A tumor inhibiting composition and method of making and using same

Abstract

The application provides a tumor inhibiting composition and a preparation method and application thereof, and belongs to the technical field of medicines. After mesoporous carbon nanotubes are oxidized and chlorinated, oleanolic acid dithiocarbamate conjugate and (2-hydroxypropyl)-beta-cyclodextrin are coupled, drugs are loaded, a protein liposome solution is added, ultrasonic treatment is carried out, centrifugal separation is carried out, unloaded drug-loaded carbon nanotubes are removed, supernatant is freeze-dried, and the tumor inhibiting composition is prepared. The tumor inhibiting composition greatly improves the drug loading capacity of the nanosystem, the synergistic effect of multiple drugs, the anti-tumor effect, realizes the effects of targeting, slow and controlled release of drugs, improves the drug utilization rate, reduces the intake amount of drugs, reduces side effects, and improves the compliance of patients.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a tumor-inhibiting composition, its preparation method, and its application. Background Technology

[0002] With the development of nanotechnology, nanomaterials, especially nanoparticles used for traditional drug delivery in vivo that can improve drug targeting, utilization, and reduce drug toxicity, represent a breakthrough in modern medicine. Carbon nanotubes (CNTs) have become a hot topic in drug carrier research due to their large specific surface area, ability to accommodate biospecific molecules and cavity-structured drugs, and excellent cell penetration. However, CNTs have poor water solubility and exhibit certain toxicity both in vivo and in vitro, limiting their application in the field of drug carriers. Functionalized CNTs, on the other hand, show improved physiological solubility and biocompatibility, laying the foundation for their application in drug carriers. Currently, CNT modification mainly falls into two categories: covalent modification and non-covalent modification. Non-covalent modification primarily improves the dispersion performance of CNTs through intermolecular π-π stacking and van der Waals forces, thereby preventing their aggregation. Covalent modification utilizes the surface defects of CNTs to connect functional groups to their surface through chemical bonds via reactions such as oxidation, cycloaddition, and amidation. Compared with non-covalent modification, CNTs with covalent modification have better stability and biocompatibility.

[0003] By using carbon nanotubes as carriers to load tumor-inhibiting compositions, a carbon nanotube-based tumor-inhibiting composition carrier system can be constructed. The purpose of constructing this type of drug carrier system is to fully utilize the performance advantages of carbon nanotubes and overcome the shortcomings of tumor-inhibiting compositions, such as poor dispersibility in the blood, rapid metabolism and clearance in vivo, and poor ability to penetrate cell barriers. This will improve the chemotherapy effect for cancer patients and minimize chemotherapy side effects, thereby alleviating patient suffering.

[0004] However, modified carbon nanotube drug carriers often suffer from problems such as low drug loading capacity, weak targeting, and limited anti-tumor effects, which restricts their development. Summary of the Invention

[0005] The purpose of this invention is to propose a tumor-inhibiting composition, its preparation method, and its application. This invention greatly increases the drug loading capacity of the nanosystem, achieves synergistic effects of multiple drugs, significantly enhances the anti-tumor effect, and simultaneously realizes targeted and controlled-release drug delivery, thereby improving drug utilization, reducing drug intake, minimizing side effects, and improving patient compliance.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing a tumor-inhibiting composition. Mesoporous carbon nanotubes are oxidized and chlorinated, then coupled with oleanolic acid dithiocarbamate conjugate and (2-hydroxypropyl)-β-cyclodextrin, loaded with drugs, added to a protein liposome solution, sonicated, centrifuged, and uncoated drug-loaded carbon nanotubes are removed. The supernatant is then freeze-dried to obtain the tumor-inhibiting composition.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Dissolve 1,2-dibromoethane in N,N-dimethylformamide, add oleanolic acid and potassium carbonate, heat and stir to react, quench the reaction, purify, and obtain the coupling compound with the following structure: ;

[0010] S2. Carbon disulfide, tetrahydropyrrole, and potassium phosphate were added to tetrahydrofuran, and the mixture was activated by stirring in an ice-water bath. The coupling compound and tetrahydropyrrole were then added, and the reaction was quenched by stirring at room temperature. The mixture was purified to obtain the oleanolic acid dithiocarbamate conjugate; its structure is as follows: ;

[0011] S3. Mesoporous carbon nanotubes were oxidized with concentrated acid, added to a solution of N,N-dimethylformamide containing thionyl chloride, heated under reflux and stirred, and unreacted thionyl chloride was removed under reduced pressure. (2-hydroxypropyl)-β-cyclodextrin, oleanolic acid dithiocarbamate conjugate and triethylamine were added, and the mixture was heated under reflux and stirred. The mixture was then filtered, washed and dried to obtain cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes.

[0012] S4. Add the cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and the drug to ethanol, sonicate, centrifuge, wash and dry to obtain drug-loaded carbon nanotubes.

[0013] S5. Dissolve 1,2-dimyristoylphosphatidylglycerol and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in ethanol to obtain an organic phase; dissolve polypeptide H7K(R2)2 in water to obtain an aqueous phase; add the organic phase dropwise to the aqueous phase under stirring, sonicate, remove ethanol under reduced pressure, filter, and freeze-dry to obtain protein liposomes;

[0014] S6. Add protein liposomes to water, add drug-loaded carbon nanotubes under ice-water bath conditions, sonicate, centrifuge, remove unencapsulated drug-loaded carbon nanotubes, freeze-dry the supernatant to obtain the tumor-inhibiting composition.

[0015] As a further improvement of the present invention, the molar ratio of 1,2-dibromoethane, oleanolic acid and potassium carbonate in step S1 is 2-4:1:2-4, and the heating and stirring reaction temperature is 35-45℃ and the time is 3-5h.

[0016] As a further improvement of the present invention, the molar ratio of carbon disulfide, tetrahydropyrrole, potassium phosphate, coupling compound and tetrahydropyrrole in step S2 is 1.5-2:0.5-1.5:0.5-1:0.3-0.5:0.3-0.5, and the reaction is carried out at room temperature with stirring for 10-15 hours.

[0017] As a further improvement of the present invention, the concentrated acid in step S3 is a mixture of concentrated nitric acid and concentrated sulfuric acid with a volume ratio of 1:2-4; the solid-liquid ratio of the mesoporous carbon nanotubes and sulfoxide is 1:80-120 g / mL; the heating, reflux, and stirring reaction time is 20-28 h; the mass ratio of the mesoporous carbon nanotubes, (2-hydroxypropyl)-β-cyclodextrin, and oleanolic acid dithiocarbamate conjugate is 1:0.5-1:0.3-0.7; and the mass ratio of the cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and the drug is 1:0.5-1.

[0018] As a further improvement of the present invention, the drug in step S4 includes hydroxycamptothecin and resveratrol in a mass ratio of 1-3:2-4, and the ultrasonic stirring power is 150-250W, the time is 10-15min, and the stirring is on for 3s and off for 3s.

[0019] As a further improvement of the present invention, in step S5, the mass ratio of 1,2-dimyristoyl phosphatidylglycerol, folic acid-polyethylene glycol-distearate phosphatidylethanolamine, and polypeptide H7K(R2)2 is 8-10:1:0.2-0.5, the ultrasonic power is 150-250W, the time is 1-3min, with 3s on and 3s off.

[0020] As a further improvement of the present invention, the mass ratio of protein liposomes to drug-loaded carbon nanotubes in step S6 is 2-4:1, the power of the ultrasound is 150-250W, the time is 2-4h, the centrifugation speed is 2500-3500r / min, and the time is 10-20min.

[0021] The present invention further protects an antitumor composition prepared by the above-described preparation method.

[0022] The present invention further protects the use of the above-described tumor-inhibiting composition in the preparation of an antitumor medicament.

[0023] Oleanolic acid possesses various biological activities, including antitumor, anti-inflammatory, antioxidant, and hepatoprotective effects. However, its poor water solubility, poor pharmacokinetic properties, low cell selectivity, and limited bioavailability hinder its further clinical application. This invention modifies oleanolic acid to obtain dithiocarbamate conjugates. Dithiocarbamate compounds can effectively chelate heavy metals and scavenge NO free radicals in vivo, further enhancing the antitumor properties of oleanolic acid. Furthermore, the synthesis method of this invention is simple, efficient, and mild.

[0024] This invention utilizes mesoporous carbon nanotubes as a carrier, which not only possesses the inherent advantages of mesoporous materials but also allows for the adsorption of antitumor compositions into the carbon nanotubes through capillary action or direct passage through the mesopores. This increases the drug loading capacity of the carrier and enables controlled-release drug delivery. The surface of the mesoporous carbon nanotubes is modified with acyl chloride groups, which react with the hydroxyl groups of (2-hydroxypropyl)-β-cyclodextrin and oleanolic acid dithiocarbamate conjugates to achieve chemical modification. This significantly improves the water solubility and biocompatibility of the carbon nanotubes and enhances their antitumor activity. The oleanolic acid dithiocarbamate conjugates loaded on the carbon nanotube surface exert a good antitumor effect upon release. Furthermore, the cyclodextrin on the surface can provide cavities for the antitumor compositions, allowing for the loading of larger quantities of these compositions and the combination of different antitumor compositions. This not only increases the drug loading capacity but also enhances drug diversity, resulting in a synergistic antitumor effect.

[0025] Hydroxycamptothecin possesses broad-spectrum anticancer activity, but its solubility is poor. Although it rapidly undergoes ring-opening at pH > 7.4, increasing solubility, its anticancer activity decreases by 90% after being converted into a water-soluble sodium salt, and its stability deteriorates, leading to increased adverse reactions. Resveratrol has certain therapeutic effects in antitumor activity and in treating cardiovascular diseases, obesity, and diabetes; however, its poor water solubility, short half-life, and poor stability result in very low bioavailability and limited clinical application. This invention utilizes a wet chemical method, driven by capillary forces, to load the drug into functionalized carbon nanotubes. In the acidic tumor microenvironment, due to the reduced solubility of hydroxycamptothecin and its poor compatibility with the inner wall of the carbon nanotube lumen, it is released from the lumen under dewetting action, thus achieving targeted release. Furthermore, by adsorbing the drug onto the cyclodextrin cavity and the surface of the carbon nanotubes, its water solubility and stability are improved, significantly enhancing its bioavailability. The synergistic effect of multiple drugs further improves the antitumor effect.

[0026] This invention employs liposomes to encapsulate drug-loaded carbon nanotubes. Under ultrasonic conditions, phospholipid molecules within the liposomes rearrange through hydrophilic-hydrophobic interactions. The hydrophobic tails of the phospholipids tend towards the surface of the equally hydrophobic mesoporous carbon nanotubes, while the hydrophilic heads face outwards, thus blocking the drug and preventing premature release. Simultaneously, the liposome surface is modified with targeted folic acid and the tumor-specific, pH-responsive, and cell-penetrating peptide H7K(R2)2, enhancing the tumor targeting of the drug system. Furthermore, the acidic tumor microenvironment opens the nanostructure, releasing the drug and exerting a highly effective anti-tumor effect.

[0027] The present invention has the following beneficial effects:

[0028] 1. This invention achieves synergistic release of multiple drugs by modifying the surface of mesoporous carbon nanotubes with an antitumor-active oleanolic acid dithiocarbamate conjugate, loaded with hydroxycamptothecin and resveratrol, thereby greatly improving the antitumor effect.

[0029] 2. This invention uses liposomes to block carbon nanotubes, preventing premature drug release. Under the targeting effect of folic acid and peptide H7K(R2)2, the drug reaches the tumor microenvironment. The acidic conditions promote the release of the drug from the nanosystem, thereby achieving targeted and controlled drug release. This greatly improves drug utilization, reduces drug intake, reduces side effects, and improves patient compliance.

[0030] 3. This invention, through the modification and application of a nano-drug delivery system, greatly increases the drug loading capacity of the nano-system by loading drugs inside and on the surface of the tube, and by grafting cyclodextrin onto the tube and using cyclodextrin for further drug loading. This significantly improves the application effect of carbon nanotubes and enhances their anti-tumor efficacy. Attached Figure Description

[0031] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 XRD pattern of mesoporous carbon nanotubes and cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes in Example 1.

[0033] Figure 2 This is a TEM image of the tumor-inhibiting composition prepared in Example 1. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0035] Mesoporous carbon nanotubes, with a diameter of 100 nm, a length of <10 μm, and a purity of >95%, were obtained either by purchasing or by self-production. The self-production method is described in the reference "Study on the Preparation of Tungsten Nanowires and Tungsten Carbide Nanowires Using Mesoporous Carbon Nanotubes as Templates," author: Ren Xiaona, PhD dissertation, Beijing University of Science and Technology, 2017. The specific steps are as follows: Polytetrafluoroethylene and ferrous chloride were mixed and dried, then rapidly heated to 650 °C using a tubular rapid heating furnace and held at that temperature for 0.5 h. The sample was then cooled to room temperature to obtain a black powder. After soaking in hydrochloric acid for 72 h or refluxing for 48 h, the powder was repeatedly centrifuged with deionized water until the liquid became colorless. The resulting black powder was then treated in a hot press furnace at 1200 °C under vacuum for 2 h to obtain mesoporous carbon nanotubes.

[0036] Example 1

[0037] This embodiment provides a method for preparing a tumor-inhibiting composition, comprising the following steps:

[0038] S1. Dissolve 20 mmol of 1,2-dibromoethane in 200 mL of N,N-dimethylformamide, add 10 mmol of oleanolic acid and 20 mmol of potassium carbonate, heat to 40 °C, stir for 4 h, cool to room temperature, quench the reaction in 500 mL of ice water, filter, extract the filtrate with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, filter, and purify by column chromatography to obtain the conjugate; ESI-MS calculated value: C 32 H 52 BrO3(M+H) + 563.75, measured value: 563.8, yield: 85%. NMR results: 1 H NMR (300MHz, CDCl3) δ5.29 (m, 1H), 4.62 (t, 2H), 3.54 (t, 2H), 3.12 (t, 1H), 2.7 1 (t, 1H), 1.8-2 (m, 3H), 1.6-1.7 (m, 6H), 1.3-1.5 (m, 14H), 1.1-1.25 (m, 21H).

[0039] The synthesis route is as follows:

[0040]

[0041] S2. 15 mmol carbon disulfide, 5 mmol tetrahydropyrrole, and 5 mmol potassium phosphate were added to 100 mL tetrahydrofuran. The mixture was activated by stirring in an ice-water bath for 30 min. 3 mmol of the conjugate and 3 mmol tetrahydropyrrole were added, and the mixture was stirred at room temperature for 12 h. The reaction was quenched in 200 mL of ice water. The mixture was filtered, and the filtrate was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and purified by column chromatography to obtain the oleanolic acid dithiocarbamate conjugate. ESI-MS calculated value: C 37 H 60 NO3S2(M+H) + 631.02, measured value: 631.0, yield: 81%. NMR results: 1 H NMR (300MHz, CDCl3) δ5.22 (m, 1H), 4.75 (t, 2H), 3.15 (t, 2H), 2.82 (t, 4H), 2.74 (t , 1H), 1.8-2.2 (m, 3H), 1.61-1.75 (m, 10H), 1.32-1.57 (m, 15H), 1.12-1.27 (m, 21H)

[0042] The synthesis route is as follows:

[0043]

[0044] S3. Add 1g of mesoporous carbon nanotubes to 200mL of mixed acid, sonicate at room temperature for 7h, dilute with 800mL of water, let stand overnight, filter through a 0.15μm filter membrane, wash until neutral, dry, add the product to a mixed solution of 100mL of sulfoxide and 10mL of N,N-dimethylformamide, heat under reflux and stir for 24h, remove unreacted sulfoxide under reduced pressure, add 0.5g of (2-hydroxypropyl)-β-cyclodextrin, 0.3g of oleanolic acid dithiocarbamate conjugate and 5mL of triethylamine, heat under reflux and stir for 24h, filter, wash, dry, to obtain cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes; Figure 1 The image shows the XRD pattern of mesoporous carbon nanotubes and cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes. As can be seen from the image, cyclodextrin and oleanolic acid dithiocarbamate conjugate were grafted onto the surface of the mesoporous carbon nanotubes. However, both showed characteristic peaks at the same position, indicating that there was no significant change in the X-ray diffraction peaks of the carbon nanotubes before and after modification. After modification, there was no significant change in the crystal form.

[0045] The concentrated acid is a mixture of concentrated nitric acid and concentrated sulfuric acid in a volume ratio of 1:2;

[0046] S4. Add 1g of cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and 0.5g of drug to 100mL of ethanol, sonicate at 250W for 10min (on for 3s, off for 3s), then magnetically stir for 24h, centrifuge, wash, and dry to obtain drug-loaded carbon nanotubes.

[0047] The drug comprises hydroxycamptothecin and resveratrol in a mass ratio of 1:2;

[0048] The encapsulation efficiency of hydroxycamptothecin was 42.6%, and the drug loading rate was 5.25%. The encapsulation efficiency of resveratrol was 36.8%, and the drug loading rate was 9.07%.

[0049] S5. Dissolve 0.8 g of 1,2-dimyristoyl phosphatidylglycerol and 0.1 g of folic acid-polyethylene glycol-distearate phosphatidylethanolamine in 200 mL of ethanol to obtain the organic phase; dissolve 0.02 g of polypeptide H7K(R2)2 in 100 mL of water to obtain the aqueous phase; under stirring, add the organic phase dropwise to the aqueous phase, sonicate at 150 W for 1 min (on for 3 s, off for 3 s), remove ethanol under reduced pressure, filter twice through a 0.22 μm polycarbonate membrane, and freeze-dry to obtain protein liposomes;

[0050] S6. Add 0.2g of protein liposomes to 100mL of water, add 0.1g of drug-loaded carbon nanotubes under ice-water bath conditions, sonicate at 150W for 2h, centrifuge at 2500r / min for 10min to remove uncoated drug-loaded carbon nanotubes, freeze-dry the supernatant to obtain the tumor-inhibiting composition. Figure 2 The image shows a transmission electron microscope (TEM) image of the prepared tumor-inhibiting composition. As can be seen from the image, the width is approximately 100 nm.

[0051] Example 2

[0052] This embodiment provides a method for preparing a tumor-inhibiting composition, comprising the following steps:

[0053] S1. Dissolve 40 mmol of 1,2-dibromoethane in 200 mL of N,N-dimethylformamide, add 10 mmol of oleanolic acid and 40 mmol of potassium carbonate, heat to 40 °C, stir for 4 h, cool to room temperature, pour into 500 mL of ice water to quench the reaction, filter, extract the filtrate with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, filter, and purify by column chromatography to obtain the conjugate;

[0054] S2. Add 20 mmol carbon disulfide, 15 mmol tetrahydropyrrole, and 10 mmol potassium phosphate to 100 mL tetrahydrofuran, stir in an ice-water bath for 30 min to activate, add 5 mmol of the conjugate and 5 mmol tetrahydropyrrole, stir at room temperature for 12 h, pour into 200 mL ice water to quench the reaction, filter, extract the filtrate with ethyl acetate, add anhydrous magnesium sulfate to the organic phase to dry, filter, and purify by column chromatography to obtain oleanolic acid dithiocarbamate conjugate.

[0055] S3. Add 1g of mesoporous carbon nanotubes to 200mL of mixed acid, sonicate at room temperature for 7h, dilute with 800mL of water, let stand overnight, filter through a 0.15μm filter membrane, wash until neutral, dry, add the product to a mixed solution of 100mL of sulfoxide and 10mL of N,N-dimethylformamide, heat under reflux and stir for 24h, remove unreacted sulfoxide under reduced pressure, add 1g of (2-hydroxypropyl)-β-cyclodextrin, 0.7g of oleanolic acid dithiocarbamate conjugate and 5mL of triethylamine, heat under reflux and stir for 24h, filter, wash, dry, and obtain cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes;

[0056] The concentrated acid is a mixture of concentrated nitric acid and concentrated sulfuric acid in a volume ratio of 1:4;

[0057] S4. Add 1g of cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and 1g of drug to 100mL of ethanol, sonicate at 150W for 15min (on for 3s, off for 3s), then stir magnetically for 24h, centrifuge, wash, and dry to obtain drug-loaded carbon nanotubes.

[0058] The drug comprises hydroxycamptothecin and resveratrol in a mass ratio of 3:4;

[0059] The encapsulation efficiency of hydroxycamptothecin was 40.3%, and the drug loading rate was 2.92%. The encapsulation efficiency of resveratrol was 38.2%, and the drug loading rate was 11.07%.

[0060] S5. Dissolve 1g of 1,2-dimyristoylphosphatidylglycerol and 0.1g of folic acid-polyethylene glycol-distearate phosphatidylethanolamine in 200mL of ethanol to obtain the organic phase; dissolve 0.05g of polypeptide H7K(R2)2 in 100mL of water to obtain the aqueous phase; under stirring, add the organic phase dropwise to the aqueous phase, sonicate at 250W for 3min (on for 3s, off for 3s), remove ethanol under reduced pressure, filter twice through a 0.22μm polycarbonate membrane, and freeze-dry to obtain protein liposomes;

[0061] S6. Add 0.4g of protein liposomes to 100mL of water, add 0.1g of drug-loaded carbon nanotubes under ice-water bath conditions, sonicate at 250W for 4h, centrifuge at 3500r / min for 20min to remove uncoated drug-loaded carbon nanotubes, freeze-dry the supernatant to obtain the tumor-inhibiting composition.

[0062] Example 3

[0063] This embodiment provides a method for preparing a tumor-inhibiting composition, comprising the following steps:

[0064] S1. Dissolve 30 mmol of 1,2-dibromoethane in 200 mL of N,N-dimethylformamide, add 10 mmol of oleanolic acid and 30 mmol of potassium carbonate, heat to 40 °C, stir for 4 h, cool to room temperature, pour into 500 mL of ice water to quench the reaction, filter, extract the filtrate with ethyl acetate, dry the organic phase with anhydrous magnesium sulfate, filter, and purify by column chromatography to obtain the conjugate;

[0065] S2. 18 mmol carbon disulfide, 10 mmol tetrahydropyrrole, and 7 mmol potassium phosphate were added to 100 mL tetrahydrofuran and activated by stirring in an ice-water bath for 30 min. 4 mmol of the conjugate and 4 mmol tetrahydropyrrole were added and stirred at room temperature for 12 h. The reaction was quenched by pouring the mixture into 200 mL of ice water. The mixture was filtered, and the filtrate was extracted with ethyl acetate. The organic phase was dried with anhydrous magnesium sulfate, filtered, and purified by column chromatography to obtain the oleanolic acid dithiocarbamate conjugate.

[0066] S3. Add 1g of mesoporous carbon nanotubes to 200mL of mixed acid, sonicate at room temperature for 7h, dilute with 800mL of water, let stand overnight, filter through a 0.15μm filter membrane, wash until neutral, dry, add the product to a mixed solution of 100mL of sulfoxide and 10mL of N,N-dimethylformamide, heat under reflux and stir for 24h, remove unreacted sulfoxide under reduced pressure, add 0.7g of (2-hydroxypropyl)-β-cyclodextrin, 0.5g of oleanolic acid dithiocarbamate conjugate and 5mL of triethylamine, heat under reflux and stir for 24h, filter, wash, dry, and obtain cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes;

[0067] The concentrated acid is a mixture of concentrated nitric acid and concentrated sulfuric acid, with a volume ratio of 1:3;

[0068] The encapsulation efficiency of hydroxycamptothecin was 41.2%, and the drug loading rate was 3.57%. The encapsulation efficiency of resveratrol was 37.1%, and the drug loading rate was 9.64%.

[0069] S4. Add 1g of cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and 0.7g of drug to 100mL of ethanol, sonicate at 200W for 12min (on for 3s, off for 3s), then magnetically stir for 24h, centrifuge, wash, and dry to obtain drug-loaded carbon nanotubes.

[0070] The drug comprises hydroxycamptothecin and resveratrol in a mass ratio of 2:3;

[0071] S5. Dissolve 0.9 g of 1,2-dimyristoyl phosphatidylglycerol and 0.1 g of folic acid-polyethylene glycol-distearate phosphatidylethanolamine in 200 mL of ethanol to obtain the organic phase; dissolve 0.035 g of polypeptide H7K(R2)2 in 100 mL of water to obtain the aqueous phase; while stirring, add the organic phase dropwise to the aqueous phase, sonicate at 200 W for 2 min (on for 3 s, off for 3 s), remove ethanol under reduced pressure, filter twice through a 0.22 μm polycarbonate membrane, and freeze-dry to obtain protein liposomes;

[0072] S6. Add 0.3g of protein liposomes to 100mL of water, add 0.1g of drug-loaded carbon nanotubes under ice-water bath conditions, sonicate at 200W for 3h, centrifuge at 3000r / min for 15min to remove uncoated drug-loaded carbon nanotubes, freeze-dry the supernatant to obtain the tumor-inhibiting composition.

[0073] Example 4

[0074] The only difference from Example 3 is that the drug is hydroxycamptothecin.

[0075] The encapsulation efficiency of hydroxycamptothecin was 65.2%, and the drug loading rate was 8.52%.

[0076] Example 5

[0077] The only difference from Example 3 is that the drug used is resveratrol.

[0078] The encapsulation efficiency of resveratrol was 54.1%, and the drug loading rate was 13.38%.

[0079] Comparative Example 1

[0080] The only difference from Example 3 is that in step S3, the oleanolic acid dithiocarbamate conjugate is replaced by an equal mass of (2-hydroxypropyl)-β-cyclodextrin.

[0081] Comparative Example 2

[0082] The only difference from Example 3 is that in step S3, (2-hydroxypropyl)-β-cyclodextrin is replaced by an equal mass of oleanolic acid dithiocarbamate conjugate.

[0083] The encapsulation efficiency of hydroxycamptothecin was 32.5%, and the drug loading rate was 2.56%. The encapsulation efficiency of resveratrol was 26.7%, and the drug loading rate was 6.31%.

[0084] Comparative Example 3

[0085] The only difference from Example 3 is that the mesoporous carbon nanotubes are replaced by an equal mass of multi-walled carbon nanotubes (diameter 100 nm, length < 10 μm, purity > 95%).

[0086] The encapsulation efficiency of hydroxycamptothecin was 22.5%, and the drug loading rate was 2.01%. The encapsulation efficiency of resveratrol was 31.2%, and the drug loading rate was 8.36%.

[0087] Comparative Example 4

[0088] The only difference from Example 3 is that steps S1 to S3 were not performed, and in step S4, the cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes were replaced by mesoporous carbon nanotubes of equal mass.

[0089] Comparative Example 5

[0090] The only difference from Example 3 is that step S4 was not performed, and in step S6, the drug-loaded carbon nanotubes were replaced by an equal mass of cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes.

[0091] Comparative Example 6

[0092] The only difference from Example 3 is that folic acid-polyethylene glycol-distearate phosphatidylethanolamine and polypeptide H7K(R2)2 were not added in step S5. The liposome preparation method is as follows:

[0093] Dissolve 1g in 200mL of ethanol to obtain an organic phase; while stirring, add the organic phase dropwise to 100mL of water, sonicate at 200W for 2min (on for 3s, off for 3s), remove ethanol under reduced pressure, filter twice through a 0.22μm polycarbonate membrane, and freeze-dry to obtain liposomes.

[0094] Test Example 1

[0095] Sunitinib was selected as the positive control drug. The antitumor activity of oleanolic acid and oleanolic acid dithiocarbamate conjugate was tested using the MTT assay. A549 and SGC7901 cells were selected as test targets. The results are shown in Table 1.

[0096] Table 1

[0097]

[0098] As shown in the table above, the oleanolic acid dithiocarbamate conjugate prepared in this invention has a significantly better inhibitory effect on human lung cancer cells A549 and human gastric adenocarcinoma cells SGC7901 than the parent molecule oleanolic acid.

[0099] Test Example 2

[0100] A suspension of human breast cancer MCF-7 cells in the exponential growth phase was injected into the peritoneal cavity of female BALB / c nude mice. Ten days later, breast cancer cells were collected from the ascites fluid of the nude mice. After centrifugation to remove the supernatant, the cells were diluted with physiological saline to a cell concentration of 2.0 × 10⁻⁶. 7 Cells / mL. 200 μL of cell suspension was subcutaneously inoculated into the abdomen of female BALB / c nude mice to establish a subcutaneous tumor model. The tumor was allowed to grow to approximately 100 mm². 3 In this study, tumor-bearing nude mice were randomly divided into 12 groups of 6 mice each. The control group received a tail vein injection of saline, while groups 1-5 and 1-6 (comparative examples) received a tail vein injection of a tumor-inhibiting composition (prepared with saline, 2 mg / kg). All groups received a single daily dose for 14 consecutive days. On day 15, the long diameter (L) and short diameter (W) of the tumor were measured using calipers.

[0101] Tumor volume V = (L × W) 2 ) / 2;

[0102] Relative tumor volume RTV = V t / V0;

[0103] Where V0 is the tumor volume measured at the beginning of the experiment, V t This represents the tumor volume measured at the end of the experiment.

[0104] Relative tumor proliferation inhibition rate (%) = (1-RTV) 实验 / RTV 对照 ) × 100%;

[0105] Among them, RTV 实验 The relative tumor volume of the experimental group, RTV 对照 This represents the relative tumor volume of the control group.

[0106] The results are shown in Table 2.

[0107] Table 2

[0108]

[0109] As can be seen from the table above, the tumor-inhibiting compositions prepared in Examples 1-3 of the present invention have good anti-tumor effects.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tumor-inhibiting composition, characterized in that, Mesoporous carbon nanotubes were oxidized and chlorinated, then coupled with oleanolic acid dithiocarbamate conjugate and (2-hydroxypropyl)-β-cyclodextrin to load the drug. The mixture was then added to a protein liposome solution, sonicated, centrifuged, and the unencapsulated drug-loaded carbon nanotubes were removed. The supernatant was freeze-dried to obtain the tumor-inhibiting composition.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Dissolve 1,2-dibromoethane in N,N-dimethylformamide, add oleanolic acid and potassium carbonate, heat and stir to react, quench the reaction, purify, and obtain the coupling compound; S2. Carbon disulfide, tetrahydropyrrole, and potassium phosphate were added to tetrahydrofuran and activated by stirring in an ice-water bath. The coupling compound and tetrahydropyrrole were added, and the reaction was quenched by stirring at room temperature. The mixture was then purified to obtain the oleanolic acid dithiocarbamate conjugate. S3. Mesoporous carbon nanotubes were oxidized with concentrated acid, added to a solution of N,N-dimethylformamide containing thionyl chloride, heated under reflux and stirred, and unreacted thionyl chloride was removed under reduced pressure. (2-hydroxypropyl)-β-cyclodextrin, oleanolic acid dithiocarbamate conjugate and triethylamine were added, and the mixture was heated under reflux and stirred. The mixture was then filtered, washed and dried to obtain cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes. S4. Add the cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and the drug to ethanol, sonicate, centrifuge, wash and dry to obtain drug-loaded carbon nanotubes. S5. Dissolve 1,2-dimyristoylphosphatidylglycerol and folic acid-polyethylene glycol-distearate phosphatidylethanolamine in ethanol to obtain an organic phase; dissolve polypeptide H7K(R2)2 in water to obtain an aqueous phase; add the organic phase dropwise to the aqueous phase under stirring, sonicate, remove ethanol under reduced pressure, filter, and freeze-dry to obtain protein liposomes; S6. Add protein liposomes to water, add drug-loaded carbon nanotubes under ice-water bath conditions, sonicate, centrifuge, remove unencapsulated drug-loaded carbon nanotubes, freeze-dry the supernatant to obtain the tumor-inhibiting composition.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of 1,2-dibromoethane, oleanolic acid, and potassium carbonate is 2-4:1:2-4, and the heating and stirring reaction is carried out at a temperature of 35-45°C for 3-5 hours.

4. The preparation method according to claim 2, characterized in that, In step S2, the molar ratio of carbon disulfide, tetrahydropyrrole, potassium phosphate, coupling compound and tetrahydropyrrole is 1.5-2:0.5-1.5:0.5-1:0.3-0.5:0.3-0.5, and the reaction is carried out at room temperature with stirring for 10-15 hours.

5. The preparation method according to claim 2, characterized in that, In step S3, the concentrated acid is a mixture of concentrated nitric acid and concentrated sulfuric acid, with a volume ratio of 1:2-4. The solid-liquid ratio of the mesoporous carbon nanotubes and sulfoxide is 1:80-120 g / mL. The heating, reflux, and stirring reaction time is 20-28 h. The mass ratio of the mesoporous carbon nanotubes, (2-hydroxypropyl)-β-cyclodextrin, and oleanolic acid dithiocarbamate conjugate is 1:0.5-1:0.3-0.

7. The mass ratio of the cyclodextrin / oleanolic acid dithiocarbamate conjugate-mesoporous carbon nanotubes and the drug is 1:0.5-1.

6. The preparation method according to claim 2, characterized in that, The drugs mentioned in step S4 include hydroxycamptothecin and resveratrol in a mass ratio of 1-3:2-4. The ultrasonic stirring power is 150-250W, the time is 10-15min, and the cycle is 3s on and 3s off.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of 1,2-dimyristoyl phosphatidylglycerol, folic acid-polyethylene glycol-distearate phosphatidylethanolamine, and polypeptide H7K(R2)2 is 8-10:1:0.2-0.

5. The ultrasonic power is 150-250W, the duration is 1-3min, with an on-time of 3s and an off-time of 3s.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of protein liposomes to drug-loaded carbon nanotubes is 2-4:1, the ultrasonic power is 150-250W, the time is 2-4h, and the centrifugation speed is 2500-3500r / min for 10-20min.

9. A tumor-inhibiting composition prepared by any one of claims 1-8.

10. The use of the tumor-inhibiting composition as described in claim 9 in the preparation of an antitumor medicament.

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