Multi-component self-assembled strychnos nanoparticles, preparation method and application thereof

Abstract

The application discloses a kind of multi-component self-assembly strychnos nanoparticles and its preparation method and application, the strychnos nanoparticles the nanoparticle includes hydrophilic component strychnine nitroxide and strychnine nitroxide, hydrophobic component strychnine and strychnine, hydrophilic and hydrophobic components gather each other and form spherical nanoparticle, particle size is 140-160nm;Its preparation method is that dimethyl sulfoxide is heated and stirred to make medicinal material component dissolution, then it is prepared by cooling dialysis membrane preparation means of nanometer one step directly to nanoparticle, preparation method is simple, cost is low, without complex monomer component extraction purification process;The above-mentioned strychnos nanoparticle is applied in antitumor drug, overcomes the shortcoming that single drug treatment is easy to produce drug resistance and plays the role of reversing tumor drug resistance, reaches excellent antitumor effect.

Description

Technical Field

[0001] This invention relates to strychnine nanoparticles, particularly to a multi-component self-assembled strychnine nanoparticle, and also to the preparation method and application of the above-mentioned strychnine nanoparticles. Background Technology

[0002] Chemotherapy is one of the main methods for treating tumors; however, multidrug resistance frequently occurs during chemotherapy, greatly increasing the difficulty of tumor treatment. Existing common strategies for reversing tumor drug resistance include the use of chemosensitizers and immunotherapy. However, their clinical application is greatly limited due to drawbacks such as single-site action, numerous adverse reactions, and insignificant efficacy. In recent years, studies have shown that many traditional Chinese medicines can effectively reverse multidrug resistance in tumors, possessing advantages such as multiple action sites, significant efficacy, and low cost, and are expected to provide new ideas for reversing tumor drug resistance and improving the effectiveness of chemotherapy in clinical practice.

[0003] Strychnos nux-vomica is the dried, mature seed of the Strychnos nux-vomica plant (family Loganiaceae). It is bitter, warm in nature, and highly toxic. It can relieve pain, dissipate nodules, and reduce swelling. In traditional Chinese medicine, Strychnos nux-vomica exhibits strong medicinal power and rapid onset of action, and is used for malignant tumors, various types of pain, arthralgia, neurosis, orthopedic diseases, and rheumatoid arthritis. However, its high toxicity, narrow therapeutic window, and short half-life lead to numerous toxic side effects in clinical application, limiting its widespread use.

[0004] Traditional strychnine medicinal dosage forms mainly include pills, tablets, powders, capsules, and ointments. However, these have drawbacks such as low drug utilization, the need for combination therapy due to strong toxic side effects, and limited application, primarily as topical preparations. Existing technologies for preparing strychnine mainly involve water decoction or organic solvent extraction to obtain a single component. These methods suffer from poor water solubility, low bioavailability, and strong toxic side effects. Novel strychnine preparations include liposomes, microemulsions, microcapsules, or chitosan nanoparticles, all of which involve additional excipients encapsulating the monomeric drug. However, these methods suffer from drawbacks such as lack of affinity of the carrier material, premature drug release, and drug resistance in monotherapy. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a multi-component self-assembled strychnine nanoparticle with strong water solubility, high bioavailability, and low toxicity, and also to provide a method for preparing and applying the above-mentioned strychnine nanoparticle.

[0006] Technical solution: This invention discloses a multi-component self-assembled strychnine nanoparticle, wherein the nanoparticle contains hydrophilic components strychnine nitride oxide and strychnine nitride, and hydrophobic components strychnine and strychnine. The hydrophilic and hydrophobic components aggregate with each other to form spherical nanoparticles.

[0007] The strychnine nanoparticles have a particle size of 140-160 nm and include 28-32% strychnine, 2-6% strychnine nitrogen oxides, 55-58% strychnine and 6-10% strychnine nitrogen oxides; specifically, they contain 30.86% strychnine, 4% strychnine nitrogen oxides, 57.14% strychnine and 8% strychnine nitrogen oxides.

[0008] The preparation method of the above-mentioned strychnine nanoparticles includes the following steps: (1) Strychnos nux-vomica is crushed and sieved. The obtained Strychnos nux-vomica powder is mixed with the extraction solvent, heated and stirred, cooled and centrifuged to remove the residue to obtain the supernatant. (2) Dialyze the supernatant, centrifuge the liquid obtained after dialysis to remove residue, and then filter the supernatant through a membrane to obtain multi-component self-assembled nux vomica nanoparticles.

[0009] In step (1), the extraction solvent is dimethyl sulfoxide (DMSO).

[0010] In step (1), the mass ratio of the nux vomica powder to the extraction solvent is 1:12~48.

[0011] In step (1), the heating temperature is 80~90℃; the stirring speed is 600~800rpm and the stirring time is 4~8h.

[0012] In step (2), the dialysis solution is water, and the dialysis time is 36-48 hours.

[0013] In step (2), the filter membrane is a 0.22~0.80µm aqueous filter membrane.

[0014] The present invention also discloses the application of the above-mentioned strychnine nanoparticles in the preparation of antitumor drugs.

[0015] The multi-component self-assembled strychnine nanoparticles play a role in reversing tumor drug resistance; the tumors include breast cancer or melanoma, specifically doxorubicin-resistant breast cancer, doxorubicin-resistant melanoma, or paclitaxel-resistant melanoma.

[0016] Invention Principle: The multi-component self-assembled strychnine nanoparticles of this invention are composed of pure drug molecules. Through the combination of various drug components of strychnine—strychnine, strychnine nitride, strychnine nitride, and strychnine nitride—the hydrophilic and hydrophobic components are cleverly utilized to form self-assembled spherical nanoparticles. No additional carrier is required, effectively solving the disadvantages of using carrier materials, such as premature drug release, carrier toxicity, and low drug loading. At the same time, it can also overcome the disadvantage of drug resistance easily generated by monotherapy. It enters cells through multiple endocytic pathways, consumes ATP, and reduces the efflux of chemotherapy drugs by ABC transporters, thereby playing a role in reversing tumor drug resistance.

[0017] The preparation method of this invention uses DMSO heating and stirring to dissolve the medicinal components, and then directly prepares nanoparticles in one step by cooling and dialysis through a membrane. It does not require complicated extraction and purification processes of monomer components. By heating and stirring the solvent, the active ingredients are effectively dissolved, and the hydrophilic and hydrophobic components combine to form self-assembled nanoparticles with multiple components. The preparation method is simple and low in cost.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Multi-component self-assembled strychnine nanoparticles do not require additional carrier loading, have strong water solubility and high bioavailability; (2) The preparation method is simple, low cost, and suitable for large-scale production; (3) When applied to anti-tumor drugs, it overcomes the disadvantage of easy drug resistance in monotherapy and plays a role in reversing tumor drug resistance. Attached Figure Description

[0019] Figure 1 The figure shows the results of molecular dynamics simulation of strychnine nanoparticles. The red clusters represent strychnine, the green clusters represent strychnine nitrogen oxides, the yellow clusters represent strychnine nitrogen oxides, and the blue clusters represent strychnine nitrogen oxides. Figure 2 The root mean square deviation (A), radius of rotation (B), and solvent-accessible surface area (C) of strychnine nanoparticles are obtained from molecular dynamics simulations. Figure 3 Particle size distribution of Strychnos nux-vomica nanoparticles in different extraction solvents; Figure 4 This is a particle size distribution diagram of strychnine nanoparticles dispersed in water; Figure 5 Transmission electron microscopy image of strychnos nux-vomica nanoparticles; Figure 6 Figure showing the composition and relative content of strychnos nux-vomica nanoparticles; Figure 7 Comparison of the resistance folds of strychnine nanoparticles and doxorubicin to breast cancer (4T1) cell lines after multiple passages of treatment; Figure 8 Figure showing the cytotoxicity of strychnine nanoparticles against common breast cancer cell lines (4T1) and doxorubicin-resistant cell lines (4T1 / DOX); Figure 9 To evaluate the in vivo antitumor effect of strychnine nanoparticles in a breast cancer drug resistance model, where a is a graph showing the change in tumor volume over time in different experimental groups of mice; and b is a graph showing the change in body weight in different experimental groups of mice. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[0021] Example 1

[0022] The multi-component self-assembled strychnine nanoparticles of the present invention have a particle size of 160±20 nm. The preparation of the above-mentioned multi-component self-assembled strychnine nanoparticles includes the following steps: (1) Strychnos nux-vomica powder was crushed and sieved. Strychnos nux-vomica powder and DMSO were mixed in a 1:12 ratio and placed in a round-bottom flask. The mixture was heated and stirred at 90°C and 800 rpm for 4 hours. After cooling, the residue was removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 36 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0023] Molecular dynamics simulations were performed on the synthesized multi-component self-assembled strychnine nanoparticles to analyze the bonding between their components. The simulation results are as follows: Figure 1 As shown, the red clusters represent strychnine, the green clusters represent strychnine nitrogen oxides, the yellow clusters represent strychnine nitrogen oxides, and the blue clusters represent strychnine nitrogen oxides. These clusters undergo gradual internal recombination over time during molecular dynamics simulations, subsequently merging into a large aggregate to form a spherical structure.

[0024] To evaluate the equilibrium properties of the spherical structure, the inventors calculated the root mean square deviation (RMSD), radius of gyration (Rg), and solvent-accessible surface area (SASA) throughout the simulation. The results are as follows: Figure 2 As shown, the RMSD fluctuates between 2.0 and 2.8 nm in the first 25 ns, marking the beginning of the assembly process, before plateauing. Similarly, the Rg value also fluctuates, eventually decreasing to below 2 nm within 25 ns, indicating the equilibrium and stability of the strychnine nanoparticles. SASA represents the available surface area of ​​the hydrophobic region interacting with water. The hydrophobic SASA decreases significantly in the first 25 ns, then remains relatively constant throughout the simulation time, indicating that the hydrophilic and hydrophobic components aggregate to form spherical nanoparticles.

[0025] Example 2

[0026] The multi-component self-assembled strychnine nanoparticles of the present invention have a particle size of 160±20 nm. The preparation of the above-mentioned multi-component self-assembled strychnine nanoparticles, compared with Example 1, involves changing the amount of strychnine powder and DMSO fed, and includes the following steps: (1) Strychnos nux-vomica powder was crushed and sieved. Strychnos nux-vomica powder and DMSO were mixed in a 1:36 ratio and placed in a round-bottom flask. The mixture was heated and stirred at 90°C and 800 rpm for 4 hours. After cooling, the residue was removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 36 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0027] Example 3

[0028] The multi-component self-assembled strychnine nanoparticles of the present invention have a particle size of 160±20 nm. The preparation of the above-mentioned multi-component self-assembled strychnine nanoparticles, compared with Example 1, involves changing the heating and stirring conditions in step (1), and includes the following steps: (1) Strychnos nux-vomica powder is crushed and sieved. Strychnos nux-vomica powder and DMSO are mixed in a 1:12 ratio and placed in a round-bottom flask. The mixture is heated and stirred at 80°C and 800 rpm for 4 hours. After cooling, the residue is removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 36 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0029] Example 4

[0030] The multi-component self-assembled strychnine nanoparticles of the present invention have a particle size of 160±20 nm. The preparation of the above-mentioned multi-component self-assembled strychnine nanoparticles, compared with Example 1, involves changing the dialysis time in step (2), and includes the following steps: (1) Strychnos nux-vomica powder was crushed and sieved. Strychnos nux-vomica powder and DMSO were mixed in a 1:12 ratio and placed in a round-bottom flask. The mixture was heated and stirred at 90°C and 800 rpm for 4 hours. After cooling, the residue was removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 48 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0031] Comparative Example 1

[0032] A type of strychnine nanoparticle with a particle size of 160±20 nm. The preparation of the above strychnine nanoparticles, compared with Example 1, involves replacing the extraction solvent DMSO in step (1) with water, and includes the following steps: (1) Strychnos nux-vomica powder is crushed and sieved. Strychnos nux-vomica powder and water are mixed in a 1:12 ratio and placed in a round-bottom flask. The mixture is heated and stirred at 90°C and 800 rpm for 4 hours. After cooling, the residue is removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 36 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0033] Results of different extraction solvents for Strychnos nux-vomica nanoparticles are as follows: Figure 3 As shown, the nanoparticles with the smallest particle size are obtained when DMSO is used as the extraction solvent.

[0034] Comparative Example 2

[0035] A type of strychnine nanoparticle with a particle size of 160±20 nm. The preparation of the above strychnine nanoparticles, compared with Example 1, involves replacing the extraction solvent DMSO in step (1) with ethanol, and includes the following steps: (1) Strychnos nux-vomica powder was crushed and sieved. Strychnos nux-vomica powder and ethanol were mixed in a 1:12 ratio and placed in a round-bottom flask. The mixture was heated and stirred at 90°C and 800 rpm for 4 hours. After cooling, the residue was removed by centrifugation. (2) Take the supernatant and dialyze it with ultrapure water for 36 hours. Centrifuge the dialysate to remove the residue, and filter the supernatant through a 0.22µm aqueous filter membrane.

[0036] The experimental results of Comparative Example 2 show that, under the same feed ratio, the yield of ethanol preparation is only about one percent of that of DMSO.

[0037] Verification of the effect of the multi-component self-assembled strychnine nanoparticles of the present invention on cell survival: Take cells in the logarithmic growth phase and resuspend them in complete culture medium (8 × 10⁻⁶). 3 Cells (100 μL / well) were seeded into 96-well plates. The plates were incubated overnight at 37°C with 5% CO2. After cell attachment, the culture medium was aspirated. 100 μL of serum-free medium was added to the blank wells, and gradient concentrations of the drug were added to the drug-treated wells. After 72 h, the drug solution was aspirated, and 100 μL of CCK-8 reagent was added. The plates were incubated at 37°C with 5% CO2 for 1 h. The absorbance (A) was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0038] Cell viability = [A 给药 -A 空白 / (A 对照 -A 空白 )]×100% Table 1. Validation of multi-component self-assembled strychnine nanoparticles, doxorubicin, and paclitaxel

[0039] The results are shown in Table 1, where the content of strychnine nanoparticles is defined as the content of strychnine. The data show that the therapeutic effect of strychnine nanoparticles was not affected in treating ordinary cell lines and their corresponding chemotherapy-resistant cell lines. In fact, the therapeutic effect on doxorubicin-resistant melanoma cells and paclitaxel-resistant melanoma cells was superior to that on ordinary cells. These therapeutic effects are attributed to the multi-component nature of strychnine nanoparticles.

[0040] To compare the resistance folds of strychnine nanoparticles and doxorubicin to breast cancer (4T1) cell lines after multiple passages: When the cell density was approximately 60-70%, the culture medium was aspirated from the culture flasks, the cells were washed twice with PBS, and then cultured in drug-containing medium until the cell density reached approximately 90% before passage. The drug concentration was gradually increased in each passage. The concentrations of strychnine nanoparticles (mg / mL) were 0.5, 1, 2, and 4 mg / mL, respectively; the concentrations of doxorubicin (ng / mL) were 50, 100, 150, and 200 ng / mL, respectively.

[0041] Cells from each generation in the logarithmic growth phase were resuspended in complete culture medium (8 × 10⁻⁶). 3 Cells (100 μL / well) were seeded into 96-well plates. The plates were incubated overnight at 37°C with 5% CO2. After cell attachment, the culture medium was aspirated. 100 μL of serum-free medium was added to the blank wells, and gradient concentrations of the drug were added to the drug-treated wells. After 72 h, the drug solution was aspirated, and 100 μL of CCK-8 reagent was added. The plates were incubated at 37°C with 5% CO2 for 1 h. The absorbance (A) was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0042] Cell viability = [A 给药 -A 空白 / (A 对照 -A 空白 )]×100%

[0043] The results are as follows Figure 7 As shown, compared with chemotherapy drugs, strychnine nanoparticles showed significant resistance to breast cancer cells after 11 consecutive generations of treatment, while the chemotherapy drug doxorubicin began to induce resistance from the initial treatment. This is because chemotherapy drugs have a single structure and target, and are easily pumped out of the body by ABC transporters, leading to chemotherapy resistance. Strychnine nanoparticles, due to their multi-component characteristics, can effectively overcome these shortcomings.

[0044] Verification of the cytotoxicity of strychnine nanoparticles against common breast cancer cell lines (4T1) and doxorubicin-resistant cell lines (4T1 / DOX): Take cells in the logarithmic growth phase and resuspend them in complete culture medium (8 × 10⁻⁶). 3 Cells (100 μL / well) were seeded into 96-well plates. The plates were incubated overnight at 37°C with 5% CO2. After cell attachment, the culture medium was aspirated. 100 μL of serum-free medium was added to the blank wells, and strychnine nanoparticles, doxorubicin, and SNP+DOX containing the same concentration of nanoparticles and doxorubicin were added to the drug-treated wells, respectively. After 72 h, the drug solutions were aspirated, 100 μL of CCK-8 reagent was added, and the plates were incubated at 37°C with 5% CO2 for 1 h. The absorbance (A) was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0045] Cell viability = [A 给药 -A 空白 / (A 对照 -A 空白 )]×100%

[0046] The results are as follows Figure 8As shown, treatment with SNPs significantly increased the cytotoxic effect of doxorubicin. This is because SNPs can effectively consume ATP and reduce the pumping of chemotherapeutic drugs by ABC transporters.

[0047] Evaluation of the in vivo antitumor effect of strychnine nanoparticles in a breast cancer drug resistance model: Laboratory animals: Balb / c mice, female, 6-8 weeks old, weighing 20-22 g (1) Prepare 4T1 / DOX cells for modeling. 4T1 / DOX cells are cultured in DMEM medium containing 10% (v / v) FBS and 100 U / mL penicillin-100 μg / mL streptomycin in a carbon dioxide incubator (37℃, 5% CO2). Change the medium every other day. When the cells have grown to 80-90% of the field of view, digest them with 0.25% trypsin and resuspend the cells in fresh medium for cell experiments. (2) On the day of tumor bearing, 4T1 / DOX cells were digested with 0.25% trypsin, centrifuged to collect the cells, counted them with a cell counter, and resuspended in sterile PBS to form 1×10⁻⁶ cells. 7 Prepare a 100 μL / mL cell suspension and keep it on ice. Invert the cells every 3-5 minutes to mix them and prevent cell deposition that could lead to hypoxia and cell death in the lower layers. Shave the fur on the right back of the mouse beforehand, gently invert the cell suspension to mix, and inject 100 μL of the cell suspension subcutaneously into the right side of the mouse using a 1 mL syringe. Approximately two weeks after injection, mice showed significant solid tumor growth. When the average tumor volume reached approximately 50 mm³, the mice were randomly divided into four groups (n=8): 1) Control group: PBS; 2) Chemotherapy group: 0.3 mg / kg / dose; 3) Nanoparticle group: 5 mg / kg / dose; 4) Drug combination group: SNP 5 mg / kg / dose, DOX 0.3 mg / kg / dose. The SNP, DOX, and drug combination groups received intratumoral injections every 3 days for a total of 6 times; the control group received a simultaneous injection of 100 μL PBS. Mouse body weight and tumor size were recorded on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 after grouping. Tumor size was determined by measuring the length (L) and width (W) of the mouse tumor with calipers and calculating the tumor volume using the following formula: .

[0048] The results are as follows Figure 9 As shown, SNPs combined with the chemotherapy drug doxorubicin can effectively inhibit the growth of doxorubicin-resistant breast cancer tumors.

Claims

1. A multi-component self-assembled strychnine nanoparticle, characterized in that, The nanoparticles contain hydrophilic components strychnine nitrogen oxides and strychnine nitrogen oxides, and hydrophobic components strychnine and strychnine. The hydrophilic and hydrophobic components aggregate to form spherical nanoparticles. The nanoparticles are prepared by the following steps: (1) Strychnos nux-vomica is crushed and sieved. The obtained Strychnos nux-vomica powder is mixed with dimethyl sulfoxide at a mass ratio of 1:12-48. The mixture is heated and stirred at 80-90℃. After cooling, the residue is removed by centrifugation to obtain the supernatant. (2) Dialyze the supernatant. The dialysate is water. After dialysis, centrifuge the liquid to remove the residue, and then filter the supernatant through a membrane to obtain multi-component self-assembled strychnine nanoparticles.

2. The strychnine nanoparticles according to claim 1, characterized in that, The strychnine nanoparticles have a particle size of 140-160 nm and include 28-32% strychnine, 2-6% strychnine nitrogen oxides, 55-58% strychnine and 6-10% strychnine nitrogen oxides.

3. The strychnine nanoparticles according to claim 1, characterized in that, In step (1), the stirring speed is 600-800 rpm and the stirring time is 4-8 h.

4. The strychnine nanoparticles according to claim 1, characterized in that, In step (2), the dialysis time is 36 to 48 hours.

5. The strychnine nanoparticles according to claim 1, characterized in that, In step (2), the filter membrane is a 0.22-0.80µm aqueous filter membrane.

6. The application of the strychnine nanoparticles according to claim 1 in the preparation of antitumor drugs, characterized in that, The tumor is either breast cancer or melanoma.

7. The application according to claim 6, characterized in that, The multi-component self-assembled strychnine nanoparticle drug exerts an effect of reversing tumor drug resistance, wherein the tumor is breast cancer.

Images