SOD anti-photoaging sustained-release microcapsules based on two polysaccharide modifications and preparation method thereof

Abstract

The application relates to the technical field of pharmaceutical preparations, and particularly discloses SOD anti-photoaging sustained-release microcapsules based on two polysaccharide modifications and a preparation method thereof. The SOD anti-photoaging sustained-release microcapsules are prepared from the following raw materials: superoxide dismutase, low-anticoagulant heparin, oligo-arginine peptide, chitooligosaccharide, beta-cyclodextrin, astaxanthin, anthocyanin, polyphenol, alginic acid derivative and calcium ion crosslinking agent; the preparation method comprises the following steps: S1, preparing a heparin-superoxide dismutase combination; S2, preparing a heparin-superoxide dismutase-oligo-arginine co-combination; S3, preparing chitooligosaccharide-beta-cyclodextrin pre-assembled nanoparticles; S4, preparing an alginic acid derivative containing a sulfur ketal bond; S5, forming an inner layer agglomerate; S6, preparing double-layer microcapsules and performing solidification treatment, washing and drying. The sustained-release microcapsules can be used for preparing anti-photoaging products, and have the advantages of avoiding inactivation and non-absorption of orally taken superoxide dismutase and realizing efficient delivery of active proteins.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical formulation technology, and more specifically, to SOD anti-photoaging sustained-release microcapsules based on two polysaccharides and their preparation method. Background Technology

[0002] Pharmaceutical formulation technology is applied to the research, development, production, and improvement of various drugs, including oral, injectable, topical, sustained-release, targeted drug delivery, and traditional Chinese medicine compound preparations. It covers numerous medical scenarios such as treatment of common diseases, management of chronic diseases, critical care, and emergency epidemic prevention. This technology optimizes drug form based on the drug's physicochemical properties and the body's absorption and metabolism patterns. This improves drug solubility, stability, and bioavailability, reduces ineffective drug loss, effectively lowers drug toxicity and side effects, precisely controls drug release rhythm and site of action, adapts to the differences in medication use among patients of different ages and constitutions, extends drug shelf life, simplifies dosing frequency, and effectively balances drug safety, therapeutic efficacy, and convenience of daily use.

[0003] Related drug formulations are made into oral capsules of superoxide dismutase. However, superoxide dismutase is easily inactivated by gastric acid and intestinal proteases. At the same time, large protein molecules have difficulty passing through the intestinal mucosal barrier on their own, resulting in insufficient number of active enzymes entering the bloodstream after oral administration, and thus the anti-photoaging effect is difficult to exert effectively. Summary of the Invention

[0004] To address the problem that related pharmaceutical formulations, which use superoxide dismutase in oral capsules, result in insufficient amounts of active enzyme entering the bloodstream after oral administration, thus hindering the anti-photoaging effect, this application provides SOD anti-photoaging sustained-release microcapsules modified with two polysaccharides and their preparation method.

[0005] In the first aspect, this application provides SOD anti-photoaging sustained-release microcapsules based on two types of polysaccharide modification, using the following technical solution: The SOD anti-photoaging sustained-release microcapsules based on two polysaccharides are made from the following raw materials in parts by weight: 10-50 parts superoxide dismutase, 5-30 parts low anticoagulant heparin, 1-10 parts oligoarginine peptide, 20-100 parts chitosan oligosaccharide, 30-150 parts β-cyclodextrin, 1-20 parts astaxanthin, 1-20 parts anthocyanin, 1-20 parts polyphenols, 50-200 parts alginic acid derivative containing thioketal bonds, and 5-50 parts calcium ion crosslinking agent. In this process, the superoxide dismutase, low-anticoagulation heparin, and oligoarginine peptide are covalently linked to form a co-conjugate; the chitosan oligosaccharide and β-cyclodextrin containing astaxanthin, anthocyanins, and polyphenols form pre-assembled nanoparticles; the alginate derivative is cross-linked under the action of calcium ions to form the outer gel of the microcapsule; and the co-conjugate and the pre-assembled nanoparticles form the inner condensate of the microcapsule through electrostatic interaction.

[0006] By employing the above technical solution, the superoxide dismutase (SOD) is dually covalently modified with low-anticoagulant heparin and oligomeric arginine peptides. The low-anticoagulant heparin polysaccharide chain introduces a large number of sulfate and carboxyl groups, forming a stereoprotective and local buffering microenvironment on the SOD molecule surface. This enhances the SOD's resistance to gastric acid, heat, and protease degradation, while also prolonging its circulating half-life in vivo. The addition of oligomeric arginine peptides endows the co-conjugate with the ability to actively transport across the intestinal epithelium, enabling SOD to efficiently cross the mucosal barrier and enter the bloodstream after release from the intestine. The pre-assembled chitosan oligosaccharide and β-cyclodextrin inclusion complex into nanoparticles utilizes the chitosan oligosaccharide's own functions of absorbing ultraviolet light, scavenging free radicals, and activating skin cells to combat photoaging, thus improving the cell wall... The material itself becomes an organic component of the efficacy system. On the other hand, antioxidant excipients such as astaxanthin, anthocyanins, and polyphenols are uniformly anchored inside the nanoparticles in a molecular inclusion form, overcoming the problems of uneven dispersion and easy degradation of lipid-soluble or sensitive components in the microcapsule system. Alginic acid derivatives containing thioketal bonds serve as the outer gel layer. The thioketal bonds on their molecular chains undergo specific cleavage in the reactive oxygen species microenvironment induced by ultraviolet light. This allows the microcapsules to not only achieve initial release under intestinal pH conditions but also undergo secondary disintegration and release when the local reactive oxygen species level in the skin tissue increases, thereby further extending the anti-photoaging effect of the oral formulation to the skin target. The above multi-level structures work together to form a complete system with synergistic effects of molecular-level protection, delivery-level protection, and response-level release.

[0007] Preferably, the oligoarginine peptide is composed of 6 to 8 arginine residues and has a cysteine ​​residue at its terminal; the chitosan oligosaccharide has a molecular weight of 1000 to 5000 Da and a degree of deacetylation ≥ 85%.

[0008] By adopting the above technical solution, the number of residues of oligoarginine peptides is controlled to 6 to 8, and its terminal cysteine ​​provides a reaction site for site-directed coupling, achieving directional linkage without affecting the active center of superoxide dismutase. The chitosan oligosaccharide is selected with a low molecular weight and maintains a high degree of deacetylation, which not only ensures its good solubility in aqueous solution and efficient electrostatic binding with negatively charged co-conjugates, but also fully utilizes the inherent biological functions of low molecular weight chitosan oligosaccharide in absorbing ultraviolet rays, inhibiting harmful bacteria on the skin surface, and preventing skin roughness and aging. Thus, the delivery function of the microcapsule inner layer material and the anti-photoaging activity are highly unified.

[0009] Preferably, the microcapsules have a particle size of 10–300 μm and an SOD encapsulation rate of not less than 75%.

[0010] By adopting the above technical solution, the microcapsule particle size is controlled within the range of 10 to 300 micrometers, which facilitates the subsequent processing of oral dosage forms and enables effective bioadhesion and retention in the intestine, thus promoting the full absorption of the contents. The encapsulation rate of not less than 75% ensures that superoxide dismutase and other active ingredients are minimally lost during the preparation process, guaranteeing the actual efficacy of the formulation. It also reflects that the electrostatic assembly between heparin modification and chitosan oligosaccharide pre-assembled nanoparticles has high binding efficiency and structural compactness, providing a basis for the stable existence of microcapsules in the gastrointestinal tract and subsequent responsive release.

[0011] Secondly, this application provides a method for preparing SOD anti-photoaging sustained-release microcapsules based on two types of polysaccharide modification, using the following technical solution: The preparation method of SOD anti-photoaging sustained-release microcapsules based on two polysaccharides includes the following steps: S1. Dissolve low-anticoagulation heparin in buffer solution, add sodium periodate to activate the reaction, and obtain activated heparin after termination and dialysis; mix activated heparin with superoxide dismutase solution, react under alkaline conditions to form Schiff base, and then add reducing agent to reduce to obtain heparin-superoxide dismutase conjugate. S2. Add a heterobifunctional cross-linking agent to the heparin-superoxide dismutase conjugate solution prepared in S1 for activation, and then add oligoarginine peptide containing cysteine ​​to react and obtain heparin-superoxide dismutase-oligoarginine coconjugate. S3. Astaxanthin, anthocyanins, and polyphenols are mixed and dissolved, and then mixed with β-cyclodextrin solution for inclusion to obtain an antioxidant inclusion complex; chitosan oligosaccharide is dissolved in acidic aqueous solution, the antioxidant inclusion complex is added, and after stirring evenly, an ionic crosslinking agent solution is added dropwise to obtain chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles through crosslinking. S4. Dissolve sodium alginate in water, add sodium periodate for partial oxidation to obtain oxidized sodium alginate; add a diamine compound containing a thioketal bond to the oxidized sodium alginate solution, and after reaction and reduction, obtain an alginate derivative containing a thioketal bond. S5. The co-conjugate of S2 is mixed with the pre-assembled nanoparticles of S3 and electrostatically self-assembled under specific pH and ionic strength to form an inner layer condensate. S6. Mix the inner layer aggregate of S5 with the alginate derivative solution of S4, disperse them, and then add calcium ion crosslinking agent to initiate gelation and form bilayer microcapsules; cure the obtained bilayer microcapsules, wash and dry them to obtain SOD anti-photoaging sustained-release microcapsules based on two polysaccharides.

[0012] By adopting the above technical solution, since each step follows the route of first constructing functional units separately and then assembling them step by step, it effectively ensures that the intermediate products formed in each step have a clear structure and function, avoiding mutual interference and activity loss that occurs when multiple active components are directly mixed; Steps S1 and S2, through heparin covalent modification and oligoarginine linkage, enable superoxide dismutase to acquire multiple capabilities at the molecular level, including acid resistance, enzymatic resistance, and transmembrane transport, providing a structurally controllable substrate with suitable surface charge for subsequent microencapsulation. Simultaneously, heparin itself has anti-photoaging properties, which allows the heparin-superoxide dismutase-oligoarginine co-conjugate to further enhance the anti-photoaging performance of the sustained-release microcapsules; Step S3 utilizes β- Cyclodextrin inclusion and chitosan oligosaccharide crosslinking are completed simultaneously, stably dispersing small molecule antioxidants in the form of pre-assembled nanoparticles. This protects sensitive components and provides positively charged building blocks for the formation of the inner condensate. The alginate derivative containing thioketal bonds synthesized in step S4 endows the outer gel with additional reactive oxygen species responsiveness beyond conventional pH response, expanding the release triggering conditions. Steps S5 and S6 combine functional units in an orderly manner through electrostatic self-assembly and calcium ion crosslinking, first forming the inner condensate and then encapsulating the outer gel. This integrates multiple functions such as gastrointestinal pH response, intestinal adhesion and penetration, and targeted disintegration in the skin microenvironment within the same formulation, ultimately obtaining a sustained-release microcapsule with a dense structure, synergistic function, and cascade release characteristics.

[0013] Preferably, in step S1, the sodium periodate activation reaction is carried out in the dark at 2-8°C for 1-6 hours; the reducing agent is sodium cyanoborohydride or sodium borohydride, and the pH of the reduction reaction is 7.0-8.5.

[0014] By adopting the above technical solution, since sodium periodate activation is carried out under low temperature and light-proof conditions, the vicinal diol group on the heparin sugar ring is selectively oxidized to generate an aldehyde group, while avoiding excessive oxidation and polysaccharide chain breakage, thus preserving the molecular skeleton of heparin. The reduction step uses sodium cyanoborohydride or sodium borohydride and is carried out under weakly alkaline conditions to stably reduce the Schiff base to a strong carbon-nitrogen single bond, ensuring that a stable covalent bond is formed between heparin and superoxide dismutase, and avoiding dissociation during subsequent processing and in vivo transport.

[0015] Preferably, in step S2, the heteromorphic bifunctional crosslinking agent is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the molar ratio of the crosslinking agent to the heparin-superoxide dismutase conjugate is 5:1 to 20:1; the molar ratio of the oligomeric arginine peptide to the heparin-superoxide dismutase conjugate is 3:1 to 10:1.

[0016] By adopting the above technical solution, since succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester is selected as a heterogeneous bifunctional crosslinking agent, the succinimide ester group at one end reacts with the free amino group remaining on the surface of superoxide dismutase, and the maleimide group exposed at the other end is specifically linked to the thiol group of oligoarginine peptide containing cysteine, thereby achieving sequentially controlled heterogeneous coupling, reducing the occurrence of self-polymerization or homopolymerization side reactions of the crosslinking agent, and improving the structural uniformity of the co-conjugate.

[0017] Preferably, in step S3, the ionic crosslinking agent is sodium tripolyphosphate, the mass ratio of chitosan oligosaccharide to sodium tripolyphosphate is 3:1 to 8:1; the concentration of the chitosan oligosaccharide solution is 0.5 to 5 mg / mL; and the average particle size of the obtained chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles is 50 to 500 nm.

[0018] By adopting the above technical solution, since the ionic crosslinking uses sodium tripolyphosphate to physically crosslink with the amino group of chitosan oligosaccharide, nanoparticles are formed under relatively mild conditions, avoiding the destruction of the active ingredients contained in the β-cyclodextrin inclusion complex; the resulting pre-assembled nanoparticles have a particle size in the nanoscale, which helps to form a uniform and dense inner layer aggregate with the co-conjugate during subsequent electrostatic self-assembly, and also facilitates the dissolution and synergistic absorption of each component at a similar rate after the microcapsules are released in the intestine.

[0019] Preferably, in step S4, the degree of oxidation of sodium alginate is 5% to 30%; the diamine compound containing a thioketal bond is a 2,2-dimethoxypropane-1,3-diamine thioketal derivative, and the molar ratio of its aldehyde group to oxidized sodium alginate is 0.5:1 to 2:1.

[0020] By adopting the above technical solution, sodium alginate generates active aldehyde groups on the sugar ring after partial oxidation, providing chemical sites for side chain modification. The degree of oxidation is controlled within an appropriate range, maintaining the gelling ability of the alginate backbone while introducing responsive groups. Grafting with diamine compounds containing thioketal bonds makes the alginate derivative stable under normal conditions, while the thioketal bonds break in an environment with increased reactive oxygen species concentration, leading to the gradual degradation of the outer gel and realizing the secondary responsive release of microcapsules in skin tissue.

[0021] Preferably, in step S5, the mass ratio of the co-conjugate to the pre-assembled nanoparticles is 1:0.5 to 1:5, the pH for electrostatic self-assembly is 4.0 to 6.0, and the stirring time is 10 to 60 minutes.

[0022] By adopting the above technical solution, since electrostatic self-assembly is carried out under weakly acidic pH conditions, the amino groups of chitosan oligosaccharide are fully protonated and carry positive charges, while the heparin sulfate and carboxyl groups on the surface of the co-conjugate carry negative charges. The interaction between positive and negative charges drives the two components to aggregate in an orderly manner, forming a dense inner layer aggregate. The setting of the stirring time ensures that the assembly process is fully completed, while avoiding excessive aggregation or loss of activity of the aggregate due to excessive time.

[0023] Preferably, in step S6, the calcium ion crosslinking agent is selected from calcium chloride, calcium acetate, or calcium lactate, and its concentration is 0.1–1.0 mol / L; the drying is freeze drying or low-temperature spray drying.

[0024] By adopting the above technical solution, since the calcium ion crosslinking agent is selected from calcium chloride, calcium acetate or calcium lactate, it can undergo ionic crosslinking with the carboxyl groups of alginic acid derivatives to form a stable gel network, thereby completely encapsulating the inner layer aggregate and isolating it from the external environment; the drying method adopts freeze drying or low temperature spray drying, which removes moisture through low temperature and rapid dehydration, maintains the active conformation of superoxide dismutase and the stability of other temperature-sensitive components, and obtains a solid microcapsule formulation that is easy to store and use.

[0025] In summary, this application has the following beneficial effects: 1. This application uses low-anticoagulation heparin and oligoarginine peptides to covalently modify superoxide dismutase (SOD). The heparin polysaccharide chain constructs a three-dimensional shielding layer and a local buffer microenvironment on the surface of the SOD molecule, enhancing the SOD's ability to resist gastric acid, heat, and protease degradation, and prolonging its in vivo circulating half-life. The oligoarginine peptides endow the co-conjugate with the ability to actively transport across the intestinal epithelium, enabling the SOD to efficiently pass through the mucosal barrier and enter the bloodstream after being released in the intestine. This solves the problems of easy inactivation and poor absorption of orally administered SOD, achieving the effect of efficient delivery of active protein.

[0026] 2. In this application, chitosan oligosaccharide and β-cyclodextrin inclusion complex are pre-assembled into nanoparticles, and antioxidant excipients such as astaxanthin, anthocyanins and polyphenols are uniformly anchored inside the nanoparticles in a supramolecular inclusion form. Chitosan oligosaccharide serves as both a delivery carrier and a functional active ingredient. Its own ultraviolet absorption and free radical scavenging capabilities complement the enzymatic antioxidant function of superoxide dismutase. After the amino groups of chitosan oligosaccharide are protonated under weakly acidic conditions, they undergo multi-site electrostatic cross-linking with the negatively charged groups on the surface of the co-conjugate, thereby achieving the effect of combining wall material delivery function and anti-photoaging, and enhancing the synergistic anti-photoaging performance of each component.

[0027] 3. The method of this application involves first constructing three functional units: heparin-superoxide dismutase-oligoarginine co-conjugate, chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles, and alginate derivatives containing thioketal bonds. These units are then assembled sequentially via electrostatic self-assembly and calcium ion cross-linking, avoiding interference between active components. The inner condensate gradually dissociates under the weakly alkaline conditions of the intestine and achieves efficient absorption through mucosal adhesion of chitosan oligosaccharide and membrane penetration of oligoarginine. The thioketal bonds in the outer gel undergo specific breakage in the highly reactive oxygen species environment induced by ultraviolet radiation in skin tissue, triggering secondary disintegration and release of the microcapsules at the target site. Simultaneously, heparin itself has anti-photoaging properties, which allows the heparin-superoxide dismutase-oligoarginine co-conjugate to further enhance the anti-photoaging performance of the sustained-release microcapsules, thereby achieving a cascade delivery and synergistic anti-photoaging effect from the gastrointestinal tract to the skin tissue. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the preparation method of SOD anti-photoaging sustained-release microcapsules based on two polysaccharide modifications proposed in this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Technical concept: Related drug formulations are made into oral capsules of superoxide dismutase. However, superoxide dismutase is easily inactivated by gastric acid and intestinal proteases. At the same time, large protein molecules have difficulty passing through the intestinal mucosal barrier on their own, resulting in insufficient number of active enzymes entering the bloodstream after oral administration, and thus the anti-photoaging effect is difficult to exert effectively.

[0031] This application discloses SOD anti-photoaging sustained-release microcapsules modified with two polysaccharides and their preparation method. The microcapsules are made from the following raw materials: superoxide dismutase, low-anticoagulant heparin, oligoarginine peptides, chitosan oligosaccharides, β-cyclodextrin, astaxanthin, anthocyanins, polyphenols, alginate derivatives, and a calcium ion crosslinking agent. The preparation method is as follows: S1, preparing a heparin-superoxide dismutase conjugate; S2, preparing a heparin-superoxide dismutase-oligoarginine co-conjugate; S3, preparing chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles; S4, preparing an alginate derivative containing thioketal bonds; S5, forming an inner layer aggregate; S6, recognizing the bilayer microcapsules and performing solidification, washing, and drying.

[0032] This application employs low-anticoagulation heparin and oligoarginine peptides to covalently modify superoxide dismutase (SOD). The heparin polysaccharide chain constructs a three-dimensional shielding layer and a local buffer microenvironment on the surface of the SOD molecule, enhancing the SOD's ability to resist degradation by gastric acid, heat, and proteases, and prolonging its in vivo circulating half-life. The oligoarginine peptides endow the co-conjugate with active transepithelial transport capability, enabling SOD to efficiently cross the mucosal barrier and enter the bloodstream after release from the intestine. This solves the problems of easy inactivation and poor absorption of orally administered SOD, achieving the effect of efficient delivery of active protein.

[0033] Example 1: This example provides SOD anti-photoaging sustained-release microcapsules based on two polysaccharides, which are made from the following raw materials in parts by weight: 10 parts superoxide dismutase, 5 parts low anticoagulant heparin, 1 part oligoarginine peptide, 20 parts chitosan oligosaccharide, 30 parts β-cyclodextrin, 1 part astaxanthin, 1 part anthocyanin, 1 part polyphenol, 50 parts alginic acid derivative containing thioketal bonds, and 5 parts calcium ion crosslinking agent; Among them, superoxide dismutase, low anticoagulant heparin and oligoarginine peptide are covalently linked to form a co-conjugate; chitosan oligosaccharide and β-cyclodextrin containing astaxanthin, anthocyanins and polyphenols form pre-assembled nanoparticles; alginic acid derivatives are cross-linked under the action of calcium ions to form the outer gel of the microcapsule, and the co-conjugate and the pre-assembled nanoparticles form the inner condensate of the microcapsule through electrostatic interaction.

[0034] Among them, the oligoarginine peptide is composed of 6 arginine residues and contains cysteine ​​at its terminal; the molecular weight of the chitosan oligosaccharide is 1000 Da and the degree of deacetylation is ≥85%.

[0035] The microcapsules have a particle size of 10 μm and an SOD encapsulation efficiency of no less than 75%.

[0036] The preparation method of the above-mentioned SOD anti-photoaging sustained-release microcapsules based on two polysaccharides includes the following steps: S1. Dissolve low-anticoagulation heparin in buffer solution, add sodium periodate to activate the reaction, and obtain activated heparin after termination and dialysis; mix activated heparin with superoxide dismutase solution, react under alkaline conditions to form Schiff base, and then add reducing agent to reduce to obtain heparin-superoxide dismutase conjugate. The sodium periodate activation reaction was carried out in the dark at 2°C for 1 hour; the reducing agent was sodium cyanoborohydride, and the pH of the reduction reaction was 7.0.

[0037] S2. Add a heterobifunctional cross-linking agent to the heparin-superoxide dismutase conjugate solution prepared in S1 for activation, and then add oligoarginine peptide containing cysteine ​​to react and obtain heparin-superoxide dismutase-oligoarginine coconjugate. Among them, the heteromorphic bifunctional crosslinking agent is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the molar ratio of the crosslinking agent to the heparin-superoxide dismutase conjugate is 5:1; the molar ratio of oligoarginine peptide to heparin-superoxide dismutase conjugate is 3:1.

[0038] S3. Mix and dissolve astaxanthin, anthocyanins, and polyphenols, and mix with β-cyclodextrin solution for inclusion to obtain an antioxidant inclusion complex; dissolve chitosan oligosaccharide in acidic aqueous solution, add the antioxidant inclusion complex, stir evenly, and then add ionic crosslinking agent solution dropwise to obtain chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles through crosslinking. The ionic crosslinking agent was sodium tripolyphosphate, and the mass ratio of chitosan oligosaccharide to sodium tripolyphosphate was 3:1; the concentration of the chitosan oligosaccharide solution was 0.5 mg / mL; and the average particle size of the obtained chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles was 50 nm.

[0039] S4. Dissolve sodium alginate in water, add sodium periodate for partial oxidation to obtain oxidized sodium alginate; add a diamine compound containing a thioketal bond to the oxidized sodium alginate solution, and after reaction and reduction, obtain an alginate derivative containing a thioketal bond. The degree of oxidation of sodium alginate is 5%; the diamine compound containing a thioketal bond is a 2,2-dimethoxypropane-1,3-diamine thioketal derivative, and its molar ratio of aldehyde group to oxidized sodium alginate is 0.5:1.

[0040] S5. The co-conjugate of S2 is mixed with the pre-assembled nanoparticles of S3 and electrostatically self-assembled under specific pH and ionic strength to form an inner layer condensate. The mixing mass ratio of the co-conjugate to the pre-assembled nanoparticles was 1:0.5, the pH for electrostatic self-assembly was 4.0, and the stirring time was 10 minutes.

[0041] S6. Mix the inner layer aggregate of S5 with the alginate derivative solution of S4, disperse them, and then add calcium ion crosslinking agent to initiate gelation and form bilayer microcapsules; cure the obtained bilayer microcapsules, wash and dry them to obtain SOD anti-photoaging sustained-release microcapsules based on two polysaccharides.

[0042] The calcium ion crosslinking agent is selected from calcium chloride, and its concentration is 0.1 mol / L; the drying method is freeze drying.

[0043] Example 2: This example provides SOD anti-photoaging sustained-release microcapsules based on two polysaccharides, made from the following raw materials in parts by weight: 30 parts superoxide dismutase, 17.5 parts low anticoagulant heparin, 5.5 parts oligoarginine peptide, 60 parts chitosan oligosaccharide, 90 parts β-cyclodextrin, 10.5 parts astaxanthin, 10.5 parts anthocyanins, 10.5 parts polyphenols, 125 parts alginic acid derivative containing thioketal bonds, and 27.5 parts calcium ion crosslinking agent; Among them, superoxide dismutase, low anticoagulant heparin and oligoarginine peptide are covalently linked to form a co-conjugate; chitosan oligosaccharide and β-cyclodextrin containing astaxanthin, anthocyanins and polyphenols form pre-assembled nanoparticles; alginic acid derivatives are cross-linked under the action of calcium ions to form the outer gel of the microcapsule, and the co-conjugate and the pre-assembled nanoparticles form the inner condensate of the microcapsule through electrostatic interaction.

[0044] Among them, the oligoarginine peptide is composed of 7 arginine residues and has a cysteine ​​residue at its end; the chitosan oligosaccharide has a molecular weight of 3000 Da and a degree of deacetylation ≥85%.

[0045] The microcapsules have a particle size of 155 μm and an SOD encapsulation efficiency of no less than 75%.

[0046] The preparation method of the above-mentioned SOD anti-photoaging sustained-release microcapsules based on two polysaccharides includes the following steps: S1. Dissolve low-anticoagulation heparin in buffer solution, add sodium periodate to activate the reaction, and obtain activated heparin after termination and dialysis; mix activated heparin with superoxide dismutase solution, react under alkaline conditions to form Schiff base, and then add reducing agent to reduce to obtain heparin-superoxide dismutase conjugate. The sodium periodate activation reaction was carried out in the dark at 5°C for 3.5 hours; the reducing agent was sodium borohydride, and the pH of the reduction reaction was 7.75.

[0047] S2. Add a heterobifunctional cross-linking agent to the heparin-superoxide dismutase conjugate solution prepared in S1 for activation, and then add oligoarginine peptide containing cysteine ​​to react and obtain heparin-superoxide dismutase-oligoarginine coconjugate. Among them, the heteromorphic bifunctional crosslinking agent is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the molar ratio of the crosslinking agent to the heparin-superoxide dismutase conjugate is 12.5:1; the molar ratio of oligoarginine peptide to the heparin-superoxide dismutase conjugate is 6.5:1.

[0048] S3. Mix and dissolve astaxanthin, anthocyanins, and polyphenols, and mix with β-cyclodextrin solution for inclusion to obtain an antioxidant inclusion complex; dissolve chitosan oligosaccharide in acidic aqueous solution, add the antioxidant inclusion complex, stir evenly, and then add ionic crosslinking agent solution dropwise to obtain chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles through crosslinking. The ionic crosslinking agent was sodium tripolyphosphate, and the mass ratio of chitosan oligosaccharide to sodium tripolyphosphate was 5.5:1; the concentration of the chitosan oligosaccharide solution was 2.75 mg / mL; and the average particle size of the obtained chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles was 275 nm.

[0049] S4. Dissolve sodium alginate in water, add sodium periodate for partial oxidation to obtain oxidized sodium alginate; add a diamine compound containing a thioketal bond to the oxidized sodium alginate solution, and after reaction and reduction, obtain an alginate derivative containing a thioketal bond. The degree of oxidation of sodium alginate is 17.5%; the diamine compound containing thioketal bonds is a 2,2-dimethoxypropane-1,3-diamine thioketal derivative, and its molar ratio of aldehyde groups to oxidized sodium alginate is 1.25:1.

[0050] S5. The co-conjugate of S2 is mixed with the pre-assembled nanoparticles of S3 and electrostatically self-assembled under specific pH and ionic strength to form an inner layer condensate. The mixing mass ratio of the co-conjugate to the pre-assembled nanoparticles was 1:2.75, the pH for electrostatic self-assembly was 5.0, and the stirring time was 35 minutes.

[0051] S6. Mix the inner layer aggregate of S5 with the alginate derivative solution of S4, disperse them, and then add calcium ion crosslinking agent to initiate gelation and form bilayer microcapsules; cure the obtained bilayer microcapsules, wash and dry them to obtain SOD anti-photoaging sustained-release microcapsules based on two polysaccharides.

[0052] The calcium ion crosslinking agent is selected from calcium acetate, with a concentration of 0.55 mol / L; the drying method is low-temperature spray drying.

[0053] Example 3: This example provides SOD anti-photoaging sustained-release microcapsules based on two polysaccharides, which are made from the following raw materials in parts by weight: 50 parts superoxide dismutase, 30 parts low anticoagulant heparin, 10 parts oligoarginine peptide, 100 parts chitosan oligosaccharide, 150 parts β-cyclodextrin, 20 parts astaxanthin, 20 parts anthocyanin, 20 parts polyphenols, 200 parts alginic acid derivative containing thioketal bonds, and 50 parts calcium ion crosslinking agent; Among them, superoxide dismutase, low anticoagulant heparin and oligoarginine peptide are covalently linked to form a co-conjugate; chitosan oligosaccharide and β-cyclodextrin containing astaxanthin, anthocyanins and polyphenols form pre-assembled nanoparticles; alginic acid derivatives are cross-linked under the action of calcium ions to form the outer gel of the microcapsule, and the co-conjugate and the pre-assembled nanoparticles form the inner condensate of the microcapsule through electrostatic interaction.

[0054] Among them, the oligoarginine peptide is composed of 8 arginine residues and contains cysteine ​​at its terminal; the chitosan oligosaccharide has a molecular weight of 5000 Da and a degree of deacetylation ≥85%.

[0055] The microcapsules have a particle size of 300 μm and an SOD encapsulation rate of not less than 75%.

[0056] The preparation method of the above-mentioned SOD anti-photoaging sustained-release microcapsules based on two polysaccharides includes the following steps: S1. Dissolve low-anticoagulation heparin in buffer solution, add sodium periodate to activate the reaction, and obtain activated heparin after termination and dialysis; mix activated heparin with superoxide dismutase solution, react under alkaline conditions to form Schiff base, and then add reducing agent to reduce to obtain heparin-superoxide dismutase conjugate. The sodium periodate activation reaction was carried out in the dark at 8°C for 6 hours; the reducing agent was sodium borohydride, and the pH of the reduction reaction was 8.5.

[0057] S2. Add a heterobifunctional cross-linking agent to the heparin-superoxide dismutase conjugate solution prepared in S1 for activation, and then add oligoarginine peptide containing cysteine ​​to react and obtain heparin-superoxide dismutase-oligoarginine coconjugate. Among them, the heteromorphic bifunctional crosslinking agent is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the molar ratio of the crosslinking agent to the heparin-superoxide dismutase conjugate is 20:1; the molar ratio of oligoarginine peptide to heparin-superoxide dismutase conjugate is 10:1.

[0058] S3. Mix and dissolve astaxanthin, anthocyanins, and polyphenols, and mix with β-cyclodextrin solution for inclusion to obtain an antioxidant inclusion complex; dissolve chitosan oligosaccharide in acidic aqueous solution, add the antioxidant inclusion complex, stir evenly, and then add ionic crosslinking agent solution dropwise to obtain chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles through crosslinking. The ionic crosslinking agent was sodium tripolyphosphate, and the mass ratio of chitosan oligosaccharide to sodium tripolyphosphate was 8:1; the concentration of the chitosan oligosaccharide solution was 5 mg / mL; and the average particle size of the obtained chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles was 500 nm.

[0059] S4. Dissolve sodium alginate in water, add sodium periodate for partial oxidation to obtain oxidized sodium alginate; add a diamine compound containing a thioketal bond to the oxidized sodium alginate solution, and after reaction and reduction, obtain an alginate derivative containing a thioketal bond. The oxidation degree of sodium alginate is 30%; the diamine compound containing thioketal bonds is a 2,2-dimethoxypropane-1,3-diamine thioketal derivative, and its molar ratio with the aldehyde group of oxidized sodium alginate is 2:1.

[0060] S5. The co-conjugate of S2 is mixed with the pre-assembled nanoparticles of S3 and electrostatically self-assembled under specific pH and ionic strength to form an inner layer condensate. The mass ratio of the co-conjugate to the pre-assembled nanoparticles was 1:5, the pH for electrostatic self-assembly was 6.0, and the stirring time was 60 minutes.

[0061] S6. Mix the inner layer aggregate of S5 with the alginate derivative solution of S4, disperse them, and then add calcium ion crosslinking agent to initiate gelation and form bilayer microcapsules; cure the obtained bilayer microcapsules, wash and dry them to obtain SOD anti-photoaging sustained-release microcapsules based on two polysaccharides.

[0062] The calcium ion crosslinking agent is selected from calcium lactate, with a concentration of 1.0 mol / L; the drying method is low-temperature spray drying.

[0063] Comparative Example 1: This comparative example is based on the content of Example 1, except that the molecular weight of chitosan oligosaccharide is 800 Da, and the rest is the same as Example 1.

[0064] Comparative Example 2: This comparative example is the same as that in Example 1, except that the oxidation degree of sodium alginate is 40%, and the rest is the same as that in Example 1.

[0065] Comparative Example 3: This comparative example is based on the content of Example 1, except that the oligomeric arginine peptide is composed of 4 arginine residues, and the rest is the same as in Example 1.

[0066] Comparative Example 4: This comparative example is the same as that in Example 1, except that the mass ratio of the co-conjugate to the pre-assembled nanoparticles is 1:0.3. The rest of the contents are the same as those in Example 1.

[0067] Comparative Example 5: This comparative example is the same as that in Example 1, except that the concentration of the calcium ion crosslinking agent is 0.06 mol / L. The rest of the contents are the same as those in Example 1.

[0068] Comparative Example 6: This comparative example is the same as that in Example 1, except that the weight of low anticoagulant heparin is 2 parts, and the rest is the same as that in Example 1.

[0069] Performance testing Sample preparation: Microcapsule samples were prepared under the same conditions according to the formulations and preparation methods of the examples and comparative examples. Three batches of each sample were prepared in parallel. Each batch was independently fed and all steps were completed. The resulting solid microcapsule formulations were sealed and stored at 4°C in the dark. Before testing, each batch of samples was taken out of the storage environment, equilibrated at room temperature in a desiccator, and then used for subsequent performance tests to ensure that the test results reflect the inherent properties of the microcapsules rather than differences in storage conditions.

[0070] Detection of SOD activity retention rate in simulated gastric juice: Appropriate amounts of microcapsule samples from the examples and comparative examples were weighed and placed in simulated gastric juice at pH 1.2. The samples were incubated at 37°C with constant shaking for 2 hours. After incubation, the microcapsules were separated from the simulated gastric juice and transferred to phosphate buffer at pH 7.4. The samples were then shaken at 37°C until the microcapsules disintegrated, and the SOD activity of the released liquid was measured. The residual activity of an equal amount of unencapsulated free SOD treated under the same simulated gastric juice conditions was used as a control. The SOD activity retention rate of each group of microcapsules was calculated. SOD activity was determined using the xanthine oxidase-cytochrome C method, with the test standard referring to GB / T5009.171.

[0071] Detection of transmembrane transport efficiency in simulated intestinal fluid: The transmembrane transport efficiency of SOD in microcapsules was evaluated using a Caco-2 cell monolayer model. Caco-2 cells were seeded in Transwell chambers and cultured until the transmembrane resistance reached the preset standard, forming a dense monolayer. Microcapsules prepared in Examples 1-3 and Comparative Examples 3 and 4 were pre-incubated in simulated gastric fluid at pH 1.2 for 2 hours, and then transferred to simulated intestinal fluid at pH 6.8 for further incubation. The drug-containing solution on the intestinal fluid side was collected and added to the top side of the Transwell chamber. The cells were cultured at 37°C and 5% carbon dioxide for 4 hours, and the culture medium on the basal side was collected to determine SOD activity. The transmembrane transport efficiency was expressed as the percentage of SOD activity on the basal side relative to the initial drug-added activity on the top side. The transport efficiency test standard referred to the relevant provisions of the Chinese Pharmacopoeia, Part IV, regarding the Caco-2 cell monolayer permeability test.

[0072] Detection of reactive oxygen species (ROS) responsive disintegration and release: Microcapsules prepared in the examples and comparative examples were weighed and placed in phosphate buffer at pH 7.4, and the mixture was kept at a constant temperature of 37°C with shaking. Hydrogen peroxide was added to the buffer as a source of ROS to maintain an appropriate concentration in the system to simulate the ROS microenvironment of skin tissue under high UV exposure. Samples were taken at different time points after the addition of hydrogen peroxide, and the SOD activity released in the buffer was measured to calculate the cumulative release rate. The cumulative release rate of an equal amount of the same batch of microcapsules under the same conditions in pH 7.4 phosphate buffer without hydrogen peroxide was used as a control. The determination of SOD activity in the release solution was performed according to GB / T5009.171.

[0073] UV-induced free radical scavenging ability test: Microcapsule samples from the examples and comparative examples were weighed and sequentially processed with simulated gastric and intestinal fluids, and the release liquid was collected. The release liquid was added to the reaction solution containing a superoxide anion free radical generation system. The system continuously generated free radicals under UV irradiation. After irradiation, a free radical scavenger was added immediately, and the residual free radical signal intensity was detected by electron spin resonance spectroscopy. The free radical scavenging rate of each group of release liquids was calculated by comparing the free radical signal intensity of the blank system without release liquid after the same UV irradiation. The test standard referred to the relevant provisions of GB / T39100 on the detection of antioxidant activity.

[0074] Anti-photoaging performance testing: Cells were seeded in 96-well plates and cultured to the logarithmic growth phase. Microcapsule release solutions of each example and comparative example, sequentially treated with simulated gastric and intestinal fluids, were added. A release solution containing an equal amount of free SOD served as a positive control, and a blank culture medium without samples served as a negative control. Cells were then irradiated with a UVB light source for a single time and cultured for 24 hours after irradiation. Cell viability was detected using the MTT assay, and the content of matrix metalloproteinase-1 and the amount of type I collagen synthesis in cells were detected using a kit method. The comprehensive anti-photoaging score was calculated based on the three indicators (maximum score of 100 points, with higher scores indicating more significant anti-photoaging effects). The testing standards were based on the testing specifications for photoprotective efficacy in the "Guidelines for the Evaluation of Cosmetic Efficacy".

[0075] In vivo anti-photoaging efficacy test: SPF-grade hairless mice were randomly divided into a model group, a normal control group, and sample groups for each example and comparative example. Mice in the model group and sample groups were exposed to combined UVA (dose 10 J / cm²) and UVB (dose 0.5 J / cm²) irradiation daily, 5 times a week, for 8 consecutive weeks to establish a skin photoaging model. From the first day of modeling, each sample group was administered the corresponding microcapsule sample by gavage daily at a dose of 1000 U / kg body weight (SOD). The normal control group and model group were given the same volume of pure water. After the experiment, the back skin of each group of mice was taken for Masson's trichrome staining to observe the morphology of collagen fibers in the dermis, and the percentage of collagen fiber area was calculated using image analysis software. At the same time, the skin elasticity index was assessed using a skin elasticity meter. With the normal control group as the improvement reference and the model group as the damage reference, the increase rate of skin collagen fibers and the improvement rate of elasticity in each treatment group were calculated, and the in vivo anti-photoaging efficacy score was finally obtained. The testing standards referred to the testing principles for anti-hair loss and anti-wrinkle efficacy in the "Cosmetic Efficacy Claim Evaluation Specification".

[0076] Detection of reactive oxygen species (ROS) and MAPK / AP-1 pathway inhibition in fibroblasts under UVB irradiation: Human skin fibroblasts were seeded in 6-well plates and cultured to 80% confluence. Microcapsule release solutions from each example and comparative example, sequentially treated with simulated gastric and intestinal fluids, were added. A release solution containing an equal amount of free SOD served as a positive control, and a blank culture medium without samples served as a negative control. After 12 hours of pre-incubation, a single irradiation with a UVB light source at a dose of 30 mJ / cm² was performed. After irradiation, cells were cultured for another 6 hours, and then collected. Intracellular ROS levels were detected using the DCFH-DA fluorescent probe method and analyzed using Western spectroscopy. The expression levels of phosphorylated c-Jun protein (pc-Jun) and matrix metalloproteinase-1 (MMP-1) were detected by blot, and grayscale analysis was performed using GAPDH as an internal reference. The expression levels of pc-Jun and MMP-1 in the negative control group were taken as 100%, and the protein expression inhibition rate of each group was calculated. The UVB pathway inhibition comprehensive score was calculated by combining three indicators: intracellular reactive oxygen species scavenging rate, pc-Jun inhibition rate, and MMP-1 inhibition rate. The maximum score is 100 points, and the higher the score, the stronger the blocking ability of the photoaging signaling pathway. The test standards refer to the detection principles of photoprotective efficacy in the "Cosmetic Efficacy Claim Evaluation Specification".

[0077] Table 1: Results of Microcapsule Delivery Performance Testing Table 2: Results of Microcapsule Anti-Photoaging Efficacy Test Example Conclusion: Based on Examples 1-3 and Comparative Example 1, and in conjunction with Tables 1 and 2, it can be seen that the molecular weight of chitosan oligosaccharide is a factor affecting the overall performance of the microcapsules. When the molecular weight of chitosan oligosaccharide is lower than the preferred range of this application, its molecular chain is too short, making it difficult to effectively encapsulate the β-cyclodextrin inclusion complex through hydrogen bonds and hydrophobic interactions. This results in a loose structure of the pre-assembled nanoparticles, which not only reduces the loading stability of the antioxidant excipients but also weakens its shielding and protective effect on SOD activity in simulated gastric juice. This manifests as a decrease in the retention rate of SOD activity in simulated gastric juice and the scavenging rate of UV-induced free radicals, ultimately leading to a reduction in the overall anti-photoaging effect and UVB pathway inhibition ability.

[0078] Based on Examples 1-3 and Comparative Example 2, and in conjunction with Table 1, it can be seen that the oxidation degree of the alginate derivative affects the reactive oxygen species-responsive disintegration and release behavior of the microcapsules. Although excessive oxidation introduces more broken thioketal bonds into the alginate backbone, the rigidity of the over-oxidized sodium alginate molecular chain is enhanced, and the density of the gel network formed with calcium ions decreases. Even in a normal environment without reactive oxygen species, non-specific leakage is more likely to occur, manifested as an increased cumulative release rate under reactive oxygen species conditions. This premature leakage disrupts the cascade delivery sequence, leading to the loss of pharmacologically active substances before reaching the target site, thereby affecting the scavenging efficiency of UV-induced free radicals.

[0079] Based on Examples 1-3 and Comparative Example 3, and in conjunction with Tables 1 and 2, it can be seen that the number of arginine residues in oligoarginine peptides affects the transmembrane transport efficiency of microcapsules. If the number of arginine residues is too small, sufficient positive charge density cannot be formed to effectively interact with the negatively charged groups on the cell membrane surface, and it is also difficult to trigger the transmembrane mechanism. This reduces the transintestinal epithelial transport capacity of the co-conjugate. Even if the SOD activity is retained in the gastric juice, it cannot smoothly enter the bloodstream to exert a systemic anti-photoaging effect, ultimately resulting in low transmembrane transport efficiency and low in vivo anti-photoaging efficacy scores.

[0080] Based on Examples 1-3 and Comparative Example 4, and in conjunction with Tables 1 and 2, it can be seen that the mixing mass ratio of the co-conjugate to the pre-assembled nanoparticles determines the assembly integrity and delivery efficiency of the inner layer aggregate. When the proportion of pre-assembled nanoparticles is too low, there are not enough positively charged chitosan oligosaccharides to perform multi-site electrostatic cross-linking with the negatively charged groups on the surface of the co-conjugate, resulting in a loose structure and poor encapsulation of the inner layer aggregate. This not only reduces the SOD activity retention rate under simulated gastric juice conditions, but also affects the dissociation and release of the aggregate in the intestine, impairing transmembrane transport efficiency and ultimately weakening the overall protective performance under ultraviolet and reactive oxygen species induced conditions.

[0081] Based on Examples 1-3 and Comparative Example 5, and in conjunction with Table 1, it can be seen that the concentration of the calcium ion crosslinking agent affects the barrier function of the outer alginate gel. When the concentration of the crosslinking agent is too low, there are insufficient ion crosslinking sites between the molecular chains of the alginate derivative, resulting in a sparse outer gel network with excessively large pores. This weakened physical barrier cannot restrain the inner aggregates, and leakage occurs even under normal conditions without reactive oxygen species stimulation, manifested as a high cumulative release rate under reactive oxygen species conditions. This not only destroys the sustained-release and targeted disintegration characteristics of the microcapsules, but also reduces their protective effect in simulated gastric juice.

[0082] Based on Examples 1-3 and Comparative Example 6, and in conjunction with Tables 1 and 2, it can be seen that the dosage of low-anticoagulation heparin is fundamental to ensuring the oral delivery efficiency of SOD. When the dosage of heparin is too low, a complete and dense three-dimensional shielding layer cannot be formed on the surface of SOD molecules, and it is also difficult to construct a local buffer microenvironment that resists degradation by gastric acid and proteases. This structural defect leads to the inactivation of SOD in simulated gastric juice, manifested as a decrease in the activity retention rate. The loss of active ingredients in the early stage of delivery causes a decline in subsequent performance indicators, ultimately resulting in the failure of the overall anti-photoaging efficacy.

[0083] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. SOD anti-photoaging sustained-release microcapsules based on two polysaccharide modifications, characterized in that, It is made from the following raw materials in parts by weight: 10-50 parts superoxide dismutase, 5-30 parts low anticoagulant heparin, 1-10 parts oligoarginine peptide, 20-100 parts chitosan oligosaccharide, 30-150 parts β-cyclodextrin, 1-20 parts astaxanthin, 1-20 parts anthocyanin, 1-20 parts polyphenols, 50-200 parts alginic acid derivatives containing thioketal bonds, and 5-50 parts calcium ion crosslinking agent; In this process, the superoxide dismutase, low-anticoagulation heparin, and oligoarginine peptide are covalently linked to form a co-conjugate; the chitosan oligosaccharide and β-cyclodextrin containing astaxanthin, anthocyanins, and polyphenols form pre-assembled nanoparticles; the alginate derivative is cross-linked under the action of calcium ions to form the outer gel of the microcapsule; and the co-conjugate and the pre-assembled nanoparticles form an inner condensate of the microcapsule through electrostatic interaction.

2. The SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 1, characterized in that, The oligoarginine peptide is composed of 6 to 8 arginine residues and has a cysteine ​​residue at its terminal; the chitosan oligosaccharide has a molecular weight of 1000 to 5000 Da and a degree of deacetylation ≥ 85%.

3. The SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 1, characterized in that, The microcapsules have a particle size of 10–300 μm and an SOD encapsulation rate of not less than 75%.

4. A method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharide modifications, characterized in that, The method for using the SOD anti-photoaging sustained-release microcapsules based on two polysaccharides as described in any one of claims 1-3 includes the following steps: S1. Dissolve low-anticoagulation heparin in buffer solution, add sodium periodate to activate the reaction, and obtain activated heparin after termination and dialysis; mix activated heparin with superoxide dismutase solution, react under alkaline conditions to form Schiff base, and then add reducing agent to reduce to obtain heparin-superoxide dismutase conjugate. S2. Add a heterobifunctional cross-linking agent to the heparin-superoxide dismutase conjugate solution prepared in S1 for activation, and then add oligoarginine peptide containing cysteine ​​to react and obtain heparin-superoxide dismutase-oligoarginine coconjugate. S3. Astaxanthin, anthocyanins, and polyphenols are mixed and dissolved, and then mixed with β-cyclodextrin solution for inclusion to obtain an antioxidant inclusion complex; chitosan oligosaccharide is dissolved in acidic aqueous solution, the antioxidant inclusion complex is added, and after stirring evenly, an ionic crosslinking agent solution is added dropwise to obtain chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles through crosslinking. S4. Dissolve sodium alginate in water, add sodium periodate for partial oxidation to obtain oxidized sodium alginate; add a diamine compound containing a thioketal bond to the oxidized sodium alginate solution, and after reaction and reduction, obtain an alginate derivative containing a thioketal bond. S5. The co-conjugate of S2 is mixed with the pre-assembled nanoparticles of S3 and electrostatically self-assembled under specific pH and ionic strength to form an inner layer condensate. S6. Mix the inner layer aggregate of S5 with the alginate derivative solution of S4, disperse them, and then add calcium ion crosslinking agent to initiate gelation and form bilayer microcapsules; cure the obtained bilayer microcapsules, wash and dry them to obtain SOD anti-photoaging sustained-release microcapsules based on two polysaccharides.

5. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S1, the sodium periodate activation reaction is carried out in the dark at 2–8°C for 1–6 hours; the reducing agent is sodium cyanoborohydride or sodium borohydride, and the pH of the reduction reaction is 7.0–8.

5.

6. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S2, the heteromorphic bifunctional crosslinking agent is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, and the molar ratio of the crosslinking agent to the heparin-superoxide dismutase conjugate is 5:1 to 20:1; the molar ratio of the oligomeric arginine peptide to the heparin-superoxide dismutase conjugate is 3:1 to 10:

1.

7. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S3, the ionic crosslinking agent is sodium tripolyphosphate, and the mass ratio of chitosan oligosaccharide to sodium tripolyphosphate is 3:1 to 8:1; the concentration of the chitosan oligosaccharide solution is 0.5 to 5 mg / mL; and the average particle size of the obtained chitosan oligosaccharide-β-cyclodextrin pre-assembled nanoparticles is 50 to 500 nm.

8. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S4, the degree of oxidation of sodium alginate is 5% to 30%; the diamine compound containing thioketal bonds is a 2,2-dimethoxypropane-1,3-diamine thioketal derivative, and its molar ratio of aldehyde groups to oxidized sodium alginate is 0.5:1 to 2:

1.

9. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S5, the mass ratio of the co-conjugate to the pre-assembled nanoparticles is 1:0.5 to 1:5, the pH for electrostatic self-assembly is 4.0 to 6.0, and the stirring time is 10 to 60 minutes.

10. The method for preparing SOD anti-photoaging sustained-release microcapsules based on two polysaccharides modified according to claim 4, characterized in that, In step S6, the calcium ion crosslinking agent is selected from calcium chloride, calcium acetate, or calcium lactate, and its concentration is 0.1–1.0 mol / L; the drying is freeze drying or low-temperature spray drying.

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