CN106924312B

CN106924312B - Application of ginseng-derived nanoparticles in preparation of medicine for treating tumors

Info

Publication number: CN106924312B
Application number: CN201710248242.6A
Authority: CN
Inventors: 曹萌; 曹鹏; 颜怀江
Original assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Current assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Priority date: 2017-04-17
Filing date: 2017-04-17
Publication date: 2021-04-27
Anticipated expiration: 2037-04-17
Also published as: CN106924312A

Abstract

The invention discloses an application of ginseng-derived nano-particles in preparing a medicament for treating tumors. The ginseng-derived nanoparticles have a membrane structure, the particle size range is 150-500nm, and the peak particle size is 280-350 nm; the ginseng-derived nano-particles are prepared by centrifuging and filtering for many times to remove impurities; the ginseng-derived nanoparticles can effectively reverse polarize Tumor-associated macrophages (TAMs) in a Tumor microenvironment, so that M2 type macrophages promoting Tumor invasion and metastasis polarize the Tumor-killing M1 type macrophages, thereby improving the Tumor microenvironment and killing tumors. Has good application prospect in the aspect of developing the medicine for preparing natural anti-tumor medicine.

Description

Application of ginseng-derived nanoparticles in preparation of medicine for treating tumors

Technical Field

The invention belongs to the field of antitumor drugs, and particularly relates to application of ginseng-derived nanoparticles in preparation of a drug for treating tumors.

Background

Tumor microenvironment (tumor micro environment) refers to a local steady-state environment formed by tumor cells, stromal cells (including fibroblasts, immune and inflammatory cells, adipocytes, glial cells, smooth muscle cells, some vascular endothelial cells and the like) and extracellular matrix and the like in the process of tumor growth, and provides a necessary material basis for the occurrence, development, invasion, metastasis and the like of tumors. The tumor microenvironment has important supporting and promoting effects on the tumor. How to intervene and improve the tumor microenvironment is the core problem of preventing tumor development and metastasis.

Recent studies have shown that Tumor-associated macrophages (TAMs), which are macrophages growing in the Tumor microenvironment, are important constituents of the Tumor microenvironment and play a key role in various stages of Tumor development and metastasis. | A

Macrophages (macrophages, or the like,

) Is one of the important immune cells of the body, and plays an important role in the aspects of host immune defense and maintaining tissue homeostasis. Macrophages are activated mainly into 2 subtypes according to functional characteristics: classically activated M1 type (classically activated macroporous) and alternatively activated M2 type(alternatively activated macro). The M1 type is regulated by cytokines secreted by Th1, such as interferon- γ (interferon- γ, INF- γ), bacterial Lipopolysaccharide (LPS), and Toll-like receptor (TLR), and is characterized by secreting proinflammatory factors including IL-6, IL-12, IL-23, and tumor necrosis factor- α (TNF- α). In addition, M1-type macrophages also highly express major histocompatibility complex (major histocompatibility complex) class I and class II molecules for efficient antigen presentation. Therefore, M1-type macrophages are thought to have the ability to kill bacteria and tumor cells and secrete a variety of proinflammatory cytokines. The M2-type macrophage cell has a function in contrast to that of M1, and is recognized as a cell having a function of promoting the growth, invasion and metastasis of tumors. The vast majority of TAMs belong to the M2-type macrophages, the most prominent tumor-infiltrating leukocytes in humans and mice. In the early stage of tumor invasion and metastasis, tumor cells release chemokines to recruit macrophages and other inflammatory cells to reach a stroma area around the tumor, and then TAM can penetrate through the basement membrane, so that the tumor cells escape from the restraint of the basement membrane to reach the stroma of surrounding normal tissues, and simultaneously both TAM and the tumor cells can stimulate angiogenesis, thereby improving the invasiveness and the motility of the cells^[11]. TAMs can also promote angiogenesis and transport nutrients for tumor growth by releasing angiogenesis-regulating enzymes, such as matrix metalloproteinases (MMP 2), MMP-9, MMP-12, and cyclooxygenase 2 (COX 2), among others. In addition, TAMs can also release a series of cytokines and growth factors that promote tumor cell infiltration and metastasis, such as Vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bEGF).

Based on the important role of TAM in tumor microenvironment, how to play the heterogeneity and plasticity of macrophages promotes TAM to be polarized to M1 type, thereby improving tumor microenvironment and possibly becoming an important target of tumor immunotherapy.

Ginseng is one of the most important Chinese medicinal materials in China, is known as the king of all herbs, and has very high medicinal value. The nanoparticle-grade microvesicles generated by ginseng carry various active ingredients of ginseng, and may be one of important ways for exerting the drug effect of ginseng. The development of a novel drug having an anti-tumor function based on ginseng has not been effectively developed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a Ginseng-derived nano-particles (English name: Ginseng derived nano-particles, GDNPs for short) which can be produced quantitatively and is applied to the preparation of the medicine for treating tumors.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

application of Ginseng radix-derived nanoparticles in preparing medicine for treating tumor is provided.

The method of treating a tumor is to reverse polarize tumor-associated macrophages.

The method for reverse polarizing tumor-associated macrophages comprises the following steps: the surface marker molecules of M2 type macrophages are down-regulated and the surface marker molecules of M1 type macrophages are up-regulated, cytokines secreted by the M1 type macrophages are up-regulated, the proportion of the M1/M2 type macrophages in a tumor microenvironment is changed, and therefore the tumor microenvironment is improved, and tumors are killed.

The surface marker molecule related to the M2 type macrophage is leukocyte differentiation antigen 206(CD 206); the M1 type macrophage related surface marker molecule is one or more of Toll-like receptor 2/4(TLR2/4), leukocyte differentiation antigen 80(CD80), leukocyte differentiation antigen 86(CD86) and major tissue complex 2 (MHC-II); the macrophage related gene M2 is one or more of arginase 1(Arg-1), leukocyte differentiation antigen 206 (also called macrophage mannose receptor (CD206), interleukin 10(IL-10) and chitinase-like 3 molecule (CHI 313); the macrophage related gene of M1 type is one or more of interleukin 6(IL-6), tumor necrosis factor alpha (TNF-alpha), leukocyte differentiation antigen 80(CD80), chemokine 9(CXCL9), chemokine 3(CCL3), Inducible Nitric Oxide Synthase (iNOS) and the like; the M1 type macrophage related immune activity cytokine is one or more of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6).

The ginseng-derived nanoparticles have a membrane structure, the particle size range is 150-500nm, and the peak particle size is 280-350 nm.

The ginseng-derived nanoparticles are prepared by the following steps:

(1) squeezing fresh and clean ginseng into raw juice by low-speed screw extrusion;

(2) filtering the raw stock obtained in the step (1) by using a screen to remove impurities, and collecting filtrate;

(3) sequentially carrying out low-speed, medium-speed, high-speed and ultra-speed centrifugation on the filtrate obtained in the step (2), discarding the precipitate after each centrifugation, collecting the supernatant, carrying out next centrifugation, wherein the last centrifugation is carried out, and collecting the precipitate;

(4) resuspending the precipitate collected in the last centrifugation in the step (3) by using a buffer solution, then ultracentrifuging for 1 time, and collecting the precipitate; re-suspending the precipitate by using a buffer solution, then centrifuging at a high speed, and collecting supernatant; and (4) passing the supernatant through a sterilization grade filter membrane to obtain the ginseng-derived nano-particles.

Preferably, the operation is carried out in an environment of 4-25 ℃.

In the step (1), the rotating speed of the low-speed spiral extrusion is 30-60 revolutions per minute.

In the step (2), the screen is 200-2000 meshes, preferably 200-1000 meshes.

In the step (3), the centrifugal force of the low-speed centrifugation is 100-500 Xg, and the centrifugation time is 5-10 minutes; the centrifugal force of the medium-speed centrifugation is 1000-5000 Xg, and the centrifugation time is 10-30 minutes; the centrifugal force of the high-speed centrifugation is 8000-12000 Xg, and the centrifugation time is 30-60 minutes; the centrifugal force of the ultracentrifugation is 100000-200000 Xg, the centrifugation time is 60-120 minutes, and the times of the low speed, the medium speed, the high speed and the ultracentrifugation are at least 1 time.

Preferably, the centrifugal force of the low-speed centrifugation is 200-500 Xg; the centrifugal force of the medium-speed centrifugation is 2000-5000 Xg; the centrifugal force of the high-speed centrifugation is 100000-12000 Xg; the centrifugal force of the ultracentrifugation is 100000-120000 Xg.

In the step (4), the buffer solution is a phosphate buffer solution, and the pH range of the buffer solution is pH7.2-7.4; the centrifugal force of the ultracentrifugation is 100000-200000 Xg, and the centrifugation time is 60-120 minutes; the centrifugal force of the high-speed centrifugation is 8000-12000 Xg, and the centrifugation time is 30-60 minutes; the aperture of the sterilization grade filter membrane is 0.45 mu m.

The ginseng-derived nano-particle material has wide sources and can be ginseng, American ginseng, pseudo-ginseng, codonopsis pilosula, radix pseudostellariae, salvia miltiorrhiza and radix scrophulariae; the preparation method has the advantages of easy operation, less time consumption and the like. The preparation method of the ginseng-derived nano-particle material of the present invention is described in reference to the method of patent application No. 2017100944577.

Has the advantages that: the application of the ginseng-derived nanoparticles in preparing the medicine for treating the tumor can effectively polarize tumor-related macrophages from M2 type for promoting tumor growth to M1 type for resisting the tumor, simultaneously up-regulate various surface active molecules such as TLR2/4, CD80 and the like related to M1 type, and secrete cytokines such as TNF-a, IL-6 and the like, thereby improving the tumor microenvironment and having good application prospect in the aspect of developing and preparing natural antitumor medicines.

Drawings

FIG. 1 is an electron microscope observation of the morphology of GDNPs;

FIG. 2 is a particle size and distribution of ginseng-derived nanoparticles analyzed by a Malvern particle sizer;

FIG. 3 shows phagocytosis of GDNPs by monocyte-macrophages;

FIG. 4 shows that GDNPs down-regulate M2-type macrophage-associated surface marker molecules in vitro, while up-regulate M1-type associated surface marker molecules;

FIG. 5 shows that GDNPs down-regulate the transcription of macrophage associated M2-type gene in vitro, while up-regulating the transcription of macrophage associated M1-type gene;

FIG. 6 shows that GDNPs promote M1 cytokines such as TNF-alpha and IL-6 secretion from M2 type macrophages in vitro;

FIG. 7 is a schematic diagram of in vivo experiments of GDNPs for treating melanoma;

FIG. 8 is a graph of tumor growth during the experiment in each group of mice;

FIG. 9 shows the final tumor volume for each group of mice;

FIG. 10 shows the final tumor weights of the mice in each group;

FIG. 11 shows the final tumor sizes of the mice in each group;

FIG. 12 shows the ratio of macrophages to lymphocytes in tumor tissues of each group of mice;

FIG. 13 change in the number of M1/M2-type macrophages in tumor tissues of various groups of mice.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: preparation of ginseng-derived nanoparticles

(1) Cleaning fresh Ginseng radix with clear water, and squeezing at 20 deg.C to obtain raw juice;

(2) filtering the squeezed raw pulp by a 500-mesh screen at 20 ℃ to remove impurities, and collecting filtrate;

(3) centrifuging the collected filtrate at 200 Xg for 10 min, removing the precipitate, and collecting the supernatant; centrifuging the supernatant at 2000 Xg for 30 min, removing the precipitate, and collecting the supernatant; centrifuging the supernatant at 10000 Xg for 60 min, removing the precipitate, and collecting the supernatant; finally, centrifuging the supernatant at 120000 Xg for 60 minutes, and collecting the precipitate;

(4) resuspending the pellet in phosphate buffer (pH7.2), centrifuging at 120000 Xg for 60 min, and collecting the pellet; resuspending the precipitate again with phosphate buffer solution of pH7.2, centrifuging at 10000 Xg for 60 min, and collecting supernatant; finally, the nano-scale particles are obtained after passing through a sterilization grade filter membrane with the aperture of 0.45 mu m. Storing at-20 deg.C to-80 deg.C.

Example 2: morphology of GDNPs observed by electron microscope

And (3) carrying out ultracentrifugation on the extracted GDNPs at 120000 Xg for 60 minutes to precipitate and compress the GDNPs tightly, discarding the supernatant, fixing the precipitate by using 2.5% (v/v) glutaraldehyde, carrying out treatment in an endoscope chamber, and observing the form of the GDNPs on an endoscope. As shown in fig. 1: as can be seen by an electron microscope, GDNPs are nanoparticles with membrane structures, and the size of the GDNPs is between 150 and 500nm, which proves that the ginseng-derived nanoscale microbodies are effectively recruited.

Example 3: particle size and distribution of ginseng-derived nanoparticles analyzed by Malvern particle sizer

The extracted GDNPs are subjected to particle size detection by a Malvern particle size analyzer, as shown in FIG. 2, the peak uniformity of the GDNPs is good.

Example 4: GDNPs promote formation and proliferation of monocyte-macrophage colony in vitro

1. Acquisition of Bone marrow derived mononuclear-macrophages (BMDM)

Firstly, removing the neck of a C57/BL6 mouse, killing the mouse, soaking the mouse in alcohol for 5 minutes, peeling the fur on the front part of the hind limb of the mouse after fixing the mouse, separating the muscle blunt, cutting off the thigh, and putting the thigh into a culture dish containing sterile PBS.

Secondly, the leg bone is clamped by tweezers, the rest muscles are removed by scissors, and the leg bone is cut off at the joint.

And thirdly, sucking PBS in the culture dish by using a 1mL injector, penetrating a needle into a marrow cavity, repeatedly washing marrow until the marrow turns white, and filtering the marrow washing liquid by using a screen.

Taking the bone marrow of the mouse, cracking the red blood cells, and adjusting the cell concentration to 1 multiplied by 10⁶Seed/ml, medium: DMEM (Gibco) + 10% FBS (Gibco) +20ng/ml M-CSF (Peprotech).

Fifthly, taking bone marrow on day 0, and adding complete culture medium containing 20ng/ml M-CSF into each dish on day 3.

Sixthly, after 5 days, each dish is added with complete culture medium of 20ng/ml M-CSF.

Seventhly, after 7 days, pancreatin digests BMDM, adds into 6-hole cell culture plate, each hole is 2X 10⁶For each cell, 1ml or 2ml of medium was added.

Uptake of GDNPs by BMDM

One day before BMDM was stained with FITC-labeled Anti-mouseF4/80antibody and added to a petri dish with a slide to allow adherent growth.

② 100ul GDNPs are diluted to 250ul by diluent.

③ 1ul of PKH26 was added to 250ul of the dilution as dye.

And fourthly, adding the diluted GDNPs into the dye, and quickly mixing.

Fifthly, incubating for 2-5 minutes at 25 ℃, and slightly inverting the centrifuge tube at regular time to ensure that the mixture is fully mixed at 25 ℃.

Sixthly, adding an equal amount of serum to stop the dyeing reaction and incubating for 1 minute.

120000 Xg, 60 min ultracentrifugation.

Eighthly, sucking away the supernatant, and resuspending the dyed GDNPs with 100ul PBS.

Ninthly, GDNPs were added to the BMDM cultured at a concentration of 20ug/ml and cultured for 24 hours.

And taking out the climbing tablet at the red body, and observing phagocytosis of the mononuclear-macrophage on the GDNPs by laser confocal observation.

As shown in fig. 3: f4/80 is a specific surface marker molecule of mouse monocyte-macrophage, mouse BMDM is provided with green fluorescence by FITC labeled Anti-mouseF4/80antibody, and PKH26 is a red membrane dye and can be combined with the membrane on the surface of GDNPs. Under a laser confocal microscope, a plurality of red particles can be obviously observed in the mononuclear-macrophage cells, and the mononuclear-macrophage cells can effectively phagocytize GDNPs.

Example 5: GDNPs down-regulate M2 type macrophage surface marker molecules in vitro and up-regulate M1 type related surface marker molecules simultaneously

Obtaining BMDM of C57/BL6 mice by inducing M-CSF (20ng/ml), and adding IL-4(20ng/ml) and IL-13(20ng/ml) to differentiate into M2 type macrophages.

② GDNPs (20ug/ml) was added, after 72 hours, the culture supernatant was aspirated and washed once with PBS to trypsinize the cells.

③ adding culture solution to terminate digestion, centrifuging at 1200rpm to collect macrophages, and blocking Fc blocking agent (room temperature, 20 min).

And fourthly, monoclonal antibodies such as anti-mouse CD206, CD80, CD86, TLR2, TLR4, MHC-II and the like are respectively added for staining (room temperature for 30 minutes), and the cells are washed for 2 times by PBS. Flow cytometry was used to identify the expression of the above molecules.

As shown in fig. 4: the GDNPs can remarkably down-regulate the surface marker molecule CD206 of M2 type macrophages, and up-regulate the surface marker molecules (CD80, CD86, TLR2, TLR4, MHC-II and the like) of M1 type macrophages. From the results of surface molecular markers, it was confirmed that the GDNPs were able to polarize M2 type macrophages to M1 type macrophages.

Example 6: GDNPs down-regulate the transcription of M2 type macrophage related gene in vitro and up-regulate the transcription of M1 type macrophage related gene

1. Extraction of macrophage Total RNA

Part of the cells from example 3 were added to 1ml Trizol, aspirated into a 1.5ml EP tube, blown up to a clear liquid cell-free mass, mixed by inversion for 10 minutes and left at room temperature for 5 minutes.

② adding 200ul chloroform, shaking vigorously for 15 seconds, extracting RNA, and standing for 3 minutes at room temperature.

③ centrifugation is carried out at 12000 RPM for 15 minutes at 4 ℃.

Fourthly, absorbing the upper aqueous phase into a new 1.5ml EP tube, adding isopropanol with the same volume, reversing and mixing evenly, and standing for 10 minutes at room temperature.

Centrifuge at 12000 RPM for 10 min at 4 deg.C.

Sixthly, the supernatant is discarded, and the precipitate is washed by adding pre-cooled 0.5ml of 75% ethanol (prepared by DEPC water), and after uniform mixing, the mixture is centrifuged for 5 minutes at 4 ℃ and 7500 RPM.

And seventhly, removing the supernatant, and drying for 5-10 minutes at room temperature.

Adding 20-60ul deionized water to dissolve RNA, blowing and mixing uniformly, and placing in a 56 ℃ water bath kettle for 10 minutes.

Ninthly, rapidly oscillating and centrifuging, and detecting the RNA concentration by an enzyme labeling instrument.

Synthesis of cDNA

(ii) the procedure was carried out according to the Rever Tra Ace qPCR RT Kit instruction

② 1ng-5 mug total RNA is taken as template according to the adjustment of RNA concentration. The reaction system is as follows:

thirdly, the above materials are added into a PCR tube without nuclease, after a proper volume of total RNA template is added, the rest is added with deionized water to complement to 20ul, and all the operations are carried out on ice.

Reaction conditions: the cDNA was obtained after reaction at 42 ℃ for 15 minutes → 85 ℃ for 5 seconds → 4 ℃ and ∞, and was stored at-20 ℃ for further use.

3. Fluorescent quantitative PCR reaction

The expression of mRNAs of M1/M2 macrophage-related genes IL-6, TNF-alpha, CD80, CXCL9, CCL3, iNOS/Arg-1, CD206, IL-10, CHI313 and the like was measured using the above cDNA as a template and GAPDH as an internal reference by referring to the SYBR Green Realtime PCR MasterMIX protocol.

Preparing reaction liquid according to the instruction of the Real-Time PCR kit:

③ reaction conditions: 95 ℃ for 30 seconds → PCR cycle (X40 cycle): 95 ℃ for 5 seconds; annealing at 55 ℃ for 10 seconds; 72 ℃ for 15 seconds extension → preparation of statistical analysis of melting curves

Taking GAPDH as an internal reference, confirming the amplification curve and the melting curve of Real-time PCR after the reaction is finished, detecting the Ct value of each template, repeating all samples for 3 times, and using 2-^△△CTThe method calculates the relative expression level of mRNA in the cells.

As shown in FIG. 5, GDNPs were able to decrease the transcription level of macrophage associated gene M2 type and simultaneously up-regulate the transcription level of cell associated gene M1 type, and it was confirmed from the results of the transcription level of M1/M2 associated gene that GDNPs polarize macrophage M2 type to M1 type.

Example 7: GDNPs promote M2 type macrophage to secrete TNF-alpha, IL-6 and other cytokines in vitro

TNF-. alpha.and IL-6 detection (ELISA method) in the culture supernatant of example 3

(ii) diluting the capture antibody (anti-mouse TNF-. alpha.or IL-6) as required by the specification, coating the antibody in a 96-well plate at 100 ul/well, and standing overnight at 4 ℃.

② wash the plate 3 times with PBST (PBS containing 0.5% Tween), 3 minutes each time. Then blocking with 2% goat serum blocking solution at 37 deg.C for 2 hr.

③ PBST wash plate 3 times. The culture supernatant samples and the standard dilutions were added to the sealed 96-well plate in order and incubated at 37 ℃ for 1 hour.

And (iv) washing the plate 5 times by PBST, adding HRP-antimouse-TNF-alpha or IL-6 (1: 10000), and incubating for 1 hour at 37 ℃.

PBST wash the board 5 times, add TMB color development liquid, incubate 15 minutes at 37 ℃.

Sixthly, adding H₂SO₄The OD value was measured at 450nm in the stop solution.

And seventhly, calculating the concentration of the TNF-alpha or the IL-6 according to the OD value.

As shown in FIG. 6, GDNPs were able to induce M1-type associated cytokines (TNF-. alpha.and IL-6) to be secreted from M2-type macrophages, and from the viewpoint of the secreted cytokines, they were able to polarize M2-type macrophages toward M1-type macrophages.

Example 8: the GDNPs polarize M2 type macrophages in the tumor microenvironment into M1 type in vivo, thereby inhibiting the growth of tumors

The male C57/BL6 mice, weighing 18-20 g, were purchased from the animal testing center of Yangzhou university. Adaptive growth for 1 week.

② mice right axilla subcutaneous inoculation mouse melanoma cell-B16F 10 (2.5X 10)⁵One/only), observed day by day.

③ 7 days after the tumor is planted, the average value of the sizes of the transplanted tumors of the mice reaches 50 to 120mm³Tumor-bearing mice were randomly divided into 3 groups: tumor-bearing model group (i.p. PBS), gavage treatment group (GDNPs 150 ug/tube), i.p. treatment group (GDNPs 100 ug/tube), treatment interval was 3 days (fig. 7).

Fourthly, the growth state of each group of mice is observed day by day, and the weight and the tumor volume of the mice are measured every 2 days (the tumor volume is calculated as length multiplied by width)²/2)

Fifthly, after 11 days of treatment, blood is collected from the orbit after measuring the body weight, the mouse is killed by introducing the neck, the tumor tissue is stripped, the volume is measured and weighed, and the formaldehyde is fixed after the separation of the other main organs.

Sixthly, taking part of mouse tumor tissue, digesting the mouse tumor tissue by collagenase for 30 minutes, grinding the mouse tumor tissue, filtering the mouse tumor tissue by a 200-mesh filter screen to obtain a tumor tissue single cell suspension, and sealing the tumor tissue single cell suspension by an Fc blocking agent (room temperature for 20 minutes).

Seventhly, monoclonal antibodies such as anti-mouse CD45, CD11b, CD206, CD80 and the like are respectively added for staining (room temperature and 30 minutes), and the cells are washed for 2 times by PBS. Flow cytometry was used to identify the expression of the above molecules.

The results show that: both gavage and intraperitoneal injection treatments of GDNPs can inhibit the growth of mouse melanoma (figures 8-11); analysis of the lymphocyte ratio of the tumor tissues of each group of mice shows that the ratio of macrophages in the tumor tissues of mice in the group of the gavage treatment and intraperitoneal injection treatment of GDNPs is obviously higher than that of the tumor-bearing model group (figure 12); meanwhile, the ratio of M1/M2 macrophages in the tumor tissues of the mice in the gavage and intraperitoneal injection treatment groups is significantly higher (FIG. 13). These results suggest that we: the GDNPs can effectively polarize M2 type macrophages in tumor tissues to M1 type in vivo, improve tumor microenvironment and inhibit tumor growth.

Claims

1. Application of a Ginseng radix-derived nanoparticle in preparing medicine for treating tumor is provided; wherein, the ginseng-derived nano-particles have a membrane structure, and the peak particle size is 280-350 nm; wherein the method of treating a tumor is reverse polarizing tumor-associated macrophages; wherein the reverse polarized tumor associated macrophages are surface marker molecules for down-regulating M2 type macrophages and surface marker molecules for up-regulating M1 type macrophages;

wherein the ginseng-derived nanoparticles are prepared by the following steps:

(4) resuspending the precipitate collected in the last centrifugation in the step (3) by using a buffer solution, then ultracentrifuging for 1 time, and collecting the precipitate; re-suspending the precipitate by using a buffer solution, then centrifuging at a high speed, and collecting supernatant; the supernatant is obtained by passing through a sterilization grade filter membrane;

in the step (3), the centrifugal force of the low-speed centrifugation is 100-500 Xg, and the centrifugation time is 5-10 minutes; the centrifugal force of the medium-speed centrifugation is 1000-5000 Xg, and the centrifugation time is 10-30 minutes; the centrifugal force of the high-speed centrifugation is 8000-12000 Xg, and the centrifugation time is 30-60 minutes; the centrifugal force of the ultracentrifugation is 100000-200000 Xg, the centrifugation time is 60-120 minutes, and the times of the low speed, the medium speed, the high speed and the ultracentrifugation are at least 1 time;

in the step (4), the centrifugal force of the ultracentrifugation is 100000-200000 Xg, and the centrifugation time is 60-120 minutes; the centrifugal force of the high-speed centrifugation is 8000-12000 Xg, and the centrifugation time is 30-60 minutes; the aperture of the sterilization grade filter membrane is 0.45 mu m.

2. The use of claim 1, wherein the method of reverse polarizing tumor-associated macrophages comprises: the cytokine secreted by the M1 type macrophage is up-regulated, and the proportion of the M1/M2 type macrophage in a tumor microenvironment is changed, so that the tumor microenvironment is improved, and the tumor is killed.

3. The use according to claim 1, wherein the macrophage-associated surface marker molecule of M2 type is leukocyte differentiation antigen 206; the M1 type macrophage related surface marker molecule is one or more of Toll-like receptor 2/4, leukocyte differentiation antigen 80, leukocyte differentiation antigen 86 and major histocompatibility complex 2.

4. The use according to claim 1, wherein in the step (4), the buffer solution is a phosphate buffer solution, and the pH value of the buffer solution is in the range of pH 7.2-7.4.