A nanometer preparation of bufalin for treating colon cancer and a preparation method thereof

Abstract

The application discloses a nanometer preparation of bufonin for treating colon cancer, which has a core-shell structure, wherein the inner core is a nanocrystal composed of bufonin and a stabilizer, and the outer shell is a pH-responsive material and a pore-forming agent; the nanometer preparation of bufonin for treating colon cancer is prepared by using the stabilizer, the bufonin and the pH-responsive material; the in-vitro dissolution characteristics of the poorly soluble drug bufonin are improved by nanometerization treatment, and the dissolution characteristics are obviously improved compared with the raw drug; the pH responsiveness of the Eudragit L-100 protects the bufadienolides in the bufonin from being destroyed by the strong acidic condition of gastric juice, thereby improving the stability and effectiveness of the drug, reducing the initial release of bufalin, cinobufagin and resibufogenin in the stomach, making the drug target the intestinal site, making the three kinds of bufonin components achieve effective and rapid synergistic release in the intestinal site, increasing the utilization rate of the drug in the intestinal tract, and being beneficial to the treatment of colon cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically to a toad venom ligand nano-formulation for treating colon cancer and its preparation method. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common cancers worldwide, with a poor prognosis and high mortality rate after metastasis to lymph nodes or distant organs. Chemotherapy is currently the most common treatment for colorectal cancer; however, traditional single-drug chemotherapy has a single target, low bioavailability, and is prone to drug resistance and severe toxic side effects. Currently, combination therapy with two or more drugs is commonly used in clinical practice. However, due to factors such as drug targets, physicochemical properties, and absorption and metabolism, very few paired drugs (4-10%) produce synergistic effects in combination chemotherapy, resulting in a severe lack of combination therapy regimens. Traditional Chinese medicine (TCM) has unique advantages in treating tumors due to its multi-component, multi-target, and multi-pathway characteristics. Multi-component TCM therapy for colorectal cancer has become a hot topic of research both domestically and internationally.

[0003] Toad venom, a traditional Chinese medicine, has been used to treat tumors in my country for nearly a thousand years. Modern research suggests that the anticancer activity of toad venom is achieved through the synergistic effect of traditional Chinese medicine in supporting the body's resistance (regulating immune function) and expelling pathogenic factors (inhibiting tumor cells). Bufotoxin is a natural cardiac steroid isolated from the traditional Chinese medicine toad venom, possessing high antitumor activity, such as against human astrocytoma, lung cancer, and gastric cancer. Among them, bufotoxin, bufotoxin glycoside, and bufotoxin liposide are three active components with strong antitumor activity and high content. However, bufotoxin components belong to the steroid class of compounds, possessing acetyl and ether bonds, making them poorly soluble in water; furthermore, bufotoxin liposide is prone to hydrolysis under acidic gastric conditions, leading to a decrease in its activity. Therefore, the current oral route cannot effectively deliver all three components to the colon tumor site, which significantly limits the multi-component synergistic antitumor effect of bufotoxin and restricts its further application.

[0004] With the development of nanotechnology, nanocrystals can effectively improve the dissolution rate and saturation solubility of poorly soluble drugs, thereby improving their oral absorption and achieving higher bioavailability compared to other formulations. However, the small size and large surface area of ​​drug nanocrystals can easily lead to premature drug leakage in the stomach or even digestive degradation, resulting in reduced drug concentration in the intestines, affecting therapeutic efficacy, and causing adverse side effects. Therefore, preventing the rapid dissolution of drug nanocrystals in the upper gastrointestinal tract, avoiding premature drug release, and maximizing drug contact with intestinal tissues to promote release in the proximal colon are problems that must be addressed. Summary of the Invention

[0005] The present invention addresses the aforementioned problems by providing a bufotoxin nanoparticle that stabilizes bufotoxin, bufotoxin lipotoxin, and bufotoxin bufotoxin in a gastric acid environment, targets the colon, and synergistically releases the three bufotoxin components, thereby improving their bioavailability and treating colon cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A bufotoxin-based nano-formulation for treating colon cancer, characterized in that the nano-formulation has a core-shell structure, with the core being nanocrystals composed of bufotoxin ligands and stabilizers, and the shell being a pH-responsive material.

[0008] The mass ratio of the bufotoxin ligand, stabilizer, pH-responsive material and porogen is (6-12):(1-8):(6-12):1.

[0009] Preferably, the mass ratio of the bufotoxin ligand, stabilizer, pH-responsive material and porogen is (8-11):(2-6):(8-11):1.

[0010] The nano-formulation has a toad venom ligand loading of 1% to 30% and an encapsulation rate of 30% to 96%; the nano-formulation has a particle size range of 50 to 500 nm and a zeta potential of -40 to +54 mV.

[0011] The bufotoxin ligand is an amorphous or microcrystalline mixture with a purity of 32-98%, consisting of 10-25% bufotoxin, 20-35% bufotoxin ligand and 10-70% bufotoxin ligand.

[0012] The stabilizer is one or more of chitosan quaternary ammonium salt, sodium dodecyl sulfate, poloxamer 407, soybean lecithin, chitosan, and sodium alginate; preferably, the stabilizer is chitosan quaternary ammonium salt, and its stabilizer mass-volume concentration is 0.01% to 10%.

[0013] The pH-responsive material is one or more of Eutec L-100, hydroxypropyl methylcellulose phthalate HP50, hydroxypropyl methylcellulose acetate succinate, and Kollicoat MAE100P; preferably, the pH-responsive material is Eutec L-100, and its mass-volume concentration is 0.01% to 20%.

[0014] The porogen is one or more of sorbitan oleate, polyethylene glycol 400, polyethylene glycol 4000, and polysorbate 80; preferably, the porogen is sorbitan oleate with a mass-volume concentration of 0.01-1.0%.

[0015] The bufotoxin ligand nano-formulation for treating colon cancer encapsulates pH-responsive materials on the surface of bufotoxin ligand nanocrystals through electrostatic interactions.

[0016] The bufotoxin ligand nano-formulation for treating colon cancer is pH-responsive, with a pH response range of pH > 6.

[0017] In the transfer and release media at pH 1.2 (0-2h) and pH 6.8 (2-6h), the in vitro release of bufotoxin, bufotoxin ligand, and bufotoxin ligand reached synchronous release. The cumulative release within 2h was <10%, the release within 4h was 30%-60%, and the cumulative release within 6h was 70%-90%.

[0018] The bufotoxin nano-formulation for treating colon cancer can prevent bufotoxin, especially lipofuscin, from being destroyed by the acidic environment of the stomach, thereby achieving the synergistic release of bufotoxin, bufotoxin, and lipofuscin in the intestinal tract.

[0019] The bufotoxin nano-formulation for treating colon cancer has a tumor inhibition rate of 40-70%, which is 1.5-4.5 times higher than that of bufotoxin nanocrystals and 3-6 times higher than that of bufotoxin crude suspension, demonstrating good therapeutic effects for colon cancer.

[0020] The bufotoxin nano-formulation for treating colon cancer showed significantly improved bioavailability compared to crude suspensions of bufotoxin, bufotoxin, and bufotoxin. The area under the pharmacokinetic curve (AUC) was increased by 5.0–7.0, 2.2–4.5, and 2.5–4.5 times, respectively, while the maximum plasma concentration was increased by 5.8–10.5, 4.6–7.5, and 10.2–15.0 times, respectively.

[0021] The toad venom ligand nanoparticles for treating colon cancer induce tumor cell apoptosis in colon cancer through a mitochondrial-dependent pathway. Furthermore, they induce tumor cell apoptosis by upregulating tumor cell ROS levels, depolarizing tumor cell mitochondrial membrane potential, upregulating the expression of Bax and Csapse-3, and inhibiting the expression of Bcl-2.

[0022] The preparation method of the above-mentioned toad venom ligand nano-formulation for treating colon cancer is as follows:

[0023] The nanocrystals were prepared by antisolvent precipitation-ultrasonic method;

[0024] The nano-formulation was prepared by electrostatic self-assembly of the outer shell and the core.

[0025] The preparation method of the above-mentioned toad venom ligand nano-formulation for treating colon cancer is characterized by the following process steps:

[0026] 1) Dissolve bufotoxin ligand, pH-responsive material and porogen in an organic solvent to prepare a bufotoxin ligand drug solution;

[0027] 2) Dissolve the stabilizer in an aqueous medium to prepare a stabilizer solution;

[0028] 3) Under the condition of probe ultrasound, add the drug solution to the stabilizer solution;

[0029] 4) The organic solvent in the mixed solution obtained in step 3) is evaporated under magnetic stirring to obtain pH-responsive bufotoxin nanocrystals.

[0030] The pH-responsive material is Utec L-100, with a mass-volume concentration of 0.01–5%; the porogen is sorbitan oleate, with a mass-volume concentration of 0.01–0.5%; and the organic solvent is one or more of methanol, ethanol, acetonitrile, and chloroform.

[0031] Preferably, the organic solvent is methanol.

[0032] The stabilizer is a chitosan quaternary ammonium salt, and the aqueous medium is purified water or PBS solution; the concentration of the stabilizer solution is 0.01-5%.

[0033] The preferred aqueous medium is purified water, and the stabilizer solution concentration is 0.01–2%.

[0034] The rate at which the bufotoxin ligand drug solution is added to the stabilizer solution is (1 ml / s) to (1 ml / 2 min).

[0035] Preferably, the rate at which the bufotoxin ligand drug solution is added to the stabilizer solution is 1 ml / s to 3 ml / min.

[0036] The probe has an ultrasonic power of 5-80%; an ultrasonic temperature of 0℃, 4℃, or 25℃; and an ultrasonic time of 5-20 minutes.

[0037] Preferably, the ultrasonic power of the probe is 50% to 70%.

[0038] Preferably, the ultrasonic temperature of the probe is 0°C.

[0039] Preferably, the ultrasound time is 8-14 minutes.

[0040] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. This invention provides bufotoxin nanocrystals for treating colon cancer, prepared using stabilizers, bufotoxin ligands, and pH-responsive materials. Nano-sizing improves the in vitro dissolution characteristics of the poorly soluble bufotoxin ligand, resulting in a significant improvement compared to the raw material. The pH-responsiveness of Eucerin L-100 protects the lipofobufotoxin ligand within the bufotoxin ligand from the strong acidity of gastric juice, thereby enhancing drug stability and efficacy. Furthermore, it reduces the initial release of the three components (bufotoxin, bufotoxin ligand, and lipofobufotoxin ligand) in the stomach, allowing the drug to target the intestinal tract and achieve effective and rapid synergistic release of the three bufotoxin ligand components in the intestinal tract, increasing drug utilization and benefiting colon cancer treatment.

[0042] 2. The chitosan quaternary ammonium salt used as a stabilizer in this invention is a basic, positively charged polysaccharide with good biocompatibility and adhesion. Its positive charge can interact with the negative charge of the intestinal mucus layer, which helps to increase the retention time of the drug in the mucus layer. Therefore, after the three components of the bufotoxin ligand are synergistically released in the intestine, the retention time in the intestine is increased. At the same time, the positively charged chitosan quaternary ammonium salt can also interact with the negatively charged mucin in the colonic mucosa, thereby increasing the adhesion and aggregation of bufotoxin ligand nanocrystals in colonic tumor tissue, exhibiting a certain degree of colonic targeting. Furthermore, the pH-responsive bufotoxin ligand nanoparticles have a particle size between 50 and 500 nm, which can easily penetrate the cell membrane of intestinal epithelial cells and the network and porous structure of the mucus layer, which is conducive to the absorption of bufotoxin ligand in the intestine and beneficial to the treatment of colon cancer. Pharmacokinetic experiments show that the bioavailability of bufotoxin ligand is significantly improved, providing a good foundation for the treatment of colon cancer.

[0043] 3. This invention, through cell and animal models, validated that pH-responsive bufotoxin nanoparticles can more effectively treat colon cancer, with significantly better results than bufotoxin nanocrystals and crude bufotoxin suspensions. The nanoparticles induce tumor cell apoptosis in colon cancer via a mitochondrial-dependent pathway; furthermore, they induce tumor cell apoptosis by upregulating tumor cell ROS levels, depolarizing tumor cell mitochondrial membrane potential, upregulating Bax and Csapse-3 expression, and inhibiting Bcl-2 expression. Therefore, they have great potential in the clinical anti-tumor field.

[0044] 4. Other advantages of the present invention will be further described in conjunction with the following drawings and specific embodiments. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0046] Figure 1 This is a particle size distribution diagram of the bufotoxin ligand nano-formulation for treating colon cancer according to the present invention. (a is the particle size diagram of the pH-responsive bufotoxin ligand nano-formulation; b is the potential diagram of the pH-responsive bufotoxin ligand nano-formulation.)

[0047] Figure 2 These are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the bufotoxin ligand nanoformulation for treating colon cancer according to the present invention. (a is an SEM image of the bufotoxin ligand raw material, b is an SEM image of the bufotoxin ligand nanoformulation, c is a TEM image of the bufotoxin ligand raw material, and d is a TEM image of the bufotoxin ligand nanoformulation.)

[0048] Figure 3 This is a dissolution curve of the nano-formulation of the present invention in media with pH 1.2 and pH 6.8.

[0049] Figure 4 XPS images of the nano-formulations of this invention are shown. (a) pH-responsive bufotoxin ligand nano-formulation group; b) bufotoxin ligand nanocrystals; c) bufotoxin ligand active pharmaceutical ingredient.

[0050] Figure 5 This is the Raman spectrum of the nano-formulation of the present invention. (a is the bufotoxin ligand active pharmaceutical ingredient; b is the stabilizer; c is the pH-responsive bufotoxin ligand nano-formulation; d is the pH-responsive coating material.)

[0051] Figure 6 These are IVIS images showing the biodistribution of the nano-formulation of this invention in the gastrointestinal tract of mice at different time points.

[0052] Figure 7 The results show the effect of the nano-formulation of this invention on ROS in tumor cells. (a, b are CT-26 cells; c, d are HT-29 cells.)

[0053] Figure 8 The results show the effect of the nano-formulation of this invention on the membrane potential of tumor cells. (a, c are CT-26 cells; b, d are HT-29 cells.)

[0054] Figure 9 This image shows tumor tissue images illustrating the tumor growth inhibition effect of the nano-formulation of this invention. (a is the control group, b is the bufotoxin ligand raw material group, c is the bufotoxin ligand nanocrystal group, and d is the pH-responsive bufotoxin ligand nano-formulation group.)

[0055] Figure 10 The figures show tumor size and mouse body weight in response to the tumor growth inhibition of the nano-formulation of this invention. (A represents tumor volume; B represents mouse body weight; a represents the control group; b represents the bufotoxin ligand raw material group; c represents the bufotoxin ligand nanocrystal group; d represents the pH-responsive bufotoxin ligand nano-formulation group.)

[0056] Figure 11 This is an immunohistochemical image of the nano-formulation of the present invention against apoptosis-related proteins in tumor tissue. (In Figure B, a is Bax protein; b is Caspase-3 protein; c is Bcl-2 protein.) Detailed Implementation

[0057] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Example 1: Preparation of a bufotoxin ligand nanocrystal

[0059] 6 mg of chitosan quaternary ammonium salt was dissolved in 10 ml of purified water and fully dissolved and swollen under magnetic stirring to obtain a stabilizer solution. 30 mg of bufotoxin ligand raw material was dissolved in 1 ml of methanol to obtain a drug solution. The stabilizer solution was placed in an ice bath at 0°C under an ultrasonic probe with an ultrasonic power of 70%, a working time of 3 seconds, an interval of 2 seconds, and an ultrasonic time of 10 minutes. The drug solution was added to the stabilizer solution at a rate of 1 ml / 1 minute. The ultrasonic solution was then evaporated under magnetic stirring to obtain bufotoxin ligand nanocrystals.

[0060] Example 2: Preparation of a pH-responsive toad venom ligand nanoformulation

[0061] 6 mg of chitosan quaternary ammonium salt was weighed and dissolved in 10 ml of purified water. The solution was fully dissolved and swollen under magnetic stirring to obtain a stabilizer solution. 30 mg of bufotoxin ligand raw material, 30 mg of Eutrich L-100, and 3 mg of sorbitan oleate were weighed and dissolved in 1 ml of methanol to obtain a drug solution. The stabilizer solution was placed in an ice bath at 0°C under an ultrasonic probe. The ultrasonic power was set to 70%, the working time was 5 seconds, the interval was 2 seconds, and the ultrasonic time was 10 minutes. The drug solution was added to the stabilizer solution at a rate of 1 ml / min. The organic solvent in the ultrasonic solution was evaporated under magnetic stirring to obtain a pH-responsive bufotoxin ligand nano-formulation. The particle size was measured to be 247.1 ± 8.52 nm, and the Zeta potential was 247.1 ± 8.52 mV.

[0062] Example 3: Preparation of a pH-responsive bufotoxin ligand nanoformulation

[0063] 8 mg of chitosan quaternary ammonium salt was weighed and dissolved in 10 ml of purified water. The solution was fully dissolved and swollen under magnetic stirring to obtain a stabilizer solution. 30 mg of bufotoxin ligand raw material, 30 mg of Eutrich L-100, and 3 mg of sorbitan oleate were weighed and dissolved in 1 ml of methanol to obtain a drug solution. The stabilizer solution was placed in an ice bath at 0°C under an ultrasonic probe. The ultrasonic power was set to 65%, the working time was 5 s, the interval was 2 s, and the ultrasonic time was 12 min. The drug solution was added to the stabilizer solution at a rate of 1 ml / 30 s. The organic solvent in the ultrasonic solution was evaporated under magnetic stirring to obtain a pH-responsive bufotoxin ligand nano-formulation. The particle size was measured to be 289.7 ± 7.41 nm, and the zeta potential was 42.0 ± 0.36 mV.

[0064] Example 4: Preparation of a pH-responsive bufotoxin ligand nanoformulation

[0065] 6 mg of chitosan quaternary ammonium salt was weighed and dissolved in 10 ml of purified water. The solution was fully dissolved and swollen under magnetic stirring to obtain a stabilizer solution. 30 mg of bufotoxin ligand raw material, 30 mg of Eutrich L-100, and 3 mg of sorbitan oleate were weighed and dissolved in 1 ml of methanol to obtain a drug solution. The stabilizer solution was placed in an ice bath at 0°C under an ultrasonic probe. The ultrasonic power was set to 50%, the working time was 3 seconds, the interval was 2 seconds, and the ultrasonic time was 14 minutes. The drug solution was added to the stabilizer solution at a rate of 1 ml / 30 seconds. The organic solvent in the ultrasonic solution was evaporated under magnetic stirring to obtain a pH-responsive bufotoxin ligand nano-formulation. The particle size was measured to be 289.7 ± 7.41 nm, and the Zeta potential was 42.0 ± 0.36 mV.

[0066] Example 5: Preparation of a pH-responsive toad venom ligand nanoformulation

[0067] 12 mg of chitosan quaternary ammonium salt was weighed and dissolved in 10 ml of purified water. The solution was fully dissolved and swollen under magnetic stirring to obtain a stabilizer solution. 30 mg of bufotoxin ligand raw material, 30 mg of Eutrich L-100, and 3 mg of sorbitan oleate were weighed and dissolved in 1 ml of methanol to obtain a drug solution. The stabilizer solution was placed in an ice bath under an ultrasonic probe with an ultrasonic power of 65%, a working time of 5 seconds, an interval of 2 seconds, and an ultrasonic time of 12 minutes. The drug solution was added to the stabilizer solution at a rate of 1 ml / 30 seconds. The ultrasonic solution was then evaporated under magnetic stirring to obtain a pH-responsive bufotoxin ligand nano-formulation with a particle size of 230.1 ± 1.77 nm and a zeta potential of 47.3 ± 0.24 mV.

[0068] Example 6: Determination of release rate

[0069] Separately, transfer crude suspension of bufotoxin ligand and pH-responsive bufotoxin ligand nanoparticles into dialysis bags (MW = 8000–14000 Da), tie both ends tightly, and place them in 40 ml of pH 1.2 medium for 2 hours of release. Then transfer them to pH 6.8 medium for another 4 hours of release. Take 2 ml samples at 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 hours, respectively. Filter the samples through a 0.22 μm filter membrane, and use the filtrate as the test solution. Add an equal volume of blank medium. Separately, accurately weigh 2.2 mg, 3.0 mg, and 5.2 mg of bufotoxin, bufotoxin ligand, and bufotoxin ligand standards into 10 mL volumetric flasks. Add 1.0 mL of the stock solution of each single-component standard to the same 10 mL volumetric flask, dilute to the mark, and shake well to obtain a mixed standard solution of bufotoxin, bufotoxin ligand, and bufotoxin ligand. The content was determined by high performance liquid chromatography (HPLC). The release rates of bufotoxin, bufotoxin ligand, and bufotoxin ligand in the bufotoxin ligand raw material and pH-responsive bufotoxin ligand nano-formulation were calculated. The results are shown in [Figure number missing]. Figure 5 .

[0070] according to Figure 5 The results showed that the cumulative release of the crude bufotoxin suspension at pH 1.2 and pH 6.8 was less than 20% throughout the release process. Using HACC as a stabilizer, the nanocrystals significantly improved the cumulative solubility of bufotoxin and bufotoxin ligands. However, the lipofuscin ligand, after being released for 2 hours in pH 1.2 and then transferred to pH 6.8 for 4 hours, was undetectable. The main reason for this phenomenon is likely that acidic media would damage its structure. For pH-responsive bufotoxin ligand nanocrystals, the characteristic of L-100—insoluble in acidic media but soluble in media with pH > 6.0—significantly protects the structure of the lipofuscin ligand from destruction by the acidic environment and enables the synergistic and sustained release of the three components in the intestine.

[0071] Example 7: Colon-targeting experiment of the toad venom ligand nano-formulation for treating colon cancer according to the present invention.

[0072] Hydrophobic near-infrared dye (DiR) was used as a fluorescent probe to replace the bufotoxin ligand in pH-responsive nanoparticles. DiR-loaded nanocrystals and pH-responsive nanoparticles were prepared using the same concentration of DiR and the same preparation process. Mice were then divided into free DiR group, DiR nanocrystal group, and pH-responsive DiR nanoparticle group. The prepared DiR-loaded nanoparticles were administered to mice by gavage. Mice were euthanized by cervical dislocation at 2, 6, 8, and 12 hours after oral administration. Their gastrointestinal tracts were collected in a dark environment, removed, and laid flat on a black panel. The fluorescence signal distribution of the pH-responsive DiR nanoparticles in the mouse gastrointestinal tract was observed in vitro using an IVIS spectral imaging system. The results are shown in [Figure number missing]. Figure 6 .

[0073] Figure 6 In the culture dish, a pouch-like upper region can be seen representing the stomach, with the small intestine forming an S-shape in the middle. The distended portion of the intestine, including the cecum, colon, and rectum, is called the large intestine. It is evident that free DiR and DiR nanocrystals exhibit strong fluorescence signals in the stomach and small intestine during the first 4 hours, while no significant fluorescence signal is observed in the colon. This indicates that the maximum amount of fluorescent dye is released or absorbed in the small intestine before reaching the colon. By 12 hours, the fluorescence intensity of free DiR and DiR nanocrystals weakens or disappears, indicating that the surface dye has been completely absorbed or cleared from the gastrointestinal tract. In contrast, the pH-responsive nanocrystals loaded with DiR show some fluorescence signals in the stomach and small intestine, possibly due to the presence of DiR fragments on the nanocrystal surface or a small amount leaking from the core. At 8 and 12 hours, the pH-responsive nanocrystals loaded with DiR show significant fluorescence signals in the colon, indicating that the pH-responsive shell provides stability to the system under acidic conditions, allowing for maximum dissolution and release of the dye near the colon, demonstrating the specificity of drug targeted delivery to the colon. Furthermore, a weak fluorescent signal remained in the colon after 12 hours. This is due to the interaction between the positively charged nanocrystals and the negatively charged mucin in the colon caused by the shedding of the pH-responsive shell, resulting in a longer drug retention time in the colon. This indicates that pH-responsive bufotoxin ligand nanoformulations overcome the limitation of rapid drug release in the stomach and upper intestine, allowing the drug to target and release near the colon.

[0074] Example 8: Effect of the pH-responsive bufotoxin nanoparticles prepared in the above-described embodiments on ROS levels in tumor cells.

[0075] Two types of colon cancer cells, HT-29 and CT-26, in the logarithmic growth phase were collected, digested, centrifuged, and their density adjusted to 2 × 10⁻⁶. 5 Cells were seeded at a concentration of [number] cells / mL in 6-well plates and cultured until adherence. Culture medium containing bufotoxin raw material, bufotoxin nanocrystals, and pH-responsive bufotoxin nanoparticles was added, with no drug-free medium serving as a control. The drug concentration for HT-29 cells was set at 0.5 μg / mL, and for CT-26 cells at 20 μg / mL. Cells were cultured for 24 h after drug administration. After culture, the culture medium was discarded, cells were washed twice with PBS, digested with a small amount of trypsin, centrifuged, and the cells were collected. Cells were resuspended in culture medium, and 500 μL of LCFH-DA solution was added to each well and incubated at 37°C for 30–60 min. After incubation, cells were centrifuged at 1000 rpm for 5 min, washed twice with PBS, centrifuged again, and resuspended. Flow cytometry analysis was performed, and the results are shown below. Figure 7 .

[0076] Figure 7 The results indicate that, for the two types of colon cancer cells mentioned above, both the bufotoxin nanocrystal group and the pH-responsive bufotoxin nanoparticle group significantly increased the ROS level in tumor cells during the same incubation period. The ROS level of the pH-responsive bufotoxin nanoparticle group was slightly higher than that of the bufotoxin nanocrystal group. The pH-responsive bufotoxin nanoparticle group showed a significant difference in ROS level increase compared to the raw material group, indicating that bufotoxin can kill tumor cells by upregulating ROS in tumor cells.

[0077] Example 9: Effect of the pH-responsive bufotoxin nanoparticles prepared in the above-described embodiments on the mitochondrial membrane potential of tumor cells.

[0078] Two types of colon cancer cells, HT-29 and CT-26, in the logarithmic growth phase were collected, digested, centrifuged, resuspended, and their density adjusted to 2 × 10⁻⁶. 5 Cells were seeded at a concentration of [number] cells / mL in 6-well plates and cultured until adherence. Then, culture medium containing bufotoxin API, bufotoxin nanocrystals, and pH-responsive bufotoxin nanoparticles was added. Drug-free medium served as a control. The drug concentration for HT-29 cells was set at 0.5 μg / mL, and for CT-26 cells at 20 μg / mL. Cells were cultured for 24 h after drug administration. After culture, cells were washed twice with PBS, digested with a small amount of trypsin, and collected by centrifugation. Cells were resuspended in JC-1 working solution and incubated for 15-20 min. After incubation, cells were collected by centrifugation, washed with 1×Incubation Buffer, centrifuged again, and resuspended. Fluorescence analysis was performed by flow cytometry. Results are shown in [figure missing]. Figure 7 .

[0079] Figure 8 The results showed that, in CT-26 colon cancer cells, within the same incubation period, bufotoxin raw material, bufotoxin nanocrystals, and pH-responsive bufotoxin nanoparticles all depolarized the mitochondrial transmembrane potential, decreasing it by (5.41±0.72)%, (10.92±0.31)%, and (15.00±0.28)%, respectively. In HT-29 colon cancer cells, bufotoxin raw material, bufotoxin nanocrystals, and pH-responsive bufotoxin nanoparticles decreased the mitochondrial transmembrane potential by (5.05±0.56)%, (8.20±1.20)%, and (12.75±2.19)%, respectively. The pH-responsive bufotoxin nanoparticles enhanced the ability of bufotoxin to depolarize the mitochondrial membrane potential and increased its ability to induce tumor cell death.

[0080] Example 10: Effects of the pH-responsive bufotoxin nanoparticles prepared in the above-described embodiments on animal tumor models.

[0081] Log-growing CT-26 colon cancer cells were digested, centrifuged, resuspended, counted, and the cell concentration was adjusted to 6 × 10⁻⁶. 7 Cells / mL, disinfect the mouse armpits with alcohol, and inoculate approximately 6 × 10⁶ cells (about 0.1 mL) into the armpits to establish a colon cancer tumor-bearing mouse model. Two days after inoculation, the size of the tumors in the mice was measured daily, and the tumor volume was increased to 100 mm². 3 Mice were randomly assigned to either side for the experiment. Colon cancer-bearing mice were randomly divided into four groups: bufotoxin raw material group, bufotoxin nanocrystal group, pH-responsive bufotoxin nanoparticle group, and saline group. The dosage was 6 mg / kg, administered by gavage, once every two days for a total of seven administrations. Body weight and tumor volume were recorded every two days. Results are shown below. Figure 8 and Figure 9 .

[0082] Differences in tumor volume and mouse body weight were compared among the groups. The growth curves of tumor tissue and tumor volume over time were presented. Figure 9 and mouse weight Figure 10 It was found that all three treatment groups—bufotoxin raw material, bufotoxin nanocrystals, and pH-responsive bufotoxin nanoparticles—exhibited tumor growth to some extent compared to the saline group. Notably, the pH-responsive bufotoxin nanoparticles showed a more pronounced inhibitory effect on tumor growth compared to the raw material and nanocrystal groups, with smaller tumor volumes, indicating a stronger tumor-inhibiting ability.

[0083] Example 11: Immunohistochemistry of pH-responsive bufotoxin nanoparticles prepared in the above-described embodiments on apoptosis-related proteins in animal tumor tissues.

[0084] Tumor tissue was paraffin-embedded, sectioned, and dewaxed to water in the following order: ① Sections were incubated in xylene I for 15 min; ② Sections were transferred to xylene II for 15 min; ③ Sections were transferred to xylene III for 15 min; ④ Sections were transferred to anhydrous ethanol I for 5 min; ⑤ Sections were transferred to anhydrous ethanol II for 5 min; ⑥ Sections were transferred to 85% ethanol for 5 min; ⑦ Sections were transferred to 75% ethanol for 5 min; ⑧ Finally, sections were rinsed with distilled water. Antigen retrieval: Sections were placed in citrate antigen retrieval buffer (pH 6.0) for antigen retrieval. After cooling, sections were placed in PBS (pH 7.4) solution and washed three times on a shaker. Serum blocking: 3% BSA solution was added and blocked at room temperature for 30 min. Primary antibody addition: After serum blocking, primary antibody PBS solution was added and incubated overnight at 4°C. Secondary antibody addition: Place tissue sections in PBS (pH 7.4) solution and wash three times on a shaker. After washing, spin dry the sections, then add the corresponding species' secondary antibody to the tissue circle and incubate at room temperature for 50 min. AEC staining: Place the sections in PBS (pH 7.4) solution and wash three times on a shaker. After washing, spin dry the sections, add the AEC staining solution to the tissue circle, and incubate in the dark for 10-25 min. After incubation, rinse the sections with tap water to stop the staining. Counterstaining cell nuclei: Counterstain the sections with hematoxylin for about 3 min, rinse the sections with tap water, use hematoxylin blue solution to return to blue, rinse the sections with running water, and mount: After nuclear microscopic examination confirms that there are no errors, place the sections in pure water, let them dry slightly, mount them with glycerol gelatin, and finally air dry the tissue sections. Microscopic examination: Observe the tissue sections and acquire images. The results are shown in […]. Figure 10 .

[0085] Figure 11 The results showed that, compared with the saline group, both the bufotoxin ligand raw material and related nanocrystal formulations significantly downregulated Bcl-2 protein expression and upregulated Bax and Caspase-3 protein expression. The pH-responsive bufotoxin ligand nano-formulations exhibited stronger effects than the bufotoxin ligand nanocrystals and the raw material. The pH-responsive bufotoxin ligand nano-formulations enhanced the ability of bufotoxin ligands to induce tumor cell apoptosis.

[0086] Example 12: In vivo pharmacokinetics of the pH-responsive bufotoxin nanoparticles prepared in the above-described embodiments of the invention.

[0087] Twenty-four healthy SD rats, with an average weight of 220-240g, were randomly divided into four groups after acclimatization for three days: a saline group, a free bufotoxin group, a bufotoxin nanocrystal group, and a pH-responsive bufotoxin nanoparticle group. The rats were fasted and deprived of water for one day before administration. Except for the saline group, the other two groups were administered bufotoxin nanoparticles at a concentration of 6 mg / kg via gavage. Blood samples of 0.3 mL were collected from the inner canthus of the eye at 5, 10, 15, 30, 45, 60, 120, 240, 360, 480, and 720 min. The blood samples were placed in heparin-treated 1.5 mL conical centrifuge tubes and centrifuged at 4000 rpm for 10 min. The separated plasma was stored at -80℃. Liquid-liquid extraction of the plasma samples was then performed, and analysis was performed using HPLC / MS / MS. The results are shown in Table 1.

[0088] Table 1. Pharmacokinetic parameters of bufotoxin ligands

[0089]

[0090]

[0091] Table 1 shows that the area under the pharmacokinetic curve (AUC) of the pH-responsive bufotoxin nanoparticles was significantly higher than that of the bufotoxin nanocrystal group and the free bufotoxin group. The maximum plasma concentration of the pH-responsive bufotoxin nanoparticles was also significantly higher than that of the bufotoxin nanocrystal group and the free bufotoxin group. The time to peak concentration of the pH-responsive bufotoxin nanoparticles was also faster than that of the bufotoxin nanocrystal group and the free bufotoxin group, indicating a faster onset of action. The bioavailability was significantly improved because the pH-responsive bufotoxin nanoparticles facilitated co-release of the drug in the intestine, and the positively charged nanocrystals enhanced the binding of the drug to the negatively charged mucin in the colon, which is beneficial for drug absorption.

[0092] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to in the method section.

[0093] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nanofomulation of bufonidine for the treatment of colon cancer, characterized in that, The nanometer preparation has a core-shell structure, the inner core is a nanocrystal composed of bufadienolides, a stabilizer, the outer shell is a pH-responsive material and a pore-forming agent; The mass ratio of the bufadienolides, the stabilizer, the pH-responsive material and the pore-forming agent is (6-12):(1-8):(6-12):1; The bufadienolides are an amorphous or microcrystalline mixture with a purity of 32-98% and consist of 10-25% bufalin, 20-35% cinobufaglycosides and 10-70% resibufogin by mass percentage; The stabilizer is chitosan quaternary ammonium salt, and the mass-volume concentration of the stabilizer is 0.01%-10%; The pH-responsive material is one or more of Eudragit L-100, hydroxypropyl methylcellulose phthalate HP50, hydroxypropyl methylcellulose acetate succinate and Kollicoat MAE100P, and the mass-volume concentration of the pH-responsive material is 0.01%-20%; The pore-forming agent is sorbitan oleate, and the mass-volume concentration of the pore-forming agent is 0.01-1.0%.

2. The bufadienolide nanometer preparation for treating colon cancer according to claim 1, wherein the drug loading of the bufadienolide is 1%-30%, and the encapsulation efficiency is 30%-96%; The particle size of the nanometer preparation is 50-500 nm, and the Zeta potential is-40-+54 mV.

3. The bufadienolide nanometer preparation for treating colon cancer according to claim 1, wherein the nanometer preparation has pH responsiveness, and the pH-responsive range of the nanometer preparation is pH>6, so that colon-specific release can be achieved; The in vitro release of bufalin, cinobufaglycosides and resibufogin reaches synchronous release in 0-2 h pH1.2, 2-6 h pH6.8 transfer release medium, the cumulative release amount in 2 h is less than 10%, the release amount in 4 h is 30%-60%, and the cumulative release amount in 6 h is 70%-90%.

4. The bufadienolide nanometer preparation for treating colon cancer according to claim 1, wherein the tumor inhibition rate of the nanometer preparation is 40-70%, which is 1.5-4.5 times higher than that of the bufadienolide nanocrystal and 3-6 times higher than that of the bufadienolide coarse suspension; The nanometer preparation induces tumor cell apoptosis in colon cancer through a mitochondria-dependent pathway. The nanocrystal is prepared by an anti-solvent precipitation-ultrasonic method, and the inner core and the outer shell are prepared by an electrostatic self-assembly method. The process steps are as follows: 1) Dissolve the bufadienolides, the pH-responsive material and the pore-forming agent in an organic solvent as a bufadienolide drug solution; 5. A process for the preparation of a nanformulation of bufonidine for the treatment of colon cancer according to any one of claims 1 to 4, characterized in that, 2) Dissolve the stabilizer in an aqueous medium as a stabilizer solution; 6. The method of claim 5, wherein the preparation of the nanofomulation of the bufonin for the treatment of colon cancer is characterized by, 3) Under the condition of probe ultrasonication, add the drug solution to the stabilizer solution; 4) Under the condition of magnetic stirring, volatilize and remove the organic solvent from the mixed solution obtained in step 3) to obtain the bufadienolide nanometer preparation for treating colon cancer. In step 2), the aqueous medium is purified water or a PBS solution, and the concentration of the stabilizer solution is 0.01%-5%. ​ ​ 7. The method of claim 6, wherein the preparation of the nanofomulation of the bufonin for the treatment of colon cancer is characterized by, ​ 8. The method of claim 6, wherein the preparation of the nanod formulation of the bufonidine for the treatment of colon cancer is characterized by, The speed of adding the stabilizer solution into the drug solution in step 3) is (1ml / s)~(1ml / 2min); The ultrasonic power of the probe in step 3) is 5%~80%; the ultrasonic temperature of the probe is one of 0℃, 4℃ and 25℃; and the ultrasonic time of the probe is 5min~20min.

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