Traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticle and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine nano preparations, in particular to traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticles and a preparation method thereof.The preparation method comprises the following steps that 1, a chitosan-based carrier and traditional Chinese medicine polysaccharide are dissolved in a buffer solution, and the chitosan-based carrier is selected from chitosan, carboxymethyl chitosan or quaternized chitosan; step 2, putting the chitosan-based carrier solution into a reaction container, and dropwise adding glacial acetic acid to adjust the pH value of the system; 3, a cross-linking agent solution is dropwise added at a constant speed, a cross-linking agent is a multivalent anion / cation compound, and stirring is performed to form a core-shell structure; step 4, dropwise adding a traditional Chinese medicine polysaccharide solution into a mixed system in the step 3, and continuously stirring; step 5, filtering and granulating the obtained solution to obtain nanoparticles; according to the traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticles and the preparation method thereof, not only is radiotherapy sensitization achieved, but also the immunoregulation function is achieved, and the synergistic interaction of radiotherapy and immunotherapy is achieved by activating dendritic cells and T cells.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine nano preparations, in particular to a traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticle and a preparation method thereof. Background Art

[0002] Radiotherapy is a commonly used method for tumor treatment in clinical practice. It kills tumor cells by destroying cancer cell DNA or generating free radicals through high-energy ion radiation, and has obvious therapeutic effects on early tumors and locally invasive tumors. However, tumor cells often exist in an oxygen-deficient environment, and hypoxic cells are tolerant to radiotherapy, which makes it difficult to completely eradicate the tumor with radiotherapy, and leads to radiotherapy resistance and recurrence and metastasis. In order to improve the effect of radiotherapy, researchers have developed various radiosensitizers. Common sensitizers include chemical small molecules and natural macromolecules. For example, nitroimidazole small molecule drugs (such as misonidazole and etanercept) can simulate the oxygen effect and increase the sensitivity of hypoxic tumor cells to radiation, but such drugs are often accompanied by adverse reactions such as neurotoxicity and gastrointestinal irritation, and their clinical use is limited.

[0003] Natural herbal polysaccharides (such as Ganoderma lucidum polysaccharides and Astragalus polysaccharides) have low toxicity and immunomodulatory bioactivities, which can assist in enhancing the effectiveness of radiotherapy. However, due to their weak targeting and easy degradation in the body, their radiosensitization effect still needs further verification. Therefore, in view of the above situation, there is an urgent need to develop a radiosensitization nanoparticle of herbal polysaccharide and its preparation method to overcome the shortcomings of current practical applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticles and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticles comprises the following steps:

[0007] Step 1, dissolving a chitosan-based carrier and a traditional Chinese medicine polysaccharide in a buffer solution respectively, wherein the chitosan-based carrier is selected from chitosan, carboxymethyl chitosan or quaternized chitosan;

[0008] Step 2: placing the chitosan-based carrier solution in a reaction vessel, and adding glacial acetic acid dropwise to adjust the pH of the system;

[0009] Step 3, adding a crosslinker solution at a constant rate, wherein the crosslinker is a multivalent anionic / cationic compound, and stirring to form a core-shell structure;

[0010] Step 4: add the Chinese medicinal polysaccharide solution dropwise to the mixed system of step 3 and continue stirring;

[0011] Step 5, filtering the obtained solution to obtain nanoparticles;

[0012] The mass ratio of the Chinese medicinal polysaccharide, the cross-linking agent and the chitosan-based carrier is 4:(1-2):(3-6).

[0013] As a further solution of the present invention: the chitosan-based carrier meets the following conditions: molecular weight 10-500 kDa; deacetylation degree 70%-95%; viscosity 100-200 mPa·s.

[0014] As a further embodiment of the present invention: the cross-linking agent is selected from at least one of polyethyleneimine, sodium tripolyphosphate, sodium alginate or calcium citrate.

[0015] As a further embodiment of the present invention: the Chinese medicinal polysaccharide is selected from angelica polysaccharide, ginseng polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, yam polysaccharide or atractylodes polysaccharide.

[0016] As a further solution of the present invention: in step 1, the buffer solution is deionized water.

[0017] As a further solution of the present invention: in step 2, the pH of the system is adjusted to 4.5-5.0.

[0018] As a further solution of the present invention: in step 5, filtration is performed using a 0.45 μm filter membrane.

[0019] A Chinese herbal polysaccharide radiotherapy sensitization nanoparticle, prepared by the above-mentioned preparation method of the Chinese herbal polysaccharide radiotherapy sensitization nanoparticle;

[0020] The particle size of the nanoparticles is 200-750 nm, and PDI is less than 0.5.

[0021] As a further solution of the present invention: the nanoparticles are used as tumor radiotherapy sensitizers.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) In addition to radiosensitization, it also has immune regulation function, achieving synergistic effect of radiotherapy and immunotherapy by activating dendritic cells and T cells;

[0024] (2) Utilizing cascade ionic crosslinking and adsorption of various chemically modified chitosan carriers and traditional Chinese medicine polysaccharides, the drug loading efficiency is significantly improved, resulting in preferential accumulation of nanoparticles in the tumor microenvironment and improved hypoxia tolerance;

[0025] (3) Based on the principle of self-assembly, it does not rely on complex equipment and uses chitosan-based carriers. It has good biocompatibility and degradability, is suitable for large-scale production, and is easy to industrialize and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of transmission electron microscopy (TEM) characterization of the nanoparticles in Example 1 of the present invention;

[0027] Among them, the left picture is an electron microscope image of ASP-NPs, and the right picture is an enlarged image of ASP-NPs.

[0028] Figure 2 Schematic diagram of the efficacy evaluation of nanoparticles in treating in situ, recurrent and metastatic tumors in Test Example 1 of the present invention;

[0029] Among them, A is the tumor growth curve, B is the tumor mass, C is the tumor photos of different treatment groups, D is the weight change of mice during treatment, and E is the lung tissue photos of different treatment groups; "*" indicates significant differences compared with the PBS group (control group) *, P < 0.05; **; P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0030] Figure 3 Schematic diagram of the evaluation of the effects of nanoparticles on serum cytokines TNF-α (Figure A) and IL-6 (Figure B) in Test Example 2 of the present invention;

[0031] Figure 4 Schematic diagram of spleen index of mice in each treatment group in the examples of the present invention.

[0032] Figure 5 Figure 2 is the TUNEL staining results of tumor tissues in different treatment groups in the examples of the present invention (left column is 2×; right column is 200×);

[0033] Among them, “*” indicates significant difference compared with the PBS group (control group)*, P<0.05; **; ns indicates no significant difference.

[0034] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0036] See also Figure 1-Figure 5The present invention provides a Chinese herbal polysaccharide radiosensitizing nanoparticle. Based on the principle of chemically modified chitosan polymers and ionic crosslinking cascade adsorption, the nanoparticles are prepared by self-assembly to exhibit both radiosensitization and immunomodulatory functions. The carriers used include chitosan, carboxymethyl chitosan, and quaternization, among other modified forms, to enhance the carrier's water solubility, biocompatibility, and surface positive charge. The Chinese herbal polysaccharide is pre-dispersed and mixed with the carrier as an immune adjuvant. The auxiliary crosslinker is a polyvalent cation / anion such as polyethyleneimine (PEI), sodium tripolyphosphate (TPP), sodium alginate (SA), and calcium citrate (CaCl2). Through gradient addition and physical methods such as ultrasound or high-speed stirring, ionic crosslinking and cascade adsorption are sequentially achieved under different pH and ionic strength conditions, thereby enhancing the carrier's adsorption efficiency for different Chinese herbal polysaccharides and controlling the size, zeta potential, and distribution uniformity of the nanoparticles. The resulting nanoparticles have a controllable particle size, a PDI < 0.2, a moderate surface charge, and excellent stability.

[0037] The chitosan-based carrier is composed of alternating β-(1→4)-N-acetyl-D-glucosamine and D-glucosamine units. The molecular chain is rich in amino (–NH2) and hydroxyl (–OH) functional groups, making it easy to chemically modify and ionically cross-link. Its molecular weight can be controlled within the range of 10-500 kDa, its viscosity is 100-200 mPa, and its degree of deacetylation (DDA) is within the range of 70%-95%.

[0038] When preparing Chinese herbal polysaccharide radiotherapy sensitizing nanoparticles, first, PEI, TPP, SA and CaCl2 are weighed separately, dissolved in deionized water and fixed to volume to prepare a cross-linker mother solution of known concentration; then, the Chinese herbal polysaccharide and chitosan-based carrier are dissolved in a suitable buffer solution in a predetermined proportion, pre-dispersed at a certain speed for 5-20 minutes, and gently stirred by adding glacial acetic acid dropwise (20 μL / time), and the pH is measured in batches until it reaches the predetermined value; finally, the cross-linker stock solution is added dropwise at a constant rate under continuous stirring conditions, and stirring is continued for 10-30 minutes. The Chinese herbal polysaccharide nanoparticles (ASP-NPs) with uniform particle size are self-assembled by ionic cross-linking and cascade adsorption.

[0039] Therefore, the preparation method of the Chinese medicine polysaccharide radiotherapy sensitization nanoparticles of the present invention mainly comprises the following steps:

[0040] The Chinese herbal polysaccharide, cross-linking agent and carrier were dissolved in ultrapure water to prepare a 10 mg / mL stock solution, which was stored at 4°C in the dark for later use. The mass ratio of the Chinese herbal polysaccharide and cross-linking agent to the carrier was 4:(1-2):(3-6);

[0041] Accurately measure 4 mL of the carrier mother solution to a 10 mL sterile penicillin bottle, add ultrapure water to make up to 10 mL, adjust the pH to the target range with glacial acetic acid, and then slowly add the cross-linker solution of the same concentration with a 1 mL syringe. After magnetic stirring at room temperature for a certain period of time, a core-shell structure is formed. Subsequently, under magnetic stirring, the traditional Chinese medicine polysaccharide solution is slowly added with a 1 mL syringe. After magnetic stirring at room temperature for a certain period of time, the nano solution is filtered through a 0.45 μm filter membrane for granulation to obtain traditional Chinese medicine polysaccharide nanoparticles.

[0042] After intravenous injection, this traditional Chinese medicine polysaccharide radiotherapy sensitizing nanoparticle can accumulate in the tumor microenvironment, improve the sensitivity of hypoxic cells to radiation, and activate dendritic cells and T lymphocytes, combining radiosensitization and immunomodulation functions, thereby significantly enhancing tumor cell apoptosis during radiotherapy, overcoming radiotherapy tolerance, and achieving better tumor inhibition effects.

[0043] Example 1: When the Chinese herbal polysaccharide is Angelica sinensis polysaccharide (ASP), the preparation process of the nanoparticles is as follows: (1) Preparation of ionic crosslinker: Accurately pipette 100 μL each of SA and CaCl2 stock solutions (concentration of 10 mg / mL) into a 2 mL volumetric flask, and dilute to the mark with ultrapure water (solution A); (2) Preparation of chitosan solution: Accurately measure 100 μL of CS stock solution (concentration of 10 mg / mL) and place it in a 1 mL volumetric flask, and dilute to the mark with ultrapure water (solution B); (3) Preparation of Chinese herbal polysaccharide solution: Accurately weigh 40 mg of Angelica sinensis polysaccharide into a 5 mL volumetric flask, and dilute to the mark with ultrapure water (solution C); Under magnetic stirring, solution A is slowly dripped into solution B, and then 1 mL of solution C is slowly dripped into the mixed system, the pH is adjusted to 5.0, and stirring is continued for 10-30 min to obtain ASP-NPs.

[0044] Example 2: The difference from Example 1 is that the Chinese medicinal polysaccharide is ginseng polysaccharide.

[0045] Example 3: The difference from Example 1 is that the Chinese medicinal polysaccharide is astragalus polysaccharide.

[0046] Example 4: The difference from Example 1 is that the Chinese medicine polysaccharide is wolfberry polysaccharide.

[0047] Example 5: The difference from Example 1 is that the Chinese medicinal polysaccharide is yam polysaccharide.

[0048] Example 6: The difference from Example 1 is that the Chinese medicine polysaccharide is Atractylodes macrocephala polysaccharide.

[0049] Example 7: The difference from Example 1 is that the carrier is carboxymethyl chitosan.

[0050] Example 8: The difference from Example 1 is that the Chinese medicinal polysaccharide is ginseng polysaccharide and the carrier is carboxymethyl chitosan.

[0051] Example 9: The difference from Example 1 is that the Chinese medicinal polysaccharide is astragalus polysaccharide and the carrier is carboxymethyl chitosan.

[0052] Example 10: The difference from Example 1 is that the Chinese medicine polysaccharide is wolfberry polysaccharide and the carrier is carboxymethyl chitosan.

[0053] Example 11: The difference from Example 1 is that the Chinese medicinal polysaccharide is yam polysaccharide and the carrier is carboxymethyl chitosan.

[0054] Example 12: The difference from Example 1 is that the Chinese medicinal polysaccharide is Atractylodes macrocephala polysaccharide and the carrier is carboxymethyl chitosan.

[0055] Example 13: The difference from Example 1 is that the cross-linking agent is sodium tripolyphosphate.

[0056] Example 14: The difference from Example 1 is that the Chinese medicinal polysaccharide is ginseng polysaccharide and the cross-linking agent is sodium tripolyphosphate.

[0057] Example 15: The difference from Example 1 is that the Chinese medicinal polysaccharide is astragalus polysaccharide and the cross-linking agent is sodium tripolyphosphate.

[0058] Example 16: The difference from Example 1 is that the Chinese medicine polysaccharide is wolfberry polysaccharide and the cross-linking agent is sodium tripolyphosphate.

[0059] Example 17: The difference from Example 1 is that the Chinese medicinal polysaccharide is yam polysaccharide and the cross-linking agent is sodium tripolyphosphate.

[0060] Example 18: The difference from Example 1 is that the Chinese medicinal polysaccharide is Atractylodes macrocephala polysaccharide and the cross-linking agent is sodium tripolyphosphate.

[0061] Example 19: The difference from Example 1 is that the carrier is carboxymethyl chitosan and the cross-linking agent is sodium tripolyphosphate.

[0062] Example 20: The difference from Example 1 is that the Chinese medicinal polysaccharide is ginseng polysaccharide, the carrier is carboxymethyl chitosan, and the cross-linking agent is sodium tripolyphosphate.

[0063] Example 21: The difference from Example 1 is that the Chinese medicinal polysaccharide is astragalus polysaccharide, the carrier is carboxymethyl chitosan, and the cross-linking agent is sodium tripolyphosphate.

[0064] Example 22: The difference from Example 1 is that the Chinese medicine polysaccharide is wolfberry polysaccharide, the carrier is carboxymethyl chitosan, and the cross-linking agent is sodium tripolyphosphate.

[0065] Example 23: The difference from Example 1 is that the Chinese medicinal polysaccharide is yam polysaccharide, the carrier is carboxymethyl chitosan, and the cross-linking agent is sodium tripolyphosphate.

[0066] Example 24: The difference from Example 1 is that the Chinese medicinal polysaccharide is Atractylodes macrocephala polysaccharide, the carrier is carboxymethyl chitosan, and the cross-linking agent is sodium tripolyphosphate.

[0067] Experimental example: single factor experiment;

[0068] This experiment examined three factors: carrier dosage, reaction system pH, and cross-linker dosage. The experimental design employed a fixed TCM polysaccharide to cross-linker ratio of 4:1, and a single-factor variable analysis was used to explore the optimal range for the chitosan-carrier ratio. Particle size results indicate that a chitosan-carrier to TCM polysaccharide ratio between 4:3 and 6 (often ranging from 3 to 6) produces TCM polysaccharide nanoparticles with a uniform and well-defined particle size.

[0069] Table 1 Particle size results of different chitosans

[0070] Concentration / mg / mL Z-Average(d.nm) PDI 0.2 731.8±71.0 0.431±0.063 0.4 311.3±78.4 0.285±0.031 0.8 375.7±62.6 0.470±0.042 1.0 717.7±46.7 0.711±0.101 1.5 746.5±65.4 0.452±0.071

[0071] Then, on the basis of fixing the ratio of traditional Chinese medicine polysaccharides and chitosan carrier at 4:5, a single-factor optimization strategy was used to systematically investigate the pH of the reaction system. Combined with the comprehensive evaluation of the dosage form, chitosan-based carrier characteristics and nanoparticle physical and chemical properties, it was found that the reaction system had a good average particle size and dispersion uniformity at a pH of around 4.5.

[0072] Table 2 Particle size results of different crosslinker pH

[0073]

[0074]

[0075] Finally, the effect of crosslinker concentration on nanoparticle formability was investigated. Using the aforementioned ratio of TCM polysaccharide to chitosan, and maintaining the optimal reaction system pH, the effects of varying crosslinker concentrations on the average particle size and dispersion uniformity of the resulting nanoparticles were examined. The optimal mass ratio of TCM polysaccharide to crosslinker to carrier was ultimately determined to be 4:(1-2):(3-6).

[0076] Table 3 Particle size results of different crosslinker concentrations

[0077] Cross-linker concentration (mg / mL) Z-Average(d.nm) PDI 0.25 324.0±56.6 0.241±0.064 0.167 316.0±30.9 0.352±0.054 0.125 302.8±11.1 0.451±0.110 0.1 230.1±27.9 0.544±0.116

[0078] The particle size results are shown in Table 1 and Table 2. The particle size of the obtained tumor nanoparticles is between 300-400nm. The Chinese medicine polysaccharide nanoparticles have a spherical structure. Figure 1 shown.

[0079] The anti-tumor efficacy evaluation test process of traditional Chinese medicine polysaccharide nanoparticles is as follows:

[0080] Test Example 1: Antigen-site tumor efficacy experiment in 4T1 tumor-bearing mouse model;

[0081] 4T1 cells in the logarithmic growth phase were digested and prepared into 1×10 7 100 μL of cell suspension was inoculated into the right back of each mouse to establish a 4T1 orthotopic tumor model. The tumor volume reached approximately 50 ± 20 mm. 3 The tumor-bearing mice were randomly divided into 4 groups (n=6), namely model group (PBS), radiotherapy group (RT), polysaccharide synergistic radiotherapy group (GP-NPs+RT), and free polysaccharide synergistic radiotherapy group (free-GP+RT). 60 A single radiotherapy treatment was performed using gamma rays from a gamma irradiation device with a dose rate of 1.2 Gy / min and a dose of 6 Gy. Before irradiation, mice were anesthetized with 0.5% sodium pentobarbital. The anesthetized mice were placed prone in a mouse fixation device and fixed on the operating table of the radiotherapy instrument. In order to avoid radiation damage to other parts of the mouse, lead blocks were used to block the non-tumor areas of the mouse, exposing only the tumor in the irradiated area and starting radiotherapy. The polysaccharide synergistic radiotherapy group and the free drug group (both 200 μL / mouse) were injected intravenously. The day before radiotherapy was marked as day 0. The drugs were administered on days 2, 4, 6, and 8, for a total of four times from day 2 to the day before sampling, and once a day. The tumor volume was measured every two days after administration. The experiment ended on day 14, and the samples were collected the next day. The efficacy of drug treatment is as follows. Figure 2 As shown, the tumor in the PBS group tended to grow rapidly, the RT group slowed down slightly, and the drug treatment group significantly increased the tumor inhibition rate.

[0082] Test Example 2: Cytokines in serum;

[0083] ELISA kits were used to detect the levels of IFN-γ, TNF-α, and IL-6 in serum. TNF-α is a natural immune serum mediator that can produce tumor hemorrhagic necrosis, while IL-6 is a key regulator of tumor accumulation and activation, and is also a factor that promotes tumor cell survival, proliferation, metastasis, and invasion. Figure 3 As shown, compared with the PBS group and RT, GP-NPs+RT promoted the secretion of TNF-α and IFN-γ in the peripheral blood of mice, and reduced the secretion of IL-6 in the peripheral blood of mice.

[0084] It should be noted that, in the present invention, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing Chinese medicine polysaccharide radiotherapy sensitization nanoparticles, characterized in that: The following steps are involved: Step 1, dissolving a chitosan-based carrier and a traditional Chinese medicine polysaccharide in a buffer solution respectively, wherein the chitosan-based carrier is selected from chitosan, carboxymethyl chitosan or quaternized chitosan; Step 2: placing the chitosan-based carrier solution in a reaction vessel, and adding glacial acetic acid dropwise to adjust the pH of the system; Step 3, adding a crosslinker solution at a constant rate, wherein the crosslinker is a multivalent anionic / cationic compound, and stirring to form a core-shell structure; Step 4: add the Chinese medicinal polysaccharide solution dropwise to the mixed system of step 3 and continue stirring; Step 5, filtering the obtained solution to obtain nanoparticles; The mass ratio of the Chinese medicinal polysaccharide, the cross-linking agent and the chitosan-based carrier is 4:(1-2):(3-6).

2. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: The chitosan-based carrier meets the following conditions: molecular weight of 10-500 kDa; deacetylation degree of 70%-95%; and viscosity of 100-200 mPa·s.

3. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: The cross-linking agent is selected from at least one of polyethyleneimine, sodium tripolyphosphate, sodium alginate or calcium citrate.

4. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: The Chinese medicinal polysaccharide is selected from angelica polysaccharide, ginseng polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, yam polysaccharide or atractylodes polysaccharide.

5. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: In step 1, the buffer was deionized water.

6. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: In step 2, the pH of the system is adjusted to 4.5-5.

0.

7. The method for preparing the radiosensitization nanoparticles of traditional Chinese medicine polysaccharide according to claim 1, characterized in that: In step 5, filtration is performed using a 0.45 μm filter membrane.

8. A Chinese herbal polysaccharide radiotherapy sensitization nanoparticle, characterized in that: Prepared by the preparation method of the traditional Chinese medicine polysaccharide radiotherapy sensitization nanoparticles according to any one of claims 1 to 7; The particle size of the nanoparticles is 200-750 nm, and PDI is less than 0.

5.

9. The Chinese medicine polysaccharide radiotherapy sensitization nanoparticles according to claim 8, characterized in that: The nanoparticles are used as tumor radiotherapy sensitizers.

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