Preparation method of water-soluble alkaloid and terpenoid co-assembled nanoparticles

Abstract

The application relates to a preparation method of water-soluble alkaloid and terpenoid co-assembly nanoparticles, and belongs to the field of nanometer materials. Through a co-assembly strategy, a pure natural co-assembly nanometer transmission system, i.e. water-soluble alkaloid and terpenoid co-assembly nanoparticles, with novel morphological characteristics and multiple biological activities is prepared, and a NSMs compound with good biological activity but unable to be used for drug transmission can be applied. The terpenoid compound is dissolved in an organic phase, and the matrine and sophoridine are dissolved in water. The organic phase solution is added into the water phase, stirring, centrifugation, and the water-soluble alkaloid and terpenoid co-assembly nanoparticles are obtained. The water-soluble alkaloid and terpenoid co-assembly nanoparticles are prepared by a simple and convenient method, the toxic side effects are reduced, and the drug loading capacity is improved. Therefore, a carrier-free, biocompatible, biodegradable and low-cost nanometer self-assembly is constructed in the application.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials, specifically relating to a method for preparing nanoparticles co-assembled with water-soluble alkaloids and terpenoid compounds. Background Technology

[0002] Nanoparticle-based drug delivery systems (DDS) have been widely used in disease treatment. Nanoparticles possess characteristics such as low toxicity, good biocompatibility, and long blood retention time. In pharmaceuticals, nanoparticles can be divided into two categories: nanocarriers and nanomedicines. Nanocarriers refer to those in which drugs are encapsulated or dispersed, such as nanoliposomes, nanospheres, and polymer micelles. Nanomedicines, on the other hand, refer to nanoparticles directly processed from raw drug substances. There are many methods for preparing nanoparticles, including ultrasonic self-assembly, vapor deposition, precipitation, hydrothermal synthesis, sol-gel methods, and microemulsion methods. In recent years, natural small molecule compounds with self-assembly and co-assembly properties have been discovered and have attracted considerable attention in the field of drug delivery. Among them, natural small molecule compounds have become a research hotspot due to their wide range of pharmacological effects.

[0003] Natural products refer to small molecules synthesized by organisms, also known as secondary metabolites. They are widely available, possess unique structures and multiple modification sites, and exhibit good biocompatibility and degradability. Alkaloids, in particular, are a class of nitrogen-containing compounds derived from nature, mostly alkaline, and often possess significant biological activity. For example, water-soluble alkaloids such as matrine and sophoridine have antiarrhythmic effects. Currently, existing matrine formulations, including injections, tablets, and capsules, are used in clinical research and treatment. However, due to deficiencies in dosage form design or drug release mechanisms, these existing formulations often suffer from short durations of efficacy, requiring frequent administration, large fluctuations in blood drug concentration, and increased toxic side effects and complications. Furthermore, due to inherent design flaws and deficiencies, when using these matrine formulations clinically, doctors may increase the dosage to achieve better clinical therapeutic effects, leading to increased toxic side effects and adverse reactions, poor patient compliance, low drug bioavailability, and failure to achieve the intended therapeutic goal.

[0004] To overcome the above shortcomings, researchers have prepared liposomes, nanoemulsions, and gels for matrine and sophoridine. For example, the preparation and properties of magnetic polylactic acid-glycolic acid oxidized matrine nanoparticles were studied in *Biomedical Engineering and Clinical*, 2012, Vol. 3; the in vitro drug release and liver distribution study of oxidized matrine polycyanoacrylate n-butyl nanoparticles was published in *Journal of China Pharmaceutical University* (CSCD), 2010, Vol. 6. However, the preparation methods of nano-preparations are complex, and the drug loading is low, limiting their application. In recent years, researchers have isolated terpenes and other small-molecule natural products with self-assembly capabilities from traditional Chinese medicine. Studies have shown that these natural products can undergo self-assembly in various solvents, forming non-covalent interactions such as hydrogen bonding, hydrophobic interactions, π-π stacking interactions, and van der Waals interactions, promoting the formation of regularly arranged aggregates between molecules. Summary of the Invention

[0005] The purpose of this invention is to address the technical problems of existing methods, such as complex procedures, long processing times, low drug loading capacity, and significant toxic side effects. It provides a method for preparing nanoparticles co-assembled from water-soluble alkaloids and terpenoids and loaded with the small-molecule inhibitor sorafenib. This invention constructs a carrier-free, biocompatible, biodegradable, and low-cost nano-co-assemblies. This attempt will provide a reference for the discovery and optimization of supramolecular self-delivery systems, thus offering opportunities to find optimal unmodified nanomedicine combinations.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing water-soluble alkaloids and terpenoids co-assembled nanoparticles, the method comprising:

[0008] Step 1: Dissolve oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) in dimethyl sulfoxide;

[0009] Step 2: Dissolve matrine (MA) in an aqueous solution;

[0010] Step 3: Add the solutions of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid to the matrine aqueous solution;

[0011] Step 4: Stir at 400 rpm to 600 rpm for 10 to 60 minutes;

[0012] Step 5: Centrifuge at 10,000 to 15,000 rpm for 10 to 30 minutes.

[0013] Furthermore, in step one, the concentrations of oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) in dimethyl sulfoxide are each 5 mg / mL to 50 mg / mL.

[0014] Furthermore, in step two, the concentration of matrine in water is 1 mg / mL to 50 mg / mL.

[0015] Furthermore, in step three, the volume ratio of the oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) solution to the matrine (MA) aqueous solution is 50~100:1.

[0016] A method for preparing water-soluble alkaloids and terpenoids co-assembled nanoparticles, the method comprising:

[0017] Step 1: Dissolve oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) in dimethyl sulfoxide;

[0018] Step 2: Dissolve sorafenib (S) in dimethyl sulfoxide;

[0019] Step 3: Dissolve matrine (MA) in an aqueous solution;

[0020] Step 4: Mix the solutions of oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA) and glycyrrhetinic acid (GA) with the sorafenib (S) solution respectively, and then add them to the aqueous solution;

[0021] Step 5: Stir at 400 rpm to 600 rpm for 10 to 60 minutes;

[0022] Step 6: Centrifuge at 10,000 to 15,000 rpm for 10 to 30 minutes.

[0023] Furthermore, in step one, the concentrations of oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) in dimethyl sulfoxide are each 5 mg / mL to 50 mg / mL.

[0024] Furthermore, in step two, the concentration of sorafenib (S) in dimethyl sulfoxide is 5 mg / mL to 50 mg / mL.

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

[0026] Further, in step four, the volume ratio of the oleanolic acid (OA), ursolic acid (UA), betulinic acid (BA), and glycyrrhetinic acid (GA) solution, the sorafenib (S) solution, and the matrine (MA) aqueous solution is 50~100:50~100:1.

[0027] The advantages of this invention over the prior art are as follows:

[0028] (1) The triterpenoids in this invention are a large class of natural products composed of isoprene as structural unit. Oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid are the main active ingredients of traditional Chinese medicine and natural plant medicine, with multiple therapeutic and tissue protection effects, including anti-tumor, antioxidant, anti-inflammatory and cardioprotective effects.

[0029] (2) The preparation method of this invention is simple and convenient, and the preparation of water-soluble alkaloids and terpenoids co-assembled nanoparticles can be achieved at room temperature, saving energy. At the same time, no organic reagents are added during the preparation process, eliminating the need for subsequent impurity removal steps. In addition, oleanolic acid, ursolic acid, betulinic acid, glycyrrhetinic acid, and matrine all have good biocompatibility, are widely available, and have low preparation costs.

[0030] (3) This invention broadens the horizons of multi-component NSMs synergistic cancer treatment and expands the application of bioactive NSMs in medicine. Attached Figure Description

[0031] Figure 1 Scanning electron microscope (SEM) images of the co-assembled nanoparticles of Examples 1 and 2; a - Example 1, b - Example 2;

[0032] Figure 2 The figures show the particle size and Zeta results of the co-assembled nanoparticles in Examples 1 and 2.

[0033] Figure 3 The image shows the UV spectrum of the MAOAS co-assembled nanoparticles from Example 2.

[0034] Figure 4 The UV spectrum of the MAUAS co-assembled nanoparticles in Example 2 is shown below.

[0035] Figure 5 The image shows the UV spectrum of the MABAS co-assembled nanoparticles from Example 2.

[0036] Figure 6 The image shows the UV spectrum of the MAGAS co-assembled nanoparticles from Example 2.

[0037] Figure 7 The image shows the TEM experimental results of MAGAS co-assembled nanoparticles in Example 2.

[0038] Figure 8 The figure shows the AFM experimental results of MAGAS co-assembled nanoparticles in Example 2;

[0039] Figure 9 CD chromatography of MAGAS co-assembled nanoparticles in Example 2;

[0040] Figure 10 The XRD pattern of the MAGAS co-assembled nanoparticles in Example 2;

[0041] Figure 11 This is a diagram showing the in vitro release results of MAGAS co-assembled nanoparticles in Example 2;

[0042] Figure 12 The figure shows the MTT experimental results of MAGAS co-assembled nanoparticles in Example 2;

[0043] Figure 13 This is a diagram showing the in vivo distribution results of MAGAS co-assembled nanoparticles in Example 2;

[0044] Figure 14 This is a graph showing the experimental results of the antitumor activity of MAGAS co-assembled nanoparticles in Example 2;

[0045] Figure 15 H&E staining of liver tumors to demonstrate the antitumor effect of MAGAS co-assembled nanoparticles in Example 2; red borders indicate the original tumor region;

[0046] Figure 16 This is a cross-sectional image of a tumor cell showing the antitumor effect of MAGAS co-assembled nanoparticles in Example 2.

[0047] Figure 17 The figure shows the in vivo safety test results of MAGAS co-assembled nanoparticles in Example 2. Detailed Implementation

[0048] 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.

[0049] As an important research direction in modern chemistry, supramolecular chemistry plays a crucial role not only in unraveling the mysteries of the origin of life but also in the preparation of novel nanostructured materials. A significant challenge is the ability to regulate and design assembly modules to form novel nanostructured materials that respond to external stimuli. Inspired by the combination of traditional Chinese medicine drugs, a natural self-assembly mode has been identified between water-soluble alkaloids and lipid-soluble terpenoid small molecules. Therefore, this invention prepares a carrier-free, pure drug self-sufficient nanosystem composed of a water-soluble alkaloid (matrine), a triterpenoid compound, and a small molecule inhibitor (sorafenib). This invention constructs a nanocomposite system with multiple functions including chemotherapy and targeted therapy. Driven by weak bonds, the self-assembly of the active ingredient exhibits greater advantages than nanocarriers, including improved drug loading efficiency and avoidance of metabolic difficulties and toxicity associated with nanocarriers. Furthermore, natural small molecules do not require modification, and their reaction conditions are harsh or the synthesis process complex, making them more economical and conducive to their transformation into clinical drugs. In addition, it has been reported that the self-assembly of small natural phytochemicals remains a serious challenge. Because it largely depends on molecular configuration, intermolecular forces, etc., its development is largely dependent on chance. This invention, inspired by traditional Chinese medicine combinations, provides a promising paradigm for designing and preparing adjuvant-free self-assembled nanostructures. It also presents a simple method for using water-soluble and lipid-soluble drugs in a synergistic tumor therapy platform for drug-dependent anaerobic digestion (DDS), and offers a promising combination strategy to expand the medical applications of water-soluble non-alkaline nanoparticles (NSMs). Meanwhile, no reports have been found regarding the co-assembly of nanoparticles with water-soluble alkaloids and terpenoids.

[0050] This invention utilizes a co-assembly strategy to prepare a novel, all-natural co-assembled nanoparticle delivery system—water-soluble alkaloids and terpenoids—with diverse morphological characteristics and bioactivities. This allows for the application of non-molecular-weighted molecular weight transport (NSM) compounds with good bioactivity but unsuitable for drug delivery. Terpenoids are dissolved in an organic phase, while matrine and sophoridine are dissolved in water. The organic phase solution is added to the aqueous phase, stirred, and centrifuged to obtain the water-soluble alkaloid and terpenoid co-assembled nanoparticles. This invention provides a simple and convenient method for preparing water-soluble alkaloid and terpenoid co-assembled nanoparticles, reducing toxic side effects and increasing drug loading. Therefore, this invention constructs a carrier-free, biocompatible, biodegradable, and low-cost nano-self-assembly. This invention designs the self-assembly of water-soluble and lipid-soluble drugs, an attempt that will provide a reference for the discovery and optimization of supramolecular self-delivery systems, thus offering opportunities to find optimal unmodified nanomedicine combinations.

[0051] Example 1

[0052] A method for preparing water-soluble alkaloids and terpenoids co-assembled nanoparticles, wherein the alkaloid is matrine, and the terpenoids are oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid, wherein the volume ratio of water to dimethyl sulfoxide is 1:100, the content of matrine is 10 mg / mL, and the content of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid is 15 mg / mL.

[0053] Step 1: Dissolve 15 mg of oleanolic acid, 15 mg of ursolic acid, 15 mg of betulinic acid and 15 mg of glycyrrhetinic acid in 1 mL of dimethyl sulfoxide and sonicate until the solid powder is completely dissolved.

[0054] Step 2: Take 0.01 mL of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid solutions respectively and add them to 1 mL of 10 mg / mL matrine aqueous solution. Stir at 400 rpm for 10 min and centrifuge at 10000 rpm for 20 min.

[0055] Example 2

[0056] A method for preparing nanoparticles co-assembled with water-soluble alkaloids, terpenoids, and small molecule inhibitors, wherein the alkaloid is matrine, the terpenoids are oleanolic acid, ursolic acid, betulinic acid, and glycyrrhetinic acid, the small molecule inhibitor is sorafenib, the volume ratio of water to dimethyl sulfoxide is 1:100, the content of matrine is 10 mg / mL, the content of oleanolic acid, ursolic acid, betulinic acid, and glycyrrhetinic acid is 15 mg / mL, and the content of sorafenib is 10 mg / mL.

[0057] Step 1: Dissolve 15 mg of oleanolic acid, ursolic acid, betulinic acid, and glycyrrhetinic acid, and 10 mg of sorafenib in 1 mL of dimethyl sulfoxide, and sonicate until the solid powder is completely dissolved.

[0058] Step 2: Take 0.01 mL of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid and sorafenib solution respectively and add them to 1 mL of 10 mg / mL matrine aqueous solution. Stir at 400 rpm for 10 min and centrifuge at 10000 rpm for 20 min.

[0059] Testing and experimentation:

[0060] Experiment 1: Microstructure Testing

[0061] The oleanolic acid, ursolic acid, betulinic acid, and glycyrrhetinic acid obtained in Examples 1-2 were co-assembled with matrine into nanoparticles. After the 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.

[0062] Depend on Figure 1 It can be seen that all these nanomaterials can co-assemble with water-soluble matrine. The results show that MA co-assembles with OA and UA to form spherical nanoparticles, and with BA and GA to form nanofibers.

[0063] Experiment 2: Particle size test

[0064] The nanoparticles co-assembled with matrine and the oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid obtained in Examples 1-2 were then uniformly dispersed in water and measured using a Malvern particle size analyzer.

[0065] Depend on Figure 2 It can be seen that MAOA and MAUA form spherical nanofibers with diameters of 400~470 nm; MABA and MAGA form nanofibers with diameters of ~25 nm and ~68 nm, respectively. All NPs exhibit negatively charged surfaces with zeta potentials ranging from -15.6 to -34.7 mV.

[0066] Experiment 3: Ultraviolet Characterization

[0067] The oleanolic acid, ursolic acid, betulinic acid and glycyrrhetinic acid obtained in Examples 1-2 were co-assembled with matrine into nanoparticles. After the nanoparticles were uniformly dispersed in water, a full-wavelength scan was performed using an ultraviolet spectrometer.

[0068] Depend on Figure 3 As can be seen in the UV / Vis spectrum, sorafenib exhibits an absorption peak at 277 nm. After assembling oleanolic acid and sorafenib into co-assembled nanoparticles with matrine, the absorption peak of sorafenib exhibits a red shift. This is due to the π-π* conjugation effect between oleanolic acid and sorafenib, resulting in the red shift.

[0069] Depend on Figure 4As can be seen in the UV / Vis spectrum, sorafenib exhibits an absorption peak at 277 nm. After assembling ursolic acid and sorafenib into co-assembled nanoparticles with matrine, the absorption peak of sorafenib exhibits a red shift. This is due to the π-π* conjugation effect between ursolic acid and sorafenib, resulting in the red shift.

[0070] Depend on Figure 5 As can be seen in the UV / Vis spectrum, sorafenib exhibits an absorption peak at 277 nm. After assembling betulinic acid and sorafenib into co-assembled nanoparticles with matrine, the absorption peak of sorafenib exhibits a red shift. This is due to the π-π* conjugation effect between betulinic acid and sorafenib, resulting in the red shift.

[0071] Depend on Figure 6 As can be seen in the UV / Vis spectrum, sorafenib exhibits an absorption peak at 277 nm. After assembling glycyrrhetinic acid and sorafenib into co-assembled nanoparticles with matrine, the absorption peak of sorafenib exhibits a red shift. This is due to the π-π* conjugation effect between glycyrrhetinic acid and sorafenib, resulting in the red shift.

[0072] Experiment 4: Transmission Electron Microscopy Characterization

[0073] After uniformly dispersing the glycyrrhetinic acid and sorafenib co-assembled with matrine obtained in Example 2 in water, a drop of the solution was added to aluminum foil. After evaporating the water, the microstructure of the sample nanoparticles was observed using a transmission electron microscope. Figure 7 As shown.

[0074] This invention selects MAGAS NPs as in vivo drug delivery carriers. Figure 7 TEM analysis revealed that the glycyrrhetinic acid, sorafenib, and matrine-co-assembled nanoparticles prepared in this invention exhibit a bilayer core-shell structure. The average particle size of the MAGAS NPs is approximately 144.16 ± 4.14 nm. The sizes obtained from SEM, TEM, and DLS were largely consistent.

[0075] Experiment 5, AFM test:

[0076] After the glycyrrhetinic acid and sorafenib co-assembled with matrine obtained in Example 2 were evenly dispersed in water, a drop of the solution was added onto a mica sheet. After evaporating the water, the microstructure of the sample nanoparticles was observed using an atomic force microscope, such as... Figure 8 As shown.

[0077] Figure 8 The results confirmed the three-dimensional (3D) AFM images of MAGAS NPs, with the magnified portion showing a spherical surface of MAGAS NPs with a diameter of 145 nm, consistent with previous particle size results.

[0078] Experiment 6: Circular Dichroism Chromatography Test

[0079] The chiral signals formed by MAGAS NPs in water were further investigated using the samples obtained in Examples 1-2. To study the molecular assembly process in greater depth, CD was measured using a Pistar π-180 spectrometer. The light source was a 150 W xenon lamp.

[0080] Figure 9 The results confirmed that GA NFs form a right-handed helical structure, with a positive Cotton effect around 259 nm in their CD spectra. MAGA NFs form a mixed left- and right-handed helical structure, with positive Cotton effects around 220 nm and 262 nm and a negative Cotton effect around 240 nm in their CD spectra. MAGAS NPs produced broad and flat peaks, indicating weakened chirality. These results suggest that chiral molecules form ordered assemblies through intermolecular interactions, with molecular packing exhibiting a fibrous structure, indicating that molecules can arrange themselves in an orderly manner to form well-ordered assemblies. Conversely, when molecules pack into a spherical shape, it indicates that the molecules forming the assemblies are arranged amorphously.

[0081] Experiment 7, XRD test:

[0082] The co-assembled nanoparticle powder samples obtained in Examples 1-2 were placed on a zero-background plate and then subjected to Cu Kα radiation generated at 40 kV and 38 mA at 5 o Scanning speed 5-60 / min o The diffraction angle 2θ within the range.

[0083] The experimental results are as follows Figure 10 As shown, the diffraction pattern of MAGA NFs exhibits six diffraction peaks of different intensities: 1.52 nm, 1.26 nm, 0.89 nm, 0.63 nm, 0.60 nm, and 0.59 nm, indicating the presence of layered structures among the molecules. The 0.59 nm peak has the highest intensity, suggesting a large number of aggregates at this size, which is attributed to the formation of dimers at the molecular level. The 1.26 nm and 1.52 nm peaks are the next most intense, representing the pitch of helical structures at the molecular level. MAGASNPs show only one relatively broad and weak diffraction peak at approximately 0.59 nm, indicating the presence of a small amount of MAGA dimers within the nanoparticles, and also suggesting that other molecules form amorphous structures during NP assembly.

[0084] Experiment 8: In vitro release test:

[0085] The co-assembled nanoparticles obtained in Examples 1-2 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 r / min. 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] Figure 11 The results confirmed that at pH 7.4, the release efficiencies of GA in MAGA NFs were 35.4% and 67.2% after 24 h, respectively. The release efficiencies of GA in MAGAS NPs were 33.1% and 57.3%, respectively. This means that MAGA NFs and MAGAS NPs remain stable while releasing GA slowly under phosphate-buffered saline conditions. At 48 h, MAGAS NPs released only about 2% (pH 7.4) and 6% (pH 6.5), further confirming their stability. At pH 7.4 and 6.5, the release rates of free GA at 12 h were 64.5% and 72.9%, respectively, showing rapid release behavior. Therefore, MAGAS NPs exhibit better stability and dispersibility, can prolong blood circulation, and accumulate in tumor tissue, thus ensuring their potential for in vivo application.

[0087] Experiment 9, MTT Test:

[0088] The specific experimental method is as follows:

[0089] (1) Cell culture: HepG2 cells in logarithmic growth phase were digested with trypsin to obtain a cell suspension. The suspension was seeded into 96-well plates with a seeding volume of 200 µL and cultured overnight at 37°C and 5% CO2 to allow the cells to adhere and grow.

[0090] (2) Preparation of solution of co-assembled nanoparticles of glycyrrhetinic acid, sorafenib and matrine: 1640 medium was added to the nanoparticle sample according to the ratio of 1 mg of co-assembled nanoparticles in Example 2 to 1 mL of culture medium to prepare a solution with a concentration of 1 mg / mL.

[0091] (3) Mix MTT solid with PBS solution to prepare a 5 mg / mL solution, then add 1640 medium to dilute to obtain a 0.5 mg / mL MTT solution;

[0092] (4) The co-assembled nanoparticle solutions of 1 mg / mL from Examples 1 and 2 were diluted to 2000 μg / mL, 1500 μg / mL, 1000 μg / mL, 500 μg / mL, 100 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. The culture medium in the 96-well plate was discarded, and 200 µL of the diluted nanoparticle solutions of different concentrations were added. Six replicates were set for each concentration. The control group was set up by adding 200 µL of 1640 culture medium. The plates were cultured at 37°C and 5% CO2 for 24 h. The laser group was incubated for 4 h, irradiated with laser for 10 min, and then incubated for another 20 h. Then, the solution in the 96-well plate was aspirated, and 200 µL of the 0.5 mg / mL MTT solution obtained in step (3) was added. The plate was incubated at 37°C for 4 h. The liquid in the wells was then aspirated to terminate the culture. 150 µL of DMSO was added to each well, and the plate was incubated for 10 min. The absorbance of each well at 492 nm was measured using a microplate reader, and the cell viability was calculated. The experimental results are as follows: Figure 12 As shown.

[0093] Figure 12 The study showed that different concentrations of MAGA NPs and MAGAS NPs inhibited cell proliferation. It was observed that the survival rate of HepG2 cells decreased rapidly with increasing GA concentration. Specifically, when the equivalent GA concentration in the culture medium for MAGA NPs and MAGAS NPs was 100 μg / mL, the cell survival rates were 41% and 37%, respectively. The combined chemotherapy efficacy of MAGAS NPs was significantly higher than that of GA chemotherapy alone and single-dose chemotherapy with MAGA NPs, demonstrating a synergistic effect in tumor treatment.

[0094] Experiment 10: In vivo distribution test

[0095] The glycyrrhetinic acid and sorafenib obtained in Example 2 were co-assembled with matrine into nanoparticles and dispersed evenly in physiological saline. Mice were randomly divided into two groups (n = 3): (1) free FITC group (equal amount of FITC in each group: 4.5 mg / kg), (2) MAGASNPs group. The tail vein injection volume was 150 μL. The biodistribution of liver tumors in mice was observed at 1, 4, 6, 12, 24, 24 h, 48 h, and 72 h. The fluorescence signals of tumors and organs were obtained using the Master In Vivo Optical Imaging System. The experimental results are as follows. Figure 13 As shown.

[0096] from Figure 13It can be seen that the fluorescently labeled MAGAS NPs exhibit time-dependent accumulation characteristics in liver tumors, rapidly accumulating in liver tumor tissue and maintaining strong fluorescence signals for a long time. This liver tumor accumulation ability is mainly attributed to the active targeting of GA. Even until the end of the observation period (72 hours), weak fluorescence emission remained at the liver tumor site, verifying the effective accumulation of the nanocomponents at the tumor site. The same dose of fluorescently labeled MAGAS NPs was injected into mice via the tail vein. Mice were euthanized by cervical dislocation at 1 h, 4 h, 6 h, 12 h, 24 h, 48 h, and 72 h after administration, and vital organs were collected for fluorescence imaging to analyze the tissue distribution of the nanoparticles. The in vitro biological distribution map shows that at 1 h after injection, a large number of fluorescently labeled NPs accumulated in the liver and kidney tissues. In contrast, the fluorescence in the spleen and heart was negligible. At 6 h after injection, the fluorescence signal in the liver tumor was the strongest. At 12 h after injection, the decline in fluorescence in the liver tumor and kidney was slow; based on the strong fluorescence signal in the kidney, it is inferred that at least some NPs were excreted through the kidney.

[0097] Experiment 11: In vivo anti-tumor test:

[0098] The glycyrrhetinic acid and sorafenib obtained in Examples 1-2 were co-assembled with matrine into nanoparticles, which were then uniformly dispersed in physiological saline. Since subcutaneous solid tumors are ectopically implanted in other sites rather than the highly vascularized liver, ectopic transplantation is difficult to reflect the actual liver microenvironment. Therefore, an H22 orthotopic xenograft mouse model was established to further evaluate the in vivo antitumor activity of MAGAS NPs and their potential damage to normal liver. All tumors were harvested along with surrounding liver tissue 14 days after treatment. Figure 14 The in vivo experimental procedure included in situ liver injection of H22 cells, drug administration, and tissue analysis. Liver morphological evaluation showed that most of the livers in the drug-loaded groups (S group, MAGA NFs group, and MAGAS NPs group) maintained a normal appearance. However, in the GA group, MA group, and control group, the tumors were larger, protruding from the liver surface and occupying a large area of ​​the liver.

[0099] Experiment 12: Histological analysis of antitumor effects:

[0100] To comprehensively and clearly evaluate the antitumor efficacy of MAGAS NPs in orthotopic xenograft mice, hematoxylin and eosin (H&E) staining was performed on liver sections from each lobe.

[0101] Figure 15-16 Histological analyses of tumor-loaded liver lobes from different groups are shown. The boundaries between tumor tissue and normal liver tissue are well-defined. The cross-sectional area of ​​the tumor in each group was analyzed using Adobe Photoshop CS6 (USA). Figure 15 (16). Compared with the control group, the drug-loaded group reduced the cross-sectional area of ​​the tumor more effectively. Among them, the MAGAS NPs group had the smallest cross-sectional area of ​​liver sections, exhibiting the best anti-tumor properties. H&E staining of the liver and tumor sites showed that drug accumulation in the tumor induced significant necrosis and apoptosis.

[0102] Experiment Thirteen, In Vivo Safety Testing:

[0103] To investigate the toxicity of the nanoparticles, tissues and organs of tumor-bearing mice were stained at the end of treatment. Major organs, including tumors, heart, liver, spleen, kidneys, and lungs, were collected and routinely stained with H&E. The experimental results are as follows: Figure 17 As shown.

[0104] The results showed that, compared with the control group, no significant inflammatory response or damage was observed in the MAGAS NPs group, further indicating that MAGAS NPs have low in vivo toxicity. The combination therapy demonstrated biocompatibility in vivo. In conclusion, MAGAS NPs exhibit better biocompatibility and biodegradability, and low in vivo toxicity, showing great potential as a promising synergistic anti-tumor drug.

Claims

1. A method for preparing nanoparticles co-assembled with water-soluble alkaloids and terpenoid compounds, characterized in that: The method is: Step one: dissolve oleanolic acid, ursolic acid, betulinic acid and glycyrrhetic acid in dimethyl sulfoxide; Step two: dissolve sorafenib in dimethyl sulfoxide; Step three: dissolve matrine in aqueous solution; Step four: mix oleanolic acid, ursolic acid, betulinic acid and glycyrrhetic acid solution with sorafenib solution respectively, and then add them into the aqueous solution; Step five: stir at 400rmp~600rmp for 10min~60min; Step six: centrifuge at 10000~15000rmp for 10~30min.

2. The method according to claim 1, wherein the method is characterized by: In step one, the concentration of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetic acid in dimethyl sulfoxide is 5 mg / mL~50 mg / mL respectively.

3. The method according to claim 1, wherein the method is characterized by: In step two, the concentration of sorafenib in dimethyl sulfoxide is 5 mg / mL~50 mg / mL.

4. The method according to claim 1, wherein the method is characterized by: In step three, the concentration of matrine in water is 1 mg / mL~50 mg / mL.

5. The method according to claim 1, wherein the method is characterized by: In step four, the volume ratio of oleanolic acid, ursolic acid, betulinic acid and glycyrrhetic acid solution, sorafenib solution and matrine aqueous solution is 50~100: 50~100: 1.

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