An oxidation fucoidan modified ros response type nano delivery system and a preparation method and application thereof

Abstract

The application discloses an oxidized fucoidan modified ROS response type nano delivery system and a preparation method and application thereof, and belongs to the technical field of biomedical nanomaterials and drug delivery. The application forms the oxidized fucoidan modified ROS response type nano delivery system with a core-shell structure by using dopamine grafted oxidized fucoidan as a shell and using a ROS response polymer loaded with a hydrophobic drug as a core. The dopamine related structure in the shell of the formed nano delivery system has a free radical / ROS scavenging capacity, and can reduce local oxidative stress to a certain extent; the peroxalate bond in the core can be broken and depolymerized under the condition of ROS stimulation to trigger drug release. The formed cascade action path of "outer layer ROS regulation-in-core response drug release" is helpful to inhibit the risk of non-specific triggering and passive leakage under the condition of relatively low ROS in non-lesions, and has the mechanism basis of triggering drug release under the condition of high ROS stimulation in lesions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials and drug delivery technology, specifically relating to a ROS-responsive nanodelivery system modified with oxidized fucoidan, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is a cardiovascular disease characterized by lipid deposition, chronic inflammation, and endothelial dysfunction. It is a crucial pathological basis for major cardiovascular and cerebrovascular events such as coronary heart disease and stroke. Numerous studies have shown that oxidative stress and reactive oxygen species (ROS) play key roles in the occurrence, development, and plaque instability of AS, participating in multiple processes including endothelial dysfunction, low-density lipoprotein oxidation, and inflammatory cascade amplification, thereby promoting lesion progression. Therefore, targeted interventions targeting abnormally elevated ROS in the lesion microenvironment, and achieving effective drug accumulation and release at the lesion site, are important directions for AS treatment research.

[0003] Currently, clinical treatment of atherosclerosis mainly relies on long-term use of drugs such as statins. However, traditional drug delivery methods often suffer from problems such as high systemic exposure, insufficient effective concentration at the lesion site, low solubility of some hydrophobic drugs, and widespread non-specific distribution in the body, resulting in limited treatment efficiency and potential systemic side effects. To improve drug accumulation at plaque sites and reduce non-specific distribution, nanodelivery systems such as liposomes, polymer nanoparticles, and micelles have been extensively studied for targeted delivery and controlled release therapy of atherosclerosis.

[0004] Among existing polymer nanodelivery systems, nanocarriers constructed from PLGA, PEG, and their derivatives have attracted attention due to their biocompatibility and tunability. However, they still face several challenges in practical applications: on the one hand, after entering the body, nanocarriers may undergo protein adsorption, aggregation, or clearance by the mononuclear phagocytic system, resulting in unstable lesion enrichment effects; on the other hand, carriers may exhibit structural instability and passive drug leakage in the body fluid environment, leading to premature drug release and difficulty in achieving selective lesion treatment. Furthermore, strategies relying solely on passive enrichment or single-target ligand modification often fail to achieve stable and reliable targeting effects under complex blood flow environments and plaque heterogeneity.

[0005] To improve selective drug release from lesions, stimulus-responsive delivery systems have been proposed, with ROS-responsive delivery systems gaining attention due to their compatibility with the high-ROS microenvironment of atherosclerotic lesions. Some existing technologies achieve stimulus-triggered release by introducing chemical bonds in the carrier that can be broken by ROS. However, relying solely on a single mechanism of ROS-triggered drug release often fails to address the tissue-damaging effects of ROS within the lesion microenvironment; in a high-ROS environment, endothelial damage and inflammatory amplification may still persist, thus affecting efficacy. Therefore, a delivery strategy is needed that can both regulate the ROS microenvironment of the lesion and achieve effective drug release under ROS stimulation, making the therapeutic effect more closely aligned with the pathological characteristics of atherosclerotic lesions. Furthermore, existing technologies present an inherent contradiction when intervening in the high-ROS microenvironment of atherosclerosis: using only ROS-scavenging materials, such as simple dopamine coatings, while alleviating oxidative stress, fails to achieve intelligent, on-demand drug release; conversely, using ROS-sensitive fracture-prone materials may lead to premature degradation or drug leakage in complex environments such as the bloodstream or non-lesion sites, hindering selective lesion treatment. Current technologies have not yet provided a delivery strategy that effectively coordinates the spatiotemporal differences between ROS regulation outside the lesion and ROS-triggered drug release within the lesion, making it difficult to simultaneously reduce oxidative stress damage and improve selective lesion release. Therefore, a delivery strategy that structurally achieves functional zoning and mechanistically reconciles the aforementioned contradictions is urgently needed.

[0006] In terms of targeting, P-selectin participates in processes such as inflammation and adhesion and is associated with plaque-related pathological changes, thus being considered a potential molecular target for targeted interventions related to atherosclerosis. Fucoidan, a sulfate-containing polysaccharide, has been used in research exploring P-selectin-related targeted delivery. However, stabilizing hydrophilic polysaccharides onto the surface of hydrophobic polymer nanoparticles and preventing their detachment in body fluid environments remains a practical challenge. Commonly used surface modification methods in existing technologies include electrostatic adsorption, physical coating, and chemical coupling. Electrostatic adsorption or physical coating is relatively simple to operate, but it is prone to desorption under the influence of salt ions, resulting in insufficient stability. Chemical coupling can improve the stability of the connection, but it usually requires additional activating / coupling reagents and multi-step reaction processes, making the process complex and potentially affecting ligand activity, which is not conducive to simplified preparation and scale-up production.

[0007] In summary, existing technologies for nanodelivery in the field of atherosclerosis still have the following shortcomings: ① The targeted enrichment effect of lesions is easily affected by the complex intracellular environment and is unstable; ② Hydrophobic drug loading systems may experience unintended release, making it difficult to achieve higher lesion selectivity; ③ A single ROS-responsive drug release strategy cannot take into account the impact of lesion oxidative stress on tissue and inflammatory processes; ④ When using hydrophilic polysaccharide ligands such as fucoidan to achieve P-selectin-related targeting, there is still a contradiction between how to construct a continuous and stable surface shell during O / W granulation and reduce the risk of desorption in the body fluid environment, and how to avoid the process complexity caused by multiple post-modification steps; at the same time, when introducing antioxidant components to regulate ROS, there may be a potential conflict with the weakening of the stimulation signal when triggering drug release by core ROS, making it difficult to achieve a balance between targeted enrichment, structural stability, process simplification, and responsive drug release. Therefore, there is an urgent need to provide a new nanodelivery system and its preparation method that can achieve P-selectin-related targeted enrichment while maintaining process simplification and reproducibility, and obtain controllable drug release performance that matches the high ROS microenvironment, so as to enhance its potential application value in the treatment of atherosclerosis. Summary of the Invention

[0008] To address the problems of unstable targeted enrichment efficiency, unintended release after hydrophobic drug loading, and lack of stimulus-response release mechanism that matches the high ROS microenvironment of lesions in existing atherosclerosis-related nanodelivery systems, this invention provides an oxidized fucoidan-modified ROS-responsive nanodelivery system, its preparation method, and its application.

[0009] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a method for preparing a ROS-responsive nanodelivery system modified with oxidized fucoidan, comprising the following steps: Fucoidan was moderately oxidized to introduce aldehyde functional groups onto the polysaccharide chain, resulting in oxidized fucoidan with an oxidation degree of 20-35%. The oxidized fucoidan was then grafted with dopamine to obtain dopamine-grafted oxidized fucoidan. Polyethylene glycol and triethylamine were dissolved in a solvent to obtain solution 1. Solution 1 was then added to an oxalyl chloride solution in an ice bath. After reacting in an ice bath and under an inert atmosphere, an oxalyl chloride PEG intermediate was obtained. The oxalyl chloride PEG intermediate and triethylamine were dissolved in a solvent to obtain solution 2. A polylactic acid-glycolic acid copolymer (3S-PLGA) solution was added dropwise to solution 2 under an inert atmosphere. After reacting, a ROS-responsive polymer was obtained. An oil-in-water emulsion was prepared using an aqueous solution of dopamine-grafted oxidized fucoidan as the aqueous phase and an organic solution of the ROS-responsive polymer and a hydrophobic drug as the oil phase. The organic solvent was then removed to solidify the oil-in-water emulsion, thereby obtaining the ROS-responsive nanodelivery system modified with oxidized fucoidan.

[0010] Under moderate oxidation conditions, this invention can introduce aldehyde functional groups onto fucoidan while preserving the sulfation structure characteristics of fucoidan as much as possible, so as to maintain its role as a P-selectin-related ligand.

[0011] The multiple aldehyde sites introduced into the oxidized fucoidan can undergo condensation reactions with the amino groups in the dopamine molecule to form Schiff bases, thereby introducing multiple dopamine side chains onto the same polysaccharide backbone to obtain dopamine-grafted oxidized fucoidan. Schiff base linkage is a dynamic covalent connection, and its stability can be affected by conditions such as pH. In the system of this invention, dopamine-grafted oxidized fucoidan is assembled in situ at the emulsion interface and simultaneously solidifies during particle formation. Simultaneously, the multi-point interaction between the polysaccharide backbone and dopamine-related structures at the interface contributes to the stable construction of the shell. Thus, multiple dopamine side chains are introduced onto the same polysaccharide backbone to obtain dopamine-grafted oxidized fucoidan. During O / W emulsification, dopamine-grafted oxidized fucoidan, possessing both a hydrophilic polysaccharide backbone and dopamine-related structural units, can accumulate at the oil-water interface and self-assemble in situ to coat the hydrophobic core surface. Simultaneously, since multiple dopamine side chains can be carried on the same polysaccharide backbone, multi-site synergistic anchoring can be formed between the shell and the core surface. This facilitates the formation of a more continuous and dense shell coating during granulation and solidification, and reduces the risk of desorption that may occur in body fluid environments through simple physical adsorption / electrostatic adsorption.

[0012] Preferably, the specific steps of grafting the oxidized fucoidan with dopamine include: mixing the oxidized fucoidan with dopamine hydrochloride in water or a buffer solution, adjusting the pH of the system to 5.0-7.0, and reacting in the dark to obtain the dopamine-grafted oxidized fucoidan.

[0013] Preferably, in the preparation step of the ROS-responsive polymer, the solvent for dissolving the raw materials and intermediates is dichloromethane.

[0014] Preferably, in the preparation step of the ROS-responsive polymer, the ratio of oxaloyl chloride PEG intermediate to polylactic acid-glycolic acid copolymer is 1~2g:0.03~0.04mmol.

[0015] Preferably, the reaction temperature of solution 2 with the polylactic acid-glycolic acid copolymer solution is 30~60℃, and the reaction time is 0.5~4h.

[0016] Preferably, the weight-average molecular weight (M) of the polyethylene glycol is... w The range is 1000~8000.

[0017] Preferably, the aqueous phase further contains polyvinyl alcohol (PVA), and the mass-volume concentration of the polyvinyl alcohol in the aqueous phase is 0.5~5.0% w / v.

[0018] The addition of polyvinyl alcohol to the aqueous phase in this invention helps to improve emulsion stability and obtain an emulsion system with a more uniform particle size distribution.

[0019] Preferably, the solvent of the oil phase is dichloromethane or trichloromethane.

[0020] Preferably, the concentration of ROS-responsive polymer in the oil phase is 15-25 mg / mL; the concentration of dopamine-grafted oxidized fucoidan in the aqueous phase is 0.5-1 mg / mL; and the volume ratio of the oil phase to the aqueous phase is 1:5.

[0021] The second technical solution of the present invention provides an oxidized fucoidan-modified ROS-responsive nanodelivery system prepared according to the above-mentioned preparation method of the oxidized fucoidan-modified ROS-responsive nanodelivery system.

[0022] The third technical solution of the present invention provides the application of the above-mentioned oxidized fucoidan-modified ROS-responsive nanodelivery system in the preparation of drugs for treating atherosclerosis.

[0023] The beneficial technical effects of the present invention are as follows: (1) Functional partitioning and cascade action: The present invention adopts a core-shell structure to achieve functional partitioning. The dopamine-related structure in the shell has free radical / ROS scavenging ability, which can reduce local oxidative stress to a certain extent; the peroxate bond in the core can be broken / depolymerized under ROS stimulation to trigger drug release. This forms a cascade action pathway of "outer layer regulating ROS - core responding to drug release", which helps to suppress the risk of non-specific triggering and passive leakage under relatively low ROS conditions in non-lesion areas, and has the mechanism basis for triggering drug release under high ROS stimulation in lesions, so as to match the delivery with the characteristics of the lesion microenvironment.

[0024] (2) Balancing Shell Construction Stability and Process Simplification: Dopamine is grafted onto multiple sites based on aldehyde sites introduced by oxidation, enabling the shell to form multi-site synergistic anchoring and dense coating on the core surface during emulsification and granulation. This improves the stability of the shell structure and reduces the risk of desorption in body fluid environments. Furthermore, the shell is formed by in-situ self-assembly at the O / W emulsified oil-water interface and simultaneous shaping during curing. This facilitates continuous coating of hydrophilic polysaccharides on the hydrophobic core surface and reduces the risk of desorption. It also avoids the multi-step post-surface modification process after granulation, making the process route simpler and providing good reproducibility and a basis for scale-up preparation. Compared to post-modification methods such as electrostatic adsorption / physical coating after nanoparticle formation, the in-situ shell construction method of this invention is more conducive to forming continuous coating and reducing the risk of desorption.

[0025] (3) Rationality and applicability of targeted enrichment: Fucoidan as a P-selectin-related ligand has a reasonable basis for targeted enrichment; combined with ROS response core and outer ROS regulation, it can further realize the intervention and triggering of drug release on the oxidative stress microenvironment of lesions on the basis of enrichment, which is suitable for the delivery of drugs related to atherosclerosis. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the synthesis route for steps one (a), two (b), and three (c) in Example 1.

[0028] Figure 2 The particle size distribution diagram (a) of the sample prepared in Example 1 of Experimental Example 1 and the zeta potential comparison diagram (b) of the samples prepared in Example 1 and Comparative Example 1 are shown.

[0029] Figure 3 Fluorescence micrographs of reactive oxygen species (ROS) levels in cells of different treatment groups in Experiment Example 2.

[0030] Figure 4 These are representative immunofluorescence staining images of the M1 polarization marker iNOS and the M2 polarization marker Arg-1 in macrophages from different treatment groups in Experiment Example 3.

[0031] Figure 5 The nanodelivery system prepared in Example 1 of Experimental Example 4 was used to test the effects of different concentrations of DPPH·(a) and ABTS· +(b) Bar chart of free radical scavenging rate. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0033] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0034] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0038] Example 1 Preparation steps of ROS-responsive nanodelivery system modified with oxidized fucoidan: Step 1: Synthesis of dopamine-grafted oxidized fucoidan: (1) Preparation of oxidized fuc: Fucoidan was dissolved in distilled water, sodium periodate (NaIO4) was added, and the reaction was stirred at room temperature in the dark for 6 h. After the reaction was completed, ethylene glycol was added and stirring was continued for 1 h to terminate the reaction. The reaction solution was put into a dialysis bag (MWCO of 3500 Da) and dialyzed in distilled water for 3 days. After the dialysis was completed, it was freeze-dried to obtain oxidized fuc with an oxidation degree of 25%.

[0039] (2) Preparation of dopamine-grafted oxidized fucoidan: 100 mg Ox-Fuc and 30 mg dopamine hydrochloride were dissolved in deionized water respectively, and the pH of the system was adjusted to 6.0 after mixing. The reaction was stirred for 12 h under light-protected conditions to allow the amino group of dopamine to undergo a condensation reaction with the aldehyde group of Ox-Fuc to form a Schiff base. The reaction solution was placed in a dialysis bag (MWCO of 3500 Da) and dialyzed with deionized water for 3 days to remove unreacted small molecule dopamine. Subsequently, it was freeze-dried to obtain dopamine-grafted oxidized fucoidan (Ox-Fuc-DA).

[0040] Step 2: Synthesis of ROS-responsive polymers: Weigh out racemic lactide (30 mmol), glycolide (10 mmol), glycerol (0.4 mmol), and 10 mL of stannous octoate solution (30.56 mmol·L⁻¹). -1 The mixture was added to a thick-walled, pressure-resistant polymerization tube (Schlenk tube) with a PTFE (polytetrafluoroethylene) sealed valve and mixed thoroughly. Ring-opening polymerization was carried out at 160 °C for 8 h. After the reaction, the crude product was dissolved in dichloromethane and purified by precipitation with anhydrous methanol. This dissolution-precipitation purification was repeated three times. The product was dried to obtain polylactic acid-glycolic acid copolymer (3S-PLGA). Oxaloyl chloride (3.28 mmol) was weighed and dissolved in dichloromethane and stirred under ice bath conditions. Separately, PEG 4000 (0.328 mmol) and triethylamine (0.361 mmol) were weighed and dissolved in dichloromethane and mixed thoroughly. The PEG / triethylamine solution was slowly added dropwise to the oxaloyl chloride dichloromethane solution. After the addition was complete, the reaction was continued under ice bath and nitrogen protection for 3 h with stirring. After the reaction, residual dichloromethane and excess oxaloyl chloride were removed from the system by vacuum distillation or rotary evaporation to obtain the oxaloyl chloride PEG intermediate (PO-PEG). 1.40 g of PO-PEG was dissolved in 10 mL of dichloromethane, and triethylamine (0.164 mmol) was added. Separately, 0.036 mmol of 3S-PLGA was dissolved in dichloromethane. Under a nitrogen atmosphere, this 3S-PLGA dichloromethane solution was added dropwise to an excess of PO-PEG dichloromethane solution. The mixture was then heated to 60 °C and reacted for 1 h. After the reaction, the organic phase was washed three times each with 1 mol / L hydrochloric acid and saturated sodium chloride solution to remove triethylamine salt and water-soluble impurities. The organic phase was dried over anhydrous magnesium sulfate and filtered. The filtrate was added dropwise to cold anhydrous diethyl ether for reprecipitation. The precipitate was collected and washed with cold methanol to remove PO-PEG. Finally, the obtained solid was dried to constant weight in a vacuum drying oven to obtain the ROS-responsive polymer (3S-PLGA-PO-PEG).

[0041] Step 3: Prepare an Ox-Fuc-DA-coated hydrophobic drug-loaded ROS-responsive nanodelivery system using an oil / water (O / W) emulsification-solvent evaporation method. Using curcumin (Cur) as the drug, 50 mg of 3S-PLGA-PO-PEG and 5 mg of curcumin were weighed and added to 2.0 mL of dichloromethane, a volatile organic solvent. The mixture was vortexed and magnetically stirred until completely dissolved to obtain the oil phase. 10 mg of Ox-Fuc-DA was weighed and added to 10 mL of 2% (w / v) PVA aqueous solution and stirred until uniformly dispersed to obtain the secondary aqueous phase. Under ice bath conditions, the oil phase was slowly added dropwise to the aqueous phase, and a primary emulsion was formed under magnetic stirring. Subsequently, the primary emulsion was emulsified using probe-type ultrasound to form an oil / water (O / W) emulsion. During the ultrasonic emulsification process, the dopamine-related groups in Ox-Fuc-DA underwent in-situ interfacial anchoring at the oil-water interface, causing Ox-Fuc-DA to accumulate at the interface and self-assemble to coat the surface of the hydrophobic core, forming a precursor emulsion of a core-shell structured nanodelivery system. The obtained core-shell nanoparticle precursor emulsion was stirred at room temperature for 4 h to evaporate and remove organic solvents and promote polymer solidification into particles, resulting in a core-shell nanoparticle dispersion. Subsequently, the obtained core-shell nanoparticle dispersion was centrifuged at 12000 rpm for 20 min, the supernatant was discarded, and the nanoparticle precipitate was resuspended in deionized water and washed three times to remove free Ox-Fuc-DA, PVA, and unencapsulated curcumin, yielding a ROS-responsive nanoparticle delivery system dispersion modified with oxidized fucoidan.

[0042] The schematic diagrams of the synthesis routes for steps one (a), two (b), and three (c) in Example 1 are shown below. Figure 1 .

[0043] Comparative Example 1 Preparation of hydrophobic drug-loaded nanoparticles without Ox-Fuc-DA coating: Except for not adding Ox-Fuc-DA to the aqueous phase, the other steps are the same as in Example 1, and a dispersion of core nanoparticles without Ox-Fuc-DA coating is obtained.

[0044] Experimental Example 1 The samples from Example 1 and Comparative Example 1 were diluted with water to achieve a nanoparticle concentration of 0.5 mg / mL. The nanoparticles were then dispersed in an ultrasonic water bath for 10 min and allowed to stand to remove air bubbles. The hydrated particle size was determined using dynamic light scattering (DLS) at 25 °C. Each sample was measured in at least three parallel measurements, and the average value was recorded and a particle size distribution graph was plotted. The Zeta potential was determined using electrophoretic light scattering. Measurements were performed in deionized water, and each sample was measured in at least three parallel measurements, and the average value was recorded and a Zeta potential change graph was plotted.

[0045] The particle size distribution diagram (a) of the sample prepared in Example 1 and the comparison diagram (b) of the zeta potential of the samples prepared in Example 1 and Comparative Example 1 are shown below. Figure 2 .

[0046] like Figure 2 As shown in Figure a, the nano-delivery system obtained in Example 1 has a relatively concentrated particle size distribution and good unimodality; the particle size after Ox-Fuc-DA coating is approximately 257.35±36.34 nm. The microstructure of the nanoparticles was observed using transmission electron microscopy (TEM) to further confirm its core-shell structure. Figure 2 The Zeta potential results in b show that the surface potential of Example 1 after coating with Ox-Fuc-DA shifts to a more negative direction compared to Comparative Example 1, changing from -7.18±0.74 mV to -13.36±0.56 mV. This is generally related to the introduction of sulfate / anionic groups of fucose, indicating that Ox-Fuc-DA was successfully constructed on the surface of nanoparticles and formed a stable coating layer.

[0047] Experimental Example 2 The intracellular ROS level was detected using the DCFH-DA fluorescent probe method to evaluate the regulatory effect of the oxidized fucoidan-modified ROS-responsive nanodelivery system of this invention on cellular oxidative stress. Specifically, a cellular oxidative stress model was established by treating cells with the pro-oxidative stimulant hydrogen peroxide (H2O2), which increased the intracellular ROS level. After the model was established, the cells were divided into at least the following groups: blank control group (oxidative stimulation without the addition of materials), comparative example 1 group (oxidative stimulation with the addition of comparative example 1 sample), and example 1 group (oxidative stimulation with the addition of example 1 sample). Each group was incubated with the corresponding sample at a preset concentration (100 μg / mL based on the total concentration of drug-loaded nanoparticles) for a preset time (24 h). The culture medium was then discarded, and the cells were washed with PBS. DCFH-DA working solution was added and incubated in the dark for 20 min, allowing the probe to enter the cells and be oxidized under the action of ROS to generate a product with green fluorescence. After incubation, the cells were washed with PBS to remove probes that had not entered the cells. Subsequently, green channel images of each group of cells were acquired using a fluorescence microscope, and the fluorescence intensity was compared. The green fluorescence intensity was positively correlated with the intracellular ROS level.

[0048] Fluorescence micrographs of reactive oxygen species (ROS) levels in cells from different treatment groups are shown below. Figure 3 (Scale bar: 100μm).

[0049] Figure 3 The results showed that the blank control group and the control group 1 cells exhibited strong green fluorescence signals, while the green fluorescence intensity of the cells in the Example 1 group was significantly reduced, indicating that the nanodelivery system of the present invention can effectively reduce the intracellular ROS level, thereby alleviating oxidative stress damage.

[0050] Experimental Example 3 To evaluate the regulatory effect of the ROS-responsive nanodelivery system modified with oxidized fucoidan on the inflammatory phenotype of macrophages, an in vitro co-culture evaluation was conducted using a macrophage polarization model, and the expression changes of M1 / M2 polarization-related markers were detected by immunofluorescence staining. RAW264.7 macrophages were cultured to 80% confluence using standard methods, and an M1 polarization model was established by inducing it with pro-inflammatory stimuli such as lipopolysaccharide (LPS); an M2 polarization model was established by inducing it with anti-inflammatory stimuli such as IL-4. After the models were established, the cells were divided into the following groups: blank group (polarization induced but no material added), comparative example 1 group (polarization induced and comparative example 1 sample added), and example 1 group (polarization induced and example 1 sample added). Each group was incubated with the corresponding sample at a preset concentration (100 μg / mL for the total concentration of drug-loaded nanoparticles and approximately 10 μg / mL for curcumin equivalent) for a preset time (24 h). The culture medium was then discarded, and the cells were washed with PBS and fixed with fixative. Immunofluorescence staining was then performed on the M1 polarization-related marker iNOS and the M2 polarization-related marker Arg-1, and the cell nuclei were counterstained with DAPI. Images of each group were acquired using a fluorescence microscope, and the fluorescence signal intensity was compared.

[0051] Representative immunofluorescence staining images of the M1 polarization marker iNOS and the M2 polarization marker Arg-1 in macrophages from different treatment groups are shown below. Figure 4 (Scale bar: 30 μm), where cell nuclei are stained with DAPI (blue), and iNOS or Arg-1 are the corresponding fluorescence channel signals.

[0052] The results are as follows Figure 4 As shown, compared with the blank group and Comparative Example 1, the iNOS fluorescence signal of Example 1 group was reduced and the Arg-1 fluorescence signal was enhanced, suggesting that the nanodelivery system of the present invention has the effect of inhibiting pro-inflammatory phenotypes and promoting the expression of anti-inflammatory related phenotypes.

[0053] Test Example 4 To evaluate the in vitro antioxidant capacity of the ROS-responsive nanodelivery system modified with oxidized fucoidan of this invention, the DPPH radical scavenging assay and ABTS were used. + The free radical scavenging assay was performed. The Ox-Fuc-DA coated nanoparticle dispersion prepared in the example was used as the test sample, and the sample concentration gradient was set to 5, 10, 20, 40, 80, and 120 μg / mL.

[0054] (1) DPPH· free radical scavenging experiment: Prepare a 0.1 mM ethanol working solution of DPPH, use immediately and store in the dark. Disperse the DPPH working solution by sonication for about 20 min before use. Use the mixture of ethanol and DPPH working solution as a control (negative control). Take a certain volume of DPPH working solution and mix it with sample solutions of different concentrations, and incubate at room temperature in the dark for 30 min. After incubation, add each reaction system to a 96-well plate and measure the absorbance at 517 nm using an ELISA reader. To correct the background absorption of the samples, set up sample blank wells (sample + ethanol, without DPPH) for each concentration of sample, and use "absorbance of sample reaction wells - absorbance of sample blank wells" as the corrected sample absorbance, and then calculate the DPPH· scavenging rate.

[0055] (2) ABTS· + Free radical scavenging experiment: ABTS was prepared as a 7 mM solution and mixed with a 2.45 mM potassium persulfate solution to generate ABTS· + The free radical cation stock solution was incubated at room temperature in the dark for approximately 16 hours before use. ABTS· + Dilute the stock solution as needed to a working solution with a suitable initial absorbance at 734 nm. Take ABTS· + The working solution was mixed with sample solutions of different concentrations and reacted at room temperature in the dark for approximately 10 minutes. Each reaction system was then added to a 96-well plate, and the absorbance was measured at 734 nm using a microplate reader. For colored samples containing curcumin, blank wells were also prepared (sample + solvent, without ABTS). + ABTS is calculated after correction according to "sample reaction well absorbance - sample blank well absorbance". + Clearance rate.

[0056] DPPH· and ABTS· respectively + The absorbance of the control well of the system was used as A. control The corrected sample absorbance was used as A. sample The free radical scavenging rate is calculated as follows: Scavenging rate (%) = [1 − (A sample / A control []×100%. At least one parallel duplicate hole should be set in each group and the average value should be calculated.

[0057] The nanodelivery system prepared in Example 1 showed different concentrations of DPPH·(a) and ABTS· + (b) The free radical scavenging rate histogram is shown below. Figure 5 .

[0058] like Figure 5As shown, within the concentration range of 5, 10, 20, 40, 80, and 120 μg / mL, the nanodelivery system prepared in this invention exhibits good performance for DPPH· and ABTS·. + All three exhibited significant scavenging effects, and the scavenging rate increased with increasing sample concentration, demonstrating a certain concentration dependence.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a ROS-responsive nanodelivery system modified with oxidized fucoidan, characterized in that, Includes the following steps: Fucoidan was moderately oxidized to introduce aldehyde functional groups onto the polysaccharide chain, resulting in oxidized fucoidan with an oxidation degree of 20-35%. The oxidized fucoidan was then grafted with dopamine to obtain dopamine-grafted oxidized fucoidan. Polyethylene glycol and triethylamine were dissolved in a solvent to obtain solution 1. Solution 1 was then added to an oxalyl chloride solution in an ice bath. After reacting in an ice bath and under an inert atmosphere, an oxalyl chloride PEG intermediate was obtained. The oxalyl chloride PEG intermediate and triethylamine were dissolved in a solvent to obtain solution 2. A polylactic acid-glycolic acid copolymer solution was added dropwise to solution 2 under an inert atmosphere. After reacting, a ROS-responsive polymer was obtained. An oil-in-water emulsion was prepared using an aqueous solution of dopamine-grafted oxidized fucoidan as the aqueous phase and an organic solution of the ROS-responsive polymer and a hydrophobic drug as the oil phase. The organic solvent was then removed to solidify the oil-in-water emulsion, thereby obtaining the ROS-responsive nanodelivery system modified with oxidized fucoidan.

2. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The specific steps of grafting the oxidized fucoidan with dopamine include: mixing the oxidized fucoidan with dopamine hydrochloride in water or a buffer solution, adjusting the pH of the system to 5.0-7.0, and reacting in the dark to obtain the dopamine-grafted oxidized fucoidan.

3. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, In the preparation steps of the ROS-responsive polymer, the solvent for dissolving the raw materials and intermediates is dichloromethane; and / or, the ratio of the oxaloyl chloride PEG intermediate to the polylactic acid-glycolic acid copolymer is 1~2g:0.03~0.04mmol.

4. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The reaction temperature of solution 2 with the polylactic acid-glycolic acid copolymer solution is 30~60℃, and the reaction time is 0.5~4h.

5. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The weight-average molecular weight of the polyethylene glycol is 1000~8000.

6. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The aqueous phase also contains polyvinyl alcohol, and the mass-volume concentration of polyvinyl alcohol in the aqueous phase is 0.5~5.0% w / v.

7. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The solvent for the oil phase is dichloromethane or trichloromethane.

8. The method for preparing the ROS-responsive nanodelivery system modified with oxidized fucoidan according to claim 1, characterized in that, The concentration of ROS-responsive polymer in the oil phase is 15-25 mg / mL; the concentration of dopamine-grafted oxidized fucoidan in the aqueous phase is 0.5-1 mg / mL; and the volume ratio of the oil phase to the aqueous phase is 1:

5.

9. A ROS-responsive nanodelivery system modified with oxidized fucoidan, prepared by the method of any one of claims 1 to 8.

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