Preparation method and application of 10-hydroxycamptothecine ginger vesicle nano preparation

Abstract

The present application takes ginger as raw material, and obtains natural vesicles (Ginger Vesicles, GV) from ginger by gradient centrifugation combined with pH regulation extraction and purification, the average particle size of which is 86.83±6.29nm, has a typical phospholipid bilayer structure, and is rich in 6-gingerol active ingredients. The ginger vesicles are used as carriers, 10-hydroxycamptothecine (HCPT) is loaded by co-incubation method, and the drug loading efficiency is optimized by introducing Ca²⁺ to prepare GV-HCPT nano-preparation, the drug loading rate of which reaches 21.32±0.38%, and the encapsulation rate reaches 80.26%. The nano-preparation has pH-responsive slow-release characteristics, the release rate is significantly accelerated under acidic conditions, and can effectively enrich in tumor tissues. In vitro experiments show that the preparation has a significant proliferation inhibitory effect on breast cancer cells 4T1 and colon cancer cells CT26; in vivo experiments show that the preparation has an inhibition rate of 50.28±2.54% on the transplanted tumor of breast cancer in mice, and has good biocompatibility, and can reduce the toxic side effects of HCPT. The present application provides a new solution for the targeted delivery of hydrophobic antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine formulation technology, specifically to a 10-hydroxycamptothecin ginger vesicle nanoformation, its preparation method, and its application. Background Technology

[0002] 10-hydroxycamptothecin (HCPT) is a natural alkaloid derived from the fruit, roots, and bark of the camptothecin tree (Campanula spp.), a member of the Davidia involucrata family. It is a topoisomerase I inhibitor, specifically acting on topoisomerase I to inhibit DNA synthesis and replication, prevent tumor cell proliferation and division, and induce tumor cell apoptosis. It exhibits significant inhibitory effects on various tumor cells, including liver cancer, gastric cancer, colon cancer, breast cancer, and bladder cancer. However, HCPT has poor water solubility and rapid metabolism in vivo, greatly limiting its full anti-tumor efficacy. Clinically, HCPT is often administered via intravenous injection or perfusion, frequently accompanied by adverse reactions such as hematologic toxicity (bone marrow suppression phenomena such as leukopenia and thrombocytopenia), gastrointestinal reactions (nausea, vomiting, diarrhea, etc.), and urinary tract irritation.

[0003] In recent years, the rapid development of nanotechnology has brought new hope for improving drug efficacy and delivery. Nanoparticles, liposomes, polymeric micelles, and other nanocarrier-based drug delivery systems have played an important role in precision drug delivery and treatment of tumors. However, these synthetic materials generally have certain toxic side effects and are easily recognized and cleared by the phagocytic cell system after entering the human body, limiting their application.

[0004] Plant-derived vesicles are tiny sac-like structures secreted by plant cells, containing a phospholipid layer. They typically range in size from 50 to 500 nm and carry bioactive molecules such as proteins, lipids, and nucleic acids, exhibiting good biocompatibility and low immunogenicity. Ginger (Zingiber officinale), as both a food and a medicinal plant, is commonly used in medicine to alleviate digestive tract diseases, control inflammatory responses, scavenge free radicals, and regulate immune function. Natural vesicles extracted from ginger not only possess good biocompatibility but also exhibit various biological activities, making them an important advantage as drug delivery carriers. However, there are currently no reports on using ginger vesicles as carriers to load 10-hydroxycamptothecin. Summary of the Invention

[0005] Purpose of the invention: This invention addresses the technical problems of poor water solubility, low bioavailability, and high toxicity of HCPT in the prior art by providing a 10-hydroxycamptothecin ginger vesicle nanoformation, its preparation method, and its application.

[0006] Technical solution:

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a 10-hydroxycamptothecin ginger vesicle nanoformation, comprising natural vesicles derived from ginger and 10-hydroxycamptothecin loaded within the natural vesicles derived from ginger.

[0009] Preferably, the ginger-derived natural vesicles have a phospholipid bilayer structure, an average particle size of 80–120 nm, and a zeta potential of -30–-20 mV.

[0010] Preferably, the ginger-derived natural vesicles are rich in the active ingredient 6-shogaol.

[0011] Preferably, the drug loading rate of the nano-formulation is 15% to 25%, and the encapsulation rate is 70% to 85%.

[0012] Preferably, the average particle size of the nano-formulation is 100–120 nm.

[0013] Secondly, this invention provides a method for preparing a 10-hydroxycamptothecin-based ginger vesicle nanoparticle formulation, comprising the following steps:

[0014] (1) Extraction of ginger vesicles: After washing and cutting ginger into pieces, crush it and add pre-cooled phosphate buffer. After filtering through gauze, centrifuge at 4000g, 8000g, 12000g and 16000g in sequence at 4℃. Collect the precipitate, resuspend it and add polyethylene glycol 6000 for precipitation. Collect the precipitate to obtain crude ginger vesicles.

[0015] (2) Purification of ginger vesicles: The crude ginger vesicles obtained in step (1) were placed in a sucrose density gradient medium for ultracentrifugation purification, and the purified ginger vesicles were collected.

[0016] (3) Loading of 10-hydroxycamptothecin: The purified ginger vesicles obtained in step (2) are mixed with 10-hydroxycamptothecin at a mass ratio of 20 to 30:1 and incubated at 35 to 40°C for 10 to 14 hours. The precipitate is collected by centrifugation to obtain 10-hydroxycamptothecin ginger vesicle nanoformulation.

[0017] Preferably, in step (1), the pH of the supernatant is adjusted to 4-5 before adding polyethylene glycol 6000 to improve the vesicle yield.

[0018] Preferably, in step (2), the sucrose density gradient medium is a sucrose solution of 10%, 15%, 30% and 50%.

[0019] Preferably, in step (3), 0.05-0.15M of Ca²⁺ is added to improve drug loading efficiency.

[0020] More preferably, in step (3), the mass ratio of ginger vesicles to 10-hydroxycamptothecin is 25:1, the incubation temperature is 37°C, the incubation time is 12 hours, and the Ca²⁺ concentration is 0.1M.

[0021] Thirdly, the present invention provides the application of the above-mentioned 10-hydroxycamptothecin ginger vesicle nanoformation in the preparation of antitumor drugs.

[0022] Preferably, the tumor is breast cancer or colon cancer.

[0023] Beneficial effects:

[0024] (1) This invention is the first to use natural ginger vesicles as a carrier to load 10-hydroxycamptothecin, making full use of the advantages of ginger vesicles being naturally sourced, having good biocompatibility, low immunogenicity, and being able to be prepared on a large scale.

[0025] (2) By optimizing the extraction pH conditions (pH 4-5), the present invention significantly increases the vesicle yield from 2.4 g / kg under neutral conditions to 12.5 g / kg, greatly improving the feasibility of large-scale production.

[0026] (3) By optimizing the drug loading conditions (GV:HCPT=25:1, incubation at 37℃ for 12h, introduction of 0.1M Ca²⁺), the present invention achieves a drug loading rate of 21.32±0.38% and an encapsulation rate of 80.26%, which significantly improves the drug loading efficiency.

[0027] (4) The GV-HCPT nano-formulation prepared by the present invention has pH-responsive sustained-release characteristics. Under acidic conditions (simulating the tumor microenvironment), the release rate reaches 60% in 24 hours, which can effectively maintain a high drug concentration. Under normal physiological conditions (pH 7.2), the release rate is less than 50% in 48 hours, and the release is slower, which is beneficial to reduce the toxicity of HCPT to normal tissues.

[0028] (5) In vitro experiments showed that GV-HCPT inhibited 4T1 breast cancer cells and CT26 colon cancer cells by 57% and 44.32%, respectively, which was significantly better than HCPT alone (17.69% and 26.93%).

[0029] (6) In vivo experiments showed that GV-HCPT inhibited mouse breast cancer xenografts by 50.28±2.54%, which was significantly better than HCPT alone (36.33±3.25%). Furthermore, histopathological examination revealed no significant toxicity in major organs, indicating good biocompatibility. Attached Figure Description

[0030] Figure 1 Schematic diagram of ginger vesicle extraction process

[0031] Figure 2 Transmission electron microscopy image of ginger vesicles

[0032] Figure 3 Particle size distribution of ginger vesicles

[0033] Figure 4 Graph showing changes in ginger vesicle yield and potential under different pH conditions

[0034] Figure 5 Transmission electron microscope images of GV-HCPT nanoparticles

[0035] Figure 6 Particle size distribution and zeta potential of GV-HCPT nanoparticles

[0036] Figure 7 In vitro sustained-release curves of GV-HCPT under different pH conditions

[0037] Figure 8 Figure 1 shows the toxicity test results of GV-HCPT on 4T1 breast cancer cells (a) and CT26 colon cancer cells (b).

[0038] Figure 9 Image showing the tumor-suppressing effect of GV-HCPT on mouse breast cancer xenografts. Detailed Implementation

[0039] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

[0040] Example 1: Extraction and purification of ginger vesicles

[0041] Thoroughly wash fresh ginger with deionized water, weigh it, cut it into pieces, and place it in a clean blender. Add pre-prepared pre-cooled phosphate buffer (PBS) until the ginger is completely submerged. Select the cold-press mode, blend for 1 minute, pause for 1 minute, and repeat five times to obtain ginger juice. Filter the extracted ginger juice through gauze to remove the residue. Pour the filtrate into centrifuge tubes and centrifuge at 4000g for 60 minutes at 4°C, collecting the supernatant. Further centrifuge the supernatant at 8000g and 12000g for 60 minutes each at 4°C, collecting the supernatant each time. Finally, centrifuge the obtained supernatant at 16000g for 60 minutes at 4°C, collecting the precipitate. Resuspend the precipitate in PBS solution, adjust the pH of the supernatant to 4-5, add 10% polyethylene glycol 6000 (PEG6000), and centrifuge at 16000g for 60 minutes at 4°C, collecting the precipitate. The precipitate was resuspended in PBS solution, and the pH was adjusted to around 7 to obtain a crude vesicle extract solution.

[0042] Sucrose was dissolved in PBS to prepare sucrose solutions with concentrations of 10%, 15%, 30%, and 50%, respectively. After pre-cooling, the sucrose solutions were added sequentially to ultracentrifuge tubes, and crude vesicle extract solution was added at the same time to balance the solution. The tubes were then placed in an ultracentrifuge at 20,000g for 1 hour. The supernatant was discarded, and the bottom precipitate was resuspended in PBS and filtered through a 0.45μm filter membrane to obtain the purified ginger vesicle solution.

[0043] Transmission electron microscopy revealed that the obtained ginger vesicles possessed a typical phospholipid bilayer structure, exhibiting spherical or near-spherical shapes with intact and smooth vesicle membranes. Nanoparticle size analysis showed an average particle size of 86.83 ± 6.29 nm and a zeta potential of -25 ± 3.28 mV. High-performance liquid chromatography-mass spectrometry analysis indicated that the vesicles were rich in the active ingredient 6-shogaol.

[0044] Example 2: Effect of different pH conditions on vesicle yield

[0045] Before PEG precipitation, the pH of the supernatant was adjusted to 4, 5, 6, 7, 8, and 9, respectively. The supernatant was then centrifuged at 16000g for 60 min at 4°C, and the precipitates were collected and accurately weighed. The results showed that the vesicle yield was highest at pH 4–5, reaching 12.5 g / kg, while the yield was only 2.4 g / kg under neutral conditions (pH 7). This indicates that acidic conditions (pH 4–5) can significantly improve the extraction efficiency of ginger vesicles.

[0046] Example 3: Stability Study of Ginger Vesicles

[0047] Ginger vesicle samples were stored under freeze-drying, -80℃, -20℃, 4℃, and room temperature conditions, respectively. Samples were taken at 2, 4, 6, 8, 10, and 12 hours, and particle size was measured using a laser particle size analyzer. The results showed that the particle size change of vesicles under freeze-drying and 4℃ refrigeration conditions was relatively gradual, indicating good stability. Freeze-dried vesicles were more uniformly dispersed in the system, confirming freeze-drying as the optimal storage method. A 10% trehalose aqueous solution was added as a freeze-drying protectant during freeze-drying.

[0048] Ginger vesicles were added to simulated gastric and intestinal fluids preheated to 37°C, and samples were taken at 0, 30 min, 1 h, 2 h, and 4 h to determine particle size. The results showed that the particle size of the vesicles in the gastrointestinal fluids varied little, indicating that the vesicles have good stability in the gastrointestinal fluids.

[0049] Example 4: Establishment of the HCPT Standard Curve

[0050] Accurately weigh 3.2 mg of HCPT into a 10 mL volumetric flask, and dilute to 10 mL with chromatographic grade DMSO. Dissolve by sonication. Transfer 1 mL of HCPT solution into a 10 mL volumetric flask and dilute to 10 mL with methanol. Dilute using the two-fold dilution method to obtain standard solutions of 32, 16, 8, 4, and 2 μg / mL. Detection was performed using high-performance liquid chromatography (HPLC) under the following conditions: C18 column (5 μm, 4.6 mm × 250 mm), mobile phase: methanol:water = 55:45 (V / V) isocratic elution for 10 min, column temperature: 25 °C, detection wavelength: 265 nm, flow rate: 1.0 mL / min, injection volume: 20 μL. A standard curve was plotted with the peak area integral value Y as the ordinate and the sample concentration X (μg / mL) as the abscissa. The regression equation was Y=163892291.6124X-4586.3451 (R²=0.999), showing good linearity in the range of 2 to 32 μg / mL.

[0051] Example 5: Preparation and Optimization of Drug Loading Conditions for GV-HCPT Nanoformulations

[0052] The freeze-dried ginger vesicles were diluted with PBS to a concentration of 500 μg / mL. HCPT was accurately weighed, dissolved in DMSO, and added to GV solution. The mixture was incubated in a 37°C water bath with shaking for 12 h. The precipitate was collected by centrifugation, and the concentration of free HCPT in the supernatant was determined by HPLC. The drug loading and encapsulation efficiency were calculated.

[0053] (1) Effect of different drug-to-material ratios on drug loading: GV:HCPT ratios of 1:1, 10:1, 15:1, 20:1, 25:1, and 30:1 were set, and the mixture was incubated at 37°C for 12 hours. The results showed that the highest drug loading rate (17.65%) and encapsulation efficiency (79.20%) were achieved when the GV:HCPT ratio was 25:1.

[0054] (2) Effect of different incubation temperatures on drug loading: Under the condition of GV:HCPT=25:1, the drugs were incubated at 4℃, 20℃, and 37℃ for 12h. The results showed that the drug loading rate was the highest at 37℃, reaching 17.28%, and the encapsulation rate was 75.22%.

[0055] (3) Effect of different incubation times on drug loading: Under the conditions of GV:HCPT=25:1 and 37℃, the drugs were incubated for 2, 4, 6, 8, 10 and 12 hours, respectively. The results showed that the drug loading rate was 4.38% after 12 hours and decreased to 2.64% after 24 hours, thus determining 12 hours as the optimal incubation time.

[0056] (4) Effect of Ca²⁺ on drug loading: Under the above optimal conditions (GV:HCPT=25:1, 37℃, 12h), 0.1M, 0.2M, and 0.3M Ca²⁺ were added respectively. The results showed that the drug loading rate was the highest under the 0.1M Ca²⁺ condition, reaching 21.32±0.38%, and the encapsulation rate was 80.26%.

[0057] Transmission electron microscopy revealed that GV-HCPT exhibited a spherical structure with a slightly increased particle size and a significantly increased internal electron density. The average particle size was measured to be 110 ± 3.28 nm using a nanoparticle size potentiometer. UV-Vis absorption spectroscopy showed characteristic absorption peaks at both 216 nm (GV characteristic peak) and 378 nm (HCPT characteristic peak), confirming successful HCPT loading.

[0058] Example 6: In vitro release experiment of GV-HCPT

[0059] Release media with pH values ​​of 2.0, 7.2, and 8.0 were prepared. HCPT and GV-HCPT at concentrations of 20 μg / mL were placed in dialysis bags (MW=8000), immersed in 100 mL of release media, and dialyzed at 37 °C and 180 rpm. 1 mL samples were taken at predetermined time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 96 h) for HPLC analysis, and an equal volume of fresh media was added after each analysis.

[0060] The results showed that GV-HCPT achieved a release rate of 60% within 24 hours at pH 2.0; the release rate was less than 50% after 48 hours at pH 7.2; and the release was even slower at pH 8.0. This indicates that GV-HCPT exhibits significant pH-responsive release characteristics, with a significantly accelerated release rate under acidic conditions (simulating the tumor microenvironment), which is beneficial for drug accumulation at the tumor site.

[0061] Example 7: In vitro cytotoxicity assay of GV-HCPT

[0062] The toxicity of GV-HCPT to 3T3 mouse embryonic cells, 4T1 breast cancer cells and CT26 colon cancer cells was detected by the CCK8 assay.

[0063] 3T3 cells were seeded in 96-well plates and cultured for 24 h. Different concentrations (5, 10, 15, 20, 25 μg / mL) of GV were then added, and incubation continued for another 24 h. After incubation with CCK-8 for 4 h, absorbance at 450 nm was measured. The results showed that GV at a concentration of 25 μg / mL maintained a cell viability of over 95% for 3T3 cells, indicating that GV has good biocompatibility.

[0064] 4T1 and CT26 cells were seeded into 96-well plates and cultured for 24 h. Different concentrations (3.125, 6.25, and 12.5 μM) of GV-HCPT and free HCPT were then added, and incubation continued for another 24 h. Cell viability was assessed using the CCK-8 assay. The results showed that at 12.5 μM, GV-HCPT inhibited 4T1 cells by 57 ± 3.35%, significantly higher than the 17 ± 3.26% inhibition rate of free HCPT; and inhibited CT26 cells by 54.32 ± 3.38%, significantly higher than the 26 ± 2.35% inhibition rate of free HCPT.

[0065] Example 8: Cellular uptake assay of GV-HCPT

[0066] 4T1 cells were seeded in 24-well plates and cultured for 24 h. Different concentrations (3.125, 6.25, 12.5 μM) of GV-HCPT and free HCPT were then added and incubated for another 24 h. Cells were then fixed and observed using a high-content cell imaging system. The results showed that the fluorescence intensity in the GV-HCPT-treated group was significantly higher than that in the free HCPT-treated group, indicating that GV can significantly enhance the uptake of HCPT by tumor cells.

[0067] Example 9: Apoptosis detection using GV-HCPT

[0068] 4T1 and CT26 cells were seeded into 6-well plates and cultured until 70% confluence. Then, GV, HCPT, and GV-HCPT (12.5 μM) were added for 24 h, respectively. Cells were collected, stained with Annexin V-FITC / PI double staining, and the apoptosis rate was detected by flow cytometry.

[0069] The results showed that the apoptosis rate of 4T1 cells in the GV-HCPT treatment group was 55.26±3.28%, which was significantly higher than that in the free HCPT group (17±3.26%); the apoptosis rate of CT26 cells in the GV-HCPT treatment group was 52.3±2.43%, which was significantly higher than that in the free HCPT group (26±2.35%).

[0070] Example 10: Evaluation of the in vivo antitumor activity of GV-HCPT

[0071] Thirty-five female 4-6 week old BALB / c mice were randomly divided into 5 groups (n=6 per group, with the remaining mice serving as a control group or as backup): negative control group (saline), GV group (200 mg / kg), HCPT group (100 mg / kg), GV-HCPT group (administered as described above), and CPT-11 positive control group (100 mg / kg). The number of 4T1 cells in the logarithmic growth phase was adjusted to 1 × 10⁻⁶. 6 100 μL of cell suspension was subcutaneously injected into the fat pad of the second pair of mammary glands on the right side of mice. After one week, once tumors had grown, drug administration began, and mouse weight and tumor size were recorded every 3 days. Tumor volume was calculated using the formula V = π / 6 × (a × b²) (where a is tumor length and b is tumor width). After drug administration, the mice were sacrificed, and the tumors and vital organs such as the heart, liver, spleen, lungs, and kidneys were isolated for H&E staining pathological examination and hematological analysis.

[0072] The results showed that the tumor inhibition rate in the GV-HCPT group was 50.28±2.54%, significantly better than that in the HCPT monotherapy group (36.33±3.25%). The GV monotherapy group also showed a certain tumor-suppressing effect (24.25±2.33%). H&E staining results showed no obvious toxic reactions in the major organs of mice in the GV-HCPT group, including the heart, liver, spleen, lungs, and kidneys. Hematological analysis showed an increase in lymphocyte count in the GV-HCPT group, suggesting that GV may have an immunoprotective effect and can alleviate the bone marrow toxicity induced by HCPT.

Claims

1. A 10-hydroxycamptothecin-based ginger vesicle nanoformation, characterized in that, Includes ginger-derived natural vesicles and 10-hydroxycamptothecin loaded within the ginger-derived natural vesicles; The natural vesicles derived from ginger have a phospholipid bilayer structure, with an average particle size of 80-120 nm and a ζ-potential of -30 to -20 mV. The drug loading rate of the nano-formulation is 15%–25%, and the encapsulation rate is 70%–85%.

2. The 10-hydroxycamptothecin ginger vesicle nanoformation according to claim 1, characterized in that, The ginger is derived from natural vesicles and is rich in the active ingredient 6-shogaol.

3. The 10-hydroxycamptothecin ginger vesicle nanoformation according to claim 1, characterized in that, The average particle size of the nano-formulation is 100–120 nm.

4. A method for preparing a 10-hydroxycamptothecin ginger vesicle nanoformation as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Extraction of ginger vesicles: After washing and cutting ginger into pieces, crush it and add pre-cooled phosphate buffer. After filtering through gauze, centrifuge at 4000g, 8000g, 12000g and 16000g in sequence at 4℃. Collect the precipitate, resuspend it and add polyethylene glycol 6000 for precipitation. Collect the precipitate to obtain crude ginger vesicles. (2) Purification of ginger vesicles: The crude ginger vesicles obtained in step (1) were placed in a sucrose density gradient medium for ultracentrifugation purification, and the purified ginger vesicles were collected. (3) Loading of 10-hydroxycamptothecin: The purified ginger vesicles obtained in step (2) are mixed with 10-hydroxycamptothecin at a mass ratio of 20 to 30:1 and incubated at 35 to 40°C for 10 to 14 hours. The precipitate is collected by centrifugation to obtain 10-hydroxycamptothecin ginger vesicle nanoformulation.

5. The preparation method according to claim 4, characterized in that, In step (1), the pH of the supernatant is adjusted to 4-5 before adding polyethylene glycol 6000.

6. The preparation method according to claim 4, characterized in that, In step (2), the sucrose density gradient medium is a sucrose solution of 10%, 15%, 30% and 50%.

7. The preparation method according to claim 4, characterized in that, In step (3), 0.05-0.15M Ca²⁺ is added to improve drug loading efficiency.

8. The preparation method according to claim 4, characterized in that, In step (3), the mass ratio of ginger vesicles to 10-hydroxycamptothecin is 25:1, the incubation temperature is 37°C, the incubation time is 12 hours, and the Ca²⁺ concentration is 0.1M.

9. The use of the 10-hydroxycamptothecin ginger vesicle nanoformation as described in any one of claims 1 to 3 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is either breast cancer or colon cancer.

Images