Hyaluronic acid hydrophobic material, hyaluronic acid hydrophobic self-assembly nano composite material and preparation method of hyaluronic acid hydrophobic self-assembly nano composite material

Abstract

The invention discloses a hyaluronic acid hydrophobic material, a hyaluronic acid hydrophobic self-assembly nano composite material and a preparation method thereof, and belongs to the technical field of drug carrier preparation. The preparation method of the hyaluronic acid hydrophobic material provided by the invention comprises the following steps: taking N, N-dimethylformamide as a solvent, adding C15-C25 long-chain fatty acid, N, N '-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and adjusting the pH value to 9-10 to obtain an organic phase solution; the preparation method comprises the following steps: by taking water as a solvent, adding hyaluronic acid and a Tween surfactant, and uniformly stirring to obtain a water-phase solution; and mixing the aqueous phase solution and the organic phase solution, carrying out esterification reaction on hydroxyl of hyaluronic acid in the aqueous phase solution and carboxyl in the hydrophobic carboxylic acid compound, and obtaining the hyaluronic acid hydrophobic material by utilizing an alcohol precipitation method after the reaction is finished.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carrier preparation, and more specifically to a hyaluronic acid hydrophobic material, a hyaluronic acid hydrophobic self-assembled nanocomposite material, and a preparation method thereof. Background Art

[0002] Neurons are the most basic structural and functional units of the nervous system, used to receive stimuli, generate excitement and conduct excitement. Neurons are one of the cells in the human body that are most vulnerable to oxidative stress. Oxidative stress refers to the excessive production of reactive oxygen species (ROS) in the intracellular and extracellular environments, leading to an oxidative stress response. When ROS accumulates excessively, it will cause damage to the function of nerve cells and increase the risk of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0003] Flavonoid glycoside natural products have attracted extensive attention in neuroprotection. For example, Polygonum cuspidatum, as a traditional Chinese medicine, its extract polydatin has been found to have a variety of biological activities, and can reduce the damage of oxidative stress to neurons by scavenging free radicals, inhibiting ROS generation, enhancing the activity of antioxidant enzymes, etc. In addition, polydatin can also protect nerve cells from inflammation-mediated damage by regulating the expression of inflammatory factors and inhibiting neuroinflammatory responses.

[0004] In addition to polydatin, icariin is also a widely used natural compound. Icariin is the main active ingredient of Epimedium brevicornu, belonging to 8-isopentenyl flavonoid glycoside compounds. It can be extracted from the dried stems and leaves of Epimedium sagittatum, Epimedium pubescens, Epimedium wushanense, Epimedium koreanum, etc. Research shows that icariin can enhance the antioxidant defense ability of cells and reduce the accumulation of ROS by activating the Nrf2 / ARE signaling pathway. In addition, icariin can also protect nerve cells from double damage of oxidative stress and inflammation by inhibiting the NF-κB signaling pathway and reducing the inflammatory response.

[0005] Polydatin and icariin have the potential for antioxidant, anti-inflammatory and neuroprotection. However, poor water solubility and fast metabolism in vivo are the main problems restricting the clinical application of flavonoid glycoside natural products such as polydatin and icariin. These problems can be solved by formulating them into nano-formulations. However, surfactants are added in the film hydration step in traditional nano-formulations, resulting in certain cytotoxicity of the nanoparticles. Therefore, it is necessary to develop a low-toxic nano-formulation for the drug delivery of polydatin and icariin. Summary of the Invention

[0006] In view of the above problems, the present invention provides a hyaluronic acid hydrophobic material, a hyaluronic acid hydrophobic self-assembled nanocomposite material, and a preparation method thereof. The present invention utilizes C 15 ~C 25The long-chain fatty acid is used to modify hyaluronic acid, and the prepared hyaluronic acid hydrophobic material can be used for drug loading.

[0007] The first object of the present invention is to provide a preparation method of a hyaluronic acid hydrophobic material, comprising the following steps: Using N,N-dimethylformamide as a solvent, adding C 15 ~C 25 long-chain fatty acid, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, adjusting the pH to 9-10 to obtain an organic phase solution; for example, the pH is 9, 9.2, 9.4, 9.6, 9.8, 10, etc., but not limited to the listed values, and other unlisted values within the above value range are also applicable.

[0008] Using water as a solvent, adding hyaluronic acid and a Tween surfactant, and stirring evenly to obtain an aqueous phase solution.

[0009] Mixing the aqueous phase solution and the organic phase solution, and the hydroxyl group of hyaluronic acid in the aqueous phase solution reacts with the carboxyl group of the hydrophobic carboxylic acid compound to obtain a hyaluronic acid hydrophobic material.

[0010] In a preferred embodiment of the present invention, the reaction temperature of the esterification reaction is 30°C to 35°C, for example, the reaction temperature is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc.

[0011] The reaction time is 40h to 48h, for example, the reaction time is 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, etc., but not limited to the listed values, and other unlisted values within the above value range are also applicable.

[0012] In a preferred embodiment of the present invention, C 15 ~C 25 The long-chain fatty acid is stearic acid, all-trans retinoic acid or docosahexaenoic acid.

[0013] In a preferred embodiment of the present invention, the molar ratio of hyaluronic acid to C 15 ~C 25 long-chain fatty acid is 1:125 to 130; for example, the molar ratio of hyaluronic acid to C 15 ~C 25 long-chain fatty acid is 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, etc.

[0014] The molar ratio of hyaluronic acid to N,N'-dicyclohexylcarbodiimide is 1:125 to 130. For example, the molar ratio of hyaluronic acid to N,N'-dicyclohexylcarbodiimide is 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, etc.

[0015] The molar ratio of hyaluronic acid to 4-dimethylaminopyridine is 1:625 to 750; for example, the molar ratio of hyaluronic acid to 4-dimethylaminopyridine is 1:625, 1:650, 1:675, 1:700, 1:725, 1:750, etc.

[0016] The mass ratio of hyaluronic acid to Tween surfactant is 1:4 to 4.5. For example, the mass ratio of hyaluronic acid to Tween surfactant is 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, etc., but is not limited to the listed values, and other unlisted values within the above numerical range are equally applicable.

[0017] In a preferred embodiment of the present invention, the mass ratio of N,N'-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 16 to 17:1. For example, the mass ratio of N,N'-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 16:1, 16.2:1, 16.4:1, 16.6:1, 16.7:1, 16.8:1, 17:1, etc., but is not limited to the listed values, and other unlisted values within the above numerical range are equally applicable.

[0018] The second object of the present invention is to provide a hyaluronic acid hydrophobic material prepared by the above preparation method.

[0019] The third object of the present invention is to provide a hyaluronic acid hydrophobic self-assembled nanocomposite material. Using the above hyaluronic acid hydrophobic material as a carrier, a flavonoid natural product is loaded to obtain a hyaluronic acid hydrophobic self-assembled nanocomposite material.

[0020] The fourth object of the present invention is to provide a preparation method of the above hyaluronic acid hydrophobic self-assembled nanocomposite material, including the following steps: Dissolve the hyaluronic acid hydrophobic material in methanol to obtain a first solution.

[0021] Dissolve the flavonoid natural product and the fragrance in methanol to obtain a second solution.

[0022] Mix the first solution and the second solution and perform rotary evaporation to obtain a film.

[0023] Dissolve the film in water and perform ultrasonic treatment to obtain a hyaluronic acid hydrophobic self-assembled nanocomposite material.

[0024] In a preferred embodiment of the present invention, the mass ratio of the hyaluronic acid hydrophobic material to the flavonoid natural product is 15:0.6 to 0.7; for example, the mass ratio of the hyaluronic acid hydrophobic material to the flavonoid natural product is 15:0.6, 15:0.62, 15:0.64, 15:0.66, 15:0.68, 15:0.7, etc.

[0025] The mass ratio of the flavonoid natural product to the fragrance is 15:0.3 to 0.4. For example, the mass ratio of the flavonoid natural product to the fragrance is 15:0.3, 15:0.32, 15:0.34, 15:0.36, 15:0.38, 15:0.4, etc., but it is not limited to the listed values, and other unlisted values within the above numerical range are equally applicable.

[0026] In a preferred embodiment of the present invention, the flavonoid natural product is polydatin or icariin, and the fragrance is menthol or borneol.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (0) The present invention uses animal-derived polysaccharide hyaluronic acid as a carrier, which has good biocompatibility. The modified carrier has low toxicity and can be used for drug delivery. When the present invention is prepared, C 15 ~C 25 long-chain fatty acids are used as modifiers, and hyaluronic acid is modified into an ester by chemical modification to slow down its decomposition rate and achieve the purpose of drug loading. Compared with the traditional nanoformulation that adds surfactants in the thin film hydration step, the present invention adds Tween surfactants in the synthesis step, and the Tween surfactants will be removed by alcohol precipitation after the esterification reaction. Therefore, there are no surfactants in the subsequent synthesis of nanoparticles step, and there is no problem of cytotoxicity caused by surfactants.

[0028] (2) The present invention uses the hyaluronic acid hydrophobic material to load the flavonoid natural product and adds a fragrance, which can enhance the blood-brain barrier permeability and achieve brain delivery. The prepared hyaluronic acid hydrophobic material of the present invention has better stability. The hyaluronic acid hydrophobic self-assembled nanocomposite can be prepared by the thin film hydration method, and its structure is spherical and evenly distributed. By loading polydatin and menthol to construct drug-loaded nanoparticles, the encapsulation efficiency and drug loading measured with polydatin as the index are 95.25% and 3.6% respectively.

[0029] (3) Taking the flavonoid glycoside natural product loaded with polydatin as an example of the present invention, cell and molecular biology experiments show that the prepared hyaluronic acid hydrophobic self-assembled nanocomposite has no cytotoxicity at a concentration of 1 μmol / L, exhibits significant antioxidant activity, and up-regulates the expression of key antioxidant genes such as Nrf2, SOD, and HO-1. This hyaluronic acid hydrophobic self-assembled nanocomposite demonstrates good antioxidant potential and provides new possibilities for protecting nerve cells. DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a comparative FTIR spectrum of HASA and HA.

[0031] Figure 2 It is a comparative NMR spectrum of different samples, where a is HASA and b is HA.

[0032] Figure 3 It is a comparative FTIR spectrum of HARA and HA.

[0033] Figure 4 It is the UV spectra of ATRA, HARA, and HA.

[0034] Figure 5 It is the TEM image and particle size distribution map of HASA nanoparticles, where a is the TEM image and b is the particle size distribution map.

[0035] Figure 6 It is the effect of samples with different concentrations on the survival rate of HT22 cells, where a is polydatin with different concentrations, b is HASA / PD-Men with different concentrations, c is HASA self-assembled nanoparticles with different concentrations, and d is menthol with different concentrations.

[0036] Figure 7 It is the effect of HASA / PD-Men on the expression of different genes, where a is Nrf2, b is SOD, and c is HO-1.

[0037] Figure 8 It is the effect of samples with different concentrations on the survival rate of BV2 cells, where a is borneol with different concentrations, b is icariin with different concentrations, c is HARA with different concentrations, and d is HARA / ICA-BO with different concentrations.

[0038] Figure 9 It is the effect of HARA / ICA-BO on the expression of different genes, where a is IL-6, b is COX-2, c is iNOS-2, and d is TNF-α. DETAILED DESCRIPTION OF THE INVENTION

[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0040] The sodium hyaluronate and polydatin (PD) used in the present invention were purchased from Dalian Meilun Biotechnology Co., Ltd.; stearic acid was purchased from Tianjin Yongda Chemical Reagent Co., Ltd.; N,N'-dicyclohexylcarbodiimide (DCC) was purchased from Beijing Bailingwei Technology Co., Ltd.; 4-dimethylamino-pyridine (DMAP), menthol (Men), N,N'-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; triethylamine and Tween 80 were purchased from Sinopharm Chemical Reagent Co., Ltd.; DMEM medium and phosphate buffer solution were purchased from Zhongsheng Aobang Biotechnology Research Co., Ltd.; the reverse transcription kit was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0041] The mouse neuron cell line HT22 cells were purchased from Zhongsheng Aobang Biotechnology Research Co., Ltd.

[0042] The R-1005 rotary evaporator used in the present invention was purchased from Zhengzhou Great Wall Scientific and Industrial Co., Ltd.; the JY96-IIN ultrasonic crusher was purchased from Dongguan Luotai Precision Instruments Co., Ltd.; the JEM-2100F transmission electron microscope was purchased from Hitachi High-Technologies Corporation; the Thermo Nicolet IS5 Fourier transform infrared spectrometer was purchased from Thermo Fisher Scientific, USA; the Bruker AVANCE III 400M nuclear magnetic resonance spectrometer was purchased from Bruker, Germany; the Waters Alliance e2695 high-performance liquid chromatography was purchased from Waters, USA; the NanoBrook 90Plus Zeta particle size analyzer was purchased from Brookhaven, USA; the Infinite F50 microplate reader was purchased from Tecan, Switzerland; the polymerase chain reaction (PCR) was purchased from Bio-Rad Laboratories, Inc.

[0043] Example 1 This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, comprising the following steps: Precisely weigh 1420 mg of stearic acid, 1030 mg of N,N'-dicyclohexylcarbodiimide, and 48.8 mg of 4-dimethylaminopyridine, add them to 20 mL of N,N-dimethylformamide (DMF), dissolve them by ultrasonic treatment, add 1 mL of triethylamine to adjust the pH to 9, and obtain an organic phase solution.

[0044] Take 500 mg of sodium hyaluronate, dissolve it in 50 mL of pure water, add 2 g of Tween 80, and stir evenly to obtain an aqueous phase solution.

[0045] Under the condition of 30 °C, dropwise add the organic phase solution into the aqueous phase solution, and react for 48 h. After the reaction is completed, filter the obtained solution, transfer the filtrate to a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, dialyze with pure water for 48 h, and change the water every 8 h. The dialysate is precipitated with 95% ethanol solution, left to stand overnight at 4 °C, the precipitate is collected and freeze-dried to obtain HASA.

[0046] Example 2 This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, comprising the following steps: Take 500 mg of sodium hyaluronate, dissolve it in 25 ml of pure water, add 2 g of Tween 80, and stir evenly to obtain an aqueous phase solution.

[0047] Take 600 mg of all-trans retinoic acid (ATRA), 1030 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 61 mg of 4-dimethylaminopyridine (DMAP), dissolve them in 20 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and dissolve them by ultrasonic treatment to obtain an organic phase solution.

[0048] Drop the organic phase solution into the aqueous phase solution, and react at 40 °C for 48 h. After the reaction is completed, filter, collect the filtrate part; transfer it to a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, dialyze with pure water for 48 h, and filter again; collect the filtrate part, precipitate with 95% ethanol at 4 °C; take the precipitate part, wash it with a small amount of absolute ethanol, redissolve it in water, and freeze-dry to obtain the modified product HARA.

[0049] Example 3 This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, comprising the following steps: Take 200 mg of sodium hyaluronate, dissolve it in 25 ml of pure water, add 1 g of Tween 80, and stir evenly to obtain an aqueous phase solution.

[0050] Take 311.1 mg of all - trans retinoic acid (ATRA), 534 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 25.3 mg of 4 - dimethylaminopyridine (DMAP), dissolve them in 15 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and dissolve by ultrasonic treatment to obtain an organic - phase solution.

[0051] Drop the organic - phase solution into the aqueous - phase solution and react at 40.4 °C for 46.7 h. After the reaction, filter and collect the filtrate part; transfer it to a dialysis bag with a molecular - weight cut - off of 8000 - 14000 Da, dialyze with pure water for 48 h, and filter again; collect the filtrate part, precipitate with 95% ethanol at 4 °C; take the precipitate part, wash it with a small amount of absolute ethanol, redissolve it in water, and lyophilize to obtain the modifier HARA. The grafting rate of HARA is 0.905 μg / mg.

[0052] Example 4 This example provides a preparation method of a hyaluronic - acid hydrophobic material, which includes the following steps: Take 500 mg of sodium hyaluronate and dissolve it in 25 ml of pure water, add 2 g of Tween 80 and stir evenly to obtain an aqueous - phase solution.

[0053] Take 300 μl of docosahexaenoic acid (DHA), 1030 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 61 mg of 4 - dimethylaminopyridine (DMAP), dissolve them in 20 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and dissolve by ultrasonic treatment to obtain an organic - phase solution.

[0054] Drop the organic - phase solution into the aqueous - phase solution, charge nitrogen for protection, and react at 40 °C for 48 h. After the reaction, filter and collect the filtrate part; transfer it to a dialysis bag, dialyze with pure water for 48 h, and filter again; collect the filtrate part, precipitate with 95% ethanol at 4 °C; take the precipitate part, wash it with a small amount of absolute ethanol, redissolve it in water, and lyophilize to obtain the modifier.

[0055] Example 5 This example provides a preparation method of a hyaluronic - acid hydrophobic self - assembled nanocomposite material, which includes the following steps: Precisely weigh 10 mg of HASA prepared in Example 1 and dissolve it in 20 mL of pure methanol solution. Subsequently, add a methanol solution containing 0.4 mg of polydatin and 0.1 mg of menthol to make the total volume 30 ml. Transfer the mixed solution to a round-bottom flask and perform rotary evaporation at 40 °C and a pressure of 0.01 MPa for 40 min to form a uniform white film at the bottom of the flask. Add 5 mL of pure water and rotate and heat for 40 min to completely dissolve the film, and then perform ultrasonic probe treatment for 5 min to obtain HASA / PD-Men self-assembled nanoparticles. The prepared solution is stored at 4 °C.

[0056] Example 6 This example provides a method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite, which includes the following steps: Weigh 15 mg of HASA prepared in Example 1, add 30 ml of methanol, and dissolve it by ultrasonic treatment for 20 min to obtain a HASA solution. Prepare solutions of polydatin (PD) with a concentration of 1 mg / ml and menthol (Men) with a concentration of 1 mg / ml in methanol respectively. Add 0.66 ml of the PD solution and 0.34 ml of the Men solution to the HASA solution, mix well and set aside. Transfer the mixed solution to a round-bottom flask, perform rotary evaporation at 30 °C and a pressure of 0.01 MPa for 30 min to evaporate the solvent, then add 5 ml of pure water, rotate and heat at 40 °C for 30 min, and perform ultrasonic probe treatment for 5 min to obtain HASA / PD-Men.

[0057] The particle size of HASA / PD-Men detected by a particle size analyzer is 181.86 nm ± 1.33 nm, the PDI is 0.04 ± 0.02, and the potential is -18.18 mV ± 0.09 mV. The encapsulation efficiency and drug loading of HASA / PD-Men detected by the membrane filtration method are 84.44 % ± 0.35% and 5.27 % ± 0.04 % respectively

[0058] Example 7 This example provides a method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite, which includes the following steps: Weigh 15 mg of HARA prepared in Example 2, add 30 ml of methanol, and dissolve it by ultrasonic treatment for 20 min to obtain a HARA solution. Prepare solutions of icariin (ICA) with a concentration of 1 mg / ml and borneol (BO) with a concentration of 1 mg / ml in methanol respectively. Add 0.66 ml of the ICA solution and 0.34 ml of the BO solution to the HARA solution, mix well and set aside. Transfer the mixed solution to a round-bottom flask, perform rotary evaporation at 30 °C and a pressure of 0.01 MPa for 30 min to evaporate the solvent, then add 5 ml of pure water, rotate and heat at 40 °C for 30 min, and perform ultrasonic probe treatment for 5 min to obtain HARA / ICA-BO.

[0059] The particle size of HARA / ICA-BO detected by the particle size analyzer was 296.52 nm ± 4.56 nm, the PDI was 0.255 ± 0.021, and the potential was -15.07 mV ± 0.04 mV. The encapsulation efficiency and drug loading of HARA / ICA-BO detected by the membrane filtration method were 79.61% ± 0.95% and 4.09% ± 0.04% respectively.

[0060] Example 8 Weigh 10 mg of HARA prepared in Example 3 and dissolve it in 30 mL of methanol. Ultrasonic for 20 min to dissolve and obtain the HARA solution; prepare solutions of icariin (ICA) with a concentration of 1 mg / ml and borneol (BO) with a concentration of 1 mg / ml in methanol respectively, and add 0.5 ml of the ICA solution and 0.5 ml of the BO solution to the HARA solution. Mix evenly and ultrasonic for 20 min, transfer the solution to a round-bottom flask, rotary evaporate at 30 °C and a pressure of 0.01 MPa for 30 min to evaporate the solvent completely, then add 5 mL of pure water, rotate and heat at 40 °C for 30 min to dissolve the film, and then ultrasonic with a probe for 5 min to obtain HARA / ICA-BO.

[0061] Comparative Example 1 Precisely weigh 10 mg of HASA prepared in Example 1 and dissolve it in 30 mL of pure methanol solution to obtain the HASA solution, and transfer the solution to a 50 mL round-bottom flask. Rotate and evaporate at a temperature of 40 °C and a pressure of 0.01 MPa for 40 min to form a uniform white film at the bottom of the flask. Then add 5 mL of pure water, rotate and heat for 40 min to completely dissolve the film, and ultrasonic with a probe for 5 min to obtain HASA self-assembled nanoparticles. The prepared solution was stored at 4 °C.

[0062] Comparative Example 2 Precisely weigh 10 mg of HASA prepared in Example 1 and dissolve it in 30 mL of pure methanol solution to obtain the HASA solution, and then add a methanol solution of 0.4 mg of polydatin to make the total volume 30 ml. Transfer the solution to a 50 mL round-bottom flask. Rotate and evaporate at a temperature of 40 °C and a pressure of 0.01 Mpa for 30 min to form a uniform white film at the bottom of the flask. Then add 5 mL of pure water, rotate and heat for 30 min to completely dissolve the film, and ultrasonic with a probe for 5 min to obtain HASA self-assembled nanoparticles. The prepared solution was stored at 4 °C. Denote it as HASA / PD.

[0063] Comparative Example 3 Weigh 10 mg of HARA prepared in Example 3 and dissolve it in 30 mL of methanol. Sonicate for 20 min to dissolve and obtain a HARA solution. Transfer the HARA solution to a round-bottom flask, rotary evaporate at 30 °C under a pressure of 0.01 MPa for 30 min to dry the solvent, then add 5 mL of pure water, rotate and heat at 40 °C for 30 min to dissolve the film, and then probe sonicate for 5 min to obtain HARA self-assembled nanoparticles. The samples prepared in the present invention were characterized and tested according to the following methods.

[0064] (1)Morphological characterization of nanoparticles The particle size and polydispersity index (PDI) of the samples were measured by a particle size analyzer. The morphology of the nanoparticles was observed by transmission electron microscopy (TEM). Take 30 μL of the newly prepared nanoparticle solution and drop it on a copper grid, let it stand and evaporate the moisture, dropwise add phosphotungstic acid solution for staining, and dry it under an infrared lamp for 10 min, then observe under the microscope.

[0065] (2)Determination of the content of polydatin The content of polydatin was analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 chromatographic column (4.6 mm × 250 mm, 5 μm), flow rate 1 mL / min, mobile phase A was 23% acetonitrile, B was 77% pure water, column temperature 30 °C, detection wavelength 306 nm, injection volume 10 μL, running time 20 min.

[0066] Preparation and detection of the standard solution: Accurately weigh 10.07 mg of polydatin standard product and dissolve it in 10 mL of methanol, and dilute it stepwise to obtain a standard solution with a concentration of 1.12 - 56 μg / mL. The standard solution was injected into the liquid phase according to the above chromatographic method. The concentration of the standard solution was the independent variable X (μg / mL), and the peak area was the dependent variable Y, and a standard curve was established.

[0067] Preparation and detection of the sample solution: Take 1 mL of the drug-loaded nanoparticle solution, add methanol for lysis at a ratio of 1:1, filter with a 0.45 μm filter membrane, and inject a part of the filtrate into the liquid phase according to the above chromatographic conditions. Substitute the obtained peak area into the standard curve to calculate the mass of polydatin in the sample solution.

[0068] (3)Determination of encapsulation efficiency and drug loading 3.1 Determination of polydatin Using polydatin as an antioxidant drug, the encapsulation efficiency of the drug-loaded nanoparticles was determined by centrifugation. Take 1 mL of the drug-loaded nanoparticle solution, add 1 mL of methanol for lysis, and determine the total mass of polydatin (m). Take another 1 mL of the drug-loaded nanoparticle solution, centrifuge at 12000 r / min for 30 minutes, collect the supernatant, and determine the mass of free polydatin (m 0 )

[0069] Encapsulation efficiency of polydatin = [(total mass of polydatin - mass of free polydatin) / mass of polydatin] × 100%.

[0070] Drug loading = [(total mass of polydatin - mass of free polydatin) / (mass of carrier + mass of drug)] × 100%.

[0071] 3.2 Determination of icariin Using icariin as the investigation standard, the encapsulation efficiency of HARA / ICA - BO was investigated. The content of icariin was detected by HPLC. The chromatographic conditions were as follows: using a C18 chromatographic column, the mobile phase was acetonitrile and 30 mM potassium dihydrogen phosphate solution, with gradient elution (0 - 10 min, 20% acetonitrile; 11 - 60 min, 35% acetonitrile), the flow rate was 1 mL / min, the wavelength was 270 nm, the column temperature was 30 °C, and the injection volume was 10 μL. Standard solutions of ICA with different concentrations were prepared, and the peak areas were measured using the above method to plot a standard curve, and the content of ICA was calculated from the standard curve.

[0072] Using ICA as the investigation standard, the encapsulation efficiency and drug loading of the drug - loaded nanoparticles were evaluated. Take 1 mL of HARA / ICA - BO solution, filter it through a 0.45 μm filter membrane, add methanol in a 1:1 ratio, ultrasonically lyse for 5 min, and then inject it into the liquid phase to detect the content of ICA, denoted as m. Take 1 mL of HARA / ICA - BO solution, add methanol in a 1:1 ratio, ultrasonically lyse for 5 min, inject it into the liquid phase to detect the content of ICA, denoted as M. Encapsulation efficiency = [m / M] × 100%; Drug loading = [m / total mass] × 100%

[0073] (4)Cell viability test 4.1 Determination of polydatin HT22 cells were selected as the model, and the MTT method was used to evaluate the cytotoxicity of blank nanoparticles, HASA / PD - Men, polydatin, and menthol. The cells were seeded in a 96 - well plate at a density of 10000 cells per well, and the plate was placed in an incubator with the culture conditions of 37 °C and 5% CO 2 . After 24 h, it was taken out, and the culture medium containing different concentrations of drugs was added to each well, and then it was placed back into the incubator. After 24 h, it was taken out, 0.5% MTT solution was added, incubated for 4 h, the solution was aspirated, and DMSO solution was added to dissolve the crystals, and the absorbance was measured at 490 nm.

[0074] Cell viability = [(absorbance of sample well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100% 4.2 Determination of icariin The culture conditions of BV2 cells were DMEM medium + 10% fetal bovine serum (FBS) + 1% double antibody, and it was placed at 37 °C and 5% CO 2The incubator. The BV2 cells in the logarithmic growth phase were digested and centrifuged, and then seeded in a 96-well plate at a density of 1×10 4 cells per well and cultured overnight. The groups were divided as follows: The blank group only contained an equal volume of culture medium solution; the control group contained only cells without drugs; the experimental groups contained cells and drugs, and the drug administration concentrations were as follows:

[0075] a. The administration concentrations of free polydatin were: 0.15 μM, 0.35 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM.

[0076] b. The administration concentration of free menthol was: 0.15 μM, 0.35 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM.

[0077] c. The administration concentrations of HASA self-assembled nanoparticles were: 0.79 μg / mL, 1.57 μg / mL, 3.15 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL.

[0078] d. The administration concentrations of HASA / PD-Men were: 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM.

[0079] After culturing for 24 h, 10 μL of CCK-8 solution was added to each well. After standing in the incubator for 1 hour, the optical density (OD value) was measured at a wavelength of 450 nm. Calculate the cell viability = (experimental group OD value - blank group OD value) / (control group OD value - blank group OD value) × 100%

[0080] (5)Polydatin antioxidant mRNA expression test The cells were seeded in a 6-well plate at a density of 60W / well and incubated in the incubator for 24 h. They were divided into 5 groups for experiments. The blank group only contained cells and culture medium. The control group was induced with a certain concentration of glutamate. The experimental groups were divided into high, medium, and low reagent groups, which were added with the inducer glutamate and the drug reagent HASA / PD-Men. After adding the induction reagents to the control group and the experimental groups, they were incubated in the incubator for 6 h and then different concentrations of drug reagents were added.

[0081] Total cellular RNA was extracted using the Trizol method and reverse transcribed into cDNA. The expression changes of antioxidant genes such as nuclear factor-erythroid 2-related factor 2 (Nrf2), superoxide dismutase gene (SOD), and heme oxygenase 1 (HO-1) were analyzed by RT-qPCR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference for quantification.

[0082] (6)Detection of the expression level of icariin anti-inflammatory factor After culturing the cells according to the method described in 4.2, an inflammatory model was established using LPS. BV2 cells in the logarithmic growth phase were taken, digested and centrifuged, and then inoculated into a 6-well plate at a density of 6×10 5 cells / well and cultured in an incubator for 12 h. Then, the cells were divided into 5 groups for experiments. The grouping was as follows: the control group was cultured normally for 12 h; the model group was treated with 1 μg / mL LPS for 6 h; the experimental group was divided into three dosage groups of low, medium, and high doses, and the concentration gradients were 0.1 μmol / L, 1 μmol / L, and 10 μmol / L respectively. After treatment for 6 h, 1 μg / mL LPS was added and co-incubated for 6 h.

[0083] Cell RNA was extracted using the Trizol method. After measuring the RNA concentration, it was reverse transcribed into cDNA. Using GAPDH as an internal reference, the prepared solution was placed on a PCR instrument for reaction, and the mRNA expression levels of inflammatory genes such as IL-6, COX-2, TNF-α, and iNOS-2 were detected by fluorescence quantitative PCR.

[0084] (7)Statistical analysis The results of cell viability tests and antioxidant mRNA expression tests were analyzed using Graphpad prism software, and the results were expressed as mean ± standard deviation. Univariate analysis of variance was used for comparison of two groups of data, and P < 0.05 indicated that the difference was statistically significant.

[0085] The carboxyl group in stearic acid reacts with the hydroxyl group of the N-acetylglucosamine unit in HA to form hyaluronic acid stearate (HASA). Figure 1 is the infrared absorption spectrum of HA and HASA prepared in Example 1. A new absorption peak appeared at 1735 cm -1 in HASA, which belongs to the stretching vibration of the carbonyl group, indicating that an ester group was formed in the reaction. Compared with unmodified HA, the C-H stretching vibration absorption peak in the 2800 - 2950 cm -1 region of HASA was significantly enhanced, indicating the successful introduction of long-chain alkyl groups.

[0086] Figure 2 is the 1 H-NMR spectrum of HASA prepared in Example 1 and HA. In the HA spectrum, the proton peak of the methyl group was at δ 2.02 ppm, and the protons belonging to the polysaccharide carbon skeleton were in the range of δ 3.2 - 4.0 ppm. In the HASA spectrum, a new peak appeared at δ 0.87 ppm, which belongs to the methyl hydrogen signal at the end of the stearic acid chain, and δ 1.27 ppm corresponds to the methylene hydrogen signal in the stearic acid chain. The above results indicate that stearic acid was successfully grafted onto the HA molecule.

[0087] Figure 3 This is the infrared spectrum of HA and HARA prepared in Example 3. In the left figure, the characteristic peaks representing ATRA are the characteristic absorption peaks of methyl at 2932 cm -1 and the characteristic absorption peak of C=O in the carboxyl group at 1658 cm -1 ; in the right figure, the stretching vibration peak of CH in HA is at 2855 cm -1 , and the peak belonging to the hydroxyl group OH is at 3392 cm -1 . By comparison, it is found that the peak area of the OH peak decreases after the reaction, indicating that the reaction has occurred successfully and a large number of OH groups have been consumed.

[0088] Figure 4 This is the ultraviolet spectrum of ATRA, HARA prepared in Example 3, and HA at 350 nm. Among them, ATRA has a maximum absorption peak at 350 nm; HARA also shows an absorption peak at 350 nm; HA has no absorption peak at 350 nm. Thus, it can be seen that when ATRA reacts with HA, an absorption peak appears at 350 nm, indicating that the synthesis reaction has occurred successfully.

[0089] The HASA self-assembled nanoparticles of Comparative Example 1 were prepared by the thin film hydration method, and the average particle size and PDI of the blank nanoparticles were measured using a particle size analyzer. The results showed that the average particle size of the blank nanoparticles was 234 nm and the PDI was 0.249, indicating that the nanoparticles were evenly distributed and had good dispersibility, as shown in Table 1.

[0090] Table 1 Measurement results of the particle size and PDI of the nanoparticles The TEM images further revealed the morphological characteristics of the nanoparticles. It was observed that the blank nanoparticles presented a regular spherical structure with a particle size of about 15 nm (see Figure 5 ). This is different from the particle size measured by the particle size analyzer, probably because the sample was in a dry state during TEM analysis, while the particle size analyzer measures the hydrated state in solution. In addition, the TEM images showed that the surface of the nanoparticles was smooth and there was no obvious aggregation phenomenon, further verifying the high homogeneity of the nanoparticles.

[0091] Compared with the blank nanoparticles, the average particle sizes of HASA / PD and HASA / PD-Men increased, reaching 276 nm and 388 nm respectively. This indicates that the encapsulation of the drug increased the size of the hydrophobic core of the nanoparticles and also affected the morphology and stability. The slight change in PDI also implies the influence of drug encapsulation on the dispersibility of the nanoparticles.

[0092] The high encapsulation efficiency and appropriate drug loading of HASA / PD-Men prepared in Example 5 indicate that HASA effectively isolates the hydrophobic drug from the external environment, which plays an important role in improving the bioavailability of the drug and prolonging the duration of drug efficacy.

[0093] The quality of polydatin in HASA / PD-Men prepared in Example 5 was determined by HPLC. The peak area (y) of the standard curve showed a good linear relationship with the concentration (x), and the regression equation was y = 267315x - 320991. The linear range was 1.12 - 56 μg / mL, and the correlation coefficient R² = 0.9998.

[0094] Using polydatin as the antioxidant drug, the encapsulation efficiency and drug loading were determined by centrifugation. The results showed that the encapsulation efficiency of HASA / PD was 96.02% and the drug loading was 4.64%; while the encapsulation efficiency of HASA / PD-Men prepared in Example 5 was 95.25% and the drug loading was 3.66% (see Table 2). The high encapsulation efficiency and drug loading demonstrated the effectiveness and stability of HASA / PD-Men in encapsulating hydrophobic drugs.

[0095] Table 2 Encapsulation efficiency and drug loading of HASA / PD and HASA / PD-Men The cytotoxicity of HT22 cells was tested by the MTT method to evaluate the effects of different concentrations of polydatin, menthol, blank nanoparticles, and HASA / PD-Men prepared in Example 5 on cell viability. The results showed that when the concentration of polydatin in HASA / PD-Men reached 16 μmol / L, the cell viability exceeded 85%; while in the blank nanoparticles and polydatin at the same concentration, the cell viabilities were above 85% and 95% respectively (see Figure 6 ). These data indicate that HASA / PD-Men has no significant toxicity to cells within the concentration range of 16 μmol / L, showing good safety.

[0096] Taking HASA / PD-Men prepared in Example 5 as an example, the antioxidant mechanism of HASA / PD-Men was further explored, and the expression levels of antioxidant-related genes such as Nrf2, SOD, and HO-1 were analyzed by RT-qPCR. The results showed that compared with the control group, at low to medium concentrations, HASA / PD-Men significantly upregulated the mRNA expression levels of Nrf2, SOD, and HO-1, showing a significant antioxidant gene activation effect (see Figure 7 ). Especially at a concentration of 1 μmol / L, the expression of these genes reached the peak, indicating that HASA / PD-Men at this concentration had the best antioxidant effect.

[0097] Nrf2 is the main antioxidant response regulator in cells, and its activation can enhance the antioxidant capacity of cells and reduce cell damage caused by ROS. HASA / PD-Men significantly upregulated the expression of Nrf2 at a concentration of 1 μmol / L, which may be due to the effective activation of the Nrf2 pathway by the polydatin released from the nanoparticles, enhancing the antioxidant defense ability of cells. SOD is an antioxidant enzyme that can convert the reactive oxygen species produced by cell metabolism into hydrogen peroxide, thereby reducing the damage of ROS to cells. HO-1 further protects cells from oxidative stress by metabolizing products with antioxidant capacity, such as bilirubin and carbon monoxide. These data together indicate that HASA / PD-Men can enhance the antioxidant capacity of cells through multiple pathways and has potential neuroprotective effects.

[0098] Taking HARA / ICA-BO prepared in Example 8 as an example, the cell viability was studied. From Figure 8 It can be seen that the effect of icariin aromatic nanoparticles on the activity of BV2 cells is as Figure 8 shown. It can be seen that icariin did not show toxicity to BV2 cells at 0-20 μmol / L; borneol did not show toxicity to BV2 cells at the highest administration concentration. HARA and HARA / ICA-BO did not produce obvious toxic side effects on cells within the experimental range.

[0099] To further explore the anti-inflammatory mechanism of HARA / ICA-BO, RT-qPCR was used to analyze the mRNA expression levels of anti-inflammatory genes such as IL-6, COX-2, TNF-α, and iNOS-2. The results are as Figure 9 shown. Compared with the control group, the expression of inflammatory factors in the model group was significantly increased. Compared with the model group, different dose groups of HARA / ICA-BO nanoparticles significantly inhibited the mRNA expression of IL-6, COX-2, TNF-α, and iNOS-2; when the administration concentration was 10 μmol / L, the inhibitory effect on inflammation induced by LPS was the strongest, indicating that HARA / ICA-BO at this concentration had the best anti-inflammatory effect.

[0100] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0101] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a hyaluronic acid hydrophobic material, characterized in that: The following steps are involved: Using N,N-dimethylformamide as solvent, add C 15 ~C 25 long-chain fatty acids, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, adjusting the pH to 9-10 to obtain an organic phase solution; Using water as solvent, adding hyaluronic acid and Tween surfactant, stirring evenly to obtain an aqueous phase solution; The aqueous solution and the organic solution are mixed, and the hydroxyl groups of the hyaluronic acid in the aqueous solution react with the carboxyl groups in the hydrophobic carboxylic acid compound to undergo an esterification reaction. After the reaction is completed, an alcohol precipitation method is used to obtain a hyaluronic acid hydrophobic material.

2. The method for preparing a hyaluronic acid hydrophobic material according to claim 1, characterized in that: The reaction temperature of the esterification reaction is 30°C~35°C, and the reaction time is 40h~48h.

3. The method for preparing a hyaluronic acid hydrophobic material according to claim 1, characterized in that: C 15 ~C 25 Long-chain fatty acids are stearic acid, all-trans retinoic acid or docosahexaenoic acid.

4. The method for preparing a hyaluronic acid hydrophobic material according to claim 1, characterized in that: Hyaluronic acid and C 15 ~C 25 The molar ratio of long-chain fatty acids is 1:125-130; The molar ratio of hyaluronic acid to N,N'-dicyclohexylcarbodiimide is 1:125-130; The molar ratio of hyaluronic acid to 4-dimethylaminopyridine is 0.04:25-30; the mass ratio of hyaluronic acid to Tween surfactant is 1:4-4.

5.

5. The method for preparing a hyaluronic acid hydrophobic material according to claim 1, characterized in that: The mass ratio of N,N'-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 16-17:

1.

6. A hyaluronic acid hydrophobic material prepared by the preparation method according to any one of claims 1 to 5.

7. A hyaluronic acid hydrophobic self-assembled nanocomposite material, characterized in that: The hyaluronic acid hydrophobic material according to claim 6 is used as a carrier to load the flavonoid glycoside natural product to obtain a hyaluronic acid hydrophobic self-assembled nanocomposite material.

8. A method for preparing the hyaluronic acid hydrophobic self-assembled nanocomposite material according to claim 7, characterized in that: The following steps are involved: dissolving a hyaluronic acid hydrophobic material in methanol to obtain a first solution; dissolving the flavonoid glycoside natural product and the aromatic agent in methanol to obtain a second solution; The first solution and the second solution are mixed and then subjected to rotary evaporation to obtain a film; The film was dissolved in water and subjected to ultrasonic treatment to obtain a hyaluronic acid hydrophobic self-assembled nanocomposite material.

9. The method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite material according to claim 8, characterized in that: The mass ratio of hyaluronic acid hydrophobic material and flavonoid glycoside natural product is 15:0.6~0.7; The mass ratio of flavonoid glycosides natural products to aromatics is 15:0.3~0.

4.

10. The method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite material according to claim 8, characterized in that: The natural flavonoid glycosides are polydatin or icariin, and the aromatic agent is menthol or borneol.

Images