Hydrophobic hyaluronic acid material, hydrophobic self-assembled nanocomposite of hyaluronic acid and preparation method thereof

Abstract

The application discloses a hyaluronic acid hydrophobic material, a hyaluronic acid hydrophobic self-assembled nanocomposite and a preparation method thereof, and belongs to the technical field of drug carrier preparation. 15 ~C 25 Long-chain fatty acid, N,N'-dicyclohexyl carbodiimide, 4-dimethylaminopyridine, adjust pH to 9-10 to obtain an organic phase solution; water is used as a solvent, hyaluronic acid and a Tween surfactant are added, and stirring is uniformly carried out to obtain an aqueous phase solution; the aqueous phase solution and the organic phase solution are mixed, the hydroxyl group of the hyaluronic acid in the aqueous phase solution and the carboxyl group in the hydrophobic carboxylic acid compound are subjected to esterification reaction, and after the reaction is completed, the hyaluronic acid hydrophobic material is obtained by using an alcohol precipitation method.

Description

Technical Field

[0001] This invention relates to the field of drug carrier preparation technology, and more specifically to hyaluronic acid hydrophobic materials, hyaluronic acid hydrophobic self-assembled nanocomposites and their preparation methods. Background Technology

[0002] Neurons are the most basic structural and functional units of the nervous system, responsible for receiving stimuli, generating excitation, and transmitting it. Neurons are among the most susceptible cells in the human body to oxidative stress. Oxidative stress refers to the excessive production of reactive oxygen species (ROS) in the intracellular and extracellular environment, leading to an oxidative stress response. Excessive accumulation of ROS can damage nerve cell function, increasing the risk of neurodegenerative diseases such as Alzheimer's and Parkinson's.

[0003] Flavonoid glycosides have attracted widespread attention for their neuroprotective effects. For example, Polygonum cuspidatum, a traditional Chinese medicine, has been found to contain resveratrol, an extract with various biological activities. These activities include mitigating neuronal damage caused by oxidative stress by scavenging free radicals, inhibiting ROS generation, and enhancing the activity of antioxidant enzymes. Furthermore, resveratrol can also protect nerve cells from inflammation-mediated damage by regulating the expression of inflammatory factors and inhibiting neuroinflammatory responses.

[0004] Besides citric acid, icariin is another widely used natural compound. Icariin is the main active ingredient of Epimedium, belonging to the 8-isopentenyl flavonoid glycoside class. It can be extracted from the dried stems and leaves of Epimedium sagittatum, Epimedium pubescens, Epimedium wushanense, and Epimedium koreanum. Studies have shown that icariin can enhance the antioxidant defense capacity of cells and reduce the accumulation of ROS by activating the Nrf2 / ARE signaling pathway. In addition, icariin can also reduce the inflammatory response by inhibiting the NF-κB signaling pathway, thereby protecting nerve cells from the dual damage of oxidative stress and inflammation.

[0005] Polygonum cuspidatum glycoside and icariin possess potential for antioxidant, anti-inflammatory, and neuroprotective effects. However, their poor water solubility and rapid metabolism in vivo are major limitations on the clinical application of flavonoid glycosides such as polygonum cuspidatum glycoside and icariin. These issues can be addressed by formulating them into nanoparticles, but traditional nanoparticle formulations involve the addition of surfactants during the film hydration step, resulting in some cytotoxicity of the nanoparticles. Therefore, it is essential to develop a low-toxicity nanoparticle formulation for the drug delivery of polygonum cuspidatum glycoside and icariin. Summary of the Invention

[0006] To address the above problems, this invention provides hydrophobic hyaluronic acid materials, hydrophobic self-assembled hyaluronic acid nanocomposites, and their preparation methods. This invention utilizes C 15 ~C 25Long-chain fatty acids are used to modify hyaluronic acid, and the resulting hydrophobic hyaluronic acid material can be used for drug delivery.

[0007] The first objective of this invention is to provide a method for preparing a hyaluronic acid hydrophobic material, comprising the following steps:

[0008] Using N,N-dimethylformamide as a solvent, add C 15 ~C 25 Long-chain fatty acids, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine are used to adjust the pH to 9-10 to obtain an organic phase solution; for example, pH values ​​of 9, 9.2, 9.4, 9.6, 9.8, 10, etc., but not limited to the listed values, other unlisted values ​​within the above range are also applicable.

[0009] Aqueous solution was obtained by adding hyaluronic acid and Tween-type surfactants to water as a solvent and stirring until homogeneous.

[0010] Aqueous and organic phase solutions are mixed, and the hydroxyl groups of hyaluronic acid in the aqueous solution undergo esterification with the carboxyl groups in the hydrophobic carboxylic acid compound to obtain a hydrophobic hyaluronic acid material.

[0011] 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.

[0012] The reaction time is 40h to 48h, for example, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, etc., but it is not limited to the listed values. Other unlisted values ​​within the above range are also applicable.

[0013] In a preferred embodiment of the present invention, C 15 ~C 25 Long-chain fatty acids include stearic acid, all-trans retinoic acid, or docosahexaenoic acid.

[0014] In a preferred embodiment of the present invention, hyaluronic acid and C 15 ~C 25 The molar ratio of long-chain fatty acids is 1:125~130; for example, hyaluronic acid and C. 15 ~C 25 The molar ratios of long-chain fatty acids are 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, etc.

[0015] The molar ratio of hyaluronic acid to N,N'-dicyclohexylcarbodiimide is 1:125~130. 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.

[0016] The molar ratio of hyaluronic acid to 4-dimethylaminopyridine is 1:625~750; 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.

[0017] 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 it is not limited to the listed values. Other unlisted values ​​within the above range are also applicable.

[0018] 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. Other unlisted values ​​within the above range are also applicable.

[0019] The second objective of this invention is to provide a hyaluronic acid hydrophobic material prepared by the above-described preparation method.

[0020] The third objective of this invention is to provide a hyaluronic acid hydrophobic self-assembled nanocomposite material, which uses the above-mentioned hyaluronic acid hydrophobic material as a carrier to load flavonoid glycoside natural products to obtain the hyaluronic acid hydrophobic self-assembled nanocomposite material.

[0021] The fourth objective of this invention is to provide a method for preparing the above-mentioned hyaluronic acid hydrophobic self-assembled nanocomposite material, comprising the following steps:

[0022] The hyaluronic acid hydrophobic material was dissolved in methanol to obtain the first solution.

[0023] The flavonoid glycosides and aromatics were dissolved in methanol to obtain a second solution.

[0024] The first and second solutions were mixed and then subjected to rotary evaporation to obtain a thin film.

[0025] After dissolving the film in water and ultrasonically treating it, a hyaluronic acid hydrophobic self-assembled nanocomposite material was obtained.

[0026] In a preferred embodiment of the present invention, the mass ratio of hyaluronic acid hydrophobic material to flavonoid glycoside natural product is 15:0.6~0.7; for example, the mass ratio of hyaluronic acid hydrophobic material to flavonoid glycoside natural product is 15:0.6, 15:0.62, 15:0.64, 15:0.66, 15:0.68, 15:0.7, etc.

[0027] The mass ratio of flavonoid glycosides to fragrances is 15:0.3 to 0.4. For example, the mass ratio of flavonoid glycosides to fragrances 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 values ​​listed. Other unlisted values ​​within the above range are also applicable.

[0028] In a preferred embodiment of the present invention, the flavonoid glycoside natural product is polydipsia glycoside or icariin, and the fragrance is menthol or borneol.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (0) This 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. In the preparation of this invention, C 15 ~C 25 Long-chain fatty acids are used as modifiers to chemically modify hyaluronic acid into esters, slowing its decomposition and achieving drug loading. Compared to traditional nanoparticle formulations that add surfactants during the film hydration step, this invention adds Tween-type surfactants during the synthesis step. After esterification, the Tween-type surfactants are removed by alcohol precipitation. Therefore, no surfactants are present in the subsequent nanoparticle synthesis steps, eliminating the cytotoxicity issues caused by surfactants.

[0031] (2) This invention uses hydrophobic hyaluronic acid as a loading agent for flavonoid glycosides and adds a fragrance to enhance blood-brain barrier permeability and achieve brain delivery. The hydrophobic hyaluronic acid material prepared by this invention has better stability. Hyaluronic acid hydrophobic self-assembled nanocomposites can be prepared by thin-film hydration method, and its structure is spherical and uniformly distributed. Drug-loaded nanoparticles are constructed by loading polysaccharide and menthol. The encapsulation efficiency and drug loading, measured with polysaccharide as an indicator, are 95.25% and 3.6%, respectively.

[0032] (3) Taking flavonoid glycosides loaded with polygalactosin as an example, cell and molecular biology experiments showed that the prepared hyaluronic acid hydrophobic self-assembled nanocomposite material did not exhibit cytotoxicity at a concentration of 1 μmol / L, but showed significant antioxidant activity, upregulating the expression of key antioxidant genes such as Nrf2, SOD, and HO-1. This hyaluronic acid hydrophobic self-assembled nanocomposite material demonstrated good antioxidant potential, providing new possibilities for protecting nerve cells. Attached Figure Description

[0033] Figure 1 This is a comparison of the FTIR spectra of HASA and HA.

[0034] Figure 2 The image shows a comparison of NMR spectra of different samples, where a represents HASA and b represents HA.

[0035] Figure 3 This is a comparison of the FTIR spectra of HARA and HA.

[0036] Figure 4 The images show the UV spectra of ATRA, HARA, and HA.

[0037] Figure 5 The images show TEM images and particle size distributions of HASA nanoparticles, where a is the TEM image and b is the particle size distribution.

[0038] Figure 6 The effect of different concentrations of the sample on the survival rate of HT22 cells is shown in the figure. Among them, a represents different concentrations of polygalactoside, b represents different concentrations of HASA / PD-Men, c represents different concentrations of HASA self-assembled nanoparticles, and d represents different concentrations of menthol.

[0039] Figure 7 The effects of HASA / PD-Men on the expression of different genes are shown, where a represents Nrf2, b represents SOD, and c represents HO-1.

[0040] Figure 8 The effect of different concentrations of samples on the survival rate of BV2 cells is shown in the figure. Among them, a represents different concentrations of borneol, b represents different concentrations of icariin, c represents different concentrations of HARA, and d represents different concentrations of HARA / ICA-BO.

[0041] Figure 9 The effects of HARA / ICA-BO on the expression of different genes are shown, where a represents IL-6, b represents COX-2, c represents iNOS-2, and d represents TNF-α. Detailed Implementation

[0042] 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.

[0043] Sodium hyaluronate and polydatin (PD) used in this 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, 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 culture medium and phosphate buffer solution were purchased from Sinopharm Aobang Biotechnology Research Co., Ltd.; and the reverse transcription kit was purchased from Wuhan Saiwei Biotechnology Co., Ltd.

[0044] HT22 mouse neuronal cells were purchased from Sinopharm Biotechnology Co., Ltd.

[0045] The R-1005 rotary evaporator used in this invention was purchased from Zhengzhou Great Wall Science & Industry Co., Ltd.; the JY96-IIN ultrasonic pulverizer was purchased from Dongguan Luotai Precision Instruments Co., Ltd.; the JEM-2100F transmission electron microscope was purchased from Hitachi High-Tech Co., Ltd.; the Thermo Nicolet IS5 Fourier transform infrared spectrometer was purchased from Thermo Fisher Scientific, USA; the Bruker AVANCEIII 400M nuclear magnetic resonance spectrometer was purchased from Bruker GmbH, Germany; the Waters Alliance e2695 high performance liquid chromatograph was purchased from Waters Corporation, USA; the NanoBrook 90Plus Zeta particle size analyzer was purchased from Brookhaven, USA; the Infinite F50 microplate reader was purchased from Tecan, Switzerland; and the polymerase chain reaction (PCR) instrument was purchased from Bio-Rad Biomedical Products Co., Ltd.

[0046] Example 1

[0047] This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, including the following steps:

[0048] Accurately weigh 1420 mg stearic acid, 1030 mg N,N'-dicyclohexylcarbodiimide and 48.8 mg 4-dimethylaminopyridine and add them to 20 mL N,N-dimethylformamide (DMF) to dissolve by sonication. Add 1 mL triethylamine to adjust the pH to 9 to obtain an organic phase solution.

[0049] Dissolve 500 mg of sodium hyaluronate in 50 mL of pure water, add 2 g of Tween 80, and stir until homogeneous to obtain an aqueous solution.

[0050] The organic phase solution was added dropwise to the aqueous phase solution at 30°C, and the reaction was allowed to proceed for 48 h. After the reaction, the resulting solution was filtered, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da. Dialysis was performed with pure water for 48 h, with the water changed every 8 h. The dialysate was precipitated with 95% ethanol solution, allowed to stand overnight at 4°C, and the precipitate was collected and freeze-dried to obtain HASA.

[0051] Example 2

[0052] This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, including the following steps:

[0053] Dissolve 500 mg of sodium hyaluronate in 25 ml of pure water, add 2 g of Tween 80 and stir until homogeneous to obtain an aqueous solution.

[0054] Dissolve 600 mg of all-trans retinoic acid (ATRA), 1030 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 61 mg of 4-dimethylaminopyridine (DMAP) in 20 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and sonicate to dissolve, thus obtaining an organic phase solution.

[0055] The organic phase solution was added dropwise to the aqueous phase solution, and the reaction was carried out at 40°C for 48 h. After the reaction was completed, the mixture was filtered, and the filtrate was collected. The filtrate was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, dialyzed with pure water for 48 h, and filtered again. The filtrate was collected and precipitated with 95% ethanol at 4°C. The precipitate was taken, washed with a small amount of anhydrous ethanol, reconstituted with water, and lyophilized to obtain the modified compound HARA.

[0056] Example 3

[0057] This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, including the following steps:

[0058] Dissolve 200 mg of sodium hyaluronate in 25 ml of pure water, add 1 g of Tween 80 and stir until homogeneous to obtain an aqueous solution.

[0059] Dissolve 311.1 mg of all-trans retinoic acid (ATRA), 534 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 25.3 mg of 4-dimethylaminopyridine (DMAP) in 15 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and sonicate to dissolve, thus obtaining an organic phase solution.

[0060] The organic phase solution was added dropwise to the aqueous phase solution, and the reaction was carried out at 40.4 °C for 46.7 h. After the reaction was completed, the mixture was filtered, and the filtrate was collected. The filtrate was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, dialyzed with pure water for 48 h, and filtered again. The filtrate was collected and precipitated with 95% ethanol at 4 °C. The precipitate was washed with a small amount of anhydrous ethanol, reconstituted with water, and lyophilized to obtain the modified compound HARA. The grafting rate of HARA was 0.905 μg / mg.

[0061] Example 4

[0062] This embodiment provides a method for preparing a hyaluronic acid hydrophobic material, including the following steps:

[0063] Dissolve 500 mg of sodium hyaluronate in 25 ml of pure water, add 2 g of Tween 80 and stir until homogeneous to obtain an aqueous solution.

[0064] Dissolve 300 μl of docosahexaenoic acid (DHA), 1030 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 61 mg of 4-dimethylaminopyridine (DMAP) in 20 ml of DMF, add 2 ml of triethylamine to adjust the pH to 9, and sonicate to dissolve, thus obtaining an organic phase solution.

[0065] The organic phase solution was added dropwise to the aqueous phase solution, and the mixture was purged with nitrogen for protection. The reaction was carried out at 40°C for 48 hours. After the reaction was completed, the mixture was filtered, and the filtrate was collected. The filtrate was transferred to a dialysis bag and dialyzed with pure water for 48 hours. The mixture was then filtered again, and the filtrate was collected and precipitated with 95% ethanol at 4°C. The precipitate was then washed with a small amount of anhydrous ethanol, reconstituted with water, and lyophilized to obtain the modified product.

[0066] Example 5

[0067] This embodiment provides a method for preparing hyaluronic acid hydrophobic self-assembled nanocomposite materials, including the following steps:

[0068] 10 mg of HASA prepared in Example 1 was accurately weighed and dissolved in 20 mL of pure methanol solution. Then, a methanol solution containing 0.4 mg of polygalactoside and 0.1 mg of menthol was added to bring the total volume to 30 mL. The mixture was transferred to a round-bottom flask and rotary evaporated at 40 °C and 0.01 MPa for 40 min, forming a uniform white film at the bottom of the flask. 5 mL of pure water was added, and the mixture was rotary heated for 40 min to completely dissolve the film. The solution was then sonicated for 5 min to obtain HASA / PD-Men self-assembled nanoparticles. The prepared solution was stored at 4 °C.

[0069] Example 6

[0070] This embodiment provides a method for preparing hyaluronic acid hydrophobic self-assembled nanocomposite materials, including the following steps:

[0071] Weigh 15 mg of the HASA prepared in Example 1, add 30 ml of methanol, and sonicate for 20 min to dissolve, obtaining a HASA solution. Prepare solutions of 1 mg / ml polysaccharide (PD) and 1 mg / ml menthol (Men) with methanol. Add 0.66 ml of PD solution and 0.34 ml of Men solution to the HASA solution and mix well. Transfer the mixture to a round-bottom flask, evaporate the solvent at 30°C and 0.01 MPa for 30 min, then add 5 ml of pure water and heat at 40°C for 30 min using a rotary evaporator. Sonicate with a probe for 5 min to obtain HASA / PD-Men.

[0072] The particle size of HASA / PD-Men, as determined by a particle size analyzer, was 181.86 nm ± 1.33 nm, with a PDI of 0.04 ± 0.02 and a potential of -18.18 mV ± 0.09 mV. The encapsulation efficiency and drug loading of HASA / PD-Men, as determined by the permeabilization method, were 84.44% ± 0.35% and 5.27% ± 0.04%, respectively.

[0073] Example 7

[0074] This embodiment provides a method for preparing hyaluronic acid hydrophobic self-assembled nanocomposite materials, including the following steps:

[0075] Weigh 15 mg of HARA prepared in Example 2, add 30 ml of methanol, and sonicate for 20 min to dissolve, obtaining a HARA solution. Prepare solutions of 1 mg / ml icariin (ICA) and 1 mg / ml borneol (BO) with methanol, respectively. Add 0.66 ml of ICA solution and 0.34 ml of BO solution to the HARA solution and mix well. Transfer the mixture to a round-bottom flask, evaporate the solvent at 30°C and 0.01 MPa for 30 min, then add 5 ml of pure water and heat at 40°C for 30 min using a rotary evaporator. Sonicate with a probe for 5 min to obtain HARA / ICA-BO.

[0076] The particle size of HARA / ICA-BO, as determined by a particle size analyzer, was 296.52 nm ± 4.56 nm, with a PDI of 0.255 ± 0.021 and a potential of -15.07 mV ± 0.04 mV. The encapsulation efficiency and drug loading of HARA / ICA-BO, as determined by the permeabilization method, were 79.61% ± 0.95% and 4.09% ± 0.04%, respectively.

[0077] Example 8

[0078] 10 mg of HARA prepared in Example 3 was weighed and dissolved in 30 mL of methanol. The solution was sonicated for 20 min to obtain a HARA solution. Icariin (ICA) and borneol (BO) solutions with concentrations of 1 mg / mL and 1 mg / mL respectively were prepared using methanol. 0.5 mL of ICA solution and 0.5 mL of BO solution were added to the HARA solution. The mixture was thoroughly mixed and sonicated for 20 min. The solution was transferred to a round-bottom flask and evaporated to dryness by rotary evaporation at 30°C and 0.01 MPa for 30 min. Then, 5 mL of pure water was added, and the mixture was heated by rotary heating at 40°C for 30 min to dissolve the film. Finally, the film was sonicated for 5 min to obtain HARA / ICA-BO.

[0079] Comparative Example 1

[0080] 10 mg of the HASA prepared in Example 1 was accurately weighed and dissolved in 30 mL of pure methanol solution to obtain a HASA solution, which was then transferred to a 50 mL round-bottom flask. The solution was rotary evaporated at 40 °C and 0.01 MPa for 40 min, forming a uniform white film at the bottom of the flask. Subsequently, 5 mL of pure water was added, and the mixture was rotary heated for 40 min to completely dissolve the film. The solution was then treated with an ultrasonic probe for 5 min to obtain HASA self-assembled nanoparticles. The prepared solution was stored at 4 °C.

[0081] Comparative Example 2

[0082] 10 mg of HASA prepared in Example 1 was accurately weighed and dissolved in 30 mL of pure methanol solution to obtain an HASA solution. Then, 0.4 mg of a methanol solution of polygalactosidine was added to bring the total volume to 30 mL. The solution was transferred to a 50 mL round-bottom flask. The solution was rotary evaporated at 40 °C and 0.01 MPa for 30 min, forming a uniform white film at the bottom of the flask. Then, 5 mL of pure water was added, and the mixture was rotary heated for 30 min to completely dissolve the film. The solution was then treated with an ultrasonic probe for 5 min to obtain HASA self-assembled nanoparticles. The prepared solution was stored at 4 °C. This solution is denoted as HASA / PD.

[0083] Comparative Example 3

[0084] Weigh 10 mg of HARA prepared in Example 3 and dissolve it in 30 mL of methanol. Sonicate the solution for 20 min to obtain a HARA solution. Transfer the HARA solution to a round-bottom flask and evaporate the solvent by rotary evaporation at 30°C and 0.01 MPa for 30 min. Then add 5 mL of pure water and heat at 40°C for 30 min to dissolve the film. Finally, sonicate the film with a probe for 5 min to obtain HARA self-assembled nanoparticles.

[0085] The samples prepared in this invention were characterized and tested according to the following methods.

[0086] (1) Morphological characterization of nanoparticles

[0087] The particle size and polydispersity index (PDI) of the samples were determined using a particle size analyzer. The morphology of the nanoparticles was observed by transmission electron microscopy (TEM). 30 μL of freshly prepared nanoparticle solution was dropped onto a copper grid, allowed to stand and evaporate the water, stained with phosphotungstic acid solution, and dried under an infrared lamp for 10 min before being observed under a microscope.

[0088] (2) Determination of Polygonin content

[0089] The content of polysaccharide was analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm), flow rate 1 mL / min, mobile phase A was 23% acetonitrile, mobile phase B was 77% pure water, column temperature 30℃, detection wavelength 306 nm, injection volume 10 μL, and run time 20 min.

[0090] Preparation and detection of standard solutions: Accurately weigh 10.07 mg of polygalactoside standard and dissolve it in 10 mL of methanol. Perform serial dilutions to obtain standard solutions with concentrations ranging from 1.12 to 56 μg / mL. Inject the standard solutions into the liquid chromatography system using the chromatographic method described above. Establish a standard curve with the standard solution concentration as the independent variable X (μg / mL) and the peak area as the dependent variable Y.

[0091] Sample solution preparation and detection: Take 1 mL of drug-loaded nanoparticle solution, add methanol at a 1:1 ratio for pyrolysis, filter through a 0.45 μm filter membrane, and inject the filtrate into the liquid chromatogram under the chromatographic conditions described above. Substitute the obtained peak area into the standard curve to calculate the mass of polygalactosidine in the sample solution.

[0092] (3) Determination of encapsulation efficiency and drug loading

[0093] 3.1 Determination of Polygonin

[0094] Using polygalactoside as an antioxidant, the encapsulation efficiency of drug-loaded nanoparticles was determined by centrifugation. 1 mL of the drug-loaded nanoparticle solution was taken, and 1 mL of methanol was added for lysis. The total mass of polygalactoside (m) was determined. Another 1 mL of the drug-loaded nanoparticle solution was taken, centrifuged at 12000 r / min for 30 minutes, and the supernatant was collected to determine the mass of free polygalactoside (m0).

[0095] Encapsulation efficiency of polydipsia glycoside = [(total mass of polydipsia glycoside - mass of free polydipsia glycoside) / mass of polydipsia glycoside] × 100%.

[0096] Drug loading = [(total mass of polydipsia glycoside - mass of free polydipsia glycoside) / sum of mass of carrier and drug] × 100%.

[0097] 3.2 Determination of Icariin

[0098] The encapsulation efficiency of HARA / ICA-BO was investigated using icariin as the evaluation standard. The icariin content was determined by HPLC under the following chromatographic conditions: a C18 column, a mobile phase of acetonitrile and 30 mM potassium dihydrogen phosphate solution, gradient elution (0–10 min, 20% acetonitrile; 11–60 min, 35% acetonitrile), a flow rate of 1 mL / min, a wavelength of 270 nm, a column temperature of 30℃, and an injection volume of 10 μL. ICA standard solutions of different concentrations were prepared, and a standard curve was plotted by determining the peak area using the above method. The ICA content was then calculated from the standard curve.

[0099] Using ICA (inductively coupled plasma) as the evaluation standard, the encapsulation efficiency and drug loading of drug-loaded nanoparticles were assessed. 1 mL of HARA / ICA-BO solution was filtered through a 0.45 μm filter membrane, methanol was added at a 1:1 ratio, and the mixture was sonicated for 5 min to lyse. The ICA content was then analyzed by liquid chromatography and recorded as m. 1 mL of HARA / ICA-BO solution was also filtered, methanol was added at a 1:1 ratio, and the mixture was sonicated for 5 min to lyse. The ICA content was then analyzed by liquid chromatography and recorded as M. Encapsulation efficiency = [m / M] × 100%; Drug loading = [m / total mass] × 100%.

[0100] (4) Cell viability test

[0101] 4.1 Determination of Polygonin

[0102] HT22 cells were used as a model, and the cytotoxicity of blank nanoparticles, HASA / PD-Men, polygalactoside, and menthol was evaluated using the MTT assay. Cells were seeded in 96-well plates at a density of 10,000 cells / well and incubated at 37°C with 5% CO2 for 24 h. After 24 h, the plates were removed, and culture medium containing different concentrations of the drug was added to each well. The plates were then returned to the incubator. After another 24 h, 0.5% MTT solution was added, and the plates were incubated for 4 h. The solution was then aspirated, and DMSO solution was added to dissolve the crystals. The absorbance was measured at 490 nm.

[0103] Cell viability = [(sample well absorbance - blank absorbance) / (control well absorbance - blank absorbance)] × 100%

[0104] 4.2 Determination of Icariin

[0105] BV2 cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin-drug antibiotics, and incubated at 37°C in a 5% CO2 incubator. BV2 cells in the logarithmic growth phase were digested, centrifuged, and seeded into 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 Cells were seeded at 1000 cells per well and cultured overnight. Grouping was as follows: Blank group contained only an equal volume of culture medium; control group contained only cells and no drug; experimental groups contained cells and drug concentrations as shown below:

[0106] a) The dosage concentrations of free polygalactoside were 0.15 μM, 0.35 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM.

[0107] b. The dosage concentrations of free menthol are: 0.15 μM, 0.35 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM.

[0108] c. The drug 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, and 100 μg / mL.

[0109] d. The dosage concentrations of HASA / PD-Men were: 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, and 64 μM.

[0110] After culturing for 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 1 hour. The optical density (OD value) was then measured at 450 nm. Cell viability was calculated as: (Experimental group optical density - Blank group optical density) / (Control group optical density - Blank group optical density) × 100%.

[0111] (5) Expression test of polygalactoside antioxidant mRNA

[0112] Cells were seeded at a density of 60 W / well in 6-well plates and incubated for 24 h. The cells were divided into 5 groups for the experiment: a blank control group containing only cells and culture medium; a control group receiving a certain concentration of glutamate for induction; and experimental groups receiving high, medium, and low reagents, which included both the inducing agent glutamate and the drug HASA / PD-Men. After adding the inducing agents, both the control and experimental groups were incubated for 6 h before the different concentrations of the drug were added.

[0113] Total RNA was extracted from cells using the Trizol method and reverse transcribed into cDNA. The expression changes of antioxidant genes such as nuclear factor-erythrocyte kinase 2-related gene 2 (Nrf2), superoxide dismutase (SOD) gene, and heme oxygenase 1 (HO-1) were analyzed by RT-qPCR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for quantification.

[0114] (6) Detection of the expression level of icariin anti-inflammatory factor

[0115] After culturing cells according to the method described in section 4.2, an inflammation model was established using LPS. BV2 cells in the logarithmic growth phase were harvested, digested, centrifuged, and then subjected to a 6×10⁻⁶ ppm solution. 5 Cells were seeded per well in 6-well plates and incubated for 12 h. The cells were then divided into 5 groups for the experiment, as follows: Control group: cells cultured normally for 12 h; Model group: treated with 1 μg / mL LPS for 6 h; Experimental group: three dosage groups (low, medium, and high) with concentration gradients of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, respectively, treated for 6 h and then incubated with 1 μg / mL LPS for another 6 h.

[0116] Cellular RNA was extracted using the Trizol method, and after determining the RNA concentration, it was reverse transcribed into cDNA. Using GAPDH as an internal control, 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 using quantitative real-time PCR.

[0117] (7) Statistical analysis

[0118] Cell viability and antioxidant mRNA expression assay results were analyzed using Graphpad Prism software, and the results are expressed as mean ± standard deviation. One-way ANOVA was used to compare the two groups of data, and P < 0.05 was considered statistically significant.

[0119] The carboxyl group in stearic acid undergoes an esterification reaction with the hydroxyl group of the N-acetylglucosamine unit in HA to produce hyaluronic acid stearate (HASA). Figure 1 Infrared absorption spectra of HA and HASA prepared in Example 1. HASA at 1735 cm⁻¹. -1 A new absorption peak appeared at 2800-2950 cm⁻¹, attributed to the stretching vibration of the carbonyl group, indicating the formation of an ester group. Compared to unmodified HA, HASA showed a higher absorption peak in the 2800-2950 cm⁻¹ range. -1 The CH stretching vibration absorption peak in the region was significantly enhanced, indicating that long-chain alkyl groups were successfully introduced.

[0120] Figure 2 The HASA and HA prepared in Example 1 1 ¹H-NMR spectra. In the HA spectrum, the peak at δ 2.02 ppm represents the proton peak of the methyl group, and the peaks at δ 3.2-4.0 ppm correspond to the protons on the polysaccharide carbon backbone. In the HASA spectrum, a new peak appears at δ 0.87 ppm, representing the methyl hydrogen signal at the end of the stearic acid chain, while the peak at δ 1.27 ppm corresponds to the methylene hydrogen signal within the stearic acid chain. These results indicate that stearic acid was successfully grafted onto the HA molecule.

[0121] Figure 3 Infrared spectra of HA and HARA prepared in Example 3. The characteristic peak representing ATRA in the left image is at 2932 cm⁻¹. -1 The characteristic absorption peak of methyl group and 1658 cm⁻¹ -1 The characteristic absorption peak of C=O in the carboxyl group; 2855 cm⁻¹ in the right figure. -1 The peak value for the stretching vibration of CH in HA is 3392 cm⁻¹. -1 The peak at this location belongs to the hydroxyl group (OH). Comparison revealed that the peak area of ​​the OH peak decreased after the reaction, indicating that the reaction occurred successfully and consumed a large number of OH groups.

[0122] Figure 4 The UV spectra of ATRA, HARA prepared in Example 3, and HA at 350 nm are shown. ATRA exhibits a maximum absorption peak at 350 nm; HARA also shows an absorption peak at 350 nm; HA shows no absorption peak at 350 nm. Therefore, the appearance of an absorption peak at 350 nm after the reaction of ATRA and HA indicates that the synthesis reaction was successful.

[0123] HASA self-assembled nanoparticles (Comparative Example 1) were prepared by thin-film hydration. The average particle size and particle size distribution (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 uniformly distributed and had good dispersibility (see Table 1).

[0124] Table 1. Particle size and PDI measurement results of nanoparticles

[0125]

[0126] TEM images further revealed the morphological characteristics of the nanoparticles. Observations showed that the blank nanoparticles exhibited a regular spherical structure with a particle size of approximately 15 nm (see...). Figure 5 This differs from the particle size measured by the particle size analyzer, possibly because the sample was in a dry state during TEM analysis, while the particle size analyzer measures the hydrated state in solution. Furthermore, the TEM images show that the nanoparticles have smooth surfaces and no obvious aggregation, further verifying the high uniformity of the nanoparticles.

[0127] Compared to the blank nanoparticles, the average particle sizes of HASA / PD and HASA / PD-Men increased to 276 nm and 388 nm, respectively. This indicates that drug encapsulation increased the hydrophobic core size of the nanoparticles, while also affecting their morphology and stability. The slight change in PDI also suggests the influence of drug encapsulation on nanoparticle dispersibility.

[0128] The high encapsulation efficiency and suitable drug loading of the HASA / PD-Men prepared in Example 5 demonstrate that HASA effectively isolates hydrophobic drugs from the external environment, which plays an important role in improving drug bioavailability and prolonging the duration of drug efficacy.

[0129] The mass of polygalactosin in the 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), with a regression equation of y = 267315x - 320991, a linear range of 1.12-56 μg / mL, and a correlation coefficient R² = 0.9998.

[0130] Using polygalactoside as an antioxidant, 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 demonstrate the effectiveness and stability of HASA / PD-Men in encapsulating hydrophobic drugs.

[0131] Table 2 Encapsulation efficiency and drug loading of HASA / PD and HASA / PD-Men

[0132]

[0133] The MTT assay was used to conduct cytotoxicity tests on HT22 cells to evaluate the effects of different concentrations of resveratrol, menthol, blank nanoparticles, and HASA / PD-Men prepared in Example 5 on cell viability. The results showed that when the resveratrol concentration in HASA / PD-Men reached 16 μmol / L, the cell viability exceeded 85%; while in the same concentrations of blank nanoparticles and resveratrol, the cell viability was above 85% and 95%, respectively (see [link to study]). Figure 6 These data indicate that HASA / PD-Men exhibits no significant toxicity to cells within a concentration range of 16 μmol / L, demonstrating good safety.

[0134] The antioxidant mechanism of HASA / PD-Men, prepared in Example 5, was further explored below using HASA / PD-Men as an example. The expression levels of antioxidant-related genes such as Nrf2, SOD, and HO-1 were analyzed using RT-qPCR. The results showed that, compared with the control group, HASA / PD-Men significantly upregulated the mRNA expression levels of Nrf2, SOD, and HO-1 at low to moderate concentrations, demonstrating a significant antioxidant gene activation effect (see [link to relevant documentation]). Figure 7 Especially at a concentration of 1 μmol / L, the expression of these genes reached its peak, indicating that HASA / PD-Men at this concentration has the best antioxidant effect.

[0135] Nrf2 is a major regulator of intracellular antioxidant responses; its activation enhances cellular antioxidant capacity and reduces ROS-induced cellular damage. HASA / PD-Men significantly upregulated Nrf2 expression at a concentration of 1 μmol / L, likely because the resveratrol released from the nanoparticles effectively activated the Nrf2 pathway, enhancing cellular antioxidant defense. SOD, an antioxidant enzyme, converts reactive oxygen species produced by cellular metabolism into hydrogen peroxide, thereby reducing ROS-induced cellular damage. HO-1, through metabolism, produces antioxidant products such as bilirubin and carbon monoxide, further protecting cells from oxidative stress. These data collectively indicate that HASA / PD-Men can enhance cellular antioxidant capacity through multiple pathways and possesses potential neuroprotective effects.

[0136] The cell viability was studied below using HARA / ICA-BO prepared in Example 8 as an example. Figure 8 It can be seen that the effect of icariin aromatic nanoparticles on the activity of BV2 cells is as follows: Figure 8 As shown, icariin did not exhibit toxicity to BV2 cells at concentrations ranging from 0 to 20 μmol / L; borneol also showed no toxicity to BV2 cells at the highest administered concentration. HARA and HARA / ICA-BO did not produce significant toxic side effects on cells within the experimental range.

[0137] 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 follows: Figure 9 As shown, the expression of inflammatory factors in the model group was significantly increased compared with the control group. Compared with the model group, different doses of HARA / ICA-BO nanoparticles significantly inhibited the mRNA expression of IL-6, COX-2, TNF-α, and iNOS-2. The inhibitory effect on LPS-induced inflammation was strongest at a drug concentration of 10 μmol / L, indicating that HARA / ICA-BO at this concentration has the best anti-inflammatory effect.

[0138] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0139] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite material, characterized in that, Includes the following steps: Using N,N-dimethylformamide as a solvent, add C 15 ~C 25 Long-chain fatty acids, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were mixed, and the pH was adjusted to 9-10 to obtain an organic phase solution. Using water as a solvent, hyaluronic acid and Tween-type surfactants were added and stirred until homogeneous to obtain an aqueous solution; When an aqueous solution and an organic solution are mixed, the hydroxyl groups of hyaluronic acid in the aqueous solution react with the hydrophobic carboxylic acid compound C. 15 ~C 25 The carboxyl groups in long-chain fatty acids undergo esterification, and after the reaction is completed, hyaluronic acid hydrophobic materials are obtained by alcohol precipitation. The hyaluronic acid hydrophobic material was dissolved in methanol to obtain a first solution; The flavonoid glycosides and aromatics were dissolved in methanol to obtain a second solution; The first and second solutions were mixed and then subjected to rotary evaporation to obtain a thin film; After dissolving the film in water and ultrasonically treating it, a hyaluronic acid hydrophobic self-assembled nanocomposite material was obtained. The flavonoid glycosides are polygalactoside or icariin, and the aromatics are menthol or borneol.

2. The method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite material according to claim 1, characterized in that, The esterification reaction is carried out at a temperature of 30℃~35℃ for 40h~48h.

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

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

1.

6. The method for preparing a hyaluronic acid hydrophobic self-assembled nanocomposite material according to claim 1, characterized in that, The mass ratio of hyaluronic acid hydrophobic material to flavonoid glycoside natural products is 15:0.6~0.7; the mass ratio of flavonoid glycoside natural products to fragrance is 15:0.3~0.

4.

7. A hyaluronic acid hydrophobic self-assembled nanocomposite material prepared by the preparation method according to any one of claims 1-6.

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