Preparation method of spleen extract

Abstract

The invention relates to the technical field of polypeptide preparation, and discloses a spleen extract preparation method, which comprises: carrying out pretreatment, homogenization, freeze thawing crushing, precipitation separation and ultrafiltration on a mammal spleen tissue to obtain a spleen polypeptide extract; coupling the spleen polypeptide extract with ADC through a chemical coupling bridge to form a targeting compound; preparing an antioxidant-basic amino acid compound; mixing the coupling compound with a wall material solution containing a functional filling agent and an antioxidant-alkaline amino acid compound, and carrying out microencapsulation embedding; carrying out freeze drying on the microcapsules; and spraying a chitosan-sodium carboxymethyl cellulose polyelectrolyte compound on the surface of the freeze-dried microcapsule to carry out hydrophilic modification. The antioxidant-basic amino acid compound is used for blocking intermolecular chemical crosslinking, the cyclodextrin inclusion effect is used for preventing aggregation, and the polysaccharide composite hydrophilic layer is used for providing environmental tolerance, so that the chemical crosslinking rate of the product is reduced by 80% or above, and the aggregation rate is reduced to 5% or below.

Description

Technical Field

[0001] This invention relates to the field of polypeptide preparation technology, and more specifically, to a method for preparing a spleen extract. Background Technology

[0002] The spleen is an important immune organ in mammals, containing abundant bioactive polypeptides. Current techniques typically involve extracting spleen polypeptides through tissue pretreatment, homogenization, freeze-thaw disruption, precipitation separation, and ultrafiltration, followed by chemical conjugation to form targeted complexes with an anti-inflammatory drug (ADC). To address the issues of poor stability and difficulties in storage and transportation of liquid-conjugated complexes, existing techniques employ microencapsulation and freeze-drying to prepare solid formulations of the conjugated complexes.

[0003] However, existing microencapsulated lyophilized formulations still have the following technical problems: First, the peptides in the spleen polypeptide-ADC coupling complex contain a large number of free amino and carboxyl groups. During storage, these active groups undergo uncontrolled intermolecular cross-linking reactions, including Maillard reactions (reaction of amino groups with carboxyl or reducing sugars) and Schiff base formation (condensation of amino and aldehyde groups), causing the coupling complex to aggregate into large polymer molecules and become inactive. Even with physical dilution using inert fillers, it is difficult to completely prevent this chemical cross-linking reaction.

[0004] Second, the coupling complex is susceptible to oxidative degradation by various reactive oxygen species during storage. Although existing technologies add antioxidants, traditional single antioxidants mainly target oxygen free radicals and have limited ability to scavenge other reactive oxygen species such as hydrogen peroxide and hypochlorous acid. Moreover, the antioxidants themselves are "sacrificial" protectants and are gradually consumed, resulting in insufficient long-term protection.

[0005] Third, when the concentration of active ingredients within the microcapsules is high, the shortened intermolecular distance leads to an increased collision frequency, making physical aggregation more likely. Existing technologies using inert fillers such as lactose and microcrystalline cellulose only provide simple spatial dilution and lack active anti-aggregation functions.

[0006] Fourth, the strong hydrophobicity of the surface of freeze-dried microcapsules makes resolvability difficult. Existing technologies improve resolvability by spraying a single hydrophilic polymer such as PEG or sodium carboxymethyl cellulose onto the surface. However, such a single hydrophilic modified layer is not stable enough in high-salt or pH-changing application environments. It will swell or shrink due to changes in ionic strength or pH, resulting in unstable resolvability. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing a spleen extract, comprising pretreatment of mammalian spleen tissue, homogenization, freeze-thaw disruption, precipitation separation, and ultrafiltration to obtain a spleen polypeptide extract. The method further includes the following steps: [Further details on chemically coupling the spleen polypeptide extract with an ADC to form a spleen polypeptide-ADC coupling complex are also provided.] An antioxidant-basic amino acid complex was prepared, wherein the complex was formed by vitamin C and lysine, vitamin E and arginine, and tea polyphenols and basic amino acids, respectively. An embedding solution was prepared by mixing the spleen polypeptide-ADC coupling complex with a wall material solution, a functional filler combination, and the antioxidant-basic amino acid complex. The functional filler combination included cyclodextrin, lactose, and microcrystalline cellulose, wherein cyclodextrin accounted for 40-60% of the total filler. The embedding solution was made into microcapsules and then freeze-dried to obtain lyophilized microcapsules; a chitosan-carboxymethyl cellulose sodium polyelectrolyte complex was sprayed onto the surface of the lyophilized microcapsules to form a hydrophilic modified layer.

[0008] Preferably, the preparation steps of the antioxidant-basic amino acid complex include: mixing vitamin C powder and L-lysine powder at a mass ratio of 1:0.5-2:1, adding deionized water and stirring to dissolve at room temperature for 30-60 minutes to form a vitamin C-lysine complex.

[0009] Preferably, the preparation steps of the antioxidant-basic amino acid complex further include: mixing vitamin E and L-arginine at a mass ratio of 1:1-2:1, adding 5-10% of the mass of vitamin E as an emulsifier, and treating under high-speed stirring conditions for 10-20 minutes to form a vitamin E-arginine emulsion complex.

[0010] Preferably, in the functional filler combination, cyclodextrin accounts for 40-60%, lactose accounts for 20-30%, and microcrystalline cellulose accounts for 10-30%.

[0011] Preferably, the cyclodextrin is β-cyclodextrin.

[0012] Preferably, the preparation steps of the chitosan-sodium carboxymethyl cellulose polyelectrolyte complex include: dissolving chitosan with a degree of deacetylation ≥85% in dilute acetic acid solution to prepare a chitosan solution with a concentration of 0.5-2%; dissolving sodium carboxymethyl cellulose with a degree of substitution ≥0.8 in deionized water to prepare a sodium carboxymethyl cellulose solution with a concentration of 0.5-2%; mixing the chitosan solution and the sodium carboxymethyl cellulose solution at a mass ratio of 1:0.8-1.5:1 and stirring for 10-20 minutes to form a polyelectrolyte complex suspension.

[0013] Preferably, the total amount of the antioxidant-basic amino acid complex added is 5-15% of the mass of the spleen polypeptide-ADC coupling complex.

[0014] Preferably, the total amount of the functional filler combination is 2-5 times the mass of the spleen polypeptide-ADC conjugate complex.

[0015] Preferably, the amount of the polyelectrolyte complex suspension sprayed is 3-8% of the mass of the lyophilized microcapsules.

[0016] Preferably, the wall material solution is selected from sodium alginate solution, chitosan solution or gelatin solution; the microcapsule forming is performed by drop addition, spraying or emulsification.

[0017] The beneficial effects of this invention are as follows: Breakthrough in chemical stability: Basic amino acids competitively block the cross-linking reaction between spleen polypeptide molecules, reducing the chemical cross-linking rate by more than 80% (from 43.5% to 11.2%), with a free group retention rate of 87-89%, fundamentally solving the problem of chemical aggregation and inactivation. Full-spectrum antioxidant protection: Water-soluble vitamin C, fat-soluble vitamin E, and tea polyphenols form a triple synergistic system, achieving a scavenging rate of over 90% for various reactive oxygen species such as oxygen free radicals, hydrogen peroxide, and hypochlorous acid. Antioxidant stability is 5-7 times higher than that of a single antioxidant and 10-15 times higher than that without a protective agent. Functional anti-aggregation: β-cyclodextrin actively prevents molecular aggregation through host-guest inclusion complexation, reducing the aggregation rate from 30-40% using traditional methods to below 5%, a reduction of over 85%, far superior to the passive dilution effect of inert fillers. Strong environmental adaptability: The polyelectrolyte complex forms a highly stable network through electrostatic cross-linking, exhibiting stable performance over a wide range of pH 4-9 and salt concentrations of 0-500 mM (coefficient of variation only 6.8%), overcoming the environmental sensitivity defects of single hydrophilic polymers. Excellent reconstitution performance: Surface tension is significantly reduced, shortening the reconstitution time from 15-20 minutes to 3-5 minutes, a reduction of 70-80%. Clarity after reconstitution is >95%, greatly improving ease of use. Ultra-long shelf life: Multiple synergistic protections extend the shelf life from 6-12 months to over 24 months (an extension of 100-200%). After 24 months of storage at room temperature, the activity retention rate is >85%, the chemical degradation rate is reduced by over 90%, and the integrity of the coupling complex is >98%. Attached Figure Description

[0018] Figure 1 These are the chemical crosslinking rate change curves of different samples of the present invention during storage at 40°C; Figure 2 This is a comparison of the free radical retention rates of various samples after 28 days of storage according to the present invention; Figure 3 This describes the change in aggregation rate over time after reconstitution of different samples according to the present invention. Figure 4 This invention describes the change in turbidity over time after reconstitution of different samples. Figure 5 This is a thermogram of the resolution time of sample G under different pH and salt concentration conditions according to the present invention; Figure 6 This is a comparison of the reconstitution time of different samples in the pH range of 4-9. Detailed Implementation

[0019] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0020] Example 1 This embodiment proposes a method for preparing a spleen extract, including pretreatment of mammalian spleen tissue, homogenization, freeze-thaw disruption, precipitation separation, and ultrafiltration to obtain a spleen polypeptide extract. The method further includes the following steps: Chemically coupling the spleen polypeptide extract with an ADC to form a spleen polypeptide-ADC coupling complex. An antioxidant-basic amino acid complex was prepared, wherein the complex was formed by vitamin C and lysine, vitamin E and arginine, and tea polyphenols and basic amino acids, respectively. An embedding solution was prepared by mixing the spleen polypeptide-ADC coupling complex with a wall material solution, a functional filler combination, and the antioxidant-basic amino acid complex. The functional filler combination included cyclodextrin, lactose, and microcrystalline cellulose, wherein cyclodextrin accounted for 50% of the total filler. The embedding solution was made into microcapsules and then freeze-dried to obtain lyophilized microcapsules; a chitosan-carboxymethyl cellulose sodium polyelectrolyte complex was sprayed onto the surface of the lyophilized microcapsules to form a hydrophilic modified layer.

[0021] in: The preparation steps of the antioxidant-basic amino acid complex include: mixing vitamin C powder and L-lysine powder at a mass ratio of 1:0.8, adding deionized water and stirring to dissolve at room temperature for 45 minutes to form a vitamin C-lysine complex.

[0022] The preparation steps of the antioxidant-basic amino acid complex also include: mixing vitamin E and L-arginine at a mass ratio of 1.5:1, adding 8% by mass of vitamin E emulsifier, and treating under high-speed stirring conditions for 15 minutes to form a vitamin E-arginine emulsion complex.

[0023] In the functional filler composition, cyclodextrin accounts for 50%, lactose accounts for 25%, and microcrystalline cellulose accounts for 20%.

[0024] The cyclodextrin is β-cyclodextrin.

[0025] The preparation steps of the chitosan-sodium carboxymethyl cellulose polyelectrolyte complex include: dissolving chitosan with a degree of deacetylation ≥85% in dilute acetic acid solution to prepare a chitosan solution with a concentration of 1.2%; dissolving sodium carboxymethyl cellulose with a degree of substitution ≥0.8 in deionized water to prepare a sodium carboxymethyl cellulose solution with a concentration of 1.2%; mixing the chitosan solution and the sodium carboxymethyl cellulose solution at a mass ratio of 1.2:0.9 and stirring for 15 minutes to form a polyelectrolyte complex suspension.

[0026] The total amount of antioxidant-basic amino acid complex added is 10% of the mass of the spleen polypeptide-ADC conjugate complex.

[0027] The total amount of the functional filler combination added is 3 times the mass of the spleen polypeptide-ADC conjugate complex.

[0028] The amount of polyelectrolyte complex suspension sprayed is 5% of the mass of the lyophilized microcapsules.

[0029] The wall material solution is selected from sodium alginate solution; the microcapsule forming is performed by drop addition.

[0030] Example 2 The difference between this embodiment and Embodiment 1 is that: Cyclodextrin accounts for 40% of the total filler.

[0031] Vitamin C powder and L-lysine powder were mixed at a mass ratio of 1:0.5, and deionized water was added and stirred and dissolved at room temperature for 30 minutes to form a vitamin C-lysine complex.

[0032] Vitamin E and L-arginine were mixed at a mass ratio of 1:1, and an emulsifier of 5% of the mass of vitamin E was added. The mixture was then treated under high-speed stirring for 10 minutes to form a vitamin E-arginine emulsion complex.

[0033] In the functional filler composition, cyclodextrin accounts for 40%, lactose accounts for 20%, and microcrystalline cellulose accounts for 10%.

[0034] A 0.5% chitosan solution was prepared by dissolving chitosan with a degree of deacetylation ≥85% in dilute acetic acid solution; a 0.5% sodium carboxymethyl cellulose solution was prepared by dissolving sodium carboxymethyl cellulose with a degree of substitution ≥0.8 in deionized water; the chitosan solution and the sodium carboxymethyl cellulose solution were mixed at a mass ratio of 1:0.8 and stirred for 10 minutes to form a polyelectrolyte complex suspension.

[0035] The total amount of antioxidant-basic amino acid complex added is 5% of the mass of the spleen polypeptide-ADC coupling complex.

[0036] The total amount of the functional filler combination added is twice the mass of the spleen polypeptide-ADC conjugate complex.

[0037] The amount of polyelectrolyte complex suspension sprayed is 3% of the mass of the lyophilized microcapsules.

[0038] The wall material solution is selected from chitosan solution.

[0039] The microcapsule formation is performed using a spray method.

[0040] Example 3 The difference between this embodiment and Embodiment 1 is that: Cyclodextrin accounts for 60% of the total filler.

[0041] Vitamin C powder and L-lysine powder were mixed at a mass ratio of 12:1, and deionized water was added and stirred at room temperature for 60 minutes to form a vitamin C-lysine complex.

[0042] Vitamin E and L-arginine were mixed at a mass ratio of 2:1, and an emulsifier of 10% of the mass of vitamin E was added. The mixture was then treated under high-speed stirring for 20 minutes to form a vitamin E-arginine emulsion complex.

[0043] In the functional filler composition, cyclodextrin accounts for 60%, lactose accounts for 30%, and microcrystalline cellulose accounts for 30%.

[0044] A 2% chitosan solution was prepared by dissolving chitosan with a degree of deacetylation ≥85% in dilute acetic acid solution; a 2% sodium carboxymethyl cellulose solution was prepared by dissolving sodium carboxymethyl cellulose with a degree of substitution ≥0.8 in deionized water; the chitosan solution and the sodium carboxymethyl cellulose solution were mixed at a mass ratio of 1.5:1 and stirred for 20 minutes to form a polyelectrolyte complex suspension.

[0045] The total amount of antioxidant-basic amino acid complex added is 15% of the mass of the spleen polypeptide-ADC conjugate complex.

[0046] The total amount of the functional filler combination added is 5 times the mass of the spleen polypeptide-ADC conjugate complex.

[0047] The amount of polyelectrolyte complex suspension sprayed is 8% of the mass of the lyophilized microcapsules.

[0048] The wall material solution is selected from gelatin solution.

[0049] Microcapsule formation employs an emulsification method.

[0050] Example 4 This embodiment presents a method for preparing spleen extract, including the following specific implementation steps: Step 1: Extraction and purification of spleen polypeptides This step uses conventional tissue extraction technology to obtain high-purity spleen polypeptide extract, including the following operations: (1) Tissue pretreatment: Fresh mammalian spleen tissue was taken, preferably porcine spleen tissue (due to its stable source and high polypeptide content). Non-target tissues such as fat and fascia were removed. The tissue was rinsed with physiological saline (0.9% NaCl solution) until no blood was found, and then drained for later use. The treatment process was carried out at 4°C to prevent enzymatic hydrolysis and oxidation.

[0051] (2) Homogenization: The pretreated spleen tissue was chopped into pieces about 5-10 mm in size and added to phosphate-buffered saline (PBS, pH 7.2-7.4, preferably pH 7.3). The tissue weight to buffer volume ratio was 1:3-5 (preferably 1:4, i.e., 100 g of tissue to 400 mL of buffer). The homogenate was homogenized in a homogenizer at 8000-12000 rpm (preferably 10000 rpm) for 2-3 minutes (preferably 2.5 minutes) to obtain a homogenized tissue homogenate. The homogenization process was carried out under ice bath conditions to prevent peptide degradation caused by temperature rise.

[0052] (3) Freeze-thaw disruption: Freeze the homogenate at -80°C for 4-6 hours (preferably 5 hours), then thaw it in a 37°C water bath for 30-45 minutes (preferably 40 minutes). This constitutes one freeze-thaw cycle. Repeat 3-5 freeze-thaw cycles (preferably 4 cycles) to fully disrupt cell membranes and organelles through the formation and melting of ice crystals, releasing intracellular polypeptides. Gently mix after each freeze-thaw cycle to ensure uniform freeze-thaw effect.

[0053] (4) Separation of sedimentation: Centrifuge the homogenate after freeze-thaw disruption at 4°C and 8000-12000 rpm (preferably 10000 rpm) for 30-45 minutes (preferably 35 minutes) to remove cell debris, insoluble proteins and macromolecular impurities, and collect the supernatant. To avoid resuspension of the sediment, keep the nozzle of the suction tube 1-2 cm away from the sediment layer when aspirating the supernatant.

[0054] (5) Ultrafiltration purification: The supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (preferably a polyethersulfone membrane) at a pressure of 0.1-0.3 MPa (preferably 0.2 MPa). Large molecular impurities (proteins with a molecular weight > 10 kDa) are retained, and small molecule active peptides with a molecular weight less than 10 kDa are enriched in the permeate. The ultrafiltration process is carried out at 4°C. The permeate is periodically replenished to 1 / 3-1 / 2 of its original volume for concentration and washing, and this process is repeated 3-5 times (preferably 4 times) to improve the recovery rate and purity of small molecule peptides.

[0055] (6) Further purification: Dialyze the ultrafiltration permeate (preferably by dialysis) using a dialysis bag with a molecular weight cutoff of 1 kDa. Dialyze with deionized water at 4°C for 24-48 hours (preferably 36 hours), changing the dialysis solution every 8-12 hours (preferably 10 hours) to remove salts and small molecule impurities with a molecular weight less than 1 kDa. The dialyzed solution is freeze-dried to obtain a high-purity spleen polypeptide extract powder with a polypeptide content ≥85%. This powder should be stored at -20°C protected from light and reconstituted in PBS buffer (pH 7.2-7.4) before use.

[0056] Step 2: Preparation of ADC Coupling Complex The spleen polypeptide extract powder obtained in step one was reconstituted in PBS buffer to prepare a spleen polypeptide solution with a concentration of 5-10 mg / mL (preferably 8 mg / mL). This solution was then chemically coupled with an ADC (antibody-drug conjugate) to form a spleen polypeptide-ADC targeting complex.

[0057] Commonly used coupling methods include EDC / NHS chemical coupling, maleimide-thiol coupling, and click chemical coupling. EDC / NHS chemical coupling is preferred because it offers mild reaction conditions, high coupling efficiency, and easy removal of byproducts.

[0058] The preferred EDC / NHS chemical coupling method is operated as follows: (1) Activation step: Adjust the pH of the spleen polypeptide solution to 5.0-6.0 (preferably pH 5.5, using 0.5M MES buffer), add EDC (molar ratio of EDC to carboxyl group is 2-5:1, preferably 3:1) and NHS (molar ratio of NHS to carboxyl group is 2-5:1, preferably 3:1), and stir the reaction at 4-25℃ (preferably room temperature 20℃) for 30-60 minutes (preferably 45 minutes). EDC first reacts with the carboxyl group of the spleen polypeptide to form an unstable O-acyl isourea intermediate, and NHS then replaces the isourea to form a stable NHS active ester.

[0059] (2) Coupling step: Add ADC solution (ADC concentration of 2-5 mg / mL, preferably 3 mg / mL) to the activated spleen polypeptide solution, with a spleen polypeptide to ADC mass ratio of 1-3:1 (preferably 2:1). Adjust the pH to 7.0-7.5 (preferably pH 7.2) and react slowly with stirring at 4-25℃ (preferably 4℃) for 2-4 hours (preferably 3 hours). The amino groups on the ADC nucleophilically attack the active NHS ester, removing NHS to form a stable amide bond, thus completing the coupling reaction. Low temperature reaction can reduce side reactions and protein denaturation.

[0060] Purification steps: After the coupling reaction is complete, the coupling complex is purified by the following methods, with dialysis being the preferred method: The coupling reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 30 kDa (the molecular weight of the spleen polypeptide-ADC coupling complex is usually greater than 30 kDa). Dialysis was performed with PBS buffer (pH 7.2-7.4) at 4°C for 48-72 hours (preferably 60 hours), changing the dialysis buffer every 12 hours to remove unreacted spleen polypeptide, EDC, NHS, and their byproducts. The dialyzed solution was then filtered through a 0.22 μm sterile filter to obtain a purified spleen polypeptide-ADC coupling complex solution. This solution concentration is generally 3-8 mg / mL (preferably 5 mg / mL) and should be used immediately for subsequent steps, or stored at 4°C for no more than 24 hours.

[0061] Coupling control: The coupling reaction typically employs moderate reaction conditions to control the coupling of only a portion of the active groups (amino or carboxyl groups) on the spleen polypeptide molecule. Depending on the molar ratio of spleen polypeptide to ADC (preferably 2:1) and the reaction conditions, an average of 1-3 (preferably 2) spleen polypeptide molecules are coupled to each ADC molecule, preserving most of the free amino and carboxyl groups to maintain the bioactivity of the coupling complex and the blocking effect of basic amino acids in subsequent steps. Therefore, the coupled spleen polypeptide-ADC complex still contains a large number of unreacted amino and carboxyl groups.

[0062] This step employs coupling techniques known in the art, and those skilled in the art can select appropriate coupling strategies based on the specific structural characteristics of the spleen polypeptide and ADC.

[0063] Step 3: Preparation of antioxidant-basic amino acid complex This step is one of the core steps. By forming a complex with antioxidants and basic amino acids, it achieves the dual protective function of "antioxidation + anti-crosslinking".

[0064] (I) Technical Principles The polypeptide moiety in the spleen polypeptide-ADC coupling complex contains a large number of free amino and carboxyl groups. During storage, these active groups undergo uncontrolled intermolecular cross-linking reactions: Dehydration condensation: Under certain conditions, amino and carboxyl groups undergo dehydration to form amide bonds (peptide bonds), leading to cross-linking between polypeptide molecules; Maillard reaction: Amino groups react with reducing sugars or aldehydes produced by oxidation in the storage environment to generate brown polymers; Schiff base formation: Amino groups condense with aldehydes produced by lipid oxidation to form C=N double bonds, leading to cross-linking between molecules.

[0065] The basic amino acids (lysine and arginine) introduced in this step contain multiple amino groups. For example, lysine has two amino groups, α-amino and ε-amino, while arginine has an α-amino group and a guanidinyl group. When basic amino acids form complexes with antioxidants, they mainly bind through hydrogen bonds and electrostatic interactions. This non-covalent interaction does not completely occupy all the active sites of the basic amino acids. Therefore, some free amino groups of the basic amino acids can still interact with the carboxyl groups of the spleen polypeptide to form ion pairs or hydrogen bonds, thereby competitively occupying the reaction sites of the polypeptide carboxyl groups and preventing covalent cross-linking reactions between polypeptide molecules (Maillard reaction and Schiff base formation). At the same time, basic amino acids themselves have electron donor properties, which can scavenge free radicals and produce a synergistic antioxidant effect with antioxidants.

[0066] Pre-forming a complex between antioxidants and basic amino acids allows for tight spatial binding, improving synergistic efficiency and facilitating uniform dispersion during subsequent microencapsulation. The basic amino acids in the complex maintain their interaction with both the antioxidant and spleen peptides, thus achieving dual functions of anti-oxidation and anti-crosslinking.

[0067] (2) Specific operations Preparation of Vitamin C-Lysine Complex Take vitamin C (ascorbic acid) powder and L-lysine powder, and mix them at a mass ratio of 1:0.5-2:1 (preferably 1:1). Add an appropriate amount of deionized water (the amount should be such that the final vitamin C concentration in the solution is 50-100 mg / mL, preferably 75 mg / mL), and stir at 300-500 rpm (preferably 400 rpm) at room temperature (20-25℃, preferably 22℃) to dissolve and form a mixed solution. The carboxyl group of vitamin C and the amino group of lysine form a complex through hydrogen bonding and electrostatic interaction. After stirring for 30-60 minutes (preferably 45 minutes), the solution becomes slightly yellow and transparent, with a pH of approximately 5.5-6.5, indicating that the complex has formed. This complex is water-soluble and can be used directly in subsequent steps.

[0068] Lysine was chosen based on the presence of two amino groups (α-amino and ε-amino), with the ε-amino group having a higher pKa (approximately 10.5). At neutral pH, it exists primarily in a protonated form, which can form ion pairs with the carboxyl groups of spleen polypeptides, effectively blocking cross-linking reactions.

[0069] Preparation of Vitamin E-Arginine Complex Take vitamin E (α-tocopherol) oily liquid and L-arginine powder, and mix them at a mass ratio of 1:1-2:1 (preferably 1:1.5). Since vitamin E is fat-soluble, an emulsifier (preferably Tween-80, at 5-10% of the mass of vitamin E, preferably 8%) needs to be added. First, mix vitamin E and Tween-80 and stir at room temperature for 5-10 minutes (preferably 8 minutes) to ensure thorough mixing. Dissolve L-arginine powder in deionized water to prepare an arginine aqueous solution with a concentration of 50-100 mg / mL (preferably 80 mg / mL). Slowly add the arginine aqueous solution dropwise to the vitamin E-Tween-80 mixture (dropping rate 1-3 mL / min, preferably 2 mL / min), while stirring at high speed (8000-10000 rpm, preferably 9000 rpm), and treat for 10-20 minutes (preferably 15 minutes) to form a milky white O / W emulsion.

[0070] If ultrasound-assisted emulsification is used, mild ultrasound conditions should be used: power 100-200W (preferably 150W), frequency 20-40kHz (preferably 30kHz), intermittent ultrasound (on for 5 seconds and off for 5 seconds), processing time 5-10 minutes (preferably 8 minutes). Avoid excessive ultrasound which may cause degradation of arginine or vitamin E. At the same time, control the temperature during emulsification to not exceed 30℃ (preferably 25℃).

[0071] In the resulting emulsion, vitamin E is dispersed in the oil phase as tiny oil droplets (0.1-1 μm in diameter), while arginine is dissolved in the continuous aqueous phase. Tween-80 molecules are oriented at the oil-water interface, with their hydrophobic ends (fatty acid chains) facing the oil phase and their hydrophilic ends (polyoxyethylene chains) facing the aqueous phase, stabilizing the emulsion system. Arginine molecules interact with the polar groups of Tween-80 through their positively charged guanidine groups and are spatially close to the vitamin E oil droplets, forming a stable vitamin E-arginine emulsion complex. The pH of the emulsion is approximately 7.5-8.5.

[0072] Arginine was chosen because it has a guanidinium side chain (pKa of about 12.5), is almost completely protonated at physiological pH, carries a positive charge, and can interact with the negatively charged region of spleen polypeptide to block cross-linking; at the same time, arginine has strong antioxidant capacity and can scavenge peroxides and hydroxyl radicals.

[0073] Preparation of tea polyphenol-basic amino acid complex Take tea polyphenol powder (mainly catechin compounds, purity ≥95%) and basic amino acids (preferably L-lysine, due to its stronger hydrogen bonding with tea polyphenols) and mix them at a mass ratio of 1:0.8-1.5:1 (preferably 1:1). Add deionized water (to achieve a tea polyphenol concentration of 30-60 mg / mL, preferably 45 mg / mL), and dissolve the polyphenols by stirring at 200-400 rpm (preferably 300 rpm) for 30-45 minutes (preferably 38 minutes) under gentle heating at 50-60℃ (preferably 55℃). Hydrogen bonds form between the phenolic hydroxyl groups in the tea polyphenol molecules and the amino groups of lysine, while hydrophobic interactions exist between the aromatic rings of the polyphenols and the alkyl side chains of lysine, forming a stable complex. The solution is brownish-yellow and transparent, with a pH of approximately 6.0-7.0. Cool to room temperature (20-25℃) before use to avoid prolonged high-temperature heating that could oxidize the tea polyphenols.

[0074] The selection of tea polyphenols is based on their multiple phenolic hydroxyl groups, which have strong hydrogen donation and free radical scavenging capabilities. They can scavenge reactive oxygen species such as hydroxyl radicals, superoxide anions, and hydrogen peroxide, and can supplement the antioxidant spectrum that vitamin C and vitamin E cannot cover.

[0075] The three antioxidant-basic amino acid complexes prepared should be used immediately, or stored for a short period (not exceeding 24 hours) under refrigeration at 4°C, and protected from light to prevent antioxidant degradation.

[0076] (III) Synergistic effect of the complex The three antioxidant-basic amino acid complexes formed have the following synergistic effects: Anti-crosslinking effect: The free amino groups of basic amino acids competitively bind to the carboxyl groups of spleen polypeptides, blocking the intermolecular Maillard reaction and Schiff base formation, reducing the chemical crosslinking rate by more than 80%.

[0077] Full-spectrum antioxidant effect: Vitamin C (water-soluble) protects the hydrophilic region of the coupling complex, Vitamin E (lipid-soluble) protects the hydrophobic region, and tea polyphenols scavenge various reactive oxygen species, forming a three-dimensional antioxidant network of "water phase-oil phase-reactive oxygen spectrum" with a scavenging rate of over 90%.

[0078] Synergistic effect: The antioxidant capacity of basic amino acids themselves is combined with that of traditional antioxidants, resulting in an overall antioxidant capacity that is 5-7 times higher than that of a single antioxidant.

[0079] Stabilizing and dispersing effect: The formation of the complex enhances the stability and dispersibility of the antioxidant in the aqueous phase or emulsion, facilitating uniform distribution during subsequent microencapsulation.

[0080] (iv) Differences from existing technologies Existing technologies typically involve directly adding antioxidants (such as vitamin C and vitamin E) to microencapsulation systems, which only provide antioxidant function and cannot prevent chemical cross-linking reactions between peptide molecules. This innovative step forms a complex with antioxidants and basic amino acids. The free amino groups of the basic amino acids achieve anti-cross-linking function, while the antioxidant capacity of the basic amino acids enhances the overall antioxidant effect, achieving a synergistic effect of "1+1>2". This bifunctional complex is not found in existing technologies.

[0081] Step 4: Microencapsulation and embedding This step employs microencapsulation technology to embed the spleen polypeptide-ADC coupling complex within the wall material, forming a microcapsule structure. The innovation lies in the introduction of a functional filler combination primarily composed of cyclodextrin, and the simultaneous embedding of the antioxidant-basic amino acid complex prepared in step three.

[0082] (1) Preparation of wall material solution Natural polymer materials such as sodium alginate, chitosan, or gelatin are selected as wall materials. Sodium alginate (molecular weight 200,000-400,000 Da, preferably medium viscosity sodium alginate of 300,000 Da) is preferred because it has good biocompatibility, fast gelation speed, and good microcapsule formation properties.

[0083] Sodium alginate powder is slowly added to deionized water (addition rate 0.5-1 g / min, preferably 0.8 g / min) while stirring at 200-400 rpm (preferably 300 rpm). The solution is fully dissolved at room temperature (20-25℃, preferably 22℃) for 2-4 hours (preferably 3 hours) to prepare a 2-4% (w / v, preferably 3% w / v) sodium alginate solution. The solution is stirred until it is uniformly transparent, free of obvious particles or lumps, and has a uniform viscosity. If a small number of bubbles are present, they can be allowed to stand for 30-60 minutes (preferably 45 minutes) to allow them to disappear naturally, or left to stand overnight in a refrigerator at 4℃. Sodium alginate, as a wall material, can rapidly gel through an "egg-box" structure under the action of calcium ions, forming a stable microcapsule structure. The prepared wall material solution should be used within 24 hours and stored at 4℃.

[0084] (ii) Addition of functional fillers This step is one of the core steps, using a combination of functional fillers, primarily cyclodextrin, to replace traditional inert fillers.

[0085] Filler composition The functional filler mixture is prepared according to the following mass ratios: cyclodextrin (α-cyclodextrin or β-cyclodextrin): 40-60% (preferably 50%); lactose: 20-30% (preferably 25%); microcrystalline cellulose: 10-30% (preferably 25%).

[0086] Selection and Mechanism of Action of Cyclodextrin Innovation: Cyclodextrin is selected as the main filler (accounting for 40-60%), which is different from the existing technology that only uses inert fillers such as lactose and microcrystalline cellulose.

[0087] Cyclodextrins are cyclic oligosaccharides formed by glucose units linked by α-1,4-glycosidic bonds, possessing a unique truncated conical-cylindrical structure: a hydrophilic outer surface (hydroxyl groups facing outwards) and a hydrophobic inner lumen (hydrogen atoms facing inwards). This structure endows cyclodextrins with inclusion-encapsulating capabilities. Hydrophobic inclusion complex: The hydrophobic cavity of cyclodextrin can encapsulate lipid-soluble molecules (such as vitamin E) and hydrophobic fragments of spleen polypeptides (such as regions containing hydrophobic amino acids such as phenylalanine, tryptophan, and tyrosine), forming stable host-guest inclusion complexes. The encapsulated molecules are locked within the cavity, significantly reducing direct contact and collisions between molecules, thus preventing aggregation.

[0088] Functional anti-aggregation: Unlike lactose and microcrystalline cellulose, which only provide passive spatial dilution, cyclodextrin actively prevents aggregation through host-guest interactions. When the hydrophobic region of the spleen polypeptide-ADC coupling complex is encapsulated by cyclodextrin, the hydrophobicity of the molecular surface decreases, the hydrophilicity increases, the hydration layer thickens, the hydrophobic aggregation driving force between molecules weakens, and the aggregation rate can be reduced to below 5%.

[0089] Enhanced stability: The formation of inclusion compounds provides physical protection to the included molecules, isolating them from external oxygen and reactive oxygen species, thus enhancing their oxidation stability; at the same time, the thermal and light stability of inclusion compounds is also superior to that of free molecules.

[0090] α-Cyclodextrin (composed of 6 glucose units, with a cavity diameter of approximately 5-8 Å) is suitable for encapsulating small molecules and short peptide chains; β-cyclodextrin (composed of 7 glucose units, with a cavity diameter of approximately 6-10 Å) is suitable for encapsulating medium-sized molecules and hydrophobic amino acid side chains. This embodiment prefers β-cyclodextrin because its cavity size matches well with the molecular size of the hydrophobic fragment of the spleen polypeptide and vitamin E.

[0091] Lactose and microcrystalline cellulose, as traditional inert fillers, play a supporting role: Lactose (20-30%): It dissolves rapidly and readily during reconstitution, creating a local osmotic pressure gradient that promotes water diffusion into the microcapsules and accelerates the overall reconstitution rate. Simultaneously, lactose fills the spaces not covered by cyclodextrin, providing additional dilution.

[0092] Microcrystalline cellulose (10-30%): an insoluble fiber that provides mechanical support for the microcapsules, enhances their compressive strength and breakage resistance, and prevents them from collapsing or breaking during freeze-drying and storage.

[0093] The combined use of the three fillers achieves a synergistic effect of "functional inclusion (cyclodextrin) + rapid dissolution (lactose) + mechanical support (microcrystalline cellulose)," providing both active anti-aggregation function and maintaining good physical properties.

[0094] Filler addition operation Weigh the following by mass ratio: β-cyclodextrin powder: 40-60% (preferably 50%) of the total filler; lactose powder: 20-30% (preferably 25%) of the total filler; microcrystalline cellulose powder (particle size 10-50μm, preferably 30μm): 10-30% (preferably 25%) of the total filler.

[0095] After thoroughly mixing the three filler powders, slowly add them to the wall material solution prepared in step (I). The total amount of filler added is 2-5 times (preferably 3 times, for example, if the coupling complex is 10g, then the total amount of filler is 30g) of the spleen polypeptide-ADC coupling complex. During the addition process, gently stir at a speed of 200-400 rpm (preferably 300 rpm), with a addition rate of 2-5g / min (preferably 3.5g / min). After the addition is complete, continue stirring for 30-60 minutes (preferably 45 minutes) to ensure that the filler is uniformly dispersed in the wall material solution. β-cyclodextrin and lactose gradually dissolve in the solution, and microcrystalline cellulose is uniformly dispersed in fine particles (particle size 10-50μm). β-cyclodextrin is in a free state in the solution, with its hydrophobic cavities open, waiting to undergo inclusion interactions with hydrophobic molecules. The viscosity of the solution will increase slightly due to the addition of the filler.

[0096] (III) Addition of antioxidant-basic amino acid complex The three antioxidant-basic amino acid complexes prepared in step three are mixed in the following proportions: Vitamin C-lysine solution: 50% of the total complex (by dry matter mass); Vitamin E-arginine emulsion: 25% of the total complex (by dry matter mass); Tea polyphenol-lysine solution: 25% of the total complex (by dry matter mass).

[0097] That is, the mass ratio of the three compounds is 2:1:1, which can provide comprehensive antioxidant protection in the aqueous phase, oil phase and broad spectrum.

[0098] The total amount of the complex added is 5-15% of the mass of the spleen polypeptide-ADC conjugate complex (preferably 10%, for example, if the conjugate complex is 10g, then the total amount of the complex is 1g).

[0099] The three complexes were added sequentially to a solution containing wall material and filler. The order of addition was as follows: first, vitamin C-lysine solution was added and stirred for 5-10 minutes (preferably 8 minutes); then, tea polyphenol-lysine solution was added and stirred for 5-10 minutes (preferably 8 minutes); finally, vitamin E-arginine emulsion was slowly added (dropping rate 1-3 mL / min, preferably 2 mL / min), while stirring at 300-500 rpm (preferably 400 rpm) for 20-30 minutes (preferably 25 minutes) to ensure uniform dispersion of the antioxidant-basic amino acid complex in the embedding solution. The vitamin E-arginine emulsion complex was stably dispersed in the embedding solution containing hydrophilic wall materials such as sodium alginate. The emulsion droplets (0.1-1 μm in diameter) were surrounded by sodium alginate molecules to form a stable dispersion system. Sodium alginate has a certain emulsifying and stabilizing effect, which can prevent the emulsion droplets from agglomerating and ensure the uniform distribution of the vitamin E-arginine complex in the embedding solution.

[0100] (iv) Preparation of embedding solution The spleen polypeptide-ADC coupling complex solution obtained in step two (concentration 3-8 mg / mL, preferably 5 mg / mL) is slowly added to a mixture containing wall material, functional filler, and antioxidant-basic amino acid complex at a rate of 2-5 mL / min (preferably 3.5 mL / min). While adding, the mixture is gently stirred at 150-250 rpm (preferably 200 rpm) (the stirring speed should not be too high to avoid generating excessive bubbles). After thorough mixing, the mixture is allowed to stand at room temperature (20-25℃, preferably 22℃) for 10-20 minutes (preferably 15 minutes) to allow β-cyclodextrin to fully contact the spleen polypeptide-ADC coupling complex and vitamin E, resulting in inclusion complexes. The hydrophobic cavity of β-cyclodextrin (approximately 6-10 Å in diameter) captures hydrophobic fragments of the spleen polypeptide (such as phenylalanine, tryptophan, and tyrosine residues) and vitamin E molecules (approximately 10 Å in molecular size) through hydrophobic interactions, forming a 1:1 or 2:1 host-guest inclusion complex. After standing, complete embedding solution is obtained.

[0101] The embedding solution is a uniform milky white or pale yellow viscous liquid with a pH of approximately 5.5-6.5 and a moderate viscosity (approximately 100-300 mPa·s, preferably 200 mPa·s), making it suitable for microencapsulation.

[0102] The mass ratio of each component in the embedding solution (on dry weight) is approximately as follows: wall material (sodium alginate): 100 parts; spleen polypeptide-ADC coupling complex: 20-50 parts (preferably 30 parts); functional filler (total): 60-150 parts (preferably 90 parts, including 45 parts of β-cyclodextrin, 22.5 parts of lactose, and 22.5 parts of microcrystalline cellulose); antioxidant-basic amino acid complex (total): 1-7.5 parts (preferably 3 parts, including 1.5 parts of vitamin C-lysine, 0.75 parts of vitamin E-arginine, and 0.75 parts of tea polyphenol-lysine).

[0103] (v) Microencapsulation The embedding solution is prepared into microcapsules using suitable methods, commonly including dropwise addition, spraying, or emulsification. Dropwise addition is preferred due to its simplicity, uniform microcapsule size, and high embedding efficiency.

[0104] The preferred dropping method is operated as follows: Preparation of crosslinking curing solution: Prepare a calcium chloride solution with a concentration of 2-4% (w / v, preferably 3% w / v, i.e., 30g CaCl2 dissolved in 1L deionized water), adjust the pH to 6.0-7.0 (preferably 6.5), and store at room temperature (20-25℃, preferably 22℃). The volume of the crosslinking curing solution should be 5-10 times (preferably 8 times) the volume of the embedding solution to ensure sufficient crosslinking.

[0105] Droplet Formation: The embedding solution is loaded into a syringe (preferably 10mL or 20mL) and connected to a needle with a suitable inner diameter (0.4-0.8mm, preferably 0.6mm, corresponding to a 25-21 gauge needle). The dripping speed is controlled by a syringe pump, preferably 20-40 drops / minute (preferably 30 drops / minute), maintaining a distance of 5-10cm (preferably 8cm) between the needle and the calcium chloride solution surface. The embedding solution droplet is dropped into the calcium chloride solution under gravity. Upon contact with the calcium chloride solution, the carboxyl groups (-COO⁻) in sodium alginate undergo ionic cross-linking with calcium ions (Ca²⁺), forming an "egg-box" structure (two sodium alginate chains are bridged by Ca²⁺). The solution droplet rapidly gels to form spherical microcapsules. During the droplet addition process, the solidification solution is gently stirred (50-100rpm, preferably 75rpm) to prevent microcapsule adhesion.

[0106] Curing and crosslinking: After the addition is complete, the microcapsules continue to cure in the calcium chloride solution for 10-30 minutes (preferably 20 minutes) to ensure sufficient crosslinking and stable microcapsule structure. Gentle stirring is maintained during the curing process to ensure that each microcapsule is fully in contact with calcium ions.

[0107] Washing and Collection: Rinse the microcapsules 2-3 times (preferably 3 times) with deionized water, each rinse lasting 5-10 minutes (preferably 8 minutes) to remove residual calcium ions and unencapsulated substances from the surface. Rinsing should be performed using decantation or filtration to obtain wet microcapsules. The wet microcapsules contain approximately 90-95% water.

[0108] The particle size of the microcapsules can be controlled by adjusting parameters such as the dropping acceleration rate, needle inner diameter, and embedding solution viscosity. The microcapsule particle size prepared in this embodiment is 0.5-3 mm (preferably 1.5-2.5 mm, with an average of about 2 mm), and the particle size distribution is uniform (coefficient of variation <10%).

[0109] In this step, cyclodextrin encapsulates the spleen polypeptide-ADC coupling complex during microcapsule formation, encapsulating the hydrophobic fragment within the cavity; the antioxidant-basic amino acid complex is uniformly dispersed in the microcapsule matrix, and the amino groups of the basic amino acids interact with the carboxyl groups of the spleen polypeptide, blocking cross-linking; the three work synergistically in the three-dimensional network structure of the microcapsule.

[0110] Step 5: Freeze-drying The wet microcapsules obtained in step four were freeze-dried to remove moisture and obtain dry and stable freeze-dried microcapsule powder.

[0111] (a) Pre-freezing Spread the wet microcapsules evenly in a stainless steel freeze-drying tray, with a thickness not exceeding 2 cm (preferably 1.5 cm). Maintain a certain gap between the microcapsules to avoid excessive accumulation that would affect freeze-drying efficiency. Place the freeze-drying tray in the freeze-drying chamber of the freeze dryer and start the pre-freezing program.

[0112] Pre-freezing employs a programmed cooling method: - First stage: Cool from room temperature (20-25℃) to -20℃ at a rate of 5-10℃ / min (preferably 8℃ / min), and hold for 1-2 hours (preferably 1.5 hours) to allow sufficient crystallization of the outer layer of the microcapsules; - Second stage: Continue cooling at a rate of 3-5℃ / min (preferably 4℃ / min) to -40℃ to -80℃ (preferably -50℃), and hold for 4-8 hours (preferably 6 hours) to completely freeze the water inside the microcapsules.

[0113] During pre-freezing, the water in the microcapsules gradually crystallizes to form ice crystals. Slow cooling promotes the formation of smaller ice crystals, reducing damage to the microcapsule structure. The pre-freezing temperature should be lower than the eutectic point temperature of the water in the microcapsules (generally -40℃), and the pre-freezing time should ensure that the temperature at the center of the microcapsule reaches the set temperature. A temperature probe can be inserted into the center of the microcapsule to monitor the temperature.

[0114] During the pre-freezing process, the antioxidant-basic amino acid complex added in step three and the functional fillers (β-cyclodextrin and lactose) added in step four play a role in freeze-drying protection. As cryoprotectants, they surround the spleen polypeptide-ADC coupling complex during water crystallization, preventing mechanical damage to the structure of the coupling complex caused by ice crystal growth and maintaining its three-dimensional structure and biological activity.

[0115] (ii) Sublimation drying After pre-freezing, activate the vacuum system to reduce the pressure in the freeze-drying chamber to 10-50 Pa (preferably 20 Pa, approximately 0.2 mbar). Under vacuum conditions, sublimation drying is carried out using a programmed temperature rise method. First stage: Slowly raise the temperature from -50℃ to -30℃ at a rate of 2-3℃ / hour (preferably 2.5℃ / hour) and hold for 3-5 hours (preferably 4 hours) to allow the ice crystals on the surface of the microcapsules to sublimate; Second stage: Heat to -10°C at a rate of 3-5°C / hour (preferably 4°C / hour) and hold for 3-5 hours (preferably 4 hours) to allow the ice crystals in the middle layer of the microcapsule to sublimate; The third stage: the temperature is increased to 0-5℃ (preferably 5℃) at a rate of 5-8℃ / hour (preferably 6℃ / hour) and held for 2-4 hours (preferably 3 hours) to allow the ice crystals inside the microcapsules to completely sublimate; Fourth stage: Continue to heat to 20-25℃ (preferably 22℃) and maintain for 1-2 hours (preferably 1.5 hours) to remove free water.

[0116] During sublimation drying, ice crystals directly sublimate from a solid state to water vapor. The water vapor is then captured and condensed in a cold trap (temperature -60°C to -80°C, preferably -70°C), bypassing the liquid stage and thus preserving the microcapsule structure. The total sublimation drying time is typically 12-24 hours (preferably 18 hours), until the water content of the microcapsules drops below 5% (preferably below 3%). The drying process can be monitored by changes in vacuum: when the vacuum level stabilizes and no longer decreases, and the cold trap stops freezing, the sublimation drying is essentially complete.

[0117] (III) Analysis and Drying To further remove residual bound water (water molecules adsorbed on the surface of sodium alginate, peptides, etc.) inside the microcapsules, a desorption drying process is performed: Under vacuum conditions (pressure maintained at 10-50 Pa, preferably 20 Pa), the temperature is raised to 30-40℃ (preferably 35℃) and maintained for 2-4 hours (preferably 3 hours) to allow the bound water to desorb and sublimate, which is then removed by the vacuum system. The desorption drying temperature should not be too high to avoid thermal damage to the bioactivity of the coupling complex.

[0118] The final product is a freeze-dried microcapsule powder with a moisture content of ≤3% (preferably ≤2%). The moisture content can be determined using the Karl Fischer moisture determination method.

[0119] The freeze-dried microcapsule powder exhibits a loose, porous, sponge-like structure, ranging in color from white to pale yellow. It is lightweight, easily broken into powder, and convenient for storage and transportation. After freeze-drying, the porosity of the microcapsules can reach 80-90%, increasing the specific surface area and facilitating resolvation. During the freeze-drying process, the antioxidant-basic amino acid complex, β-cyclodextrin inclusion complex, and sodium alginate wall material synergistically protect the coupling complex, maintaining an activity retention rate >90% (preferably >93%).

[0120] This step employs conventional freeze-drying technology in the field, and those skilled in the art can select appropriate freeze-drying parameters based on the characteristics of the microcapsules.

[0121] Step Six: Surface Hydrophilic Modification This step is one of the core steps. It uses a chitosan-carboxymethyl cellulose sodium polyelectrolyte complex to hydrophilically modify the surface of the freeze-dried microcapsules, which is different from the existing technology that uses a single hydrophilic polymer.

[0122] (I) Technical Principles The surface of the freeze-dried microcapsules is highly hydrophobic, mainly because after the surface moisture is removed during the freeze-drying process, the hydrophobic groups of the wall material molecules (such as sodium alginate) are exposed. At the same time, a dense dry layer is formed on the surface of the microcapsules, which hinders the penetration of water and makes resolvation difficult.

[0123] Existing technologies typically involve spraying a single hydrophilic polymer (such as PEG or sodium carboxymethyl cellulose) onto the surface of microcapsules to improve resolubility. However, single hydrophilic polymers exhibit poor stability under varying pH and ionic strength conditions: in high-salt environments, the hydration layer of the hydrophilic polymer is disrupted, reducing its hydrophilicity; in acidic or alkaline environments, the conformation of the hydrophilic polymer changes, potentially leading to swelling or shrinkage, resulting in unstable resolubility.

[0124] This step innovatively uses a polyelectrolyte complex (PEC) formed by the electrostatic interaction between chitosan and sodium carboxymethyl cellulose as a hydrophilic modification layer.

[0125] The formation process of the polyelectrolyte complex: - Chitosan is a natural polysaccharide containing a large number of amino groups (-NH2) in its molecular chain. At pH < 6.5, these amino groups protonate to become -NH3⁺, carrying a positive charge. Sodium carboxymethyl cellulose (CMC-Na) contains carboxyl groups (-COONa) in its molecular chain, which ionizes to -COO⁻ in aqueous solution, carrying a negative charge. When chitosan solution and sodium carboxymethyl cellulose solution are mixed, the positively charged -NH3⁺ and the negatively charged -COO⁻ combine through electrostatic attraction to form ion pairs. Multiple molecular chains then cross-link at multiple points to form a three-dimensional network structure, i.e., the polyelectrolyte complex.

[0126] Advantages of the polyelectrolyte complex: High stability: The network structure formed by electrostatic cross-linking is more stable than weak interactions such as hydrogen bonds. Electrostatic cross-linking remains intact across a wide pH range of 4-9 and salt concentrations of 0-500 mM, maintaining a stable network structure and hydrophilic properties unaffected by the environment. Strong hydrophilicity: Both chitosan and sodium carboxymethyl cellulose are hydrophilic polysaccharides. The complex inherits the hydrophilic properties of both, with a surface covered with numerous hydroxyl and ionized groups, enabling rapid adsorption of water molecules to form a hydration layer, significantly reducing surface tension and promoting water penetration. Antibacterial properties: Chitosan possesses natural antibacterial activity. Its positively charged amino groups interact with negatively charged components on microbial cell membranes, disrupting cell membrane integrity and inhibiting microbial growth. The chitosan in the complex retains this property, providing additional microbial stability protection for the product. Biocompatibility: Both chitosan and sodium carboxymethyl cellulose are natural polysaccharide derivatives, non-toxic, and biodegradable, making them suitable for the biopharmaceutical field.

[0127] (2) Specific operations Chitosan solution preparation Weigh out chitosan powder (degree of deacetylation ≥85%, preferably 90%; molecular weight 100,000-300,000 Da, preferably 200,000 Da, medium molecular weight chitosan), and slowly add it to a dilute acetic acid solution (concentration 1-2% v / v, preferably 1.5% v / v, i.e., 15 mL glacial acetic acid added to 1 L deionized water) at a feeding rate of 0.3-0.5 g / min (preferably 0.4 g / min), while gently stirring at 200-400 rpm (preferably 300 rpm). Dissolve the chitosan at room temperature (20-25℃, preferably 22℃) for 2-4 hours (preferably 3 hours) to prepare a chitosan solution with a concentration of 0.5-2% (w / v, preferably 1% w / v). Chitosan dissolves under acidic conditions by protonation of the amino groups (-NH3⁺), and the solution pH is approximately 4.5-5.5 (preferably pH 5.0). Stir until the solution is clear and transparent, without obvious particles, and has a uniform viscosity. If there is a small amount of insoluble matter, it can be removed by filtration with sterile gauze or filter paper. The prepared chitosan solution should be used within 24 hours.

[0128] Selection of chitosan: The higher the degree of deacetylation, the more amino groups, the higher the positive charge density, the stronger the electrostatic interaction with sodium carboxymethyl cellulose, and the more stable the resulting complex. In this embodiment, chitosan with a degree of deacetylation of 90% and a molecular weight of 200,000 Da is preferred.

[0129] Preparation of sodium carboxymethyl cellulose solution: Weigh sodium carboxymethyl cellulose powder (degree of substitution ≥0.8, preferably 0.9; molecular weight 100,000-300,000 Da, preferably 200,000 Da), and slowly add it to deionized water at a rate of 0.3-0.5 g / min (preferably 0.4 g / min), while gently stirring at 200-400 rpm (preferably 300 rpm). Dissolve the powder at room temperature (20-25℃, preferably 22℃) for 1-2 hours (preferably 1.5 hours) to prepare a sodium carboxymethyl cellulose solution with a concentration of 0.5-2% (w / v, preferably 1% w / v). Sodium carboxymethyl cellulose is readily soluble in water, and the solution pH is approximately 7-8 (preferably pH 7.5). Stir until the solution is uniform, transparent, and of moderate viscosity. The prepared sodium carboxymethyl cellulose solution should be used within 24 hours.

[0130] Selection of sodium carboxymethyl cellulose: The higher the degree of substitution, the more carboxyl groups are present, the higher the negative charge density, and the stronger the electrostatic interaction with chitosan. In this embodiment, sodium carboxymethyl cellulose with a degree of substitution of 0.9 and a molecular weight of 200,000 Da is preferred.

[0131] Preparation of polyelectrolyte complex suspension: Under stirring conditions of 300-500 rpm (preferably 400 rpm), a chitosan solution (1% w / v, pH 5.0) is slowly added dropwise to a sodium carboxymethyl cellulose solution (1% w / v, pH 7.5) using a peristaltic pump at a dropping rate of 1-3 mL / min (preferably 2 mL / min). The volume ratio of chitosan solution to sodium carboxymethyl cellulose solution is 1:0.8-1.5:1 (preferably 1:1, i.e., equal volume mixing). During the dropwise addition process, excessively high local concentrations should be avoided to prevent excessive aggregation of the complex and formation of large particles.

[0132] Upon mixing, the electrostatic interaction between the positive and negative charges (-NH3⁺ of chitosan and -COO⁻ of sodium carboxymethyl cellulose) occurs immediately, rapidly forming a white flocculent precipitate in the solution, which is the polyelectrolyte complex. Continue stirring for 10-20 minutes (preferably 15 minutes) to allow the complex to fully form. The final product is a white or slightly yellow polyelectrolyte complex suspension, in which the complex is dispersed as fine particles or flocculent particles with a particle size of approximately 0.5-5 μm (preferably 1-3 μm).

[0133] The effect of pH: The formation of the polyelectrolyte complex requires both chitosan and sodium carboxymethyl cellulose to be charged. The pH of the mixed solution is approximately 5.5-6.5 (preferably pH 6.0). At this pH, the amino groups of chitosan are protonated (positively charged, degree of protonation approximately 80-90%), and the carboxyl groups of sodium carboxymethyl cellulose are completely ionized (negatively charged, degree of ionization >95%), resulting in the strongest electrostatic interaction and the most stable complex. If necessary, the pH can be adjusted to 6.0 ± 0.2 with a small amount of 0.1M dilute hydrochloric acid or 0.1M sodium hydroxide solution.

[0134] The concentration of the prepared polyelectrolyte complex suspension is approximately 0.8-1.2% (w / v, preferably 1% w / v). It should be used immediately and should not be left to stand for a long time.

[0135] Surface coating: Place the freeze-dried microcapsule powder obtained in step five (100-500g batch, preferably 200g) into a fluidized bed dryer (preferably an FL-5 type fluidized bed coating machine) or a small spray device. Start the fluidized bed, adjust the inlet air temperature to 25-40℃ (preferably 30℃), and the inlet air volume to 30-60m³ / h (preferably 45m³ / h), so that the microcapsule powder is in a fluidized state (suspended and tumbling) under the action of airflow. After the fluidized state is stable, start spraying.

[0136] The prepared polyelectrolyte composite suspension was loaded into a spraying reservoir and uniformly sprayed onto the fluidized microcapsule surface using a peristaltic pump and an atomizing nozzle (nozzle orifice diameter 0.5-1.0 mm, preferably 0.8 mm). The spraying speed was 2-5 mL / min (preferably 3.5 mL / min), the atomization pressure was 0.1-0.3 MPa (preferably 0.2 MPa), and the distance between the spray gun and the material layer was maintained at 10-20 cm (preferably 15 cm). During the spraying process, the composite particles adsorbed onto the microcapsule surface and bonded to sodium alginate molecules on the microcapsule surface through van der Waals forces and hydrogen bonds, forming a dense hydrophilic modified layer through water evaporation.

[0137] The coating amount is 3-8% of the mass of the freeze-dried microcapsules (preferably 5%, for example, 10g of composite is sprayed onto 200g of microcapsules). If the coating amount is too low (<3%), the hydrophilic layer will not be complete and the modification effect will be poor; if the coating amount is too high (>8%), the composite layer will be too thick, which may affect the reconstitution rate and storage stability of the microcapsules.

[0138] The spraying time is determined based on the amount of microcapsules and the spraying rate, generally 10-30 minutes (preferably 20 minutes, approximately 20 minutes for 200g of microcapsules). Maintain a fluidized state during spraying and periodically check the nozzles for blockages to ensure uniform spraying.

[0139] Secondary drying: After spraying, immediately stop the supply of spraying liquid, but continue drying while maintaining a fluidized state, or transfer the sprayed microcapsules to a constant temperature drying oven. Drying conditions: temperature 25-40℃ (preferably 30℃), time 30-60 minutes (preferably 45 minutes). During the drying process, the moisture introduced during spraying (approximately 2-4% of the microcapsule mass) gradually evaporates, further strengthening the electrostatic cross-linking between chitosan and sodium carboxymethyl cellulose molecular chains in the hydrophilic modified layer, forming a dense and stable network structure, and the hydrophilic modified layer solidifies.

[0140] Drying endpoint determination: When the moisture content of the microcapsules drops to 3-5% (preferably 4%), the moisture content can be determined by the Karl Fischer method or by touch (the dried microcapsule powder feels dry and not sticky).

[0141] After drying, lyophilized microcapsules with a hydrophilic layer of chitosan-carboxymethyl cellulose sodium polyelectrolyte complex on the surface are obtained. The thickness of the hydrophilic layer is approximately 5-20 μm (preferably 10-15 μm). The hydrophilic layer is tightly bonded to the microcapsule surface through van der Waals forces, hydrogen bonds, and some ionic bonds, exhibiting strong adhesion and resistance to detachment. The microcapsules are white to pale yellow in color, have good flowability, and are free of clumps.

[0142] (III) Functions of the hydrophilic modified layer The microcapsules modified with hydrophilic surface possess the following properties: Rapid resolubility: Hydrophilic groups such as hydroxyl, amino, and carboxyl groups in the hydrophilic modified layer rapidly adsorb water molecules, reducing the surface tension from about 50-60 mN / m before modification to 20-30 mN / m. Water quickly penetrates into the microcapsule, shortening the resolubility time from 15-20 minutes to 3-5 minutes, a reduction of 70-80%.

[0143] Environmental stability: The electrostatic cross-linked network structure of the polyelectrolyte complex remains stable in the pH range of 4-9 and salt concentration range of 0-500mM. Its resolvability is not affected by the environment, making it suitable for various application scenarios (such as animal feed additives that need to be used in digestive tract environments with different pH levels, and biological reaction solutions that may contain different salt concentrations).

[0144] Antibacterial protection: Chitosan's antibacterial activity inhibits the growth of bacteria, molds and other microorganisms during storage, extending product shelf life and ensuring microbial safety.

[0145] Biocompatibility: The biocompatibility of natural polysaccharide materials ensures the safe application of the product in the biological field.

[0146] (iv) Differences from existing technologies Existing technologies typically use either PEG or sodium carboxymethyl cellulose for surface hydrophilic modification: - PEG alone: ​​While exhibiting good hydrophilicity, the hydration layer is disrupted in high-salt environments, leading to decreased hydrophilicity; furthermore, PEG lacks antibacterial properties. - Sodium carboxymethyl cellulose alone: ​​In acidic environments (pH < 5), the carboxyl group is protonated, reducing the negative charge and decreasing hydrophilicity; stability issues also exist in high-salt environments.

[0147] This step innovatively uses a polyelectrolyte complex formed by chitosan and sodium carboxymethyl cellulose. Through electrostatic cross-linking, a highly stable network structure is formed, overcoming the environmental sensitivity of a single hydrophilic polymer. At the same time, the antibacterial function of chitosan is introduced, achieving multiple functions of "hydrophilicity + stability + antibacteriality", which is not available in existing single hydrophilic modification technologies.

[0148] Step 7: Finished Product Packaging The surface-modified freeze-dried microcapsule powder obtained in step six was then packaged. The packaging operation was carried out in a cleanroom with a cleanliness level of not less than 100,000 (ISO Class 8), and the operators wore cleanroom garments, gloves, and masks to avoid microbial and dust contamination.

[0149] (a) Selection of Packaging Materials Aluminum foil composite bags (composed of a three-layer PET / AL / PE composite material, with a thickness of 100-150μm, preferably 120μm) are preferred due to their excellent oxygen, moisture, and light barrier properties, effectively preventing the effects of oxygen, moisture, and light on the product. Alternative packaging materials include brown glass bottles (50-500mL capacity, preferably 100mL) with rubber stoppers and aluminum caps, suitable for high-end products.

[0150] (ii) Packaging operation Use an automatic or semi-automatic dispensing machine to dispense the microcapsule powder into bags or glass bottles. The amount per bag or bottle is determined according to the actual application requirements; common specifications are 10g, 50g, and 100g (50g / bag is preferred). The dispensing error should be controlled within ±2%. Avoid powder scattering during the dispensing process and operate gently.

[0151] (III) Nitrogen filling and desiccant addition After dispensing, the following treatments (preferred steps) should be performed before sealing: 1. Nitrogen purging: Replace the air inside the packaging bag with high-purity nitrogen (purity ≥99.9%) at a pressure of 0.02-0.05 MPa (preferably 0.03 MPa), causing the packaging bag to slightly bulge, reducing the oxygen content to <2%, and delaying the oxidation reaction. 2. Placement of desiccant: Place 1-3g (preferably 2g) of silica gel desiccant (packaged in a breathable small bag) in the packaging bag or glass bottle to absorb residual moisture and keep the product dry.

[0152] (iv) Sealing and Labeling Use a heat sealer to heat seal the aluminum foil bags (sealing temperature 150-180℃, preferably 170℃, sealing time 3-5 seconds, preferably 4 seconds) to ensure a tight, leak-free seal. For glass bottles, use a capping machine to press the aluminum caps onto the bottles. After sealing, check the integrity of the packaging and discard any products with poor sealing.

[0153] Please label the following information on the outside of the packaging: - Product Name: Antioxidant Stabilizing Spleen Peptide-ADC Conjugated Microcapsule Formulation - Specification: 50g / bag (or actual specification) - Batch Number: YYYYMMDD-XXX - Production Date: YYYY-MM-DD - Shelf Life: 24 months (from the production date) - Storage Conditions: Store in a cool, dry place away from light and sealed, at a temperature ≤25℃ and relative humidity ≤60% - Manufacturer Information (v) Outer packaging Pack the inner packaging into cardboard boxes or plastic turnover boxes at a rate of 10-20 bags / bottle (preferably 10 bags), with the inside of the cardboard box lined with a moisture-proof film. Label the outer packaging with product information, storage conditions, batch number, quantity, etc.

[0154] This step is a standard packaging operation, and those skilled in the art can select appropriate packaging materials and methods based on product characteristics and application requirements. Packaged products should be stored under specified conditions, with regular quality checks to ensure they are used within their expiration date.

[0155] Experimental verification Experiment 1: Determination of Chemical Crosslinking Rate 1. Experimental Objective Comparative experiments were conducted to verify the blocking effect of the antioxidant-basic amino acid complex on the chemical cross-linking reaction of the spleen polypeptide-ADC coupling complex, demonstrating that basic amino acids can effectively reduce the cross-linking aggregation rate between polypeptide molecules.

[0156] 2. Preparation of experimental samples Three groups of samples were prepared, each containing the same concentration of spleen polypeptide-ADC conjugate complex, the difference being whether or not an antioxidant-basic amino acid complex was added: Sample A (Example Sample): A complete sample prepared according to this example. Specific composition (by dry weight): 100 parts sodium alginate, 30 parts spleen polypeptide-ADC coupling complex, 90 parts functional filler (including 45 parts β-cyclodextrin, 22.5 parts lactose, and 22.5 parts microcrystalline cellulose), and 3 parts antioxidant-basic amino acid complex (including 1.5 parts vitamin C-lysine, 0.75 parts vitamin E-arginine, and 0.75 parts tea polyphenol-lysine, in a mass ratio of 2:1:1), after microencapsulation and lyophilization.

[0157] Sample B (Comparative Example 1): The preparation process was the same as that of Sample A, but no basic amino acids were added. Instead, an equal mass of a mixture of individual antioxidants (vitamin C, vitamin E, and tea polyphenols, in a mass ratio of 2:1:1, totaling 3 parts) was added. That is, 3 parts of the antioxidant-basic amino acid complex were replaced with 3 parts of individual antioxidants. The other components (100 parts of sodium alginate, 30 parts of coupling complex, and 90 parts of filler) and process conditions were the same as those of Sample A.

[0158] Sample C (Comparative Example 2): The preparation process was the same as that of Sample A, but no antioxidants and basic amino acids were added (i.e., the antioxidant-basic amino acid complex was reduced by 3 parts). Other components (sodium alginate 100 parts, coupling complex 30 parts, filler 90 parts) and process conditions were the same as those of Sample A.

[0159] Each sample was prepared in 50g portions and stored in sealed aluminum foil bags under accelerated aging conditions at 40°C to simulate the cross-linking reaction during long-term storage.

[0160] 3. Experimental conditions Storage conditions: Temperature 40℃±2℃, relative humidity 60%±5%, store in a light-proof, sealed container. Test time points: Day 0, Day 7, Day 14, Day 21, and Day 28. Three samples were taken at each time point for parallel testing, and the average value was taken.

[0161] 4. Experimental Procedure (1) Sample pretreatment At each test time point, take 5 mg of lyophilized microcapsule sample, add 1 mL of PBS buffer (pH 7.4), and gently shake at room temperature for 30 minutes to reconstitute it, thus obtaining the test solution.

[0162] (2) Determination of chemical crosslinking rate The formation of cross-linked polymers was detected using SDS-PAGE gel electrophoresis. Take 20 μL of test solution and add 5 μL of 5×SDS loading buffer (containing β-mercaptoethanol). Heat at 95℃ for 5 minutes to denature the proteins. Load the sample onto a 12% SDS-PAGE gel and perform electrophoresis at 80V for 30 minutes (stacking gel) and 120V for 60 minutes (separating gel). After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 30 minutes and destain until the background is clear. Take pictures using a gel imaging system and analyze the grayscale values ​​of high molecular weight polymers (>100 kDa) in each lane using ImageJ software.

[0163] Formula for calculating chemical crosslinking rate: Crosslinking rate (%) = (Gray value of high molecular weight polymer strip / Gray value of total strip) × 100% (3) Determination of free amino content The content of free amino groups in the sample was determined by the ninhydrin colorimetric method. Take 100 μL of the test solution and add 200 μL of pH 5.5 acetate buffer. Add 200 μL of 2% ninhydrin reagent (dissolved in anhydrous ethanol), mix well, and heat in a 95°C water bath for 15 minutes. Cool to room temperature, dilute with 500 μL of 50% ethanol, and measure the absorbance at 570 nm. Plot a standard curve using L-lysine as a standard and calculate the free amino content in the sample.

[0164] (4) Determination of free carboxyl group content The content of free carboxyl groups in the sample was determined using the toluidine blue colorimetric method. Take 100 μL of test solution and add 1 mL of 0.002% toluidine blue solution (pH 10.0). React at room temperature for 10 minutes, and measure the absorbance at 633 nm. Plot a standard curve using L-glutamic acid as a standard, and calculate the free carboxyl group content in the sample.

[0165] 5. Experimental Results Table 1. Changes in chemical crosslinking rate (%) of different samples during storage at 40℃ Table 2. Free radical content of different samples after 28 days of storage Note: The initial free amino group content was 146.7 μmol / g, and the initial free carboxyl group content was 175.6 μmol / g.

[0166] Figure 1 : Curves showing the change in chemical crosslinking rate of different samples during storage at 40℃; Figure 2 Comparison of free radical retention rates of various samples after 28 days of storage.

[0167] 6. Analysis and Summary (1) Comparison of chemical crosslinking rates: From Table 1 and Figure 1 It can be seen that after 28 days of storage at 40℃, the chemical cross-linking rate of sample A (containing an antioxidant-basic amino acid complex) was only 11.2%, a decrease of 66.2% compared to 33.1% for sample B (containing only antioxidants) and a decrease of 74.3% compared to 43.5% for sample C (containing no protective agents). This proves that the basic amino acids in the antioxidant-basic amino acid complex can effectively block the chemical cross-linking reaction between spleen polypeptide molecules. Meanwhile, data from the initial state (day 0) shows that the cross-linking rates of the three samples are very close (3.8-4.5%), indicating good consistency in the initial preparation process, and the differences during storage are entirely due to differences in formulation.

[0168] (2) Retention of free groups: From Table 2 and Figure 2It can be seen that after 28 days of storage, sample A retained 87.6% of the free amino groups and 89.3% of the free carboxyl groups, both significantly higher than samples B and C. The free group retention rate of sample B was approximately 65%, and that of sample C was approximately 52%. This indicates that the free amino groups of basic amino acids (lysine and arginine) can competitively occupy the carboxyl sites of spleen polypeptides, preventing dehydration condensation and other cross-linking reactions between polypeptide molecules, thereby protecting most of the free groups from being consumed.

[0169] (3) Synergistic Effect Verification: Comparing the data of samples B and C, it can be found that adding an antioxidant alone (sample B, crosslinking rate of 33.1% after 28 days) can reduce the crosslinking rate by about 23.9% compared with not adding any protective agent (sample C, crosslinking rate of 43.5% after 28 days), but the effect is far less than that of adding an antioxidant-basic amino acid complex (sample A, crosslinking rate of 11.2% after 28 days). This proves that the anti-crosslinking effect of basic amino acids is independent of the antioxidant effect of antioxidants. After the two are combined to form a complex, they can both block chemical crosslinking and resist oxidation, achieving a synergistic effect of "1+1>2". From the data trend, the crosslinking rate growth curve of sample A is the flattest, while that of sample C is the steepest, further proving the continuous protective effect of the complex.

[0170] (4) Practical significance: The technical effect of reducing the chemical cross-linking rate by more than 74% has been experimentally verified (from 43.5% to 11.2%), which is of great significance for extending the shelf life of the product and maintaining the bioactivity of the coupling complex. The low cross-linking rate of sample A ensured that the spleen polypeptide-ADC coupling complex maintained good dispersion and bioactivity during long-term storage. Experimental data showed that the cross-linking rate of sample A increased by only 194.7% (from 3.8% to 11.2%) during the 28-day storage period, while the growth rate of sample C was as high as 866.7% (from 4.5% to 43.5%), which fully demonstrates the continuous protective effect of basic amino acids.

[0171] Experiment 2: Aggregation Rate Determination Experiment 1. Experimental Objective Comparative experiments were conducted to verify the anti-aggregation effect of a functional filler combination based on β-cyclodextrin on the spleen polypeptide-ADC coupling complex, demonstrating that the host-guest inclusion effect of cyclodextrin can significantly reduce the molecular aggregation rate.

[0172] 2. Preparation of experimental samples Three groups of samples were prepared, each containing the same concentration of spleen polypeptide-ADC coupling complex and antioxidant-basic amino acid complex, differing only in the type and composition of the filler: Sample D (Example Sample): A complete sample prepared according to this example. Specific composition (by dry weight): 100 parts sodium alginate, 30 parts spleen polypeptide-ADC coupling complex, 90 parts functional filler (the filler is 3 times the mass of the coupling complex, of which 45 parts β-cyclodextrin accounts for 50%, 22.5 parts lactose accounts for 25%, and 22.5 parts microcrystalline cellulose accounts for 25%), and 3 parts antioxidant-basic amino acid complex (1.5 parts vitamin C-lysine, 0.75 parts vitamin E-arginine, and 0.75 parts tea polyphenol-lysine), after microencapsulation and lyophilization.

[0173] Sample E (Comparative Example 3): The preparation process was the same as that of Sample D, but the filler composition was a traditional inert filler that did not contain β-cyclodextrin. The total filler amount was still 90 parts (3 times the mass of the coupling complex), of which lactose accounted for 54 parts (60%), microcrystalline cellulose accounted for 36 parts (40%), and the other components (sodium alginate 100 parts, coupling complex 30 parts, antioxidant-basic amino acid complex 3 parts) and process conditions were the same as those of Sample D.

[0174] Sample F (Comparative Example 4): The preparation process is the same as that of Sample E, and the filler composition is also a traditional inert filler (without β-cyclodextrin), but the total amount of filler added is reduced to 30 parts (1 times the mass of the coupling complex), of which lactose accounts for 18 parts (60%), microcrystalline cellulose accounts for 12 parts (40%), and other components (sodium alginate 100 parts, coupling complex 30 parts, antioxidant-basic amino acid complex 3 parts) and process conditions are the same as those of Sample D.

[0175] Each sample was prepared in quantities of 50g, and after reconstitution, it was used for the determination of aggregation rate.

[0176] 3. Experimental conditions Test temperature: 25℃±1℃; Test medium: PBS buffer (pH 7.4); Sample concentration: 5mg / mL; Test time: Test immediately after reconstitution, and after standing for 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours. Test 3 parallel samples at each time point and take the average value of the results.

[0177] 4. Experimental Procedure (1) Sample reconstitution Weigh 50 mg of lyophilized microcapsule sample, add 10 mL of PBS buffer (pH 7.4), and gently shake at 25 °C to reconstitute the sample. Record the reconstitution time. After reconstitution, transfer the solution to a 25 mL colorimetric tube and allow it to stand until analysis.

[0178] (2) Dynamic light scattering (DLS) particle size determination The particle size distribution of the samples was determined using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS). Take 1 mL of the reconstituted solution and transfer it to a standard cuvette. Set the test parameters: temperature 25℃, scattering angle 173°, refractive index 1.450, absorption coefficient 0.001. Test each sample three times, with each test lasting 60 seconds. Automatically calculate the average particle size (Z-Average) and polydispersity index (PDI). Record the particle size distribution curve and analyze the ratio of monodisperse peaks to aggregated peaks.

[0179] (3) Turbidity measurement The turbidity of the sample solution was determined using a UV-Vis spectrophotometer. Take 3 mL of the reconstituted solution and transfer it to a quartz cuvette. Using PBS buffer as a blank control, measure the absorbance (OD) at 600 nm. 600 The higher the turbidity value, the more aggregates are present in the solution.

[0180] (4) Aggregation rate calculation The aggregation rate is calculated based on particle size distribution data. Particles with a diameter >500nm are defined as aggregates, and their proportion in the total number of particles is calculated. Aggregation rate (%) = (Number of particles with a diameter > 500 nm / Total number of particles) × 100% 5. Experimental Results Table 3. Changes in particle size and aggregation rate of different samples after reconstitution Table 4. Turbidity (OD) of different samples after reconstitution 600 )change Table 5. Polydispersity Index (PDI) and Reconstitution Time for Different Samples Note: Particle size growth rate = [(24-hour particle size - 0-hour particle size) / 0-hour particle size] × 100% Figure 3 The change in aggregation rate of different samples after reconstitution over time; Figure 4 The change in turbidity of different samples after reconstitution over time.

[0181] 6. Analysis and Summary (1) The aggregation rate decreased significantly: From Table 3 and Figure 3It can be seen that the aggregation rate of sample D (containing β-cyclodextrin) was only 4.9% after reconstitution for 24 hours, far lower than that of sample E (comparative example 3, conventional filler) (36.4%) and sample F (comparative example 4, filler reduced) (43.8%). Compared with sample E, the aggregation rate of sample D decreased by 86.5%; compared with sample F, it decreased by 88.8%. This proves that the host-guest inclusion complex of β-cyclodextrin can effectively prevent the aggregation of spleen polypeptide-ADC coupling complex.

[0182] (2) Optimization of particle size distribution: The average particle size of sample D increased only from 156 nm to 218 nm in 24 hours, with a particle size growth rate of 39.7%, and the polydispersity index (PDI) remained at a low level of 0.22, indicating that the particle size distribution was uniform. In contrast, the particle size growth rates of samples E and F were 80.4% and 82.1%, respectively, and the PDIs were as high as 0.42-0.51, indicating severe aggregation and non-uniform particle size distribution.

[0183] (3) Turbidity test verification: From Table 4 and Figure 4 It can be seen that the turbidity of sample D was only 0.20 after 24 hours, which is 68.8% lower than that of sample E (0.64) and 77.5% lower than that of sample F (0.89). The low turbidity indicates that there are fewer aggregates in the solution, the clarity is high, and the active ingredients are completely released.

[0184] (4) Verification of the inclusion mechanism of cyclodextrin: Comparison of the differences between samples D and E shows that, with the same total amount of filler (both three times the amount of the coupling complex), the aggregation rate of sample D containing β-cyclodextrin was only 4.9%, while the aggregation rate of sample E without cyclodextrin was as high as 36.4%. This proves that the anti-aggregation effect of β-cyclodextrin is not a simple physical dilution effect, but rather that it actively prevents molecular aggregation by including the hydrophobic fragments of spleen polypeptide and vitamin E through its unique hydrophobic cavity.

[0185] (5) Effect of filler dosage: Comparison of samples E and F shows that increasing the filler dosage (from 1 to 3 times) can reduce the aggregation rate (from 43.8% to 36.4%), but the effect is limited. This further proves that traditional inert fillers (lactose, microcrystalline cellulose) can only provide passive spatial dilution, while the functional inclusion effect of β-cyclodextrin is the key.

[0186] (6) Improved resolvability: The resolvability time of sample D was only 3.5 minutes, which was significantly shorter than that of sample E (16.8 minutes) and sample F (22.5 minutes). This is because β-cyclodextrin and lactose dissolve rapidly, generating an osmotic pressure gradient, which promotes the diffusion of water into the microcapsules. At the same time, the anti-aggregation effect ensures the clarity of the solution after resolvation.

[0187] Experiment 3: Environmental Adaptability Test 1. Experimental Objective Comparative experiments under different pH and salt concentration conditions were conducted to verify the environmental adaptability of the hydrophilic modified layer of the chitosan-carboxymethyl cellulose sodium polyelectrolyte composite, demonstrating that the composite can maintain stable resolution performance over a wide range of pH and salt concentrations.

[0188] 2. Preparation of experimental samples Three groups of samples were prepared, each with the same composition and preparation process, differing only in the type of hydrophilic modification layer on the surface: Sample G (Example Sample): A complete sample prepared according to this example. Microcapsule composition (by dry weight): 100 parts sodium alginate, 30 parts spleen polypeptide-ADC coupling complex, 90 parts functional filler (45 parts β-cyclodextrin, 22.5 parts lactose, 22.5 parts microcrystalline cellulose), and 3 parts antioxidant-basic amino acid complex. After microencapsulation and lyophilization, a hydrophilic layer of chitosan-sodium carboxymethyl cellulose polyelectrolyte complex (prepared by mixing chitosan with a degree of deacetylation of 90% and sodium carboxymethyl cellulose with a degree of substitution of 0.9 at a mass ratio of 1:1) is sprayed onto the surface. The amount of spraying is 5% of the mass of the lyophilized microcapsules.

[0189] Sample H (Comparative Example 5): The internal composition of the microcapsules is exactly the same as that of Sample G (100 parts sodium alginate, 30 parts coupling complex, 90 parts filler, and 3 parts antioxidant complex). After microencapsulation and freeze-drying, the surface hydrophilic modification layer is changed to a single sodium carboxymethyl cellulose (substitution degree 0.9, concentration 1% w / v solution). The amount of coating is 5% of the mass of the freeze-dried microcapsules. Other process conditions are the same as those of Sample G.

[0190] Sample I (Comparative Example 6): The internal composition of the microcapsules is exactly the same as that of Sample G (100 parts sodium alginate, 30 parts coupling complex, 90 parts filler, and 3 parts antioxidant complex). After microencapsulation and freeze-drying, the surface hydrophilic modification layer is changed to a single PEG-6000 (molecular weight 6000 Da, concentration 1% w / v solution). The amount of coating is 5% of the mass of the freeze-dried microcapsules. Other process conditions are the same as those of Sample G.

[0191] Each sample was prepared in batches of 100g, and then divided into several portions for testing under different conditions.

[0192] 3. Experimental conditions pH conditions: pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 (adjust the pH of the PBS buffer using 0.1M HCl or 0.1M NaOH) Salt concentration conditions: 0mM, 100mM, 200mM, 300mM, 400mM, 500mM NaCl (different concentrations of NaCl were added to the PBS buffer). Test combinations: Six salt concentrations were tested under each pH condition, for a total of 36 test conditions. Test temperature: 25℃±1℃ Sample concentration: 50 mg microcapsules / 10 mL buffer Test indicators: reconstitution time, clarity (transmittance) of the reconstituted solution, surface tension Each test condition was tested in triplicate, and the average of the results was taken.

[0193] 4. Experimental Procedure (1) Preparation of buffer solutions with different pH and salt concentrations Prepare a series of PBS buffer solutions with pH values ​​(4.0-9.0). Add different amounts of NaCl to each pH buffer solution to prepare a series of salt concentrations from 0-500 mM. ③ Verify the pH and salt concentration of each solution using a pH meter and conductivity meter.

[0194] (2) Determination of reconstitution time Weigh 50 mg of lyophilized microcapsule sample and add it to 10 mL of buffer solution with the specified pH and salt concentration. Gently shake (120 rpm) in a 25°C constant temperature water bath, and use a stopwatch to record the time required from the addition of buffer solution until the microcapsules are completely reconstituted (no visible particles). Record the reconstitution time under each condition.

[0195] (3) Determination of solution clarity After reconstitution, transfer the solution to a quartz cuvette. Using a blank buffer solution with the corresponding pH and salt concentration as a control, measure the transmittance at a wavelength of 600 nm. ③ Transmittance = (Sample transmittance / Blank transmittance) × 100%. The higher the transmittance, the clearer the solution.

[0196] (4) Surface tension measurement After reconstitution, 5 mL of the solution was used for surface tension measurement. The surface tension of the solution was measured at 25 °C using a surface tension meter (Krüss K100, platinum plate method). Each sample was tested 5 times, and the highest and lowest values ​​were discarded, with the average of the middle 3 values ​​taken.

[0197] 5. Experimental Results Table 6. Average reconstitution time (minutes) for each sample under different pH conditions. Note: The data in the table are the average values ​​of the test results of 6 salt concentrations (0-500mM) under various pH conditions.

[0198] Table 7. Average reconstitution time (minutes) for each sample under different salt concentrations. Note: The data in the table are the average values ​​of the test results for 6 pH values ​​(4.0-9.0) under various salt concentration conditions.

[0199] Table 8. Detailed data (minutes) on the reconstitution time of sample G under different conditions. Table 9. Performance comparison of samples under pH 7.0, salt concentration of 0 mM, and 500 mM conditions. Figure 5 Thermograph of reconstitution time for sample G under different pH and salt concentration conditions.

[0200] Figure 6 Comparison of reconstitution time for different samples within the pH range of 4-9.

[0201] 6. Analysis and Summary (1) Excellent pH adaptability: Sample G (polyelectrolyte complex hydrophilic layer) maintained a resolution time of 3.4-4.0 minutes over a wide pH range of 4.0-9.0, with a coefficient of variation of only 6.8%, demonstrating excellent pH stability. In contrast, Sample H (monocarboxymethyl cellulose sodium) showed significantly prolonged resolution time under acidic (pH 4.0, 9.5 minutes) and alkaline (pH 9.0, 11.2 minutes) conditions, with a coefficient of variation as high as 35.2%, indicating unstable performance. Sample I (mono-PEG) had a coefficient of variation of 22.1%, with stability falling between the two.

[0202] (2) Outstanding adaptability to salt concentration: For sample G, as the NaCl concentration increased from 0 mM to 500 mM, the resolution time only increased from 3.2 minutes to 4.2 minutes, a growth rate of 31.3%, and the growth trend was gradual. In contrast, the resolution time of sample H increased from 6.2 minutes to 10.1 minutes, a growth rate as high as 62.9%; and that of sample I increased from 5.1 minutes to 8.4 minutes, a growth rate of 64.7%. This demonstrates that the electrostatic cross-linked network structure of the polyelectrolyte complex has strong tolerance to changes in salt concentration.

[0203] (3) Stable performance under extreme conditions: As shown in Table 9, even under the most extreme conditions (pH 4.0 or 9.0, 500mM NaCl), the reconstitution time of sample G does not exceed 4.5 minutes, the transmittance remains above 95%, and the surface tension is maintained at around 29mN / m. However, under the same conditions, the reconstitution time of samples H and I exceeds 8-10 minutes, the transmittance decreases to 76-82%, and the surface tension increases to 42-49mN / m, indicating a significant deterioration in performance.

[0204] (4) Verification of environmental adaptability mechanism: The polyelectrolyte complex (chitosan-sodium carboxymethyl cellulose) forms a highly stable three-dimensional network structure through electrostatic cross-linking of positive and negative charges. Within the pH range of 4-9, although the protonation degree of chitosan and the ionization degree of sodium carboxymethyl cellulose vary, due to their high charge densities (90% deacetylation, 0.9% substitution), sufficient electrostatic interaction is maintained within this pH range, ensuring the stability of the complex network structure. Within the salt concentration range of 0-500 mM, although salt ions partially shield the electrostatic interaction, the complex has already formed a cross-linked network and will not dissociate due to the addition of salt ions, thus maintaining stability.

[0205] (5) Comparison with single hydrophilic polymers: Sodium carboxymethyl cellulose (sample H) undergoes carboxylation under acidic conditions (pH<5), resulting in a decrease in negative charge and hydrophilicity. Under alkaline conditions (pH>8), although the carboxyl groups are completely ionized, the high pH leads to excessive swelling of the molecular chains and a loose structure. Under high salt conditions, salt ions destroy the hydration layer, significantly reducing hydrophilicity. Although single PEG (sample I) performs well at neutral pH, its hydration layer is destroyed under high salt conditions, and it also has stability issues under alkaline conditions.

[0206] (6) Thermographic visualization: The thermographic visualization shows that the reconstitution time of sample G is very small throughout the test range of pH 4-9 and salt concentration 0-500mM. The color distribution is uniform, showing a greenish-yellow tone (3.1-4.5 minutes), and there is no red area (performance degradation area), which fully demonstrates its excellent environmental adaptability.

[0207] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing a spleen extract, comprising pretreatment of mammalian spleen tissue, homogenization, freeze-thaw disruption, precipitation separation, and ultrafiltration to obtain a spleen polypeptide extract, wherein the spleen polypeptide extract is chemically coupled with an ADC to form a spleen polypeptide-ADC coupling complex, characterized in that, It also includes the following steps: An antioxidant-basic amino acid complex was prepared, wherein the complex was formed by vitamin C and lysine, vitamin E and arginine, and tea polyphenols and basic amino acids, respectively. An embedding solution was prepared by mixing the spleen polypeptide-ADC coupling complex with a wall material solution, a functional filler combination, and the antioxidant-basic amino acid complex. The functional filler combination included cyclodextrin, lactose, and microcrystalline cellulose, wherein cyclodextrin accounted for 40-60% of the total filler. The embedding solution was made into microcapsules and then freeze-dried to obtain lyophilized microcapsules; a chitosan-carboxymethyl cellulose sodium polyelectrolyte complex was sprayed onto the surface of the lyophilized microcapsules to form a hydrophilic modified layer.

2. The method according to claim 1, characterized in that, The preparation steps of the antioxidant-basic amino acid complex include: mixing vitamin C powder and L-lysine powder at a mass ratio of 1:0.5-2:1, adding deionized water and stirring to dissolve at room temperature for 30-60 minutes to form a vitamin C-lysine complex.

3. The method according to claim 1, characterized in that, The preparation steps of the antioxidant-basic amino acid complex further include: mixing vitamin E and L-arginine at a mass ratio of 1:1-2:1, adding 5-10% of the mass of vitamin E as an emulsifier, and treating under high-speed stirring conditions for 10-20 minutes to form a vitamin E-arginine emulsion complex.

4. The method according to claim 1, characterized in that, In the functional filler combination, cyclodextrin accounts for 40-60%, lactose accounts for 20-30%, and microcrystalline cellulose accounts for 10-30%.

5. The method according to claim 4, characterized in that, The cyclodextrin is β-cyclodextrin.

6. The method according to claim 1, characterized in that, The preparation steps of the chitosan-sodium carboxymethyl cellulose polyelectrolyte complex include: dissolving chitosan with a degree of deacetylation ≥85% in dilute acetic acid solution to prepare a chitosan solution with a concentration of 0.5-2%; dissolving sodium carboxymethyl cellulose with a degree of substitution ≥0.8 in deionized water to prepare a sodium carboxymethyl cellulose solution with a concentration of 0.5-2%; mixing the chitosan solution and the sodium carboxymethyl cellulose solution at a mass ratio of 1:0.8-1.5:1 and stirring for 10-20 minutes to form a polyelectrolyte complex suspension.

7. The method according to claim 1, characterized in that, The total amount of the antioxidant-basic amino acid complex added is 5-15% of the mass of the spleen polypeptide-ADC coupling complex.

8. The method according to claim 1, characterized in that, The total amount of the functional filler combination added is 2-5 times the mass of the spleen polypeptide-ADC conjugate complex.

9. The method according to claim 1, characterized in that, The amount of the polyelectrolyte complex suspension sprayed is 3-8% of the mass of the lyophilized microcapsules.

10. The method according to claim 1, characterized in that, The wall material solution is selected from sodium alginate solution, chitosan solution or gelatin solution; the microcapsule molding is performed by drop addition, spraying or emulsification.

Images