A licorice protein-doxorubicin nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a glycyrrhizin protein-doxorubicin nanoparticle as well as a preparation method and application thereof. The glycyrrhizin protein-doxorubicin nanoparticle comprises glycyrrhizin protein and doxorubicin; wherein the doxorubicin is dispersed in a matrix of the glycyrrhizin protein, and the glycyrrhizin protein molecules are crosslinked by glutaraldehyde. The glycyrrhizin protein-doxorubicin nanoparticle prolongs the terminal elimination half-life of free DOX, reduces the clearance rate, and increases the in-vivo exposure amount; tissue distribution research shows that, compared with free DOX, the glycyrrhizin protein-doxorubicin nanoparticle is more accumulated in tumor tissues, is eliminated more slowly, and is less distributed in heart tissues.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a glycyrrhizin-doxacin nanoparticle, its preparation method, and its application. Background Technology

[0002] Among numerous antitumor drugs, doxorubicin (DOX), as a representative anthracycline antibiotic, has become one of the most widely used first- or second-line chemotherapy drugs in clinical practice since its discovery in the 1960s, due to its broad-spectrum and highly effective antitumor activity. However, the clinical application of DOX is limited by serious adverse reactions, among which dose-dependent and cumulative cardiotoxicity is the most prominent. Clinical data show that when the cumulative dose of DOX exceeds 400-550 mg / m²... 2 At this time, the incidence of congestive heart failure rises sharply to 5-20%, and this cardiac damage often does not appear for months or even years after drug discontinuation, exhibiting delayed and irreversible characteristics. In addition, systemic adverse reactions such as bone marrow suppression, hepatotoxicity, and nephrotoxicity also seriously affect patients' treatment compliance and quality of life.

[0003] To overcome the limitations of traditional doxorubicin formulations and achieve targeted drug delivery while reducing toxicity and enhancing efficacy, researchers have developed various doxorubicin nanoformulations. Among them, polyethylene glycol-modified liposomal doxorubicin is the first FDA-approved antitumor nanoformulation. It achieves passive targeting through a high-permeability, long-retention effect, significantly prolonging the drug's circulation time in vivo and reducing cardiotoxicity. However, clinical practice has shown that the ultra-long retention time of polyethylene glycol-modified liposomal doxorubicin in vivo can cause new adverse reactions, including hand-foot syndrome, stomatitis, and nephrotoxicity, seriously affecting patient safety and tolerability. Furthermore, while existing polymer nanoparticles possess good biocompatibility, they are often rapidly recognized and cleared by organs rich in phagocytes, such as the liver and spleen, resulting in a short distribution half-life in vivo, making it difficult to achieve ideal tumor targeting effects. Therefore, developing a novel doxorubicin nanodelivery system that combines good tumor targeting, suitable circulation time in vivo, and high multi-organ safety remains of significant clinical importance and urgent need.

[0004] In recent years, protein nanocarriers have attracted widespread attention due to their unique biodegradability advantages. Studies have shown that albumin, silk fibroin, and whey protein can all be prepared into nanoparticles through self-assembly or desolvation methods for encapsulating and delivering antitumor drugs. These protein nanoparticles not only improve drug solubility and stability but also enable EPR-mediated passive targeting through particle size control. Furthermore, their abundant surface active groups provide possibilities for functional modification.

[0005] Licorice is one of the most frequently used medicinal herbs in Traditional Chinese Medicine (TCM) clinical practice, possessing the functions of tonifying the spleen and replenishing qi, clearing heat and detoxifying, relieving spasms and pain, and harmonizing other herbs. For a long time, research on licorice has focused on small molecule components such as triterpenoid saponins and flavonoids, with relatively little attention paid to its large molecule component—protein. Recent studies have found that licorice protein has good biocompatibility and can encapsulate small molecule drugs such as curcumin, astragaloside IV, paeoniflorin, and aconitine, achieving the goals of reducing toxicity and enhancing efficacy, as well as improving bioavailability. It is a promising delivery material.

[0006] However, there are currently no reports on the preparation of nanoparticles using glycyrrhizin (GP) as a carrier to encapsulate doxorubicin, nor are there any studies on the application of these nanoparticles in cancer treatment. Whether doxorubicin encapsulated with glycyrrhizin can achieve reduced toxicity and enhanced efficacy requires further investigation. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing glycyrrhizin-doxacin nanoparticles, their preparation method, and applications.

[0008] Specifically, the present invention is achieved through the following technical solutions: The glycyrrhizin-doxacin nanoparticles (DNPs) provided by the present invention comprise: glycyrrhizin and doxorubicin; wherein the doxorubicin is dispersed within the matrix of the glycyrrhizin, and the glycyrrhizin molecules are cross-linked by glutaraldehyde.

[0009] like Figure 1 As shown, the glycyrrhizin-doxorubicin nanoparticles of the present invention are red loose powders. The micromorphology of the nanoparticles was observed using TEM, and the results showed that DNPs are spherical nanoparticles with a round appearance and uniform size distribution.

[0010] In some preferred embodiments, the glycyrrhizin-doxacin nanoparticles have a particle size of 130-140 nm and a drug loading of 15%-25%.

[0011] Further preferably, the size of the glycyrrhizin-doxacin nanoparticles prepared according to the optimal preparation process is 132.5~134.3 nm. Figure 2 The drug loading is 21.67 ± 2.98%. The glycyrrhizin-doxacin nanoparticles of this invention have a narrow particle size distribution, falling within the ideal range of 50-200 nm. This allows the nanoparticles to passively accumulate at the tumor site by utilizing the high permeability of tumor blood vessels and the obstruction of lymphatic return, while avoiding rapid filtration by the kidneys and massive phagocytosis by hepatic and splenic macrophages. The drug loading of 21.67 ± 2.98% means that each unit mass of nanoparticle carries a sufficient amount of doxorubicin, achieving a therapeutic dose without requiring excessive excipients.

[0012] The licorice protein used in this invention is a protein with a molecular weight of 25-35 kDa extracted from licorice, or a dimer protein linked by disulfide bonds.

[0013] The glycyrrhizin-doxacin nanoparticles of the present invention exhibit good stability. After being placed at 4°C for 30 days, the changes in particle size, zeta potential, and drug loading are all less than 5%, and the polydispersity index remains <0.3. This provides a reliable guarantee for the industrial production and long-term storage of the formulation, while ensuring the quality consistency between different batches and improving the safety and convenience of clinical medication.

[0014] On the other hand, the present invention also provides a method for preparing the above-mentioned glycyrrhizin-doxorubicin nanoparticles, using glycyrrhizin as a carrier material and glutaraldehyde as a crosslinking agent, and preparing a glycyrrhizin nanoparticle formulation co-loaded with DOX using a solvent removal method. Specifically, the method for preparing glycyrrhizin-doxorubicin nanoparticles of the present invention includes the following steps: (1) Dissolve glycyrrhizin in PBS to obtain a glycyrrhizin solution; (2) Add an ethanol suspension of doxorubicin to the glycyrrhizin solution while stirring, and stir to allow doxorubicin and glycyrrhizin to self-assemble into nanoparticle precursors; (3) Add glutaraldehyde solution to carry out cross-linking reaction, so that the glycyrrhizin molecules are cross-linked by glutaraldehyde; (4) Dialyze the cross-linked reaction solution to remove ethanol, glutaraldehyde and free small molecules; (5) The dialysis solution was freeze-dried to obtain glycyrrhizin-doxorubicin nanoparticles.

[0015] PBS is phosphate-buffered saline.

[0016] In some preferred embodiments, the concentration of glycyrrhizin is 10-30 mg / mL, the volume ratio of ethanol to PBS is (0.5-4):1, the mass of doxorubicin is 10%-50% of the glycyrrhizin mass, the amount of glutaraldehyde relative to glycyrrhizin is 20-50 μg / mg, and the cross-linking reaction time is 2-24 hours.

[0017] Further preferably, the optimal preparation process of the present invention is as follows: the concentration of glycyrrhizin is 15 mg / mL, the volume ratio of ethanol to PBS is 2:1, the mass of doxorubicin is 50% of the glycyrrhizin mass, the amount of glutaraldehyde relative to glycyrrhizin is 20 μg / mg, and the cross-linking reaction time is 2 hours. Upon testing, the DNPs prepared according to the optimal process are a loose red powder with a drug loading of 21.67 ± 2.98%, a particle size of 132.5~134.3 nm, and TEM shows that the DNPs are uniformly sized regular spheres under a microscopic scale. Figure 2After being stored at 4 ℃ for 30 days, the particle size, potential, and LC% of DNPs all changed by less than 5%, indicating their stability. In vitro release studies showed that within 24 h, the cumulative release rates of DNPs were 75.59 ± 5.78% (pH 5.0) and 51.45 ± 2.13% (pH 7.4), respectively, significantly higher than 5.0%. P The concentration of DNPs (<0.05) is lower than the cumulative release rate of free DOX under the same pH conditions, indicating that DNPs have a significant sustained-release effect and pH-sensitive release characteristics.

[0018] In another aspect, the present invention also provides a pharmaceutical composition comprising the above-described glycyrrhizin-doxacin nanoparticles and a pharmaceutically acceptable carrier.

[0019] The pharmaceutically acceptable carriers include, but are not limited to, fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.

[0020] In some preferred embodiments, the pharmaceutical composition is an injection, a lyophilized powder for injection, or an oral preparation, more preferably a lyophilized powder for injection.

[0021] Finally, the present invention also provides the application of the above-mentioned glycyrrhizin-doxacin nanoparticles in the preparation of antitumor drugs.

[0022] In some preferred embodiments, the tumor includes, but is not limited to, acute leukemia, malignant lymphoma, breast cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, bone and soft tissue sarcoma, nephroblastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, endometrial cancer, urothelial carcinoma, pleomorphic lung cancer, esophageal cancer, Hodgkin's lymphoma, and more preferably non-small cell lung cancer.

[0023] The technical solution of the present invention has the following beneficial effects: (1) The glycyrrhizin-doxacin nanoparticles provided by this invention have uniform particle size, narrow distribution, good drug loading capacity, and good stability. In vitro release studies show that the glycyrrhizin-doxacin nanoparticles have pH-responsive release characteristics. The release rate is significantly faster under acidic conditions (pH 5.0) simulating the tumor microenvironment than under physiological conditions (pH 7.4). At the same time, the glycyrrhizin-doxacin nanoparticles slow down the release of free DOX, laying the foundation for selective drug release and sustained release of DOX at the tumor site in vivo.

[0024] (2) The glycyrrhizin-doxacin nanoparticles provided by this invention achieve "detoxification and enhancement" of free DOX at both in vivo and in vitro levels. At the cellular level, the glycyrrhizin-doxacin nanoparticles enhance the selective killing of tumor cells by DOX. At the animal level, the glycyrrhizin-doxacin nanoparticles enhance DDR by activating the γ-H2A.X / ATR / CHK1 signaling axis and downstream P53 protein in tumor tissue to achieve stronger tumor tissue killing activity; at the same time, they significantly reduce the systemic toxicity and organ pathological damage caused by DOX, and reduce cardiotoxicity by maintaining the Bax / Bcl-2 balance in cardiac tissue.

[0025] (3) The glycyrrhizin-doxacin nanoparticles provided by the present invention prolong the terminal elimination half-life of free DOX, reduce the clearance rate, and increase the in vivo exposure. Tissue distribution studies show that, compared with free DOX, glycyrrhizin-doxacin nanoparticles accumulate more in tumor tissue, are eliminated more slowly, and are distributed less in heart tissue.

[0026] (4) Compared with doxorubicin hydrochloride injection (ADM), the glycyrrhizin-doxorubicin nanoparticles provided by this invention exhibit stronger antitumor activity and higher cardiac and hepatic safety. While the antitumor activity of the glycyrrhizin-doxorubicin nanoparticles is weaker than that of doxorubicin hydrochloride liposome injection (Lip-ADM), it effectively improves the nephrotoxicity caused by the prolonged exposure time of Lip-ADM. Therefore, the efficacy-safety characteristics of the glycyrrhizin-doxorubicin nanoparticles give them good potential for clinical application. Attached Figure Description

[0027] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0028] Figure 1 Photograph of the glycyrrhizin-doxacin nanoparticles of this invention; Figure 2 TEM image (50,000 ×) of the glycyrrhizin-doxacin nanoparticles of the present invention; Figure 3This is a graph showing the effect of an orthogonal experiment. Figure 4 The Tyndall effect of the glycyrrhizin-doxacin nanoparticle solution of the present invention; Figure 5 The particle size distribution of the glycyrrhizin-doxacin nanoparticles of this invention is shown in the diagram (n = 3). Figure 6 Zeta potential diagram of the glycyrrhizin-doxacin nanoparticles of the present invention (n = 3); Figure 7 The stability results of the glycyrrhizin-doxacin nanoparticles of the present invention after 30 days of storage (Mean ± SD, n=3); Figure 8 Results of in vitro release of DOX and glycyrrhizin-doxorubicin nanoparticles; Figure 9 The effect of GNPs on the viability of LLC and H9C2 cells (n = 3); Figure 10 LLC cell killing curve (n = 3); Figure 11 The killing curve of H9C2 cells (n = 3); Figure 12 LLC cell migration results (50 × 10⁻⁶); Figure 13 For LLC cell migration statistics (n = 3); Note: P<0.05; Figure 14 Fluorescence image of mitochondrial membrane potential in LLC cells (400 ×). Figure 15 Changes in mitochondrial membrane potential in LLC cells (n = 3); Note: P<0.05, P<0.01; Figure 16 The changes in the LLC cell cycle are shown below; where A: normal group; B: DOX group; C: DNPs group; D: statistical analysis of cell cycle distribution in each group. Figure 17 Kill curves for the LLC-3D spherical model (n = 3); Figure 18 The curve shows the change in tumor volume over time (n = 8). Figure 19 For tumor weight (n = 8), note: P<0.05; P<0.01; Figure 20For mouse body weight and organ index (n = 8), note: P<0.05; P<0.01; Figure 21 A diagram showing the morphology of a tumor; Figure 22 HE staining and TUNEL staining for tumor pathological sections; Figure 23 The apoptosis positivity rate in tumor sections (n ​​= 3) is shown in the figure. P<0.05; P<0.01; Figure 24 The results of serum biochemical marker detection in mice (n = 8) are shown. Note: P<0.05; Figure 25 HE staining of mouse liver, heart, spleen, lung, and kidney (liver, heart, and kidney: 400×; spleen and lung: 200×); Figure 26 Serum CK-MB and cTnI levels in mice (n = 8), Note: P<0.05; P<0.01. Detailed Implementation

[0029] To fully understand the purpose, features, and effects of this invention, the invention will be described in detail through the following embodiments. Except as described below, the process methods of this invention employ conventional methods or apparatus in the art. Unless otherwise specified, the terms used below have the meanings commonly understood by those skilled in the art.

[0030] Example 1: Preparation of DNPs 1. Reagents and Instruments The main experimental reagents, consumables and instruments are shown in Table 1 and Table 2.

[0031] Table 1. Reagents and Consumables Used in the Experiment

[0032] Table 2. Experimental Instruments

[0033] 2. Experimental Methods Glycyrrhizin-doxacin nanoparticles were prepared by a desolvation method. An appropriate amount of glycyrrhizin was dissolved in a PBS solution at pH 7.4. Doxorubicin ethanol suspension was slowly added dropwise while stirring. After stirring for 1 h, 8% glutaraldehyde solution was added to continue cross-linking. After cross-linking was complete, ethanol, glutaraldehyde, and free small molecules were removed by dialysis, and the nanoparticles were then lyophilized to obtain DNPs.

[0034] Protein concentration (A), the ratio of dehydrating agent (ethanol) to PBS (B), the ratio of small molecule (DOX) to protein mass (C), the amount of cross-linking agent (glutaraldehyde) (D), and the cross-linking time (E) were selected as factors for nanoparticle preparation. An orthogonal experiment was conducted using an L16(45) orthogonal array. Four different levels were selected for each factor, with encapsulation efficiency (EE%) and drug loading (LC%) as evaluation indicators. The factor levels are shown in Table 3. A total of 16 experiments were conducted according to the orthogonal experimental design table. Three batches of samples were prepared in parallel for each experiment. LC% and EE% were calculated, and range analysis was performed. The analysis results and effect curves for each factor are shown in Table 4. Figure 3 The calculation methods for LC% and EE% are as follows: EE% = (Amount of DOX in DNPs / Amount of DOX input) × 100%; LC% = (Amount of DOX in DNPs / Amount of DNPs) × 100%.

[0035] Table 3. Factor Level Table for Orthogonal Experiment

[0036] Table 4 Orthogonal experimental design and results ( n = 3)

[0037] As shown in Table 3, the optimal preparation process for the DNPs of this invention is A2B3C4D1E1, namely: GP concentration of 15 mg / mL, ethanol:PBS = 2:1, DOX feed ratio of 50%, glutaraldehyde dosage of 20 μg / mg, and crosslinking time of 2 h.

[0038] The DNPs used in the following embodiments were all prepared according to the optimal preparation process.

[0039] Example 2: Properties of DNPs 1. Particle size distribution and morphology characterization Dissolve an appropriate amount of DNPs lyophilized powder in deionized water and observe the Tyndall effect under laser illumination. Use a Malvern particle size analyzer to determine the particle size, polydispersity index (PDI), and Zeta potential of the DNPs. Prepare a 1 mg / mL DNPs aqueous solution by dissolving an appropriate amount of DNPs lyophilized powder in deionized water, pour it into a Malvern four-way cuvette, control the page height at 10–15 mm, select water as the dispersion medium, set the temperature to 25 °C, and determine the particle size distribution of the sample. Prepare three batches of samples in parallel.

[0040] Drop the sample onto a copper grid, let it stand for 3-5 minutes, use filter paper to absorb excess liquid from the edge of the droplet, let it dry naturally, and then observe the morphology of the decoction under a transmission electron microscope (TEM).

[0041] DNPs are a red, loose powder. Figure 1 When dissolved in water, it exhibits a significant Tyndall effect. Figure 4 The microstructure of the nanoparticles was observed using TEM, and the results were as follows ( Figure 2 The results showed that DNPs were spherical nanoparticles with a uniform appearance and size distribution, and the nanoparticle size was approximately 130 nm as observed under a microscope. The DNPs were analyzed using a Malvern laser particle size analyzer, and the results were... Figure 5 , Figure 6 The results showed that the DNPs had a particle size of 132.5 ± 0.8 nm and a PDI of 0.245 ± 0.005, consistent with the TEM observations; the Zeta potential was -18.3 ± 0.4 mV.

[0042] 2. Stability Study The prepared DNPs were placed at 4 °C. After 0, 1, 3, 7, 14 and 30 days, a certain amount of samples were taken and their particle size, PDI value and Zeta potential were determined using a Malvern particle size analyzer. The DOX concentration was determined according to the analytical method validation guidelines of Chinese Pharmacopoeia 2025 Edition 9101 and its EE % and LC % were calculated.

[0043] The stability results of DNPs are shown in [the table]. Figure 7 Within 30 days, the particle size ranged from 132.5 to 134.3 nm, the PDI ranged from 0.245 to 0.262, the Zeta potential ranged from -18.3 to -17.4 mV, and the LC% ranged from 22.29 to 23.31%. After being placed at 4 °C for 30 days, the DNPs formulation showed changes of less than 5% in particle size, potential, and LC% and a PDI that remained below 0.3, indicating good stability of the DNPs.

[0044] 3. In vitro release studies The in vitro release of nanoparticles was studied using dialysis. A PBS solution with pH 7.4 simulated the in vivo physiological environment, while a PBS solution adjusted to pH 5.0 with phosphate simulated the tumor microenvironment. Two mL of 1 mg / mL DOX solution and DNPs containing an equivalent concentration of DOX were placed in separate dialysis bags (molecular weight cutoff 8000–10000), and the ends were sealed. The dialysis bags were then placed in 38 mL of PBS solutions at different pH values ​​and incubated at 37 °C with constant shaking at 300 rpm / min. Four mL of the extradialysate was collected at 0, 1, 2, 4, 8, 12, and 24 h for lyophilization, and the volume was replenished with fresh PBS solution of the same pH value. The DOX content in the lyophilized powder was determined according to the analytical method validation guidelines of the 2025 edition of the Chinese Pharmacopoeia, 9101, and the cumulative release rate was calculated.

[0045]

[0046] Where t is the sampling time, M is the total amount of DOX, M RT M is the cumulative DOX release at time t. t t is the amount of DOX removed at time t, v is the sampling volume, and V is the total volume of externally released liquid.

[0047] Results of in vitro release studies are shown in Figure 8 The results showed that the release curves of DOX and DNPs exhibited similar overall trends, with rapid release in the first 8 hours followed by a gradual slowdown. At 24 hours, the cumulative release rates of free DOX were 97.74 ± 0.35% (pH 5.0) and 81.94 ± 4.38% (pH 7.4), while the cumulative release rates of DNPs were 75.59 ± 5.78% (pH 5.0) and 51.45 ± 2.13% (pH 7.4), respectively. These results indicate that DNPs have a significantly faster in vitro release rate compared to free DOX. P <0.05) slowed down, with DNPs showing a significant release rate at pH 5.0 ( P <0.05) when pH is above 7.4.

[0048] 4. Conclusion Experimental results show that the DNPs of the present invention are stable and have the potential to achieve targeted release and sustained release at tumor sites in vivo.

[0049] Example 3: In vitro activity assay of GNPs 1. Reagents and Instruments The main experimental reagents, consumables and instruments are shown in Tables 5 and 6.

[0050] Table 5. Reagents and Consumables Used in the Experiment

[0051] Table 6. Experimental Instruments

[0052] 2. Experimental Methods 2.1 LLC and H9C2 cell culture Both LLC and H9C2 cells were purchased from Wuhan Pronosei Life Sciences Co., Ltd. LLC cells were cultured in LLC-specific medium, while H9C2 cells were cultured in DMEM complete medium (DMEM medium + 10% FBS). Cell culture was conducted in a constant temperature and humidity incubator at 37℃ and 5% CO2.

[0053] 2.2 LLC and H9C2 cell viability assay Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent to evaluate the toxic effects of DOX, GNPs, and DNPs on H9C2 cells and their killing effect on LLC.

[0054] After LLC cells and H9C2 cells reached the logarithmic growth phase, they were digested with trypsin at a concentration of 5 × 10⁻⁶. 3 Cells were seeded at a density of 100 μL of cell suspension per well in 96-well plates. After incubation at 37 °C for 24 h, the original culture medium was discarded, and 200 μL of serum-free DMEM medium containing different concentrations of DOX, DNPs, and GNPs (the DOX concentration in DNPs was consistent with that of free DOX) was added to each well. Control wells and cell-free control wells were also prepared. After incubation for 48 h, 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated in the dark for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the blue-purple formazan. The absorbance of the 96-well plates at 490 nm was measured using a microplate reader, and the cell inhibition rate was calculated.

[0055]

[0056] in, This represents the absorbance value of the administration well. The absorbance of the uninoculated wells is shown. This represents the absorbance value of the well without drug delivery.

[0057] The results of the effect of GNPs on cell viability are shown in Figure 9The results showed that when the concentration of drug-free GNPs was below 500 μg / mL, the survival rate of LLC cells was greater than 90%; and when the concentration was below 250 μg / mL, the survival rate of H9C2 cells was greater than 90%. Therefore, the safe dose of GNPs for LLC is ≤ 500 μg / mL, and the safe dose for H9C2 is ≤ 250 μg / mL.

[0058] The cytotoxic activity of DOX and DNPs against LLC and H9C2 cells was evaluated within a safe and non-toxic concentration range for GNPs. Results for LLC cell experiments are shown below. Figure 10 48 h after administration, DNPs and DOX reduced the IC50 of LLC. 50 The IC50 values ​​for DNPs were 0.1561 and 0.2282 μg / mL, respectively. 50 The concentration of free DOX was reduced to 68.40%. Results of the H9C2 cell viability assay are shown below. Figure 11 48 h after administration, the IC50 of DNPs and DOX on H9C2 was [not specified]. 50 The IC50 values ​​for DNPs were 0.1900 and 0.1670 μg / mL, respectively. 50 The concentration of DNPs was increased to 113.77% of that of free DOX. The results showed that DNPs enhanced the selective killing ability of LLC cells compared to free DOX.

[0059] 2.3 LLC cell migration ability assay The effects of DOX and DNPs on the migration ability of LLC cells were evaluated using a scratch assay.

[0060] Beforehand, draw equidistant parallel lines on the bottom of the 6-well plate using a marker pen as positioning marks. After the LLC cells grow to the logarithmic growth phase, they are digested with trypsin at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of 500 μL of cell suspension per well in 6-well plates. Cells were cultured at 37°C until the cell density reached >90%. Using a 200 μL pipette tip, a straight line perpendicular to the positioning mark was drawn along the bottom of each well. Each well was washed three times with 500 μL PBS to remove floating cells. 1 mL of serum-free DMEM medium containing DOX and DNPs (DOX concentrations of 0.02 and 0.04 μg / mL, respectively) was added. A blank medium control well was also included. Each concentration was used in triplicate. Samples were taken and photographed at the same location at 0, 24, and 48 h. The results were analyzed using Image J 1.54g to calculate cell migration rate (MR).

[0061]

[0062] Where D0 is the initial intercellular distance at 0 h, and D t The distance between cells is th.

[0063] The results of the scratch test are shown below. Figure 12 , Figure 13 The results showed that the scratch widths were basically the same in all groups at 0 h; after culturing for 24 h, cells in all groups showed varying degrees of migration; after culturing for 48 h, the mean mitochondrial (MR) of the Control group was 20.50 ± 2.66%, the MR of the DOX-0.004 μg / mL group was 17.15 ± 2.23%, the MR of the DOX-0.002 μg / mL group was 18.93 ± 2.98%, the MR of the DNPs-0.004 μg / mL group was 14.94 ± 1.86%, and the MR of the DNPs-0.002 μg / mL group was 16.33 ± 2.07%. Different concentrations of DOX and DNPs inhibited LLC cell migration, but at the same DOX concentration, the migration rate of the DNPs group was lower than that of the DOX group. Specifically, 0.04 μg / mL DNPs significantly inhibited LLC cell migration (…). P <0.05). This indicates that DNPs have a stronger ability to inhibit cell invasion compared to DOX.

[0064] 2.4 Detection of mitochondrial membrane potential in LLC cells The effects of DOX and DNPs on mitochondrial membrane potential in LLC cells were detected using a mitochondrial membrane potential detection kit (JC-1).

[0065] Drug administration: LLC cells were digested with trypsin after reaching the logarithmic growth phase, and then administered at a dose of 5 × 10⁻⁶ mg / L. 5 Cells were seeded at a density of 1 mL of cell suspension per well in 6-well plates. After incubation at 37 °C for 24 h, the original culture medium was discarded, and 2 mL of serum-free DMEM medium containing DOX and DNPs (DOX concentration of 0.195 μg / mL) was added to each well. A control well containing blank culture medium was also included. After incubation for another 24 h, the apoptosis inducer CCCP was added to one of the untreated wells to a final CCCP concentration of 0.1 mmol / L. After 1 h of treatment, the mitochondrial membrane potential of LLC cells completely disappeared, serving as the positive control group.

[0066] Staining: Follow the instructions for the mitochondrial membrane potential assay kit (JC-1): Discard the original culture medium and wash once with 1 mL PBS. Add 1 mL of serum-free DMEM medium and 1 mL of JC-1 working solution to each well, mix thoroughly, and incubate at 37 ℃ for 20 min. Discard the supernatant and wash twice with ice-cold JC-1 staining buffer (1×). Add 2 mL of serum-free DMEM medium and observe the cells under a fluorescence microscope. Observe the green fluorescence emitted by JC-1 monomers under excitation light of 490 nm and emission light of 530 nm, and observe the red fluorescence emitted by JC-1 aggregates under excitation light of 525 nm and emission light of 590 nm.

[0067] JC-1 staining results are shown in […]. Figure 14 , Figure 15 The results showed that after administration of CCCP, the red fluorescence of JC-1 polymer in LLC cells was essentially extinguished, and the mitochondrial membrane potential decreased significantly. P <0.01). Administration of 0.195 μg / mL DOX and DNPs of equal concentrations of DOX also significantly decreased the mitochondrial membrane potential in LLC cells. P <0.01, but the mitochondrial membrane potential in the DNPs-treated group was significantly lower than that in the DOX-treated group ( P <0.05). This indicates that DNPs have a stronger effect on downregulating LLC mitochondrial membrane potential compared to DOX.

[0068] 2.5 LLC cell cycle detection Effects of DOX and DNPs on LLC cell cycle.

[0069] Drug administration: LLC cells were digested with trypsin after reaching the logarithmic growth phase, and then administered at a dose of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 6-well plates, with 1 mL of cell suspension in each well. After 12 h, the cells were replaced with serum-free DMEM for synchronization for another 12 h. 2 mL of LLC-specific medium containing DOX and DNPs (DOX concentration of 1.5625 μg / mL) was added to each well.

[0070] Fixation: After 12 h, trypsin digests cells in each well, counts are performed, and 5 × 10⁶ cells are collected per concentration. 5 One cell. Wash once with 1 mL PBS, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells in 300 μL PBS. Add 700 μL of pre-cooled anhydrous ethanol at -20 ℃ to the resuspended cells, mixing dropwise while adding. After thorough mixing, fix at -20 ℃ overnight.

[0071] Staining: Centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cells in 1 mL PBS, and incubate at room temperature for 15 min. Centrifuge at 300 g for 5 min, discard the supernatant, add 100 μL RNase A Reagent and thoroughly pipette to resuspend the cells, incubate at 37 ℃ for 30 min. Add 400 μL Cycle Blue Reagent and mix thoroughly, incubate at 2–8 ℃ in the dark for 30 min. Analyze the results.

[0072] Cell cycle detection results are shown below Figure 16 Compared with the control group, the proportion of cells in the G2 / M phase increased in both the DOX and DNPs groups. Statistical results showed that the proportion of cells in the G2 / M phase in the DNPs group (22.90 ± 1.34%) was higher than that in the DOX group (18.93 ± 2.12%), and higher than that in the control group (8.87 ± 0.37%). These results indicate that DNPs have a stronger effect in inducing cell arrest in the G2 / M phase and possess a stronger inhibitory effect on cell mitosis.

[0073] 2.6 LLC Cell 3D Spheroid Viability Assay After LLC cells reached the logarithmic growth phase, they were digested with trypsin at a concentration of 5 × 10⁻⁶. 3 Seeds were generated at a density of cells / well in BeyoGold culture medium treated with 3D cell culture coating. TM 100 μL of cell suspension was seeded into each well of a 96-well plate. After 48 h, the cells were observed under a microscope to form compact cell spheroids. The original medium in each well was replaced three times with half the original volume of serum-free DMEM, and then 100 μL of serum-free DMEM containing different concentrations of DOX and DNPs (with the DOX concentration kept constant) was added. The plates were incubated at 37 ℃ for another 96 h. 20 μL of medium containing cell spheroids was carefully aspirated from each well and added to a standard 96-well plate. 180 μL of serum-free DMEM and 20 μL of CCK-3D solution were added, and the plates were incubated for 1 h. The absorbance of the 96-well plate at 450 nm was measured using a microplate reader, and the cell inhibition rate was calculated.

[0074] The cytotoxic activity of DOX and DNPs against the LLC-3D spheroid model was evaluated within the safe and non-toxic concentration range of GNPs. Figure 17 LLC cells were seeded in ultra-low adsorption round-bottom 96-well plates for 48 h, and dense, non-adherent spheres were observed under a microscope. 96 h after drug administration, DNPs and DOX showed an effect on the IC50 of LLC-3D spheres. 50 The IC50 values ​​for DNPs were 0.5211 and 0.7187 μg / mL, respectively. 50 The concentration was reduced to 72.51% of DOX. The results indicate that DNPs enhanced the killing activity of DOX against the LLC-3D spheroid model.

[0075] 3. Conclusion Based on the above results, DNPs achieve enhanced killing of tumor cells and attenuated protection of normal cardiomyocytes at the in vitro level, and preliminarily verify the application potential of the novel licorice protein nano-delivery system DNPs in tumor therapy.

[0076] Example 4: Clinical application value of DNPs DNPs are compared with clinically used doxorubicin hydrochloride injection (ADM) and its liposome preparation, namely doxorubicin hydrochloride liposome injection (Lip-ADM). By establishing a subcutaneous LLC tumor mouse model, the anti-tumor efficacy of the three agents is comprehensively evaluated in terms of tumor growth inhibition, tumor histopathological changes, and apoptosis-inducing ability; by monitoring mouse body weight changes, organ indexes, serum biochemical indicators (ALT, AST, LDH, BUN, CRE) and HE-stained pathological sections of major organs (heart, liver, spleen, lung, kidney), the systemic safety and organ toxicity differences of the three agents are systematically compared. Meanwhile, a blank licorice protein nanoparticle (GNPs) control group is additionally set to clarify the effect of the carrier itself on the efficacy and safety.

[0077] 1. Reagents and Instruments Main experimental reagents, consumables and instruments are shown in Table 7 and Table 8.

[0078] Table 7 Instruments for experiment

[0079] Table 8 Reagents and consumables for experiment

[0080] 2. Experimental Methods 2.1 Animal feeding Forty-eight 6-week-old male C57BL / 6 mice (weighing approximately 20 g) were purchased from Beijing Spford Biotechnology Co., Ltd., with the license number SCXK (Jing) 2024-0001. The mice were raised in the animal room of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences, with an ambient temperature of 22 ± 2 °C, an ambient humidity of 55 ± 5 %, a light cycle of 12 h light / 12 h darkness, and free access to food and water. All animal experiments were approved by the Ethics Committee of the Laboratory Animal Center of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences, with the approval number IBTCMCACMS21-2503-08.

[0081] 2.2 Modeling, administration and sample collection Forty-eight C57 mice were acclimatized for 7 days and then randomly divided into a normal control group (n=8) and a model group. LLC cells in the logarithmic growth phase were collected, digested, and resuspended in PBS to obtain an LLC cell suspension. The suspension was then subcutaneously injected into the left axilla of mice in the model group with 3.5 × 10⁻⁶ LLC cells. 6 (each 0.2 mL of PBS).

[0082] Forty-eight mice were divided into six groups of eight. The day of tumor cell inoculation was recorded as day 0. The drug was administered via tail vein injection every three days for a total of three administrations. The dosage was 0.1 mL / 10 g. The specific grouping and administration regimen are shown in Table 9.

[0083] Table 9. Grouping and treatment of C57 mice

[0084] 2.3 Evaluation of antitumor activity and safety Tumor growth was continuously monitored throughout the experiment. Tumor dimensions (L being the longest diameter of the tumor, W being the maximum diameter perpendicular to the longest diameter) were measured using calipers, and tumor volume was calculated. The formula for calculating tumor volume is as follows:

[0085] After the last administration, mice were fasted for 24 hours, then euthanized by cervical dislocation. Serum, liver, heart, spleen, lungs, and kidneys were obtained. Tumor weight was measured, and the tumor growth inhibition rate (TGI) was calculated as: TGI = (1 - tumor weight in the treatment group / average tumor weight in the control group) × 100%. The dissected tumor tissue was embedded in paraffin, prepared into pathological sections, and stained with eosin-hematoxylin (HE) and TUNEL to observe tumor cell apoptosis.

[0086] Throughout the experiment, the living conditions and food intake of C57 mice were continuously observed. Mice were fasted for 12 hours after the last administration, and their weight was recorded. The heart and spleen were weighed, and organ indices were calculated. The dissected liver, heart, spleen, lungs, and kidneys were embedded in paraffin, prepared as pathological sections, and stained with hematoxylin and eosin (HE) to observe tissue condition. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) levels were measured according to the kit instructions to analyze organ damage in each treatment group.

[0087] The efficacy results indicate that GNPs affect the development process of tumors within the body ( Figure 18 No significant effect was observed; tumor weight at the end of the experiment ( Figure 19 There was no significant difference compared to the Model group. Weight and organ index results indicated ( Figure 20 During the treatment period, the body weight of the GNPs-H group steadily increased. At the end of the experiment, there were no significant differences in body weight, cardiac index, and spleen index between the GNPs-H group and the Model group. The results indicate that GNPs have no significant effect on tumor growth, body weight, or organ health in mice.

[0088] 2.3 Tumor volume changes and TGI results The efficacy results of the drug against in vivo ectopic tumors in LLC are shown in [the table below]. Figure 18 , Figure 19 , Figure 21 . Figure 18 The results showed that 6 days after tumor inoculation (i.e., two doses), there was a difference in tumor volume between the Model group and the drug-treated group. The tumor development in the Lip-ADM group and the DNPs-H group was slower, with the Lip-ADM group even showing a decreasing trend in tumor volume, while the ADM group showed no significant decrease in tumor volume. At the end of the experiment ( Figure 19 , Figure 21 The TGI in the ADM group was 33.20 ± 10.29%, the TGI in the Lip-ADM group was 67.64 ± 13.47%, and the TGI in the DNPs-H group was 47.52 ± 22.11%. Tumor weight was significantly different in all three groups. P <0.01) is less than that of the Model group. The above results indicate that ADM, Lip-ADM and DNPs can all significantly inhibit the growth of ectopic tumors in LLC, and the inhibitory effect is ranked as follows: Lip-ADM is better than DNPs-H is better than ADM.

[0089] 2.4 Tumor histopathological results The results of HE staining and TUNEL staining of tumor tissues in each group are shown in the figure. Figure 22 HE staining results showed that the tumor cells in the Model group were neatly and tightly arranged with intact nuclei and abundant blood vessels; the tumor cells in the ADM group showed vacuoles and increased nuclear volume; the tumor cells in the Lip-ADM group and the DNPs-H group showed a large number of vacuoles, the tumor tissue structure was destroyed, and fibrosis appeared in the intercellular spaces.

[0090] TUNEL staining results showed that under low magnification (100 ×), the Model group and ADM group appeared blue overall, while the Lip-ADM group sections were generally yellowish, with a large number of yellowish-brown apoptotic bodies observed. The DNPs-H group showed a large area of ​​yellowish-brown apoptotic tissue. Under high magnification (400 ×), three fields of view were randomly selected from each section for quantitative analysis of positive areas, and the results showed ( Figure 23The positive rates were 4.26 ± 2.53% in the Model group, 6.86 ± 2.78% in the ADM group, 11.35 ± 2.96% in the Lip-ADM group, and 9.81 ± 1.51% in the DNPs group. The positive rates of apoptotic cells in the Lip-ADM group (P<0.01) and the DNPs-H group (P<0.05) were significantly higher than those in the Model group.

[0091] 2.5 Results of mouse body weight and organ index Throughout the experiment, the mice maintained good mental condition and had smooth fur. In the later stages of the experiment, some mice experienced mobility impairment due to large tumors, but their overall condition remained good. No mice experienced stress or death due to tail vein injection or drug toxicity during the experimental period.

[0092] Mouse body weight and organ index results are shown in Figure 20 . Figure 20 A showed that after the first dose (Day 3) and the second dose (Day 6) of Lip-ADM and ADM, respectively, body weight began to decrease. The body weight of the DNPs-H group remained higher than that of the ADM and Lip-ADM groups, consistent with the model group. At the end of the experiment, there was no significant difference in body weight between the Model group and the Control group, while the body weight of the ADM and Lip-ADM groups ( P <0.01) Compared with the Control group, the body weight decreased, while the body weight of the DNPs-H group was significantly lower ( P <0.01) was higher than that of the Lip-ADM group, but not significantly different from the Control and Model groups; Figure 20 B shows that there was no significant difference in cardiac index between the Control group and the Model group, while the cardiac index was significantly different between the ADM group and the Lip-ADM group. P <0.01) was higher than that of the Model group, while the DNPs-H cardiac index was not significantly different from that of the Model group, which was significantly ( P The value of DNPs was <0.01, which was lower than that of the Lip-ADM group. These results indicate that the formation and development of the ectopic tumor model had no effect on the body weight and cardiac index of mice during the experimental period. Compared with the Control and Model groups, the ADM and Lip-ADM groups showed a decrease in body weight and an increase in cardiac index, while the DNPs group showed no significant difference in body weight and cardiac index compared with the Control and Model groups.

[0093] Figure 20 C showed that the spleen index in the Model group was greater than that in the Control group. After drug administration, the spleen index in all groups decreased compared to the Model group, with the spleen indices in the Lip-ADM group and the DNPs group being significantly smaller than those in the Model group. P <0.01) and ADM group ( P<0.05) 2.6 Serum biochemical index test results Serum biochemical index test results are shown in Figure 24 . Figure 24 A showed that there was no significant difference in ALT activity between the Control group and the Model group, while ALT activity was significantly different in the ADM group. P <0.05) was higher than that of the Model group. There was no significant difference in ALT activity between the Lip-ADM group and the DNPs-H group and the Model group. The ALT activity in the DNPs-H group was significantly higher ( P The AST / ALT ratio in the ADM group was <0.05, lower than that in the ADM group. Meanwhile, the AST / ALT ratio in the ADM group showed a decreasing trend compared to the Model group. Figure 24 C). Figure 24 B. Figure 24 C Figure 24 The results showed no significant differences in AST activity, CRE activity, and BUN activity between the Control group and the Model group, and between the drug-treated group and the Model group. Figure 24 F shows that the LDH content in the Model group is significantly higher than that in the Control group. P <0.01) increases, and after administration, LDH levels decrease to varying degrees (Lip-ADM group). P <0.01; other groups P <0.05).

[0094] 2.7 Pathological analysis of major organs HE staining results of major organs in each group of mice are shown in the figure. Figure 25 .

[0095] Liver section staining results showed that the liver lobule structure was intact and the hepatic cords were clear and neatly arranged in the Control, Model, and Lip-ADM groups; the hepatic cord cells in the ADM group were significantly damaged, and a large number of vacuoles appeared in the cytoplasm; the hepatic cord cells in the DNPs-H group had loose and lightly stained cytoplasm, and a few fatty vacuoles appeared. Heart section staining results showed that the cardiomyocytes in the ADM group were deformed, disordered, and had widened intercellular matrix; the cardiomyocytes in other groups were neatly arranged, with clear striations, and no obvious abnormal pathological manifestations were observed. Spleen section staining results showed that the red and white pulp boundaries were clear in the Control, Lip-ADM, and DNPs-H groups, with the white pulp appearing as islands; the boundaries between the Model and ADM groups were blurred, with the white pulp connected and diffusely distributed, and the normal physiological structure was destroyed. Lung section staining results showed that in the Control group and DNPs-H, the bronchial epithelial cells were tightly and neatly arranged, the alveolar septa were thin, and the alveolar cavities were empty and clean; in the Model group, the bronchial epithelial cells were disordered, and pink secretions appeared in the alveolar cavities (indicated by blue arrows); in the ADM group and Lip-ADM group, the bronchial epithelial cells were disordered, the alveolar septa were thickened, but the overall alveolar structure was intact. Kidney section staining results showed that in the Lip-ADM group, the renal tubular epithelial cells were significantly detached, and there was obvious exudate in the tubules, appearing as cellular casts (indicated by blue arrows); in other groups, the renal corpuscles and renal tubules were clear and intact.

[0096] In summary, the above results indicate that the establishment of the tumor model led to structural abnormalities in the spleen and lungs. Lip-ADM and DNPs-H effectively reversed the histopathological state of the spleen and lungs, but the spleen histopathological changes were more severe in the ADM group. In addition, the ADM group showed more severe liver and heart tissue damage; the Lip-ADM group showed more severe kidney pathological damage; and the DNPs-H group showed mild liver damage. Other organs did not show significant pathological changes.

[0097] 2.8 Determination and Results of Serum Cardiac Injury Markers CK-MB and cTnI Levels The levels of creatine kinase isoenzyme MB (CK-MB) and cardiac troponin I (cTnI) in the serum of the Control, Model, DOX-H and DNPs groups were detected by ELISA to assess cardiac damage.

[0098] CK-MB Detection Method: Take 25 μL of serum obtained in Example 2.2, add 25 μL of standard & sample diluent and mix well to obtain the sample detection solution. Set up blank wells, zero wells, standard wells, and sample wells. Place the blank wells last, and add chromogenic reagents A and B and stop solution to the other wells for zeroing. Add 50 μL of standard working solutions of different concentrations, standard & sample diluent (zero well), and sample detection solution to the microplate, then add 50 μL of biotin antigen working solution. After sealing the plate, gently shake to mix, and incubate at 37 ℃ for 30 min. Discard the liquid in the plate and wash 5 times. Add 50 μL of avidin-HRP working solution to each well and incubate at 37 ℃ for 30 min. Discard the liquid in the plate and wash 5 times. Add chromogenic reagents A and B and incubate at 37 ℃ for 10 min. Add 50 μL of stop solution to each well. Detect the absorbance at 450 nm.

[0099] cTnI content detection method: Take 40 μL of serum obtained in Example 2.2, add 60 μL of standard & sample diluent and mix well to obtain the sample detection solution. Add 100 μL of standard working solution, standard & sample diluent and sample detection solution of different concentrations to the microplate. After sealing the plate, gently shake to mix and incubate at 37 ℃ for 90 min. Discard the liquid in the plate, immediately add 100 μL of biotinylated antibody working solution and incubate at 37 ℃ for 60 min. Discard the liquid in the plate and wash the plate 3 times. Add 100 μL of HRP enzyme conjugate working solution to each well and incubate at 37 ℃ for 30 min. Discard the liquid in the plate and wash the plate 5 times. Add 90 μL of substrate solution to each well and incubate at 37 ℃ for 15 min. Add 50 μL of stop solution to each well. Immediately measure the absorbance at 450 nm.

[0100] The results of serum CK-MB and cTnI content measurements are shown in the figure. Figure 26 .

[0101] Compared with the control group, there was no significant difference in CK-MB levels in the model group. After administration of ADM and Lip-ADM, CK-MB levels were significantly higher than in the model group. P <0.05); the CK-MB content in the DNPs-H group was significantly lower than that in the ADM group ( P <0.05) and Lip-ADM group ( P <0.01), with no significant difference from the model group.

[0102] Compared with the control group, there was no significant difference in cTnl levels in the model group. After administration, cTnl levels were significantly higher in both groups than in the model group. P <0.01); the content of DNPs-H in the group was lower than that in the other two groups, and there was a significant difference compared with the ADM group ( P<0.05).

[0103] The above results indicate that the establishment of the tumor model has no significant effect on the levels of serum cardiac markers. Compared with the clinical preparations ADM and Lip-ADM, DNPs significantly reduced the levels of serum cardiac injury markers CK-MB and cTnI.

[0104] 3. Conclusion DNPs were compared with clinically used DOX formulations ADM and Lip-ADM. The pharmacodynamic and toxicological differences among the three DOX formulations were systematically evaluated using an LLC mouse subcutaneous tumor model. A GNPs administration group was also added to clarify the carrier effect.

[0105] Firstly, in vivo experiments on GNPs showed that they do not possess LLC tumor-killing activity, have good safety profile for tail vein injection, and are a good drug carrier for DOX.

[0106] In vivo experiments of three DOX formulations showed that Lip-ADM had the best antitumor effect, followed by DNPs, while ADM was relatively weaker. This is because the EPR effect of the nano-formulations promotes their accumulation at the tumor site, while also reducing their distribution in cardiac tissue and cardiotoxicity. The long retention effect of PEG-modified Lip-ADM further enhanced its in vivo antitumor activity, but the prolonged in vivo exposure time also led to stronger systemic and nephrotoxicity. In summary, DNPs showed better tumor-suppressive effects than ADM but less than Lip-ADM, but exhibited higher overall safety. This result provides encouraging evidence for the clinical application of DNPs, suggesting that DNPs, as a novel DOX nano-formulation, have unique clinical application value.

[0107] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0108] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0109] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A glycyrrhizin-doxacin nanoparticle, characterized in that, include: Glycyrrhizin and doxorubicin; The doxorubicin is dispersed within the glycyrrhizin matrix, and the glycyrrhizin molecules are cross-linked by glutaraldehyde.

2. The glycyrrhizin-doxacin nanoparticles according to claim 1, characterized in that, The nanoparticles have a particle size of 130-140 nm and a drug loading of 15%-25%.

3. The glycyrrhizin-doxacin nanoparticles according to claim 1, characterized in that, The licorice protein is a protein with a molecular weight of 25-35 kDa extracted from licorice, or a dimer protein linked by disulfide bonds.

4. The glycyrrhizin-doxacin nanoparticles according to claim 1, characterized in that, After being placed at 4°C for 30 days, the nanoparticles showed changes of less than 5% in particle size, zeta potential, and drug loading.

5. A method for preparing glycyrrhizin-doxacin nanoparticles according to any one of claims 1 to 4, characterized in that, include: (1) Dissolve glycyrrhizin in PBS to obtain glycyrrhizin solution; (2) While stirring, add doxorubicin ethanol suspension dropwise to the glycyrrhizin solution and stir. (3) Add glutaraldehyde solution to carry out cross-linking reaction; (4) Dialyze the cross-linked reaction solution to remove ethanol, glutaraldehyde and free doxorubicin; (5) The dialysis solution was freeze-dried to obtain glycyrrhizin-doxacin nanoparticles.

6. The preparation method according to claim 5, characterized in that, The concentration of the glycyrrhizin solution is 10-30 mg / mL, the volume ratio of ethanol to PBS is (0.5-4):1, the mass of doxorubicin is 10%-50% of the glycyrrhizin protein, the amount of glutaraldehyde relative to glycyrrhizin is 20-50 μg / mg, and the cross-linking reaction time is 2-24 hours.

7. A glycyrrhizin-doxacin nanoparticle, characterized in that, It is prepared by the preparation method described in any one of claims 5 to 6.

8. A pharmaceutical composition, characterized in that, Includes the glycyrrhizin-doxacin nanoparticles and pharmaceutically acceptable carriers as described in any one of claims 1 to 5 and 7.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is an injection, a lyophilized powder injection, or an oral preparation.

10. The use of glycyrrhizin-doxacin nanoparticles according to any one of claims 1 to 4 and 7 in the preparation of antitumor drugs.

Images