PH-sensitive norcantharidin solid self-microemulsion as well as preparation method and application thereof

Abstract

The invention discloses a pH-sensitive norcantharidin solid self-microemulsion as well as a preparation method and application thereof, and belongs to the technical field of medicines, the pH-sensitive norcantharidin solid self-microemulsion comprises the following components in parts by weight: 0.1-3 parts of norcantharidin, 1-10 parts of a pH-sensitive polymer, 30-80 parts of a solid adsorbent and 87-98.9 parts of a blank self-microemulsion. The pH-sensitive norcantharidin solid self-microemulsion disclosed by the invention can improve the solubility and permeability of a drug, and because an amido bond of a pH-sensitive polymer is broken under an acidic condition, the drug is regularly released from the interior of the nano solid self-microemulsion, so that targeted release at a tumor site is caused. Animal experiments show that the pH-sensitive norcantharidin solid self-microemulsion can effectively improve the absorption rate constant and apparent permeability coefficient of norcantharidin at a colon tumor part, can improve the ability of inhibiting tumor growth, migration and invasion, and further can significantly improve the treatment effect on colon tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a pH-sensitive norcantharidin solid self-microemulsion and a preparation method and application thereof. Background Art

[0002] Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies in clinical practice, and its global incidence ranks third among cancers. The disease is difficult to treat, has a high mortality rate, and its incidence rate is increasing year by year, which has posed a serious threat to human life and health. For this disease, patients rely on surgery and chemotherapy to control the disease, but the high recurrence rate and high complication rate after surgery have always been at the forefront of various cancers.

[0003] Norcantharidin (NCTD) is a synthetic derivative of cantharidin, an effective ingredient extracted from insect cantharidin. Clinical and experimental studies have confirmed that it has effective anti-tumor effects on a variety of tumor cells such as esophageal cancer, bile duct cancer, lung cancer and colorectal cancer. The results of related experimental studies show 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 improving leukocyte levels, regulating immunity, and not producing bone marrow suppression. Currently, the commercially available NCTD drug formulations are mainly injection formulations and tablet formulations. Due to the poor water solubility and permeability of the drug, the irritation to the injection is relatively large, and the oral bioavailability is relatively low. In addition, traditional oral drugs or injections cannot guarantee the effective concentration and bioavailability of the tumor site, and produce serious systemic toxicity. Therefore, it is necessary to design a new formulation to improve the solubility and permeability of the drug in the present invention to achieve tumor-targeted drug delivery. Summary of the invention

[0004] The main purpose of the present invention is to provide a pH-sensitive norcantharidin solid self-microemulsion and its preparation method and application. The pH-sensitive norcantharidin solid self-microemulsion prepared by the present invention can improve the solubility and permeability of the drug and realize tumor-targeted drug delivery. In addition, the outer layer of the pH-sensitive norcantharidin solid self-microemulsion of the present invention can be targeted to the tumor site according to the surface charge reversal characteristics triggered by pH, so that they can interact with the negatively charged mucin in the colon mucosa, thereby enhancing the accumulation of the solid self-microemulsion in the acidic tumor site to exert a good therapeutic effect.

[0005] Norcantharidin has a strong anti-tumor effect on a variety of cancer cells. The present invention first prepares a pH-sensitive norcantharidin self-microemulsion, then adds micropowder silica gel to solidify the self-microemulsion to obtain a pH-sensitive norcantharidin solid self-microemulsion, and examines its in vitro release results.

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

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

[0008] Furthermore, the solid adsorbent is micro-powder silica gel.

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

[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 self-microemulsion has an average particle size of 70 to 80 nm, a polydispersity index of 0.18 to 0.24, a surface charge of -4.2 to -3.7 mV, and a drug loading and encapsulation efficiency of 92 to 94% and 0.9 to 1.2%, respectively.

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

[0013] The chitosan is dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and a catalyst triethylamine are added to react, and after the reaction is completed, the chitosan is dialyzed, purified and freeze-dried to obtain a pH-sensitive polymer.

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

[0015] Dissolving the pH-sensitive polymer and norcantharidin in the blank self-microemulsion, stirring and dissolving to obtain a pH-sensitive norcantharidin self-microemulsion;

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

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

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

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

[0020] Another aspect of the present invention provides a use of the aforementioned pH-sensitive norcantharidin solid self-microemulsion in the preparation of a drug for treating colon cancer.

[0021] Another aspect of the present invention provides the aforementioned pH-sensitive norcantharidin solid self-microemulsion for use in treating colon cancer.

[0022] Compared with the prior art, 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 preparation, which can improve the solubility and permeability of the drug, avoid the release of the drug in the upper gastrointestinal tract (stomach and small intestine), and release it at the tumor site. Due to the presence of CS-DMMA, the amide bond breaks under acidic conditions (pH is 6.5-6.8), resulting in the outer layer of the pH-sensitive norcantharidin solid self-microemulsion being unstable, and the drug is regularly released from the inside of the nano solid self-microemulsion. At the same time, the presence of solid adsorbent micropowder silica gel improves the stability of the preparation, so that the drug is targeted for release at the tumor site.

[0023] In addition, according to the in situ intestinal absorption test, the pH-sensitive norcantharidin solid self-microemulsion of the present invention can effectively improve the absorption rate constant K of norcantharidin in the colon tumor site. a and the apparent permeability coefficient P app The in vitro antitumor effect showed that pH-sensitive norcantharidin solid self-microemulsion could improve the ability to inhibit tumor growth, migration and invasion; the in vivo efficacy showed that treatment with pH-sensitive norcantharidin solid self-microemulsion could increase the proportion of CD4+T and CD8+T cells, inhibit the proliferation of tumor cells, promote apoptosis and thus exert good in vivo antitumor activity, indicating that this preparation can significantly improve the therapeutic effect of colon tumors. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 The in vitro release curve of the pH-sensitive norcantharidin solid self-microemulsion under different pH conditions in Example 1 of the present invention is shown;

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

[0027] Figure 4 The FTIR analysis diagram of the maleamidated chitosan synthesized in Example 1 of the present invention is shown: wherein, (a) CS; (b) DMMA; (c) CS+DMMA (physical mixing); (d) CS-DMMA;

[0028] Figure 5 The DSC analysis diagrams of the maleamidated chitosan synthesized in Example 1 of the present invention are shown: (a) CS; (b) DMMA; (c) CS+DMMA (physical mixing); (d) CS-DMMA;

[0029] Figure 6 The absorption rate constants 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 are shown. a and the 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 is a diagram showing the analysis results of the in vitro tumor effect experiment of the pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention:

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

[0033] Figure 8 The following are a schematic diagram of the experimental scheme of the in vivo antitumor activity experiment of the pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention, a bioluminescent imaging diagram of CT-26 tumor-bearing mice, a tumor anatomy diagram, and a comparison diagram 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 scheme; (B) is the in vivo bioluminescence imaging of three different CT-26 tumor-bearing mice; (C) is the situation of tumors removed 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] Fig. 9 The analysis diagram of the in vivo antitumor activity experiment of the pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention is shown:

[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 administration;

[0037] Fig.10 The main organ tissues and tumor tissue pathological sections of CT-26 tumor-bearing mice in the in vivo antitumor activity experiment of the pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention are shown;

[0038] Fig.11 The graphs showing the comparative analysis of apoptosis and proliferation of tumor cells in CT-26 carcinoma in situ mice under the action of different drugs in the in vivo anti-tumor activity experiment of the pH-sensitive norcantharidin solid self-microemulsion in Example 1 of the present invention are shown, and the scale bar is 1:50 μm;

[0039] Among them, (A) is the TUNEL method to detect the apoptosis of tumor cells in CT-26 carcinoma in situ mice in each drug administration group; (B) is the Ki67 immunohistochemistry method to detect the proliferation of tumor cells in CT-26 carcinoma in situ mice in each drug administration group;

[0040] Fig.12 The schematic diagram shows the preparation process of the pH-sensitive norcantharidin solid self-microemulsion of the present invention and its tumor site targeted release. DETAILED DESCRIPTION

[0041] It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or the intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. The present invention will be described in detail below in conjunction with the embodiments.

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

[0043] Solid self-emulsifying drug delivery system (SSME) is a combination of self-microemulsifying drug delivery system (SME) and solid dosage form, and the selection of suitable solid carriers makes the self-microemulsifying drug delivery system solid. Solid self-microemulsifying drug delivery system can solve the shortcomings of self-emulsifying drug delivery system as liquid preparation, inconvenient storage and carrying, high production cost, low stability, few dosage form selection, etc., and can improve drug efficacy and reduce drug side effects.

[0044] In order to implement the above-mentioned purpose, the first aspect of the embodiment 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 of norcantharidin, 1 to 10 parts of pH-sensitive polymer, 30 to 80 parts of solid adsorbent and 87 to 98.9 parts of blank self-microemulsion.

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

[0046] Maleamidated chitosan (CS-DMMA) is synthesized from low molecular weight chitosan and 2,3-dimethylmaleic anhydride. Chitosan (CS) is a natural polymer compound with abundant resources and low price. It is obtained by N-deacetylation of chitin. It has good biodegradability, biocompatibility, bioadhesion and film-forming properties, so it is widely used as a drug carrier. CS is a natural cationic polymer containing a large number of amine groups. It can react with 2,3-dimethylmaleic anhydride (DMMA) to form 2,3-dimethylmaleic anhydride amide bonds. It is very easy to hydrolyze in a weakly acidic environment (pH 6.5-6.8). Therefore, CS-DMMA can be used as a carrier to release drugs in a tumor-specific pH environment.

[0047] Based on the fact that the tumor tissue (pH=6.5-6.8) is more acidic than the normal tissue (pH=7.4) under the tumor microenvironment, the present invention introduces the synthetic pH-sensitive polymer maleamidated chitosan (CS-DMMA) into the self-microemulsification formula to prepare a pH-sensitive norcantharidin solid self-microemulsion with the ability to release drugs in targeted tumor tissues, so as to improve the solubility and permeability of the drug while avoiding the premature release of the drug in the stomach and small intestine, thereby increasing the drug concentration in the colon tumor lesion, thereby improving the 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 micropowder silica gel. The present invention uses micropowder silica gel as a solid adsorbent, which has the advantages of large specific surface area, high porosity, strong stability, stable chemical properties, etc., and is widely used in the field of pharmaceuticals. Its relatively increased surface area, when in a liquid environment, greatly increases wettability and enhances adsorption capacity, provides a strong guarantee for the quality and efficacy of the drug, and can effectively improve the stability and therapeutic effect of the pH-sensitive norcantharidin solid self-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. In order 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-propylene glycol; further 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 self-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 self-microemulsion of the present invention has a nanometer-scale particle size and a uniform particle size distribution, and has a relatively high surface charge. Based on the above-mentioned excellent surface properties, the outer layer of the pH-sensitive norcantharidin solid self-microemulsion can be targeted to the tumor site according to the pH-triggered surface charge reversal characteristics, so that they can interact with negatively charged mucins in the colon mucosa, thereby enhancing the accumulation of the solid self-microemulsion in the acidic tumor site and exerting a good therapeutic effect.

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

[0053] The chitosan is dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and a catalyst triethylamine are added to react, and after the reaction is completed, the chitosan is dialyzed, purified and freeze-dried to obtain a pH-sensitive polymer.

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

[0055] Dissolving the pH-sensitive polymer and norcantharidin in the blank self-microemulsion, stirring and dissolving to obtain a pH-sensitive norcantharidin self-microemulsion;

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

[0057] The invention prepares pH-sensitive polymer maleamidated chitosan (CS-DMMA) by reacting 2,3-dimethylmaleic anhydride and chitosan, mixes the maleamidated chitosan with an oil phase, a surfactant and a co-surfactant to prepare a pH-sensitive self-microemulsion, then adds norcantharidin to prepare a pH-sensitive norcantharidin self-microemulsion, and finally uses micropowder silica gel for adsorption to obtain a pH-sensitive norcantharidin solid self-microemulsion (NCTD@CS-DMMA SSME).

[0058] The present invention adopts a solid adsorption method, adopts a solid powder material (micropowder silica gel) with good adsorption to lipid oil droplets, good self-density and good fluidity as an adsorbent, adsorbs liquid pH-sensitive norcantharidin self-microemulsion, and solidifies the liquid pH-sensitive norcantharidin self-microemulsion into solid powder. The adsorption and solidification process is simple and the types of auxiliary materials are few. The shortcomings of liquid preparations such as inconvenient storage and carrying, high production cost, low stability, and few dosage form options are solved. At the same time, the production cost can be reduced as much as possible, and the preparation is suitable for large-scale production of preparations.

[0059] In order to further improve the comprehensive performance of the pH-sensitive norcantharidin solid self-microemulsion, in some preferred embodiments, the raw material ratio of the chitosan and the 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; the mass ratio of the pH-sensitive norcantharidin self-microemulsion to the solid adsorbent is 1:0.3-0.8.

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

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

[0062] The present application is further described in detail below in conjunction with specific embodiments. These embodiments 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 of norcantharidin API (Dalian Meilun Biotechnology Co., Ltd., batch number: 20171107, purity ≥ 98%), 4 parts of maleamidated chitosan, 60 parts of micropowdered silica gel, and 95 parts of blank self-microemulsion. The blank self-microemulsion is composed of ethyl oleate, polyoxyethylene hydrogenated castor oil and 1,2-propylene glycol, and the mass ratio is 3:4:3.

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

[0066] A. Synthesis of maleamidated chitosan:

[0067] CS and DMMA were weighed at a material ratio of 5:4. CS was first added to 10 mL of DMSO (dimethyl sulfoxide) and stirred until completely dissolved. Then DMMA and 1 mL of catalyst triethylamine were added to perform a light-shielded sealing reaction under the action of the catalyst.

[0068] After the reaction, 10% NaHCO was added to the system.3 The solution was transferred into a dialysis bag (MW=1000), and the dialysis bag was placed in a NaOH aqueous solution with a pH value of 8-9 for dialysis. The dialysate was changed every 2 hours to remove excess DMMA. After purification, the obtained CS-DMMA solution was freeze-dried to obtain maleamidated chitosan, and the obtained product was stored at a low temperature of 4°C for future use. It dissolved when the pH value was 6.5-6.8.

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

[0070] Ethyl oleate (oil phase): polyoxyethylene hydrogenated castor oil (emulsifier): 1,2-propylene glycol (co-emulsifier) ​​were mixed in a mass percentage of 30%:40%:30%, and stirred until clear to obtain a blank self-microemulsion.

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

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

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

[0074] The pH-sensitive norcantharidin self-microemulsion and micropowdered silica gel were mixed in a mass ratio of 1:0.6, added by stirring, stirred and mixed evenly, and then dried to obtain a 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 of norcantharidin API, 1 part of maleamidated chitosan, 30 parts of micropowdered silica gel and 98.9 parts of a blank self-microemulsion. The blank self-microemulsion is composed of ethyl oleate, polyoxyethylene hydrogenated castor oil and 1,2-propylene glycol in a mass ratio of 3:4:3. The preparation method thereof is referred to Example 1, except for the addition ratio of the raw materials.

[0077] Example 3

[0078] A pH-sensitive norcantharidin solid self-microemulsion comprises the following components in parts by weight: 3 parts of norcantharidin API, 10 parts of maleamidated chitosan, 80 parts of micropowder silica gel and 87 parts of blank self-microemulsion. The preparation method and raw materials thereof refer to those in Example 1, and the only difference is the addition ratio of the raw materials.

[0079] Characterization experiments

[0080] (I) Nuclear Magnetic Resonance Proton Spectrum Test

[0081] The CS-DMMA synthesized in Example 1 was treated with heavy water (D 2 O) as a solvent, and the hydrogen spectrum of CS-DMMA was recorded at 400MHz using a nuclear magnetic resonance hydrogen spectrometer. In the hydrogen spectrum, a new chemical shift appeared at d = 1.90ppm. This chemical shift was attributed to the typical 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 1 .

[0082] (II) Average particle size, polydispersity index and Zeta potential test

[0083] The NCTD@CS-DMMA SSME in Example 1 was prepared in parallel for 3 times, and the prepared solid self-microemulsion was a light white powder. Figure 3 A. The average particle size, PDI and Zeta potential were measured respectively. The average particle size of the prepared NCTD@CS-DMMA SSME was 75.88±0.85nm, PDI was 0.22±0.02, the surface charge was -3.9±0.12mV, the drug loading and encapsulation efficiency were 93.15±0.56% and 1.01±0.05%, respectively. In the experiment, the prepared NCTD@CS-DMMA SSME showed a light blue opalescence after emulsification, and the appearance was clear and transparent, and the experimental operation was reproducible and stable.

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

[0085] The NCTD@CS-DMMA SSME in 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 the NCTD contained in the pH-sensitive norcantharidin solid self-microemulsion in the NCTD@CS-DMMA SSME in Example 1 was rapidly released in the first two hours in the release medium at pH 6.5, and the cumulative release within 12 hours was 52.3%. When released in the medium at pH 7.4, the cumulative release within 12 hours was only 26.9%, indicating that the release of the preparation showed a relatively obvious pH responsiveness. The results are shown in FIG. Figure 2 .

[0086] (IV) Transmission electron microscopy observation

[0087] The pH-sensitive norcantharidin solid self-microemulsion (NCTD@CS-DMMA SSME) in Example 1 was observed using a transmission electron microscope. The results showed that the solid self-microemulsion had a uniform spherical structure. Figure 3 B.

[0088] (V) FT-IR analysis test

[0089] The CS-DMMA and NCTD@CS-DMMA SSME synthesized in Example 1 were subjected to FT-IR analysis. That is, CS, DMMA, a physical mixture of CS and DMMA (CS+DMMA), and CS-DMMA and KBr were accurately weighed in sequence and mixed in a ratio of 1:100, and thin slices were prepared by a tabletting method. The samples were then analyzed by a Fourier transform infrared spectrometer at 500-3500 cm -1 The FT-IR spectrum is shown in Figure 4 ; Referring to the above method, CS-DMMA, micropowder silica gel, NCTD API, blank solid self-microemulsion and NCTD@CS-DMMA SSME were scanned and analyzed in turn, and the FT-IR spectrum was shown in Figure 3 C.

[0090] (VI) DSC analysis test

[0091] The CS-DMMA and NCTD@CS-DMMA SSME in Example 1 were subjected to DSC analysis. That is, CS, DMMA, a physical mixture of CS and DMMA (CS+DMMA), and CS-DMMA were accurately weighed in an aluminum crucible in sequence, and the crucible was covered and placed on a press, and a small hole was pierced in the crucible cover. A blank aluminum crucible was used as a reference, and scanning analysis was performed in the range of 50-250°C under nitrogen protection, and the heating rate was set to 15°C / min. The results are shown in FIG. Figure 5 ; Referring to the above method, CS-DMMA, micropowder silica gel, NCTD API, blank solid self-microemulsion (Blank SSME) and NCTD@CS-DMMA SSME were scanned and analyzed in turn. The results are shown in Figure 3 D.

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

[0093] 1) Healthy male SD rats with an average body weight of about 200-220 g were taken and adapted to feeding at room temperature for one week. The rats were randomly divided into three 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 was housed in the same cage;

[0094] 2) Rats were fasted for 16 hours and were allowed to drink water freely. First, the rats were anesthetized by intraperitoneal injection of 5 ml / kg of 20% urethane solution, fixed on a rat board, and then opened layer by layer along the midline of the abdomen. Next, each segment of the intestine to be perfused was separated, a rubber tube was inserted and tied with surgical sutures. The perfusion fluid was injected into the intestinal segment at a flow rate of 0.2 mL / min for 60 minutes. During this period, the next fluid supply bottle and collection bottle were replaced every 15 minutes. Finally, the rats were killed, and the length (l) and radius (r) of the perfusion segment were accurately measured. The absorption rate constant (K) was calculated by the weight method according to the following formula: a ) and the apparent absorption coefficient (P app ), which is calculated as follows:

[0095] v=πr 2 l

[0096]

[0097] Where v is the volume of the perfused intestinal segment, Ci n and Cou t are the drug concentrations of the intestinal inlet and outlet perfusates, Vi n and Vou t are the volumes of the inlet and outlet perfusion fluids of the intestine respectively; Q is the flow rate. The inlet and outlet volumes of the intestine are calculated by the weight method, that is, according to the actual mass before and after perfusion and the density of the liquid before and after perfusion, the test results are shown in Figure 6 , NCTD@CS-DMMA SSME-6.5, NCTD@CS-DMMA-SSME-7.4 were compared with NCTD-7.4, *P<0.05.

[0098] (VIII) In vitro anti-tumor effect experiment

[0099] 1) HT29 colon cancer cells were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-streptomycin. The cells were incubated at 5% CO 2 The cells were cultured at 37°C and 95% relative humidity, digested with 0.25% trypsin, and washed with PBS. When the cells grew to 80%-90%, cells in the logarithmic growth phase were taken for subsequent research.

[0100] 2) NCTD@CS-DMMA SSME was added to phosphate buffer 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 by MTT method. The results are shown in Figure 7 A. HT29 cells were seeded in 96-well plates, with 4×10 3 Cells were incubated with 200 μL of culture medium for 24 h. NCTD-6.5, NCTD-7.4, SSME-6.5 and SSME-7.4 were added at different concentrations of 5 to 80 μg / mL and removed after 24 h of incubation. Then, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 4 h. The crystals were dissolved in 150 μL of DMSO. Finally, the optical density (OD) value was measured at 490 nm using a microplate reader to calculate the cell activity. The results are shown in Figure 7 E and 7F.

[0101] Cell activity (%) = 0D (experimental group) / 0D (control group) × 100%

[0102] 3) HT29 cells were taken from a 6-well plate at a density of 1.0×10 6 Cells were cultured overnight in McCoy's 5A medium. HT29 cell monolayers were scraped with a p200 pipette tip and washed with PBS to create scratches. Cells were incubated in medium containing NCTD-6.5, NCTD-7.4, SSME-6.5, and SSME-7.4 (40 μg / mL) and their migration was observed under a microscope at predetermined time points (12 h and 24 h). The results are shown in Figure 7 B.

[0103] 4) The cells were digested with 0.25% trypsin and adjusted to 1×10 6 mL. Take 200 μL of cell suspension from the upper chamber and treat with NCTD-6.5, NCTD-7.4, SSME-6.5 and SSME-7.4 respectively, and take 600 μL of fresh culture medium containing 10% FBS from the lower chamber. After incubation for 24 hours, fix with 4% paraformaldehyde for 20 minutes, remove the non-migrated cells on the surface with a cotton swab, and count the migrated cells under an inverted microscope after staining with 0.1% crystal violet for 10 minutes. The analysis results are shown in Figure 7C and 7E, each drug-treated group was compared with the control group, ****P<0.0001; SSME-6.5 was compared with other drug-treated groups, ****P<0.0001. The Transwell invasion assay was the same as the Transwell migration assay. 100 μL of matrix gel (0.25 μg / mL) was pre-coated in the upper chamber 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 7 D and 7F, each drug-treated group was compared with the control group, ****P<0.0001; SSME-6.5 was compared with other drug-treated groups, ****P<0.0001.

[0104] (IX) In vivo anti-tumor activity experiment

[0105] 1) Healthy male SPF Balb / c mice with an average body weight of about 16-18 g were obtained and adaptively raised at room temperature for one week. The mice were randomly divided into 5 groups, including a model control group (untreated group), a NCTD SSME group, a NCTD-GLSO@CS-DMMA SSME group (Ganoderma lucidum spore oil as the oil phase), a commercially available NCTD tablet group, and a NCTD@CS-DMMA SSME group, with 3 mice in each group, and each group was raised in the same cage; the diagram of the experimental scheme is shown in Figure 8 A.

[0106] 2) CT-26Luc colon cancer cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin. The cells were incubated at 5% CO 2 , 37°C, 95% relative humidity, digested with 0.25% trypsin, and washed with PBS. When the cells grew to 80%-90%, cells in the logarithmic growth phase were taken for subsequent research;

[0107] 3) The mice were fasted for 24 hours, with free access to water, and anesthetized by intraperitoneal injection of 20% urethane (5 mL / kg). After disinfection with iodine, a midline incision was made along the abdomen of the mice, and the cecum was carefully removed with sterile forceps. A 30G needle with a microsyringe was used to inject a CT-26Luc cell suspension (50 μL, 1×10 6 ) was injected into the outer wall tissue layer of the cecum. Subsequently, the cecum was returned to the abdominal cavity, and the peritoneum and skin were sutured. The 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 given oral administration every two days starting from the 5th day for 14 consecutive days. Except for the low-dose group NCTD-GLSO@CS-DMMA SSME group, which was given a dose of 2.6 mg / kg, the NCTD doses of the other groups were all 5.2 mg / kg. During the administration period, the weight changes of the mice were observed every day. The weight of the mice in each group showed a slow increase trend, indicating that there was no obvious systemic toxicity after oral administration. The results are shown in Figure 8 C. Tumor size was detected by IVIS once every 3 days for a total of 5 times. The tumor in the model control group continued to grow over time, while the tumors in each drug-treated group showed a trend of shrinking. The fluorescence intensity of the NCTD@CS-DMMA SSME group was the weakest. There was no significant difference in tumor growth between the NCTD SSME group and the saline group. The results are shown in Figure 8 B.

[0109] 5) Tumor-bearing mice were killed, and tumors from each group were collected and weighed on the second day after the last administration. 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 weights of NCTD tablets and NCTD@CS-DMMA SSME were 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 Figure 8 D and 8E. Immunophenotyping of tumor tissues was performed by flow cytometry analysis Figure 8 The ratio of CD3 / CD4+T and CD3 / CD8+T in model A. After NCTD@CS-DMMA SSME treatment, the ratio of CD4+T and CD8+T cells in tumor tissue increased. Fig. 9 A. After administration, CD8+T cells in the tumor tissues of CT-26 orthotopic colon cancer mice in each group were subjected to immunofluorescence. The fluorescence intensity of NCTD@CS-DMMA SSME CD8+T cells was the highest in the tumor, indicating that the preparation promoted T cell upregulation in immune regulation. The results are shown in Fig. 9 B.

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

[0111] The schematic diagram of the preparation process of the pH-sensitive norcantharidin solid self-microemulsion and its tumor-targeted release is as follows: Fig.12 As shown in the in situ intestinal absorption test, the pH-sensitive norcantharidin solid self-microemulsion of the present invention can effectively improve the absorption rate constant K of norcantharidin in the colon tumor site. a and the apparent permeability coefficient P app The in vitro antitumor effect showed that pH-sensitive norcantharidin solid self-microemulsion could improve the ability to inhibit tumor growth, migration and invasion; the in vivo efficacy showed that treatment with pH-sensitive norcantharidin solid self-microemulsion could increase the proportion of CD4+T and CD8+T cells, inhibit the proliferation of tumor cells, promote apoptosis and thus exert good in vivo antitumor activity, indicating that this preparation can significantly improve the therapeutic effect of colon tumors.

[0112] The above-described embodiments only represent the embodiments of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. The present invention can also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described embodiments should be regarded as illustrative rather than restrictive in any respect. The scope of the present invention should be described by the attached claims, and any changes equivalent to the intention and scope of the claims should also be included in the scope of the present invention.

Claims

1. A pH-sensitive norcantharidin solid self-microemulsion, characterized in that: The invention comprises the following components in parts by weight: 0.1 to 3 parts of norcantharidin, 1 to 10 parts of pH-sensitive polymer, 30 to 80 parts of solid adsorbent and 87 to 98.9 parts of blank self-microemulsion.

2. The pH-sensitive norcantharidin solid self-microemulsion according to claim 1, characterized in that: The pH sensitive polymer is maleamidated chitosan.

3. The pH-sensitive norcantharidin solid self-microemulsion according to claim 1, characterized in that: The solid adsorbent is micro powder silica gel.

4. The pH-sensitive norcantharidin solid self-microemulsion according to claim 1, characterized in that: The blank self-microemulsion consists of an oil phase, an emulsifier and an auxiliary emulsifier; Wherein, the oil phase is ethyl oleate, the emulsifier is polyoxyethylene hydrogenated castor oil, and the co-emulsifier is 1,2-propylene glycol.

5. The pH-sensitive norcantharidin solid self-microemulsion according to claim 4, 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).

6. The pH-sensitive norcantharidin solid self-microemulsion according to any one of claims 1 to 5, characterized in that: The pH-sensitive norcantharidin solid self-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 rate of 92-94% and 0.9-1.2% respectively.

7. A method for preparing the pH-sensitive norcantharidin solid self-microemulsion according to any one of claims 1 to 6, characterized in that: The following steps are involved: The chitosan is dissolved in a solvent, and then 2,3-dimethylmaleic anhydride and a catalyst triethylamine are added to react, and after the reaction is completed, the chitosan is dialyzed, purified and freeze-dried to obtain a pH-sensitive polymer. The oil phase, emulsifier and co-emulsifier are mixed and stirred in proportion until a clear state is obtained to obtain a blank self-microemulsion; Dissolving the pH-sensitive polymer and norcantharidin in the blank self-microemulsion, stirring and dissolving to obtain a pH-sensitive norcantharidin self-microemulsion; The pH-sensitive norcantharidin self-microemulsion is mixed with a solid adsorbent to obtain the pH-sensitive norcantharidin solid self-microemulsion.

8. The method for preparing the pH-sensitive norcantharidin solid self-microemulsion according to claim 7, characterized in that: The raw material ratio of the chitosan to the 2,3-dimethylmaleic anhydride is 1-1.5:

1.

9. The method for preparing the pH-sensitive norcantharidin solid self-microemulsion according to claim 7, characterized in that: 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.

10. Use of the pH-sensitive norcantharidin solid self-microemulsion according to any one of claims 1 to 5 in the preparation of a drug for treating colon cancer.

Images