Preparation method and application of hyaluronic acid-naringenin self-assembled nanoparticles

Abstract

The invention discloses a preparation method of anti-tumor hyaluronic acid-naringenin self-assembled nanoparticles, and belongs to the field of medicines. According to the method, carboxyl on hyaluronic acid and hydroxyl on naringenin are combined to form an ester group, and the hyaluronic acid-naringenin polymer is obtained. Hydrophilic hyaluronic acid is combined with hydrophobic naringenin to form a hydrophilic and hydrophobic polymer, then under the action of ultrasonic waves in an aqueous solution, the hydrophobic end repels water molecules and is mutually gathered, and the hydrophilic end wraps the periphery of the hydrophobic end and is in contact with water, so that the structure that the core is hydrophobic naringenin and the core is hydrophobic naringenin is formed. The outer core is a nanoparticle of hydrophilic hyaluronic acid. The particle size of the nanoparticles is 140-200nm, the nanoparticles are stable and uniform, in-vitro release experiment results show that the nanoparticles can be slowly released, and cell experiments show that the preparation can obviously improve the toxicity of naringenin to tumor cells, which shows that the nanoparticles have good anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a method for preparing anti-tumor hyaluronic acid-naringenin self-assembled nanoparticles. Background Art

[0002] Naringenin (NAR) is a polyphenolic flavonoid compound extracted from natural plants such as citrus fruits, with diverse pharmacological activities. Numerous studies have shown that naringenin exhibits significant inhibitory potential against various cancers, including prostate cancer, liver cancer, and colon cancer. However, naringenin suffers from poor solubility and weak hydrophilicity. It is easily degraded in the gastrointestinal environment and undergoes significant first-pass metabolism, which greatly limits its application in clinical treatment. To improve the bioavailability of naringenin, current research focuses on developing new formulations of naringenin using nanotechnology, such as nanoliposomes, solid lipid nanoparticles, nanogels, and self-assembling polymer-drug complexes. In particular, the construction of natural polymer-naringenin conjugates with self-assembling properties has become a highly anticipated research direction in this field.

[0003] Hyaluronic acid (HA) is a naturally occurring high molecular weight acidic mucopolysaccharide and one of the main components of the extracellular matrix. An important way for HA to act on cells is by binding to HA receptors on the cell surface. Currently, a variety of receptors that exist on the cell surface and can bind to and act on HA have been discovered (Tammi R, Rilla K, Pienimäki JP, et al. Hyaluronan enters keratinocytes by a new endocytic route for catabolism [J]. Journal of Biological Chemistry, 2001, 276 (37): 3511135122.). Hyaluronic acid of different relative molecular weights interacts differently with the same hyaluronic acid receptor, and hyaluronic acid of the same relative molecular weight also produces different effects after interacting with different hyaluronic acid receptors (Cirillo N. The hyaluronan / CD44 axis: a double-edged sword in cancer [J]. International Journal of Molecular Sciences, 2023, 24(21): 15812.). Therefore, hyaluronic acid can play a role in targeting tumor cells.

[0004] Nanoparticles are excellent drug and gene delivery vehicles. When the particle diameter is between 50 and 200 nm and they carry specific surface groups, they can avoid breakdown and clearance by the liver, kidneys, or spleen, circulating in the body for extended periods. They can also evade recognition by the immune system, preventing immune responses. However, many monodisperse nanoparticle systems require demanding preparation conditions and complex methods, and sometimes require the addition of chemically or biologically toxic compounds or active substances to maintain nanoparticle stability and activity.

[0005] By combining natural polymers with hydrophobic compounds, the hydrophilic molecules are converted to amphiphilic ones, forming nanoparticles that also possess a certain degree of targeting. When used as drug and gene carriers, these nanoparticles can be delivered to cells and tissues, where they can reside and accumulate at the targeted sites, releasing the drug or promoting the immune system to repair lesions in the targeted areas. Therefore, the preparation of polysaccharide nanoparticles is a promising research direction for insoluble drug carriers. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the prior art, and based on the aforementioned hydrophilic properties and application characteristics of hyaluronic acid, the primary objective of the present invention is to provide a method for preparing self-assembled hydrophobic nanoparticles of hyaluronic acid-naringenin. This method involves the formation of ester groups by combining carboxyl groups on hyaluronic acid with hydroxyl groups on naringenin, which are then grafted onto the hyaluronic acid polysaccharide. The reaction is simple and the operation is convenient. The resulting nanoparticles have a particle size of 140-200 nm and are stable and uniform.

[0007] Another object of the present invention is to provide hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles prepared by the above method.

[0008] Another object of the present invention is to provide a use of the hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles in the preparation of anti-tumor drugs.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] A method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles mainly comprises the following steps:

[0011] (1) dissolving hyaluronic acid in solvent M to obtain solution A; dissolving naringenin in solvent N to form solution B;

[0012] (2) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is added to solution A in step (1), and then N-hydroxysuccinimide (NHS) is added. Solution B is then added to the mixed solution to react. After the reaction is completed, the resulting reaction solution is ultrasonicated in an ice bath, dialyzed, and freeze-dried to obtain hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles.

[0013] The hyaluronic acid described in step (1) is preferably hyaluronic acid with a molecular weight of 4k;

[0014] The solvents M and N described in step (1) are independently one of water and anhydrous ethanol;

[0015] The concentration of hyaluronic acid in solution A described in step (1) is 0.1-1 g / L, preferably 0.75 g / L;

[0016] The concentration of naringenin in solution B described in step (1) is 0.01-0.1 g / L, preferably 0.075 g / L;

[0017] The amounts of solution A and solution B described in step (2) satisfy the volume ratio of solution A to solution B of 10:1;

[0018] The reaction in step (2) is carried out at a stirring speed of 3000 to 8000 r / min at 40 to 70° C. for 2 to 6 hours; preferably at 6000 r / min and 60° C. for 4 hours;

[0019] The water dialysis described in step (2) refers to adding 1 to 10 L of water for every 1 g of solid, and the dialysis refers to dialysis using a 3.5 kDa cellulose dialysis bag, changing the water every 8 hours, and dialysis for 16 hours;

[0020] The ultrasonic treatment in step (2) refers to ultrasonic treatment at a power of 50 to 200 W for 5 to 15 seconds, preferably ultrasonic treatment in an ice bath at a power of 100 W for 10 seconds;

[0021] The invention relates to hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles prepared by the method.

[0022] Application of the above-mentioned hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles in the preparation of anti-tumor drugs.

[0023] The mechanism of the present invention is as follows: hydrophilic hyaluronic acid combines with hydrophobic naringenin to form a hydrophilic-hydrophobic polymer. Then, under the action of ultrasound in an aqueous solution, the hydrophobic ends repel water molecules and aggregate with each other, while the hydrophilic ends are wrapped around the hydrophobic ends and come into contact with water, thus forming nanoparticles with a hydrophobic core of naringenin and an outer core of hydrophilic hyaluronic acid.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) The hyaluronic acid-naringenin nanoparticles obtained by the method of the present invention have a particle size of 140-200 μm, and the particles are stable and uniform.

[0026] (2) The hyaluronic acid-naringenin nanoparticles obtained by the method of the present invention can be slowly released, effectively reducing toxicity in the body.

[0027] (3) The hyaluronic acid-naringenin nanoparticles obtained by the method of the present invention can significantly increase the toxicity of naringenin to tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The average particle size of HA-NAR micelle preparation.

[0029] Figure 2 These are the infrared absorption spectra of hyaluronic acid, naringenin, and HA-NAR micelle preparation.

[0030] Figure 3 Figure 2 shows the release behavior of the formulation and API at 37°C using phosphate buffered saline (PBS) containing 1% Tween-80 as the release medium. The two curves represent the release behavior of naringenin and HA-NAR formulations in PBS at pH 7.4.

[0031] Figure 4 These are the results of toxicity experiments on RM-1 cells using naringenin, HA-NAR micelle preparation, and naringenin.

[0032] Figure 5 These are the results of the toxicity experiments of naringenin, HA-NAR micelle preparation and naringenin on 4T1 cells. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0034] Example 1: Preparation of Hyaluronic Acid-Naringenin Nanoparticles

[0035] Dissolve 30 mg of hyaluronic acid (molecular weight 4k) in 40 ml of water to form a hyaluronic acid aqueous solution, then stir at room temperature for 3 minutes. Add 8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the solution and stir at room temperature for 3 minutes. Then, add 8 mg of N-hydroxysuccinimide (NHS) to the solution and stir at room temperature for 20 minutes. Then, slowly add 3 mg of naringenin in 5 ml of anhydrous ethanol to form an ethanolic solution of naringenin. Stir at room temperature for 10 minutes, cover, transfer to a 60°C water bath, and heat at approximately 600 rpm for 4 hours. After completion of the reaction, sonicate in an ice bath at 100 W for 10 seconds, dialyze using a 3.5 kDA cellulose dialysis bag for 16 hours, changing the water every 8 hours, and freeze-dry. Store at low temperatures.

[0036] 10 mg of hyaluronic acid-naringenin prepared in Example 1 was added to 10 mL of distilled water to prepare an aqueous solution of hyaluronic acid-naringenin nanoparticles with a particle size of 140-150 nm.

[0037] Take an appropriate amount of the micelle preparation prepared above and measure its particle size distribution by dynamic light scattering. The interfacial potential of the liposome sample is measured by interfacial potential analyzer. The average particle size is 142.3 nm ( Figure 1 ), the interfacial potential was -20.3 mV. The drug loading was measured by UV spectrophotometry to be 5.4% (w / w).

[0038] Example 2: Preparation of Hyaluronic Acid-Naringenin Nanoparticles

[0039] Dissolve 24 mg of hyaluronic acid (molecular weight 4k) in 40 ml of water to form a hyaluronic acid aqueous solution, then stir at room temperature for 3 minutes. Add 7.5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the solution, stirring at room temperature for 3 minutes. Then, add 5 mg of N-hydroxysuccinimide (NHS) to the solution, stirring at room temperature for 20 minutes. Then, slowly add 2 mg of naringenin to the solution in 5 ml of anhydrous ethanol to form an ethanolic solution of naringenin. Stir at room temperature for 10 minutes, cover, transfer to a 60°C water bath, and heat at approximately 600 rpm for 4 hours. After completion of the reaction, sonicate in an ice bath at 100 W for 10 seconds, dialyze using a 3.5 kDa cellulose dialysis bag for 16 hours, changing the water every 8 hours, and freeze-dry. Store at low temperatures.

[0040] The particle size distribution of the micelle preparation prepared in Example 2 was measured by dynamic light scattering, and the average particle size was 172.8 nm at room temperature.

[0041] Example 3: Preparation of Hyaluronic Acid-Naringenin Nanoparticles

[0042] Dissolve 26 mg of hyaluronic acid (molecular weight 4k) in 40 ml of water to form a hyaluronic acid aqueous solution, then stir at room temperature for 3 min. Add 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to the solution and stir at room temperature for 3 min. Then, add 7.5 mg of N-hydroxysuccinimide (NHS) to the solution and stir at room temperature for 20 min. Then, slowly add 2 mg of naringenin in 5 ml of anhydrous ethanol to form an ethanolic solution of naringenin. Stir at room temperature for 10 min, cover, transfer to a 60°C water bath, and heat at approximately 600 rpm for 4 h. After completion of the reaction, sonicate in an ice bath at 100 W for 10 s, dialyze using a 3.5 kDA cellulose dialysis bag for 16 h, changing the water every 8 h, and freeze-dry. Store at low temperatures.

[0043] The particle size distribution of the micelle preparation prepared in Example 3 was measured by dynamic light scattering, and the average particle size was 194.2 nm at room temperature.

[0044] Test Example 1:

[0045] Hyaluronic acid-naringenin self-assembled nanoparticles were prepared according to Example 1. Sample tablets were prepared using the potassium bromide tableting method. Infrared scanning was performed on hyaluronic acid HA, naringenin NAR, and hyaluronic acid-naringenin (HA-NAR) nano freeze-dried powder, and their respective infrared absorption spectra were recorded.

[0046] Results: As Figure 2 As shown, HA-NAR nanoparticles have a peak at 1654 cm -1 The infrared absorption peak (-C=O stretching vibration peak in carboxyl group) is weakened, and the 1319cm -1 The addition of an infrared absorption peak (-C=O stretching vibration peak in the ester bond) indicated that the polymer HA-NAR was successfully synthesized.

[0047] Test Example 2:

[0048] Hyaluronic acid-naringenin self-assembled nanoparticles were prepared according to Example 1. 1 mL of HA-NAR micelle solution and 1 mL of NAR physiological saline solution were placed in dialysis bags, bubbles were removed, the bags were sealed, and the bags were placed in 30 mL of PBS release medium containing 1% Tween 80, pH = 7.4. The bags were shaken in a water bath at 37°C (100 rpm). -1 At the pre-set time points, 1 mL of sample was taken and 1 mL of release medium was added simultaneously. The drug concentration in the release medium was determined by HPLC, and the cumulative release rate, Q, was calculated.

[0049] Results: As Figure 3 As shown, in the PBS release medium at pH 7.4, HA-NAR micelles exhibited significant sustained-release properties compared to naringenin in saline. The cumulative drug release from the polymer micelles over 72 hours was only approximately 60%. These results indicate that the micelles significantly sustained the release of NAR, thereby reducing toxicity to normal cells. This sustained-release effect is attributed to the rigid hydrophobic core of the drug-loaded micelles, which inhibits drug diffusion.

[0050] Test Example 3:

[0051] Hyaluronic acid-naringenin self-assembled nanoparticles were prepared according to Example 1. RM-1 and 4T1 cells in the logarithmic growth phase were taken and 1×10 6Cells were seeded into 96-well plates at a density of 100 cells / well and incubated with culture medium at 37°C for 24 hours. After cell attachment, different concentrations of HA-NAR micelle preparations or naringenin solution were added, with six replicates per well. After 72 hours of incubation, 20 μL of MTT solution was added to each well and incubated for another 4 hours. Finally, the medium containing MTT was aspirated and discarded, and 100 μL of DMSO was added dropwise to fully dissolve the formazan crystals. The cells were then analyzed using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 490 nm.

[0052] Results: As Figure 4 、 5 As shown in the figure, the toxicity of HA-NAR micelles to RM-1 cells and 4T1 cells was significantly enhanced compared to that of naringenin API. Comparing the degree of toxicity enhancement of the formulation in these two cell types, it can be found that HA-NAR micelles have good selectivity and cytotoxicity for tumor cells with high expression of hyaluronic acid receptors.

[0053] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing hyaluronic acid-naringenin self-assembled nanoparticles, characterized in that The following steps are involved: 1) Dissolve hyaluronic acid in solvent M to obtain solution A; dissolve naringenin in solvent N to form solution B. 2) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added to solution A in step 1), followed by N-hydroxysuccinimide (NHS), and solution B was added to the mixed solution for reaction. After the reaction, the resulting reaction solution was sonicated in an ice bath, dialyzed, and freeze-dried to obtain hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles.

2. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, characterized in that: The hyaluronic acid described in step 1) is hyaluronic acid with a molecular weight of 4k; The solvents M and N in step 1) are independently selected from water and anhydrous ethanol.

3. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, characterized in that: The concentration of hyaluronic acid in solution A described in step 1) is 0.1-1 g / L; The concentration of naringenin in the solution B in step 1) is 0.01-0.1 g / L.

4. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, wherein: The concentration of hyaluronic acid in solution A described in step 1) is 0.75 g / L; The concentration of naringenin in the solution B described in step 1) is 0.075 g / L.

5. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, characterized in that: The amounts of solution A and solution B described in step 2) are such that the volume ratio of solution A to solution B is 10:

1.

6. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, wherein: The reaction in step 2) is carried out at a stirring speed of 3000 to 8000 r / min and a temperature of 40 to 70° C. for 2 to 6 hours.

7. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, characterized in that: The water-addition dialysis in step 2) refers to adding 1 to 10 L of water per 1 g of solid, and the dialysis refers to dialysis using a 3.5 kDa cellulose dialysis bag.

8. The method for preparing hyaluronic acid-naringenin self-assembled hydrophobic nanoparticles according to claim 1, characterized in that: The ultrasonic treatment in step 2) refers to ultrasonic treatment at a power of 50 to 200 W for 5 to 15 seconds, followed by rapid cooling for 5 to 15 seconds.

9. Hyaluronic acid-naringenin self-assembled nanoparticles prepared by the method according to any one of claims 1 to 8.

10. Use of the hyaluronic acid-naringenin self-assembled nanoparticles according to claim 9 in the preparation of anti-tumor drugs.

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