A pH-sensitive norcantharidin solid self-microemulsion, and a preparation method and application thereof

Abstract

This invention discloses a pH-sensitive norcantharidin solid self-microemulsion, its preparation method, and its applications, belonging to the field of pharmaceutical technology. It comprises the following components in parts by weight: 0.1–3 parts norcantharidin, 1–10 parts pH-sensitive polymer, 30–80 parts solid adsorbent, and 87–98.9 parts blank self-microemulsion. The pH-sensitive norcantharidin solid self-microemulsion of this invention can improve the solubility and permeability of the drug. Due to the amide bond cleavage of the pH-sensitive polymer under acidic conditions, the drug is released systematically from the interior of the nano-solid self-microemulsion, resulting in targeted release at the tumor site. Animal experiments show that the pH-sensitive norcantharidin solid self-microemulsion of this invention can effectively improve the absorption rate constant and apparent permeability coefficient of norcantharidin at the colon tumor site, thereby enhancing its ability to inhibit tumor growth, migration, and invasion, and thus significantly improving the therapeutic effect of colon tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a pH-sensitive norepinephrine solid microemulsion, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the gastrointestinal system, ranking third in global incidence among cancers. It is a challenging disease to treat, with a high mortality rate, and its incidence is increasing year by year, posing a serious threat to human life and health. Patients rely on surgery and chemotherapy to control the disease, but the high recurrence rate and high complication rate after surgery remain among the highest of all cancers.

[0003] Norcantharidin (NCTD) is a synthetic derivative of cantharidin, the active ingredient extracted from insect cantharidin. Clinical and experimental studies have confirmed its effective anti-tumor effects against various tumor cells, including esophageal cancer, cholangiocarcinoma, lung cancer, and colorectal cancer. Related experimental studies have shown that NCTD can inhibit the growth, invasion, and metastasis of colon cancer cells. Compared with other anticancer drugs used clinically for gastrointestinal tumors, it also has unique advantages such as increasing white blood cell levels, regulating immunity, and not causing bone marrow suppression. Currently, commercially available NCTD drugs are mainly in injection and tablet forms. Due to poor water solubility and permeability, injections are highly irritating, and oral bioavailability is low. Furthermore, traditional oral or injection formulations cannot guarantee effective concentrations and bioavailability at the tumor site and can produce severe systemic toxicity. Therefore, this invention necessitates the design of new dosage forms to improve drug solubility and permeability, enabling targeted tumor delivery. Summary of the Invention

[0004] The main objective of this invention is to provide a pH-sensitive norcantharidin solid microemulsion, its preparation method, and its applications. The pH-sensitive norcantharidin solid microemulsion prepared by this invention can improve the solubility and permeability of the drug, enabling targeted tumor delivery. Furthermore, the outer layer of the pH-sensitive norcantharidin solid microemulsion of this invention can target tumor sites based on pH-triggered surface charge reversal properties, allowing it to interact with negatively charged mucins in the colonic mucosa. This enhances the accumulation of the solid microemulsion at acidic tumor sites, thereby achieving a better therapeutic effect.

[0005] Norcantharidin has a strong anti-tumor effect on a variety of cancer cells. In this invention, a pH-sensitive norcantharidin self-microemulsion was first prepared, and then micronized silica gel was added to solidify the self-microemulsion to obtain a pH-sensitive norcantharidin solid self-microemulsion. Its in vitro release results were then investigated.

[0006] To achieve the above objectives, the present invention provides a pH-sensitive norcantharidin solid self-microemulsion comprising the following components in parts by weight: 0.1 to 3 parts norcantharidin, 1 to 10 parts pH-sensitive polymer, 30 to 80 parts solid adsorbent, and 87 to 98.9 parts blank self-microemulsion.

[0007] Furthermore, the pH-sensitive polymer is maleamidized chitosan (CS-DMMA). Even further, the maleamidized chitosan is prepared from chitosan modified with 2,3-dimethylmaleic anhydride.

[0008] Furthermore, the solid adsorbent is micronized silica gel.

[0009] Furthermore, the blank self-microemulsion is composed of an oil phase, an emulsifier, and a co-emulsifier; wherein the oil phase is ethyl oleate, the emulsifier is polyoxyethylene hydrogenated castor oil, and the co-emulsifier is 1,2-propanediol.

[0010] Furthermore, the mass ratio of the oil phase, the emulsifier, and the co-emulsifier is 1:(1-1.5):(0.8-1.2).

[0011] Furthermore, the pH-sensitive norcantharidin solid microemulsion has an average particle size of 70–80 nm, a polydispersity index of 0.18–0.24, a surface charge of -4.2–-3.7 mV, and drug loading and encapsulation efficiency of 92–94% and 0.9–1.2%, respectively.

[0012] Another aspect of the present invention provides a method for preparing a pH-sensitive cantharidin solid microemulsion, comprising the following steps:

[0013] Chitosan was dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and triethylamine catalyst were added to react. After the reaction was completed, the mixture was purified by dialysis and freeze-dried to obtain a pH-sensitive polymer.

[0014] The oil phase, emulsifier, and co-emulsifier were mixed and stirred in proportion until a clear state was obtained to obtain a blank self-microemulsion.

[0015] The pH-sensitive polymer and norcantharidin were dissolved in the blank microemulsion and stirred to obtain a pH-sensitive norcantharidin microemulsion.

[0016] The pH-sensitive norcantharidin self-microemulsion was mixed with a solid adsorbent to obtain the pH-sensitive norcantharidin solid self-microemulsion.

[0017] Furthermore, the ratio of chitosan to 2,3-dimethylmaleic anhydride is 1 to 1.5:1.

[0018] Furthermore, the mass concentration of the pH-sensitive norcantharidin in the microemulsion is 1–30 mg / g.

[0019] Furthermore, the mass concentration of the pH-sensitive polymer in the pH-sensitive cantharidin microemulsion is 10–100 mg / g.

[0020] In another aspect, the present invention provides the application of the aforementioned pH-sensitive norcantharidin solid microemulsion in the preparation of a drug for treating colon cancer.

[0021] In another aspect, the present invention provides the aforementioned pH-sensitive norcantharidin solid microemulsion for the treatment of colon cancer.

[0022] Compared with existing technologies, the present invention has the following beneficial effects: The pH-sensitive norcantharidin solid self-microemulsion of the present invention is an oral solid self-microemulsion formulation that can improve the solubility and permeability of the drug, avoiding release of the drug in the upper gastrointestinal tract (stomach and small intestine) and releasing it at the tumor site. Due to the presence of CS-DMMA, the amide bond breaks under acidic conditions (pH 6.5-6.8), causing instability in the outer layer of the pH-sensitive norcantharidin solid self-microemulsion. The drug is released regularly from the interior of the nano-solid self-microemulsion. At the same time, the presence of the solid adsorbent micronized silica gel improves the stability of the formulation, enabling targeted release of the drug at the tumor site.

[0023] Furthermore, according to in situ intestinal absorption experiments, the pH-sensitive norcantharidin solid self-microemulsion of the present invention can effectively increase the absorption rate constant K of norcantharidin at the colon tumor site. a and apparent transparency coefficient P app In vitro antitumor effects showed that pH-sensitive norcantharidin solid microemulsion could enhance the ability to inhibit tumor growth, migration and invasion; in vivo efficacy showed that treatment with pH-sensitive norcantharidin solid microemulsion could increase the proportion of CD4+T and CD8+T cells, inhibit tumor cell proliferation, and promote apoptosis, thus exerting good in vivo antitumor activity, indicating that this preparation can significantly improve the treatment effect of colon cancer. Attached Figure Description

[0024] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the CS-DMMA synthesized in Example 1 of the present invention is shown;

[0025] Figure 2 The in vitro release curves of pH-sensitive norcantharidin solid microemulsion under different pH conditions are shown in Example 1 of the present invention.

[0026] Figure 3Characterization diagrams of the pH-sensitive cantharidin solid microemulsion in Example 1 of the present invention are shown: (A) appearance diagram; (B) transmission electron microscopy image; (C) FTIR image and (D) DSC curve (wherein, (a) NCTD@CS-DMMA SSME, (b) BlankSSME, (c) CS-DMMA, (d) NCTD active pharmaceutical ingredient, (e) micronized silica gel;

[0027] Figure 4 The FTIR analysis chromatograms of maleamidized chitosan synthesized in Example 1 of the present invention are shown: (a) CS; (b) DMMA; (c) CS+DMMA (physical mixture); (d) CS-DMMA;

[0028] Figure 5 The following are DSC analysis chromatograms of maleamidized chitosan synthesized in Example 1 of the present invention: (a) CS; (b) DMMA; (c) CS+DMMA (physical mixture); (d) CS-DMMA;

[0029] Figure 6 The absorption rate constant K of NCTD-7.4, NCTD@CS-DMMA SSME-6.5, and NCTD@CS-DMMA-SSME-7.4 in the in situ intestinal absorption experiment of the present invention is shown. a and apparent absorption coefficient P app Comparison chart (Mean±SD, n=3):

[0030] Where (A) is the absorption rate constant K a (B) is the apparent absorption coefficient P. app ;

[0031] Figure 7 The following figure shows the analytical results of the in vitro tumor activity experiment of pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention:

[0032] Among them, (A) Cytotoxicity of NCTD-7.4 and NCTD@CS-DMMA SSME on HT29 cells under different pH conditions; (B) Microscopic observation of the healing effect of pH-sensitive norcantharidin solid self-microemulsion on CT-26 cells in the cell scratch assay; (C) Microscopic observation of the effect of the drug on HT-29 cell migration detected by the Transwell assay; (D) Microscopic observation of the effect of the drug on HT-29 cell invasion detected by the Transwell assay; (E) Comparison of the effect of different norcantharidin formulations on cell migration rate under different pH conditions (Mean±SD, n=3); (F) Comparison of the effect of different norcantharidin formulations on cell invasion rate under different pH conditions (Mean±SD, n=3).

[0033] Figure 8 The following diagrams illustrate the experimental protocol for the in vivo antitumor activity experiment of the pH-sensitive norcantharidin solid microemulsion in Example 1 of the present invention, in vivo bioluminescence imaging of CT-26 tumor-bearing mice, a schematic diagram of tumor anatomy, and a comparison of the total tumor weight and total body weight of CT-26 tumor-bearing mice in each group after drug administration:

[0034] Among them, (A) is a schematic diagram of the in vivo antitumor activity experimental protocol; (B) is an in vivo bioluminescence imaging image of three different CT-26 tumor-bearing mice; (C) is the tumor removal situation after dissection of each group of mice; (D) is the tumor weight of each group of mice (Mean±SD, n=3); (E) is the weight change of each group of mice (Mean±SD, n=3).

[0035] Figure 9 The following is an analytical diagram illustrating the in vivo antitumor activity of the pH-sensitive norcantharidin solid microemulsion in Example 1 of the present invention:

[0036] Among them, (A) is a schematic diagram of the ratio of CD3 / CD4+T and CD3 / CD8+T in the model analyzed by flow cytometry (n=3); (B) is an immunofluorescence image of CD8+T cells in the tumor tissue of CT-26 orthotopic colon cancer mice in each group after drug administration;

[0037] Figure 10 The following are pathological sections of major organs and tumor tissues from CT-26 tumor-bearing mice in the in vivo antitumor activity experiment of pH-sensitive norcantharidin solid microemulsion in Example 1 of the present invention.

[0038] Figure 11 The diagram shows a comparative analysis of tumor cell apoptosis and tumor cell proliferation in CT-26 orthotopic carcinoma mice under different drug treatments in the in vivo antitumor activity experiment of pH-sensitive norcantharidin solid microemulsion in Example 1 of the present invention. The scale bar is 1:50 μm.

[0039] Among them, (A) is the TUNEL assay for the apoptosis of CT-26 orthotopic carcinoma mice in each treatment group; (B) is the Ki67 immunohistochemistry assay for the proliferation of CT-26 orthotopic carcinoma mice in each treatment group.

[0040] Figure 12 The diagram illustrates the process of preparing the pH-sensitive norcantharidin solid self-microemulsion of the present invention and its targeted release at tumor sites. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. The invention will now be described in detail with reference to embodiments.

[0042] In the process of preparing pH-sensitive norcantharidin solid self-microemulsion, the norcantharidin can be derived from substances such as norcantharidin raw material and norcantharidin extract.

[0043] Solid self-emulsifying drug delivery systems (SSMEs) combine self-microemulsifying drug delivery systems (SMEs) with solid dosage forms, selecting a suitable solid carrier to solidify the SME. SSMEs overcome the drawbacks of liquid formulations such as inconvenience in storage and portability, high production costs, low stability, and limited dosage form options. Furthermore, they can improve drug efficacy and reduce side effects.

[0044] To achieve the above objectives, a first aspect of the present invention provides a pH-sensitive norcantharidin solid self-microemulsion comprising the following components in parts by weight: 0.1 to 3 parts norcantharidin, 1 to 10 parts pH-sensitive polymer, 30 to 80 parts solid adsorbent, and 87 to 98.9 parts blank self-microemulsion.

[0045] In some preferred embodiments of the present invention, the pH-sensitive polymer is maleamide-modified chitosan. More preferably, the maleamide-modified chitosan is prepared from chitosan modified with 2,3-dimethylmaleic anhydride; the maleamide-modified chitosan dissolves at a pH of 6.5 to 6.8.

[0046] Maleamide-modified chitosan (CS-DMMA) is synthesized from low-molecular-weight chitosan and 2,3-dimethylmaleic anhydride. Chitosan (CS) is an abundant and inexpensive natural polymer compound obtained through the N-deacetylation reaction of chitin. It possesses excellent biodegradability, biocompatibility, bioadhesion, and film-forming properties, and is therefore widely used as a drug carrier. CS is a natural cationic polymer containing a large number of amine groups, which can react with 2,3-dimethylmaleic anhydride (DMMA) to form 2,3-dimethylmaleic anhydride amide bonds. It is readily hydrolyzed under weakly acidic conditions (pH 6.5–6.8), therefore CS-DMMA can serve as a carrier to release drugs in tumor-specific pH environments.

[0047] Based on the characteristic that tumor tissue (pH=6.5-6.8) is more acidic than normal tissue (pH=7.4) in the tumor microenvironment, this invention introduces a synthetic pH-sensitive polymer, maleamide-modified chitosan (CS-DMMA), into the self-microemulsion formulation to prepare a pH-sensitive norcantharidin solid self-microemulsion that targets tumor tissue for drug release. This aims to improve drug solubility and permeability while avoiding premature drug release in the stomach and small intestine, thereby increasing the drug concentration at the colon tumor lesion site, enhancing drug efficacy, and effectively controlling the proliferation and metastasis of colon cancer cells.

[0048] In a preferred embodiment of the present invention, the solid adsorbent is micronized silica gel. Micronized silica gel, used as a solid adsorbent in this invention, possesses advantages such as large specific surface area, high porosity, strong stability, and stable chemical properties, and is widely used in the pharmaceutical field. Its relatively increased surface area significantly enhances wettability and adsorption capacity in a liquid environment, providing strong assurance for the quality and efficacy of the drug, and effectively improving the stability and therapeutic effect of pH-sensitive norcantharidin solid microemulsion.

[0049] In a preferred embodiment of the present invention, the blank self-microemulsion is composed of an oil phase, an emulsifier, and a co-emulsifier. To further improve the water solubility and permeability of the self-microemulsion, preferably the oil phase is ethyl oleate, the emulsifier is polyoxyethylene hydrogenated castor oil, and the co-emulsifier is 1,2-propanediol; more preferably, the mass ratio of the oil phase, the emulsifier, and the co-emulsifier is 1:(1-1.5):(0.8-1.2), and more preferably 3:4:3.

[0050] In a preferred embodiment of the present invention, the pH-sensitive norcantharidin solid microemulsion has an average particle size of 70–80 nm, a polydispersity index of 0.18–0.24, a surface charge of -4.2–-3.7 mV, and a drug loading and encapsulation efficiency of 92–94% and 0.9–1.2%, respectively.

[0051] The pH-sensitive norcantharidin solid microemulsion of the present invention has a nano-sized particle size and uniform particle size distribution, and also has a high surface charge. Based on the above-mentioned excellent surface properties, the outer layer of the pH-sensitive norcantharidin solid microemulsion can target the tumor site according to the pH-triggered surface charge reversal characteristic, which allows it to interact with the negatively charged mucin in the colonic mucosa, thereby enhancing the accumulation of the solid microemulsion in the acidic tumor site and exerting a good therapeutic effect.

[0052] A second aspect of the present invention provides a method for preparing the aforementioned pH-sensitive norcantharidin solid microemulsion, comprising the following steps:

[0053] Chitosan was dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and triethylamine catalyst were added to react. After the reaction was completed, the mixture was purified by dialysis and freeze-dried to obtain a pH-sensitive polymer.

[0054] The oil phase, emulsifier, and co-emulsifier were mixed and stirred in proportion until a clear state was obtained to obtain a blank self-microemulsion.

[0055] The pH-sensitive polymer and norcantharidin were dissolved in the blank microemulsion and stirred to obtain a pH-sensitive norcantharidin microemulsion.

[0056] The pH-sensitive norcantharidin self-microemulsion was mixed with a solid adsorbent to obtain the pH-sensitive norcantharidin solid self-microemulsion.

[0057] This invention prepares a pH-sensitive polymer, maleamidated chitosan (CS-DMMA), by reacting 2,3-dimethylmaleic anhydride and chitosan. The maleamidated chitosan is then mixed with an oil phase, a surfactant, and a co-surfactant to prepare a pH-sensitive self-microemulsion. Norcantharidin is then added to prepare a pH-sensitive norcantharidin self-microemulsion. Finally, the pH-sensitive norcantharidin solid self-microemulsion (NCTD@CS-DMMA SSME) is obtained by adsorption with micronized silica gel.

[0058] This invention utilizes a solid adsorption method, employing a solid powder material (micronized silica gel) with good adsorption properties for lipid droplets, high density, and good flowability as the adsorbent to adsorb liquid pH-sensitive cantharidin self-microemulsion and solidify it into a solid powder. The adsorption and solidification process is simple and requires fewer types of excipients, solving the disadvantages of liquid formulations such as inconvenient storage and portability, high production costs, low stability, and limited dosage form selection. At the same time, it can minimize production costs and is suitable for large-scale formulation production.

[0059] To further improve the overall performance of the pH-sensitive norcantharidin solid self-microemulsion, in some preferred embodiments, the ratio of chitosan to 2,3-dimethylmaleic anhydride is 1–1.5:1; the solvent includes, but is not limited to, dimethyl sulfoxide; the mass concentration of norcantharidin in the pH-sensitive norcantharidin self-microemulsion is 1–30 mg / g; the mass concentration of the pH-sensitive polymer in the pH-sensitive norcantharidin self-microemulsion is 10–100 mg / g; and the mass ratio of the pH-sensitive norcantharidin self-microemulsion to the solid adsorbent is 1:0.3–0.8.

[0060] More preferably, the ratio of chitosan to 2,3-dimethylmaleic anhydride is 5:4, the mass concentration of norcantharidin in the pH-sensitive norcantharidin microemulsion is 10 mg / g, the mass concentration of pH-sensitive polymer in the pH-sensitive norcantharidin microemulsion is 40 mg / g, and the mass ratio of pH-sensitive norcantharidin microemulsion to the solid adsorbent is 1:0.6.

[0061] A third aspect of the present invention provides an application of a pH-sensitive norcantharidin solid microemulsion in the preparation of a drug for treating colon cancer.

[0062] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0063] Example 1

[0064] This embodiment provides a pH-sensitive norcantharidin solid self-microemulsion, comprising the following components in parts by weight: 1 part norcantharidin raw material (Dalian Meilun Biotechnology Co., Ltd., batch number: 20171107, purity ≥98%), 4 parts maleamidized chitosan, 60 parts micronized silica gel, and 95 parts blank self-microemulsion. The blank self-microemulsion is composed of ethyl oleate, polyoxyethylene hydrogenated castor oil, and 1,2-propanediol in a mass ratio of 3:4:3.

[0065] Its preparation method is carried out according to the following steps:

[0066] A. Synthesis of maleamide-modified chitosan:

[0067] Weigh CS and DMMA in a material ratio of 5:4. First, add CS to 10 mL of DMSO (dimethyl sulfoxide) and stir until completely dissolved. Then add DMMA and 1 mL of triethylamine catalyst and carry out a light-protected closed reaction under the action of the catalyst.

[0068] After the reaction, a 10% NaHCO3 solution was added to the system and transferred to a dialysis bag (MW=1000). The dialysis bag was placed in a NaOH aqueous solution with pH=8-9 for dialysis. The dialysis solution was changed every 2 hours to remove excess DMMA. After purification, the obtained CS-DMMA solution was freeze-dried to obtain maleamidized chitosan. The obtained product was stored at 4°C for later use and dissolved when the pH value was 6.5-6.8.

[0069] B. Preparation of blank self-microemulsion:

[0070] A blank microemulsion was obtained by mixing ethyl oleate (oil phase), polyoxyethylene hydrogenated castor oil (emulsifier), and 1,2-propanediol (co-emulsifier) ​​in a mass percentage ratio of 30%:40%:30% and stirring until clear.

[0071] C. Preparation of pH-sensitive norcantharidin self-microemulsion:

[0072] The synthesized CS-DMMA was added to a blank self-microemulsion at a concentration of 40 mg / g and stirred. Then, the NCTD active pharmaceutical ingredient was added to the blank self-microemulsion at a concentration of 10 mg / g and stirred until completely dissolved to obtain a pH-sensitive norcantharidin self-microemulsion (NCTD@CS-DMMA SME).

[0073] D. Preparation of pH-sensitive norcantharidin solid microemulsion:

[0074] pH-sensitive norcantharidin self-microemulsion and micronized silica gel were mixed at a mass ratio of 1:0.6. The mixture was added while stirring until homogeneous and then dried to obtain pH-sensitive norcantharidin solid self-microemulsion (NCTD@CS-DMMA SSME), which was stored at 4°C for subsequent use.

[0075] Example 2

[0076] A pH-sensitive norcantharidin solid self-microemulsion comprises the following components in parts by weight: 0.1 parts norcantharidin active pharmaceutical ingredient, 1 part maleamide-modified chitosan, 30 parts micronized silica gel, and 98.9 parts blank self-microemulsion. The blank self-microemulsion is composed of ethyl oleate, polyoxyethylene hydrogenated castor oil, and 1,2-propanediol in a mass ratio of 3:4:3. Its preparation method is the same as in Example 1, differing only in the proportions of the raw materials added.

[0077] Example 3

[0078] A pH-sensitive norcantharidin solid self-microemulsion comprises the following components in parts by weight: 3 parts norcantharidin active pharmaceutical ingredient, 10 parts maleamide chitosan, 80 parts micronized silica gel, and 87 parts blank self-microemulsion. The preparation method and raw materials are the same as in Example 1, differing only in the proportions of the raw materials added.

[0079] Characterization experiment

[0080] (I) Nuclear Magnetic Resonance Hydrogen Spectroscopy

[0081] The CS-DMMA synthesized in Example 1 was dissolved in heavy water (D2O) as a solvent, and its proton NMR spectrum was recorded at 400 MHz. A new chemical shift appeared at d = 1.90 ppm in the proton NMR spectrum. This shift was attributed to the characteristic proton peak of DMMA in CS-DMMA, indicating that the amino group in chitosan successfully reacted with 2,3-dimethylmaleic anhydride to form an amide bond. The results are shown in [Figure number missing]. Figure 1 .

[0082] (II) Tests for average particle size, polydispersity index and zeta potential

[0083] Three parallel preparations of the NCTD@CS-DMMA SSME from Example 1 were performed. The prepared solid microemulsions were light white powders. The results are shown in [Figure 1]. Figure 3 A. The average particle size, PDI, and Zeta potential were measured. The results showed that the average particle size of the prepared NCTD@CS-DMMA SSME was 75.88±0.85 nm, the PDI was 0.22±0.02, the surface charge was -3.9±0.12 mV, and the drug loading and encapsulation efficiency were 93.15±0.56% and 1.01±0.05%, respectively. Furthermore, the prepared NCTD@CS-DMMA SSME exhibited a pale blue opalescent appearance after emulsification, with a clear and transparent appearance. The experimental operation also showed good reproducibility and stability.

[0084] (III) In vitro release test under different pH conditions

[0085] The NCTD@CS-DMMA SSME from Example 1 was placed in 900 mL of PBS buffer at pH 6.5 and 7.4, respectively, and the cumulative release (%) of the drug at different time points was measured. The results showed that in the pH 6.5 release medium, the pH-sensitive norcantharidin solid microemulsion containing NCTD in Example 1 was rapidly released within the first two hours, with a cumulative release rate of 52.3% within 12 hours. However, when released in the pH 7.4 medium, the cumulative release rate within 12 hours was only 26.9%, indicating that the release of this formulation exhibits a more significant pH responsiveness. (See attached figures). Figure 2 .

[0086] (iv) Observation using transmission electron microscopy

[0087] The pH-sensitive norcantharidin solid microemulsion (NCTD@CS-DMMA SSME) from Example 1 was observed using transmission electron microscopy. The results showed that the solid microemulsion exhibited a uniform spherical structure. (See attached image.) Figure 3 B.

[0088] (V) FT-IR Analysis and Testing

[0089] The CS-DMMA and NCTD@CS-DMMA SSME synthesized in Example 1 were subjected to FT-IR analysis. Specifically, CS, DMMA, a physical mixture of CS and DMMA (CS+DMMA), and CS-DMMA and KBr were precisely weighed sequentially and mixed in a 1:100 ratio. Thin films were prepared by pelleting, and the samples were analyzed using a Fourier transform infrared spectrometer in the range of 500-3500 cm⁻¹. -1 Scanning analysis was performed within the wavelength range, and its FT-IR spectrum is shown below. Figure 4 Following the above method, CS-DMMA, micronized silica gel, NCTD active pharmaceutical ingredient, blank solid self-microemulsion, and NCTD@CS-DMMA SSME were sequentially scanned and analyzed. Their FT-IR spectra are shown below. Figure 3 C.

[0090] (vi) DSC Analysis and Testing

[0091] DSC analysis was performed on CS-DMMA and NCTD@CS-DMMA SSME from Example 1. Specifically, CS, DMMA, a physical mixture of CS and DMMA (CS+DMMA), and CS-DMMA were precisely weighed sequentially into an aluminum crucible. After covering the crucible with a lid, it was placed on a press and a small hole was punched in the lid. Using a blank aluminum crucible as a reference, DSC analysis was performed in the range of 50-250°C under nitrogen protection, with a heating rate of 15°C / min. The results are shown in [Figure / Reference]. Figure 5 Following the above method, CS-DMMA, micronized silica gel, NCTD active pharmaceutical ingredient, blank solid microemulsion (Blank SSME), and NCTD@CS-DMMA SSME were sequentially scanned and analyzed. The results are shown in the table below. Figure 3 D.

[0092] (vii) In situ intestinal absorption experiment, calculation of absorption rate constant K a and apparent absorption coefficient P app

[0093] 1) Take healthy male SD rats with an average weight of about 200-220g, acclimate them at room temperature for one week, and then randomly divide them into 3 groups, including NCTD-7.4 control group, NCTD@CS-DMMA SSME-6.5 group and NCTD@CS-DMMA SSME-7.4 group, with 3 rats in each group, and each group is housed in the same cage;

[0094] 2) Rats were fasted for 16 hours but given free access to water. First, rats were anesthetized by intraperitoneal injection of 5 ml / kg of 20% urethane solution and fixed to a rat board. Then, the intestine was opened layer by layer along the midline. Next, each segment of the intestine to be perfused was separated, a rubber tube was inserted, and the tube was secured with surgical sutures. The perfusion solution was injected into the intestinal segment at a flow rate of 0.2 mL / min for 60 min, with the next infusion and collection bottle replaced every 15 min. Finally, the rats were sacrificed, and the length (l) and radius (r) of the perfused segment were accurately measured. The absorption rate constant (K) was calculated by gravimetric method according to the following formula. a ) and apparent absorption coefficient (P app The calculation formula is as follows:

[0095] v = πr 2 l

[0096]

[0097] Where v is the volume of the perfused intestinal segment, Ci n and Cou t These represent the drug concentrations in the intestinal infusion fluid at the intestinal tract inlet and outlet, respectively. n and Vou t , where represents the volume of perfusion fluid at the intestinal inlet and outlet; Q represents the flow rate. The intestinal volume was calculated using the gravimetric method, based on the actual mass and fluid density before and after perfusion. The test results are shown below. Figure 6 Compared with NCTD-7.4, NCTD@CS-DMMA SSME-6.5 and NCTD@CS-DMMA-SSME-7.4 have *P<0.05.

[0098] (viii) In vitro antitumor effect experiment

[0099] 1) HT29 colon cancer cells were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-streptomycin. Cells were cultured at 5% CO2, 37°C, and 95% relative humidity, digested with 0.25% trypsin, and washed with PBS. When the cells reached 80%-90% confluence, cells in the logarithmic growth phase were harvested for further studies.

[0100] 2) NCTD@CS-DMMA SSME was added to phosphate buffer solutions at pH 6.5 and 7.4 to obtain microemulsions of NCTD@CS-DMMA SSME-6.5 (SSME-6.5) and NCTD@CS-DMMA SSME-7.4 (SSME-7.4). The in vitro antitumor activity of the samples under different pH conditions was determined using the MTT assay, and the results are shown in [Figure number missing]. Figure 7 A. HT29 cells were seeded in 96-well plates at 4 × 10⁶ cells per well. 3 Cells were incubated with 200 μL of culture medium for 24 h. Different concentrations of NCTD-6.5, NCTD-7.4, SSME-6.5, and SSME-7.4 were added at concentrations ranging from 5 to 80 μg / mL, and incubated for 24 h before removal. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued for 4 h. The crystals were then dissolved in 150 μL of LDMSO. Finally, the optical density (OD) was measured at 490 nm using a microplate reader, and cell viability was calculated. The results are shown in [Figure number missing]. Figure 7 E and 7F.

[0101] Cell viability (%) = OD (experimental group) / OD (control group) × 100%

[0102] 3) Take HT29 cells into 6-well plates at a density of 1.0 × 10⁶ cells per well. 6 Cells were cultured overnight in McCoy's 5A medium. HT29 cell monolayers were scraped with a p200 pipette tip, washed with PBS, and scratches were created. Cells were incubated in medium containing NCTD-6.5, NCTD-7.4, SSME-6.5, and SSME-7.4 (40 μg / mL), and migration was observed under a microscope at predetermined time points (12 h and 24 h). Results are shown below. Figure 7 B.

[0103] 4) Cells were digested with 0.25% trypsin and adjusted to 1×10⁻⁶ cells with serum-free medium. 6 mL. 200 μL of cell suspension was taken from the upper chamber and treated with NCTD-6.5, NCTD-7.4, SSME-6.5, and SSME-7.4, respectively. 600 μL of fresh culture medium containing 10% FBS was taken from the lower chamber. After incubation for 24 hours, the cells were fixed with 4% paraformaldehyde for 20 minutes. Unmigrated cells were removed from the surface with cotton swabs. After staining with 0.1% crystal violet for 10 minutes, the migrating cells were counted under an inverted microscope. The analysis results are shown below. Figure 7For C and 7E, compared with the control group, the P-value was ****P<0.0001; for SSME-6.5, compared with other treatment groups, the P-value was ****P<0.0001. The Transwell invasion assay and Transwell migration assay were performed using the same method. 100 μL of matrix gel (0.25 μg / mL) was pre-coated into the upper cavity of a 24-well Transwell with a diameter of 8 μm, and incubated at 37°C for 4 hours to form a gel. The results are shown in [Figure number missing]. Figure 7 For D and 7F, compared with the control group, ****P<0.0001; for SSME-6.5, compared with other treatment groups, ****P<0.0001.

[0104] (ix) In vivo antitumor activity experiment

[0105] 1) Healthy male SPF-grade Balb / c mice, with an average weight of approximately 16–18 g, were acclimatized at room temperature for one week. The mice were then randomly divided into 5 groups: a model control group (no medication), an NCTD SSME group, an NCTD-GLSO@CS-DMMA SSME group (using Ganoderma lucidum spore oil as the oil phase), a commercially available NCTD tablet group, and an NCTD@CS-DMMA SSME group, with 3 mice in each group. Each group was housed in the same cage. The experimental protocol is illustrated in the diagram below. Figure 8 A.

[0106] 2) CT-26Luc colon cancer cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin. Cells were cultured at 5% CO2, 37°C, and 95% relative humidity, digested with 0.25% trypsin, and washed with PBS. When the cells reached 80%-90% confluence, cells in the logarithmic growth phase were harvested for further studies.

[0107] 3) Mice were fasted for 24 hours, allowed free access to water, and anesthetized by intraperitoneal injection of 20% urethane (5 mL / kg). After disinfection with povidone-iodine, the cecum was carefully removed through a midline abdominal incision using sterile forceps. A 50 μL (1 × 10⁻⁶) suspension of CT-26Luc cells containing 10 μg / μL of matrix gel was injected using a 30G needle with a microsyringe. 6 The cecum was injected into the outer wall tissue layer of the cecum. The cecum was then returned to the abdominal cavity, and the peritoneum and skin were sutured. Tumor growth was observed using a small animal imaging system.

[0108] 4) After successful modeling, except for the model control group, the other four groups were treated with oral administration of the drug every two days starting on day 5, for a total of 14 days. Except for the low-dose group NCTD-GLSO@CS-DMMA SSME group (dose 2.6 mg / kg), the NCTD dose for all other groups was 5.2 mg / kg. During the treatment period, the body weight of the mice was observed daily. The body weight of the mice in each group showed a slow increasing trend, indicating that there was no significant systemic toxicity after oral administration. The results are shown in [see attached table]. Figure 8 C. Tumor size was measured using IVIS every 3 days for a total of 5 times. The tumors in the model control group continued to increase in size over time, while the tumors in each treatment group showed a shrinking trend, with the NCTD@CS-DMMA SSME group exhibiting the weakest fluorescence intensity. There was no significant difference in tumor growth between the NCTD SSME group and the saline group; results are shown in [see attached table]. Figure 8 B.

[0109] 5) Tumor-bearing mice were sacrificed, and tumors from each group were collected on day 2 after the last administration and weighed. The average tumor weights of the model control group, NCTDSSME group, NCTD-GLSO@CS-DMMA SSME group, NCTD tablet group, and NCTD@CS-DMMA SSME group were 1.21, 0.95, 0.44, 0.27, and 0.16 g, respectively. Compared with the control group, the tumor weight of NCTD tablets and NCTD@CS-DMMA SSME was reduced by 4.44 times and 7.43 times, respectively, indicating that NCTD@CS-DMMA SSME had a significant inhibitory effect on tumor growth. The results are shown in the table below. Figure 8 D and 8E. Immunophenotypic analysis of tumor tissues was performed using flow cytometry. Figure 8 The ratios of CD3 / CD4+ T cells and CD3 / CD8+ T cells in model A. Following NCTD@CS-DMMA SSME treatment, the proportions of CD4+ T cells and CD8+ T cells in tumor tissue increased; results are shown in [Figure A]. Figure 9 A. Immunofluorescence was performed on CD8+ T cells in tumor tissues of CT-26 orthotopic colon cancer mice in each group after drug administration. The fluorescence intensity of NCTD@CS-DMMA SSME CD8+ T cells was the highest in the tumor, indicating that this agent promotes T cell upregulation in immune regulation. Results are shown in [Figure number missing]. Figure 9 B.

[0110] 6) Heart, liver, spleen, lung, kidney, colon, and tumor tissues were collected and preserved in 4% paraformaldehyde tissue fixative for hematoxylin-eosin (H&E) staining. Except for the colon tissue of the model group mice, which showed significant mucosal damage and almost complete disappearance of goblet cells, no damage or necrosis was observed in the other mouse tissues, indicating that the preparation has no systemic toxicity. The section images are shown below. Figure 10The TUNEL assay was used to detect cell apoptosis in each group. Compared with other drug-treated groups, NCTD@CS-DMMA SSME showed the largest apoptotic area, indicating that a large number of tumor cells underwent apoptosis. The results are shown in [Figure number missing]. Figure 11 A. Ki67 assay was used to detect cell proliferation in each group. Compared with the model group, the expression level of Ki67 in tumor tissues decreased in all treatment groups, with the largest decrease observed in NCTD@CS-DMMA SSME, indicating that it can significantly inhibit the proliferation of transplanted tumor cells. Results are shown in [Figure number missing]. Figure 11 B.

[0111] The schematic diagram of the preparation process of the pH-sensitive norcantharidin solid self-microemulsion of the present invention and its targeted release at the tumor site is shown below. Figure 12 As shown, in situ intestinal absorption experiments demonstrate that the pH-sensitive norcantharidin solid self-microemulsion of the present invention can effectively increase the absorption rate constant K of norcantharidin at the colon tumor site. a and apparent transparency coefficient P app In vitro antitumor effects showed that pH-sensitive norcantharidin solid microemulsion could enhance the ability to inhibit tumor growth, migration and invasion; in vivo efficacy showed that treatment with pH-sensitive norcantharidin solid microemulsion could increase the proportion of CD4+T and CD8+T cells, inhibit tumor cell proliferation, and promote apoptosis, thus exerting good in vivo antitumor activity, indicating that this preparation can significantly improve the treatment effect of colon cancer.

[0112] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. The present invention can also be implemented in other specific ways or forms without departing from its spirit or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of the present invention should be defined by the appended claims, and any variations equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A pH-sensitive norcantharidin solid microemulsion, characterized in that, The product comprises the following components in parts by weight: 0.1–3 parts norcantharidin, 1–10 parts pH-sensitive polymer, 30–80 parts solid adsorbent, and 87–98.9 parts blank self-microemulsion; wherein the pH-sensitive polymer is maleamide-modified chitosan; wherein the maleamide-modified chitosan is composed of 2,3 The blank self-microemulsion was prepared by modifying chitosan with dimethyl maleic anhydride; the blank self-microemulsion was composed of an oil phase, an emulsifier and a co-emulsifier; wherein the oil phase was ethyl oleate, the emulsifier was polyoxyethylene hydrogenated castor oil, the co-emulsifier was 1,2-propanediol; and the solid adsorbent was micronized silica gel.

2. The pH-sensitive norcantharidin solid microemulsion according to claim 1, characterized in that, The mass ratio of the oil phase, the emulsifier, and the co-emulsifier is 1:(1~1.5):(0.8~1.2).

3. The pH-sensitive norcantharidin solid microemulsion according to any one of claims 1 or 2, characterized in that, The pH-sensitive cantharidin solid microemulsion has an average particle size of 70-80 nm, a polydispersity index of 0.18-0.24, a surface charge of -4.2-3.7 mV, an encapsulation efficiency of 92-94%, and a drug loading of 0.9-1.2%.

4. A method for preparing a pH-sensitive norcantharidin solid microemulsion as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Chitosan was dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and triethylamine catalyst were added to react. After the reaction was completed, the mixture was purified by dialysis and freeze-dried to obtain a pH-sensitive polymer. The oil phase, emulsifier, and co-emulsifier were mixed and stirred in proportion until a clear state was obtained to obtain a blank self-microemulsion. The pH-sensitive polymer and norcantharidin were dissolved in the blank microemulsion and stirred to obtain a pH-sensitive norcantharidin microemulsion. The pH-sensitive norcantharidin self-microemulsion was mixed with a solid adsorbent to obtain the pH-sensitive norcantharidin solid self-microemulsion.

5. The method for preparing pH-sensitive norcantharidin solid microemulsion according to claim 4, characterized in that, The ratio of chitosan to 2,3-dimethylmaleic anhydride is 1~1.5:

1.

6. The method for preparing pH-sensitive norcantharidin solid microemulsion according to claim 4, characterized in that, The pH-sensitive norcantharidin in the microemulsion has a mass concentration of 1-30 mg / g; the pH-sensitive norcantharidin in the microemulsion has a mass concentration of 10-100 mg / g.

7. The use of a pH-sensitive norcantharidin solid microemulsion as described in any one of claims 1 to 3 in the preparation of a drug for treating colon cancer.

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