Casein modified active peptide capable of promoting bone repair and preparation method of casein modified active peptide

Abstract

The invention relates to the field of nutritional health food, and discloses a casein modified active peptide for promoting bone repair and a preparation method thereof.The structure of the active peptide sequentially comprises a bone targeting sequence with high affinity to hydroxyapatite, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions and a casein phosphopeptide core sequence capable of chelating calcium ions from the C-terminal to the N-terminal. The digestion sequence can be specifically hydrolyzed by matrix metalloproteinase, and the alkynyl functional group is used for covalent cross-linking. The active peptide can be mixed with an azido-containing multi-arm cross-linking agent, and hydrogel can be rapidly formed under physiological conditions through cycloaddition reaction. The system can be gelated in situ and anchored to a bone defect part in a targeted manner, and is triggered by a specific enzyme in a pathological microenvironment to controllably release active peptide fragments capable of chelating calcium ions, so that the concentration of local mineral ions is adjusted, the cell living environment is improved, and an effective new way is provided for promoting bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of nutritional health foods, in particular to a casein-modified active peptide capable of promoting bone repair and a preparation method thereof. Background Art

[0002] Bone defects are caused by the interruption of bone tissue continuity due to trauma, tumors, infection or congenital malformations, and are one of the major challenges facing orthopedic clinics. Promoting bone tissue regeneration and repair is the key to restoring the integrity and functionality of the patient's bone structure. In bone repair strategies, the application of bioactive factors to regulate the bone regeneration microenvironment and promote osteoblast function is an important direction. Among them, casein phosphopeptides contain multiple phosphoserine residues in their molecular structure, which can effectively bind to mineral ions such as calcium and phosphorus, thereby stabilizing mineral ions in the body fluid environment. They are considered to have the potential to promote bone tissue mineralization and regeneration.

[0003] However, when bioactive molecules such as casein phosphopeptides are directly applied to bone defect sites, their clinical application effects are limited in many aspects. First, as free small molecule polypeptides, these molecules lack an effective retention mechanism after being applied to defect sites rich in body fluids. They are easily diluted and cleared by body fluids, resulting in a short effective action time in the target area, low bioavailability, and difficulty in achieving the expected therapeutic effect. To solve this problem, existing technologies often use biomaterials such as hydrogels as carriers to encapsulate them. However, conventional physical encapsulation methods are often accompanied by obvious burst release effects, and the subsequent release process is difficult to accurately control and cannot match the physiological process of bone repair.

[0004] Furthermore, in the field of nutrition and health, even some recognized bioactive ingredients often fail to achieve the expected health benefits due to inefficient absorption and utilization within the body. For example, oral casein phosphopeptides, after being digested and absorbed and entering the circulatory system, are indiscriminately transported to various tissues and organs throughout the body, failing to prioritize and actively act on bones, which are most in need of nutritional support. This widespread distribution of nutrients results in a low effective dose that actually reaches and acts on bones, compromising the effectiveness of supplementation.

[0005] Furthermore, traditional nutritional supplements often release their ingredients in a relatively crude manner. They typically release all active ingredients rapidly and all at once upon entering the digestive tract. This burst of release results in a transient increase in the concentration of the active ingredient in the body, followed by rapid metabolic clearance, resulting in a brief window of effectiveness. This model is incompatible with the long-term, sustained nutritional needs of human bone metabolism. A more ideal nutritional supplement solution should ensure a continuous and stable supply of nutrients and even intelligently respond to the body's demand signals during specific states (such as during active bone remodeling), releasing key nutrients at critical moments to maximize the value and efficiency of the supplement. Bone repair is a dynamic process precisely regulated by multiple cells and signaling molecules, with varying requirements for active factors at different stages. For example, during inflammation and tissue remodeling, the expression of specific enzymes, such as matrix metalloproteinases, is significantly upregulated. An ideal delivery system should be able to sense and respond to these microenvironmental signals, enabling on-demand release, ensuring that the nutrients are delivered when and where they are most needed. Existing technologies generally lack such intelligent regulatory mechanisms that link the release of active factors with specific physiological signals. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a casein-modified active peptide that promotes bone repair and a preparation method thereof. The bioactive ingredients used for bone repair in the prior art usually have defects such as non-targeted delivery, easy loss in the target area, and uncontrollable release pattern, resulting in low local effective concentration and potential bioavailability problems.

[0007] In order to solve the above technical problems, the present invention provides a multi-domain casein-modified active peptide with a functionally designed structure and a standardized preparation method thereof.

[0008] The first aspect of the present invention provides a casein-modified active peptide that promotes bone repair.

[0009] The casein-modified active peptide is a linear peptide chain, and its overall structure contains four functional domains with clear functions in the order from N-terminus to C-terminus: N-terminal modified alkynyl functional group: This functional group is located at the N-terminus of the peptide chain and serves as a chemical reaction active site. Its function is to provide the entire peptide chain with a group that can be used for covalent bonding reactions, thereby fixing the peptide chain molecule to a polymer network or matrix.

[0010] Enzyme cleavage short peptide sequence: This functional domain, immediately following the alkyne group, consists of a specific amino acid sequence designed to serve as a substrate for specific proteolytic enzymes (such as matrix metalloproteinases or cathepsin K) secreted by specialized cells during the physiological processes of bone repair and remodeling. This sequence functions to be recognized and cleaved in specific biological microenvironments, forming an enzyme-controlled cleavage site.

[0011] Casein phosphopeptide core sequence: This functional domain, located in the middle of the peptide chain, is a sequence of one or more phosphoserine residues derived from natural casein. Its function is to chelate divalent metal cations (particularly calcium ions) through its phosphate groups, thereby carrying substances that promote bone mineralization.

[0012] Bone-targeting sequence: This functional domain, located at the C-terminus of the peptide chain, is a sequence of multiple consecutive acidic amino acid residues. Its function is to generate electrostatic affinity with hydroxyapatite, an inorganic salt component in bone tissue, through the dense negative charge on this sequence, thereby anchoring or enriching the entire peptide chain molecule on the bone tissue surface.

[0013] The technical concept of the present invention is to obtain a multifunctional molecular entity by linearly connecting the above four functional domains. Its mechanism of action is as follows: First, the bone-targeting sequence at the C-terminus allows the entire peptide chain to be macroscopically localized to the bone tissue interface within the bone defect. Second, the alkynyl functional group at the N-terminus allows the peptide chain to be chemically cross-linked and fixed to a carrier, preventing it from escaping the target area. In this fixed state, when the bone repair process begins, surrounding cells secrete specific enzymes into the microenvironment. These enzymes recognize and cleave the enzymatic short peptide sequence within the peptide chain, resulting in the release of the fragment connected to the casein phosphopeptide core sequence from the fixed peptide chain. In this way, the release behavior of the active fragment is regulated by the enzyme activity in the bone repair microenvironment, achieving a temporal and spatial correlation between the release and the physiological repair process.

[0014] In a specific embodiment, the bone-targeting sequence is a polyaspartic acid sequence or a polyglutamic acid sequence, and the length of the sequence is 6-12 amino acid residues.

[0015] In a specific embodiment, the enzymatic cleavage short peptide sequence is a specific substrate sequence of matrix metalloproteinase or cathepsin K.

[0016] In a specific embodiment, the amino acid sequence of the casein phosphopeptide core sequence is derived from a functional fragment containing phosphoserine in αs1-casein or β-casein.

[0017] In a specific embodiment, the N-terminus modified alkynyl functional group is formed by subjecting the amino group at the N-terminus of the peptide chain to an amidation reaction with propiolic acid, or by directly using a non-natural amino acid with an alkynyl group as the N-terminal amino acid residue during peptide chain synthesis.

[0018] The second aspect of the present invention provides a method for preparing a casein-modified active peptide that promotes bone repair.

[0019] The method is used to prepare the casein-modified active peptide described in any of the aforementioned schemes, and is characterized by comprising the following steps: First, solid-phase peptide synthesis is performed on an insoluble solid-phase resin support, with the peptide chain synthesized stepwise from the C-terminus to the N-terminus. Specifically, the amino acids that comprise the bone-targeting sequence, the casein phosphopeptide core sequence, and the enzymatically cleaved short peptide sequence are sequentially attached to the resin through cyclic deprotection and coupling reactions, following a predetermined amino acid sequence.

[0020] Next, a chemical group bearing an alkyne functional group is introduced at the N-terminus of the peptide chain.

[0021] Finally, the synthesized, intact peptide chain attached to the resin is treated with chemical reagents to cleave it from the resin and simultaneously remove all protecting groups used to protect the amino acid side chain functional groups during the synthesis process. The crude product obtained by cleavage is then purified to obtain the target peptide product.

[0022] In a preferred embodiment, in the preparation method, the phosphoserine residue constituting the core sequence of the casein phosphopeptide is introduced during solid phase synthesis by directly coupling an Fmoc-serine derivative whose side chain hydroxyl group has been pre-modified with a phosphate group carrying a protecting group.

[0023] In a specific embodiment, the step of introducing a chemical group with an alkynyl functional group at the N-terminus of the peptide chain is specifically operated as follows: after the last amino acid constituting the enzymatically cleaved short peptide sequence is coupled to the peptide chain and its N-terminal protecting group is removed, the free N-terminal amino group of the peptide chain formed and connected to the resin is subjected to an amidation reaction with a carboxylic acid containing an alkynyl group or its active ester.

[0024] In a preferred embodiment, the purification step comprises separating and purifying the crude product after cleavage using reverse phase high performance liquid chromatography.

[0025] In a specific embodiment, the cleavage and removal of protecting groups is performed by treating the resin connected with the intact peptide chain at room temperature using a cleavage reagent containing trifluoroacetic acid as a main component and a cationic capture agent.

[0026] The present invention provides a casein-modified active peptide that promotes bone repair and a preparation method thereof. It has the following beneficial effects: 1. The casein-modified active peptide provided by the present invention has a specific affinity for bone tissue hydroxyapatite through its C-terminal bone-targeting sequence, and can simultaneously covalently bond with the external matrix through its N-terminal alkynyl functional group, achieving precise targeting and long-term retention at the bone defect site. This dual fixation mechanism significantly increases the local concentration of the active peptide in the target area, avoiding loss due to rapid flushing by body fluids, thereby improving its bioavailability at the bone repair site.

[0027] 2. This invention constructs an intelligent "release on demand" system by designing a short peptide sequence within the peptide chain that can be cleaved by specific enzymes (such as matrix metalloproteinases) in the bone repair microenvironment. This enables the release of the core casein phosphopeptide sequence carrying calcium ions to be actively triggered by the physiological process of bone tissue repair, rather than simply passive diffusion. This allows the release of the active ingredient to be precisely synchronized with the physiological repair process, thereby improving the effectiveness and targeted nature of the treatment.

[0028] 3. The preparation method provided by the present invention adopts the mature solid-phase peptide synthesis (SPPS) method in the field of chemical synthesis, which can accurately control the amino acid sequence of the peptide chain and the position of each functional domain (including phosphorylation sites, enzyme cleavage sites, bone-targeting sequences and N-terminal alkynyl groups). This method avoids the common problems of product heterogeneity and low purity in biological extraction methods, and ensures that the final casein-modified active peptide product has a clear structure, high purity and good batch-to-batch reproducibility, providing a reliable technical path for subsequent quality control and large-scale production. DETAILED DESCRIPTION

[0029] The following will be combined with the present invention specification to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Experimental materials: The sources and specifications of the main raw materials and reagents used in the following examples and comparative examples are as follows. Reagents not specifically specified are all commercially available products of analytical grade or higher.

[0031] Peptide solid phase synthesis related reagents: Fmoc-Asp(OtBu)-OH:CAS:71989-14-5; Fmoc-Ser(PO(OBzl)OH)-OH:CAS:1212481-01-0; Fmoc-Glu(OtBu)-OH:CAS:71989-18-9; Fmoc-Gly-OH:CAS:29022-11-5; Fmoc-Pro-OH:CAS:71989-31-6; Fmoc-Leu-OH:CAS:35661-60-0; Fmoc-Val-OH:CAS:68858-20-8; Fmoc-Arg(Pbf)-OH:CAS:154445-77-9; 5-Hexynoic acid: CAS: 53293-00-8; N,N-Diisopropylethylamine,CAS:7087-68-5; Trifluoroacetic acid: CAS: 76-05-1; Triisopropylsilane: CAS: 6485-79-6; Reagents for polymer synthesis and modification: 2-Bromoisobutyryl bromide: CAS: 20769-85-1; N-isopropylacrylamide: CAS: 2210-25-5; N,N,N',N'',N''-pentamethyldiethylenetriamine: CAS: 3030-47-5; Cuprous bromide: CAS: 7787-70-4; Sodium azide: CAS: 26628-22-8; General solvents and conventional reagents: N,N-dimethylformamide: CAS: 68-12-2; Dichloromethane: CAS: 75-09-2; Piperidine: CAS: 110-89-4; Triethylamine: CAS: 121-44-8; Ether: CAS: 60-29-7; Toluene: CAS: 108-88-3; Tetrahydrofuran: CAS: 109-99-9; Acetonitrile: CAS: 75-05-8; Deuterated chloroform: CAS: 865-49-6; Potassium bromide, CAS: 7758-02-3; Reagents and materials related to performance testing and biological experiments: Hydroxyapatite: CAS: 12167-74-7; Calcium chloride: CAS: 22691-02-7; Example 1-3: Example 1: Preparation of casein-modified active peptides This embodiment aims to provide a specific preparation process of the casein-modified active peptide as described in the first aspect of the present invention. The amino acid sequence of the prepared peptide is designed from N-terminus to C-terminus as follows: Pra-Gly-Pro-Leu-Gly-Val-Arg-Gly-Ser(PO3H2)-Glu-Glu-Ser(PO3H2)-Glu-Glu-Ser(PO3H2)-Ala-(Asp)8-NH2. Wherein, Pra represents propargylglycine, Ser(PO3H2) represents phosphoserine, and (Asp)8 represents eight consecutive aspartic acid residues.

[0032] Solid Phase Peptide Synthesis: The Fmoc solid-phase peptide synthesis method was used, using AM resin with a RinkAmide linker (RinkAmideAMresin, degree of substitution 0.5mmol / g) as the solid phase support, and the synthesis scale was 0.1mmol. The specific synthesis steps are as follows: (1) Resin pretreatment and connection to the first amino acid: Weigh 0.2 g of the RinkAmideAM resin, place it in a solid phase reactor, swell it with dichloromethane for 30 minutes, and then wash it three times with N,N-dimethylformamide. Treat the resin with 20% (v / v) piperidine / DMF solution for 15 minutes to remove the Fmoc protecting group on the connecting arm, and then wash it thoroughly with DMF. Weigh Fmoc-Asp(OtBu)-OH (0.4 mmol, 4 equivalents), HBTU (0.4 mmol, 4 equivalents) and HOBt (0.4 mmol, 4 equivalents) and dissolve them in DMF. Add N,N-diisopropylethylamine (DIEA) (0.8 mmol, 8 equivalents) to activate it for 5 minutes, then add it to the resin and react at room temperature for 2 hours to complete the coupling of the first aspartic acid residue.

[0033] (2) Gradual extension of the peptide chain: According to the preset amino acid sequence, the amino acid coupling cycle is carried out from the C-terminus to the N-terminus. Each cycle includes the following steps: a) Deprotection: The peptide resin was treated with 20% (v / v) piperidine / DMF solution twice for 10 minutes each time to remove the Fmoc protecting group at the N-terminus of the peptide chain, and then washed with DMF 5 times and DCM 3 times.

[0034] b) Coupling: The next Fmoc-protected amino acid (0.4 mmol, 4 equiv), HBTU (0.4 mmol, 4 equiv), and HOBt (0.4 mmol, 4 equiv) were dissolved in DMF, activated with DIEA (0.8 mmol, 8 equiv), and reacted with the peptide resin for 2 h.

[0035] c) According to this cycle, the following protected amino acids were coupled in sequence: 7 Fmoc-Asp(OtBu)-OH, 1 Fmoc-Ala-OH, 1 Fmoc-Ser(PO(OBzl)2)-OH, 2 Fmoc-Glu(OtBu)-OH, 1 Fmoc-Ser(PO(OBzl)2)-OH, 2 Fmoc-Glu(OtBu)-OH, 1 Fmoc-Ser(PO(OBzl)2)-OH, 1 Fmoc-Gly-OH, 1 Fmoc-Arg(Pbf)-OH, 1 Fmoc-Val-OH, 1 Fmoc-Gly-OH, 1 Fmoc-Leu-OH, 1 Fmoc-Pro-OH, and 1 Fmoc-Gly-OH.

[0036] (3) Introduction of the N-terminal alkynyl functional group: After the last glycine was coupled and its Fmoc protecting group was removed, Fmoc-Pra-OH (0.4 mmol, 4 equivalents) was coupled to the N-terminus of the peptide chain according to the method described in the step coupling. Finally, the Fmoc protecting group of Fmoc-Pra-OH was removed using a 20% (v / v) piperidine / DMF solution.

[0037] (4) Post-treatment of the resin: After the synthesis, the peptide resin was washed with DMF, DCM and methanol in sequence and then dried under vacuum.

[0038] Cleavage of peptide chains and acquisition of crude products: Place the dried peptide resin in a round-bottom flask and add 10 mL of pre-chilled cleavage reagent (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% deionized water). Stir the reaction at room temperature for 3 hours. After the reaction is complete, filter the mixture and collect the filtrate. Slowly add the filtrate dropwise to 50 volumes of ice-cold anhydrous ether, resulting in the precipitation of a white precipitate. Let the mixture stand at 4°C for 30 minutes, then collect the precipitate by centrifugation. Wash the precipitate three times with ice-cold anhydrous ether and then dry it under vacuum to obtain the crude peptide as a white powder.

[0039] purification: The crude peptide was purified by preparative reverse-phase high-performance liquid chromatography.

[0040] Chromatographic column: C18 reverse phase column (10 μm, 250 mm × 20 mm).

[0041] Mobile phase A: aqueous solution containing 0.1% TFA.

[0042] Mobile phase B: acetonitrile solution containing 0.1% TFA.

[0043] Elution gradient: The proportion of mobile phase B increased linearly from 5% to 45% in 30 minutes.

[0044] Flow rate: 10mL / min.

[0045] The detection wavelength was 220 nm. The eluted fractions corresponding to the main peak were collected, and after removing most of the acetonitrile by rotary evaporation, the solution was freeze-dried to obtain the target product ER-TFP as a white powder with a purity greater than 95%.

[0046] Structure identification: The chemical structure of the purified product was confirmed by electrospray ionization mass spectrometry. The observed mass-to-charge ratio (m / z) signal peak was consistent with the theoretically calculated value for the target peptide. Furthermore, the distribution pattern of the isotopic peak cluster corresponding to this signal peak was highly consistent with the theoretical isotopic distribution predicted based on the elemental composition of the target peptide.

[0047] The above analysis results confirmed that the target casein-modified active peptide with correct sequence and intact structure has been successfully prepared.

[0048] Example 2: Preparation of a thermosensitive multi-arm cross-linking agent This example aims to provide a specific preparation process of a temperature-sensitive multi-arm cross-linking agent with an azide group that can undergo click chemistry cross-linking reaction with the casein-modified active peptide described in Example 1.

[0049] Preparation of four-arm polyethylene glycol-poly (N-isopropylacrylamide) copolymer: (1) Preparation of macroinitiator: Weigh four-arm polyethylene glycol (4-armPEG-OH, Mw = 10,000 Da, 1.0 g, 0.1 mmol) and dissolve it in 50 mL of anhydrous dichloromethane. Cool it in an ice bath. Under nitrogen protection, slowly add triethylamine (TEA, 0.84 mL, 6.0 mmol) and then slowly add 2-bromoisobutyryl bromide (2.0 mL, 16.2 mmol). The mixture is reacted in an ice bath for 2 hours and then warmed to room temperature for 24 hours. The reaction solution is washed with saturated sodium chloride solution, and the organic phase is separated and dried over anhydrous magnesium sulfate. After filtration, the filtrate is concentrated on a rotary evaporator and then precipitated in ice ether to collect the white solid. After vacuum drying, the four-arm polyethylene glycol-bromine macroinitiator (4-armPEG-Br) is obtained.

[0050] (2) Atom transfer radical polymerization: The macroinitiator (4-armPEG-Br, 0.5 g), N-isopropylacrylamide (NIPAM, 5.0 g) and N,N,N',N'',N''-pentamethyldiethylenetriamine obtained in step (1) were dissolved in 20 mL of toluene. The mixed solution was subjected to three freeze-vacuum-thaw cycles to remove oxygen. Under a nitrogen atmosphere, cuprous bromide was added as a catalyst and the reaction bottle was sealed. The reaction system was placed in a 60°C oil bath for 24 hours. After the reaction, the solution was diluted with tetrahydrofuran and passed through a short column filled with neutral alumina to remove the copper catalyst. The filtrate was concentrated and precipitated in a large amount of ice ether. The precipitate was collected by centrifugation. The precipitate was redissolved in THF and precipitated again in ice ether. This process was repeated twice. Finally, the obtained white solid was dried in a vacuum oven at 40°C for 24 hours to obtain 4-armPEG-g-PNIPAM copolymer.

[0051] Preparation of thermosensitive multi-arm cross-linker: The 4-armPEG-g-PNIPAM copolymer (2.0 g) prepared in Step 2.1 and sodium azide (1.0 g NaN3) were added to 50 mL of N,N-dimethylformamide. The mixture was stirred at 50°C under nitrogen for 48 hours to completely replace the bromine atoms at the copolymer arm terminals with azide groups. After the reaction, the reaction solution was placed in a dialysis bag (molecular weight cut-off 3500 Da) and dialyzed against deionized water for 3 days, with the deionized water frequently replaced. After dialysis, the solution in the dialysis bag was freeze-dried to obtain the final product, a thermosensitive multi-arm crosslinker, as a white, flocculent solid.

[0052] Structure identification: Product characterization Elemental analysis: The elemental composition of the final product, T-MAC, and its intermediate (the brominated polymer before azidation) was determined using elemental analysis. The results showed no bromine was detected in the final product, T-MAC, compared to the intermediate. However, the measured mass percentage of nitrogen was consistent with the theoretically calculated value after the introduction of terminal azide groups and PNIPAM side chains. This result confirms that the bromine atoms at the polymer terminals have been completely replaced by azide groups.

[0053] Thermosensitive phase transition behavior: The product, T-MAC, was dissolved in deionized water to prepare a 0.5% (w / v) aqueous solution. The transmittance of this solution at 500 nm was monitored using a UV-visible spectrophotometer as a function of temperature. The results showed that the lower critical solution temperature (LCST) of the polymer aqueous solution was 33.5°C. At this temperature, the solution transitioned from a clear, transparent state to an opaque, turbid state, demonstrating a significant thermosensitive phase transition. This provides direct functional evidence that the PNIPAM segments were successfully grafted onto the polymer backbone.

[0054] Example 3: Preparation of an injectable two-component hydrogel precursor solution This example is intended to illustrate how to formulate the active components prepared in Examples 1 and 2 into a two-component precursor solution that can be used for instant mixing to form a hydrogel.

[0055] Preparation of component A solution (active peptide solution): Weigh 20 mg of the purified lyophilized casein-modified active peptide powder prepared in Example 1 and place it in a sterile centrifuge tube. Add 1 mL of phosphate-buffered saline (PBS, pH 7.4) to the tube. Vortex the tube at low speed until the powder is completely dissolved, forming a clear, transparent solution. Sterilize the solution by filtering it through a 0.22 μm pore size sterile filter and collect it in a sterile storage bottle to obtain a 20 mg / mL solution of Component A. Seal the bottle and store it at 4°C until needed.

[0056] Preparation of component B solution (crosslinker solution): Weigh 20 mg of the purified, lyophilized powder of the temperature-sensitive multi-arm cross-linker prepared in Example 2 and place it in a sterile centrifuge tube. Add 1 mL of phosphate-buffered saline (PBS, pH 7.4) to the tube. Vortex the tube at low speed until the powder is completely dissolved, forming a clear, transparent solution. Sterilize the solution by filtering it through a 0.22 μm pore size sterile filter and collect it in a sterile storage bottle to obtain a 20 mg / mL solution of Component B. Seal the bottle and store it at 4°C until needed.

[0057] Comparative Examples 1-3: Comparative Example 1: Preparation of modified peptides without bone targeting sequences This comparative example differs from Example 1 in that the eight aspartic acid residues that constitute the bone-targeting sequence were not attached during solid-phase peptide synthesis. Peptide chain synthesis began directly with the C-terminal amino acid (alanine) of the casein phosphopeptide core sequence attached to the resin. All other steps, including subsequent amino acid coupling, introduction of the N-terminal alkynyl group, cleavage, purification, and identification, were identical to those described in Example 1.

[0058] Comparative Example 2: Preparation of modified peptides without enzyme-cleaved short peptide sequences Compared to Example 1, this comparative example differs in that, during the solid-phase peptide synthesis process, the original enzymatically cleavable short peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) was replaced with a scrambled sequence consisting of identical amino acids that lacks specific enzymatic cleavage activity. All other steps, including subsequent amino acid coupling, introduction of an N-terminal alkynyl group, cleavage, purification, and identification, were identical to those described in Example 1.

[0059] Comparative Example 3: Preparation of a modified peptide without an N-terminal alkynyl functional group Compared to Example 1, this comparative example differs in that the introduction of the N-terminal alkynyl functional group was omitted during the solid-phase peptide synthesis process. Specifically, after coupling the last amino acid (glycine) in the enzymatically cleaved short peptide sequence and removing its Fmoc protecting group, peptide chain elongation was terminated without any N-terminal chemical modification. All other steps, including subsequent cleavage, purification, and identification methods, were identical to those described in Example 1.

[0060] Test Example 1-5: Test Example 1: Gel Formation Ability and Mechanical Properties Test Purpose of the experiment: This test example aims to determine the gelation time and final mechanical properties of the system formed by mixing the component A solution and the component B solution described in Example 3, in order to verify its feasibility as an in situ gel scaffold.

[0061] Experimental steps: Instrument Preparation: Use a rotational rheometer with a temperature-controlled platform. Set the temperature of the parallel plate fixture (20 mm diameter) to a constant temperature of 37.0 ± 0.1 °C.

[0062] Sample preparation: Component A solution (active peptide solution) and component B solution (cross-linking agent solution) prepared in Example 3 were taken out from the 4°C storage environment and equilibrated at room temperature for 15 minutes.

[0063] Loading and Mixing: Using a micropipette, pipette a 150 μL drop of Component B onto the center of the rheometer's lower platform. Then, quickly pipette a 150 μL drop of Component A onto Component B. Immediately lower the upper fixture to the set test gap (0.5 mm) and activate the rheometer's rotation program to rapidly mix the two components.

[0064] Dynamic Time-Varying Sweep: The dynamic time-varying sweep test begins immediately upon mixing initiation. The strain is set to 1% and the frequency is 1 Hz. The storage modulus (G') and loss modulus (G") are continuously monitored over time for 30 minutes.

[0065] Data recording: Record the curve of the change in the values ​​of G' and G". The time point corresponding to the intersection of the G' and G" values ​​is recorded as the gelation time. Record the plateau value of G' at the end of the 30-minute test and record it as the final storage modulus.

[0066] Repeat the experiment: Repeat the experimental process three times according to the steps from sample preparation to data recording to obtain multiple sets of data.

[0067] Experimental data According to the above experimental steps, the data obtained are recorded in the table below.

[0068] Table 1: Rheological properties of hydrogel system Results The above experimental data show that when component A containing the active peptide of the present invention is mixed with component B containing a multi-arm crosslinker at 37°C, which simulates physiological body temperature, the storage modulus (G') of the system increases rapidly and exceeds the loss modulus (G"), indicating that the system completes the transition from liquid to solid hydrogel in about 1 minute. This result confirms that the two-component system has the ability to undergo in situ gelation under the target physiological environment.

[0069] This gelation behavior is based on an efficient covalent bonding reaction between the alkynyl functional group introduced at the N-terminus of the active peptide molecule and the azide group at the end of the multi-arm crosslinker molecule. This reaction proceeds spontaneously under mild aqueous conditions, forming stable chemical crosslinks that connect the discrete peptide molecules and crosslinker molecules into a macroscopic three-dimensional network. It is this network structure that imparts the system with a storage modulus that resists deformation and imparts the mechanical characteristics of a solid gel.

[0070] The final measured storage modulus value confirmed that the formed hydrogel possessed a certain mechanical strength. This in situ-formed, structurally stable gel network serves as a physical barrier and reservoir, retaining casein-modified active peptides covalently anchored to network nodes in specific areas. This structural feature is a prerequisite for achieving subsequent enzyme-controlled release, ensuring that the active peptide molecules are not lost from the site of action due to physical factors such as flushing by body fluids before being cut by specific enzymes.

[0071] Test Example 2: Test of the binding ability of active peptides to hydroxyapatite Purpose of the experiment: This test example aims to quantitatively compare the binding ability of the active peptide prepared in Example 1 with the peptide without bone-targeting sequence prepared in Comparative Example 1 to hydroxyapatite, so as to verify the function of the bone-targeting sequence.

[0072] Experimental steps: (1) Sample solution preparation: The active peptide prepared in Example 1 and the peptide without bone-targeting sequence prepared in Comparative Example 1 were respectively prepared into solutions with an initial concentration of 1.0 mg / mL using phosphate buffered saline solution (PBS, pH 7.4).

[0073] (2) Binding experiment: Accurately weigh 20 mg of hydroxyapatite powder and place it in a 1.5 mL centrifuge tube. Add 1.0 mL of the peptide solution prepared above to the tube. Place the centrifuge tube in a thermostatic shaker at 37°C and incubate at 100 rpm for 4 hours.

[0074] (3) Separation and sampling: After incubation, centrifuge the tube at 12,000 rpm for 10 minutes. Carefully aspirate the supernatant.

[0075] (4) Concentration determination: The concentration of peptide remaining in the supernatant was determined using a reversed-phase high-performance liquid chromatography system. The chromatographic conditions were the same as those described in Example 1.3. The peptide concentration in the supernatant was calculated using a pre-prepared peptide concentration-peak area standard curve ( ).

[0076] (5) Calculation of binding rate: According to the initial peptide concentration ( = 1.0 mg / mL) and the remaining peptide concentration in the supernatant ( ), according to the formula: Binding rate ; The binding rate of peptide to HA was calculated.

[0077] in: Binding rate (%): refers to the percentage of peptide binding to hydroxyapatite; : refers to the initial concentration of the peptide solution before incubation with hydroxyapatite (mg / mL); : Refers to the final concentration (mg / mL) of peptide remaining in the supernatant after incubation and centrifugation.

[0078] (6) Repeated experiments: Three independent binding experiments were performed for each peptide sample.

[0079] Experimental data: According to the above experimental steps, the data obtained are recorded in the table below.

[0080] Table 2: Binding rate of different peptides to hydroxyapatite Result description: The experimental data above show that the active peptide prepared in Example 1 exhibited a binding rate exceeding 80% after incubation with hydroxyapatite powder. In contrast, the peptide prepared in Comparative Example 1, in which the bone-targeting sequence was removed, exhibited a binding rate of less than 8% to hydroxyapatite. This result clearly demonstrates the high affinity of the active peptides described herein for hydroxyapatite, a major inorganic component of bone tissue.

[0081] The mechanism of producing this high affinity is that the C-terminus of the active peptide of the present invention contains a sequence composed of multiple consecutive aspartic acid residues. In a buffer environment of pH 7.4, the carboxyl groups of the side chains of this sequence are largely deprotonated and carry a high density of negative charges. This polyanionic structure produces a strong electrostatic attraction with the calcium ions exposed on the surface of the hydroxyapatite lattice, thereby effectively adsorbing and fixing the entire peptide molecule on the hydroxyapatite surface. Since the peptide of Comparative Example 1 lacks this key polyanionic sequence, it is unable to establish an effective electrostatic interaction with hydroxyapatite, and its extremely low binding rate is only due to nonspecific physical adsorption.

[0082] This specific high affinity for bone mineral components is a core feature of the present invention. It enables the active peptide to actively accumulate and anchor at the interface of bone tissue or bone defects after application in a physiological environment, rather than being diluted or rapidly cleared in body fluids. This targeted fixation provides the physical basis for the subsequent release of the active fragment at a specific site, triggered by microenvironmental enzymes, and is the key to addressing the inefficient delivery of bioactive ingredients in existing technologies.

[0083] Test Example 3: Enzyme-responsive release behavior test Purpose of the experiment: This test example aims to verify whether the release behavior of the active fragment of the active peptide described in the present invention is regulated by a specific enzyme after being immobilized in the hydrogel network.

[0084] Experimental steps: (1) Preparation of hydrogel samples: Experimental group gel: Referring to the method of Example 3, 100 μL of component A solution (containing the ER-TFP prepared in Example 1) was mixed with 100 μL of component B solution, and in situ cross-linked at 37° C. to form a hydrogel.

[0085] Comparative Example Gel: Referring to the method of Example 3, 100 μL of the solution containing the peptide prepared in Comparative Example 2 (without the enzymatically cleaved short peptide sequence) was mixed with 100 μL of Component B solution, and in situ cross-linked at 37° C. to form a hydrogel.

[0086] (2) Release experiment: Place the two hydrogel samples mentioned above in 1.5 mL centrifuge tubes respectively. Prepare the release medium: (a) Tris-HCl buffer (pH 7.4) containing matrix metalloproteinase (MMP-2, final concentration 10 μg / mL); (b) Tris-HCl buffer (pH 7.4) without enzyme. 1.0 mL of the corresponding release medium was added to each centrifuge tube containing gel. Both the experimental and comparative gels were set up with and without enzyme. All tubes were placed in a thermostatted shaker at 37°C and gently shaken at 50 rpm.

[0087] (3) Sampling and Analysis: After 24 hours, all the release medium was aspirated from each centrifuge tube and centrifuged through an ultrafiltration tube (molecular weight cutoff 3 kDa) to remove any enzymes present, and the filtrate was collected. The concentration of the released peptide fragments containing the casein phosphopeptide core sequence in the filtrate was analyzed using a reversed-phase high-performance liquid chromatography system. The chromatographic conditions were the same as those described in Example 1.3. Based on the measured concentration and the total volume of the release medium, the cumulative release amount of the peptide fragments over 24 hours was calculated, and the cumulative release rate relative to the initial total amount of peptide in the gel was calculated.

[0088] (4) Repeated experiments: Three independent release experiments were performed for each condition.

[0089] Experimental data: According to the above experimental steps, the data obtained are recorded in the table below.

[0090] Table 3: Cumulative release rate of hydrogels under different conditions Results The experimental data above show that when the gel prepared in Example 1 was placed in a medium containing MMP-2, a cumulative release rate exceeding 65% was detected over 24 hours. In contrast, when the gel was placed in an enzyme-free medium, or when the gel prepared in Comparative Example 2, which lacks a specific enzyme cleavage sequence, was used (even in an enzyme-containing medium), the cumulative release rate was less than 6%. This result confirms that the release behavior of the peptide fragment is highly dependent on the presence of the specific enzyme and the predetermined cleavage site within the peptide chain.

[0091] The release mechanism is based on the fact that the active peptide molecule contains an amino acid sequence that is recognized and hydrolyzed by MMP-2. In the presence of MMP-2, the enzyme is able to enter the hydrogel network, contact, and catalyze the cleavage of peptide bonds within this specific sequence. This cleavage process causes a molecular fragment carrying the casein phosphopeptide core sequence to separate from the peptide backbone anchored to the gel network. Because this fragment has a smaller molecular weight and is no longer covalently bound to the gel network, it can be released from the gel into the surrounding medium by diffusion. In the absence of the enzyme or the cleavage sequence, this specific hydrolysis process cannot occur, resulting in the active fragment being continuously and stably fixed within the gel, exhibiting only minimal physical leakage.

[0092] This enzyme-triggered release characteristic is a core functional element of the present invention. It directly links the release of the active ingredient to specific pathological or physiological microenvironmental signals (i.e., overexpression of a specific enzyme). In this way, the release of the active ingredient is no longer a passive, time-dependent diffusion process, but rather a process controlled by the biochemical state of the target area. This mechanism ensures the selective release of the active fragment at the point of physiological need, providing the technical foundation for achieving effective biological concentrations at a specific time and location.

[0093] Test Example 4: Calcium ion chelation ability test Purpose of the experiment: This test example aims to determine whether the peptide fragments released from the hydrogel by enzymatic cleavage have their intended biological function, namely the ability to chelate calcium ions.

[0094] Experimental steps: (1) Sample Preparation: The peptide fragment solution released from the "Gel of Example 1" in the "MMP-2-containing" condition in the release experiment of Test Example 3 and subjected to ultrafiltration was collected. The exact concentration of the peptide fragment in this solution was determined by high-performance liquid chromatography. A Tris-HCl buffer solution without peptide was also prepared as a negative control.

[0095] (2) Instrument preparation: Use an ion meter equipped with a calcium ion selective electrode (Ca²⁺-ISE) and a reference electrode. Calibrate the electrode using a series of calcium chloride (CaCl²) standard solutions of known concentrations to establish the relationship between the potential response and the free calcium ion concentration.

[0096] (3) Chelating ability determination: a) Take 1.0mL of the above peptide fragment solution and place it in a constant temperature test cup, maintaining the temperature at 37°C. b) Immerse the calibrated calcium ion selective electrode and reference electrode in the solution, and after the potential reading stabilizes, record the initial free calcium ion concentration. c) Add 100μL of 10mM CaCl2 standard solution to the solution to increase the total calcium ion concentration in the system by a certain value. d) Gently stir the mixture and continuously monitor the potential changes. After the potential reading reaches stability again, record the final free calcium ion concentration at this time. e) Replace the peptide fragment solution with the Tris-HCl buffer of the negative control group and repeat steps a) to d).

[0097] (4) Data calculation: The amount of calcium ions chelated by the peptide fragment is calculated by subtracting the final free calcium ion increment measured from the total amount of calcium ions added. The amount of calcium ions chelated per unit mass of peptide is then calculated based on the concentration and volume of the peptide fragment.

[0098] (5) Repeated experiments: Each sample was tested three times independently.

[0099] Experimental data: According to the above experimental steps, the data obtained are recorded in the table below.

[0100] Table 4: Calcium chelating ability of released peptide fragments Result description: The experimental data above show that the peptide fragments released from the hydrogel via enzymatic hydrolysis exhibit significant calcium ion chelation, while the calcium ion binding capacity of the control buffer is negligible. This result functionally validates the design of the present invention: the molecular fragments released via enzyme-responsive cleavage possess their intended biochemical activity.

[0101] The structural basis for the realization of this chelating function lies in the casein phosphopeptide core sequence contained in the released fragment. The multiple phosphoserine residues in this sequence have their phosphate groups deprotonated under the physiological environment of pH 7.4, showing a high density of negative charges. These negatively charged phosphate groups can act as a binding agent for divalent cations (such as Ca 2+ ) binding sites, forming a stable chelate structure through electrostatic attraction and coordination, thereby capturing and binding the free calcium ions in the solution.

[0102] The results of this test not only demonstrated the effectiveness of the release observed in Test Example 3 but, more importantly, confirmed the biological functionality of the released substance. This demonstrates the integrity of the entire technical solution: an inactive precursor covalently anchored to the hydrogel network can, under the action of a specific enzyme, precisely cleave and release an active molecule with a specific biochemical function (such as mineral ion chelation). This entire process, from "immobilization" to "release" to "functional realization," provides a viable technical path for executing biological functions on demand within specific physiological microenvironments.

[0103] Test Example 5: Cytocompatibility and biological effect test Purpose of the experiment: This test case aims to evaluate the effect of peptide fragments released by enzymatic hydrolysis on the survival state of osteoblasts in a high calcium environment through in vitro cell experiments, thereby verifying their preset biological functions at the cellular level.

[0104] Experimental steps: (1) Cell culture: Mouse osteoblast precursor cell lines were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cells reached the logarithmic phase, they were trypsinized, passaged, and counted.

[0105] (2) Experimental grouping and sample treatment: Cells were seeded in 96-well plates at a density of 5×10³ per well and cultured for 24 hours until the cells adhered to the wall. Subsequently, the original culture medium was discarded and replaced with the following culture media with different treatments: Negative control group: normal α-MEM culture medium. High calcium control group: CaCl2 was additionally added to the α-MEM culture medium to make the final total calcium ion concentration in the culture medium 5mM. Experimental group: The peptide fragments released after enzymatic hydrolysis collected and purified in Test Example 3 were added to the high calcium (5mM) α-MEM culture medium to make the final concentration in the culture medium 100μg / mL.

[0106] (3) Cell incubation: The treated 96-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48 hours.

[0107] (4) Cell viability assay: After incubation, add 10 μL of CCK-8 solution to each well. Return the 96-well plate to the incubator and continue incubation for 2 hours. Measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader.

[0108] (5) Data processing: Taking the average OD value of the negative control group as 100%, the relative cell viability of the other groups was calculated. The formula is: Relative cell viability (%) = (OD value of experimental group / average OD value of negative control group) × 100; (6) Repeated experiment: Three replicate wells were set up for each experimental group.

[0109] Experimental data: According to the above experimental steps, the data obtained are recorded in the table below.

[0110] Table 5: Effects of released peptide fragments on cell viability in high calcium environment Results The experimental data above showed that compared with the negative control group under normal culture conditions, the cell viability of the high-calcium control group was significantly reduced, indicating that high concentrations of calcium ions inhibit the survival of osteoblasts. After adding the enzymatically released peptide fragments of the present invention to the high-calcium culture medium, the cell viability values ​​of the experimental group recovered significantly, significantly exceeding those of the high-calcium control group.

[0111] The intrinsic mechanism of this result is that excessive extracellular calcium ion concentration can cause osmotic pressure imbalance and ion homeostasis disorder, thereby producing toxic effects on cells. The active peptide fragments released by enzymatic hydrolysis in the present invention have a core sequence containing multiple negatively charged phosphate groups and have calcium ion chelating ability verified in Test Example 4. In a cell culture environment, these peptide fragments can capture and bind to excess free calcium ions in the culture medium, effectively reducing the actual free calcium ion concentration in the microenvironment in which the cells are located. In this way, the peptide fragments alleviate the cytotoxic stress caused by high calcium, provide cells with a more suitable living environment, and thus restore cell viability.

[0112] This test case biologically validates the ultimate functionality of the present invention's technical solution. It links the physicochemical properties demonstrated in previous test cases (gelation, bone targeting, enzyme-controlled release, and ion chelation) with the ultimate biological effect, fully demonstrating the overall system workflow: upon receiving a specific biological signal (an enzyme), an immobilized precursor peptide can release an active molecule with a specific biological function (modulating the local ionic environment) on demand, positively influencing cellular behavior. This provides direct experimental evidence for utilizing this system to in situ regulate mineral ion concentrations and improve cell survival conditions in pathological microenvironments such as bone defects.

[0113] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A casein-modified active peptide that promotes bone repair, characterized in that: The casein-modified active peptide is a linear peptide chain, and its structure comprises the following functional domains from the N-terminus to the C-terminus: N-terminally modified alkynyl functional group; A short peptide sequence that responds to the cleavage of specific enzymes in the bone repair microenvironment; a casein phosphopeptide core sequence derived from casein and comprising one or more phosphoserine residues; A bone-targeting sequence with specific affinity for skeletal hydroxyapatite.

2. A casein-modified active peptide for promoting bone repair according to claim 1, characterized in that: The bone-targeting sequence is a polyaspartic acid sequence or a polyglutamic acid sequence, and the sequence length is 6-12 amino acid residues.

3. The casein-modified active peptide for promoting bone repair according to claim 1, characterized in that: The enzyme-cleaved short peptide sequence is a specific substrate sequence of matrix metalloproteinase or cathepsin K.

4. The casein-modified active peptide for promoting bone repair according to claim 1, characterized in that: The amino acid sequence of the casein phosphopeptide core sequence is derived from αs1-casein or β-casein.

5. The casein-modified active peptide for promoting bone repair according to claim 1, characterized in that: The N-terminus modified alkynyl functional group is formed by covalently linking with propiolic acid or a non-natural amino acid having an alkynyl group.

6. A method for preparing a casein-modified active peptide that promotes bone repair, for preparing a casein-modified active peptide that promotes bone repair according to any one of claims 1 to 5, characterized in that: The following steps are involved: Using a solid-phase peptide synthesis method, the amino acids constituting the bone-targeting sequence, the casein phosphopeptide core sequence, and the enzymatically cleaved short peptide sequence are sequentially coupled and connected on a solid-phase resin carrier in order from the C-terminus to the N-terminus, and a chemical group with an alkynyl functional group is introduced at the N-terminus of the peptide chain; Finally, the synthesized intact peptide chain is cleaved from the resin, deprotected, and purified.

7. The method for preparing a casein-modified active peptide for promoting bone repair according to claim 6, characterized in that: The phosphoserine residue in the core sequence of the casein phosphopeptide is introduced during the solid phase synthesis process by coupling an Fmoc-serine derivative that has been pre-protected with a phosphate group.

8. The method for preparing a casein-modified active peptide for promoting bone repair according to claim 6, characterized in that: The step of introducing a chemical group with an alkynyl functional group into the N-terminus of the peptide chain is specifically as follows: After the last amino acid constituting the enzyme-cleaved short peptide sequence is coupled and its N-terminal protecting group is removed, the free N-terminal amino group of the resulting peptide chain connected to the resin is reacted with a carboxylic acid containing an alkynyl group or its active ester.

9. The method for preparing a casein-modified active peptide for promoting bone repair according to claim 6, characterized in that: The purification step includes separation and purification using reverse phase high performance liquid chromatography.

10. The method for preparing a casein-modified active peptide for promoting bone repair according to claim 6, characterized in that: The steps of cleavage and removal of protecting groups are performed using a cleavage reagent containing trifluoroacetic acid at room temperature.

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