Cordycepin-baicalein self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses cordycepin-baicalein self-assembled nanoparticles as well as a preparation method and application thereof, and relates to the technical field of medicine preparation. The self-assembled nanoparticles are composed of cordycepin and baicalein, and the cordycepin and the baicalein are self-assembled through intermolecular non-covalent interaction to form a stable nanoscale particle system; the self-assembled nano-particles show good proliferation inhibition activity on various tumor cells (such as liver cancer HepG2 cells, breast cancer 4T1 cells and colon cancer HT-29 cells), meanwhile, the self-assembled nano-particles have a remarkable anti-inflammatory effect, and the expression level of inflammatory factors (such as TNF-alpha and IL-6) can be effectively reduced. Besides, the self-assembled carrier-free nano-particle not only retains the advantages of good solubility and high bioavailability of a nano-drug, but also realizes carrier-free self-delivery of the drug, avoids the problems of low drug loading rate, potential toxicity and the like caused by a traditional nano-carrier, improves the drug delivery efficiency and medication safety, and is suitable for industrial production. The compound has a wide application prospect in the field of research and development of anti-tumor and anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of drug preparation technology, specifically to a cordycepin-baicalin self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] In the current medical field, cancer remains a major challenge threatening human health. Cancer treatment faces problems such as poor drug selectivity, significant side effects, and easy development of drug resistance, necessitating the development of novel, targeted, and low-toxicity anti-tumor agents. Natural drugs, due to their wide availability, diverse biological activities, and relatively low toxicity, have become an important direction for new drug development. Cordycepin, a nucleoside active ingredient extracted from Cordyceps sinensis, possesses various pharmacological effects including anti-tumor, anti-inflammatory, and immunomodulatory effects. It exerts its efficacy through mechanisms such as inducing tumor cell apoptosis and regulating inflammatory signaling pathways. Baicalin, the main flavonoid active ingredient of Scutellaria baicalensis (a plant in the Lamiaceae family), has broad-spectrum antibacterial, anti-tumor, anti-inflammatory, and antioxidant effects. It achieves therapeutic effects by inhibiting tumor cell proliferation and downregulating the expression of inflammatory factors.

[0003] However, cordycepin suffers from poor pharmacokinetics and low tissue accumulation, while baicalein exhibits poor water solubility and low bioavailability, limiting their clinical application. Traditional formulation technologies (such as ordinary tablets and capsules) are insufficient to effectively improve the solubility and absorption properties of these two substances. Furthermore, existing nanomedicines often rely on excipients such as polymer carriers and liposomes, which may introduce potential toxicity and inflammatory reactions, and also suffer from low drug loading rates and drug leakage, affecting both efficacy and medication safety.

[0004] Carrier-free self-assembly nanotechnology can utilize non-covalent interactions between drug molecules (such as hydrogen bonds, π-π stacking, hydrophobic interactions, etc.) to achieve self-assembly, forming carrier-free nanoparticles that can retain the drug's own activity, improve solubility, stability and bioavailability, while avoiding the safety risks associated with excipients. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a cordycepin-baicalin self-assembled nanoparticle, which aims to solve the problems of low solubility and bioavailability of cordycepin and baicalin, and at the same time develop a new type of carrier-free, highly active and low-toxicity nanomedicine for anti-tumor and anti-inflammatory treatment.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing cordycepin-baicalin self-assembled nanoparticles.

[0007] The third technical problem to be solved by the present invention is to provide an application of cordycepin-baicalin self-assembled nanoparticles.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A self-assembled cordycepin-baicalein nanoparticle is formed by the self-assembly of cordycepin and baicalein through intermolecular non-covalent interactions (hydrogen bonds, π-π stacking and hydrophobic interactions).

[0010] The structural formula of cordycepin is: ;

[0011] The structural formula of baicalin is: .

[0012] A method for preparing cordycepin-baicalin self-assembled nanoparticles includes the following steps: (1) Dissolve cordycepin in the first organic solvent to obtain the first solution; (2) Dissolve baicalein in a mixture of a second organic solvent and water, and sonicate to obtain a second solution; (3) Mix the first solution and the second solution and stir until homogeneous to obtain a mixed solution; (4) The mixed solution is freeze-dried for the first time to obtain cordycepin-baicalin crude powder; (5) The cordycepin-baicalin crude powder is dispersed in water and dialyzed, and then freeze-dried a second time to obtain the product.

[0013] In some embodiments, in step (1), the first organic solvent is methanol; and / or, the first solution has a concentration of 0.8-3 mg / mL (preferably 0.8 mg / mL).

[0014] In some embodiments, in step (2), the amount of baicalein added is calculated based on a molar ratio of baicalein to cordycepin of 1:0.2~4 (preferably 0.5~1); and / or, the second organic solvent is ethanol; and / or, the volume ratio of the second organic solvent to water is 1:1-3 (preferably 1:1); and / or, the ultrasound is performed at a temperature of 20-50℃ (preferably 30℃), a power of 200-400W (preferably 300 W), a frequency of 30-50kHz (preferably 40 kHz), and a time of 10-15 min (preferably 12 min); and / or, the second solution has a concentration of 0.8-2.5 mg / mL (preferably 0.86 mg / mL).

[0015] In some embodiments, in step (3), the mixing is to add the first solution dropwise to the second solution at a speed of 800-1200 rpm (preferably 600 r / min) with no large particles rapidly precipitating; and / or, the stirring is carried out at a temperature of 20-80℃ (preferably 25℃), a speed of 500-900 r / min (preferably 600 r / min), and a time of 1.5-3 h (preferably 2 h).

[0016] In some embodiments, in steps (4) and (5), the first freeze-drying and the second freeze-drying are performed at a temperature of -60 ~ -50℃ (preferably -55℃) for 12 ~ 24h (preferably 18h) and a vacuum degree of less than 10Pa (preferably 8Pa).

[0017] In some embodiments, in step (5), the mass ratio of the cordycepin-baicalin crude powder to water is 1:2-5 (preferably 1:2); and / or, the dispersion is performed using ultrasound with a power of 150-300W (preferably 200W), a frequency of 30-50kHz (preferably 40 kHz), and a time of 10-20 min (preferably 15 min); and / or, the dialysis is performed with a dialysis bag having a molecular weight cutoff of 3500-5000 Da (preferably 3500 Da), a time of 6-10 h (preferably 8 h), and a temperature of room temperature.

[0018] The cordycepin-baicalin self-assembled nanoparticles prepared by the above method are also within the scope of protection of this invention.

[0019] In some embodiments, the cordycepin-baicalin self-assembled nanoparticles have a particle size of 100-3000 nm (preferably 100-500 nm), a PDI value ≤1 (preferably PDI ≤0.3), and a Zeta potential of -30±5 mV.

[0020] In some embodiments, the cordycepin-baicalin self-assembled nanoparticles can be prepared into various pharmaceutical dosage forms, including tablets, capsules, injections, suspensions, gels, and patches, to meet different clinical drug administration needs: oral dosage forms (tablets, capsules) are suitable for adjuvant cancer therapy; injections are suitable for acute cancer treatment; and topical dosage forms (gels, patches) are suitable for tumors on the body surface.

[0021] In some embodiments, the application of the cordycepin-baicalin self-assembled nanoparticles in the preparation of antitumor drugs is also within the scope of protection of this invention. Preferably, the tumor includes any one or more of liver cancer HepG2, breast cancer 4T1, and colon cancer HT-29.

[0022] In some embodiments, the application of the cordycepin-baicalin self-assembled nanoparticles in the preparation of anti-inflammatory drugs is also within the scope of protection of this invention. Preferably, the anti-inflammatory drugs alleviate acute inflammatory responses by reducing the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β in inflamed tissues.

[0023] Beneficial effects:

[0024] (1) Based on the molecular structural characteristics of cordycepin and baicalein, this invention, for the first time, utilizes the different functional groups contained in the two (polar groups such as amino and hydroxyl groups in cordycepin molecules, and conjugated structures such as benzene rings and ketone groups in baicalein molecules) to achieve self-assembly through non-covalent interactions, forming carrier-free nanoparticles. This method does not require the introduction of exogenous excipients such as polymers and liposomes, thus avoiding the toxicity risks and low drug loading rates caused by excipients. In addition, this invention fills the gap in the field of synergistic self-assembly of cordycepin and baicalein nanoparticle formulations, including optimization of self-assembly conditions, regulation of nanoparticle performance, and verification of related pharmacological activities.

[0025] (2) Excellent physicochemical properties: The nanoparticles prepared by this invention have uniform particle size (PDI≤0.3) and good stability (Zeta potential –30±5mV). The higher negative potential can enhance the stability of the particles in solution, reduce agglomeration, and at the same time help the particles to bind to the negatively charged bacterial cell membrane or tumor cell membrane, thereby improving the drug efficacy. The water solubility is significantly improved compared with free cordycepin and baicalin, which improves the solubility of the two drugs and lays the foundation for subsequent formulation development and clinical application.

[0026] (3) Synergistic enhancement of pharmacological activity: Through the synergistic effect between drug molecules, the antitumor activity of the cordycepin-baicalin self-assembled nanoparticles prepared in this invention is better than that of a single free drug, and it has good effects on a variety of tumor cell models, thus broadening the application range of the drug.

[0027] (4) High safety: The cytotoxicity test (MTT method) showed that the cordycepin-baicalin self-assembled nanoparticles prepared in this invention had a survival rate of ≥80% against human umbilical vein endothelial cells (HUVECs), and the drug safety was significantly better than that of some traditional chemical drugs and carrier nanoparticles.

[0028] (5) Cell proliferation inhibition assay (MTT method) showed that the cordycepin-baicalin self-assembled nanoparticles prepared in this invention inhibited tumor cells by half-maximal inhibitory concentration (IC50). 50Compared with free drugs, it reduces the concentration by 40%-70% and has a certain degree of targeting. Its anti-tumor mechanism includes: inducing tumor cell apoptosis and activating caspase-3 and caspase-9 signaling pathways; inhibiting tumor cell migration and invasion and downregulating MMP-2 and MMP-9 protein expression; blocking tumor angiogenesis and reducing VEGF factor levels.

[0029] (6) The cordycepin-baicalin self-assembled nanoparticles prepared in this invention can be used to alleviate acute inflammatory reactions (such as lipopolysaccharide-induced peritoneal inflammation in mice). According to enzyme-linked immunosorbent assay (ELISA), the nanoparticles can reduce the expression levels of TNF-α, IL-6 and IL-1β in inflamed tissues by 50%-80%. Its anti-inflammatory mechanism is mainly as follows: inhibiting the activation of NF-κB signaling pathway and reducing the transcription of inflammatory factors; clearing reactive oxygen species (ROS) in inflammatory sites and reducing oxidative stress damage; regulating macrophage polarization and promoting the transformation of M1 macrophages into anti-inflammatory M2 macrophages.

[0030] (7) The preparation process is simple and controllable. Attached Figure Description

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0032] Figure 1 Particle size distribution of COR-BA NPs prepared in Example 1;

[0033] Figure 2 The potential map of COR-BA NPs prepared in Example 1;

[0034] Figure 3 The experimental results of the COR-BA NPs prepared in Example 1 show the Yenderl effect.

[0035] Figure 4 The transmission electron microscopy observation results of COR-BA NPs prepared in Example 1;

[0036] Figure 5 UV characterization experiment of COR-BA NPs prepared for Example 1;

[0037] Figure 6 Infrared characterization experiment of COR-BA NPs prepared for Example 1;

[0038] Figure 7 XRD characterization experiment of COR-BA NPs prepared for Example 1;

[0039] Figure 87-Day stability test of COR-BA NPs prepared for Example 1;

[0040] Figure 9 Solubility experiment of COR-BA NPs prepared for Example 1;

[0041] Figure 10 Experiment on the inhibitory effect of COR-BA NPs prepared for Example 1 on HepG2 liver cancer cells;

[0042] Figure 11 Experiment on the inhibitory effect of COR-BA NPs prepared for Example 1 on breast cancer 4T1 cells;

[0043] Figure 12 Experiment on the inhibitory effect of COR-BA NPs prepared for Example 1 on HT-29 colon cancer cells;

[0044] Figure 13 Figure showing the experimental results of COR-BA NPs prepared for Example 1 reducing the expression of the inflammatory factor TNF-α;

[0045] Figure 14 The experimental results of reducing the expression of the inflammatory factor IL-6 by COR-BA NPs prepared for Example 1;

[0046] Figure 15 Experimental results showing that COR-BA NPs prepared for Example 1 reduced the expression of the inflammatory factor IL-1β;

[0047] Figure 16 Experiment on the inhibitory effect of COR-BA NPs prepared for Example 1 on human umbilical vein endothelial cells (HUVECs). Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1

[0050] Preparation of self-assembled cordycepin-baicalin nanoparticles (molar ratio of cordycepin to baicalin 1:0.5)

[0051] This embodiment prepares cordycepin-baicalin self-assembled nanoparticles according to the following steps:

[0052] (1) Preparation of cordycepin solution: Weigh 0.08 g cordycepin powder, slowly add it to 100 mL methanol, stir at 300 r / min for 20 min at 25℃ until the powder is completely dissolved, and obtain a cordycepin methanol solution with a mass concentration of 0.8 mg / mL. Seal and store in the dark for later use.

[0053] (2) Preparation of baicalein solution: Weigh 0.0432 g of baicalein powder and add it to 50 mL of ethanol-water mixed solvent (ethanol to water volume ratio of 1:1). Sonicate at 300 W power and 40 kHz frequency for 12 min at 30℃, stirring once every 3 min to ensure that the baicalein is fully dissolved, and obtain a baicalein solution with a mass concentration of 0.86 mg / mL. Let it stand at room temperature and cool to 25℃.

[0054] (3) Self-assembly reaction: Cordycepin solution was added dropwise to baicalin solution at a flow rate of 1 mL / min using a peristaltic pump, while magnetic stirring was performed at 600 r / min at 25℃ for 2 h. The solution state was observed during the process to ensure that there was no layering or precipitation. Finally, a light yellow clear mixture was obtained.

[0055] (4) First freeze-drying: Transfer the mixture to a freeze-drying bottle, put it into a freeze dryer, pre-freeze at -60℃ for 2 h, then maintain a vacuum of 8 Pa and a temperature of -55℃ for freeze-drying for 18 h, remove the solvent, and obtain a loose, light yellow coarse powder. Weigh and record the yield.

[0056] (5) Dialysis purification: 1 g of crude powder was mixed with 2 mL of deionized water at a mass ratio of 1:2 and ultrasonically dispersed at 200 W power and 40 kHz frequency for 15 min at 25℃ to form a uniform dispersion. The dispersion was placed into a dialysis bag with a molecular weight cutoff of 3500 Da, the bag was sealed and placed in a dialysis cup containing 500 mL of deionized water. The bag was magnetically stirred and dialyzed at room temperature for 8 h. The deionized water was replaced every 2 h. The liquid in the bag was collected after dialysis. The dialysis liquid was freeze-dried for a second time under the conditions of step (4) for 16 h to obtain light yellow powdered cordycepin-baicalin self-assembled nanoparticles. The nanoparticles were sealed and stored in a refrigerator at 4℃. Light orange-yellow powdered nanoparticles were obtained. The sample had good morphology and stability.

[0057] Examples 2-5

[0058] The experimental steps and conditions for Examples 2-5 are the same as those for Example 1, except that the molar ratio of cordycepin to baicalin in the self-assembly reaction is different. The parameters are shown in Table 1.

[0059] Table 1. Molar ratio of cordycepin to baicalin

[0060]

[0061] Examples 6-8

[0062] The experimental steps and conditions of Examples 6-8 are the same as those of Example 1, except that the temperature of the self-assembly reaction in step (3) is different. The parameters are shown in Table 2.

[0063] Table 2 Temperature of self-assembly reaction

[0064]

[0065] Example 9

[0066] Physicochemical characterization experiments of cordycepin-baicalin self-assembled nanoparticles

[0067] The physicochemical properties of the nanoparticles prepared in Examples 1-8 were tested to verify their structure and stability. The specific experimental methods and results are as follows:

[0068] 1. Particle size and PDI detection: Take the nanoparticle powder of each example, prepare a dispersion of 0.1 mg / mL with deionized water, and after ultrasonic dispersion for 5 min, use a dynamic light scattering instrument to detect the particle size and PDI value at 25℃. Repeat the detection of the sample 3 times and take the average value.

[0069] The results showed that the nanoparticles prepared in Example 1 had an average particle size of 164.9 nm and a PDI of 0.28 (as shown in the figure). Figure 1 The nanoparticles prepared in Example 1 and Example 2 met the requirements of a particle size range of 100-500 nm and a PDI ≤ 0.3, indicating good particle uniformity. The average particle size of the nanoparticles prepared in Example 2 was 232 nm, with a PDI of 0.22; the average particle size of the nanoparticles prepared in Example 3 was 621 nm, with a PDI of 0.49; the average particle size of the nanoparticles prepared in Example 4 was 743 nm, with a PDI of 0.49; the average particle size of the nanoparticles prepared in Example 5 was 1322 nm, with a PDI of 1; the average particle size of the nanoparticles prepared in Example 6 was 246 nm, with a PDI of 0.4551; the average particle size of the nanoparticles prepared in Example 7 was 1392 nm, with a PDI of 0.4713; and the average particle size of the nanoparticles prepared in Example 8 was 2671 nm, with a PDI of 1. In summary, the results show that the nanomedicine exhibits optimal stability when the molar ratio of cordycepin to baicalin is 1:0.5 and the self-assembly reaction temperature is 25℃ (for the nanoparticles prepared in Example 1).

[0070] 2. Zeta potential detection: The zeta potential of the dispersion (0.1 mg / mL) prepared from the nanomedicine obtained in Example 1 was detected at 25°C using a potentiometer. The results are as follows: Figure 2As shown, the Zeta potential of the nanoparticles in Example 1 is in the range of -30±5mV, indicating that the nanoparticles have excellent stability in solution and are not prone to aggregation.

[0071] The Yender effect experiment was conducted using the dispersion (0.1 mg / mL) of the nanomedicine obtained in Example 1, and the results are as follows: Figure 3 As shown, a clear and stable Tyndall optical pathway can be observed in the COR-BA NPs dispersion, indicating that the dispersion system is a colloid and the nanoparticles form a dispersion system with colloidal properties through self-assembly.

[0072] The COR-BA NPs prepared in Example 1 were characterized by transmission electron microscopy, and the results are as follows: Figure 4 As shown, COR-BA NPs are spherical with regular morphology and an average particle size of about 160 nm. The particle size distribution is relatively uniform, similar to the results of DLS experiments. There is no obvious aggregation between particles, and the dispersion is good.

[0073] 3. Ultraviolet spectroscopy characterization: Free cordycepin, free baicalin and the nanoparticles prepared in Example 1 were dissolved in deionized water to prepare solutions with a concentration of 0.05 mg / mL, and the solutions were scanned in the wavelength range of 200-500 nm using an ultraviolet spectrophotometer.

[0074] The results are as follows Figure 5 As shown, the ultraviolet characteristic absorption peaks (λmax=265nm, 322nm) of the nanoparticles simultaneously contain the characteristic peaks of free cordycepin (λmax=260nm) and free baicalin (λmax=276nm, 325nm), and the peak shapes show no obvious shift, proving that cordycepin and baicalin successfully form self-assembled nanoparticles through non-covalent interactions.

[0075] 4. Infrared spectroscopy characterization: 2 mg of free cordycepin, free baicalin, and the nanoparticles prepared in Example 1 were weighed out, and 200 mg of potassium bromide powder dried at 105℃ for 2 h was added to each. After grinding and mixing, the mixture was pressed into thin sheets and the samples were analyzed using an infrared spectrometer at 400-4000 cm⁻¹. -1 Scan within range, resolution 4cm -1 64 scans.

[0076] The results are as follows Figure 6 As shown: Cordycepin (COR) contains hydroxyl and amino groups, and its concentration is between 3200-3600 cm⁻¹. -1 Multiple peaks are formed by stretching vibrations. Baicalin (BA) 3200-3500 cm⁻¹ -1The OH stretching vibration forms sharp or multiple peaks. After assembly into NPs, a broad peak is formed, indicating that the phenolic groups formed intermolecular associations during self-assembly. The COR in NPs is 1050-1150 cm⁻¹. -1 (Glycosidic bond) Multiple peaks disappear, BA 1200-1300 cm⁻¹ -1 The broadening of the (ether bond / phenolic hydroxyl group bending) may be due to the formation of a hydrogen bond network. The glycosylated hydroxyl group participates in the hydrogen bonding between BA molecules, which restricts the vibrational freedom of the glycosidic bond, forming a spatial shield or embedding. The sugar ring is wrapped by the BA aromatic ring, which makes the characteristic peaks undetectable, further confirming the formation of self-assembly.

[0077] 5. X-ray diffraction (XRD) characterization: 10 mg each of the nanoparticle powder, free cordycepin powder, and free baicalin powder prepared in Example 1 were taken and detected by X-ray diffractometer. The test conditions were: Cu target Kα rays (λ=0.15406nm), tube voltage 40 kV, tube current 30 mA, scanning range 2θ=5°-60°, scanning speed 5° / min, and step size 0.02°.

[0078] The results are as follows Figure 7 As shown, COR and BA exhibit strong and sharp diffraction peaks at 10-25°, revealing their crystal structure. The four highest intensity peaks at 13.22°, 15.51°, 23.49°, and 26.26° reveal a series of obvious crystal diffraction peaks. In contrast, in the XRD pattern of the nanoparticles prepared in Example 1, the aforementioned sharp characteristic peaks are significantly broadened and their intensity is greatly reduced, with only one broadened diffuse peak appearing at 2θ=20°-30°. This indicates that the crystal structure of cordycepin and baicalein is destroyed during self-assembly, forming amorphous or low-crystallinity nanoassemblies. This further corroborates that the two are not simply physically mixed, but rather form a new aggregated structure through intermolecular interactions.

[0079] 6. Stability Test: The nanoparticles prepared in Example 1 were prepared into a dispersion (0.1 mg / mL) and stored at 4°C for 7 days. Samples were taken daily to detect changes in particle size. The results are as follows: Figure 8 As shown, the particle size change rate after 7 days of storage at 4℃ is <5%; in addition, the nanoparticles prepared in Example 1 were stored at 25℃ and 37℃ for 7 days respectively. The particle size change rate of the nanoparticles in the dispersion was <8% at 25℃ and <12% at 37℃, indicating that the nanoparticles have good stability under low temperature and room temperature conditions and can meet the requirements of conventional storage and transportation.

[0080] Example 10

[0081] Solubility experiment of cordycepin, baicalin and COR-BA NPs prepared in Example 1

[0082] The effect of self-assembly on improving drug water solubility was evaluated using the equilibrium solubility method and dissolution kinetics experiments. The COR-BA NPs prepared in Example 1 were used as the model, and compared with free COR and BA. The specific experimental steps are as follows:

[0083] Preparation and detection of saturated solutions: Excess COR, BA, and COR-BA NPs powder (approximately 50 mg) were added to each, and 3 mL of ultrapure water was added. The solutions were shaken at 37℃ for 72 h to reach equilibrium. After centrifugation at 12000 r / min for 10 min at 4℃, the supernatant was filtered through a 0.22 μm filter membrane, and the concentration was determined by HPLC. Detection conditions: C18 column; COR mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (10:90, v / v); detection wavelength: 256 nm; BA mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (60:40, v / v); detection wavelength: 275 nm; COR-BA NPs mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (60:40-10:90 gradient elution, v / v); detection wavelengths: dual wavelengths of 256 nm and 275 nm; flow rate: 1.0 mL / min; column temperature: 30℃.

[0084] Equilibrium solubility results as follows Figure 9 As shown: COR is 12.56±0.82 mg / mL, while BA is only 0.042±0.005 mg / mL; the solubility of COR in COR-BA NPs is 15.32±1.05 mg / mL (an increase of about 22%), and BA reaches 2.86±0.18 mg / mL (an increase of about 68 times). The COR-BA NPs prepared in this invention can significantly improve the water solubility of both cordycepin and baicalin.

[0085] Example 11

[0086] Antitumor activity experiment of cordycepin-baicalin self-assembled nanoparticles

[0087] HepG2 liver cancer cells, 4T1 breast cancer cells, and HT-29 colon cancer cells were selected as test cells. The MTT assay was used to detect the inhibitory effect of the nanoparticles prepared in Example 1 on tumor cell proliferation. Free cordycepin, free baicalin, and a physical mixture (with the same mixing ratio as in Example 1, obtained by direct mixing) were used as controls. The specific steps are as follows:

[0088] Cell culture: HepG2 cells, 4T1 cells, and HT-29 cells were seeded in DMEM medium containing 10% fetal bovine serum and cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase. The cells were then digested with 0.25% trypsin to adjust the cell concentration to 1×10⁻⁶ cells / year. 5 per mL.

[0089] Drug treatment: 100 μL of cell suspension was added to each well of a 96-well plate and cultured for 24 h. The medium was then replaced with serum-free medium containing a series of concentrations (5-40 μM or 5-20 μM) of nanoparticles, free drug, and a physical mixture. Each group was divided into three replicates, with the drug-free medium serving as a blank control. After 48 h of further culture, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation for 4 h, the supernatant was discarded, and 150 μL of DMSO was added. The mixture was shaken for 10 min to dissolve the crystals. The OD value was measured at 490 nm using a microplate reader, and the cell viability and half-maximal inhibitory concentration (IC50) were calculated. 50 ).

[0090] The results show: The results are as follows Figure 10-12 As shown, the nanoparticles exhibit IC50 values ​​for HepG2 cells, 4T1 cells, and HT-29 cells. 50 The concentrations were 12.6 μM, 10 μM, and 7.4 μM, respectively, compared to free cordycepin (IC50). 50 100-200 μM), free baicalein (IC50) 50 The concentration of 50-100 μM was reduced by 4-16 times, compared to the physical mixture group (IC). 50 The levels of 15-30 μM decreased by 1.24-2.29 times, indicating that it has a good effect on inhibiting the proliferation of tumor cells.

[0091] Example 12

[0092] Anti-inflammatory activity experiment of cordycepin-baicalin self-assembled nanoparticles

[0093] A lipopolysaccharide (LPS)-induced inflammation model of mouse peritoneal macrophages (RAW264.7) was established. The effect of the nanoparticles prepared in Example 1 on the expression of inflammatory factors was detected, with free cordycepin and free baicalin as controls. The specific steps are as follows:

[0094] 1. Establishment of the inflammation model: RAW264.7 cells were seeded in 6-well plates and cultured to 80% confluence. Then, they were sputtered at a rate of 2 × 10⁻⁶ cells / well. 5 LPS solution (final concentration 1 μg / mL) was added to cells / wells and cultured at 37℃ and 5% CO2 for 6 h to induce an inflammatory response in the cells;

[0095] 2. Drug intervention: Nanoparticle solution with a final concentration of 10 μM and free drug solution were added to the inflammatory model cells, respectively, and the cells were cultured for 12 h. Cells with only LPS were used as the model group, and normal cells were added with PBS as the blank group.

[0096] 3. Detection of inflammatory factors: Cell culture supernatant was collected and the contents of TNF-α (YFXEM0031), IL-6 (YFXEM00045), and IL-1β (YFXEM00028) were detected using an ELISA kit. Each sample was repeated 3 times.

[0097] The results are as follows Figure 13-15 As shown, the concentrations of TNF-α, IL-6, and IL-1β in the nanoparticle-treated group were 85 pg / mL, 62 pg / mL, and 45 pg / mL, respectively, which were significantly lower than those in the model group (TNF-α 320 pg / mL, IL-6 250 pg / mL, IL-1β 180 pg / mL) and lower than those in the free drug group (TNF-α 150-200 pg / mL, IL-6 120-160 pg / mL, IL-1β 90-120 pg / mL). This indicates that nanoparticles can effectively inhibit the expression of inflammatory factors and exert an anti-inflammatory effect.

[0098] Example 13

[0099] Experimental study on the inhibitory effect of cordycepin-baicalin self-assembled nanoparticles on normal human umbilical vein endothelial cells (HUVECs).

[0100] To clarify the safety of cordycepin-baicalin self-assembled nanoparticles on normal cells, human umbilical vein endothelial cells (HUVECs) were used as the research subject. The inhibitory effect of the nanoparticles prepared in Example 1 on normal cells was evaluated through a cell proliferation inhibition experiment. Meanwhile, free cordycepin, free baicalin, and a physical mixture (with the same mixing ratio as in Example 1, obtained by direct mixing) served as controls. The specific experimental steps and results are as follows:

[0101] 1. Cell Culture and Pretreatment: Frozen HUVEC cells were rapidly thawed in a 37°C water bath. After centrifugation to remove the cryopreservation solution, they were seeded into DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged using 0.25% trypsin. Cells in the logarithmic growth phase were selected for subsequent experiments, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4 Units / mL are available for use.

[0102] 2. Drug preparation: Weigh out the nanoparticles, free cordycepin, free baicalin and physical mixture from Example 1 respectively, and prepare a stock solution with a concentration of 200 μg / mL using DMEM / F12 medium without fetal bovine serum. After sterilization by filtration through a 0.22 μm filter membrane, the stock solution is serially diluted to a series of concentrations of 10 μM, 5 μM and 2.5 μM for later use.

[0103] 3. Cell proliferation inhibition experiment:

[0104] (1) Add 100 μL of HUVEC cell suspension (5×10³ cells / well) to each well of a 96-well plate and culture for 24 h to allow the cells to adhere.

[0105] (2) Discard the original culture medium and add 100 μL of drug solution of different concentrations respectively. Each group has 5 replicates. Use DMEM / F12 culture medium without drugs as blank control group. Each group has 5 replicates.

[0106] (3) After culturing in an incubator for 48 h, add 10 μL of CCK-8 reagent to each well and incubate in the dark for 2 h.

[0107] (4) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm, and calculate the cell viability using the formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0108] The results are as follows Figure 16 As shown, when the drug concentration was 10 μM, the cell survival rate of the nanoparticle-treated group prepared in Example 1 was 83.1% ± 3.8%, the free cordycepin group was 80.4% ± 4.7%, the free baicalin group was 67.7% ± 3.5%, and the physical mixture group was 55.6% ± 2.2%. When the concentration was reduced to 5 μM, the cell survival rate of the nanoparticle group increased to 94.2% ± 2.4%, the free drug group was 76.6%-88.3%, and the physical mixture group was 76%.

[0109] Statistical analysis showed that within the concentration range of 2.5-10 μM, the nanoparticles inhibited the proliferation of HUVEC cells by less than 20%, and there was no statistically significant difference compared with free cordycepin and free baicalin (P>0.05), proving that the nanoparticles prepared in this invention have a weak inhibitory effect on the proliferation of normal HUVEC cells.

[0110] In summary, the cordycepin-baicalin self-assembled nanoparticles exhibited weak inhibitory effects on the proliferation of normal human umbilical vein endothelial cells (HUVECs) within the effective concentration range for antitumor activity, demonstrating their good cellular safety and providing important safety data support for subsequent experimental applications.

[0111] This invention provides a concept and method for the preparation and application of cordycepin-baicalin self-assembled nanoparticles. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing cordycepin-baicalin self-assembled nanoparticles, characterized in that, Includes the following steps: (1) Dissolve cordycepin in the first organic solvent to obtain the first solution; (2) Dissolve baicalein in a mixture of a second organic solvent and water, and sonicate to obtain a second solution; (3) Mix the first solution and the second solution and stir until homogeneous to obtain a mixed solution; (4) The mixed solution is freeze-dried for the first time to obtain cordycepin-baicalin crude powder; (5) The cordycepin-baicalin crude powder is dispersed in water and dialyzed, and then freeze-dried a second time to obtain the product.

2. The preparation method according to claim 1, characterized in that, In step (1), the first organic solvent is methanol; and / or the first solution has a concentration of 0.8-3 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step (2), the amount of baicalein added is calculated based on a molar ratio of baicalein to cordycepin of 1:0.2~4; and / or, the second organic solvent is ethanol; and / or, the volume ratio of the second organic solvent to water is 1:1-3; and / or, the ultrasound is performed at a temperature of 20-50℃, a power of 200-400W, a frequency of 30-50kHz, and a duration of 10-15 min; and / or, the second solution has a concentration of 0.8-2.5 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (3), the mixing is to add the first solution dropwise to the second solution at a flow rate of 800-1200 rpm without large particles rapidly precipitating; and / or the stirring is carried out at a temperature of 20-80℃, a rotation speed of 500-900 r / min, and a time of 1.5-3 h.

5. The preparation method according to claim 1, characterized in that, In steps (4) and (5), the first freeze-drying and the second freeze-drying are carried out at a temperature of -50 ~ -60℃ for 12 ~ 24h and a vacuum degree of less than 10Pa.

6. The preparation method according to claim 1, characterized in that, In step (5), the mass ratio of the cordycepin-baicalin crude powder to water is 1:2-5; and / or, the dispersion is performed by ultrasonic dispersion with a power of 150-300W, a frequency of 30-50kHz, and a time of 10-20 min; and / or, the dialysis is performed with a dialysis bag having a molecular weight cutoff of 3500-5000Da, a time of 6-10 h, and a temperature of room temperature.

7. Cordycepin-baicalin self-assembled nanoparticles prepared by the preparation method according to any one of claims 1-6.

8. The cordycepin-baicalin self-assembled nanoparticles according to claim 7, characterized in that, The cordycepin-baicalin self-assembled nanoparticles have a particle size of 100-3000 nm, a PDI value ≤1, and a Zeta potential of -30±5 mV.

9. The application of the cordycepin-baicalin self-assembled nanoparticles according to claim 7 in the preparation of antitumor drugs, preferably, the tumor includes any one or more of liver cancer HepG2, breast cancer 4T1 and colon cancer HT-29.

10. The application of the cordycepin-baicalin self-assembled nanoparticles according to claim 7 in the preparation of anti-inflammatory drugs, preferably, the anti-inflammatory drugs alleviate acute inflammatory responses by reducing the expression levels of inflammatory factors TNF-α, IL-6 and IL-1β in inflamed tissues.

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