in the following two core issues: (I) The contradictory needs of bioprotection and infection control: Existing gel materials (such as hydrogels and organic gels) mostly rely on surface antibacterial agent coatings or drug sustained release to achieve infection control, but they have the following defects: 1) Excessive local concentration of antibacterial agents can easily cause cytotoxicity and disrupt the normal tissue repair process; 2) Drug sustained release systems are difficult to dynamically respond to the intensity of microbial invasion, which can easily lead to drug resistance; 3) Although the open porous structure is conducive to material exchange, it provides an invasion channel for exogenous pathogens (such as Staphylococcus aureus and Pseudomonas aeruginosa), especially in chronic wounds or immunodeficiency scenarios where the risk of infection is significantly increased. Although some studies have improved protective performance by constructing dense surface layers (such as electrospun membranes) or introducing photodynamic antibacterial mechanisms, these materials often sacrifice the flexibility or bioactivity of the gel, making it difficult to meet the long-term use requirements under dynamic physiological environments. (II) The dilemma of synergistic relationship between mechanical properties and biofunctionalization: The mechanical strength of traditional gels mainly depends on the regulation of cross-linking density. However, although high cross-linking can improve compressive / shear resistance, it will lead to the following problems: 1) Reduced porosity, hindering the penetration of nutrients and the discharge of metabolic waste, limiting cell migration and tissue regeneration; 2) Rigid network structures lack shear-thinning properties, making it difficult to deliver to the target site through minimally invasive methods such as injection; 3) A single phase cannot simulate the heterogeneous structure of natural tissues, and cannot provide hierarchical microenvironment support for loaded probiotics or stem cells. Although liquid-liquid phase separation technology has been used to construct porous framework structures, existing materials mostly achieve phase separation through post-processing (such as solvent replacement and freeze-drying), which has problems such as complex processes and poor structural controllability, and lacks synergistic design of surface protection and internal functions.

[0003] Therefore, it is of great significance to develop a gel material that combines dynamic biological protection with multi-level mechanical regulation capabilities. Summary of the Invention

[0004] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a biphasic gel material that combines good mechanical strength and shear-thinning ability, as well as good bioinertness and drug loading capacity.

[0005] The present invention also proposes a method for preparing the above-mentioned biphase gel material.

[0006] This invention also proposes applications of the aforementioned biphase gel materials.

[0007] According to one aspect of the present invention, a biphasic gel material is provided, the biphasic gel material being formed by the self-assembly of a first polymer and a second polymer based on hydrogen bonds, wherein the first polymer comprises a branched polymer and the second polymer comprises a long-chain linear polymer with multiple hydrogen bond sites, the branched polymer comprising at least one of the following substances: 1) a polyether polymer with hydroxyl groups at the end; 2) a polymer obtained by derivatization reaction of the hydroxyl groups of the polyether polymer in 1).

[0008] The biphasic gel material according to embodiments of the present invention has at least the following beneficial effects: The present invention cleverly utilizes the special topological characteristics of branched polymers and their derivatives. These polymers have the significant advantage of high spatial density of hydrogen bond binding sites. Based on this characteristic, they can be induced to form chain entanglement with long-chain linear polymers, thereby triggering self-assembly. During self-assembly, the polymer structure collapses and induces micro-phase separation, thereby forming a stable framework-vacuole biphasic structure inside the gel. This structure endows the gel with good mechanical strength and significant shear-thinning behavior, while also exhibiting excellent biocompatibility and drug loading capacity. The present invention utilizes the above-mentioned branched polymers and their derivatives to construct a biphasic gel system through hydrogen bond crosslinking with linear polymers. During gel formation, surface moisture evaporates to generate a dense bioinert film in situ, which can effectively block the invasion of exogenous bacteria; internally, due to liquid-liquid phase separation, a framework-vacuole structure is formed, which not only provides the mechanical properties and rheological properties required for the gel, but can also be used to load active ingredients such as probiotics or cells, making it suitable for biomedical applications.

[0009] Furthermore, this invention utilizes the characteristic of the biphasic gel to form a self-contained film on its surface during macroscopic phase separation, thereby achieving a physical barrier effect against pathogens. It still has a good bacterial isolation effect without relying on drugs, overcoming the problem of poor bacterial isolation performance of traditional gel and cream formulations.

[0010] In summary, this invention utilizes a hydrogen-bonded, nonlinear-linear chain entanglement mechanism to form a biphasic gel possessing high mechanical strength, shear-thinning properties, drug loading capacity, and antimicrobial properties. This system combines the loading capacity of hyperbranched polymers with the inert membrane structure formed by phase separation, providing a novel strategy for the development of biomedical materials.

[0011] The chain entanglement of the present invention is different from the salting-out effect of the Hofmeister effect, and the resulting spatial structure is more compact, providing the material with higher mechanical strength.

[0012] According to some embodiments of the present invention, the raw materials for preparing the biphasic gel material consist of a first polymer, a second polymer, and a solvent.

[0013] According to some embodiments of the present invention, the mass ratio of the first polymer to the second polymer is 1:0.5~2.0.

[0014] According to some embodiments of the present invention, the total mass percentage of the first polymer and the second polymer in the raw materials for preparing the biphasic gel material is 10-30%.

[0015] According to some embodiments of the present invention, the branched polymer includes at least one of a star polymer, a dendritic polymer, or a hyperbranched polymer.

[0016] According to some embodiments of the present invention, the first polymer includes at least one of hyperbranched polyglycidol and its derivatives, multi-arm polyethylene glycol (PEG) with terminal hydroxyl groups and its derivatives.

[0017] Hyperbranched polyglycerol (HPG) is a hyperbranched polymer synthesized from glycidyl monomers. Its structure is rich in hydroxyl groups and ether structures similar to polyethylene glycol (PEG), exhibiting good biocompatibility and stability. Due to its hyperbranched topology, HPG has a much smaller molecular size compared to linear polymers of the same molecular weight. This results in a higher density of hydroxyl and ether groups per unit space compared to PEG-like polymers, and a more densely packed array of hydrogen bonding sites. Furthermore, HPG can interact with some drug molecules through hydrogen bonding, enabling it to effectively load hydrophobic drugs.

[0018] According to some embodiments of the present invention, the derivative is at least one of a product in which the terminal hydroxyl group is derivatized by an aldehyde group or an ester group.

[0019] According to some embodiments of the present invention, the first polymer comprises aldehyde-modified hyperbranched polyglycidyl and hyperbranched polyglycidyl succinate.

[0020] According to some embodiments of the present invention, the long-chain linear multi-hydrogen-bonding polymer includes at least one of polyvinyl alcohol (PVA), silk fibroin, or keratin.

[0021] According to some embodiments of the present invention, the molecular weight of the first polymer is in the range of 4.5 to 12 kDa.

[0022] According to some embodiments of the present invention, the first polymer is a hyperbranched polymer, wherein the degree of branching of the hyperbranched anionic polymer is 0.5 to 0.6.

[0023] According to some embodiments of the present invention, the molecular weight of the second polymer is in the range of 10~20 kDa.

[0024] According to some embodiments of the present invention, the solvent includes at least one of water, phosphate-buffered saline (PBS), LB liquid culture medium, or DMEM culture medium. Water, PBS, etc., can be used as solvents, making it environmentally friendly.

[0025] According to another aspect of the present invention, a method for preparing the above-mentioned biphasic gel material is provided, comprising the following steps: The dispersions of the first polymer and the second polymer are mixed and reacted to obtain the final product.

[0026] According to some embodiments of the present invention, the preparation method further includes a step of mixing the dispersion of the first polymer with the dispersion of the second polymer and then subjecting the mixture to vortexing or oscillation. Vortexing or oscillation further enhances the uniformity of the mixture.

[0027] According to some embodiments of the present invention, the duration of the vortex or oscillation is 5 to 60 seconds.

[0028] According to some embodiments of the present invention, the preparation method further includes a step of vortexing followed by static treatment.

[0029] According to some embodiments of the present invention, the settling time is 30~300s.

[0030] According to some embodiments of the present invention, the mass concentration of the first polymer in the dispersion of the first polymer is 10-66%.

[0031] According to some embodiments of the present invention, the mass concentration of the second polymer in the dispersion of the second polymer is 5-25%.

[0032] According to some embodiments of the present invention, the mixing and / or reaction temperature is 5~30°C. Compared with traditional hydrogen-bonded gels, the gel material of the present invention can be rapidly obtained at room temperature in a one-step process.

[0033] According to some embodiments of the present invention, the mixing time is 1 to 5 minutes.

[0034] According to another aspect of the present invention, an application of the above-described biphasic gel material is provided, specifically, a gel composition loaded with bioactive ingredients is provided, wherein the raw materials for preparing the gel composition loaded with bioactive ingredients include the above-described biphasic gel material and bioactive substances, wherein the bioactive substances include drugs and / or microorganisms.

[0035] The biphasic gel of this invention combines mechanical strength and shear thinning ability. Due to the use of liquid-liquid phase separation method, it has a gel microstructure containing vacuoles. Since the gel skeleton contains amphiphilic structure, the gel can load hydrophobic drugs and live bacteria. The composition can be a drug delivery system or a microbial delivery system.

[0036] According to some embodiments of the present invention, the drug is a hydrophobic drug, including but not limited to at least one of curcumin, dexamethasone, or ofloxacin.

[0037] According to some embodiments of the present invention, the bioactive substance is loaded in the first polymer.

[0038] According to another aspect of the present invention, a method for preparing the above-mentioned gel composition loaded with bioactive ingredients is provided, comprising the following steps: The first polymer is prepared into first polymer-bioactive substance nanoparticles by combining the first polymer with the drug, and then mixed with a dispersion of the second polymer and reacted to obtain the final product.

[0039] According to some embodiments of the present invention, the temperature during the preparation of the drug delivery system is 20~25°C.

[0040] According to some embodiments of the present invention, the preparation process of the first polymer-bioactive substance nanoparticles includes the following steps: The first polymer and the bioactive substance are mixed and dispersed in an organic solvent to obtain dispersion I, which is then slowly added to water under high-speed stirring to obtain the final product.

[0041] According to some embodiments of the present invention, the preparation process of the first polymer-bioactive substance nanoparticles further includes a post-processing step, specifically including ultrafiltration.

[0042] According to some embodiments of the present invention, the molecular weight cutoff of the ultrafiltration treatment is 100 kDa.

[0043] According to some embodiments of the present invention, the post-processing further includes a step of ultrafiltration followed by centrifugation, wherein the centrifugation process includes at least one of the following conditions: 1) centrifugation speed of 4000~5000 rpm (preferably 4500 rpm); 2) time of 15~30 minutes; 3) centrifugation temperature of 15~25℃ (preferably 20℃).

[0044] According to some embodiments of the present invention, the particle size range of the first polymer-bioactive substance nanoparticles is 100~220 nm.

[0045] According to some embodiments of the present invention, the addition rate of the slow addition is 0.3~0.5 mL / min.

[0046] According to some embodiments of the present invention, the stirring speed of the high-speed stirring is 200~400 rpm.

[0047] According to some embodiments of the present invention, the dispersion I is slowly added to water by injection.

[0048] According to some embodiments of the present invention, the mass concentration of the first polymer in the dispersion I is 1 to 10%.

[0049] According to some embodiments of the present invention, the mass concentration of the bioactive substance in the dispersion I is 0.1-1%.

[0050] According to some embodiments of the present invention, the organic solvent includes dimethyl sulfoxide (DMSO).

[0051] According to some embodiments of the present invention, the volume ratio of organic solvent to water in the dispersion I is 1:20~50.

[0052] According to another aspect of the present invention, the above-described biphasic gel material is provided for use in the preparation of drugs for bacterial barrier or prevention of skin diseases.

[0053] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0054] Figure 1 This is a molecular weight distribution diagram of the hyperbranched polyglycidol prepared in the synthesis example of the present invention.

[0055] Figure 2 These are the reverse-gated carbon spectra of three batches of hyperbranched glycidol prepared in the synthesis examples of this invention.

[0056] Figure 3 This is a schematic diagram of the preparation process of the biphasic gel material and the control group scheme in Example 1 of the present invention.

[0057] Figure 4 This is a graph showing the quantitative results of the mass ratio and water content of the condensed layer for different concentration ratios of polyvinyl alcohol (PVA) and hyperbranched polyglycidyl glycerol (HPG) in Example 1 of the present invention.

[0058] Figure 5 This is a laser confocal image of the hydrogel formed by polyvinyl alcohol (PVA) and hyperbranched polyglycidyl (HPG) in Example 2 of the present invention.

[0059] Figure 6The left image shows the effect of different molar ratios of hyperbranched glycidyl ether (HPG) on the hydration kinetic radius of polyvinyl alcohol (PVA) in Example 2 of this invention, and the right image shows the effect of salting out after the addition of different concentrations of salt on the hydration kinetic radius of PVA.

[0060] Figure 7 This is a comparison of the tensile stress-strain curves of hydrogels formed by different concentrations of PVA gel and polyvinyl alcohol (PVA) and hyperbranched polyglycidyl (HPG) in Example 2 of the present invention.

[0061] Figure 8 The images show electron microscope (EM) images of the surface (left) and interior (right) of the hydrogel formed by polyvinyl alcohol (PVA) and hyperbranched polyglycidyl (HPG) in Example 2 of this invention.

[0062] Figure 9 The image shows the fluorescence colocalization of the cross-section of the hydrogel formed by polyvinyl alcohol (PVA) and hyperbranched polyglycidyl (HPG) in Example 2 of the invention. The left image is a bright field image, and the right image is an excitation effect image at 555 nm and 650 nm.

[0063] Figure 10 The diagram shows the effect of temperature on the gelation time of the hydrogel formed by polyvinyl alcohol (PVA) and hyperbranched polyglycidyl (HPG) in Example 2 of this invention (left) and the rheological study results (right).

[0064] Figure 11 This figure shows the results of the antibacterial study of the hydrogel formed by polyvinyl alcohol (PVA) and hyperbranched glycidyl glycerol (HPG) in Example 2 of the present invention.

[0065] Figure 12 This is a dynamic light scattering result diagram of nanoparticles formed by hyperbranched polyglycidyl (HPG) and curcumin in Example 2 of the present invention.

[0066] Figure 13 This is the drug release curve of the drug delivery system obtained in Example 3 of the present invention. Detailed Implementation

[0067] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0068] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0069] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0070] In the description of this invention, liquid-liquid phase separation (LLPS) refers to the formation of droplets with different compositions and properties by certain biological macromolecules (such as proteins and RNA) within cells through interactions. These droplets are similar to the separated state of oil droplets in water, forming a unique substructure in the aqueous phase.

[0071] The room temperature in the description of the embodiments of the present invention is 25°C.

[0072] Hydrogen-bonded crosslinking enables materials to self-assemble in aqueous environments, typically exhibiting excellent strength and shear-thinning properties. Furthermore, as the solvent decreases, the molecular chain arrangement within the system changes, resulting in a hierarchical structure. However, constructing a heterogeneous system that self-assembles entirely in water based on hydrogen bonds presents several challenges. Firstly, water molecules are polar and competitively bind to hydrogen bond sites, disrupting the stability of the crosslinking. Secondly, hydrogen bonding is a reversible force at room temperature, making it difficult for the crosslinked system to maintain high mechanical strength while retaining high water content, much like skin. Additionally, the energy of a single hydrogen bond is relatively low (approximately -40 kJ / mol), usually requiring an increase in the number of hydrogen bonds per unit space to improve the material's mechanical strength. Therefore, constructing efficient and stable hydrogen-bonded crosslinks in aqueous solutions has become a critical problem to be solved.

[0073] In view of this, this invention innovatively proposes a simple and feasible room-temperature aqueous hydrogen bond crosslinking method based on liquid-liquid phase separation. This method can form a high-strength gel material with shear-thinning ability in a single step at room temperature. Moreover, through the dehydration process, a bio-inert film can be formed in situ on the gel surface, while a framework-vacuole structure is formed internally. This internal and external biphase structure enables the gel to load a wide variety of complex contents, and it can be applied to the treatment of skin diseases, implant materials, and other fields, showing high practical value and clinical translation potential.

[0074] This invention relates to a hydrogen-bonded gel crosslinking method and mechanism based on nonlinear polymers of polyethers and linear long-chain polymers, forming an in-situ biphasic gel material through polymeric hydrogen bonding crosslinking. During the formation of this biphasic gel, an inert film is formed in situ after water loss on its surface, effectively isolating it from the invasion of exogenous pathogenic bacteria. Internally, a framework-vacuole structure appears due to microscopic liquid-liquid phase separation, providing the gel with strength, elasticity, and shear-thinning properties, and also allowing for the loading of probiotics or cells for use in the biomedical field.

[0075] Synthesis example HPG Synthesis and Purification Add 0.63 g (4.6 mmol) of 1,1,1-trihydroxypropane (TMP) to a flask, purge with argon gas, and add 410 μL (1.5 mmol) of CH3O3. Incubate for approximately 30 min. After complete activation, add 25 mL of glycidyl ether to the system using a syringe pump. This process should exceed 12 h. After the reaction is complete, dissolve the product in methanol, extract with acetone, remove the supernatant, and repeat 3-5 times. Rotary evaporate until the product becomes viscous, add an appropriate amount of water to dissolve the product, and dialyze using a 1000D dialysis tube for 12 hours, changing the water every 3 hours. Add acetone to the final dialysate at a ratio of dialysate:acetone of 1:10. Observe the product emulsification. After standing for 12 hours, a viscous, colorless, and transparent product will be obtained at the bottom of the container. Dry under vacuum at 85 °C for 4-6 hours to obtain the HPG sample. Repeat the above operation three times. The resulting sample was characterized by reverse-gated carbon NMR spectroscopy and gel permeation chromatography, yielding a weight-average molecular weight of Mw = 7469 and a PD of 1.80 (e.g., Figure 1 As shown), the average branching degree is 0.571 (as shown). Figure 2 (As shown). The mixture is then used in subsequent embodiments.

[0076] Example 1 This example provides a biphasic gel material formed by the self-assembly of a branched polymer and polyvinyl alcohol (PVA1799, Xinhua, catalog number 642807245172). The branched polymer synthesis example uses purified HPG or eight-armed PEG-OH (Shanghai Jinpan Biotechnology Co., Ltd., catalog number JPB-80086) synthesized in this example. The preparation process is as follows... Figure 3 As shown, the specific operation is as follows: Add 9 mL of deionized water and 1 g of polyvinyl alcohol 1799 to a 50 mL single-necked round-bottom flask. Gently stir and reflux at 90 °C for 1 hour, then allow to cool naturally to room temperature.

[0077] Take 500 mg of HPG (prepared by the synthetic example, molecular weight Mw=7469, PD=1.80, degree of branching=0.571), 500 mg of octagonal PEG-OH (Mw=5000), or 500 mg of glycerol into three 2 mL centrifuge tubes respectively, then add 500 mg of deionized water to each, vortex for 5 min, shake at 120 rpm until completely dissolved, and prepare 50% HPG, 50% octagonal PEG-OH and 50% glycerol aqueous solutions respectively.

[0078] 1 mL of 10% PVA aqueous solution was mixed with 0.2 mL of 50% HPG, 50% octagonal PEG-OH, and 50% glycerol aqueous solution (as a control group), respectively. The observed phenomena were as follows. Figure 3As shown, no gel phase was formed in the glycerol group, while the eight-arm PEG-OH group and HPG group could form gels with PVA. The gel formed by HPG and PVA was the most uniform.

[0079] Mixed solutions were prepared at PVA:HPG mass ratios of 2:1, 1:1, and 1:2, with total solute masses of 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg, respectively. The thickness of the coagulated layer was measured, the supernatant was removed, and the solutions were concentrated under vacuum for 4 hours. The solutions were then weighed and the water content was calculated (results are shown below). Figure 4 (As shown). When the PVA:HPG mass ratio is 1:1, the total solute concentration has the least impact on gel yield and is the most stable.

[0080] Example 2 This example provides a biphasic gel material formed by the self-assembly of HPG and PVA. The raw material source is the same as in Example 1. The preparation process is as follows: 0.5 mL of 20% PVA aqueous solution is diluted to 1 mL of 10% PVA aqueous solution and mixed with 200 μL of 50% HPG aqueous solution. After shaking for 15 s, it is allowed to stand for 5 min to obtain H / P gel.

[0081] To observe the entanglement and structural collapse that occurs between HPG and PVA, a fluorescence staining experiment was performed. The specific procedure is as follows: 1) PVA staining Add 10 mL of DMSO and 200 mg of polyvinyl alcohol (PVA) to a 50 mL single-necked round-bottom flask. Heat at 90 °C with gentle stirring and reflux for 1 hour, then allow to cool naturally to room temperature. Add 10 mg of rhodamine isothiocyanate B (RBITC) and react with stirring at room temperature for 24 hours. Add 10 mL of methanol to form a precipitate. Remove the supernatant and wash the precipitate with methanol to thoroughly remove free dye. Vacuum dry the sample, weigh it, and heat it in a 50 mL single-necked round-bottom flask to prepare a 1% aqueous solution.

[0082] 2) HPG staining HPG was dehydrated (dryed under vacuum at 85°C for 4 hours) and then protected by aeration. 400 mg of HPG was weighed and dissolved in 10 mL of N,N-dimethylformamide (DMF) by stirring. 5 mg of Cy5 dye (anthocyanin Cy5) was dissolved in 1 mL of DMF under argon protection. The two systems were mixed thoroughly, and then approximately 10 molecular sieve particles were added and shaken vigorously. Under nitrogen protection, 6.5 mg of 4-dimethylaminopyridine (DMAP) and 16.5 μL of DIC (N,N'-diisopropylcarbodiimide) were added. The reaction was carried out at 100 rpm at room temperature for 72 hours. After the reaction, the molecular sieve was removed by centrifugation, and 15 mL of acetone was added to precipitate the product. The supernatant was removed. The product was dissolved in methanol, and then acetone was added to precipitate the product. This step was repeated until the supernatant was completely colorless. The precipitate was dried under vacuum and then dissolved in a 1% aqueous solution in a 50 mL single-necked round-bottom flask.

[0083] 3) H / P gel staining Add 100 μL of 1% PVA-RBITC solution to 1 mL of 10% PVA solution. Add 20 μL of 1% HPG-Cy5 solution to 1 mL of 50% HPG solution. Then mix according to the preparation steps to obtain a gel for fluorescent staining of both components.

[0084] Laser confocal imaging was performed on this gel, with excitation at 555nm and 650nm. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, a framework-vacuole structure formed by micro-phase separation appears in the H / P gel. HPG and PVA molecules are uniformly distributed within the gel framework, and their concentrations in the vacuoles are relatively low. Single-molecule fluorescence correlation spectroscopy reveals that the addition of HPG reduces the hydration kinetic radius of PVA single molecules, indicating that the two molecules become entangled and their structure collapses.

[0085] To verify the effect of molar ratio on the hydration kinetic radius of PVA, PVA-RBITC was prepared as a 2.5 nmol / mL aqueous solution, and HPG aqueous solutions with different molar ratios (PVA:HPG molar ratios of 1:1, 1:10, 1:500, 1:1000, and 1:2000, with Control representing no HPG added) were added. The hydration kinetic radius of PVA was then measured as follows: Figure 6 The figure is shown in the middle left. It can be seen from the figure that when the molar ratio is 1:1, the hydration kinetic radius of PVA monomers does not change significantly. However, when the molar ratio is greater than 1:10, the hydration kinetic radius of PVA monomers decreases, proving that entanglement and structural collapse occur. Simultaneously, to verify that this chain entanglement differs from the salting-out behavior caused by Hofmeister precipitate, the effects of low (0.25 μM) and high (0.25 M) sulfate concentrations on the hydration kinetic radius of PVA were measured under the same conditions. The results are as follows... Figure 6 As shown in the right-middle figure, the salting-out behavior induced by Hofmeister is significantly weaker than that induced by HPG chain entanglement.

[0086] A 10% H / P gel was prepared by mixing 1 mL of 10% PVA with 1 mL of 10% HPG; a 20% H / P gel was prepared by mixing 1 mL of 20% PVA with 1 mL of 20% HPG; and a 30% H / P gel was prepared by mixing 1.5 mL of 20% PVA with 0.5 mL of 60% HPG. The prepared gels were filled into a mold, while 10% and 20% PVA aqueous solutions were used as control groups and filled into the mold. Both experimental and control groups were placed at -20℃ and allowed to solidify for 10 hours (8-12 hours is acceptable). After demolding, the gels were stretched using a universal testing instrument at a speed of 5 mm / min, and the stress-strain curves were recorded. The results are as follows: Figure 7 As shown in the figure, the gel of the present invention has good mechanical properties.

[0087] To characterize the macroscopic phase separation structure of HPG and PVA, a 10% H / P gel was placed under 40% humidity for 30 min. Subsequently, surface and cross-sectional samples of the gel were obtained by lyophilization and liquid nitrogen fracturing, and then imaged using a scanning electron microscope (SEM). The results are shown below. Figure 8 As shown. From Figure 8 As can be seen, a continuous film structure forms on the gel surface. Figure 8 (Middle left image) The gel has a porous internal structure. Figure 8 (Middle right figure). The aforementioned stained gel was frozen and sectioned to a thickness of 5 μm. The longitudinal section of the gel was observed under a laser confocal microscope, and the results are as follows. Figure 7 As shown in the figure, a significant porous-membrane gradient structure can be observed under bright field conditions. Figure 9 (Middle left image) and co-localization of components reveals phase separation at the gel surface, forming a continuous film. Figure 9 (Right image in the middle)

[0088] To characterize the rheology of H / P gels, the rheological properties of 10% HP gels were tested, and the results are as follows: Figure 10 As shown in the figure, the gel forms in approximately 40 minutes at room temperature, and can form rapidly under low-temperature conditions. Figure 10 (Middle left image), the formed gel exhibits good shear-thinning properties ( Figure 10 (Right image in the middle)

[0089] To test the antibacterial activity of the H / P gel, an antibacterial experiment was conducted. Specifically, 10% PVA solution and 10% H / P gel were placed in Transwell chambers with an 8-micron pore size. Half of the chambers were dried for 30 minutes to promote the formation of a continuous, uniform film with a thickness of approximately 50-200 μm on the surface. Then, 5 × 10⁵ μm of H / P gel was added to the chamber. 7 200 μL of CFU-containing Staphylococcus aureus suspension was added to 1.5 mL of PBS in a 6-well plate, which was then placed in the chamber and incubated at 37°C for 8 hours. After incubation, the supernatant was removed, diluted, plated, and counted. It was observed that the inert membrane formed by the H / P gel effectively blocked the invasion of pathogenic bacteria (e.g., ...). Figure 11 (As shown). The bio-inert membrane formed on the gel surface plays a crucial role in isolating exogenous bacterial invasion. Without adding any drugs to the gel, this bio-inert membrane alone provides physical protection against pathogens, effectively solving the problem of poor antibacterial properties inherent in most gel and cream formulations. This characteristic makes the biphasic gel of this invention safer and more reliable in biomedical applications, reducing the risk of treatment failure or complications due to bacterial infection.

[0090] Example 3 This example provides a drug delivery system, which is a biphasic gel material loaded with curcumin. The specific preparation process is as follows: 50 mg of HPG and 3 mg of curcumin are weighed and dissolved in 1 mL of DMSO. This DMSO mixture is slowly injected into 30 mL of ultrapure water stirred at 300 rpm using a 1 mL syringe at room temperature until complete injection. The aqueous solution is placed in a 100 kDa molecular weight cutoff ultrafiltration tube and centrifuged at 4500 rpm for 15 min at room temperature. The lower filtrate is removed, resuspended in 15 mL of ultrapure water, and centrifuged again at 4500 rpm for 15 min at room temperature. The mixture is washed with water, and the upper filtrate is collected after three washes to obtain HPG-curcumin nanoparticles. These nanoparticles are suspended in a 50% HPG aqueous solution and mixed with 10% PVA to prepare the H / P drug-loaded gel.

[0091] Dynamic light scattering experiments were conducted on the HPG-curcumin nanoparticles obtained in Example 3, and the results are as follows: Figure 12 As shown in the figure, HPG and curcumin formed nanoparticles with a particle size of 236.6 nm and a PDI of 0.1654. 300 mg of the drug-loaded gel obtained in Example 3 was placed in 30 mL of a 1 wt% Tween 80 aqueous solution for drug release studies. The release system was placed in a 37°C water bath and shaken at 200 rpm. 1 mL of the release solution was collected at 0, 1, 2, 4, 8, 12, 24, 48, and 72 h for UV spectrophotometric analysis. The drug release curves are shown in the figure. Figure 13As shown, 74.13% of the drug was released within 72 hours.

[0092] This invention utilizes the macroscopic phase separation structure generated by biphasic gels. Specifically, a bio-inert film forms on the gel surface, while an internal framework-vacuolar structure is formed. This dual-phase structure allows for the loading of a wide range of complex contents, making it suitable for applications in skin diseases and implant materials, demonstrating high practical value and clinical translational potential. During the formation process, the surface of the biphasic gel loses water, forming an in-situ inert film that effectively isolates it from the invasion of exogenous pathogenic bacteria. Internally, the microscopic liquid-liquid phase separation creates a framework-vacuolar structure, providing the gel with strength, elasticity, and shear-thinning properties. Simultaneously, it can load probiotics or cells for use in the biomedical field. By using it to isolate exogenous bacterial invasion, it achieves physical protection against pathogens without the presence of drugs inside, solving the problem of poor antibacterial properties inherent in most gel and cream formulations. The biphase system of this invention, through a hydrogen bond-based sphere-linear chain entanglement, not only prepares a gel with both strength and shear thinning ability in a one-step process at room temperature, but also utilizes the drug loading capacity of hyperbranched anionic polymers and the bio-inert membrane formed by phase separation of the gel itself, ultimately obtaining a biphase gel with mechanical strength, drug loading capacity and antibacterial ability.

[0093] In summary, the biphasic gel system of this invention is prepared by the self-assembly of HPG and PVA via hydrogen bonding. The two components can undergo chain entanglement through hydrogen bonding and induce structural collapse. Phase separation and gel formation were observed in a room-temperature aqueous solution. HPG can form nanocomposites with drugs via precipitation and further load them into the gel. Simultaneously, the bio-inert membrane formed by the dehydration phase separation of the gel can effectively block the invasion of pathogenic bacteria.

[0094] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A two-phase gel material, characterized by: The biphasic gel material is formed by self-assembly of a first polymer and a second polymer based on hydrogen bonds, wherein the first polymer comprises a branched polymer, and the second polymer comprises a long-chain linear multi-hydrogen bond site polymer, and the branched polymer comprises at least one of the following: 1) a polyether-based polymer with a terminal hydroxyl group; and 2) a polymer obtained by derivatization of the hydroxyl group of the polyether-based polymer in 1).

2. The biphasic gel material of claim 1, wherein: The raw material for preparing the biphasic gel material comprises a first polymer, a second polymer and a solvent.

3. The biphasic gel material of claim 1, wherein: The mass ratio of the first polymer to the second polymer is 1:0.5-2.

0.

4. The biphasic gel material of claim 1, wherein: The sum of the mass of the first polymer and the second polymer in the raw material for preparing the biphasic gel material accounts for 10-30%.

5. The biphasic gel material of claim 1, wherein: The first polymer comprises at least one of an ultrabranched polyglycidol and a derivative thereof, and a multi-arm polyethylene glycol with a terminal hydroxyl group and a derivative thereof, and the derivative is at least one of a product in which the terminal hydroxyl group is derivatized by an aldehyde group or an ester group.

6. The biphasic gel material of claim 1, wherein: The long-chain linear multi-hydrogen bond site polymer comprises at least one of polyvinyl alcohol, silk fibroin or keratin.

7. A process for the preparation of a biphasic gel material according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: The dispersion liquid of the first polymer is mixed with the dispersion liquid of the second polymer, and then reacted to obtain the biphasic gel material.

8. A gel composition loaded with a bioactive ingredient, characterized in that: The raw material for preparing the gel composition loaded with a bioactive component comprises the biphasic gel material according to any one of claims 1-6 and a bioactive substance, and the bioactive substance comprises a drug and / or a microorganism.

9. The method of preparing a bioactive ingredient-loaded gel composition according to claim 8, wherein: The method comprises the following steps: The first polymer is prepared into a first polymer-bioactive substance nanoparticle with a drug, and then mixed with the dispersion liquid of the second polymer, and then reacted to obtain the gel composition loaded with the bioactive component.

10. Use of the biphasic gel material according to any one of claims 1-6 in the preparation of a drug for blocking or preventing skin diseases.

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