A cordycepin-loaded protein-glycosan ternary complex nanoparticle, a preparation method and application thereof

Abstract

This invention discloses a protein-polysaccharide ternary nanoparticle loaded with cordycepin, its preparation method, and its applications. The invention employs a layer-by-layer self-assembly method to modify the surface of zein nanoparticles. Since zein has a positive surface charge, the first layer modification uses the anionic polysaccharide sodium alginate for electrostatic bonding, and the second layer modification uses the cationic polysaccharide chitosan, constructing positively charged ternary composite nanoparticles. These nanoparticles have small particle size, high encapsulation efficiency, high drug loading capacity, and a sustained-release effect, significantly reducing cytotoxicity and significantly improving the therapeutic effect on osteoarthritis. This invention develops a novel cordycepin formulation, overcoming the limitations of cordycepin's clinical use.

Description

Technical Field

[0001] This invention relates to the field of cordycepin nanoparticle drug delivery system technology, and in particular to a protein-polysaccharide ternary nanoparticle loaded with cordycepin using sodium alginate and chitosan dual-modified zein, its preparation method and application. Background Technology

[0002] Cordycepin (3′-deoxyadenosine) is a derivative of nucleoside adenosine and a major component of Cordyceps sinensis. Studies have shown that cordycepin has anti-inflammatory, immunomodulatory, anti-diabetic, anti-hyperlipidemia, and antioxidant effects, as well as cardiovascular disease prevention and anti-cancer effects. Currently, research on cordycepin is becoming an extremely active field in medicinal chemistry, anti-aging, beauty, and health products. However, because cordycepin is rapidly deaminated by adenosine deaminase (ADA) in vivo, it transforms into 3′-deoxyinosine nucleoside with very low biological activity (Li Jing, Jiang Hanying. Metabolic characteristics and pharmacological effects of cordycepin in vivo [J]. Foreign Medical Sciences (Traditional Chinese Medicine), 2005, (05): 283-285+293.), its clinical use is limited.

[0003] Nanoparticles are a common type of nanoscale delivery system, typically using polymers as wall materials to effectively encapsulate and transport natural active ingredients. Proteins and polysaccharides, as two major types of biopolymers, are widely used for constructing nanoscale carriers for the preservation and delivery of bioactive substances due to their advantages such as wide availability, safety, non-toxicity, good biocompatibility, and ease of fabrication into nanomaterials. For example, Chinese patent CN107281109A uses O-carboxymethyl chitosan as a cordycepin nanoparticle carrier, employing an ionic crosslinking method with sodium tripolyphosphate as a crosslinking agent to prepare cordycepin / O-carboxymethyl chitosan nanoparticles. Chinese patent CN116172974A uses EDC and NHS to activate the carboxyl groups of polyethylene glycol and polyethylene glycol-biotin, obtaining NHS-polyethylene glycol and NHS-polyethylene glycol-biotin intermediates. These intermediates are then covalently bonded to the amino groups of cordycepin to obtain cordycepin-polyethylene glycol and cordycepin-polyethylene glycol-biotin, respectively, which are then self-assembled to obtain cordycepin nanoparticles. The above-mentioned technical solutions all suffer from problems such as low encapsulation efficiency and complex preparation methods. Therefore, there is an urgent need to develop a cordycepin preparation with a long duration of action, high bioavailability, and suitability for clinical use. Summary of the Invention

[0004] In view of the problems raised in the background art, the present invention aims to provide a protein-polysaccharide ternary composite nanoparticle loaded with cordycepin, its preparation method and application. The present invention uses an antisolvent method combined with a layer-by-layer electrostatic deposition method to prepare ternary composite nanoparticles by attaching sodium alginate and chitosan to the surface of zein nanoparticles, so as to improve the stability of zein nanoparticles and the encapsulation rate of cordycepin.

[0005] In a first aspect, the present invention provides a protein-polysaccharide ternary composite nanoparticle loaded with cordycepin, wherein cordycepin is encapsulated in zein as the core and sodium alginate and chitosan are assembled into a shell to construct a core-shell structure for delivering cordycepin composite nanoparticles.

[0006] The nanoparticles have a diameter of about 200 nm and a potential of 40-50 mV.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned protein-polysaccharide ternary composite nanoparticles loaded with cordycepin, specifically including the following steps:

[0008] (1) Preparation of cordycepin-zein ethanol solution: Zein was dissolved in an aqueous ethanol solution, cordycepin was added, and the mixture was stirred thoroughly to obtain an ethanol solution of cordycepin-zein; wherein, in the ethanol solution of cordycepin-zein, the concentration of zein was 20 mg / mL, and the mass ratio of cordycepin to zein was 0.025-0.1:1. The volume fraction of ethanol in the aqueous ethanol solution was 80%.

[0009] (2) Preparation of a dispersion of cordycepin-loaded sodium alginate modified zein nanoparticles: The ethanol solution of cordycepin-zein obtained in step (1) was slowly added dropwise to the sodium alginate solution and stirred to obtain a dispersion. The stirring speed was 400-800 rpm and the time was 30-60 min. The concentration of the sodium alginate solution was 1-2 mg / mL and the solvent was water. The volume ratio of the ethanol aqueous solution of cordycepin-zein to the sodium alginate solution was 1:4.

[0010] (3) Preparation of the sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin: The sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin obtained in (2) was slowly added dropwise to the chitosan solution, with a stirring speed of 400-800 rpm and a stirring time of 30-60 min; then, the organic solvent was removed under reduced pressure on a rotary evaporator, and the volume was made up to 20 mL with deionized water to obtain the sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin.

[0011] The concentration of the chitosan solution is 0.1-2 mg / mL, and the solvent is a 1% (v / v) acetic acid solution.

[0012] The volume ratio of the sodium alginate-modified zein nanoparticle dispersion loaded with cordycepin to the chitosan solution was 1:1.

[0013] (4) The sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin obtained in step (3) was freeze-dried to obtain protein-polysaccharide ternary composite nanoparticles loaded with cordycepin.

[0014] Thirdly, the present invention provides the application of the above-mentioned cordycepin-loaded protein-polysaccharide ternary composite nanoparticles in the preparation of anti-inflammatory drugs.

[0015] The inflammation described in this invention is osteoarthritis.

[0016] Fourthly, the present invention also provides a pharmaceutical composition comprising the above-mentioned protein-polysaccharide ternary composite nanoparticles loaded with cordycepin.

[0017] In one embodiment, the anti-inflammatory application is to inhibit the expression of inflammatory factors in osteoarthritis.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] This application uses cordycepin as the active pharmaceutical ingredient. However, the toxicity and duration of action of cordycepin itself limit its use. This application employs an antisolvent method combined with layer-by-layer electrostatic deposition to prepare cordycepin-loaded protein-polysaccharide composite nanoparticles, reducing the toxicity of cordycepin and exhibiting better biocompatibility. Simultaneously, the cordycepin-loaded protein-polysaccharide composite nanoparticles demonstrate a sustained-release effect, increasing the duration of drug action. In vitro models have verified that the cordycepin-loaded protein-polysaccharide composite nanoparticles possess certain anti-inflammatory effects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a flowchart of the calcium alginate-chitosan microspheres loaded with cordycepin prepared in Example 1.

[0022] Figure 2 This is a microscopic image of the calcium alginate-chitosan microspheres loaded with cordycepin prepared in Example 1.

[0023] Figure 3 This is a diagram of the high-performance liquid chromatography (HPLC) method for detecting cordycepin.

[0024] Figure 4The figure shows the effect of different pH values ​​on the particle size of protein-polysaccharide composite nanoparticles loaded with cordycepin.

[0025] Figure 5 Encapsulation efficiency of protein-polysaccharide composite nanoparticles loaded with cordycepin at different dosages.

[0026] Figure 6 The figure shows the release curve of the protein-polysaccharide composite nanoparticles loaded with cordycepin, where the horizontal axis represents time and the vertical axis represents the drug release ratio.

[0027] Figure 7 Infrared spectra of various raw materials for preparing nanoparticles, synthesized samples, and mixtures of sample and raw materials.

[0028] Figure 8 The graph shows the changes in average particle size, PDI, and potential of protein-polysaccharide composite nanoparticles loaded with cordycepin under low-temperature storage conditions.

[0029] Figure 9 To investigate the effects of different concentrations of free cordycepin and cordycepin-loaded protein-polysaccharide composite nanoparticles on the activity of L929 cells.

[0030] Figure 10 The expression levels of TNF-α and IL-6 in each group of a rat osteoarthritis model are shown. Detailed Implementation

[0031] The present application will be further described in detail below with reference to specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0032] Cordycepin (3′-deoxyadenosine) is a derivative of nucleoside adenosine and a major component of Cordyceps sinensis. Studies have shown that cordycepin has anti-inflammatory, immunomodulatory, anti-diabetic, anti-hyperlipidemia, and antioxidant effects, as well as cardiovascular disease prevention and anti-cancer effects. To further explore the application potential of cordycepin, this application uses an antisolvent method combined with a layer-by-layer electrostatic deposition method to prepare cordycepin-loaded protein-polysaccharide ternary composite nanoparticles. Cell experiments confirmed that these nanoparticles can reduce the toxicity of cordycepin, and in vitro models verified that the cordycepin-loaded protein-polysaccharide composite nanoparticles have certain anti-inflammatory effects.

[0033] The following detailed description is provided in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the following embodiments are commercially available; all instruments used are commercially available; and all processes involved are conventionally selected by those skilled in the art unless otherwise specified. Example 1

[0034] This embodiment provides a method for preparing cordycepin-loaded calcium alginate / chitosan microspheres via a dual-emulsion crosslinking method, specifically including the following steps:

[0035] (1) Prepare the oil phase: Add 6% Span-80 to the liquid paraffin and stir magnetically for 30 min to make it completely emulsified (prepare 2 portions).

[0036] (2) Prepare a 1.5% (w / v) sodium alginate solution and add cordycepin to the sodium alginate solution and stir until it is completely dissolved.

[0037] (3) Prepare a 7% (w / v) calcium chloride solution for use as a crosslinking agent.

[0038] (4) Under mechanical stirring, sodium alginate solution and calcium chloride solution containing cordycepin are added dropwise to the oil phase using a syringe. The oil-water ratio is set to 3:2 and stirring is maintained at a certain speed for more than 30 minutes to form emulsions.

[0039] (5) Mix the two emulsions, maintain the crosslinking temperature, stir at 800 rpm, and cure for 4 h.

[0040] (6) After cross-linking and curing, the microspheres were centrifuged, washed three times with anhydrous ethanol and twice with aqueous solution to obtain calcium alginate microspheres loaded with cordycepin.

[0041] (7) Prepare a 0.1% chitosan solution using a 10% acetic acid solution. Soak the obtained calcium alginate microspheres in the chitosan solution for 5-10 min. Centrifuge and discard the supernatant to obtain calcium alginate-chitosan microspheres loaded with cordycepin.

[0042] 1. Through the above methods and procedures (such as...) Figure 1 Cordycepin-loaded calcium alginate / chitosan microspheres were prepared, and their morphology was observed under a microscope. Figure 2 (a) shows calcium alginate microspheres loaded with cordycepin. Figure 2 (b) are calcium alginate-chitosan microspheres loaded with cordycepin. Microscopic observation shows that the microspheres are spherical and relatively dispersed, but their size uniformity is not high.

[0043] 2. High-performance liquid chromatography (HPLC) for the detection of cordycepin content: The specific steps are as follows: Wavelength determination: For cordycepin determination, the literature generally reports a wavelength between 250-260 nm. In this experiment, we scanned the cordycepin standard solution within the wavelength range of 200-500 nm using a UV spectrophotometer. The wavelength at which the cordycepin absorption peak was most significant and the sensitivity was highest was determined, and the detector was set at this wavelength for detection. Mobile phase determination: In the literature, phosphate buffer is mostly used as the mobile phase for the analysis of cordycepin and adenosine. This experiment tested various mobile phase conditions, including water-methanol-acetic acid solution (185:14:1), methanol-10 mM potassium dihydrogen phosphate solution (15:85), acetonitrile aqueous solution, and methanol aqueous solution. The cordycepin standard was scanned at wavelengths of 200-500 nm using a UV spectrophotometer. Figure 3 (a) shows that the absorption peak of cordycepin at 260 nm was the most significant and sensitive; therefore, the detector was set at 260 nm for detection. Acetonitrile aqueous solution was ultimately selected as the mobile phase for the high-performance liquid chromatography (HPLC) determination of cordycepin, as its composition is simple and has minimal impact on the chromatographic column. Through the determination of different samples and adjustments to the mobile phase ratio, the final mobile phase ratio of 10 + 90 = acetonitrile + aqueous solution was determined. The HPLC chromatogram of the cordycepin standard solution is shown below. Figure 3 (c) The final chromatographic conditions were: column: C18 column, length 250 mm, inner diameter 4.6 mm, particle size 5 µm; temperature: 35℃; mobile phase: acetonitrile + water = 10 + 90; flow rate: 1.0 mL / min; detection wavelength: 260 nm; injection volume: 10 μL. A standard curve was plotted with the concentration of the standard cordycepin solution as the x-axis and the peak area as the y-axis, as shown below. Figure 3 As shown in (b), the standard curve equation is y = 21.10657x + 2.37645, and the correlation coefficient R0 is... 2 It is 0.9999.

[0044] 3. The cumulative drug release rate of the cordycepin-loaded calcium alginate-chitosan microspheres is as follows:

[0045] 10 mg of cordycepin-containing calcium alginate-chitosan microspheres were dissolved in 1 mL of water and placed in a dialysis bag. 15 mL of freshly prepared PBS was added to a centrifuge tube as a release medium to simulate the in vivo environment. The centrifuge tube was placed on a shaker at 37℃±0.5℃ and 100 r / min for in vitro drug release experiments. A specific volume of release medium was collected from the centrifuge tube at specified time points, and the same volume of freshly prepared PBS was immediately added after each collection. The collected release medium was filtered through a 0.22 μm disposable syringe filter, and the drug concentration was detected by high-performance liquid chromatography (HPLC). The cumulative release rate at each specified time point was calculated.

[0046] Cordycepin-loaded microspheres were subjected to sustained release in a PBS solution simulating the in vivo environment. High-performance liquid chromatography (HPLC) analysis revealed no cordycepin in the release medium. This may be due to excessive use of the release medium, resulting in an insufficient concentration, or an inadequate drug loading. Therefore, the free cordycepin content during the preparation process was measured. The results showed that the free cordycepin content was almost identical to the dosage, indicating that this preparation method failed to successfully encapsulate cordycepin and thus failed to achieve a sustained release effect. Example 2

[0047] This embodiment provides a method for preparing protein-polysaccharide composite nanoparticles loaded with cordycepin, comparing the effect of sodium alginate solution pH on the nanoparticles, and specifically includes the following steps:

[0048] (1) Dissolve 0.2 g of zein in 10 mL of 80% ethanol aqueous solution, then add 20 mg of cordycepin and stir thoroughly to dissolve, to obtain cordycepin-zein ethanol aqueous solution.

[0049] (2) Preparation of sodium alginate solution: Dissolve 4 mg of sodium alginate in 20 mL of deionized water and stir overnight to ensure complete dissolution. Adjust the pH to 3, 4, 5, 6, or 7.

[0050] (3) Preparation of sodium alginate-modified zein nanoparticle dispersion loaded with cordycepin: Take 2 mL of the cordycepin-zein ethanol solution obtained in step (1) and slowly add it dropwise to 8 mL of the sodium alginate solution obtained in (2), and stir for 60 min.

[0051] (4) Preparation of chitosan solution: Dissolve 2 mg of chitosan in 10 mL of 1% acetic acid solution and stir overnight to ensure complete dissolution.

[0052] (5) Preparation of sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin: The sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin obtained in (3) was slowly added dropwise to the chitosan solution obtained in (4) and stirred for 60 min; then, the organic solvent was removed under reduced pressure on a rotary evaporator and the volume was made up to 20 mL with deionized water to obtain the sodium alginate-chitosan dual-modified zein nanoparticle dispersion loaded with cordycepin.

[0053] The average particle size, polydispersity index (PDI), and zeta potential of the above nanoparticles were measured using a Malvern laser particle size analyzer.

[0054] The average particle size and polydispersity index (PDI) of each nanoparticle during the preparation of cordycepin-loaded nanoparticles are as follows: Figure 4 As shown, different pH values ​​of sodium alginate affect the prepared nanoparticles, with the best particle size results observed at pH 4. The particle size increases with increasing pH, ultimately leading to the selection of slightly acidic conditions for nanoparticle preparation. Example 3

[0055] This embodiment provides a method for preparing a dispersion of cordycepin-loaded zein nanoparticles modified with sodium alginate. The pH of the sodium alginate solution is 4, and the amount of sodium alginate is adjusted to 20 mg, 10 mg, 4 mg, 2 mg, and 0 mg. The remaining preparation steps and the amount of each component added are the same as in Example 2. The average particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using a Malvern laser particle size analyzer. Table 1 shows the effect of different sodium alginate concentrations on the average particle size, PDI, and zeta potential of the cordycepin-loaded zein nanoparticles. It can be seen that as the sodium alginate concentration increases, the particle size and PDI of the sodium alginate-modified cordycepin-loaded zein nanoparticles continuously increase. When the sodium alginate concentration is 0.02%, the particle size is the smallest, the dispersibility is also good, and the overall zeta potential is negative.

[0056] Table 1. Average particle size, PDI, and ζ-potential of cordycepin-zein particles coated with different concentrations of alginate.

[0057] Example 4

[0058] This example investigates the effects of different chitosan dosages on the average particle size, polydispersity index (PDI), and zeta potential of nanoparticles prepared by coating cordycepin with different sodium alginate concentrations. The results are shown in Tables 2-4.

[0059] Table 2 Average particle size (nm)

[0060]

[0061] Table 3. Polydispersion Index (PDI)

[0062]

[0063] Table 4. Zeta potential (MV)

[0064]

[0065] Table 2-4 shows the average particle size, PDI, and ζ-potential of sodium alginate-coated cordycepin zein nanoparticles modified with chitosan at different concentrations. When using chitosan for coating, excessive concentrations resulted in flocculent formations (indicated by "--" in the table). Using different concentrations of chitosan to coat different concentrations of sodium alginate, a chitosan concentration of 0.02%, and sodium alginate concentrations of 0.01% and 0.02% all showed good particle size and PDI, and the ζ-potential also exhibited positive values. Based on comprehensive comparison, protein-polysaccharide composite nanoparticles loaded with cordycepin with a sodium alginate concentration of 0.02% and a chitosan concentration of 0.02% were selected as the subject of subsequent research. Example 5

[0066] Based on the optimal sodium alginate and chitosan solutions determined in the aforementioned examples, i.e., sodium alginate dosage of 4 mg, pH of 4, and chitosan dosage of 2 mg, the amount of cordycepin added was adjusted to 20 mg, 10 mg, and 5 mg. The remaining preparation steps and the amount of each component added were the same as in Example 2.

[0067] The protein-polysaccharide composite nanoparticles loaded with cordycepin of this invention were reconstituted after being lyophilized and weighed. The reconstituted solution was centrifuged at 10,000 rpm for 40 min, and the supernatant was collected. The absorbance was measured at 260 nm using a UV spectrophotometer. The encapsulation efficiency and drug loading were calculated using the following formulas:

[0068] Encapsulation rate (%) = (Total cordycepin content (mg) - Free cordycepin content (mg)) / Total cordycepin content (mg) * 100

[0069] Drug loading (%) = (Total cordycepin content (mg) - Free cordycepin content (mg)) / Total weight of nanoparticles (mg) * 100

[0070] The drug release curve of the protein-polysaccharide composite nanoparticles loaded with cordycepin was determined by dialysis.

[0071] Encapsulation efficiency, drug loading, and drug release results of cordycepin-loaded protein-polysaccharide composite nanoparticles are as follows: Figure 5As shown, ZCSCs with different concentrations of cordycepin exhibit different encapsulation efficiencies. The best encapsulation effect was observed when the cordycepin concentration was 2 mg / mL, with an encapsulation efficiency of 78.6%. Using 2 mg / mL cordycepin, ZCSC NPs were prepared according to the optimal sodium alginate and chitosan concentrations mentioned above, achieving a drug loading of 6.4%. In summary, the optimal ZCSC preparation method using 2 mg / mL cordycepin, 0.02% sodium alginate, and 0.02% chitosan yielded the best results, with a particle size of 195.53 nm, a PDI of 0.195, a zeta potential of 49.3 mV, an encapsulation efficiency of 78.6%, and a drug loading of 6.4%. These results were used in further research. The drug release curve is shown below. Figure 6 As shown, the drug release curve indicates that ZCSC can release about 40% within 2 hours and about 70% cumulatively after 34 hours, with a stable release curve.

[0072] The FT-IR spectra of cordycepin, zein, sodium alginate, chitosan, cordycepin-loaded sodium alginate-modified zein nanoparticles, and cordycepin-loaded sodium alginate-chitosan dual-modified zein nanoparticles were analyzed using infrared spectroscopy. The FT-IR spectra of zein (Cor), sodium alginate (SA), and chitosan (CS) are shown below. Figure 7 As shown in (a), the spectra of zein-loaded nanoparticles (ZC), sodium alginate-modified zein-loaded nanoparticles (ZCSA), cordycepin-loaded protein-polysaccharide composite nanoparticles (ZCSC), and mixtures of various raw materials are as follows. Figure 7 As shown in (b), by comparing the characteristic peaks of different samples, it can be concluded that cordycepin is encapsulated in zein nanoparticles, and sodium alginate and chitosan modify the nanoparticles.

[0073] The protein-polysaccharide composite nanoparticle dispersion loaded with cordycepin was placed in a 4°C refrigerator, and the average particle size, polydispersity index (PDI), and zeta potential were measured at 1, 3, 5, and 7 days. The results showed that under one week of low-temperature storage, the average hydrated particle size, PDI, and zeta potential of the ZCSC sample remained essentially unchanged. Figure 8 This indicates that it has good stability under low temperature conditions. Example 6

[0074] Cordycepin-loaded protein-polysaccharide composite nanoparticles (ZCSCs) prepared by the optimized preparation method of the above embodiments (2 mg / mL cordycepin, 0.02% sodium alginate at pH 4 and 0.02% chitosan) were used in cell and animal experiments.

[0075] The cytotoxicity assessment of cordycepin-loaded protein-polysaccharide composite nanoparticles includes the following steps:

[0076] 1. Cytotoxicity Assay: The in vitro toxicity of cordycepin and cordycepin-loaded protein-polysaccharide composite nanoparticles to L929 cells was investigated using the MTT assay. First, L929 cells in logarithmic growth phase were seeded at a density of 8000 cells per well in 96-well plates and cultured in a CO2 incubator for 12 h. Then, different concentrations of cordycepin and cordycepin-loaded protein-polysaccharide composite nanoparticles (calculated based on the cordycepin loading in the nanoparticles) were prepared and diluted to the desired concentrations using blank DMEM medium. The original medium in the wells was discarded, and medium containing different concentrations of the samples was added, followed by culturing for 24 h. Afterward, 20 μL of MTT solution was added to each well, and the cells were incubated for another 4 h. The liquid in the wells was then discarded, and 150 μL of DMSO was added. The cells were incubated on a shaker in the dark for 15 min, and the absorbance at 570 nm was measured using a microplate reader.

[0077] 3. Results

[0078] The MTT assay was used to study the cell viability of L929 cells co-incubated with cordycepin and cordycepin-loaded protein-polysaccharide composite nanoparticles for 24 h. The results are as follows: Figure 9 As shown in the figure, when the concentration of cordycepin is 0.1 mg / mL, the cell viability of both cordycepin and cordycepin-loaded protein-polysaccharide composite nanoparticles is higher than 80%. However, as the concentration of cordycepin increases, the cell viability of L929 cells gradually decreases. In contrast, the cell viability of the cordycepin-loaded protein-polysaccharide composite nanoparticles remains consistently high. Compared with free cordycepin, the cordycepin-loaded protein-polysaccharide composite nanoparticles exhibit lower toxicity and a certain degree of cell proliferation effect. Example 7

[0079] The evaluation of the anti-inflammatory effect of cordycepin-loaded protein-polysaccharide composite nanoparticles in osteoarthritis includes the following steps:

[0080] 1. Establishment and administration of a rat model of osteoarthritis

[0081] Male SD rats weighing approximately 200 g were anesthetized with 1 mL of 7% chloral hydrate. A parapatellar incision was made on the medial side of the rat's knee joint. After separating the skin and opening the knee joint capsule, the fat was removed to expose the medial meniscus and tibial ligament. The medial meniscus and tibial ligament were then cut with a scalpel blade to destabilize the joint, and the wound was sutured. In the sham-operated group, only the ligament was exposed without cutting. Four weeks after modeling, rats were injected into the joint cavity with cordycepin-loaded nanoparticles every two weeks. Four weeks after administration, the rats were sacrificed, and blood samples were collected for analysis of inflammatory factors.

[0082] The expression levels of TNF-α and IL-6 were detected using an ELISA kit. Results showed that unilateral meniscus transection induced osteoarthritis in rats, followed by intra-articular drug administration. After four weeks of treatment, rats were sacrificed, and blood samples were collected for ELISA experiments. The results were as follows... Figure 10 As shown, the Sham group represents the sham surgery group, the PBS group represents the osteoarthritis group, the ZCSA group represents protein-polysaccharide composite nanoparticles without cordycepin loading, the ZCSC group represents protein-polysaccharide composite nanoparticles loaded with cordycepin, and the Free Cor group represents the free cordycepin group. The results showed that compared with the PBS group, ZCSA group, and Free Cor group, the expression levels of inflammatory factors TNF-α and IL-6 were significantly reduced in the ZCSC group, indicating that the protein-polysaccharide composite nanoparticles loaded with cordycepin have a certain anti-inflammatory effect.

[0083] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A protein-polysaccharide ternary composite nanoparticle loaded with cordycepin, characterized in that, A composite nanoparticle for delivering cordycepin was constructed by encapsulating cordycepin in zein as the core and assembling sodium alginate and chitosan into a shell. The preparation method of the aforementioned cordycepin-loaded protein-polysaccharide ternary composite nanoparticles specifically includes the following steps: (1) Dissolve 20 mg / mL zein in an ethanol aqueous solution, add cordycepin, stir thoroughly to dissolve, and obtain solution A; the mass ratio of cordycepin to zein is 0.025-0.1:1; (2) Add solution A dropwise to sodium alginate solution and stir to obtain dispersion B; the concentration of sodium alginate solution is 1-2 mg / mL and the pH is 3-5; the volume ratio of solution A to sodium alginate solution is 1:

4. (3) Slowly add dispersion B to the chitosan solution, stirring at 400-800 rpm for 30-60 min; remove the organic solvent by rotary evaporation under reduced pressure, and dilute with water to obtain dispersion C; the concentration of the chitosan solution is 0.1-2 mg / mL, and the solvent is 1% acetic acid solution; (4) Freeze-dry the dispersion C to obtain protein-polysaccharide ternary composite nanoparticles loaded with cordycepin.

2. The protein-polysaccharide ternary composite nanoparticles loaded with cordycepin according to claim 1, characterized in that, The composite nanoparticles have a particle size of 190-210 nm and a potential of 40-50 mV.

3. The method for preparing the protein-polysaccharide ternary composite nanoparticles loaded with cordycepin according to claim 1 or 2, characterized in that, Specifically, the following steps are included: (1) Dissolve zein in an ethanol aqueous solution, add cordycepin, stir thoroughly to dissolve, and obtain solution A; (2) Add solution A dropwise to sodium alginate solution and stir to obtain dispersion B. (3) Slowly add dispersion B to the chitosan solution, stirring at 400-800 rpm for 30-60 min; remove the organic solvent by rotary evaporation under reduced pressure, and dilute with water to obtain dispersion C; (4) Freeze-dry the dispersion C to obtain protein-polysaccharide ternary composite nanoparticles loaded with cordycepin.

4. The method for preparing cordycepin-loaded protein-polysaccharide ternary composite nanoparticles according to claim 3, characterized in that, In solution A described in step (1), the concentration of zein is 20 mg / mL, and the mass ratio of cordycepin to zein is 0.025-0.1:

1.

5. The method for preparing cordycepin-loaded protein-polysaccharide ternary composite nanoparticles according to claim 3, characterized in that, The sodium alginate solution described in step (2) has a concentration of 1-2 mg / mL and a pH of 3-5.

6. The method for preparing cordycepin-loaded protein-polysaccharide ternary composite nanoparticles according to claim 3, characterized in that, The volume ratio of solution A to sodium alginate solution in step (2) is 1:

4.

7. The method for preparing cordycepin-loaded protein-polysaccharide ternary composite nanoparticles according to claim 3, characterized in that, The concentration of the chitosan solution in step (3) is 0.1-2 mg / mL, and the solvent is 1% acetic acid solution.

8. The method for preparing cordycepin-loaded protein-polysaccharide ternary composite nanoparticles according to claim 3, characterized in that, The volume ratio of dispersion B to chitosan solution is 1:

1.

9. The application of the cordycepin-loaded protein-polysaccharide ternary composite nanoparticles as described in claim 1 or 2 in the preparation of anti-inflammatory drugs.

10. A pharmaceutical composition, characterized in that, Protein-polysaccharide ternary composite nanoparticles loaded with cordycepin as described in claim 1 or 2.

Images