Anti-tumor medicine as well as preparation method and application thereof
By preparing a complex of gold nanocages/selenium polysaccharide/thiophene modified material and paclitaxel, the problems of poor water solubility and drug resistance of paclitaxel injection were solved, enabling targeted delivery to tumors, improving therapeutic efficacy and reducing side effects.
Patent Information
- Application Number
- CN202511126898.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-13
- Publication Date
- 2025-10-31
AI Technical Summary
Existing paclitaxel injections suffer from poor water solubility, frequent allergic reactions, inaccurate drug delivery, drug resistance, and insufficient stability, which affect treatment efficacy and increase side effects.
Selenium polysaccharide was prepared by fermenting Ganoderma lucidum polysaccharide with selenium-enriched yeast. It was then reacted with 3-(3-bromo)propoxy-4-methylthiophene and gold nanocages, combined with biotin-PEG-propionaldehyde and 5-aminothiophene, to prepare a gold nanocage/selenium polysaccharide/thiophene modified compound. This compound was then combined with paclitaxel to form a polyethylene glycol/thiophene modified compound. Through ferric chloride polymerization, an anti-tumor drug with tumor-targeted delivery was generated.
It increases drug concentration in the tumor area, reduces drug resistance, has biocompatibility and degradability, enhances therapeutic effects, reduces dosage and side effects, and improves patient compliance.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to an anti-tumor drug, its preparation method, and its application. Background Technology
[0002] Paclitaxel (trade name: taxol) is an antitumor active ingredient discovered in the bark of the Pacific yew tree. In 1971, Dr. Wall and Dr. Wani determined its chemical structure using nuclear magnetic resonance (NMR) and single-crystal X-ray diffraction. However, research was halted for a time due to the low concentration of paclitaxel in the bark and its complex structure. Research resumed in 1979 after Susan Horwitz reported that paclitaxel possesses a unique mechanism that promotes microtubule polymerization and prevents microtubule depolymerization, sparking a surge in paclitaxel research. Clinical trials began in 1983, and it was approved for marketing by the FDA in 1992. It is now widely used in the treatment of various cancers, including ovarian cancer, breast cancer, and lung cancer.
[0003] The main obstacle to the clinical application of paclitaxel as an injectable chemical drug is its extremely poor water solubility (less than 0.03 mg / mL), which leads to drug transport problems. Current paclitaxel injections often use polyoxyethylene castor oil (Cremophor EL) at a concentration of 6 mg / mL to assist transport. Because the clinical therapeutic dose of paclitaxel is relatively high (generally 135 mg / mL),... 2 Patients must tolerate high concentrations of Cremophor EL. However, Cremophor EL can cause severe allergic reactions. Even if dexamethasone and H1 receptor antagonists are taken before paclitaxel administration, some patients still experience varying degrees of allergic reactions.
[0004] To increase the water solubility of paclitaxel, researchers have studied its dosage forms for many years. From the initial cyclodextrin inclusion complexes to the current nanoparticles, liposomes, and other new dosage forms, the dosage forms of paclitaxel are constantly being improved, and some dosage forms are already in clinical use.
[0005] Researchers have experimented with encapsulating paclitaxel with various cyclodextrin derivatives, resulting in varying degrees of improved water solubility. However, paclitaxel precipitation occurred after dilution in aqueous media. To address this issue, the cyclodextrin concentration needs to be increased; however, most cyclodextrins are highly toxic, and high concentrations can cause hemolysis. Hydroxypropyl-β-cyclodextrin has low toxicity, almost no hemolytic activity, and can be used to prepare oral and injectable formulations. Hydroxypropyl-β-cyclodextrin exhibits significantly better solubilizing effects on paclitaxel than β-cyclodextrin, with the solubilizing effect increasing with increasing proportion. At a proportion of 40%-60%, the solubility of paclitaxel can increase by approximately 1000-9000 times. However, at a proportion of 40%, the viscosity becomes too high, resulting in poor flowability, making it unsuitable for injection. A more suitable proportion is below 20%.
[0006] Liposomes are targeted drug carriers that use specialized technology to encapsulate drugs within lipid microparticles with diameters ranging from micrometers to nanometers. The phospholipids or cholesterol in the outer membrane of these lipid microparticles are amphiphilic, allowing them to solubilize poorly water-soluble substances. Paclitaxel liposomes for injection (trade name: Lipusu) were approved for marketing in my country in 2003. Although paclitaxel liposomes possess a certain degree of targeting, the precision of drug release in vivo is still not ideal. Some drugs may be released into non-target tissues, affecting therapeutic efficacy and increasing adverse reactions. Drug resistance is also a concern, and their stability is poor. Summary of the Invention
[0007] The purpose of this invention is to propose an anti-tumor drug, its preparation method, and its application. This drug has a tumor-targeted delivery effect, increases the drug concentration in the tumor area, reduces drug resistance, has biocompatibility and degradability, enhances therapeutic effect, reduces dosage, reduces side effects, and improves patient compliance, thus having broad application prospects.
[0008] The technical solution of this invention is implemented as follows:
[0009] This invention provides a method for preparing an antitumor drug. Ganoderma lucidum polysaccharide is fermented with selenium-enriched yeast to obtain selenium polysaccharide, which is then reacted with 3-(3-bromo)propoxy-4-methylthiophene and gold nanocages to obtain a modified gold nanocage / selenium polysaccharide / thiophene compound. Biotin-PEG-propionaldehyde and 5-aminothiophene are reacted to obtain a modified polyethylene glycol / thiophene compound, which is then reacted with the modified gold nanocage / selenium polysaccharide / thiophene compound and paclitaxel under the action of ferric chloride to obtain the antitumor drug.
[0010] As a further improvement to the present invention, the following steps are included:
[0011] S1. Preparation of selenium polysaccharide: Ganoderma lucidum polysaccharide was added to the culture medium, selenium-enriched yeast seed liquid was inoculated, fermented and cultured, the cell bodies were removed by filtration, ethanol was added to precipitate, filtered, washed, and dried to obtain selenium polysaccharide;
[0012] S2. Preparation of selenium polysaccharide / thiophene modified product: 3-(3-bromo)propoxy-4-methylthiophene, base and selenium polysaccharide were added to a mixed solution of N,N-dimethylformamide and water, heated and stirred to react, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0013] S3. Preparation of polyethylene glycol / thiophene modified products:
[0014] S4. Preparation of gold nanocages / selenium polysaccharide / thiophene modified product: Gold nanocages were dispersed in PBS buffer, selenium polysaccharide / thiophene modified product was added, the mixture was stirred by sonication, centrifuged, washed, and dried to obtain gold nanocages / selenium polysaccharide / thiophene modified product;
[0015] S5. Preparation of antitumor drugs: Polyethylene glycol / thiophene modified material was added to nitromethane, gold nanocages / selenium polysaccharide / thiophene modified material was added, and the mixture was stirred and mixed evenly. Then, paclitaxel ethanol solution was added and stirred and mixed evenly. Under inert gas protection, ferric chloride nitromethane solution was added dropwise, and the reaction was heated and stirred. The mixture was dialyzed, and the unpermeated liquid was freeze-dried to obtain the antitumor drugs.
[0016] As a further improvement of the present invention, the mass ratio of Ganoderma lucidum polysaccharide to culture medium in step S1 is 7-10:40-70, the culture medium is YEPD medium, and the bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 The inoculum concentration of the selenium-enriched yeast seed solution is 2-3 v / v%, and the fermentation conditions are 25-30℃, 50-150 r / min, and fermentation culture for 36-48 h.
[0017] As a further improvement of the present invention, the mass ratio of 3-(3-bromomethoxy)4-methylthiophene, base and selenium polysaccharide in step S2 is 2-3:3-4:10, the heating and stirring reaction temperature is 50-60℃ and the time is 5-7h, and the base is selected from at least one of triethylamine, diethylamine, NaOH, KOH and ethylenediamine.
[0018] As a further improvement of the present invention, the mass ratio of biotin-PEG-propionaldehyde and 5-aminothiophene in step S3 is 5-7:1-3, and the stirring reaction time is 3-5h.
[0019] As a further improvement of the present invention, the pH value of the PBS buffer in step S4 is 7-7.5, the mass ratio of the gold nanocages to the selenium polysaccharide / thiophene modifier is 0.5-1:10, and the ultrasonic stirring reaction time is 30-50 min.
[0020] As a further improvement of the present invention, the mass ratio of polyethylene glycol / thiophene modified material, gold nanocage / selenium polysaccharide / thiophene modified material, paclitaxel and ferric chloride in step S5 is 3-5:4-7:1-2:6-9, and the heating and stirring reaction temperature is 45-55℃ and the time is 70-75h.
[0021] This invention further protects an antitumor drug prepared by the above-described preparation method.
[0022] This invention further protects the use of the above-mentioned antitumor drug in the preparation of a drug for treating hypoxic tumors.
[0023] As a further improvement of the present invention, the hypoxic tumors include lung cancer, breast cancer, cervical cancer, liver cancer, pancreatic cancer, and colorectal cancer.
[0024] The present invention has the following beneficial effects:
[0025] This invention uses Ganoderma lucidum polysaccharide as a substrate. After fermentation with selenium-enriched yeast, selenium polysaccharide is obtained by alcohol precipitation. This enhances the body's non-specific immune function, helps the body resist tumor cells, activates intracellular apoptosis-related signaling pathways, induces apoptosis in various tumor cells, has antioxidant effects, can scavenge free radicals, reduce oxidative damage, alleviate oxidative stress, and inhibit the growth and proliferation of tumor cells.
[0026] A selenium polysaccharide / thiophene modified compound was prepared by nucleophilic substitution of 3-(3-bromo)propoxy-4-methylthiophene with the hydroxyl groups on the selenium polysaccharide molecular chain. By introducing gold nanocages, the gold atoms on the surface of the nanocages, which possess high chemical activity, were able to coordinate with atoms containing lone pairs of electrons. The sulfur atoms in the thiophene molecule also contain lone pairs of electrons, potentially coordinating with the gold atoms on the surface of the gold nanocages, thus achieving coupling. The resulting complex exhibits good stability. Therefore, the gold nanocages can form a gold-DNA complex with the DNA sequence of cancer cells via thiol groups, thereby allowing the insertion of the drug molecule paclitaxel into the cancer cell DNA, significantly improving drug efficacy, increasing drug concentration at the tumor site, avoiding the drug resistance problem associated with direct use of chemotherapy drugs, and enhancing the effect of chemotherapy.
[0027] Biotin-PEG-propionaldehyde can be coupled with 5-aminothiophene via a Schiff base reaction to prepare a polyethylene glycol / thiophene modified compound. Biotin has a tumor cell targeting effect. Tumor cells grow rapidly, increasing their demand for biotin, which leads to the overexpression of biotin receptors on the surface of various tumor cells. After biotin binds to the biotin receptors on the surface of tumor cells, it can enter the tumor cells through receptor-mediated endocytosis. Linking biotin with anti-tumor drugs can enable the drugs to specifically target tumor cells, achieving precision treatment, enhancing drug efficacy, and reducing toxicity to normal tissues.
[0028] This invention involves polymerizing polyethylene glycol / thiophene modified material and gold nanocage / selenium polysaccharide / thiophene modified material under the action of ferric chloride to generate a polythiophene complex. This complex is both hydrophilic and lipophilic, which can increase the solubility of the water-insoluble drug paclitaxel and increase its circulation time in the blood. Through the enhanced effects of penetration and retention, it passively targets the tumor site. At the same time, the presence of polyethylene glycol and polysaccharide makes it biocompatible and biodegradable, which can reduce systemic toxicity and enhance the therapeutic effect. Moreover, the material itself will not cause discomfort to patients and will be excreted from the body through metabolic degradation.
[0029] Furthermore, in hypoxic tumors, insufficient oxygen supply within the tumor tissue leads to a hypoxic state in tumor cells. The thiophene molecules on the polythiophene chain of this invention contain oxygen-loving sulfur atoms, enhancing oxygen-carrying capacity. By releasing oxygen, it reduces HIF-1α expression (decreasing ABCG2 transcription) and also improves the diffusion efficiency of paclitaxel in hypoxic regions, thereby significantly enhancing its antitumor effect. Additionally, one reason for paclitaxel resistance is the overexpression of ABC transporters, leading to active efflux of the drug and insufficient intracellular concentration. The antitumor drug prepared in this invention significantly increases the drug concentration in the tumor region, thus reducing paclitaxel resistance.
[0030] The antitumor drug prepared by this invention has a tumor-targeted delivery effect, increases the drug concentration in the tumor area, reduces drug resistance, has biocompatibility and degradability, enhances the therapeutic effect, reduces the dosage, reduces side effects, and improves patient compliance, and has broad application prospects. Detailed Implementation
[0031] 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.
[0032] YEPD culture medium formula: 10g yeast extract, 20g peptone, 20g glucose, 1L distilled water.
[0033] Preparation of selenium-enriched yeast (10 billion CFU / g) inoculum: Selenium-enriched yeast was inoculated into YEPD medium and activated at 25°C and 100 rpm for 18-24 hours to obtain a culture with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed solution.
[0034] Ganoderma lucidum polysaccharides, purity >90%.
[0035] Biotin-PEG-propionaldehyde, molecular weight 1000-2000, purity >95%.
[0036] Gold nanocages with an average particle size of 100 nm and a zeta potential of approximately -15 mV.
[0037] Example 1
[0038] This embodiment provides a method for preparing an antitumor drug, including the following steps:
[0039] S1. Preparation of selenium polysaccharide: 7g of Ganoderma lucidum polysaccharide was added to 40g of YEPD medium, and selenium-enriched yeast seed liquid was inoculated at an inoculation amount of 2v / v%, fermented at 25℃ and 50r / min for 36h, the cells were removed by filtration, ethanol was added to the system content to 70wt%, precipitation was carried out for 1h, filtered, washed, and dried to obtain selenium polysaccharide.
[0040] S2. Preparation of selenium polysaccharide / thiophene modified product: 2g of 3-(3-bromo)propoxy-4-methylthiophene, 3g of triethylamine and 10g of selenium polysaccharide were added to 300mL of a mixed solution of N,N-dimethylformamide and water (volume ratio 3:7), heated to 50℃, stirred for 5h, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0041] S3. Preparation of polyethylene glycol / thiophene modified products:
[0042] S4. Preparation of gold nanocages / selenium polysaccharide / thiophene modified product: 0.5g of gold nanocages were dispersed in 150mL of PBS buffer with pH 7, 10g of selenium polysaccharide / thiophene modified product was added, the mixture was ultrasonically stirred for 30min, centrifuged, washed, and dried to obtain gold nanocages / selenium polysaccharide / thiophene modified product.
[0043] S5. Preparation of antitumor drugs: 3g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, 4g of gold nanocage / selenium polysaccharide / thiophene modified material was added, and the mixture was stirred for 10min. Then, 50mL of ethanol solution containing 1g of paclitaxel was added, and the mixture was stirred for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 6g of ferric chloride was added dropwise. The mixture was heated to 45℃ and stirred for 70h. The mixture was dialyzed for 1 day using a dialysis bag with a pore size of 8000Da. The impurity was freeze-dried to obtain the antitumor drugs.
[0044] Example 2
[0045] This embodiment provides a method for preparing an antitumor drug, including the following steps:
[0046] S1. Preparation of selenium polysaccharide: 10g of Ganoderma lucidum polysaccharide was added to 70g of YEPD medium, and selenium-enriched yeast seed liquid was inoculated at an inoculation amount of 3v / v%, fermented at 30℃ and 150r / min for 48h, the cells were removed by filtration, ethanol was added to the system content to 70wt%, precipitation was carried out for 1h, filtered, washed, and dried to obtain selenium polysaccharide.
[0047] S2. Preparation of selenium polysaccharide / thiophene modified product: 3g of 3-(3-bromo)propoxy-4-methylthiophene, 4g of NaOH and 10g of selenium polysaccharide were added to 300mL of a mixed solution of N,N-dimethylformamide and water (volume ratio of 5:10), heated to 60℃, stirred for 7h, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0048] S3. Preparation of polyethylene glycol / thiophene modified products:
[0049] S4. Preparation of gold nanocages / selenium polysaccharide / thiophene modified product: 1g of gold nanocages were dispersed in 150mL of PBS buffer with pH 7.5, 10g of selenium polysaccharide / thiophene modified product was added, the mixture was ultrasonically stirred for 50min, centrifuged, washed, and dried to obtain gold nanocages / selenium polysaccharide / thiophene modified product.
[0050] S5. Preparation of antitumor drugs: 5g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, 7g of gold nanocage / selenium polysaccharide / thiophene modified material was added, and the mixture was stirred for 10min. Then, 50mL of ethanol solution containing 2g of paclitaxel was added, and the mixture was stirred for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 9g of ferric chloride was added dropwise. The mixture was heated to 55℃ and stirred for 75h. The mixture was dialyzed for 1 day using a dialysis bag with a pore size of 8000Da. The impurity was freeze-dried to obtain the antitumor drugs.
[0051] Example 3
[0052] This embodiment provides a method for preparing an antitumor drug, including the following steps:
[0053] S1. Preparation of selenium polysaccharide: 8.5g of Ganoderma lucidum polysaccharide was added to 55g of YEPD medium, and selenium-enriched yeast seed liquid was inoculated at an inoculation amount of 2.5v / v%, fermented at 27℃ and 100r / min for 42h, the cells were removed by filtration, ethanol was added to the system content to 70wt%, precipitation was carried out for 1h, filtered, washed, and dried to obtain selenium polysaccharide;
[0054] S2. Preparation of selenium polysaccharide / thiophene modified product: 2.4 g of 3-(3-bromo)propoxy-4-methylthiophene, 3.5 g of triethylamine and 10 g of selenium polysaccharide were added to a mixed solution of N,N-dimethylformamide and water (volume ratio 4:8), heated to 55 °C, stirred for 6 h, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0055] S3. Preparation of polyethylene glycol / thiophene modified products:
[0056] S4. Preparation of gold nanocages / selenium polysaccharide / thiophene modified product: 0.7 g gold nanocages were dispersed in 150 mL of PBS buffer with pH 7.2, 10 g of selenium polysaccharide / thiophene modified product was added, the mixture was ultrasonically stirred for 40 min, centrifuged, washed, and dried to obtain gold nanocages / selenium polysaccharide / thiophene modified product.
[0057] S5. Preparation of antitumor drugs: 4g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, and 5.5g of gold nanocage / selenium polysaccharide / thiophene modified material was added. The mixture was stirred and mixed for 10min. 50mL of ethanol solution containing 1.5g of paclitaxel was added and stirred and mixed for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 7.5g of ferric chloride was added dropwise. The mixture was heated to 50℃ and stirred for 72h. Dialysis was performed using a dialysis bag with a pore size of 8000Da for 1 day. The unpermeable solution was freeze-dried to obtain the antitumor drug.
[0058] Comparative Example 1
[0059] The difference from Example 3 is that step S1 was not performed.
[0060] Specifically as follows:
[0061] S1. Preparation of Ganoderma lucidum polysaccharide / thiophene modified product: 2.4 g of 3-(3-bromo)propoxy-4-methylthiophene, 3.5 g of triethylamine and 10 g of Ganoderma lucidum polysaccharide were added to a mixed solution of N,N-dimethylformamide and water (volume ratio 4:8), heated to 55 °C, stirred for 6 h, filtered, washed and dried to obtain Ganoderma lucidum polysaccharide / thiophene modified product;
[0062] S2. Preparation of polyethylene glycol / thiophene modified products:
[0063] S3. Preparation of gold nanocages / Ganoderma lucidum polysaccharide / thiophene modified product: 0.7g of gold nanocages were dispersed in 150mL of PBS buffer with a pH of 7.2, and 10g of Ganoderma lucidum polysaccharide / thiophene modified product was added. The mixture was stirred by sonication for 40min, centrifuged, washed, and dried to obtain the gold nanocages / Ganoderma lucidum polysaccharide / thiophene modified product.
[0064] S4. Preparation of antitumor drugs: 4g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, and 5.5g of gold nanocage / Ganoderma lucidum polysaccharide / thiophene modified material was added. The mixture was stirred and mixed for 10min. 50mL of ethanol solution containing 1.5g of paclitaxel was added and stirred and mixed for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 7.5g of ferric chloride was added dropwise. The mixture was heated to 50℃ and stirred for 72h. Dialysis was performed using a dialysis bag with a pore size of 8000Da for 1 day. The unpermeated liquid was freeze-dried to obtain the antitumor drugs.
[0065] Comparative Example 2
[0066] The difference from Example 3 is that step S4 was not performed.
[0067] Specifically as follows:
[0068] S1. Preparation of selenium polysaccharide: 8.5g of Ganoderma lucidum polysaccharide was added to 55g of YEPD medium, and selenium-enriched yeast seed liquid was inoculated at an inoculation amount of 2.5v / v%, fermented at 27℃ and 100r / min for 42h, the cells were removed by filtration, ethanol was added to the system content to 70wt%, precipitation was carried out for 1h, filtered, washed, and dried to obtain selenium polysaccharide;
[0069] S2. Preparation of selenium polysaccharide / thiophene modified product: 2.4 g of 3-(3-bromo)propoxy-4-methylthiophene, 3.5 g of triethylamine and 10 g of selenium polysaccharide were added to a mixed solution of N,N-dimethylformamide and water (volume ratio 4:8), heated to 55 °C, stirred for 6 h, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0070] S3. Preparation of polyethylene glycol / thiophene modified products:
[0071] S4. Preparation of antitumor drugs: 4g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, 5.5g of selenium polysaccharide / thiophene modified material was added, and the mixture was stirred for 10min. Then, 50mL of ethanol solution containing 1.5g of paclitaxel was added, and the mixture was stirred for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 7.5g of ferric chloride was added dropwise. The mixture was heated to 50℃ and stirred for 72h. The mixture was dialyzed for 1 day using a dialysis bag with a pore size of 8000Da. The unpermeated liquid was freeze-dried to obtain the antitumor drugs.
[0072] Comparative Example 3
[0073] The difference from Example 3 is that no polyethylene glycol / thiophene modifier was added in step S5.
[0074] Specifically as follows:
[0075] S1. Preparation of selenium polysaccharide: 8.5g of Ganoderma lucidum polysaccharide was added to 55g of YEPD medium, and selenium-enriched yeast seed liquid was inoculated at an inoculation amount of 2.5v / v%, fermented at 27℃ and 100r / min for 42h, the cells were removed by filtration, ethanol was added to the system content to 70wt%, precipitation was carried out for 1h, filtered, washed, and dried to obtain selenium polysaccharide;
[0076] S2. Preparation of selenium polysaccharide / thiophene modified product: 2.4 g of 3-(3-bromo)propoxy-4-methylthiophene, 3.5 g of triethylamine and 10 g of selenium polysaccharide were added to a mixed solution of N,N-dimethylformamide and water (volume ratio 4:8), heated to 55 °C, stirred for 6 h, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product;
[0077] S4. Preparation of antitumor drugs: 9.5g of gold nanocages / selenium polysaccharide / thiophene modified material was added to 150mL of nitromethane, and 50mL of ethanol solution containing 1.5g of paclitaxel was added. The mixture was stirred and mixed for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 7.5g of ferric chloride was added dropwise. The mixture was heated to 50℃ and stirred for 72h. The mixture was dialyzed for 1 day using a dialysis bag with a pore size of 8000Da. The unpermeable solution was freeze-dried to obtain the antitumor drugs.
[0078] Comparative Example 4
[0079] The difference from Example 3 is that no gold nanocage / selenium polysaccharide / thiophene modifier was added in step S5.
[0080] Specifically as follows:
[0081] S1. Preparation of polyethylene glycol / thiophene modified products:
[0082] S2. Preparation of antitumor drugs: 9.5g of polyethylene glycol / thiophene modified material was added to 150mL of nitromethane, and 50mL of ethanol solution containing 1.5g of paclitaxel was added. The mixture was stirred and mixed for 15min. Under nitrogen protection, 20mL of nitromethane solution containing 7.5g of ferric chloride was added dropwise. The mixture was heated to 50℃ and stirred for 72h. Dialysis was performed using a dialysis bag with a pore size of 8000Da for 1 day. The impurity was freeze-dried to obtain the antitumor drugs.
[0083] Test Example 1
[0084] The encapsulation efficiency and drug loading of the antitumor drugs prepared in Examples 1-3 and Comparative Examples 1-4 were determined.
[0085] In the final step of preparing antitumor drugs, the content of free drug in the centrifuged supernatant was measured using HPLC (high performance liquid chromatography), and the encapsulation efficiency was calculated according to the encapsulation efficiency formula:
[0086] Encapsulation efficiency = (1 − free drug amount / total drug amount) × 100%
[0087] Add 5 mg of antitumor drug powder to 1 mL of dichloromethane-tetrahydrofuran, dilute with 10 mL of methanol, shake thoroughly to allow the dichloromethane and tetrahydrofuran to evaporate rapidly, then centrifuge the solution at high speed, collect the supernatant, and measure the concentration of paclitaxel using HPLC (chromatographic conditions: octadecylsilane-bonded silica gel as the packing material; initial mobile phase: acetonitrile-water (40:60); gradient elution immediately after the main peak of paclitaxel has eluted (approximately 35 minutes); flow rate: 1.5 mL / min; column temperature: 30 °C; detection wavelength: 227 nm; injection volume: 10 μL). Calculate the drug loading using the drug loading formula.
[0088] Drug loading = (Mass of drug in antitumor drug / Mass of antitumor drug) × 100%
[0089] The results are shown in Table 1.
[0090] Table 1
[0091] Group Encapsulation rate (%) Drug loading (%) Example 1 98.99 15.25 Example 2 99.10 15.74 Example 3 99.24 16.12 Comparative Example 1 96.73 12.56 Comparative Example 2 98.87 10.11 Comparative Example 3 95.82 10.35 Comparative Example 4 95.04 9.73
[0092] As can be seen from the table above, the antitumor drugs prepared in Examples 1-3 of the present invention have high drug loading and encapsulation efficiency.
[0093] Test Example 2
[0094] Clean-grade mice weighing 22-26g were randomly divided into a blank control group, a model group, a paclitaxel group, Example 1-3 groups, and Comparative Example 1-4 groups, with 10 mice in each group. The room temperature was maintained at 18-21℃, the relative humidity at 40%-60%, and the light exposure was 12h / day. The animals had free access to food and water, and the experimental environment met national standards.
[0095] On day 7 after intraperitoneal implantation of H22 hepatocellular carcinoma cells in mice, the mice were euthanized by cervical dislocation, the abdomen was disinfected, and ascites fluid was aseptically aspirated and placed in a centrifuge tube. The fluid was diluted 10-fold with physiological saline, thoroughly mixed by pipetting, centrifuged, and the supernatant was decanted. Physiological saline was added at a specific ratio to prepare a tumor cell suspension. 8 Prepare 0.2 mL / mouse and inoculate it into the right axilla of mice (except for the blank control group, all other groups of mice were modeled using this method) to establish a liver cancer solid tumor animal model. The blank control group was given physiological saline.
[0096] Antitumor drugs were prepared into a mixture with physiological saline and aerated with oxygen for 10 min, each 0.25 mL containing 0.2 mg of paclitaxel. Additionally, paclitaxel was prepared with physiological saline to a concentration of 0.2 mg / 0.25 mL for administration. Starting on day 2 of modeling, the mice were administered daily at a dose of 0.25 mg / mouse via tail vein injection. Both the control and model groups received physiological saline. Administration continued for 10 days. On day 10, the mice were weighed and euthanized by cervical dislocation. The tumor tissue was dissected, weighed, and the tumor inhibition rate was calculated.
[0097] Tumor inhibition rate = [(average tumor mass in the model group - average tumor mass in the drug treatment group) / average tumor mass in the model group] × 100%.
[0098] Measurement of thymus index, spleen index, and liver index in tumor-bearing mice:
[0099] After the mice were euthanized, they were dissected and the tumor was removed. At the same time, the thymus, spleen and liver were removed. The mass of the thymus, spleen and liver was measured and the thymus, spleen and liver indices were calculated.
[0100] Thymus index = thymus mass (mg) / body mass (g), spleen index = spleen mass (mg) / body mass (g), liver index = liver mass (mg) / body mass (g)
[0101] The results are shown in Table 2.
[0102] Table 2
[0103]
[0104] Note: Compared with the model group, P<0.05; compared with the paclitaxel group, #P<0.05.
[0105] As can be seen from the table above, the antitumor drugs prepared in Examples 1-3 of the present invention have good antitumor effects and have little impact on organs.
[0106] 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 an antitumor drug, characterized in that, Selenium polysaccharide was obtained by fermenting Ganoderma lucidum polysaccharide with selenium-enriched yeast. It was then reacted with 3-(3-bromo)propoxy-4-methylthiophene and gold nanocages to prepare a modified gold nanocage / selenium polysaccharide / thiophene compound. A modified polyethylene glycol / thiophene compound was prepared by reacting biotin-PEG-propionaldehyde and 5-aminothiophene. The modified gold nanocage / selenium polysaccharide / thiophene compound and paclitaxel were then reacted with ferric chloride to prepare an antitumor drug.
2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of selenium polysaccharide: Ganoderma lucidum polysaccharide was added to the culture medium, selenium-enriched yeast seed liquid was inoculated, fermented and cultured, the cell bodies were removed by filtration, ethanol was added to precipitate, filtered, washed, and dried to obtain selenium polysaccharide; S2. Preparation of selenium polysaccharide / thiophene modified product: 3-(3-bromo)propoxy-4-methylthiophene, base and selenium polysaccharide were added to a mixed solution of N,N-dimethylformamide and water, heated and stirred to react, filtered, washed and dried to obtain selenium polysaccharide / thiophene modified product; S3. Preparation of polyethylene glycol / thiophene modified products: S4. Preparation of gold nanocages / selenium polysaccharide / thiophene modified product: Gold nanocages were dispersed in PBS buffer, selenium polysaccharide / thiophene modified product was added, the mixture was stirred by sonication, centrifuged, washed, and dried to obtain gold nanocages / selenium polysaccharide / thiophene modified product; S5. Preparation of antitumor drugs: Polyethylene glycol / thiophene modified material was added to nitromethane, gold nanocages / selenium polysaccharide / thiophene modified material was added, and the mixture was stirred and mixed evenly. Then, paclitaxel ethanol solution was added and stirred and mixed evenly. Under inert gas protection, ferric chloride nitromethane solution was added dropwise, and the reaction was heated and stirred. The mixture was dialyzed, and the unpermeated liquid was freeze-dried to obtain the antitumor drugs.
3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of Ganoderma lucidum polysaccharide to culture medium is 7-10:40-70, the culture medium is YEPD medium, and the bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 The inoculum concentration of the selenium-enriched yeast seed solution is 2-3 v / v%, and the fermentation conditions are 25-30℃, 50-150 r / min, and fermentation culture for 36-48 h.
4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of 3-(3-bromomethoxy)4-methylthiophene, the base, and the selenium polysaccharide is 2-3:3-4:
10. The heating and stirring reaction is carried out at a temperature of 50-60°C for 5-7 hours. The base is selected from at least one of triethylamine, diethylamine, NaOH, KOH, and ethylenediamine.
5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of biotin-PEG-propionaldehyde and 5-aminothiophene is 5-7:1-3, and the stirring reaction time is 3-5 hours.
6. The preparation method according to claim 2, characterized in that, In step S4, the pH of the PBS buffer is 7-7.5, the mass ratio of the gold nanocages to the selenium polysaccharide / thiophene modifier is 0.5-1:10, and the ultrasonic stirring reaction time is 30-50 min.
7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of polyethylene glycol / thiophene modified material, gold nanocage / selenium polysaccharide / thiophene modified material, paclitaxel, and ferric chloride is 3-5:4-7:1-2:6-9, and the heating and stirring reaction temperature is 45-55℃, and the time is 70-75h.
8. An antitumor drug prepared by the method according to any one of claims 1-7.
9. The use of the antitumor drug as described in claim 8 in the preparation of a medicament for treating hypoxic tumors.
10. The application according to claim 9, characterized in that, The hypoxic tumors include lung cancer, breast cancer, cervical cancer, liver cancer, pancreatic cancer, and colorectal cancer.