Traditional Chinese medicine source preparation for anti-tumor immunotherapy

Abstract

The invention relates to the field of micro-nano material medicine, in particular to a traditional Chinese medicine source preparation for anti-tumor immunotherapy. The preparation of the traditional Chinese medicine source preparation comprises the following steps: treating a traditional Chinese medicine decoction and an optional anti-tumor drug by a freeze-thaw method and / or a calcium adding method, and when the anti-tumor drug is adriamycin, preparing adriamycin-entrapped traditional Chinese medicine micro-nano particles by adopting the calcium adding method. The micro-nano particles of the novel traditional Chinese medicine source preparation provided by the invention can avoid side effects caused by the particles entering blood through a subdermal interstitial administration mode, and can effectively cause an immune activation effect, so that the novel traditional Chinese medicine source anti-tumor medicine preparation is prepared. The method is of great significance in research of action mechanisms of pharmacy and traditional Chinese medicine.

Description

Technical Field

[0001] The present invention relates to the field of micro-nano material medicine, and in particular to a traditional Chinese medicine-derived preparation for anti-tumor immunotherapy. Background Art

[0002] Traditional Chinese Medicine (TCM) is a treasure trove of pharmaceutical research. The complex molecular systems within TCM decoctions offer immense potential for exploration in drug and material research. Using TCM as raw materials, particles with diverse physical and chemical properties can be synthesized. However, these particles are often micro- and nano-sized and complex in composition, making intravascular administration prone to adverse reactions such as vascular inflammation and hemolysis.

[0003] Currently, there is no research on the use of micro-nanoparticles derived from traditional Chinese medicine for anti-tumor immunotherapy via the interstitial route. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a preparation derived from traditional Chinese medicine for anti-tumor immunotherapy.

[0005] In a first aspect, the present invention provides a preparation derived from traditional Chinese medicine for anti-tumor immunotherapy. The preparation of the preparation derived from traditional Chinese medicine includes treating a traditional Chinese medicine decoction and an optional anti-tumor drug by a freeze-thaw method and / or a calcium addition method. When the anti-tumor drug is doxorubicin, the calcium addition method is used to prepare traditional Chinese medicine micro-nanoparticles encapsulating doxorubicin.

[0006] The present invention uses traditional Chinese medicine as raw material to prepare a new type of anti-tumor immune preparation. The new type of anti-tumor preparation developed by the present invention has unexpectedly been found to have a certain immune activation effect by using the micro-nano particles provided by the present invention. A doxorubicin preparation based on the particles has also been developed. Doxorubicin has a broad-spectrum anti-tumor ability. The preparation prepared by loading doxorubicin into micro-nano particles also has a sustained-release ability. The micro-nano particles prepared using traditional Chinese medicine provided by the present invention have excellent water dispersibility and biocompatibility, can adsorb drugs, fluorescent molecules, etc., and have broad application prospects in the medical fields such as drug loading and tracing. At the same time, the micro-nano particles prepared using traditional Chinese medicine can be administered by interstitial injection under the dermis, avoiding adverse reactions caused by direct entry of micro-nano particles and traditional Chinese medicine components into the blood, and can produce more significant immune activation effects and changes in the mechanical properties of tumors, and can be used for anti-tumor immunotherapy.

[0007] According to the present invention, the inventors unexpectedly discovered that administering drugs through the interstitial route can promote these specific particles to enter the body without causing adverse reactions, produce immune activation effects, and also affect the mechanics of biological tissues. The micro-nanoscale particles in the traditional Chinese medicine-derived preparations provided by the present invention provide a safe and effective method for in vivo medication.

[0008] Preferably, the Chinese herbal medicine slices used in the Chinese herbal medicine decoction are selected from one or a combination of slices of licorice, notopterygium root, aster, platycodon, angelica root, kudzu root, astragalus root, angelica pubescens, raw rehmannia root, cassia twig, forsythia and Achyranthes bidentata.

[0009] Further preferably, the Chinese medicine-derived preparation is a liquid preparation; preferably, the Chinese medicine-derived preparation contains PBS buffer or physiological saline, the Chinese herbal medicine slices are preferably Achyranthes bidentata slices, and the anti-tumor drug is preferably doxorubicin.

[0010] In the present invention, the self-assembled particles obtained by combining the above-mentioned Chinese herbal medicine pieces have different physical and chemical properties, and the synergistic effect of the Achyranthes bidentata pieces and doxorubicin is particularly good.

[0011] The present invention unexpectedly discovered that based on Achyranthes bidentata granules, these particles can be taken up by RAW264.7 macrophages in vitro, digested by lysosomes, and promote cell proliferation, enhanced phagocytic activity, increased TNF-α secretion and other immune responses, and overall biocompatibility is good. Based on the particles derived from Achyranthes bidentata, the present invention mainly promotes the entry of these particles into the body through interstitial administration. Taking the interstitial injection of the upper limb as an example, it can cause enlargement of the axillary lymph nodes and maturation of the DC cells therein. It promotes the polarization of M cells in the nearby tumor tissue in the M1 direction (anti-tumor). After administration, the tumor tissue also showed softening. The above phenomenon shows that Achyranthes bidentata granules are administered through the interstitial route and have the potential to be used as an immune adjuvant, and can be transformed into an anti-tumor preparation.

[0012] Further preferably, the traditional Chinese medicine-derived preparation for anti-tumor immunotherapy contains micro- and nanoparticles; the micro- and nanoparticles are spherical or bi-spherical micron or nanometer-sized particles; preferably, the size of the micro- and nanoparticles is 200 nm to 3 μm, preferably 200 to 1000 nm or 1 to 3 μm; and / or, the average particle size of the micro- and nanoparticles obtained by the freeze-thaw method is 200 nm to 1000 nm; the average particle size of the micro- and nanoparticles obtained by the calcium addition method is 1 to 3 μm. The doxorubicin-encapsulated Achyranthes bidentata micro- and nanoparticles prepared by the calcium addition method are particularly effective.

[0013] The second aspect of the present invention also provides a method for preparing the traditional Chinese medicine-derived preparation for anti-tumor immunotherapy, comprising: providing a traditional Chinese medicine decoction, treating the traditional Chinese medicine decoction and the optional anti-tumor drug by freeze-thaw method and / or calcium addition method to obtain micro-nano particles, and suspending the micro-nano particles in PBS or physiological saline injection; preferably, the concentration of the micro-nano particles in the preparation is 5-10 mg / mL.

[0014] Preferably, the preparation of the Chinese medicine decoction comprises soaking and decocting the Chinese medicine slices in water; preferably, the soaking time is 1 to 5 hours, and the decocting time is 30 to 60 minutes. Preferably, double-distilled water is used for soaking, and the amount of water added is enough to cover the Chinese medicine slices.

[0015] Preferably, the freeze-thaw method comprises placing the supernatant of the Chinese medicine decoction at -80 to -20°C for 12 to 24 hours;

[0016] And / or, the calcium addition method includes adding 80 to 120 μL of a CaCl2 solution with a concentration of 180 to 220 mg / mL, preferably 100 μL of a CaCl2 solution with a concentration of 200 mg / mL, to the supernatant of the traditional Chinese medicine decoction; preferably, when an anti-tumor drug is added to the preparation, the anti-tumor drug is encapsulated by the calcium addition method.

[0017] Further preferably, in the freeze-thaw method, the decoction of the Chinese herbal medicine slices is centrifuged at 10,000 g to remove the residue and collect the supernatant; the supernatant is frozen at -80°C to -20°C for 12-24 hours, preferably at -20°C for 24 hours; thawed; centrifuged again at 10,000 g to collect the particles; then resuspended by blowing with double distilled water; and centrifuged again at 10,000 g to wash to obtain micro-nano particles.

[0018] Further preferably, in the calcium addition method, the decoction of the Chinese herbal medicine slices is centrifuged at 10,000 g to remove the residue and collect the supernatant; CaCl2 solution is added to make the concentration of CaCl2 in the decoction supernatant 400-600 μg / mL, preferably 500 μg / mL; preferably, 40 mL of the supernatant is aliquoted, and 100 μL of 200 mg / mL CaCl2 solution is added dropwise thereto; the directly precipitated particles are collected again by centrifugation at 10,000 g; then the particles are resuspended by blowing with double distilled water; and the particles are washed by centrifugation at 10,000 g again to obtain the micro-nano particles.

[0019] The present invention has discovered novel self-assembled particles made from traditional Chinese medicine, and found that these assembled particles themselves have immune activation capabilities. A method for loading doxorubicin into these novel particles has been successfully developed, resulting in the preparation of a novel anti-tumor immune preparation.

[0020] In a third aspect, the present invention provides the use of the Chinese medicine-derived preparation for anti-tumor immunotherapy or the Chinese medicine-derived preparation obtained by any of the above methods for preparing the Chinese medicine-derived preparation for anti-tumor immunotherapy, including any one or more of the following:

[0021] 1) Application in the preparation of immunomodulatory drugs;

[0022] 2) Application in the preparation of drugs for tumor treatment;

[0023] 3) Application in the preparation of carriers of fluorescent tracers.

[0024] In a fourth aspect, the present invention provides a Chinese herbal medicine-derived preparation for anti-tumor immunotherapy, or a Chinese herbal medicine-derived preparation obtained by a method for preparing the same, for administration via the interstitial route, preferably via subdermal interstitial injection. The present invention utilizes Chinese herbal medicine as raw material to synthesize particles with varying physical and chemical properties. These particles can be administered via interstitial injection, allowing them to enter the body. They can even induce immune activation, potentially enabling anti-tumor therapy.

[0025] The beneficial effects of the present invention are at least:

[0026] 1) The present invention is the first to use a calcium addition method to synthesize and extract spherical or bi-spherical uniform micro-nanoparticles from a Chinese herbal medicine decoction, and the calcium addition method can better encapsulate doxorubicin;

[0027] 2) The production cost of the present invention is low, the Chinese herbal medicine slices are cheap and easy to obtain, have low toxicity, and good biocompatibility;

[0028] 3) The synthesis method of the present invention is simple and can be synthesized using traditional Chinese medicine decoction;

[0029] 4) The micro-nano particles of the present invention are formed by the aggregation of components in a traditional Chinese medicine decoction and can be used directly as medicine;

[0030] 5) The product obtained by the present invention has small particles, excellent water dispersibility, and biocompatibility. It can adsorb drugs, fluorescent molecules, etc. and will be used in drug delivery, tracing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Figures 1 and 2 are data from experimental example 1 of the present invention, showing the in vitro co-incubation of Achyranthes bidentata particles with RAW264.7 cells; Figures a, b, and c are scanning electron micrographs of the cells after co-incubation with the particles and the RAW264.7 cells themselves after critical point drying; Figure d is a statistical graph showing the level of TNF-α secreted by the cells after co-incubation with the particle suspension; Figure e is a statistical graph showing the activity of phagocytosis of neutral red by the cells after co-incubation with the particles; and Figure f is a statistical graph showing the level of reactive oxygen species in the cells after co-incubation with the particles.

[0033] Figure 2The IVIS images of Experimental Example 2 of the present invention were obtained after oral administration of Achyranthes bidentata granules (NX oral), administration as in Example 1 (NX-ISI), and tail vein administration (NX-IVI);

[0034] Figure 3 The rheological properties of the interstitial tissue surrounding the tumor after administration in Experimental Example 3 of the present invention in the manner of Example 1 are shown; Figure a is a scanning diagram of the storage modulus and loss modulus; Figure b is a statistical diagram of the jump points of the storage modulus;

[0035] Figure 4 Figure 3 shows the tumor inhibition effect and immune activation effect of Experimental Example 3 of the present invention; Figure a shows the tumor weight results of each group; Figures b, c, d, and e show the statistical graphs of biochemical indicators in serum; Figure f shows the flow cytometry analysis of dendritic cell maturation after grinding of mouse lymph nodes; Figure g shows the statistical graph of the flow cytometry results of dendritic cell maturation; Figure h shows the flow cytometry analysis of macrophage polarization direction after grinding of mouse tumor tissue; Figure i shows the statistical graph of the flow cytometry results of macrophage polarization direction;

[0036] Figure 5 Schematic diagram and drug release diagram of Achyranthes bidentata particles loaded with doxorubicin prepared by the calcium addition method in Experimental Example 4 of the present invention; Figure a shows, from left to right, a doxorubicin hydrochloride aqueous solution, a precipitate of Achyranthes bidentata particles prepared by the calcium addition method, and a schematic diagram of the precipitate of Achyranthes bidentata particles loaded with doxorubicin prepared by the calcium addition method; Figure b is an SEM image of the doxorubicin aqueous solution after drying on a silicon wafer; Figure c is an SEM image of the precipitate of Achyranthes bidentata particles prepared by the calcium addition method; Figure d is an SEM image of the precipitate of Achyranthes bidentata particles loaded with doxorubicin prepared by the calcium addition method; and Figure e is an in vitro drug release curve of the Achyranthes bidentata particles loaded with doxorubicin. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] Where specific techniques or conditions are not specified in the examples of the present invention, the techniques or conditions described in the literature in the field or in the product specifications were used. For devices, instruments, reagents, etc. used, where the manufacturer is not specified, all are conventional products that can be purchased through regular channels. The raw materials used in the present invention are all readily available in the domestic product market.

[0039] Example 1

[0040] (1) Weigh 50 g of Achyranthes bidentata slices and place them in a medicine pot. Add 500 mL of double-distilled water to cover the slices and soak for 1 hour. Decoction for 1 hour and collect the decoction liquid.

[0041] (2) After collecting the decoction, centrifuge at 10,000 g immediately to remove the residue and collect the supernatant.

[0042] (3) Freeze the supernatant at -20°C for 24 h.

[0043] (4) Thaw the cells, centrifuge at 10,000 g, and collect the particles (500-1,000 nm in diameter).

[0044] (5) The micro-nano particles are resuspended in double-distilled water and blown, and then centrifuged and washed in a handheld centrifuge to obtain the Achyranthes bidentata micro-nano particles.

[0045] (6) Suspend the Achyranthes bidentata micro-nanoparticles in PBS (or normal saline injection) at a particle concentration of 5 mg / mL.

[0046] Example 2

[0047] (1) Weigh 50g of Achyranthes bidentata slices, place them in a medicine pot, add double-distilled water to cover the slices, and soak for 1 hour. Decoction for 1 hour, and collect the decoction liquid after completion.

[0048] (2) After collecting the decoction, centrifuge at 10,000 g immediately to remove the residue and collect the supernatant.

[0049] (3) Aliquot 40 mL of the supernatant and add 100 μL of 200 mg / mL CaCl2 solution to it to control the CaCl2 concentration in the decoction supernatant to 500 μg / mL.

[0050] (4) The directly precipitated particles were collected by centrifugation at 10,000 g; then they were resuspended by blowing with double-distilled water; and the micro-nanoparticles were washed by centrifugation again at 10,000 g.

[0051] (5) The micro-nano particles are resuspended in double-distilled water and blown, and then centrifuged and washed in a handheld centrifuge to obtain the Achyranthes bidentata micro-nano particles.

[0052] (6) Suspend the Achyranthes bidentata micro-nanoparticles in PBS or saline injection at a particle concentration of 5 mg / mL.

[0053] Example 3

[0054] (1) Weigh 50g of Achyranthes bidentata slices, place them in a medicine pot, add double-distilled water to cover the slices, and soak for 1 hour. Decoction for 1 hour, and collect the decoction liquid after completion.

[0055] (2) After collecting the decoction, centrifuge at 10,000 g immediately to remove the residue and collect the supernatant.

[0056] (3) Add an excess of doxorubicin hydrochloride to the supernatant to prepare a concentration of 500 μg / mL. After the doxorubicin hydrochloride is fully dissolved, centrifuge at 10,000 g for 30 minutes to remove the undissolved doxorubicin hydrochloride and retain the supernatant.

[0057] (4) 40 mL of the supernatant retained in step (3) was aliquoted and 100 μL of a 200 mg / mL CaCl2 solution was added dropwise thereto to control the CaCl2 concentration in the decoction supernatant to 500 μg / mL.

[0058] (5) The directly precipitated particles were collected by centrifugation at 10,000 g; then they were resuspended by blowing with double-distilled water; and the micro-nanoparticles were washed by centrifugation again at 10,000 g.

[0059] (6) The micro-nano particles are resuspended in double-distilled water and blown, and then centrifuged and washed in a handheld centrifuge to obtain the Achyranthes bidentata micro-nano particles loaded with doxorubicin.

[0060] (7) The Achyranthes bidentata micro-nanoparticles loaded with doxorubicin were suspended in PBS or saline injection at a particle concentration of 5 mg / mL.

[0061] Experimental Example 1 Biological Effects of Niuxi Granules on RAW264.7 Cells in Vitro

[0062] To avoid contamination, the Achyranthes bidentata granules prepared by the method of Example 1 were sterilized in a 100° C. water bath for 30 minutes before administration.

[0063] Take Raw264.7 cells and inoculate them, 3000 per dish. Cultivate until they adhere to the wall. Dilute the Achyranthes bidentata particles with culture medium to a concentration of 100 μg / mL and incubate with the cells. After 8 hours, take them out and fix them with 5% glutaraldehyde solution overnight. Then dehydrate them with ethanol gradient and dry them in a CO2 critical point dryer. After gold spraying, send them to SEM to take pictures of the morphology ( Figure 1 a). The results showed that after co-incubation of Raw264.7 cells with Achyranthes bidentata particles, the cells phagocytosed the surrounding particles very cleanly ( Figure 1 b) and compared with cells that did not phagocytize particles ( Figure 1 c) The surface morphology has changed, the external structure has become less smooth, and the number of protruding pseudopodia has increased.

[0064] Take 2.5*10 of Raw264.7 cells 5 Cells were seeded at 100 μg / well in six-well plates and cultured in a CO2 incubator at 37°C. Achyranthes bidentata particle suspensions of varying concentrations were incubated with the cells for 24 hours. The cells were then lysed and protein extracted. The relative expression of TNF-α in the protein was determined by Western blotting. Figure 1dThe results showed that as the concentration of Achyranthes bidentata granules increased, the secretion of TNF-α also increased, indicating that Achyranthes bidentata granules can stimulate cells to secrete TNF-α proinflammatory factors, and Achyranthes bidentata granules have the ability to promote the polarization of Raw264.7 macrophages towards M1.

[0065] 3000 Raw264.7 cells were seeded per well on a 96-well plate. After the cells adhered, different concentrations of Achyranthes bidentata particles were administered and incubated for 48 hours. The culture medium was aspirated, and 200 μL of 0.1% neutral red solution was added. After 3 hours of incubation, the supernatant was aspirated and 200 μL of purified water was added to lyse the cells. The cells were incubated at 4°C overnight. 100 μL of the supernatant was aspirated and placed in another 96-well plate. The absorbance of the solution at 490 nm was measured using a multifunctional microplate reader. Figure 1 eThe results showed that with the increase of the concentration of Achyranthes bidentata particles, the phagocytic activity of Raw264.7 cells increased, and reached the peak of phagocytic activity at a concentration of 1 μg / mL. Then, with the further increase of the concentration of Achyranthes bidentata particles, the phagocytic activity began to decrease, indicating that the appropriate particle concentration can achieve better phagocytic activity of Raw264.7 cells, thereby regulating the body's immune ability.

[0066] 3000 Raw264.7 cells were seeded per well in a 96-well plate. After the cells attached, NX particles were administered and incubated for 24 hours. DCFH-DA was diluted 1:1000 in serum-free culture medium, and 100 μL was added to each well. After incubation for 60 minutes in a 37°C cell culture incubator, the cells were washed with serum-free culture medium, and the fluorescence intensity of each well was measured using a multi-function microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Figure 1 fThe results showed that the increase in the concentration of Achyranthes bidentata particles had no statistical difference in the production of reactive oxygen species in Raw264.7 cells, and would not cause oxidative damage to the cells, further demonstrating its good biocompatibility.

[0067] Experimental Example 2 In vivo tracing of Niuxi granules

[0068] Achyranthes bidentata granules were prepared and collected by the method of Example 1 and stained with DiR fluorescent dye. Animals (Kunming mice) were divided into 3 groups, 2 in each group. Each group was administered by oral gavage, and waited for 6 hours, which was recorded as the NX oral group; the granules were injected from the interstitial space of the upper limb of the mouse (carpal tunnel), and the fluorescence distribution in the mouse body was observed for 12 hours, which was recorded as the NX-ISI group; the granules were injected into the tail vein of the mouse, and waited for 6 hours, which was recorded as the NX-IVI group. After the time was reached, the mice were killed and dissected, and the organs were arranged from top to bottom as heart, liver, spleen, lung, kidney, upper limb lymph node, thymus, brain, and digestive tract, and observed under small animal infrared imaging (IVIS).

[0069] Figure 2The results showed that the particles in the NX oral group were distributed in the digestive tract, which is in line with the general practice. Oral Chinese medicine enters the blood through the gastrointestinal tract and is then distributed to other organs. The particles in the NX-ISI group were concentrated in the liver, followed by the upper limb lymph nodes, indicating that Niuxi granules have the advantage of targeted drainage of lymph nodes, further suggesting that they may play a strong immunomodulatory role. The particles in the NX-IVI group were mainly distributed in the liver, followed by the spleen and lungs, indicating that Niuxi granules have a tendency to target the lungs, suggesting that they may have the ability to deliver drugs to lung diseases.

[0070] Experimental Example 3: Immune Activation Effect and Tumor Softening Effect of Niuxi Granules

[0071] The Achyranthes bidentata granules were prepared and collected using Achyranthes bidentata slices. 5 The animals were divided into 4 groups, each with 3 animals. Each group was injected with PBS through the intercostal space of the upper limb of the mouse (carpal tunnel), which was recorded as the PBS group; the Chinese medicine-derived preparation obtained in Example 1 was injected into the intercostal space of the upper limb of the mouse (carpal tunnel), which was recorded as the NX-ISI group; the Chinese medicine-derived preparation obtained in Example 1 was injected around the mouse tumor, which was recorded as the NX-peritumoral group; the Chinese medicine-derived preparation obtained in Example 1 was administered by oral gavage, which was recorded as the oral group. Starting from the third day, Achyranthes bidentata granules (5 mg / mL, 100 uL per mouse) were administered. The Dox concentration was 2 mg / mL, and 100 uL was administered per mouse. The administration was continued every other day for a total of 14 days. After the mice were sacrificed, blood, melanoma, and draining lymph nodes were collected. The melanoma was weighed. The melanoma and lymph nodes were ground and filtered through a 100 μm filter. After staining, the cells were analyzed by flow cytometry. Melanoma tumors were ground to obtain tumor stroma for rheological analysis. Blood samples were allowed to rest for 1 hour and then centrifuged at 1800 g for 10 minutes. The supernatant was then collected and sent for analysis.

[0072] The results showed that the rheological test results ( Figure 3 a. Figure 3 b) The NX-ISI group showed a softening of the interstitial structure of the tumor. Figure 4 a) This indicates that the NX-ISI group has a tendency to inhibit the proliferation of melanoma. Serological test results ( Figure 4 b- Figure 4 e) This shows that Achyranthes bidentata granules have no obvious damage to the organs of mice and have little toxicity and side effects. Figure 4 f- Figure 4 i) This indicates that the NX-ISI group can better activate dendritic cells ( Figure 4 f. Figure 4 g), thereby mobilizing the body's immune system; at the same time, the NX-ISI group can promote macrophages to tend towards M1 polarization ( Figure 4 h, 4i), the latter secrete proinflammatory cytokines and chemokines and actively participate in the immune response.

[0073] Experimental Example 4: Loading of Achyranthes bidentata granules with doxorubicin hydrochloride (DOX) and drug release

[0074] In this experimental example, the preparation was prepared using the same method as in Example 3, wherein the decoction of Achyranthes bidentata slices was centrifuged at 10,000 g to remove the residue and the supernatant was collected; doxorubicin hydrochloride was added to the supernatant until it was excessive and fully dissolved by ultrasound; the undissolved drug precipitate was removed by centrifugation at 10,000 g, and the decoction supernatant was collected again. At this time, the decoction supernatant contained a saturated solution of doxorubicin hydrochloride.

[0075] Take 40 mL of the supernatant and add 100 μL of a 200 mg / mL CaCl₂ solution. Let it stand for 15 minutes to allow the particles to precipitate, shake it, and let it stand for another 15 minutes to allow the particles to fully precipitate. Centrifuge the supernatant of the precipitated particles at 10,000 g. Once a precipitate is visible, resuspend the white precipitate in double-distilled water, centrifuge it in a handheld centrifuge, and wash it. Centrifuge for 30 seconds and wash it three times, collecting the lower precipitate. This yields doxorubicin hydrochloride-loaded Achyranthes bidentata particles.

[0076] Prepare 0.9% normal saline injection as the release medium. Centrifuge the Achyranthes bidentata granules-doxorubicin hydrochloride composite particles to remove the solvent, add the release medium to resuspend, transfer to the dialysis bag, add the same dissolution medium outside the dialysis bag, and the molecular weight cutoff of the dialysis bag is 3500Da. Stir at a constant speed under 37°C water bath conditions. Take out 3mL of the dissolution medium outside the dialysis bag at 30min, 1h, 2h, 4h, 15h, 24h, 48h, and 72h, respectively, and then immediately replenish the same amount of dissolution medium to keep the total amount of liquid in the system unchanged. Repeat the experiment in three groups. Use a multifunctional microplate reader at an excitation wavelength of 478nm and an emission wavelength of 570nm to detect the content of doxorubicin hydrochloride in the solution by fluorescence method, and calculate the cumulative dissolution rate ( Figure 5 e). The release curve was obtained by fitting the Ritger-Peppas equation.

[0077] The results showed that the Achyranthes bidentata granules loaded with doxorubicin hydrochloride were prepared by the method of this embodiment. The doxorubicin hydrochloride solution was red; the Achyranthes bidentata granules were white granules; after loading doxorubicin hydrochloride, the Achyranthes bidentata granules turned red, while the supernatant had no obvious red color ( Figure 5 a).

[0078] Under SEM observation, it can be seen that the free doxorubicin hydrochloride solution has no specific structure ( Figure 5 b). Achyranthes bidentata granules are mostly micron-sized spherical particles ( Figure 5 c). After loading doxorubicin hydrochloride, the morphology of the particles is not significantly affected ( Figure 5d). According to the release curve results, the drug release reached 40% at 72h, and then entered a slow and long-term release ( Figure 5 e).

[0079] In summary, Achyranthes bidentata granules can be used as carriers to load the anti-tumor drug doxorubicin hydrochloride, thereby increasing the solubility of the latter. In addition to doxorubicin, many other anti-tumor small molecule drugs can also be loaded using the method described in the present invention.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A preparation derived from traditional Chinese medicine for anti-tumor immunotherapy, characterized in that: The preparation of the traditional Chinese medicine preparation includes treating the traditional Chinese medicine decoction and the optional anti-tumor drug by freeze-thaw method and / or calcium addition method. When the anti-tumor drug is doxorubicin, the calcium addition method is used to prepare the traditional Chinese medicine micro-nano particles encapsulating doxorubicin.

2. The Chinese medicine-derived preparation for anti-tumor immunotherapy according to claim 1, characterized in that: The Chinese medicine decoction liquid uses one or more pieces selected from the group consisting of licorice, notopterygium root, aster, platycodon, angelica root, kudzu root, astragalus root, angelica pubescens, radix rehmanniae, cassia twig, forsythia and achyranthes.

3. The Chinese medicine-derived preparation for anti-tumor immunotherapy according to claim 1 or 2, characterized in that: The traditional Chinese medicine-derived preparation is a liquid preparation; preferably, the traditional Chinese medicine-derived preparation contains PBS buffer or physiological saline, the traditional Chinese medicine slices are preferably Achyranthes bidentata slices, and the anti-tumor drug is preferably doxorubicin.

4. The Chinese medicine-derived preparation for anti-tumor immunotherapy according to any one of claims 1 to 3, characterized in that: The traditional Chinese medicine-derived preparation for anti-tumor immunotherapy contains micro-nano particles; the micro-nano particles are spherical or bi-spherical micrometer or nanometer-sized particles; preferably, the size of the micro-nano particles is 200nm to 3μm, preferably 200 to 1000nm or 1 to 3μm; and / or, the average particle size of the micro-nano particles obtained by the freeze-thaw method is 200nm to 1000nm; the average particle size of the micro-nano particles obtained by the calcium addition method is 1 to 3μm.

5. The method for preparing the Chinese medicine-derived preparation for anti-tumor immunotherapy according to any one of claims 1 to 4, characterized in that: include: A Chinese herbal decoction is provided, and the Chinese herbal decoction and the optional anti-tumor drug are treated by freeze-thaw method and / or calcium addition method to obtain micro-nano particles, and the micro-nano particles are suspended in PBS or normal saline injection; preferably, the concentration of the micro-nano particles in the preparation is 5-10 mg / mL.

6. The method for preparing a traditional Chinese medicine-derived preparation for anti-tumor immunotherapy according to claim 5, characterized in that: The preparation of the Chinese medicine decoction comprises soaking and decocting the Chinese medicine slices in water; preferably, the soaking time is 1 to 5 hours, and the decocting time is 30 to 60 minutes. Double distilled water is preferably used for soaking, and the amount of water added is enough to cover the Chinese medicine slices.

7. The method for preparing a traditional Chinese medicine-derived preparation for anti-tumor immunotherapy according to claim 5 or 6, characterized in that: The freeze-thaw method includes placing the supernatant of the Chinese medicine decoction at -80 to -20°C for 12 to 24 hours.

8. The method for preparing a traditional Chinese medicine-derived preparation for anti-tumor immunotherapy according to claim 5 or 6, characterized in that: The calcium addition method comprises adding 80 to 120 μL of a CaCl2 solution having a concentration of 180 to 220 mg / mL to the supernatant of the traditional Chinese medicine decoction. Preferably, when an anti-tumor drug is added to the preparation, the anti-tumor drug is encapsulated by the calcium addition method.

9. Use of the Chinese medicine-derived preparation for anti-tumor immunotherapy according to any one of claims 1 to 4 or the Chinese medicine-derived preparation obtained by the method for preparing the Chinese medicine-derived preparation for anti-tumor immunotherapy according to any one of claims 5 to 8, characterized in that: Include any one or more of the following: 1) Application in the preparation of immunomodulatory drugs; 2) Application in the preparation of drugs for tumor treatment; 3) Application in the preparation of carriers of fluorescent tracers.

10. The Chinese medicine-derived preparation for anti-tumor immunotherapy according to any one of claims 1 to 4 or the Chinese medicine-derived preparation for anti-tumor immunotherapy obtained by the method for preparing the Chinese medicine-derived preparation according to any one of claims 5 to 7, characterized in that: The administration method is through the interstitial route, preferably through subdermal interstitial injection.

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