A method for preparing co-assembled nanoparticles

Abstract

The application relates to a preparation method of co-assembled nanoparticles, and belongs to the field of nanomaterials. A new type of nanoparticle (MUR NPs) is developed by taking hydrophobic core ursolic acid and hydrophilic shell matrine as a delivery system, aiming at improving the water solubility and bioavailability of resveratrol. And the NSMs compound with good biological activity but unable to be used for drug delivery can be applied. Scanning electron microscopy (SEM), x-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectrophotometry are used to characterize the properties and structure of the molecular interaction in the MUR NPs. In addition, the MUR NPs have good physicochemical stability, have sustained gastrointestinal digestion release characteristics, and improve in-vitro antioxidant and bioavailability. The study may contribute to the development of RES oral delivery systems and the application of hydrophobic active molecules in the field of functional foods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials, and particularly relates to a preparation method of co-assembled nanoparticles. BACKGROUND

[0002] Resveratrol (RES) is a natural, small-molecule bioactive compound that can be obtained from plants. Studies have shown that resveratrol has a series of biological activities such as anti-inflammatory, antioxidant, and liver protection, and resveratrol is listed as one of the "100 most popular effective anti-aging substances" internationally. Resveratrol has strong physiological activity, but due to its poor water solubility, it is easy to oxidize and decompose, and has a fast metabolism in the body, which greatly limits its bioavailability in the body. However, the application of RES in the field of functional foods is limited due to its molecular structure defects, including instability, poor water solubility, short half-life in the body, and low bioavailability. Therefore, many scholars have tried to design and construct nano delivery carriers to overcome these limitations by encapsulating and delivering resveratrol.

[0003] Nanocarrier drug delivery system is a drug transport form that uses nanoparticles as drug carriers. Nanocarrier drug delivery system is a new type of nanocarrier made of natural or synthetic polymer materials. In the nanocarrier drug delivery system, drug molecules can be integrated with the carrier to form nanoparticles through chemical methods such as covalent coupling or physical methods such as wrapping and adsorption. Due to the small size of nanoparticles, they can easily penetrate biological membranes and be transported in bulk in the gastrointestinal tract, thus effectively improving the bioavailability of poorly soluble drugs. With the development of nanotechnology, nanocarrier drug delivery system has become one of the important frontiers and hotspots in the field of medical research.

[0004] Matrine is an alkaloid extracted from the fruit or aerial part of Sophora flavescens, which has anti-inflammatory, anti-fibrosis, anti-tumor and anti-viral and other important pharmacological effects. Matrine is a typical representative of matrine alkaloids, which is used as a hepatoprotective drug and an immunomodulator. Ursolic acid (UA) is a pentacyclic triterpenoid compound. Ursolic acid is a bioactive substance widely present in Fructus Evonymi, which has obvious antioxidant activity and is a natural antioxidant. Due to its poor water solubility and low dissolution rate, the bioavailability is limited. Liu et al. prepared ursolic acid nanoparticles and freeze-dried them into powder, and compared their antioxidant properties with those of ursolic acid. By combining two compounds with pharmacological activity through co-assembly strategy, a nano-drug delivery platform based on multi-component NSMs is constructed, which is expected to obtain a drug delivery system that can synergistically play a role. Therefore, we selected triterpenoid ursolic acid and water-soluble alkaloid matrine as research objects, and studied the nanoparticles formed by co-assembly of the two components and loaded with resveratrol. We are encouraged to use co-assembly nanotechnology to improve the drug loading capacity of water-soluble drugs and improve the bioavailability of lipid-soluble drugs. The co-assembly of nanomedicine is simple in synthesis, has a high drug loading content, and can achieve highly stable drug delivery without any carrier. Matrine and ursolic acid have good functional properties, which can form stable complexes and have good effects on encapsulation and protection of RES. SUMMARY

[0005] The purpose of the present application is to solve the problems of poor water solubility, short in vivo half-life and low bioavailability of resveratrol, and to provide a preparation method of plant-derived matrine and ursolic acid co-assembly nanoparticles for packaging and transporting hydrophobic nutritional molecules resveratrol, and to construct a carrier-free, biocompatible, biodegradable and low-cost nano self-assembly. This attempt will provide a reference for the discovery and optimization of supramolecular self-delivery systems, and also provide a new idea for the effective application of RES in the field of functional foods.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of co-assembly nanoparticles, the method comprises the following steps:

[0008] Step one: dissolve ursolic acid (UA) in dimethyl sulfoxide;

[0009] Step two: dissolve matrine (MA) in an aqueous solution;

[0010] Step three: add the ursolic acid solution obtained in step one to the matrine aqueous solution obtained in step two;

[0011] Step four: stir at a speed of 400 rmp to 600 rmp for 10 min to 60 min;

[0012] Step five: centrifugation at 10000-15000 rpm for 10-30 min.

[0013] Further, in step one, the concentration of ursolic acid (UA) in dimethyl sulfoxide is 5-50 mg / mL.

[0014] Further, in step two, the concentration of matrine (MA) in water is 1-50 mg / mL.

[0015] Further, in step three, the volume ratio of the ursolic acid (UA) solution to the matrine (MA) aqueous solution is 50-100:1.

[0016] A preparation method of a co-assembled nanoparticle, the method comprising:

[0017] Step one: dissolving ursolic acid (UA) in dimethyl sulfoxide;

[0018] Step two: dissolving resveratrol (RES) in dimethyl sulfoxide;

[0019] Step three: dissolving matrine (MA) in an aqueous solution;

[0020] Step four: mixing the ursolic acid (UA) solution and the resveratrol (RES) solution, and then adding them to the matrine aqueous solution;

[0021] Step five: stirring at 400-600 rpm for 10-60 min.

[0022] Step six: centrifugation at 10000-15000 rpm for 10-30 min.

[0023] Further, in step one, the concentration of ursolic acid (UA) in dimethyl sulfoxide is 5-50 mg / mL.

[0024] Further, in step two, the concentration of resveratrol (RES) in dimethyl sulfoxide is 5-50 mg / mL.

[0025] Further, in step three, the concentration of matrine (MA) in water is 1-50 mg / mL.

[0026] Further, in step four, the volume ratio of the ursolic acid (UA) solution, the resveratrol (RES) solution, and the matrine (MA) aqueous solution is 50-100:50-100:1.

[0027] The present application has the following beneficial effects over the prior art:

[0028] (1) The triterpenoid ursolic acid and alkaloid matrine in the present application are the main effective components of traditional Chinese medicine and natural plant medicine, and have multiple therapeutic and tissue protective effects, including antioxidant, anti-inflammatory, and heart protection.

[0029] (2) The present application enhances the bioavailability of resveratrol to enhance its biological activity.

[0030] (3) The present application highlights the potential of natural small molecules in the field of nutraceuticals, food and drugs, in order to provide data support for the practical application of food and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Scanning electron micrograph of UA co-assembled nanoparticles of Example 1;

[0032] Figure 2 Scanning electron micrograph of MA co-assembled nanoparticles of Example 1;

[0033] Figure 3 Scanning electron micrograph of MU co-assembled nanoparticles of Example 1;

[0034] Figure 4 Scanning electron micrograph of MUR co-assembled nanoparticles of Example 2;

[0035] Figure 5 Contact angle diagram of MUR co-assembled nanoparticles of Examples 1, 2;

[0036] Figure 6 Contact angle measurement value diagram of MUR co-assembled nanoparticles of Examples 1, 2;

[0037] Figure 7 UV spectrum of MUR co-assembled nanoparticles of Example 2;

[0038] Figure 8 UV spectrum of MUR co-assembled nanoparticles MURNPs of Example 2 after adding SDS;

[0039] Figure 9 UV spectrum of MUR co-assembled nanoparticles MURNPs of Example 2 after adding urea;

[0040] Figure 10 SEM image of MUR co-assembled nanoparticles MURNPs of Example 2 after adding SDS;

[0041] Figure 11 SEM image of MUR co-assembled nanoparticles MURNPs of Example 2 after adding urea;

[0042] Figure 12The image shows the FT-IR spectrum of the MUR co-assembled nanoparticles from Example 2.

[0043] Figure 13 The CD chromatogram of the MUR co-assembled nanoparticles in Example 2 is shown below.

[0044] Figure 14 The XRD pattern of the MUR co-assembled nanoparticles in Example 2;

[0045] Figure 15 This is a diagram showing the in vitro release results of the MUR co-assembled nanoparticles in Example 2;

[0046] Figure 16 This is a diagram showing the in vitro gastrointestinal release results of the MUR co-assembled nanoparticles in Example 2;

[0047] Figure 17 The figure shows the experimental results of DPPH antioxidant activity of MUR co-assembled nanoparticles in Example 2;

[0048] Figure 18 The figure shows the experimental results of the ABTS antioxidant activity of MUR co-assembled nanoparticles in Example 2;

[0049] Figure 19 This is a diagram showing the results of an in vitro cellular uptake experiment of MUR co-assembled nanoparticles in Example 2; Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0051] This invention innovatively prepares RES-loaded MAUA nanoparticles (MURNPs) to improve the water solubility, bioavailability, and bioactivity of RES, and optimizes the RES addition ratio. Simultaneously, the structural characteristics and intermolecular forces of the nanoparticles were analyzed. FT-IR, XRD, UV-VIS, and SEM results indicate the presence of hydrogen bonds, hydrophobic interactions, and electrostatic interactions within the composite nanoparticles. Furthermore, its gastrointestinal digestive sustained-release properties and in vitro antioxidant activity were investigated. This provides new insights for the effective application of RES in functional foods and also offers a theoretical basis for the high-value utilization of fat-soluble and water-soluble natural small molecules.

[0052] This invention develops novel nanoparticles (MURNPs) using a hydrophobic nucleoursolic acid and a hydrophilic shell matrine as a delivery system, aiming to improve the water solubility and bioavailability of resveratrol. It also enables the application of NSMs compounds with good biological activity but unsuitable for drug delivery. The properties and structure of the molecular interactions within the MURNPs were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Furthermore, the MURNPs exhibit good physicochemical stability, sustained gastrointestinal digestion and release characteristics, and improved in vitro antioxidant and bioavailability. This research may contribute to the development of oral resveratrol delivery systems and the application of hydrophobic active molecules in functional foods. In addition, the antioxidant activity of resveratrol is significantly enhanced through nanoencapsulation, and the prepared nanoparticles can improve the stability of resveratrol in a simulated gastrointestinal environment and delay its release. In vitro cellular evaluation showed that MURNPs exhibited higher cellular uptake. This invention uses a simple and convenient method to prepare co-assembled nanoparticles, reducing toxic side effects and increasing drug loading. Therefore, this invention provides new ideas for the effective application of RES in the field of functional foods, and it also provides a theoretical basis for the high-value utilization of fat-soluble and water-soluble natural small molecules.

[0053] Example 1

[0054] A method for preparing nanoparticles co-assembled with alkaloids and terpenoids, wherein the alkaloid is matrine, the terpenoid is ursolic acid, the volume ratio of water to dimethyl sulfoxide is 1:100, the content of matrine is 1 mg / mL, and the content of ursolic acid is 15 mg / mL.

[0055] The method for preparing the matrine and ursolic acid co-assembled nanoparticles of Example 1 was carried out according to the following steps:

[0056] Step 1: Dissolve 15 mg of ursolic acid in 1 mL of dimethyl sulfoxide and sonicate until the solid powder is completely dissolved;

[0057] Step 2: Add 0.01 mL of ursolic acid solution to 1 mL of matrine solution and stir at 400 rpm for 10 min. Centrifuge at 10000 rpm for 20 min.

[0058] Example 2

[0059] A method for preparing matrine, ursolic acid and resveratrol nanoparticles, wherein the volume ratio of water to dimethyl sulfoxide is 1:100, the content of matrine is 1 mg / mL, the content of ursolic acid is 15 mg / mL, and the content of resveratrol is 10 mg / mL.

[0060] Step 1: Dissolve 15 mg of ursolic acid and 10 mg of resveratrol in 1 mL of dimethyl sulfoxide, and sonicate until the solid powder is completely dissolved;

[0061] Step 2: Add 0.01 mL of ursolic acid and resveratrol solution to 1 mL of matrine solution, and stir at 400 rpm for 10 min. Centrifuge at 10000 rpm for 20 min.

[0062] Testing and experimentation:

[0063] Experiment 1: Microstructure Testing

[0064] The matrine and ursolic acid nanoparticles obtained in Examples 1-2 were co-assembled. After the matrine, ursolic acid, and resveratrol nanoparticles were evenly dispersed in water, a drop of the solution was added to aluminum foil. After evaporating the water, the aluminum foil containing the sample was fixed with conductive adhesive and then sputtered with gold. The microstructure of the hydrogel was then observed using a scanning electron microscope. Figure 1 As shown.

[0065] Depend on Figures 1-4 It can be seen that all matrines and ursolic acid can co-assemble with curcumin. The results show that self-assembled UA can form spherical nanoparticles, while MA has no obvious shape. MAUA co-assembly forms relatively rounded spherical nanoparticles; similarly, MURNPs form relatively rounded nanospheres.

[0066] Experiment 2, Contact Angle Test:

[0067] The matrine and ursolic acid nanoparticles obtained in Examples 1-2 were co-assembled. After the matrine, ursolic acid and resveratrol nanoparticles were uniformly dispersed in water, the contact angle was measured.

[0068] Depend on Figures 5-6 It can be seen that the θ values ​​of MA, UA, and RES alone are 29.5°, 142.6°, and 133.9°, respectively, indicating that MA is a hydrophilic molecule, while the latter two are hydrophobic molecules, exhibiting complete water migration. However, when they are co-assembled, the prodrug MURNPs have an θ value of 45.4°, exhibiting hydrophilic properties. Due to the interaction between UA and RES and MA, amphiphilic nanoparticles are ultimately formed with the hydrophilic end facing outward and the hydrophobic end facing inward. One end is more compatible with polar solutes (hydrophilic end), and the other end is more compatible with nonpolar solutes (hydrophobic end).

[0069] Experiment 3: Ultraviolet Characterization

[0070] The matrine obtained in Examples 1-2 was co-assembled with ursolic acid into nanoparticles. The matrine, ursolic acid and resveratrol were co-assembled into nanoparticles. After being evenly dispersed in water, the nanoparticles were scanned at all wavelengths using an ultraviolet spectrometer.

[0071] Depend on Figure 7 It can be seen that ultraviolet absorption spectroscopy is an effective means of qualitatively studying molecular conformational changes, with the maximum absorption time of resveratrol at 312 nm. With the addition of resveratrol, the maximum absorption spectrum of resveratrol in MURNPs shifts to a shorter wavelength (from λmax = 312 nm to λmax = 300 nm), exhibiting a blue shift. UV-Vis absorption measurements reveal the interaction between resveratrol and MAUA, indicating that the steric hindrance of MAUA affects the π-π* transition of the benzene ring in the RES molecular structure. This significant change in absorption wavelength may also be due to non-covalent effects during assembly (hydrogen bonding, electrostatic and hydrophobic interactions), but more information is needed to explore structural changes and confirm the formation of the complex.

[0072] Experiment 4: Characterization of Forces

[0073] The matrine, ursolic acid, and resveratrol obtained in Example 2 were co-assembled into nanoparticles, dispersed evenly in water, and then SDS and urea were added respectively. A full-wavelength scan was performed using a UV spectroscopy instrument. A drop of the solution was then placed on aluminum foil. After evaporating the moisture, the sample was observed using a scanning electron microscope to examine the microstructure of the nanoparticles. Figures 8-11 As shown.

[0074] Depend on Figures 8-11 We can explain the forces that cause the assemblies to assemble. We disrupted the assemblies using urea and SDS. SDS is an effective anionic detergent; for example, DNA and proteins often bind through electrostatic attraction or coordinate bonds, and SDS can break these valence bonds. Urea has the ability to form hydrogen bonds; at a concentration of 8M, urea can break the hydrogen bonds in water, reducing hydrophobic interactions. A certain redshift occurred in the maximum UV absorption wavelength (from λmax 300nm to λmax 304nm), indicating that the NPs underwent a certain degree of disassembly, further proving the existence of electrostatic interactions in the assemblies. SEM results also showed that after adding SDS, MURNFs changed from nanospheres to irregular sheets, proving that electrostatic interactions were involved in the assembly of NPs. Subsequently, adding urea to the MURNFs caused a certain redshift in the maximum UV absorption wavelength (from λmax 300nm to λmax = 303nm). The SEM results were the same as those after adding SDS; the MURNFs changed from nanospheres to a mixture of spherical and irregular sheets, proving that hydrogen bonding was involved in the assemblies. In summary, we can conclude that MUR NPs exhibit weak bonding interactions, including at least electrostatic and hydrogen bonding. Therefore, we hypothesize that MAUA and RES are assembled within the nanoparticles through hydrogen bonding and hydrophobic interactions. The outer layer forms the MAUA molecular layer structure through hydrophobic interactions, hydrogen bonding, and electrostatic interactions.

[0075] Experiment 5, FT-IR test:

[0076] The sample obtained in Example 2 was pulverized into particles using potassium bromide (KBr). The Fourier transform infrared spectroscopy scan range was 500 to 4000 cm⁻¹. -1 The resolution is 4cm. -1 .

[0077] Figure 12 The results confirmed that the main absorption band of resveratrol is 3280 cm⁻¹. 1 (OH tensile test), 3019 (H-Csp3 tensile test), 1606, 1587, 1384 cm -1 These peaks correspond to three characteristic strong bands: CC aromatic double bond stretching, CC olefin stretching, and CO stretching, respectively. These peaks were observed in all MUR nanoparticles, confirming the presence of RES. At 1717 cm-1... -1 The peak at that point corresponds to the stretching vibration of the carbonyl group in the carboxyl group of UA. However, after co-assembly, the peaks of the carboxyl groups of MURNPs all shift to a lower wavenumber (1689 cm⁻¹). -1 This is likely due to the electrostatic interaction between the carboxyl group and the quaternary ammonium salt ion. The electrostatic interaction results in a weaker chemical bond, slowing down the stretching vibration frequency of the C=O double bond. The carboxyl peak shifts like a low wavenumber, further demonstrating the strengthening of intermolecular hydrogen bonds.

[0078] Experiment 6: Circular Dichroism Chromatography Test

[0079] The chiral signals formed by MURNPs in water were further investigated on the samples obtained in Example 2. To study the molecular assembly process in greater depth, CD was measured using a Pistarπ-180 spectrometer. The light source was a 150W xenon lamp.

[0080] Figure 13 The results confirmed the absence of peaks in MUR C NPs, indicating weakened chirality. These results suggest that when chiral molecules are packed into a spherical shape, the molecules forming the assembly exhibit an amorphous arrangement.

[0081] Experiment 7, XRD test:

[0082] The co-assembled nanoparticle powder sample obtained in Example 2 was placed on a zero-background plate, and then Cu Kα radiation generated at 40 kV and 38 mA was used to scan the diffraction angle 2θ in the range of 5-60° at a rate of 5° / min. The experimental results are as follows. Figure 14 As shown.

[0083] from Figure 14The XRD patterns of MA, UA, RE, and MURNPs are shown. Notably, MA, UA, and RES exhibit numerous sharp diffraction peaks, indicating their crystalline structure. Conversely, MURNPs assembled from monomers show softer diffraction peaks, suggesting a typical amorphous structure. Furthermore, after MURNP formation, the distinctive crystalline diffraction peaks of RES disappear, leaving only the amorphous diffraction pattern of the nanoparticles. Previous studies have shown that the characteristic diffraction peaks of small molecules with crystalline structures can be masked when encapsulated within amorphous macromolecules such as polysaccharides and proteins. For example, in a study involving corn protein-propylene glycol alginate-tea saponin composite nanoparticles containing RES, the disappearance of the characteristic diffraction peaks of RES confirmed its encapsulation within the nanoparticles. In conclusion, the disappearance of XRD diffraction peaks in the nanoparticles indicates the successful loading of RES within the MURNPs.

[0084] Experiment 8: In vitro release test:

[0085] The co-assembled nanoparticles obtained in Example 2 were dispersed in 2 mL of dispersion medium and then placed in a dialysis membrane bag (molecular weight cutoff = 3500 Da). 500 mL of PBS containing 0.05% Tween 80 (pH 7.4 and 5.6) was used, the temperature was maintained at 37 °C, and the mechanical stirring speed was 90 rpm. Samples (0.1 mL) were drawn from the dialysis bag at specified time intervals, and the drug concentration was determined by high-performance liquid chromatography (HPLC). Subsequently, time-concentration release curves were constructed.

[0086] from Figure 15 It can be seen that in PBS at pH 5.6 and pH 7.4, free RES degraded by approximately 50.66% after 4 h and 69.31% after 24 h. The stability of the RES-loaded composite nanoparticles was significantly improved, with a degradation rate of 32.29% after 4 h and 43.59% after 24 h. Quite similar RES degradation profiles were observed in various types of nanoparticles. The improved RES stability may be due to its being trapped within the hydrophobic interior of the nanoparticles, inhibiting deprotonation and decomposition by hindering direct contact with the aqueous phase. At pH 5.6, after 4 h, free RES and MURNPs degraded by approximately 59.50% and 30.32%, respectively, and after 48 h, by 84.17% and 48.29%, as reported in previous studies on other nutrients. This suggests that the formation of MURNPs composite nanoparticles is an effective means of inhibiting RES degradation under physiological conditions. Nanoparticles enter cells through cell division, a process in which nanoparticles compete hydrophobically with lipids on the cell membrane. They bind to cells and interact with biological amphiphilic molecules, gradually releasing these components. Compared to free RES, MURNPs are released more slowly at both pH levels.

[0087] Experiment 9: In vitro gastrointestinal release test:

[0088] Simulated gastric juice (SGF) and simulated intestinal juice (SIF) were first prepared. During the simulated digestion phase, a fresh suspension containing 3 mL of MURNPs and 3 mL of free RES was mixed with 3 mL of SGF and placed in a dialysis bag (molecular weight cutoff of 2,000 Da). This mixture was then placed in 60 mL of SGF release medium for 2 hours. Next, 6 mL of SIF was added to the dialysis bag, and the mixture was transferred to 120 mL of SIF release medium solution and continuously incubated for 4 hours. At regular intervals, 0.5 mL of release medium was collected to determine the RES content, and fresh medium was added to maintain a consistent volume. The collected solution was centrifuged at 12,000 rpm for 10 minutes to determine the RES content.

[0089] from Figure 16 It can be seen that during gastric digestion, free RES was rapidly released at 71.19±0.37%, while the release of free RES from MURNPs was only 51.73±0.06%. Compared with free RES, the release rate of RES from MURNPs was significantly reduced. This result indicates that loading RES into nanoparticles can effectively improve their gastric digestibility and promote their entry into the small intestine for digestion and absorption, laying the foundation for improving their bioavailability. Meanwhile, during intestinal digestion, free RES was released at 18.38±0.16%, while the release of RES from MURNPs was 11.51±0.53%, a significant reduction. The good gastrointestinal digestibility and sustained-release properties of MURNPs may be related to intermolecular hydrogen bonding and electrostatic interactions during assembly, which helps MURNPs maintain a more stable structure. In summary, MURNPs exhibit good in vitro stability and sustained-release properties during gastrointestinal digestion, showing potential as oral carriers to deliver hydrophobic active small molecules to improve their bioavailability.

[0090] Experiment 10: Antioxidant Activity Test

[0091] The antioxidant activity of the RES-MUR complex was evaluated by measuring the DPPH and ABTS radical scavenging abilities of RES. The concentration of free RES solution was mixed with an equal volume of DPPH solution (0.1 mM, ethanol) and then incubated in the dark at 25°C for 40 minutes. The DPPH radical scavenging rate of the free RES-MURNPs complex was then measured using a microplate reader and calculated using formula (1). Each sample was measured three times, and the results are expressed as mean ± standard deviation.

[0092] DPPH clearance rate (%) = (1 - (A2 - A1) / A0) × 100% (1)

[0093] In this context, A0 represents the absorbance of a standard sample prepared under the same conditions. A1 represents the absorbance of the complex sample without DPPH solution, while A2 represents the absorbance of the complex sample alone.

[0094] To assess the antioxidant potential of the samples, an ABTS free radical scavenging assay was performed. First, the ABTS working solution was diluted with PBS (0.1M, pH 7.4) to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Then, the samples were thoroughly mixed with the ABTS solution. After incubation at 25°C for 10 minutes, the absorbance at 734 nm was measured using a microplate reader. The scavenging activity of the RES and MURNPs complex was determined using formula (2). Each sample was measured three times, and the results are expressed as the mean ± standard deviation of the three measurements for each sample.

[0095] ABTS clearance rate (%) = (1-A / A0)×100% (2)

[0096] Where A0 is the absorbance of a standard sample prepared under the same conditions, but without any sample added; A is the absorbance of the standard model.

[0097] from Figures 17-18It can be seen that both RES and MURNPs exhibit significant DPPH radical scavenging activity in a dose-dependent manner. Specifically, at an equivalent concentration of 70 μg / mL RES, the DPPH radical scavenging rates of RES and MURNPs were 46.16 ± 5.03% and 65.87 ± 1.40%, respectively. Compared to RES, MURNPs showed a relatively higher level of free radical scavenging activity. Similarly, MURNPs also demonstrated stronger ABTS radical scavenging ability. At an equivalent concentration of 70 μg / mL RES, the ABTS radical scavenging rate of MURNPs was 88.45 ± 0.10%. The ABTS radical scavenging activity was MUR nanoparticles > RES ethanol solution in all samples. Therefore, MUR-containing nanoparticles have significant potential for scavenging ABTS radicals. During nanoparticle formation, the dispersibility of RES in water was improved, and the hydrophilic groups of MA were distributed on the surface of the nanoparticles. This structure facilitates the binding of active groups with hydrophilic DPPH and ABTS radicals and their scavenging. Subsequently, storage stability analysis provides necessary data support for determining the shelf life of the samples. Therefore, we investigated the free radical scavenging ability of MURNPs after being stored at 4°C for 7 days to assess their antioxidant activity. After 7 days, the DPPH and ABTS free radical scavenging rates of MURNPs were 51.80±1.32% and 88.12±0.80%, respectively, and their antioxidant activity did not change significantly. In summary, MURNPs exhibit good storage stability, which lays the foundation for better exertion of their efficacy.

[0098] Experiment 11: In vitro cell uptake test:

[0099] First, with 1×10 6 Cells were seeded in 6-well plates at a density of cells / mL and cultured at 37°C for 24 hours. The medium was then exchanged with fresh medium containing free RES and MURNPs (RES concentration 20 μg / mL) and cultured for 3 hours. The uptake of various NPs by L929 cells was monitored at different time intervals using an inverted fluorescence microscope.

[0100] The results are as follows Figure 19 As shown, in the cellular uptake experiment, the nanoparticles exhibited rapid uptake behavior. After 30 min of incubation in L929 cells, significant fluorescence signals were observed in the cytoplasm of the MURNPs group. Compared with the free FITC and free RES groups, the fluorescence signals of the MURNPs group were significantly enhanced. This indicates that the nanoparticles have a high affinity for the cell membrane. Negatively charged particles bind to cation sites in the form of clusters, promoting the adsorption of other free particles, thereby increasing the uptake of negatively charged nanoparticles by cells.

Claims

1. A method for preparing co-assembled nanoparticles, characterized in that: The method is as follows: Step 1: Dissolve ursolic acid in dimethyl sulfoxide; Step 2: Dissolve resveratrol in dimethyl sulfoxide; Step 3: Dissolve matrine in an aqueous solution; Step 4: Mix the ursolic acid solution and resveratrol solution and then add them to the matrine aqueous solution; Step 5: Stir at 400 rpm to 600 rpm for 10 to 60 minutes; Step 6: Centrifuge at 10,000 to 15,000 rpm for 10 to 30 minutes.

2. The method for preparing co-assembled nanoparticles according to claim 1, characterized in that: In step one, the concentration of ursolic acid in dimethyl sulfoxide is 5 mg / mL to 50 mg / mL.

3. The method for preparing co-assembled nanoparticles according to claim 1, characterized in that: In step two, the concentration of resveratrol in dimethyl sulfoxide is 5 mg / mL to 50 mg / mL.

4. The method for preparing co-assembled nanoparticles according to claim 1, characterized in that: In step three, the concentration of matrine in water is 1 mg / mL to 50 mg / mL.

5. The method for preparing co-assembled nanoparticles according to claim 1, characterized in that: In step four, the volume ratio of the ursolic acid solution, resveratrol solution, and matrine aqueous solution is 50~100:50~100:1.

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