Crosslinked astragalus polysaccharide-platinum drug polymer as well as preparation method and application thereof

Abstract

The invention provides a cross-linked astragalus polysaccharide-platinum drug polymer as well as a preparation method and application thereof, and belongs to the field of drug polymer synthesis. The cross-linked astragalus polysaccharide-platinum drug polymer provided by the invention has a chemical structure as shown in a formula I which is described in the specification. The cross-linked astragalus polysaccharide-platinum drug polymer contains the astragalus polysaccharide unit and the cis-platinum-norcantharidin unit, and the astragalus polysaccharide unit improves the enrichment capacity of the polymer on tumor cells and prolongs the residence time; the cis-platinum-norcantharidin unit improves the anti-cancer effect of the polymer and reduces the toxic and side effects of a platinum drug. The toxicity of the cross-linked astragalus polysaccharide-platinum drug polymer provided by the invention is lower than that of cis-platinum and a cis-platinum-norcantharidin small-molecule prodrug; the cross-linked astragalus polysaccharide-platinum drug polymer has IC50 of 23.65 [mu] g / mL and 15.37 [mu] g / mL on two cancer cells A549 and HepG2 respectively, and has relatively low toxic and side effects and good anti-cancer activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical polymer synthesis, and more particularly to a cross-linked astragalus polysaccharide-platinum polymer, its preparation method, and its application. Background Technology

[0002] Cancer has always been a major medical challenge, and platinum-based anticancer drugs are crucial among current anticancer medications. Cisplatin, a classic representative of small-molecule platinum-based anticancer drugs, is highly effective but also has strong toxic side effects, easily causing significant damage to the body. Furthermore, small-molecule platinum-based anticancer drugs are easily eliminated from the body, reducing their therapeutic efficacy, and after a certain period of use, tumor cells are prone to developing drug resistance, leading to a series of side effects.

[0003] Related studies have shown that cisplatin and norcantharidin can have a synergistic effect when used in combination to improve the therapeutic effect. However, cisplatin and norcantharidin are used separately and are not bonded together, which increases the inconvenience of use. Furthermore, since cisplatin and norcantharidin are used separately, the toxic side effects of cisplatin cannot be effectively reduced. Summary of the Invention

[0004] The purpose of this invention is to provide a cross-linked astragalus polysaccharide-platinum polymer, its preparation method, and its applications. The cross-linked astragalus polysaccharide-platinum polymer provided by this invention exhibits low toxicity and good anticancer effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a cross-linked astragalus polysaccharide-platinum polymer having the chemical structure shown in Formula I: Formula I; In equation I, R is... .

[0006] This invention also provides a method for preparing the cross-linked astragalus polysaccharide-platinum polymer described in the above technical solution, comprising the following steps: Under an inert atmosphere, Astragalus polysaccharide, solvent, N,N-carbonyldiimidazole and triethylamine were mixed to carry out the first activation reaction, and then ethylenediamine was added to carry out the amination modification reaction of the polysaccharide to obtain cationic Astragalus polysaccharide. Under an inert atmosphere, hydroxycisplatin, norcantharidin, and a solvent were mixed and subjected to a complexation reaction to obtain a cisplatin-norcantharidin small molecule prodrug. Under inert atmosphere and light-protected conditions, the cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent are mixed to carry out a second activation reaction, and then the cationic astragalus polysaccharide is added to carry out a cross-linking reaction to obtain cross-linked astragalus polysaccharide-platinum drug polymer.

[0007] Preferably, the mass ratio of the astragalus polysaccharide, the mass of N,N-carbonyldiimidazole, and the volume of triethylamine is (0.20~0.80) g : (0.30~0.80) g : (20~60) μL; The mass ratio of Astragalus polysaccharide to the volume ratio of ethylenediamine is (0.20~0.80) g : (40~100) μL.

[0008] Preferably, the temperature of the amination modification reaction of the polysaccharide is 35~45℃, and the time of the amination modification reaction of the polysaccharide is 22~26h.

[0009] Preferably, the molar ratio of hydroxycisplatin to norcantharidin is 1:(2~4).

[0010] Preferably, the complexation reaction is carried out at a temperature of 55-65°C for 10-14 hours.

[0011] Preferably, the molar ratio of the cisplatin-norcantharidin small molecule prodrug, the activator, and the activating adjuvant is 1:(2~4):(2~4). The molar ratio of the cisplatin-norcantharidin small molecule prodrug to the cationic astragalus polysaccharide is 1:(1~2).

[0012] Preferably, the temperature of the second activation reaction is 25~30℃, and the time of the second activation reaction is 10~14h.

[0013] Preferably, the crosslinking reaction is carried out at a temperature of 25-30°C for 22-26 hours.

[0014] The present invention also provides the application of the cross-linked astragalus polysaccharide-platinum polymer described in the above technical solution or the cross-linked astragalus polysaccharide-platinum polymer prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0015] This invention provides a cross-linked astragalus polysaccharide-platinum polymer having the chemical structure shown in Formula I: Formula I; In equation I, R is... . This invention relates to a cross-linked astragalus polysaccharide-platinum drug polymer containing astragalus polysaccharide units and cisplatin-norcantharidin units. The astragalus polysaccharide units enhance the polymer's accumulation on tumor cells and prolong its retention time; the cisplatin-norcantharidin units enhance the polymer's anticancer effect while reducing the toxic side effects of platinum drugs (cisplatin). Cross-linking the astragalus polysaccharide units and cisplatin-norcantharidin units results in a cross-linked astragalus polysaccharide-platinum drug polymer with lower toxicity and better anticancer effects. Example results show that the cross-linked astragalus polysaccharide-platinum drug polymer provided by this invention has lower toxicity compared to cisplatin and cisplatin-norcantharidin small molecule prodrugs; the cross-linked astragalus polysaccharide-platinum drug polymer has an IC50 of [missing information - likely referring to an IC50 value] against A549 and HepG2 cancer cells. 50 The concentrations were 23.65 μg / mL and 15.37 μg / mL, respectively, indicating low toxicity and good anticancer activity. Attached Figure Description

[0016] Figure 1 The image shows a SEM image of Astragalus polysaccharide (APS) in Example 1 of this invention, with the scale bar on the left image being 300 μm and the scale bar on the right image being 100 μm. Figure 2 The image shows a SEM image of cationic Astragalus polysaccharide (APS-NH2) in Example 1 of this invention, with the scale bar on the left image being 300 μm and the scale bar on the right image being 100 μm. Figure 3 The image shows a SEM image of the cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 of this invention, with the scale bar on the left image being 300 μm and the scale bar on the right image being 100 μm. Figure 4 Infrared spectra of Astragalus polysaccharide (APS) and cationic Astragalus polysaccharide (APS-NH2) in Example 1 of this invention; Figure 5 Infrared spectra of cisplatin (DDP) and cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD) in Example 1 of this invention; Figure 6 Infrared spectra of Astragalus polysaccharide (APS), cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD), and cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 of this invention; Figure 7 Astragalus polysaccharide (APS) in Example 1 of this invention 1 H-NMR spectrum; Figure 8 The cationic astragalus polysaccharide (APS-NH2) in Example 1 of this invention 1 H-NMR spectrum; Figure 9The small molecule prodrug of hydroxycisplatin and cisplatin-norcantharidin (Pt(IV)-NCTD) in Example 1 of this invention 1 H-NMR spectrum; Figure 10 The cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 of this invention 1 H-NMR spectrum; Figure 11 The Astragalus polysaccharide (APS) and cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 of this invention showed the effects of IC50 on 293A cells, A549 cells, and HepG2 cells. 50 Data bar chart. Detailed Implementation

[0017] This invention provides a cross-linked astragalus polysaccharide-platinum polymer having the chemical structure shown in Formula I: Formula I; In equation I, R is... .

[0018] The cross-linked Astragalus polysaccharide-platinum drug polymer of this invention contains Astragalus polysaccharide units and cisplatin-norcantharidin units. The Astragalus polysaccharide units are used to enhance the polymer's accumulation ability on tumor cells and prolong its retention time; the cisplatin-norcantharidin units are used to enhance the polymer's anticancer effect while reducing the toxic side effects of platinum drugs (cisplatin); by cross-linking the Astragalus polysaccharide units and cisplatin-norcantharidin units, the cross-linked Astragalus polysaccharide-platinum drug polymer can have lower toxic side effects and better anticancer effects.

[0019] This invention also provides a method for preparing the cross-linked astragalus polysaccharide-platinum polymer described in the above technical solution, comprising the following steps: Under an inert atmosphere, Astragalus polysaccharide, solvent, N,N-carbonyldiimidazole and triethylamine were mixed to carry out the first activation reaction, and then ethylenediamine was added to carry out the amination modification reaction of the polysaccharide to obtain cationic Astragalus polysaccharide. Under an inert atmosphere, hydroxycisplatin, norcantharidin, and a solvent were mixed and subjected to a complexation reaction to obtain a cisplatin-norcantharidin small molecule prodrug. Under inert atmosphere and light-protected conditions, the cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent are mixed to carry out a second activation reaction, and then the cationic astragalus polysaccharide is added to carry out a cross-linking reaction to obtain cross-linked astragalus polysaccharide-platinum drug polymer.

[0020] Unless otherwise specified, all reagents used in this invention are commercially available reagents in the field of drug synthesis and analysis that are well known to those skilled in the art.

[0021] In this invention, under inert conditions, astragalus polysaccharide, solvent, N,N-carbonyldiimidazole and triethylamine are mixed to carry out a first activation reaction, and then ethylenediamine is added to carry out an amination modification reaction of the polysaccharide to obtain cationic astragalus polysaccharide.

[0022] In one embodiment of the present invention, the inert atmosphere may be nitrogen.

[0023] As one embodiment of the present invention, the structural formula of the astragalus polysaccharide can be: .

[0024] In one embodiment of the present invention, the solvent may be anhydrous dimethyl sulfoxide.

[0025] In one embodiment of the present invention, the mixing of Astragalus polysaccharide, solvent, N,N-carbonyldiimidazole and triethylamine can be carried out by first mixing Astragalus polysaccharide with solvent and then adding N,N-carbonyldiimidazole and triethylamine.

[0026] In one embodiment of the present invention, the mass ratio of the astragalus polysaccharide to the volume ratio of the solvent can be (0.20~0.80) g: (30~80) mL, or (0.40~0.65) g: (40~60) mL, or (0.50~0.60) g: (40~50) mL; the mass ratio of the astragalus polysaccharide, the mass of N,N-carbonyldiimidazole, and the volume ratio of triethylamine can be (0.20~0.80) g: (0.30~0.80) g: (20~60) μL, or (0.40~0.65) g: (0.40~0.65) g: (30~50) μL, or (0.50~0.60) g: (0.50~0.60) g: (40~50) μL. The present invention limits the ratio of the mass of Astragalus polysaccharide, the volume of solvent, the mass of N,N-carbonyldiimidazole, and the volume of triethylamine to the above range to ensure that the first activation reaction proceeds sufficiently.

[0027] In one embodiment of the present invention, the temperature of the first activation reaction can be 20~30℃; the time of the first activation reaction can be 1~2h. The present invention limits the temperature and time of the first activation to the above ranges to ensure sufficient activation of Astragalus polysaccharide and its reaction with N,N-carbonyldiimidazole.

[0028] In one embodiment of the present invention, the mass ratio of Astragalus polysaccharide to the volume ratio of ethylenediamine can be (0.20~0.80) g:(40~100) μL, (0.40~0.65) g:(60~90) μL, or (0.50~0.60) g:(70~80) μL. Limiting the mass ratio of Astragalus polysaccharide to the volume ratio of ethylenediamine to the above range allows for sufficient amination modification reaction of the polysaccharide, utilizing ethylenediamine for cationic modification of the Astragalus polysaccharide.

[0029] In one embodiment of the present invention, the amination modification reaction of the polysaccharide can be carried out under oil bath and stirring conditions. The present invention does not have any particular limitation on the equipment used for the oil bath; any oil bath heating equipment commonly used by those skilled in the art can be used. The present invention does not have any particular limitation on the stirring speed; any speed commonly used by those skilled in the art can be used to rotate the mixture and ensure uniform reaction.

[0030] In one embodiment of the present invention, the temperature of the amination modification reaction of the polysaccharide can be 35~45℃, or even 40℃; the time of the amination modification reaction of the polysaccharide can be 22~26h, or even 24h. By limiting the temperature and time of the amination modification reaction of the polysaccharide to the above ranges, the present invention ensures that the reaction proceeds sufficiently.

[0031] As one embodiment of the present invention, after the amination modification reaction of the polysaccharide is completed, the reaction product can be precipitated, dialyzed and dried to obtain cationic astragalus polysaccharide.

[0032] In one embodiment of the present invention, the precipitation process specifically involves adding ice-cold anhydrous ethanol to the reaction product, allowing it to stand, and then centrifuging. In this embodiment, the amount of ice-cold anhydrous ethanol added is three times the volume of the reaction product; the standing time is 0.5 hours. The present invention does not have special requirements for the centrifugation speed, as long as it is sufficient to separate the precipitate and the liquid. The present invention uses the addition of ice-cold anhydrous ethanol to precipitate the cationic astragalus polysaccharides in the reaction product.

[0033] In one embodiment of the present invention, the precipitate separated by centrifugation is subjected to dialysis.

[0034] In an embodiment of the present invention, the dialysis operation specifically involves redissolving the precipitate in water and dialyzing for 3 days, changing the dialysis solution every 2 hours. In an embodiment of the present invention, the dialysis solution is deionized water.

[0035] In one embodiment of the present invention, the drying process can be freeze-drying; the freeze-drying time can be 2 days. The present invention does not specifically limit the freeze-drying temperature; freeze-drying temperatures commonly used by those skilled in the art can be employed.

[0036] In embodiments of the present invention, the reactions occurring during the preparation of the cationized astragalus polysaccharide are shown in Formula 1: Formula 1.

[0037] In this invention, hydroxycisplatin, norcantharidin, and a solvent are mixed under an inert atmosphere and subjected to a complexation reaction to obtain a cisplatin-norcantharidin small molecule prodrug.

[0038] In one embodiment of the present invention, the inert atmosphere may be nitrogen.

[0039] As one embodiment of the present invention, the method for preparing hydroxycisplatin includes: mixing cisplatin with water to obtain a cisplatin solution, adding H2O2 solution for oxidation reaction, and then sequentially performing rotary evaporation, recrystallization, washing and drying to obtain hydroxycisplatin.

[0040] In an embodiment of the present invention, the concentration of the cisplatin solution is 55.5 mmol / L; the concentration of the H2O2 solution is 30 wt%; and the molar ratio of cisplatin to H2O2 in the H2O2 solution is 1:172.

[0041] In an embodiment of the present invention, the H2O2 solution is added dropwise; the rate at which the H2O2 solution is added is 2 mL / min.

[0042] In an embodiment of the present invention, the oxidation reaction is carried out under water bath conditions; the temperature of the oxidation reaction is 70°C; and the time of the oxidation reaction is 5 hours.

[0043] In an embodiment of the present invention, the oxidation reaction is shown in Formula 2: Formula 2.

[0044] In an embodiment of the present invention, after the oxidation reaction is completed, the oxidation reaction product is subjected to rotary evaporation; the rotary evaporation is carried out in a rotary evaporator. The present invention does not impose any special limitations on the operation of the rotary evaporation; parameters commonly used by those skilled in the art for concentrating the oxidation reaction product can be employed.

[0045] In an embodiment of the present invention, the recrystallization is a cooling recrystallization; the cooling recrystallization is carried out in a refrigerator; the temperature of the cooling recrystallization is 4°C; and the cooling recrystallization time is 12 hours.

[0046] In an embodiment of the present invention, the washing liquid used for washing is deionized water; and the washing is performed three times.

[0047] In an embodiment of the present invention, centrifugation is performed after washing. The present invention does not impose special limitations on the parameters for centrifugation, as long as solid-liquid separation can be achieved.

[0048] In embodiments of the present invention, the drying is carried out in a vacuum drying oven. The present invention does not impose special limitations on the drying temperature and time, as long as the moisture in the solid obtained by centrifugation is completely dried.

[0049] In one embodiment of the present invention, the molar ratio of hydroxycisplatin to norcantharidin can be 1:(2~4), 1:(2.5~3.5), or 1:(3~3.5). Limiting the molar ratio of hydroxycisplatin to norcantharidin to the above range ensures the smooth progress of the complexation reaction and allows hydroxycisplatin to react sufficiently.

[0050] In an embodiment of the present invention, the solvent in the mixture of hydroxycisplatin, norcantharidin and solvent is N,N-dimethylformamide; the concentration of hydroxycisplatin in the mixture obtained by mixing hydroxycisplatin, norcantharidin and solvent is 0.18 mol / L.

[0051] In one embodiment of the present invention, the temperature of the complexation reaction can be 55~65℃, or even 60℃; the time of the complexation reaction can be 10~14h, or even 12h. Limiting the temperature and time of the complexation reaction to the above ranges ensures the smooth progress of the complexation reaction.

[0052] In an embodiment of the present invention, the complexation reaction is shown in Formula 3: Formula 3.

[0053] In this invention, the reaction mechanism of the complexation reaction is shown in Equation 4: Formula 4.

[0054] As one embodiment of the present invention, after the complexation reaction is completed, the complexation reaction product can be subjected to rotary evaporation, precipitation, washing and drying in sequence to obtain cisplatin-norcantharidin small molecule prodrug.

[0055] In one embodiment of the present invention, the rotary evaporation can be performed in a rotary evaporator. The present invention does not impose any particular limitations on the operation of the rotary evaporation; parameters commonly used by those skilled in the art can be employed to remove the solvent from the complexation reaction product.

[0056] In an embodiment of the present invention, the precipitation specifically involves adding anhydrous methanol to the product after the solvent has been removed by rotary evaporation, followed by precipitation with ice-cold anhydrous diethyl ether. The addition of anhydrous methanol in this invention dissolves the cisplatin-norcantharidin small molecule prodrug, ensuring the purity of the cisplatin-norcantharidin small molecule prodrug obtained by precipitation.

[0057] In an embodiment of the present invention, centrifugation is performed immediately after the sedimentation is completed to separate a pale yellow powder.

[0058] In an embodiment of the present invention, the pale yellow powder is washed; the washing liquid used is acetone; and the washing is performed three times.

[0059] In an embodiment of the present invention, centrifugation is performed after washing. The present invention does not impose special limitations on the parameters for centrifugation, as long as solid-liquid separation can be achieved.

[0060] In embodiments of the present invention, the drying is carried out in a vacuum drying oven. The present invention does not impose special limitations on the drying temperature and time, as long as the moisture in the solid obtained by centrifugation is completely dried.

[0061] After obtaining cationic astragalus polysaccharide and cisplatin-norcantharidin small molecule prodrug, the present invention carries out a second activation reaction by mixing the cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent under an inert atmosphere and light-protected conditions. Then, the cationic astragalus polysaccharide is added to carry out a cross-linking reaction to obtain cross-linked astragalus polysaccharide-platinum drug polymer.

[0062] In one embodiment of the present invention, the inert atmosphere may be nitrogen.

[0063] In one embodiment of the present invention, the molar ratio of the cisplatin-norcantharidin small molecule prodrug, the activator, and the activating adjuvant can be 1:(2~4):(2~4), 1:(2.5~3.5):(2.5~3.5), or 1:(3~3.5):(3~3.5). Limiting the molar ratio of the cisplatin-norcantharidin small molecule prodrug, the activator, and the activating adjuvant to the above ranges allows for a more complete activation reaction.

[0064] In an embodiment of the present invention, the solvent in the mixture of cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent is N,N-dimethylformamide; the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; the activating aid is N-hydroxysuccinimide; and the concentration of cisplatin-norcantharidin small molecule prodrug in the mixture obtained by mixing the cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent is 0.045 mol / L.

[0065] In one embodiment of the present invention, the temperature of the second activation reaction can be 25~30℃, or 27~30℃; the time of the second activation reaction can be 10~14h, or 12h. Limiting the temperature and time of the second activation reaction to the above ranges ensures the smooth progress of the second activation reaction.

[0066] In one embodiment of the present invention, the molar ratio of the cisplatin-norcantharidin small molecule prodrug to the cationic astragalus polysaccharide can be 1:(1~2) or 1:(1.5~2). Limiting the molar ratio of the cisplatin-norcantharidin small molecule prodrug to the cationic astragalus polysaccharide within the above range allows for better obtaining of the cross-linked astragalus polysaccharide-platinum drug polymer.

[0067] In one embodiment of the present invention, the temperature of the crosslinking reaction can be 25~30℃, or 27~30℃; the time of the crosslinking reaction can be 22~26h, or 24h. Limiting the temperature and time of the crosslinking reaction to the above ranges ensures the smooth progress of the crosslinking reaction.

[0068] As one embodiment of the present invention, after the crosslinking reaction is completed, the crosslinking reaction product can be dialyzed and dried to obtain a crosslinked astragalus polysaccharide-platinum polymer.

[0069] In one embodiment of the present invention, the dialysis operation specifically involves dialysis of the cross-linking reaction product for 48 hours, with the dialysis solution being changed every 2 hours. In this embodiment, the dialysis solution is deionized water.

[0070] In one embodiment of the present invention, the drying process can be freeze-drying; the freeze-drying time can be 2 days. The present invention does not specifically limit the freeze-drying temperature; freeze-drying temperatures commonly used by those skilled in the art can be employed.

[0071] In an embodiment of the present invention, the crosslinking reaction is shown in Formula 5: Equation 5; In Equation 5, R is... .

[0072] The present invention also provides the application of the cross-linked astragalus polysaccharide-platinum polymer described in the above technical solution or the cross-linked astragalus polysaccharide-platinum polymer prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0073] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0074] Example 1 A cross-linked astragalus polysaccharide-platinum polymer has the following chemical structure: Formula I; In equation I, R is... .

[0075] The preparation method of the cross-linked Astragalus polysaccharide-platinum polymer is as follows: Under nitrogen atmosphere, Astragalus polysaccharide and anhydrous dimethyl sulfoxide were mixed, followed by the addition of N,N-carbonyldiimidazole and triethylamine. The mixture was subjected to a first activation reaction at 25°C for 1.5 h. Then, ethylenediamine was added, and the mixture was subjected to an amination modification reaction of the polysaccharide at 40°C in an oil bath for 24 h. Three volumes of ice-cold anhydrous ethanol were added to the amination modification product, and the mixture was allowed to stand for 0.5 h before centrifugation. The precipitate and liquid were separated. The obtained precipitate was reconstituted with water and dialyzed for 3 days, with the dialysate changed every 2 h. After dialysis, the precipitate was freeze-dried for 2 days to obtain cationic Astragalus polysaccharide. The mass ratio of Astragalus polysaccharide to anhydrous dimethyl sulfoxide was 0.60 g: 40 mL; the mass ratio of Astragalus polysaccharide, N,N-carbonyldiimidazole, and triethylamine was 0.60 g: 0.60 g: 40 μL; and the mass ratio of Astragalus polysaccharide to ethylenediamine was 0.60 g: 80 μL. Under nitrogen atmosphere, 1.01 g of cisplatin was mixed with 60 mL of water to obtain a cisplatin solution (55.5 mmol / L concentration). Then, 65 mL of 30 wt% H₂O₂ solution (the molar ratio of cisplatin to H₂O₂ in the solution was 1:172, and the H₂O₂ solution was added at a rate of 2 mL / min) was added. The oxidation reaction was carried out in a 70 °C water bath for 5 h. The oxidation product was then concentrated by rotary evaporation. The concentrated product was recrystallized at 4 °C for 12 h. The recrystallized product was washed three times with deionized water and centrifuged to separate the solid. The separated solid was dried in a vacuum drying oven to obtain 0.79 g of hydroxycisplatin (70% yield). 0.60 g of hydroxycisplatin, 1.21 g of norcantharidin, and 10 mL of... The N,N-dimethylformamide mixture was subjected to a complexation reaction at 60°C for 12 hours. After the complexation reaction, the N,N-dimethylformamide was removed by rotary evaporation of the complexation product. Then, anhydrous methanol was added for precipitation, followed by precipitation with ice-cold anhydrous diethyl ether. After precipitation, the product was immediately centrifuged to obtain a pale yellow powder. The pale yellow powder was washed three times with acetone and then centrifuged. The product obtained by centrifugation was dried in a vacuum drying oven to obtain 0.77 g of cisplatin-norcantharidin small molecule prodrug (yield 64%). The molar ratio of hydroxycisplatin to norcantharidin was 1:3. The concentration of hydroxycisplatin in the mixture obtained by mixing hydroxycisplatin, norcantharidin, and N,N-dimethylformamide was 0.18 mol / L. Under nitrogen atmosphere and in the dark, 0.30 g of the cisplatin-norcantharidin small molecule prodrug, 0.26 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 0.17 g of N-hydroxysuccinimide (NHS), and 10 mL of N,N-dimethylformamide were mixed and subjected to a second activation reaction at 25 °C for 12 h. Then, 0.23 g of the cationic astragalus polysaccharide was added and a cross-linking reaction was carried out at 25 °C for 24 h. The cross-linking product was dialyzed for 48 h, with the dialysate changed every 2 h. After dialysis, the precipitate was lyophilized for 2 days to obtain 0.23 g. Cross-linked Astragalus polysaccharide-platinum polymer (yield 42.6%); the molar ratio of the cisplatin-norcantharidin small molecule prodrug, activator, and activating aid is 1:3:3; the concentration of the cisplatin-norcantharidin small molecule prodrug in the mixture obtained by mixing the cisplatin-norcantharidin small molecule prodrug, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N,N-dimethylformamide is 0.045 mol / L; the molar ratio of the cisplatin-norcantharidin small molecule prodrug to cationic Astragalus polysaccharide is 1:2.

[0076] The surface morphology of Astragalus polysaccharide (APS), cationic Astragalus polysaccharide (APS-NH2), and cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 was observed using an ultra-high resolution scanning electron microscope (EYKY-EM6200 / 6900). The testing method was as follows: the sample was fixed to the sample stage with conductive adhesive, ensuring the sample size did not exceed the stage. Excess powder or impurities on the sample surface were blown away with a bulb syringe. Then, a gold film was deposited on the sample surface using a coating instrument to ensure good conductivity.

[0077] The SEM image of Astragalus polysaccharide (APS) in Example 1 is shown below. Figure 1 As shown, the scale bar in the left image is 300 μm, and the scale bar in the right image is 100 μm. From Figure 1 It can be seen that the morphology of unmodified Astragalus polysaccharide is irregular, relatively compact blocky and coarse structure.

[0078] The SEM image of cationic astragalus polysaccharide (APS-NH2) in Example 1 is shown below. Figure 2 As shown, the scale bar in the left image is 300 μm, and the scale bar in the right image is 100 μm. From Figure 2 It can be seen that the blocky structure of Astragalus polysaccharide after ethylenediamine cationization modification disappears and the surface becomes an interwoven and complex fibrous structure. This is because water molecules are continuously lost during the preparation of APS-NH2, which leads to changes in the spatial structure.

[0079] The SEM image of the cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 is shown below. Figure 3 As shown, the scale bar in the left image is 300 μm, and the scale bar in the right image is 100 μm. From Figure 3 It can be seen that the morphology of APS-NH2 changed again after reacting with the cisplatin-norcantharidin small molecule prodrug Pt(IV)-NCTD. The morphology of PAD showed an irregular flocculent structure, indicating that APS-NH2 and Pt(IV)-NCTD cross-linked each other during the reaction, causing the network structure of APS-NH2 to continuously twist and bend into flocculent form.

[0080] Fourier transform infrared spectroscopy was used to analyze the astragalus polysaccharide (APS), cationic astragalus polysaccharide (APS-NH2), cisplatin (DDP), cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD), and cross-linked astragalus polysaccharide-platinum polymer (PAD) in Example 1. Sample preparation involved adding 2 mg of the sample to be tested and 100 mg of dried KBr to an agate mortar, grinding them into powder, and pressing them into thin sheets.

[0081] The infrared spectra of Astragalus polysaccharide (APS) and cationic Astragalus polysaccharide (APS-NH2) in Example 1 are as follows: Figure 4 As shown, from Figure 4 It can be seen that APS-NH 2, At 3421cm -1 There is an absorption peak (a), which is the stretching vibration peak of the polysaccharide -OH group. APS-NH2 shows an absorption peak at 1709 cm⁻¹. -1 1539cm -1 The presence of absorption peaks (b and c) indicates that the amide bond is a characteristic peak, demonstrating that ethylenediamine has been successfully bonded to the APS backbone.

[0082] The infrared spectra of cisplatin (DDP) and cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD) in Example 1 are as follows: Figure 5 As shown, from Figure 5 It can be seen that DDP is at 3458cm -1 and 3288cm -1 There is an absorption peak, which is the stretching vibration peak of -NH2; Pt(IV)-NCTD has an absorption peak at 3435 cm⁻¹. -1 and 3270cm -1 It exhibits an absorption peak, which is also a characteristic stretching peak of -NH2; in addition, Pt(IV)-NCTD shows an absorption peak at 1301 cm⁻¹. -1 and 1236cm -1 There is an absorption peak, which is the stretching vibration peak of the carbonyl group in ester compounds; Pt(IV)-NCTD has an absorption peak at 1718 cm⁻¹. -1 The absorption peak at [value missing] is the stretching vibration peak of the carbonyl group in carboxylic acid compounds. Due to the wavenumber shift, it is speculated that Pt(IV)-NCTD may form a dimer, and the influence of intermolecular hydrogen bonds causes the absorption position to shift to a lower wavenumber. The above results indicate that the oxidation of cisplatin and the reaction with norcantharidin successfully yielded a compound with carboxyl groups at both ends.

[0083] The infrared spectra of Astragalus polysaccharide (APS), cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD), and cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 are as follows: Figure 6 As shown, from Figure 6 It can be seen that PAD is at 3431cm -1 There is an absorption peak, which is the stretching vibration peak of the polysaccharide -OH group; at 1636 cm⁻¹ -1 1562cm -1 and 1250cm -1 There is an absorption peak, which is the stretching vibration peak of secondary amide; at 1296 cm⁻¹ -1 and 1190cm -1 The presence of an absorption peak indicates the stretching vibration of the carbonyl group in the ester compound. These results demonstrate the successful synthesis of the cross-linked astragalus polysaccharide-platinum polymer (PAD).

[0084] The astragalus polysaccharide (APS), cationic astragalus polysaccharide (APS-NH2), hydroxycisplatin, cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD), and cross-linked astragalus polysaccharide-platinum polymer (PAD) in Example 1 were characterized by 1H nuclear magnetic resonance spectroscopy (1H-NMR). Specifically, 30 mg of each sample was taken and dissolved completely in 0.5 mL of deuterated water or deuterated DMSO. Tetramethylsilane (TMS) was used as an internal standard for testing. APS and APS-NH2 were dissolved in D2O, while hydroxycisplatin and Pt(IV)-NCTD were dissolved in DMSO-d6.

[0085] Astragalus polysaccharide (APS) in Example 1 1 H-NMR spectrum as shown Figure 7 As shown; cationic astragalus polysaccharide (APS-NH2) 1 H-NMR spectrum as shown Figure 8 As shown; hydroxycisplatin and cisplatin-norcantharidin small molecule prodrug (Pt(IV)-NCTD) 1 H-NMR spectrum as shown Figure 9 As shown; cross-linked Astragalus polysaccharide-platinum polymer (PAD) 1 H-NMR spectrum as shown Figure 10 As shown.

[0086] from Figure 7 and Figure 8 As can be seen, δH=4.3-5.5 represents the anomeric proton peak of the monosaccharide molecule in APS, δH=3.2-4.3 represents the H2-H6 proton peak of the APS monosaccharide molecule, and δH=2.98 represents the proton peak of ethylenediamine (NH2-CH2-CH2-NH2). These results indicate that ethylenediamine has successfully bonded to the APS backbone.

[0087] from Figure 9 As can be seen from the spectrum of hydroxycisplatin, the chemical shift of 7.13 ppm belongs to the amino group, and the chemical shift of 4-5 ppm belongs to the axial hydroxyl group of cisplatin. From the 1H NMR spectrum of Pt(IV)-NCTD, it can be seen that a (6.23 ppm) is the characteristic peak of the amino group of cisplatin, and b (4.65 ppm), c (4.59 ppm), d (2.73-2.93 ppm), and e (1.46-1.52 ppm) are the characteristic peaks of norcantharidin. The integral area ratio and the proton ratio are close, which proves that the synthesized Pt(IV)-NCTD structure is correct.

[0088] from Figure 10As can be seen, both APS-NH2 and Pt(IV)-NCTD are included in the characteristic chemical shift diagrams. Furthermore, the chemical shift of ethylenediamine at δH=2.98 has shifted, indicating a reaction occurring at this point, and the presence of an amide structure chemical shift. These results demonstrate that APS-NH2 and Pt(IV)-NCTD successfully bond to form PAD.

[0089] PAD 1 1H-NMR spectral data: 1H NMR (400 MHz, DMSO) δ 6.03 (s, 1H), 6.10 - 5.51 (m, 1H), 5.65 (dd, J = 185.0, 88.8 Hz, 2H), 6.10 - 4.98 (m, 3H), 6.10 - 4.92 (m, 3H), 6.10 - 4.65 (m, 5H), 4.22 - 3.04 (m, 66H), 3.14 - 3.04 (m, 2H), 3.01 (s, 1H), 2.87 - 2.43 (m, 28H), 2.53 (d, J = 15.8 Hz, 15H), 2.53 (d, J = 15.8 Hz, 15H), 2.55 - 2.30 (m, 18H), 2.55 - 2.21 (m, 20H), 2.55 - 1.80 (m, 24H), 2.55 - 1.59 (m, 25H), 2.55 - 1.92 (m, 23H), 2.55 - 2.05 (m, 22H), 1.51 (s,5H), 1.51 (s, 4H), 1.39 - 1.18 (m, 3H), 1.39 - 1.13 (m, 4H).

[0090] Cytotoxicity assays were performed on the astragalus polysaccharide (APS) and cross-linked astragalus polysaccharide-platinum polymer (PAD) from Example 1.

[0091] The Astragalus polysaccharide (APS) and cross-linked Astragalus polysaccharide-platinum polymer (PAD) in Example 1 showed IC50 in 293A, A549, and HepG2 cells. 50 The data is shown in Table 1, and the bar chart is as follows: Figure 11 As shown.

[0092] Table 1. Influence of Astragalus polysaccharide (APS) and cross-linked Astragalus polysaccharide-platinum polymer (PAD) on the IC50 of 293A, A549 and HepG2 cells in Example 1. 50 data Unit: μg / mL

[0093] From Table 1 and Figure 11 It can be seen that APS causes less damage to normal 293A cells, but induces apoptosis in A549 and HepG2 cells. PAD, on the other hand, causes significant damage to both A549 and HepG2 cancer cells, and its IC50 value for both cancer cell lines is relatively high. 50 The concentrations were 23.65 μg / mL and 15.37 μg / mL, respectively, demonstrating good anticancer activity.

[0094] The cross-linked astragalus polysaccharide-platinum polymer provided by this invention has lower toxicity compared to cisplatin and cisplatin-norcantharidin small molecule prodrugs; the cross-linked astragalus polysaccharide-platinum polymer has an IC50 of [missing information - likely referring to an IC50 value] against A549 and HepG2 cancer cells. 50 The concentrations were 23.65 μg / mL and 15.37 μg / mL, respectively, indicating low toxicity and good anticancer activity.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cross-linked Astragalus polysaccharide-platinum polymer having the chemical structure shown in Formula I: Formula I; In equation I, R is... .

2. The preparation method of the cross-linked astragalus polysaccharide-platinum polymer according to claim 1, comprising the following steps: Under an inert atmosphere, Astragalus polysaccharide, solvent, N,N-carbonyldiimidazole and triethylamine were mixed to carry out the first activation reaction, and then ethylenediamine was added to carry out the amination modification reaction of the polysaccharide to obtain cationic Astragalus polysaccharide. Under an inert atmosphere, hydroxycisplatin, norcantharidin, and a solvent were mixed and subjected to a complexation reaction to obtain a cisplatin-norcantharidin small molecule prodrug. Under inert atmosphere and light-protected conditions, the cisplatin-norcantharidin small molecule prodrug, activator, activating aid and solvent are mixed to carry out a second activation reaction, and then the cationic astragalus polysaccharide is added to carry out a cross-linking reaction to obtain cross-linked astragalus polysaccharide-platinum drug polymer.

3. The preparation method according to claim 2, characterized in that, The mass ratio of Astragalus polysaccharide, N,N-carbonyl diimidazole, and triethylamine is (0.20~0.80) g : (0.30~0.80) g : (20~60) μL; The mass ratio of Astragalus polysaccharide to the volume ratio of ethylenediamine is (0.20~0.80) g : (40~100) μL.

4. The preparation method according to claim 2, characterized in that, The amination modification reaction of the polysaccharide was carried out at a temperature of 35-45°C for 22-26 hours.

5. The preparation method according to claim 2, characterized in that, The molar ratio of hydroxycisplatin to norcantharidin is 1:(2~4).

6. The preparation method according to claim 2, characterized in that, The complexation reaction is carried out at a temperature of 55-65°C for 10-14 hours.

7. The preparation method according to claim 2, characterized in that, The molar ratio of the cisplatin-norcantharidin small molecule prodrug, activator, and activating aid is 1:(2~4):(2~4). The molar ratio of the cisplatin-norcantharidin small molecule prodrug to the cationic astragalus polysaccharide is 1:(1~2).

8. The preparation method according to claim 2, characterized in that, The temperature of the second activation reaction is 25~30℃, and the time of the second activation reaction is 10~14h.

9. The preparation method according to claim 2, characterized in that, The cross-linking reaction is carried out at a temperature of 25-30°C for 22-26 hours.

10. The application of the cross-linked astragalus polysaccharide-platinum polymer of claim 1 or the cross-linked astragalus polysaccharide-platinum polymer prepared by the preparation methods of claims 2 to 9 in the preparation of anticancer drugs.

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