A traditional Chinese medicine nanoparticle with tumor targeting function and its preparation method

Abstract

The present invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine nanoparticle with tumor targeting function and a preparation method thereof. Among them, the traditional Chinese medicine nanoparticle comprises the following raw materials in parts by weight: 10-30 parts of Pep-14 peptide, 6-15 parts of traditional Chinese medicine saponin powder, and 2-10 parts of paclitaxel. The traditional Chinese medicine saponin powder is prepared from ginseng, notoginseng, licorice, and bupleurum by an ultrasonic-assisted deep eutectic solvent extraction method. The saponin in the extract has high purity and strong activity. The designed Pep-14 peptide contains a tumor targeting segment, a hydrophobic binding segment, and a hydrophilic binding segment, and has tumor targeting function. In an aqueous solution, it can simultaneously load hydrophobic and hydrophilic components of traditional Chinese medicine by non-covalent forces, and self-assemble to form nanoparticles with smaller sizes, realizing efficient delivery, precise release, and deep tumor penetration functions, and improving the tumor killing efficiency and effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine nanoparticle with tumor targeting function and a preparation method thereof. Technical Background

[0002] Cancer has become one of the major public health problems today due to its complex etiology and great difficulty in radical cure. As one of the main methods for treating cancer, long-term monotherapy with a single drug has deficiencies such as insufficient efficacy, drug resistance, and adverse drug reactions. The anti-tumor active ingredients of traditional Chinese medicine can directly act on tumor cells, inhibit tumor growth and metastasis, or relieve the adverse reactions or complications caused by treatments such as chemotherapy, radiotherapy, or surgical resection, and have advantages such as high efficiency, low toxicity, wide action range, and multiple targets. Using the active ingredients of traditional Chinese medicine as anti-tumor drugs is becoming a new treatment method. However, although many traditional Chinese medicines have significant anti-tumor effects, due to the problems of strong hydrophobicity, low solubility, poor stability, and difficulty in drug formation of their lipophilic components, and the low cell uptake efficiency and insufficient targeting of their water-soluble components, the anti-tumor efficacy of traditional Chinese medicine in vivo is not ideal.

[0003] Therefore, there is an urgent need for a new drug delivery system to achieve efficient co-loading of lipophilic and water-soluble components of traditional Chinese medicine, so as to improve the tumor targeting rate and enhance the anti-tumor effect. Summary of the Invention

[0004] In view of the above situation, the present invention provides a traditional Chinese medicine nanoparticle with tumor targeting function and a preparation method thereof. The total saponins extracted and purified from traditional Chinese medicines ginseng and notoginseng are used as the main anti-tumor components. Based on the self-assembly principle, a polypeptide sequence containing a tumor targeting segment, a hydrophobic binding segment, and a hydrophilic binding segment is designed. The hydrophilic active ingredient total saponins and the hydrophobic drug paclitaxel are loaded by non-covalent interaction to prepare a traditional Chinese medicine nanoparticle, which can achieve the co-delivery of multiple components of traditional Chinese medicine, tumor targeting and deep penetration, and at the same time respond to release in the acidic microenvironment of the tumor, avoid the loss of drugs during transportation, and enhance the anti-tumor activity.

[0005] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0006] The present invention provides a traditional Chinese medicine nanoparticle with tumor targeting function, and the traditional Chinese medicine nanoparticle comprises the following raw materials in parts by weight: 10-30 parts of Pep-14 peptide, 6-15 parts of traditional Chinese medicine saponin powder, and 2-10 parts of paclitaxel.

[0007] Further, the traditional Chinese medicine saponin powder comprises the following raw materials in parts by weight: 15-30 parts of ginseng, 20-40 parts of notoginseng, 10-15 parts of licorice, and 10-15 parts of bupleurum.

[0008] Further, the sequence of the Pep-14 peptide is:

[0009] H2N-Arg-Gly-Asp-Phe-Leu-Ile-Val-Phe-Cys-Gln-Cys-Glu-His-Lys-COOH (H2N-RGDFLIVFCQCEHK) was synthesized by solid-phase synthesis method.

[0010] The preparation method of the traditional Chinese medicine saponin powder comprises the following steps:

[0011] S1: Weigh 15 - 30 parts of ginseng, 20 - 40 parts of notoginseng, 10 - 15 parts of licorice, and 10 - 15 parts of bupleurum, wash, dry in the sun, crush and mix them to obtain traditional Chinese medicine powder;

[0012] S2: Extract the total saponins of traditional Chinese medicine by ultrasonic-assisted deep eutectic solvent extraction method. Add choline chloride, 1,4-butanediol, and purified water to the traditional Chinese medicine powder obtained in step S1. The mass ratio of the traditional Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8. Reflux and extract for 20 min at 30 °C in a water bath and an ultrasonic power of 260 W. After filtering with a filter cloth, obtain the crude saponin extract;

[0013] S3: Take D101 macroporous resin, soak the D101 macroporous resin in 95% (v / v, volume fraction) ethanol for 24 h to swell it. Pack the column by the wet method, and rinse it evenly with 95% ethanol until the dripping becomes non-linear. Rinse until there is no turbidity when adding 4 times the volume of ultrapure water to the effluent and mixing evenly. Then rinse with a large amount of ultrapure water until there is no alcohol smell in the effluent to obtain the pretreated macroporous resin;

[0014] S4: Use the pretreated macroporous resin to elute the crude saponin extract obtained in step S2. The diameter-to-height ratio is 1:3, the flow rate is 1 BV / h. After adsorption for 2 h, elute successively with 3 BV volumes of ultrapure water, 20% (v / v) ethanol, and 70% (v / v) ethanol at an elution flow rate of 1.5 BV / h. Recover the 70% ethanol eluate fraction;

[0015] S5: Rotate and evaporate to dryness the 70% ethanol eluate fraction recovered in step S4. The rotation evaporation temperature is 25 - 30 °C, and the cold trap temperature is set at -10 °C to obtain the traditional Chinese medicine saponin powder.

[0016] The present invention also provides a preparation method of traditional Chinese medicine nanoparticles with tumor targeting function, which specifically comprises the following steps:

[0017] Step 1: Weigh 10 - 30 parts of Pep-14 peptide, 6 - 15 parts of traditional Chinese medicine saponin powder, and 2 - 10 parts of paclitaxel by weight. Dissolve the Pep-14 peptide in physiological saline according to the mass-volume ratio of 1 - 3 mg:0.5 mL to obtain a peptide solution. Dissolve paclitaxel in absolute ethanol according to the mass-volume ratio of 1 - 5 mg:0.2 mL to obtain a paclitaxel solution;

[0018] Step 2: Add the weighed traditional Chinese medicine saponin powder into the peptide solution obtained in Step 1, stir magnetically at 100 rpm for 5 min. After dissolution, a peptide-saponin self-assembly solution is obtained;

[0019] Step 3: While stirring, dropwise add the paclitaxel solution obtained in Step 1 into the peptide-saponin self-assembly solution obtained in Step 2 by the emulsification-solvent evaporation method at a rate of 2 - 3 drops / s, with a stirring speed of 250 rpm. After complete dropwise addition, continue stirring for 15 min, probe sonicate at 4°C for 5 min with a power of 120 W, sonicate for 5 s and interval for 3 s, stir at room temperature at 100 rpm for 12 h to evaporate ethanol, and filter through a 0.8 μm polyethersulfone membrane to obtain a traditional Chinese medicine nanoparticle solution;

[0020] Step 4: Pre-freeze the traditional Chinese medicine nanoparticle solution obtained in Step 3 in a -80°C refrigerator for 12 h, and then perform freeze-drying to obtain traditional Chinese medicine nanoparticles.

[0021] The beneficial effects achieved by the present invention are as follows:

[0022] The traditional Chinese medicine nanoparticles with tumor targeting function prepared by the present invention can achieve the simultaneous loading and delivery of traditional Chinese medicine components with different properties by designing the Pep-14 peptide with a tumor targeting segment, a hydrophobic binding region, and a hydrophilic binding region. Among them, the RGD peptide segment can target integrin αvβ3 on the surface of tumor cells, and the FLIVF peptide segment has hydrophobicity and can self-assemble into a structure with a hydrophobic cavity in an aqueous solution. On this basis, paclitaxel or hydrophobic components in other traditional Chinese medicine active ingredients are combined through hydrophobic interaction and π-π interaction, effectively improving its bioavailability and reducing the dosage. The CQCEHK peptide segment is a hydrophilic binding region, and the ends of its peptide segments are negatively charged E (glutamic acid), positively charged H (histidine), and K (lysine), which bind water-soluble drugs through electrostatic interaction and hydrogen bond interaction of the amino acid R group. This self-assembly strategy of multi-component co-loading and co-delivery can, on the one hand, improve the stability of the drug in blood circulation and reduce the biological toxicity to normal cells, and on the other hand, its electrically neutral property can achieve the precise release of the drug at the weakly acidic tumor site, overcoming the limitations of traditional carrier systems, especially meeting the co-delivery of various components in traditional Chinese medicine. In addition, the hydrophobic drug binding region in the polypeptide sequence is located in the middle of the peptide segment, and the steric hindrance of the hydrophilic peptide segments on both sides can make the drug-loaded particles tend to form a small-size self-assembled morphology after loading the drug, and can achieve nano-scale assembly. This nano-drug delivery system of this size can penetrate through the dense intercellular spaces at the tumor site to achieve deep penetration and improve the anti-tumor effect. Description of the Drawings

[0023] Figure 1 It is a mechanism diagram of the traditional Chinese medicine nanoparticles with tumor targeting function prepared by the present invention;

[0024] Figure 2 Purity detection results of Pep-14 peptide used in Examples 1-4;

[0025] Figure 3 Mass spectrometry detection results of Pep-14 peptide used in Examples 1-4;

[0026] Figure 4 Particle size detection results of traditional Chinese medicine nanoparticles prepared in Example 1;

[0027] Figure 5 Optimal conformation molecular simulation results of Pep-14 peptide in water used in Examples 1-4;

[0028] Figure 6 Transmission electron microscope pictures of traditional Chinese medicine nanoparticles prepared in Example 1;

[0029] Figure 7 Hemolysis safety results of traditional Chinese medicine nanoparticles prepared in Example 1;

[0030] Figure 8 In vitro anti-tumor efficacy detection results of traditional Chinese medicine nanoparticles prepared in Example 1.

[0031] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. Detailed implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0033] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes and do not limit the content of this application.

[0034] In the following embodiments, the mechanism diagram of the traditional Chinese medicine nanoparticles with tumor targeting function is as Figure 1 shown. Unless otherwise specified, all are conventional methods; the materials used in the following embodiments, unless otherwise specified, the raw materials are all newly purchased materials on the market.

[0035] Example 1: This example provides a traditional Chinese medicine nanoparticle with tumor targeting function. The traditional Chinese medicine nanoparticle comprises the following raw materials in parts by weight: 25 parts of Pep-14 peptide, 10 parts of traditional Chinese medicine saponin powder, and 10 parts of paclitaxel.

[0036] The traditional Chinese medicine saponin powder comprises the following raw materials in parts by weight: 15 parts of ginseng, 40 parts of notoginseng, 10 parts of liquorice, and 10 parts of bupleurum. The specific preparation method is as follows:

[0037] S1: Weigh 15 parts of ginseng, 40 parts of notoginseng, 10 parts of liquorice, and 10 parts of bupleurum, wash, dry in the sun, crush and mix them to obtain traditional Chinese medicine powder.

[0038] S2: Extract the total saponins of traditional Chinese medicine by ultrasonic-assisted deep eutectic solvent extraction method. Add choline chloride, 1,4-butanediol, and purified water to the traditional Chinese medicine powder obtained in step S1. The mass ratio of traditional Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8. Reflux and extract for 20 min at 30 °C in a water bath and 260 W ultrasonic power. After filtering with a filter cloth, obtain the crude saponin extract.

[0039] S3: Take D101 macroporous resin, soak D101 macroporous resin in 95% ethanol for 24 h to swell it, pack the column by wet method, and rinse it evenly with 95% ethanol until the dripping is not in a line. Rinse until there is no turbidity after adding 4 times the volume of ultrapure water to the effluent and mixing evenly. Then rinse with a large amount of ultrapure water until the effluent has no alcohol smell to obtain the pretreated macroporous resin.

[0040] S4: Use the pretreated macroporous resin to elute the crude saponin extract obtained in step S2. The diameter-to-height ratio is 1:3, the flow rate is 1 BV / h, and after adsorption for 2 h, elute with 3 BV volumes of ultrapure water, 20% ethanol, and 70% ethanol respectively at an elution flow rate of 1.5 BV / h. Recover the 70% ethanol eluate fraction.

[0041] S5: Rotate and evaporate the 70% ethanol eluate fraction recovered in step S4 to dryness. The rotation evaporation temperature is 30 °C, and the cold trap temperature is set at -10 °C to obtain the traditional Chinese medicine saponin powder.

[0042] This example also provides a preparation method of a traditional Chinese medicine nanoparticle with tumor targeting function, which specifically comprises the following steps:

[0043] Step 1: Weigh 25 parts of Pep-14 peptide, dissolve Pep-14 peptide in physiological saline according to the mass-volume ratio of 2.5 mg:0.5 mL to obtain a peptide solution. Weigh 10 parts of paclitaxel, dissolve paclitaxel in absolute ethanol according to the mass-volume ratio of 5 mg:0.2 mL to obtain a paclitaxel solution.

[0044] Step 2: Weigh 10 portions of traditional Chinese medicine saponin powder and add it to the peptide solution obtained in Step 1. Stir magnetically at 100 rpm for 5 min. After dissolution, a peptide-saponin self-assembly solution is obtained;

[0045] Step 3: While stirring, use the emulsification-solvent evaporation method to dropwise add the paclitaxel solution obtained in Step 1 to the peptide-saponin self-assembly solution obtained in Step 2 at a rate of 2-3 drops / s. The stirring speed is 250 rpm. After complete dropping, continue stirring for 15 min, probe sonicate at 4°C for 5 min, with a power of 120 W, sonicate for 5 s, interval of 3 s, and stir at room temperature at 100 rpm for 12 h to evaporate ethanol, and filter through a 0.8 μm polyethersulfone membrane to obtain a traditional Chinese medicine nanoparticle solution;

[0046] Step 4: Pre-freeze the traditional Chinese medicine nanoparticle solution obtained in Step 3 in a -80°C refrigerator for 12 h, and then perform freeze-drying to obtain traditional Chinese medicine nanoparticles.

[0047] Example 2: This example provides a traditional Chinese medicine nanoparticle with tumor targeting function. The traditional Chinese medicine nanoparticle comprises the following raw materials in parts by weight: 30 parts of Pep-14 peptide, 15 parts of traditional Chinese medicine saponin powder, and 6 parts of paclitaxel.

[0048] The traditional Chinese medicine saponin powder comprises the following raw materials in parts by weight: 20 parts of ginseng, 20 parts of notoginseng, 12 parts of licorice, and 12 parts of bupleurum. The specific preparation method is as follows:

[0049] S1: Weigh 20 parts of ginseng, 20 parts of notoginseng, 12 parts of licorice, and 12 parts of bupleurum. Wash, dry in the sun, crush and mix to obtain traditional Chinese medicine powder;

[0050] S2: Use ultrasonic-assisted deep eutectic solvent extraction method to extract total saponins of traditional Chinese medicine. Add choline chloride, 1,4-butanediol, and purified water to the traditional Chinese medicine powder obtained in Step S1. The mass ratio of traditional Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8. Reflux and extract at 30°C in a water bath under the condition of 260 W ultrasonic power for 20 min, and filter with a filter cloth to obtain a crude saponin extract;

[0051] S3: Take D101 macroporous resin, soak the D101 macroporous resin in 95% ethanol for 24 h to swell, pack the column by the wet method, and wash it evenly with 95% ethanol until the dropping is not in a continuous line. Wash until there is no turbidity after adding 4 times the volume of ultrapure water to the effluent and mixing evenly, and then wash with a large amount of ultrapure water until the effluent has no alcohol smell to obtain a pretreated macroporous resin;

[0052] S4: Use macroporous resin for pretreatment to elute the crude saponin extract obtained in step S2, with a diameter-to-height ratio of 1:3, a flow rate of 1 BV / h. After adsorption for 2 h, elute successively with 3 BV volumes of ultrapure water, 20% ethanol, and 70% ethanol at an elution flow rate of 1.5 BV / h, and recover the 70% ethanol elution fraction;

[0053] S5: Rotate and evaporate to dryness the 70% ethanol elution fraction recovered in step S4. The rotary evaporation temperature is 25°C, and the cold trap temperature is set at -10°C to obtain traditional Chinese medicine saponin powder.

[0054] This example also provides a preparation method of traditional Chinese medicine nanoparticles with tumor targeting function, which specifically includes the following steps:

[0055] Step 1: Weigh 30 parts of Pep-14 peptide, dissolve the Pep-14 peptide in physiological saline according to the mass-volume ratio of 3 mg: 0.5 mL to obtain a peptide solution. Weigh 6 parts of paclitaxel, dissolve the paclitaxel in absolute ethanol according to the mass-volume ratio of 3 mg: 0.2 mL to obtain a paclitaxel solution;

[0056] Step 2: Weigh 15 parts of traditional Chinese medicine saponin powder and add it to the peptide solution obtained in step 1, stir magnetically at 100 rpm for 5 min. After dissolution, obtain a peptide-saponin self-assembly solution;

[0057] Step 3: While stirring, add dropwise the paclitaxel solution obtained in step 1 to the peptide-saponin self-assembly solution obtained in step 2 by the emulsification-solvent evaporation method at a rate of 2 - 3 drops / s, with a stirring speed of 250 rpm. After complete dropwise addition, continue stirring for 15 min, probe sonicate at 4°C for 5 min, with a power of 120 W, sonicate for 5 s, interval 3 s, stir at room temperature at 100 rpm for 12 h to volatilize ethanol, and filter with a 0.8 μm polyethersulfone membrane to obtain a traditional Chinese medicine nanoparticle solution;

[0058] Step 4: Pre-freeze the traditional Chinese medicine nanoparticle solution obtained in step 3 in a -80°C refrigerator for 12 h, and then perform freeze-drying to obtain traditional Chinese medicine nanoparticles.

[0059] Example 3: This example provides a traditional Chinese medicine nanoparticle with tumor targeting function. The traditional Chinese medicine nanoparticle includes the following raw materials in parts by weight: 12 parts of Pep-14 peptide, 8 parts of traditional Chinese medicine saponin powder, and 4 parts of paclitaxel.

[0060] The traditional Chinese medicine saponin powder includes the following raw materials in parts by weight: 30 parts of ginseng, 30 parts of notoginseng, 15 parts of licorice, and 15 parts of bupleurum. The specific preparation method is as follows:

[0061] S1: Weigh 30 parts of ginseng, 30 parts of notoginseng, 15 parts of licorice, and 15 parts of bupleurum, wash, dry in the sun, pulverize and mix to obtain traditional Chinese medicine powder;

[0062] S2: Use the ultrasonic-assisted deep eutectic solvent extraction method to extract the total saponins from traditional Chinese medicine. Add choline chloride, 1,4-butanediol, and purified water to the traditional Chinese medicine powder obtained in step S1. The mass ratio of the traditional Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8. Carry out reflux extraction at 30 °C in a water bath and an ultrasonic power of 260 W for 20 min. After filtering with a filter cloth, obtain the crude saponin extract;

[0063] S3: Take D101 macroporous resin, soak the D101 macroporous resin in 95% ethanol for 24 h to swell it, pack the column by the wet method, and rinse it evenly with 95% ethanol until the dripping is not in a continuous line. Rinse until there is no turbidity after adding 4 times the volume of ultrapure water to the effluent and mixing evenly, and then rinse with a large amount of ultrapure water until there is no alcohol smell in the effluent to obtain the pretreated macroporous resin;

[0064] S4: Use the pretreated macroporous resin to elute the crude saponin extract obtained in step S2. The diameter-to-height ratio is 1:3, the flow rate is 1 BV / h, and after adsorption for 2 h, elute successively with 3 BV volumes of ultrapure water, 20% ethanol, and 70% ethanol at an elution flow rate of 1.5 BV / h, and recover the 70% ethanol eluate fraction;

[0065] S5: Rotate and evaporate to dryness the 70% ethanol eluate fraction recovered in step S4. The rotation evaporation temperature is 25 °C, and the cold trap temperature is set at -10 °C to obtain the traditional Chinese medicine saponin powder.

[0066] This embodiment also provides a preparation method of traditional Chinese medicine nanoparticles with tumor targeting function, which specifically includes the following steps:

[0067] Step 1: Weigh 12 parts of Pep-14 peptide, dissolve the Pep-14 peptide in physiological saline according to the mass-volume ratio of 1.2 mg:0.5 mL to obtain a peptide solution. Weigh 4 parts of paclitaxel, dissolve the paclitaxel in absolute ethanol according to the mass-volume ratio of 2 mg:0.2 mL to obtain a paclitaxel solution;

[0068] Step 2: Weigh 8 parts of the traditional Chinese medicine saponin powder and add it to the peptide solution obtained in step 1. Stir magnetically at 100 rpm for 5 min. After dissolution, obtain a peptide-saponin self-assembly solution;

[0069] Step 3: Use the emulsification-solvent evaporation method to dropwise add the paclitaxel solution obtained in step 1 to the peptide-saponin self-assembly solution obtained in step 2 while stirring at 2-3 drops / s, with a stirring speed of 250 rpm. After complete dropping, continue stirring for 15 min, probe-ultrasonic at 4 °C for 5 min, with a power of 120 W, ultrasonic for 5 s, interval 3 s, and stir at room temperature at 100 rpm for 12 h to volatilize ethanol, and filter with a 0.8 μm polyethersulfone filter membrane to obtain the traditional Chinese medicine nanoparticle solution;

[0070] Step 4: After pre-freezing the Chinese medicine nanoparticle solution obtained in Step 3 in a -80 °C refrigerator for 12 h, perform freeze-drying to obtain Chinese medicine nanoparticles.

[0071] Example 4: This example provides a Chinese medicine nanoparticle with tumor targeting function. The Chinese medicine nanoparticle comprises the following raw materials in parts by weight: 10 parts of Pep-14 peptide, 6 parts of Chinese medicine saponin powder, and 2 parts of paclitaxel.

[0072] The Chinese medicine saponin powder comprises the following raw materials in parts by weight: 30 parts of ginseng, 30 parts of notoginseng, 10 parts of licorice, and 10 parts of bupleurum. The specific preparation method is as follows:

[0073] S1: Weigh 30 parts of ginseng, 30 parts of notoginseng, 10 parts of licorice, and 10 parts of bupleurum, wash, dry in the sun, crush and mix them to obtain Chinese medicine powder.

[0074] S2: Use ultrasonic-assisted deep eutectic solvent extraction method to extract total Chinese medicine saponins. Add choline chloride, 1,4-butanediol, and purified water to the Chinese medicine powder obtained in Step S1. The mass ratio of Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8. Reflux and extract for 20 min under the conditions of 30 °C water bath and 260 W ultrasonic power. After filtering with filter cloth, obtain crude saponin extract.

[0075] S3: Take D101 macroporous resin, soak D101 macroporous resin in 95% ethanol for 24 h to swell it, load the column by wet method, wash it evenly with 95% ethanol until the dripping is not in a line. When adding 4 times the volume of ultrapure water to the effluent and mixing evenly without turbidity, then wash it with a large amount of ultrapure water until the effluent has no alcohol smell to obtain pretreated macroporous resin.

[0076] S4: Use the pretreated macroporous resin to elute the crude saponin extract obtained in Step S2. The diameter-to-height ratio is 1:3, the flow rate is 1 BV / h. After adsorption for 2 h, elute successively with 3 BV volumes of ultrapure water, 20% ethanol, and 70% ethanol at an elution flow rate of 1.5 BV / h, and recover the 70% ethanol elution fraction.

[0077] S5: Rotate and evaporate the 70% ethanol elution fraction recovered in Step S4 to dryness. The rotation evaporation temperature is 25 °C, and the cold trap temperature is set at -10 °C to obtain Chinese medicine saponin powder.

[0078] This example also provides a preparation method of a Chinese medicine nanoparticle with tumor targeting function, which specifically includes the following steps:

[0079] Step 1: Weigh 10 parts of Pep-14 peptide. Dissolve the Pep-14 peptide in physiological saline according to the mass-volume ratio of 1 mg: 0.5 mL to obtain a peptide solution. Weigh 2 parts of paclitaxel. Dissolve the paclitaxel in absolute ethanol according to the mass-volume ratio of 1 mg: 0.2 mL to obtain a paclitaxel solution;

[0080] Step 2: Weigh 6 parts of traditional Chinese medicine saponin powder and add it to the peptide solution obtained in Step 1. Stir magnetically at 100 rpm for 5 min. After dissolution, a peptide-saponin self-assembled solution is obtained;

[0081] Step 3: While stirring, use the emulsification-solvent evaporation method to dropwise add the paclitaxel solution obtained in Step 1 to the peptide-saponin self-assembled solution obtained in Step 2 at a rate of 2-3 drops / s. The stirring speed is 250 rpm. After complete dropping, continue stirring for 15 min. Probe sonicate at 4°C for 5 min, with a power of 120 W, sonication for 5 s, interval of 3 s, and stir at room temperature at 100 rpm for 12 h to evaporate ethanol. Filter through a 0.8 μm polyethersulfone membrane to obtain a traditional Chinese medicine nanoparticle solution;

[0082] Step 4: Pre-freeze the traditional Chinese medicine nanoparticle solution obtained in Step 3 in a -80°C refrigerator for 12 h, and then perform freeze-drying to obtain traditional Chinese medicine nanoparticles.

[0083] Investigation of the physicochemical properties of polypeptides

[0084] Take 5 mg of the Pep-14 peptide used in Examples 1-4, add 5 mL of purified water and dissolve it by sonication to prepare a 1 mg / mL peptide solution. Use HPLC (high performance liquid chromatography), MS (mass spectrometry), DLC (nano particle size analyzer), and molecular dynamics simulation to investigate its purity, molecular weight, particle size, and molecular conformation. The HPLC chromatographic conditions are as follows: Phase A (0.1% trifluoroacetic acid water) - Phase B (0.1% trifluoroacetic acid acetonitrile), mobile phase flow rate 1 mL / min, detection wavelength 220 nm, chromatographic column is Shim-pack GIST C18 (4.6×250 mm×5 μm), injection volume 25 μL, 0.01 - 30.00 min: 15%A → 45%A, 30.00 - 33.00 min: 45%A → 100%A, 33.00 - 38.00 min: 100%A, 38.00 - 40.00 min: 100%A → 15%A, 40.00 - 50.00 min: 100%A → 15%A. The results are shown in Figure 2 and Table 1; The MS results are shown in Figure 3 ; The particle size results are shown in Figure 4 ; The peptide molecular conformation is shown in Figure 5 .

[0085] Detection of the purity of total saponins in traditional Chinese medicine

[0086] Accurately weigh 4.00, 6.00, 7.00, 7.00, 7.00, 6.00, 7.00, 7.00, 7.00 mg of reference substances of saponins Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, and Rg3 respectively, place them in 5 mL centrifuge tubes, add 5.00 mL of methanol to dissolve and label as S1. Take 2.50 mL of S1 and add 2.50 mL of methanol to dissolve and label as S2. Take 2.50 mL of S2 and add 2.50 mL of methanol to dissolve and label as S3. Take 2.50 mL of S3 and add 2.50 mL of methanol to dissolve and label as S4. Take 2.50 mL of S4 and add 2.50 mL of methanol to dissolve and label as S5. Take 2.50 mL of S5 and add 2.50 mL of methanol to dissolve and label as S6. The preparation concentrations of the series of mixed reference substance solutions are shown in Table 2. Establish an HPLC analysis method for the samples, with a detection wavelength of 203 nm. Accurately weigh 6 portions of 40.00 mg of the traditional Chinese medicine saponin powder prepared in Examples 1 - 4 respectively, add 5.00 mL of methanol and dissolve by ultrasonic treatment, filter through a 0.45 μm nylon filter membrane, place in a 5 mL volumetric flask and make up to the mark. Take 1 mL as the sample solution for detection, and the purity results are shown in Table 3.

[0087] Investigation of the drug loading of paclitaxel

[0088] The HPLC method was used to investigate the drug loading of PTX. An HPLC analysis method was established with paclitaxel reference substance. Take 40 mg of the traditional Chinese medicine nanoparticles prepared in Examples 1 - 4, dissolve them in 2 mL of 50% ethanol aqueous solution, ultrasonicate to disrupt the nanoparticle structure and release paclitaxel. Detect the contents of paclitaxel and Pep - 14 peptide in the solution respectively according to the detection methods of paclitaxel and Pep - 14 peptide. Calculate the drug loading according to the following formula: LE% = mass of paclitaxel / (mass of paclitaxel + mass of Pep - 14 peptide) × 100%, and the results are shown in Table 2.

[0089] Investigation of the morphology of traditional Chinese medicine nanoparticles

[0090] Weigh 1 mg of the traditional Chinese medicine nanoparticles prepared in Examples 1 and 4, add 1 mL of normal saline and dissolve by ultrasonic treatment to obtain a 1 mg / mL nanoparticle solution. Take 10 μL and drop it onto a copper grid, precipitate for 1 min, suck the liquid on the surface with filter paper, then place the copper grid under an electric heating lamp to dry. Stop baking when the surface liquid is about to disappear. Drop 5 μL of 2% phosphotungstic acid solution, precipitate for 1 min, suck the surface liquid dry with filter paper, dry in a cool place, and perform TEM (transmission electron microscopy) detection at 100 kV. The results are as Figure 6 shown.

[0091] Investigation of hemolysis

[0092] Weigh 30 mg of the traditional Chinese medicine nanoparticles prepared in Example 1, add 10 mL of normal saline to dissolve, obtain a 3000 μg / mL nanoparticle solution, and successively dilute it with normal saline to 1000 μg / mL and 100 μg / mL nanoparticle solutions for standby; Take the blood of healthy rabbits and put it into a conical flask containing glass beads, shake for 10 min, add 10 times the amount of normal saline, shake well, centrifuge at 1000 rpm for 15 min, remove the supernatant, and continue to wash the precipitate with normal saline until the supernatant is no longer red. Make the obtained red blood cells into a 4% red blood cell suspension with normal saline. Use purified water and normal saline as positive and negative controls respectively. Add purified water, 3000 μg / mL, 1000 μg / mL, 100 μg / mL nanoparticle solutions and normal saline to the 4% red blood cell suspension respectively. After gently shaking up and down, centrifuge at 1000 rpm for 5 min, and observe the hemolysis situation. The results are shown in Figure 7 。

[0093] In vitro anti-tumor investigation

[0094] The CCK-8 method was used to detect the in vitro anti-tumor activity of the traditional Chinese medicine nanoparticles prepared in Example 1. Take LLC (mouse lung cancer) cells in the logarithmic growth phase and inoculate 1×10 4 cells per well in a 96-well plate, incubate overnight at 37°C and 5% CO2. After the cells adhere to the wall, discard the culture medium, divide the cells into a control group, a paclitaxel group, a total saponins of traditional Chinese medicine group and a traditional Chinese medicine nanoparticle group, and set up a blank group without cells and drugs. Each group has 5 replicates. The drug concentrations in the traditional Chinese medicine nanoparticle group are set at 100 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 1 μg / mL. According to the paclitaxel and total saponins of traditional Chinese medicine contents in each concentration of the traditional Chinese medicine nanoparticle group, prepare equal amounts of paclitaxel solutions and total saponins of traditional Chinese medicine solutions with different concentration gradients for drug administration. After incubating for 24 h, discard the culture medium, wash twice with PBS, add 100 μL of CCK-8 reagent, incubate for 2 h, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well at a wavelength of 450 nm, and calculate the cell survival rate according to the following formula: Cell survival rate = (absorbance value of the experimental group - absorbance value of the blank group) / (absorbance value of the control group - absorbance value of the blank group) × 100%. The results are shown in Figure 8 。

[0095] Table 1 HPLC detection results of Pep-14 peptide

[0096]

[0097] Table 2 Preparation concentrations of a series of mixed reference substance solutions

[0098]

[0099] Table 3 Purity of Traditional Chinese Medicine Saponin Powder and Paclitaxel Drug Loading Capacity

[0100]

[0101] Table 1, Figure 2 and Figure 3 The results showed that the polypeptide purity was 98.08%, the mass spectrometry results corresponded to the polypeptide molecular weight, the target Pep-14 peptide was successfully synthesized, and the purity was qualified.

[0102] The results in Table 3 showed that the saponin purity in the traditional Chinese medicine saponin powder prepared in Examples 1-4 was high, and the method had good stability; the paclitaxel drug loading capacity was large, significantly improving the solubility of the hydrophobic drug paclitaxel.

[0103] Figure 4 The particle size results showed that the particle size of the traditional Chinese medicine nanoparticles prepared in Example 1 was uniform, about 100 nm, Figure 6 and the TEM results were also in line with it. The nanoparticles were spherical and had a small size, which was beneficial to blood circulation and penetration into deep tumors.

[0104] Figure 5 The molecular simulation results showed that the optimal conformation of Pep-14 peptide in water was that the hydrophilic segment was turned outwards, which was beneficial to binding hydrophilic drugs, and there was a certain steric hindrance, which could effectively prevent the peptide segment binding hydrophobic drugs in the middle from forming a molecular longitudinal arrangement, effectively controlling the size of traditional Chinese medicine nanoparticles.

[0105] Figure 7 The hemolysis results showed that the traditional Chinese medicine nanoparticles prepared in Example 1 had good biosafety and loaded traditional Chinese medicine saponin in a non-covalent binding form, which could effectively reduce the hemolytic toxicity of saponin.

[0106] Figure 8 The in vitro cell viability results showed that the traditional Chinese medicine nanoparticles prepared in Example 1 had high in vitro anti-tumor efficiency, and loading two drugs with different properties at the same time significantly improved the drug efficacy.

[0107] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0108] The above describes the present invention and its implementation manners. This description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar ways and embodiments without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine nanoparticle with tumor targeting function, characterized in that, The traditional Chinese medicine nanoparticles comprise the following raw materials in parts by weight: 10-30 parts of Pep-14 peptide, 6-15 parts of traditional Chinese medicine saponin powder, and 2-10 parts of paclitaxel; The sequence of the Pep-14 peptide is: H2N-Arg-Gly-Asp-Phe-Leu-Ile-Val-Phe-Cys-Gln-Cys-Glu-His-Lys-COOH; The traditional Chinese medicine saponin powder is prepared from the following raw materials in parts by weight: 15-30 parts of ginseng, 20-40 parts of notoginseng, 10-15 parts of liquorice, and 10-15 parts of bupleurum. The specific preparation method is as follows: S1: Weigh ginseng, notoginseng, liquorice, and bupleurum, wash, dry in the sun, pulverize, and mix them to obtain traditional Chinese medicine powder; S2: Add choline chloride, 1,4-butanediol, and purified water to the traditional Chinese medicine powder obtained in step S1, perform water bath and ultrasonic reflux extraction, and filter to obtain a crude saponin extract; S3: Take macroporous resin, swell the macroporous resin, and wash it successively with 95% ethanol and purified water until the effluent has no alcohol smell to obtain pretreated macroporous resin; S4: Use the pretreated macroporous resin to adsorb, elute, and recover the elution fraction from the crude saponin extract obtained in step S2; S5: Spin-dry the elution fraction recovered in step S4 to obtain traditional Chinese medicine saponin powder.

2. The Chinese medicine nanoparticles with tumor targeting function according to claim 1, characterized in that, In step S2, the mass ratio of the traditional Chinese medicine powder, choline chloride, 1,4-butanediol, and purified water is 1:6:6:8, and the reflux extraction time is 20 min; In step S3, the macroporous resin is D101 macroporous resin, and the swelling time is 24 h; In step S4, the elution process uses ultrapure water, 20% ethanol, and 70% ethanol in sequence, and the recovered elution fraction is the 70% ethanol elution fraction.

3. A preparation method of a traditional Chinese medicine nanoparticle with tumor targeting function according to any one of claims 1-2, characterized in that, The specific preparation method is as follows: Step 1: Weigh Pep-14 peptide and dissolve it in physiological saline to obtain a peptide solution. Weigh paclitaxel and dissolve it in absolute ethanol to obtain a paclitaxel solution; Step 2: Weigh traditional Chinese medicine saponin powder and add it to the peptide solution obtained in step 1, stir and dissolve it to obtain a peptide-saponin self-assembly solution; Step 3: While stirring, dropwise add the paclitaxel solution obtained in step 1 to the peptide-saponin self-assembly solution obtained in step 2. After complete addition, continue to stir, ultrasonicate, volatilize, and filter to obtain a traditional Chinese medicine nanoparticle solution; Step 4: Freeze-dry the traditional Chinese medicine nanoparticle solution obtained in step 3 to obtain traditional Chinese medicine nanoparticles.

4. The preparation method of a traditional Chinese medicine nanoparticle with tumor targeting function according to claim 3, characterized in that, In step 1, the mass-volume ratio of the Pep-14 peptide to physiological saline is 1-3 mg: 0.5 mL, and the mass-volume ratio of paclitaxel to absolute ethanol is 1-5 mg: 0.2 mL; In step 3, the ultrasonication process is carried out at 4°C, and the volatilization condition is magnetic stirring at 100 rpm at room temperature for 12 h; In step 4, the traditional Chinese medicine nanoparticle solution is pre-frozen at -80°C for 12 h before freeze-drying.

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