Magnetic nano-particles as well as preparation method and application thereof

Abstract

The invention provides magnetic nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of drug carrier materials. A linear polymer with high-tension cycloolefin at one end and other groups at the other end is used for initiating olefin ring-opening double-decomposition polymerization reaction at a cycloolefin section, so that a chain structure is formed through polymerization, carboxyl is exposed after side chains of a high-molecular monomer A are deprotected, and the high-molecular monomer A can be coordinated with multivalent transition metal ions in a solvent; after cross-linking folding of the chain segment is achieved, the chain segment is dispersed in a solvent, an alkaline substance is added, metal ions complexed with the A side chain are converted into solid nano-particles in situ through a co-precipitation method, and chain-shaped composite nano-particles are obtained. The nano-particles have magnetism, and can actively pass through some tumor microenvironments with resistance, such as pancreatic tumors, under the action of an external alternating magnetic field, so that the nano-particles can enter parts where conventional drugs cannot enter for treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carrier materials, and in particular to magnetic nanoparticles and a preparation method and application thereof. Background Art

[0002] Common magnetic nanoparticles are in the micrometer or hundreds of nanometer range in size. At this scale, particles tend to accumulate in the liver and spleen and be engulfed by macrophages. If they are made of materials that cannot be metabolized by the organism, they will be vomited out by macrophages, and the particles will remain in the body for more than 8 months, causing unpredictable mechanical damage to the organism. Studies have shown that only nanoparticles with a size of less than 50nm can pass through blood vessels and enter tumor tissues. Although smaller nano drug carriers stay in the body for a shorter time, the rod-like morphology of the magnetic nanomachines formed by bottlebrush polymers helps to prolong their residence time in the body.

[0003] The magnetic nanoparticles prepared by the prior art are usually

[0004] In recent years, with the development of ring-opening metathesis polymerization (ROMP) reaction and its catalysts, it has become easy to synthesize nanoparticles with specific structures and sizes by selecting monomers with good biocompatibility and controlling the reaction time. Due to the above advantages, small-sized nanoparticles have great application potential in drug delivery systems. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing magnetic nanoparticles. The magnetic nanoparticles prepared by this method are worm-like in shape, small in particle size, and magnetic, so that they can move in biological tissues and thus penetrate some areas with resistance.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0007] A method for preparing magnetic nanoparticles comprises the following steps:

[0008] (1) Under anhydrous and oxygen-free conditions, polymer monomer A and polymer monomer B are added to a solvent, a catalyst is added to cause a ring-opening metathesis polymerization reaction for 2-3 hours (ROMP), and after the reaction is completed, vinyl ethyl ether is added dropwise to terminate the reaction, and the obtained reaction solution is dissolved, centrifuged, and dried to obtain a chain polymer I;

[0009] (2) dissolving the chain polymer I in dichloromethane (DCM) solvent, then adding trifluoroacetic acid to react for 40-50 hours, draining the solvent, adding dichloromethane to dissolve again, and draining twice; finally adding a small amount of ether, draining with a water pump at 30-60° C. in a warm water bath to obtain a chain polymer II; this step removes the isobutyl group and other protecting groups;

[0010] (3) Under an inert environment, the chain polymer II is dissolved in a mixed solvent I of dichloromethane and methanol (DCM / MeOH) to obtain a reactant a; anhydrous ferric chloride and anhydrous ferrous chloride are dissolved in a mixed solvent II of dichloromethane and methanol (DCM / MeOH) to obtain a reactant b; then the reactants a and b are added to a mixed solvent III of dichloromethane and methanol (DCM / MeOH), and after the dropwise addition is completed, ammonia water is added and stirred to react for 3-5 hours, and the solvent is removed by reduced pressure distillation to obtain the magnetic nanoparticles. In this step, the iron is attached to the polymer by a coprecipitation method.

[0011] Preferably, the polymer monomer A in step (1) is composed of an intermediate monomer C and a cycloolefin group; one end of the intermediate monomer C is connected to the cycloolefin group, and the other end is connected to the terminal group E;

[0012] The intermediate monomer C is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl and their derivatives; the terminal group E is any one of methoxy, phenyl, vinyl, ester, amide, alkyl, benzyl, acyl halide, pyridine, ketone, naphthyl or substituted naphthyl.

[0013] Preferably, the molecular weight of the polymer monomer A is 500-50000.

[0014] More preferably, the polymer monomer A in step (1) is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol.

[0015] Preferably, the polymer monomer B in step (1) comprises an intermediate monomer D, a cycloolefin group and a tert-butyl ester group; one end of the intermediate monomer D is connected to the cycloolefin group, and the other end is connected to the tert-butyl ester group;

[0016] The intermediate monomer D is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl, and their derivatives.

[0017] Preferably, the molecular weight of the polymer monomer B is 500-50000.

[0018] More preferably, the polymer monomer B is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol-tert-butyl ester.

[0019] Preferably, the mass ratio of the polymer monomer A to the polymer monomer B in step (1) is 1:100-100:1;

[0020] The solvent is dichloromethane, and the catalyst is Grubbs third generation catalyst (purchased from Sigma-Aldrich Company).

[0021] Preferably, in the removal reaction of step (2), the ratio of chain polymer I, dichloromethane and trifluoroacetic acid is 50-55 mg: 5-6 mL: 300-350 μL;

[0022] The ratio of dichloromethane added to the chain polymer I during the re-dissolution in step (2) is 1:1; the ratio of ether to chain polymer I is 100 μL:50 mg.

[0023] Preferably, in step (3), the ratio of dichloromethane to methanol in the mixed solvent I is 1:1; the ratio of dichloromethane to methanol in the mixed solvent II is 1:1; the ratio of dichloromethane to methanol in the mixed solvent III is 1:1;

[0024] The ratio of the chain polymer I to the mixed solvent I is 5-50 mg: 3-5 ml; the ratio of the anhydrous ferric chloride, anhydrous ferrous chloride and the mixed solvent II is 0.1-3 mg: 0.1-1.5 mg: 3-5 ml;

[0025] The volume ratio of the mixed solvent I, the mixed solvent II, the mixed solvent III and the ammonia water is 3-5ml:3-5ml:40-50ml:1-2ml; the concentration of the ammonia water is 20-30%.

[0026] The present invention also provides a magnetic nanoparticle prepared by the method and application of the magnetic nanoparticle. The magnetic nanoparticle is used for preparing anti-tumor drugs.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention uses a linear polymer with a high-tension cycloolefin at one end and other groups at the other end to initiate an olefin ring-opening metathesis polymerization reaction in the cycloolefin segment, thereby polymerizing to form a chain structure, and then removing the protection of the side chain of the polymer monomer A to expose the carboxyl group, which can react with transition metal ions (such as Fe 2+ and Fe 3+ ) to coordinate, realize the cross-linking and folding of this chain segment, disperse it in a solvent, add alkaline substances, and use the "coprecipitation method" to make the metal ions complexed with the A side chain in situ converted into solid nanoparticles to obtain chain-shaped composite nanoparticles. The nanoparticles are worm-like, small in size, and magnetic, so that they can move in biological tissues and penetrate some areas with resistance.

[0029] 2. The nanoparticles of the present invention are synthesized using linear polymers that can be metabolized in vivo, and are connected to side chain-modified polyamino acids, which can be degraded in vivo, so they have good biocompatibility and are highly safe for the human body. In the subsequent application process, positive drugs, fluorescent groups, targeting groups, etc. can be introduced into the polyamino acid side chains of the nanoparticles, so that the nanoparticles become drug-carrying molecules, break through the biological barrier in a targeted manner, and deliver the drugs to the lesion site, so as to achieve the purpose of drug concentration accumulation and treat the disease.

[0030] 3. The nanoparticles of the present invention are magnetic and can actively pass through some tumor microenvironments with resistance, such as pancreatic tumors, under the action of an external alternating magnetic field, thereby entering areas that conventional drugs cannot enter for treatment.

[0031] 4. The nanomaterials provided by the present invention have broad application prospects as drug-carrying materials in the treatment of tumors and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a structural diagram of the chain polymer I prepared in Example 5;

[0033] Figure 2 This is a two-photon microscopy image of the nanoparticles prepared in Example 13 loaded with the fluorescent dye molecule Cyanine 3. DETAILED DESCRIPTION

[0034] The present invention provides a method for preparing magnetic nanoparticles, the steps of which are as follows:

[0035] (1) under anhydrous and oxygen-free condition, polymer monomer A and polymer monomer B are added to a solvent, a catalyst is added to cause a ring-opening metathesis polymerization reaction for 2-3 hours (ROMP), after the reaction is completed, 1-5 drops of vinyl ethyl ether are added to terminate the reaction, and the obtained reaction solution is dissolved, centrifuged, and dried to obtain a chain polymer I;

[0036] (2) dissolving the chain polymer I in dichloromethane (DCM) solvent, then adding trifluoroacetic acid to carry out a removal reaction for 40-50 hours, draining the solvent, adding dichloromethane to dissolve again, and repeating the process of draining twice; finally, adding ether, and draining in a warm water bath at 30-60° C. to obtain a chain polymer II;

[0037] (3) Under an inert environment, the chain polymer II is dissolved in a mixed solvent I of dichloromethane and methanol (DCM / MeOH) to obtain a reactant a; anhydrous ferric chloride and anhydrous ferrous chloride are dissolved in a mixed solvent II of dichloromethane and methanol (DCM / MeOH) to obtain a reactant b; then the reactants a and b are added to a mixed solvent III of dichloromethane and methanol (DCM / MeOH), and after the dropwise addition is completed, ammonia water is added and stirred to react for 3-5 hours, and the solvent is removed by reduced pressure distillation to obtain the magnetic nanoparticles.

[0038] Preferably, the polymer monomer A in step (1) is composed of an intermediate monomer C and a cycloolefin group; one end of the intermediate monomer C is connected to the cycloolefin group, and the other end is connected to the terminal group E;

[0039] The intermediate monomer C is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl and their derivatives; the terminal group E is any one of methoxy, phenyl, vinyl, ester, amide, alkyl, benzyl, acyl halide, pyridine, ketone, naphthyl or substituted naphthyl.

[0040] Preferably, the molecular weight of the polymer monomer A is 500-50000.

[0041] More preferably, the polymer monomer A in step (1) is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol.

[0042] Preferably, the polymer monomer B in step (1) comprises an intermediate monomer D, a cycloolefin group and a tert-butyl ester group; one end of the intermediate monomer D is connected to the cycloolefin group, and the other end is connected to the tert-butyl ester group;

[0043] The intermediate monomer D is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl, and their derivatives.

[0044] Preferably, the molecular weight of the polymer monomer B is 500-50000.

[0045] More preferably, the polymer monomer B is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol-tert-butyl ester.

[0046] Preferably, the mass ratio of the polymer monomer A to the polymer monomer B in step (1) is 1:100-100:1;

[0047] The solvent is dichloromethane, and the catalyst is Grubbs third generation catalyst (purchased from Sigma-Aldrich Company).

[0048] Preferably, in step (2), the ratio of the chain polymer I, dichloromethane and trifluoroacetic acid is 50-55 mg: 5-6 mL: 300-350 μL;

[0049] The ratio of dichloromethane added to the chain polymer I during the re-dissolution in step (2) is 1:1; the ratio of ether to chain polymer I is 100 μL:50 mg.

[0050] Preferably, in step (3), the ratio of dichloromethane to methanol in the mixed solvent I is 1:1; the ratio of dichloromethane to methanol in the mixed solvent II is 1:1; the ratio of dichloromethane to methanol in the mixed solvent III is 1:1;

[0051] The ratio of the chain polymer I to the mixed solvent I is 5-50 mg: 3-5 ml; the ratio of the anhydrous ferric chloride, anhydrous ferrous chloride and the mixed solvent II is 0.1-3 mg: 0.1-1.5 mg: 3-5 ml;

[0052] The volume ratio of the mixed solvent I, the mixed solvent II, the mixed solvent III and the ammonia water is 3-5ml:3-5ml:3-50ml:1-2ml; the concentration of the ammonia water is 20-30%.

[0053] More preferably, the method for preparing the polymer monomer A comprises the following steps:

[0054] S1-1, methoxy polyethylene glycol 5000 and toluene are mixed and dried, the solution is cooled to 0°C, and then nitrogen is introduced for 15-20 minutes under stirring, 4-(dimethylamino)pyridine and triethylamine are added under nitrogen flow, and then 2-bromo-2-methylpropionyl bromide is slowly added dropwise; after the addition is completed, the mixture is reacted under oil bath heating conditions for 48 hours; after the reaction is completed, the triethylamine salt is removed by filtration; the filtrate is then concentrated to a mass fraction of mPEG of 10%, and after heating and dissolving, the mixture is slowly added dropwise to n-hexane to obtain an mPEG-Br precipitate; the dissolution-precipitation steps are repeated once to obtain an mPEG-Br product;

[0055] S1-2, the mPEG-Br and DMF are mixed, and then sodium azide is added, and the reaction is carried out at 60° C. and a rotation speed of 600 r / min for 24 hours. After the reaction, DMF is removed; the product is dissolved in tetrahydrofuran, dripped into n-hexane, centrifuged at -20° C., the supernatant is discarded, and the steps of dissolution-precipitation-centrifugation are repeated once, and mPEG-N3 is obtained after drying;

[0056] S1-3, dissolving 6-amino-1-hexanol and cis-5-norbornene-exo-2,3-dicarboxylic anhydride in toluene, reacting in an oil bath at 130-140° C. for 30-36 hours, and purifying by column chromatography with a mixed solvent of ethyl acetate and hexane to obtain norbornene hexanol;

[0057] S1-4, dissolving the norbornene hexanol in dichloromethane (DCM), and then adding 4-pentynoic acid. First add dicyclohexylcarbodiimide (DCC) under the condition of ice water bath, and then add 4-dimethylaminopyridine (DMAP) dropwise, and then react at room temperature and 800r / min for 20-24h, and purify to obtain norbornene ester after the reaction is completed;

[0058] S1-5. Mix the mPEG-N3 in step S1-2 and the norbornene ester in step S1-4, and add PMDETA, ultra-dry tetrahydrofuran and CuBr in anhydrous, oxygen-free, nitrogen environment. Freeze at -196°C for 5 minutes and evacuate for 10 minutes, then return to room temperature, repeat three times, maintain vacuum, heat to 50°C, and then return nitrogen. React for 24 hours. After the reaction, open the lid, cool to room temperature, stir in the air for 12 hours to completely oxidize the copper, then remove the copper through a neutral alumina column, using dichloromethane as an eluent. Use rotary evaporation to remove dichloromethane, dissolve in tetrahydrofuran, precipitate in n-hexane, centrifuge at -20°C, pour off the supernatant, repeat dissolution-precipitation-centrifugation twice to obtain the product. Dry at room temperature in a vacuum oven for 24 hours to obtain Norb-mPEG, which is polymer monomer A.

[0059] More preferably, the preparation method of the polymer monomer B comprises the following steps:

[0060] S2-1. Mix methoxypolyethylene glycol 2000 and toluene, dry them, cool the solution to 0°C, then introduce nitrogen for 15-20 minutes under stirring, add 4-(dimethylamino)pyridine and triethylamine under nitrogen flow, and then slowly add 2-bromo-2-methylpropionyl bromide; after the addition is completed, react for 48 hours under oil bath heating conditions; after the reaction is completed, filter out the triethylamine salt; then concentrate the filtrate to a mass fraction of mPEG of 10%, heat to dissolve, and slowly add to n-hexane to obtain an mPEG-Br precipitate; repeat the dissolution-precipitation steps to obtain an mPEG-Br product.

[0061] S2-2, calcium hydride and tert-butyl acrylate (t-BA) were mixed, t-BA monomer was distilled off under reduced pressure, and t-BA monomer was directly added dropwise to a high vacuum reaction tube, and then PMDETA, MeOPEG-Br, ultra-dry anisole and CuBr were added in sequence. Freeze-pumped three times, heated to 80°C, nitrogen was returned after the temperature reached 80°C, and the reaction time was 6h. After the reaction, open the lid, cool to room temperature, add 5 ml of chloroform, stir in the air for more than 12 hours to completely oxidize the copper, then pass through a neutral alumina column to remove copper, use dichloromethane as the eluent to elute the column, collect the chromatographic liquid and concentrate it, drop it into n-hexane for precipitation, centrifuge at -20°C, pour out the supernatant, repeat dissolution-precipitation-centrifugation 3 times, bake in a vacuum oven at room temperature for 12 hours to obtain mPEG-PtBA-Br, then use DMF to dissolve mPEG-PtBA-Br, and then use a bull horn spoon to add three times the molar amount of sodium azide, the reaction temperature is 60°C, and the reaction time is 24 hours. After the reaction is completed, use an oil pump and a cold trap to remove DMF. Then use tetrahydrofuran to dissolve the product, drop it into a large amount of -20°C n-hexane to precipitate the product. Centrifuge at -20°C and pour out the supernatant. Repeat dissolution-precipitation-centrifugation once more. Dry in a vacuum oven at room temperature for 24 hours. PEG-PtBA-N3 product was obtained with a t-BA conversion rate of 66%.

[0062] S2-3, dissolving 6-amino-1-hexanol and cis-5-norbornene-exo-2,3-dicarboxylic anhydride in toluene, reacting in an oil bath at 130-140° C. for 30-36 hours, and purifying by column chromatography with a mixed solvent of ethyl acetate and hexane to obtain norbornene hexanol;

[0063] S2-4, dissolving the norbornene hexanol in dichloromethane (DCM), and then adding 4-pentynoic acid. First, add dicyclohexylcarbodiimide (DCC) under the condition of ice water bath, and then add 4-dimethylaminopyridine (DMAP) dropwise, and then react at room temperature and 800r / min for 20-24h, and purify to obtain norbornene ester after the reaction is completed;

[0064] S2-5. Add PEG-PtBA-N3 obtained in S2-2 and norbornene ester obtained in S2-4 to the reaction tube, and then add PMDETA, ultra-dry tetrahydrofuran and CuBr in anhydrous, oxygen-free and nitrogen environment. Continue to freeze-pump three times, maintain vacuum, heat to 50°C, and then return nitrogen to react for 24 hours. After the reaction, open the lid, cool to room temperature, stir in the air for 12 hours to completely oxidize the copper, and then remove the copper through a neutral alumina column, using dichloromethane as an eluent. Use rotary evaporation to remove dichloromethane, dissolve in tetrahydrofuran, drop into -20°C n-hexane for precipitation, centrifuge at -20°C, pour off the supernatant, repeat dissolution-precipitation-centrifugation twice to obtain the product. Dry in a vacuum oven at room temperature for 24 hours to obtain the product (Norb-mPEG-tBA, polymer monomer B).

[0065] The present invention will be further described below in conjunction with the embodiments.

[0066] Example 1

[0067] The preparation method of high molecular polymer A comprises the following steps:

[0068] The first step: in an inert environment, 500010g of allyl polyethylene glycol and 320ml of toluene are mixed, dried at 138°C and 700r / min, and the toluene is distilled for 24h to remove a large amount of water in the toluene. The solution is cooled to 0°C in isolation of water, and mPEG (allyl polyethylene glycol) is precipitated from the toluene; then high-purity nitrogen is bubbled into the solution for 15 minutes under stirring; under nitrogen conditions, 0.024g of 4-dimethylaminopyridine and 0.66g of triethylamine are first quickly added, and then 1.95g of 2-bromo-2-methylpropionyl bromide is slowly added dropwise, and the addition time needs to exceed 15min. After the addition is completed, the nitrogen is removed; then the reaction is carried out in an oil bath at 45°C and a speed of 600r / min for 48h. After the reaction is completed, it is filtered while hot to remove the triethylamine salt, and the filtrate is concentrated to 10% by mass of mPEG (i.e., 10g per 100mL of solution). mPEG) was heated to 40°C to promote dissolution, and then 50 mL was measured and slowly added dropwise to 10 times (1 L) volume of n-hexane through a filter membrane. The product mPEG-Br ester was precipitated and filtered to collect. The dissolution-precipitation process was repeated again. The product was filtered to collect the product to obtain 8.9 g of pure mPEG-Br product with a yield of 89%. The specific reaction is as follows (1):

[0069]

[0070] Step 2: Mix 2g of the mPEG-Br and 8ml of DMF (N,N-dimethylformamide), and then add 0.078g of sodium azide in a three-fold molar amount for reaction. The reaction temperature is 60°C, the speed is 600r / min, and the reaction time is 24h. After the reaction, use a cold trap protection oil pump to remove DMF (N,N-dimethylformamide); then use about 15ml of tetrahydrofuran to dissolve the product, drop it into 10 times the volume of n-hexane, centrifuge at -20°C, pour off the supernatant, repeat dissolution-precipitation-centrifugation, and dry it in a vacuum drying oven at room temperature for 12h. 1.9g of mPEG-N3 product is obtained with a yield of 95%; the specific reaction is as shown in formula (2):

[0071]

[0072] Step 3: Dissolve 7.5 g of 6-amino-1-hexanol and 10 g of cis-5-norbornene-exo-2,3-dicarboxylic anhydride in 125 mL of toluene, and react in an oil bath at 138° C. for 36 hours to obtain a reaction product; then wash the silica gel column with ethyl acetate / n-hexane = 1:2, elute the silica gel column with ethyl acetate:hexane = 1:1, and purify the product by column chromatography using wet loading and dry loading; collect 12.5 g of colorless oil, collect 15 g of norbornene hexanol, and the yield is 86%; the specific reaction is as shown in formula (3):

[0073]

[0074] Step 4: dissolve 1g of the norbornene hexanol in 20ml of DCM, add 0.410g of 4-pentynoic acid, then add 0.941g of DCC in an ice-water bath, then add 1ml of DMAP (4-dimethylaminopyridine) with a concentration of 0.1g / ml dropwise, react for 20h at room temperature and 800r / min after the addition is completed; filter out part of the generated DCU (dicyclohexylurea); use ethyl acetate / n-hexane = 1:3 as an eluent for column chromatography, wet column loading, dry loading, collect the chromatographic liquid for rotary evaporation to obtain a colorless oily liquid, dissolve the obtained product with a small amount of ethyl acetate, place it in a refrigerator at -20°C for cooling, filter it at low temperature after cooling, repeat rotary evaporation-dissolution-cooling-filtration once, and obtain 0.68g of colorless oily liquid norbornene ester, with a yield of 50%; the specific reaction is as follows (4):

[0075]

[0076] Step 5: Mix 1.8 g of mPEG-N3 obtained in the second step and 2.7 g of norbornene ester obtained in the fourth step, and then add 61 mg of PMDETA (pentamethyldiethylenetriamine), 8 mL of ultra-dry tetrahydrofuran and 51 mg of CuBr (cuprous bromide) in anhydrous, oxygen-free, nitrogen environment. Freeze at -196 ° C for 5 min and evacuate for 10 min, freeze and evacuate three times, heat to 50 ° C, and then return nitrogen. React for 24 hours. After the reaction, open the lid, cool to room temperature, stir in the air to oxidize all copper, then remove copper by column chromatography, collect the chromatographic liquid and spin dry, dissolve it in tetrahydrofuran, precipitate in -20 ° C n-hexane, centrifuge at -20 ° C, pour out the supernatant, and repeat 2 times to obtain the product. After drying at room temperature for 24 h, 1.6 g of product (cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol, Norb-mPEG, polymer monomer A, molecular weight 5000, denoted as mPEG5000) was obtained with a yield of 84%. The specific reaction is as shown in formula (5):

[0077]

[0078] Example 2

[0079] The preparation method of high molecular polymer B comprises the following steps:

[0080] Step 6: 10 g of methoxy polyethylene glycol with a molecular weight of 2000 was reacted with 3 times the molar amount of 2-bromoisobutyryl bromide according to the conditions of the first step of Example 1, and 9 g of PEG-PtBA-N3 product was obtained after purification with a yield of 89%; the specific reaction is as shown in formula (6):

[0081]

[0082] Step 7: Mix 20g calcium hydride and 120ml t-BA, stir and remove water at room temperature and 800r / min, perform vacuum distillation, and add PMDETA (pentamethyldiethylenetriamine), MeOPEG-Br, 2ml anisole and 51mgCuBr (cuprous bromide) to the purified t-BA monomer. Continue to freeze and pump three times, heat to 80℃ and store nitrogen, and the reaction time is 6h. After the reaction, open the lid, cool to room temperature, add 5ml chloroform, stir in the air to oxidize all copper, then remove copper by column chromatography, and collect the chromatographic liquid. The chromatographic liquid is concentrated to 1 / 10 volume, precipitated in -20℃ n-hexane, centrifuged at -20℃, and the supernatant is poured out, and repeated 3 times. Dry at room temperature for 12h to obtain the product mPEG-PtBA-Br. Then, DMF (N,N-dimethylformamide) was used to dissolve mPEG-PtBA-Br, and three times the molar amount of sodium azide was added. The reaction temperature was 60°C and the reaction time was 24h. After the reaction, DMF (N,N-dimethylformamide) was removed. The product was dissolved in tetrahydrofuran, and dropped into a large amount of frozen n-hexane at -20°C to precipitate the product, centrifuged, and the supernatant was discarded. Repeat once. Dry at room temperature for 24h. The PEG-PtBA-N3 product was obtained, and the t-BA conversion rate was 66%; the specific reaction is as shown in formula (7) and formula (8):

[0083]

[0084] Step 8: Under anhydrous and oxygen-free nitrogen environment, 0.3 g of norbornene ester obtained in the fourth step of Example 1 and 1.5 g of PEG-PtBA-N3 with a molecular weight of 4400 obtained in the seventh step of Example 2 were added to the reaction tube, and 59 mg of PMDETA (pentamethyldiethylenetriamine), 8 ml of ultra-dry tetrahydrofuran, and 49 mg of CuBr (cuprous bromide) were added in sequence. Continue to freeze-pump three times, heat to 50 ° C, and then return nitrogen. React for 24 hours. After the reaction, open the lid, cool to room temperature, stir in the air to completely oxidize the copper, then column chromatography to remove copper, collect the chromatographic liquid, and spin dry. Use tetrahydrofuran to dissolve the crude product, precipitate in n-hexane, centrifuge at -20 ° C, pour out the supernatant, and repeat twice to obtain the product. After drying at room temperature for 24 hours, the product (cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol-tert-butyl ester, Norb-mPEG-tBA, polymer monomer B, molecular weight 4400) was obtained; the specific reaction is as shown in formula (9):

[0085]

[0086] Example 3

[0087] The preparation method of high molecular polymer A, the second step to the fifth step are the same as in Example 1, and the specific steps of the first step are as follows:

[0088] In an inert environment, 500010g of methoxy polyethylene glycol and 320ml of toluene were mixed, dried at 138°C and 700r / min, and the toluene was distilled for 24h to remove a large amount of water in the toluene. The solution was cooled to 0°C with isolation of water, and mPEG was precipitated from the toluene; then high-purity nitrogen was bubbled into the solution for 15 minutes under stirring; under nitrogen conditions, 0.024g of 4-(dimethylamino)pyridine and 0.66g of triethylamine were quickly added first, and then 1.95g of 2-bromo-2-methylpropionyl bromide was slowly added dropwise, and the addition time needed to exceed 15min. After the addition was completed, the nitrogen was removed; then the reaction was carried out in an oil bath at 45°C and a speed of 600r / min for 48h. After the reaction was completed, it was filtered while hot to remove the triethylamine salt, and the filtrate was concentrated to 10% mass fraction of mPEG (i.e., 10g of mPEG was contained in every 100mL of solution). mPEG) was heated to 40°C to promote dissolution, and then 50 mL was slowly added dropwise to 10 times (1 L) volume of n-hexane through a filter membrane. The product mPEG-Br ester was precipitated and collected by filtration. The dissolution-precipitation process was repeated again, and the product was collected by filtration.

[0089] Step 6: Dissolve 10 g of the product in dichloromethane, place in an ice-water bath, add 0.5 g of boron tribromide, react for 1 h, remove the ice-water bath, add distilled water, keep the temperature at 35 ° C, and react for 2 h. After the reaction, use a rotary evaporator to dry the solvent and boron tribromide, and distill the product under reduced pressure to remove impurities.

[0090] Example 4

[0091] The preparation method of high molecular polymer A, the second step to the fifth step are the same as in Example 1, and the specific steps of the first step are as follows:

[0092] In an inert environment, 500010g of polyethylene glycol amine and 320ml of toluene were mixed, dried at 138°C and 700r / min, and the toluene was distilled for 24h to remove a large amount of water in the toluene. The solution was cooled to 0°C with isolation of water, and mPEG was precipitated from the toluene; then high-purity nitrogen was bubbled into the solution for 15 minutes under stirring; in a nitrogen environment, 0.024g of 4-dimethylaminopyridine and 0.66g of triethylamine were quickly added first, and then 1.95g of 2-bromo-2-methylpropionyl bromide was slowly added dropwise, and the addition time needed to exceed 15min. After the addition was completed, the nitrogen was removed; then the reaction was carried out in an oil bath at 45°C and a speed of 600r / min for 48h. After the reaction was completed, it was filtered while hot to remove the triethylamine salt, and the filtrate was concentrated to 10% mass fraction of mPEG (i.e., 10g of mPEG was contained in every 100mL of solution). mPEG) was heated to 40°C to promote dissolution, and then 50 mL was slowly added dropwise to 10 times (1 L) volume of n-hexane through a filter membrane. The product mPEG-Br ester was precipitated and collected by filtration. The dissolution-precipitation process was repeated again, and the product was collected by filtration.

[0093] Example 5

[0094] A method for preparing magnetic nanoparticles, comprising the following steps:

[0095] (1) Under anhydrous and oxygen-free conditions, 100 mg of polymer monomer B prepared in Example 2 and 500 mg of polymer monomer A prepared in Example 1 (feed ratio A:B=5:1) were added to a reaction bottle, dehydrated using a cold trap and transferred to a glove box, 5 ml of ultra-dry dichloromethane solvent and 1.5 mg of Grubbs third-generation catalyst were added to undergo a ring-opening metathesis polymerization reaction for 2 h. After the reaction was completed, a drop of vinyl ethyl ether was added to terminate the reaction, the reaction solution was dripped into 40 ml of n-hexane, centrifuged at -20°C, the supernatant was discarded, the precipitate was dissolved using tetrahydrofuran, and then dripped into n-hexane and centrifuged at -20°C, the supernatant was discarded; the dissolution-precipitation-centrifugation was repeated twice; and the chain polymer I was obtained by drying in a vacuum oven at room temperature for 24 h.

[0096] (2) 50 mg of the chain polymer I was dissolved in 5 mL of dichloromethane (DCM) solvent, and then 331 μL of trifluoroacetic acid was added to react for 48 h to remove the tert-butyl group of monomer A in the polymer to expose the carboxyl group; the solvent was drained in a warm water bath at 40° C., and 5 mL of dichloromethane was added to redissolve, and the extraction was repeated twice; finally, 100 μL of ether was added, and the trifluoroacetic acid and the tert-butyl alcohol removed by the reaction were removed in a warm water bath at 40° C. to obtain a chain polymer II containing carboxyl groups and double bonds;

[0097] (3) Under an inert environment, 50 mg of chain polymer II is dissolved in 3 ml of a 1:1 mixed solvent I (DCM / MeOH) of dichloromethane and methanol to obtain reactant a; 2.89 mg of anhydrous ferric chloride and 1.13 mg of anhydrous ferrous chloride are dissolved in 3 ml of a 1:1 mixed solvent II (DCM / MeOH) of dichloromethane and methanol to obtain reactant b; then reactant a and reactant b are slowly added to 44 ml of a 1:1 mixed solvent III (DCM / MeOH) of dichloromethane and methanol (i.e., the cross-linking concentration is 1 mg / mL), after the dropwise addition is completed, 1.00 mL of 25% ammonia water is added and stirred for 3 hours, and the solvent is removed by vacuum distillation to obtain the magnetic nanoparticles (NPs). The structure is as follows Figure 1 .

[0098] Example 6

[0099] A method for preparing magnetic nanoparticles, wherein steps (2)-(3) are the same as those in Example 5, and step (1) is as follows:

[0100] (1) Under anhydrous and oxygen-free conditions, 100 mg of polymer monomer B prepared in Example 2 and 10 g of polymer monomer A prepared in Example 1 (feed ratio A:B=100:1) were added to a reaction bottle, dehydrated using a cold trap and transferred to a glove box, 5 ml of ultra-dry dichloromethane solvent and 1.5 mg of Grubbs third-generation catalyst were added to carry out a ring-opening metathesis polymerization reaction for 2 h. After the reaction was completed, a drop of vinyl ethyl ether was added to terminate the reaction, the reaction solution was dripped into 40 ml of n-hexane, centrifuged at -20°C, the supernatant was discarded, the precipitate was dissolved using tetrahydrofuran, and then dripped into n-hexane and centrifuged at -20°C, the supernatant was discarded; the dissolution-precipitation-centrifugation was repeated twice; and the reaction was dried in a vacuum oven at room temperature for 24 h to obtain a chain polymer I.

[0101] Example 7

[0102] A method for preparing magnetic nanoparticles, wherein steps (2)-(3) are the same as those in Example 5, and step (1) is as follows:

[0103] (1) Under anhydrous and oxygen-free conditions, 100 mg of polymer monomer B prepared in Example 2 and 1 g of polymer monomer A prepared in Example 1 (feed ratio A:B=10:1) were added to a reaction bottle, dehydrated using a cold trap and transferred to a glove box, 5 ml of ultra-dry dichloromethane solvent and 1.5 mg of Grubbs third-generation catalyst were added to carry out a ring-opening metathesis polymerization reaction for 2 h. After the reaction was completed, a drop of vinyl ethyl ether was added to terminate the reaction, the reaction solution was dripped into 40 ml of normal hexane, centrifuged at -20°C, the supernatant was discarded, the precipitate was dissolved using tetrahydrofuran, and then dripped into normal hexane and centrifuged at -20°C, the supernatant was discarded; the dissolution-precipitation-centrifugation was repeated twice; and the reaction was dried in a vacuum oven at room temperature for 24 h to obtain a chain polymer I.

[0104] Example 8

[0105] A method for preparing magnetic nanoparticles, wherein steps (2)-(3) are the same as those in Example 5, and step (1) is as follows:

[0106] (1) Under anhydrous and oxygen-free conditions, 1 g of polymer monomer B prepared in Example 2 and 100 mg of polymer monomer A prepared in Example 1 (feed ratio A:B=1:10) were added to a reaction bottle, dehydrated using a cold trap and transferred to a glove box, 5 ml of ultra-dry dichloromethane solvent and 1.5 mg of Grubbs third-generation catalyst were added to carry out a ring-opening metathesis polymerization reaction for 2 h. After the reaction was completed, a drop of vinyl ethyl ether was added to terminate the reaction, the reaction solution was dripped into 40 ml of n-hexane, centrifuged at -20°C, the supernatant was discarded, the precipitate was dissolved using tetrahydrofuran, and then dripped into n-hexane and centrifuged at -20°C, the supernatant was discarded; the dissolution-precipitation-centrifugation was repeated twice; and the reaction was dried in a vacuum oven at room temperature for 24 h to obtain a chain polymer I.

[0107] Example 9

[0108] A method for preparing magnetic nanoparticles, wherein steps (2)-(3) are the same as those in Example 5, and step (1) is as follows:

[0109] (1) Under anhydrous and oxygen-free conditions, 10 g of polymer monomer B prepared in Example 2 and 100 mg of polymer monomer A prepared in Example 1 (feed ratio A:B=1:100) were added to a reaction bottle, dehydrated using a cold trap and transferred to a glove box, 5 ml of ultra-dry dichloromethane solvent and 1.5 mg of Grubbs third-generation catalyst were added to carry out a ring-opening metathesis polymerization reaction for 2 h. After the reaction was completed, a drop of vinyl ethyl ether was added to terminate the reaction, the reaction solution was dripped into 40 ml of normal hexane, centrifuged at -20°C, the supernatant was discarded, the precipitate was dissolved using tetrahydrofuran, and then dripped into normal hexane and centrifuged at -20°C, the supernatant was discarded; the dissolution-precipitation-centrifugation was repeated twice; and the reaction mixture was dried in a vacuum oven at room temperature for 24 h to obtain a chain polymer I.

[0110] Example 10

[0111] A method for preparing magnetic nanoparticles, wherein steps (1)-(2) are the same as those in Example 5, and step (3) is as follows:

[0112] (3) Under an inert environment, 5 mg of chain polymer II was dissolved in 3 ml of a 1:1 mixed solvent I (DCM / MeOH) of dichloromethane and methanol to obtain reactant a; 0.289 mg of anhydrous ferric chloride and 0.113 mg of anhydrous ferrous chloride were dissolved in 3 ml of a 1:1 mixed solvent II (DCM / MeOH) of dichloromethane and methanol to obtain reactant b; reactant a and reactant b were then slowly added to 44 ml of a 1:1 mixed solvent III (DCM / MeOH) of dichloromethane and methanol (i.e., the cross-linking concentration was 0.1 mg / mL), and after the dropwise addition was completed, 1.00 mL of 25% ammonia water was added and stirred for reaction for 3 h, and the solvent was removed by reduced pressure distillation to obtain the magnetic nanoparticles.

[0113] Embodiment 11

[0114] A method for preparing magnetic nanoparticles, wherein steps (1)-(2) are the same as those in Example 5, and step (3) is as follows:

[0115] (3) Under an inert environment, 50 mg of the chain polymer II was dissolved in 3 ml of a 1:1 mixed solvent I (DCM / MeOH) of dichloromethane and methanol to obtain a reactant a; 2.89 mg of anhydrous ferric chloride and 1.13 mg of anhydrous ferrous chloride were dissolved in 3 ml of a 1:1 mixed solvent II (DCM / MeOH) of dichloromethane and methanol to obtain a reactant b; then the reactants a and b were slowly added to 3 ml of a 1:1 mixed solvent III (DCM / MeOH) of dichloromethane and methanol (i.e., the cross-linking concentration was 10 mg / mL), and after the dropwise addition was completed, 1.00 mL of 25% ammonia water was added and stirred for reaction for 3 h, and the solvent was removed by reduced pressure distillation to obtain the magnetic nanoparticles.

[0116] Example 12

[0117] A method for preparing magnetic nanoparticles, wherein steps (1)-(2) are the same as those in Example 5, and step (3) is as follows:

[0118] (3) Under an inert environment, 50 mg of the chain polymer II was dissolved in 3 ml of a 1:1 mixed solvent I (DCM / MeOH) of dichloromethane and methanol to obtain a reactant a; 2.89 mg of anhydrous ferric chloride and 1.13 mg of anhydrous ferrous chloride were dissolved in 3 ml of a 1:1 mixed solvent II (DCM / MeOH) of dichloromethane and methanol to obtain a reactant b; then the reactants a and b were slowly added to 19 ml of a 1:1 mixed solvent III (DCM / MeOH) of dichloromethane and methanol (i.e., the cross-linking concentration was 2 mg / mL), and after the dropwise addition was completed, 1.00 mL of 25% ammonia water was added and stirred for reaction for 3 h, and the solvent was removed by reduced pressure distillation to obtain the magnetic nanoparticles.

[0119] Embodiment 13

[0120] A method for preparing nanoparticles carrying fluorescent dye molecules Cyanine 3, the steps are as follows:

[0121] 50 mg of the nanoparticles prepared in Example 1 were dispersed in 10 mL of DCM, and Cy3-PEG1000-SH (the ratio of the thiol group to the CC double bond in the nanoparticles was 1:5) was added thereto, followed by the addition of 5 mg of AIBN, and the mixture was stirred at 50 °C for 3 h. After the reaction, the solvent was removed by vacuum distillation, and the mixture was redissolved in DCM and precipitated in ether to remove impurities to obtain a red solid product, which was the nanoparticle-loaded fluorescent dye molecule Cyanine 3.

[0122] The following experiment was performed on the nanoparticles prepared in Example 13 carrying the fluorescent dye molecule Cyanine 3:

[0123] Panc-1 and HPSC mixed cell spheres with fluorescence labels were cultured in vitro (green fluorescence is Calcein AM, marking HPSC cells; blue fluorescence is DAPI, marking Panc-1 cells), and then the nanoparticles with Cyanine 3 red fluorescence labeling obtained in Example 13 were dissolved in DMSO and added to the microporous culture dish for culturing the cell spheres at a concentration of 0.5 mg / mL. The control group was cultured under incubator conditions without adding external field strength. The experimental group used a homemade external alternating field strength and incubated the cell spheres under the same culture conditions (37°C, 5% CO2) for 48 hours, and then observed with a two-photon microscope. The results are as follows: Figure 2 ,from Figure 2 It can be seen that the red fluorescence intensity fills the entire blue-green area, indicating that the nanoparticles successfully penetrate the matrix barrier and enter the cell sphere.

[0124] The anti-tumor cell proliferation effect of the nanoparticles prepared in Example 5 was evaluated, and the specific method was as follows:

[0125] (1) Experimental Materials

[0126] Norb-allyl mPEG5000, Norb-mPEG2000-tBA mixed polymer, ferrosoferric oxide (10 nm, Jiangsu Xianfeng Nanotechnology Co., Ltd.), PEG-Ome5k (Sigma), PBS buffer (Jiangsu KeyGene Biotechnology Co., Ltd.), MTT (Jiangsu KeyGene Biotechnology Co., Ltd.), RPMI-1640 medium (Jiangsu KeyGene Biotechnology Co., Ltd.), DMEM medium (Jiangsu KeyGene Biotechnology Co., Ltd.), primary cell culture medium (Hefei Wanwu Biotechnology Co., Ltd.), trypsin-EDTA digestion solution (Jiangsu KeyGene Biotechnology Co., Ltd.), fetal bovine serum (Shanghai Darthill Biotechnology Co., Ltd.), doxorubicin (B&K Technology Co., Ltd.), DMSO (Tianjin Fuyu Fine Chemical Co., Ltd.), penicillin (Beijing Zhongsheng Aobang Biotechnology Co., Ltd.), and streptomycin (Beijing Zhongsheng Aobang Biotechnology Co., Ltd.).

[0127] (2) Experimental instruments:

[0128] Microplate reader (Molecular Devices SpectraMax), microscope (Leica), 0.22 μm filter membrane (Millex), high pressure steam sterilizer (TOMY SX-700), cell incubator (Nuaire), centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.), clean bench (Labgard), 96-well cell culture plate (Costar), 25 cm2 culture flask (Costar).

[0129] (3) Cell lines

[0130] A549 (human lung cancer cells), HT1080 (human fibrosarcoma cells), Panc-1 (human pancreatic cancer cells), MCF-7 (human breast cancer cells), HPSC (human pancreatic stellate cells, purchased from Hefei Wanwu Biotechnology), and S180 (mouse sarcoma cells).

[0131] (4) Grouping and dosage

[0132] Positive control: doxorubicin (DOX), prepared into target concentrations (5.00 μM, 2.50 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM) in PBS buffer containing 0.5% DMSO; compound A (mPEG5000, step five of Example 1), chain polymer I (Example 5), nanoparticles (NPs), and 10 nm Fe3O4 of the present invention were prepared into target concentrations (4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml) in PBS buffer containing 0.5% DMSO; blank control: PBS buffer containing 0.5% DMSO.

[0133] (5) Experimental operation

[0134] Step 1. Adherent cell culture method: Adherent cells A549, HT1080, Panc-1, MCF-7, and HPSC were cultured to the logarithmic growth phase and 1×10 4 The cells were seeded on a 96-well plate at a density of 100 / well and cultured in an incubator at 37°C and 5% CO2 until the cells adhered to the wall. According to the preset grouping and dosing concentration, 25 μL of the test compound and DOX were added to the 96-well plate. The blank group was added with 25 μL of PBS buffer containing 0.5% DMSO. After continuing to culture in the incubator for 24 hours, the supernatant was removed with a pipette, and 25 μL of MTT solution was added to each well and placed in the incubator for 4 hours. The supernatant was removed again with a pipette, and 100 μL of DMSO was added to each well. The plate was shaken for 15 minutes to fully dissolve the generated purple crystals, and the OD (absorbance) value was immediately detected with an enzyme marker at a wavelength of 490 nm.

[0135] Step 2: Suspension cell culture method: culture suspension cells S180 to the logarithmic growth phase and culture at 1×10 4 The cells were seeded on a 96-well plate at a density of 100 / well and cultured in an incubator at 37°C and 5% CO2 until the cells adhered to the wall. 25 μL of the test compound and DOX were added to the 96-well plate according to the preset grouping and dosing concentration. 25 μL of PBS buffer containing 0.5% DMSO was added to the blank group. After continuing to culture in the incubator for 48 hours, 25 μL of MTT solution was added to each well. After being placed in the incubator for 4 hours, the plates were centrifuged at 3000 rpm for 5 minutes, the supernatant was carefully removed with a pipette, 100 μL of DMSO was added to each well, and the plates were shaken for 15 minutes to fully dissolve the generated purple crystals. The OD (absorbance) value was immediately detected with an enzyme marker at a wavelength of 490 nm.

[0136] Step 3: Statistical methods:

[0137] The inhibition rate of tumor cells at different concentrations of the test samples was calculated as follows:

[0138] Growth inhibition rate = [(average OD value of blank group - average OD value of sample group) / average OD value of blank group] × 100%. The experiment was repeated 6 times, and a growth curve was plotted with inhibition rate versus test compound concentration on Origin software to calculate the IC50 (half effective inhibitory concentration) value of the test compound.

[0139] (6) Experimental results

[0140] The experimental results are shown in Table 1. The tested compounds showed different anti-tumor cell proliferation effects. Nanoparticles and 10nmFe3O4 showed good activity in vitro. Norb-allyl mPEG5000 and Norb-mPEG2000-tBA mixed polymers showed certain anti-tumor cell proliferation activity. PEG5k did not show obvious anti-tumor cell proliferation activity at the given concentration. Subsequently, nanoparticles with good anti-tumor cell proliferation activity were selected to evaluate the anti-tumor activity in mice at a dose of 4 mg / kg, and the mixed polymer was evaluated at a dose of 10 mg / kg for anti-tumor activity in mice.

[0141] Table 1 In vitro antitumor activity (DOX: μM; other groups: mg / mL)

[0142]

[0143]

[0144] There were 3 independent samples in each group in the above experiments.

[0145] The anti-tumor effect of the nanoparticles prepared in Example 5 was evaluated, and the specific steps were as follows:

[0146] (1) Experimental Materials

[0147] Compounds: doxorubicin hydrochloride (B&K Technology Co., Ltd.), physiological saline, nanoparticles and Norb-allyl mPEG5000, Norb-mPEG2000-tBA mixed polymer (synthesized according to Example 1)

[0148] Tumor source: Mouse S180 sarcoma (Animal Experiment Center, Peking University Health Science Center, self-transplanted and maintained).

[0149] Mouse species: ICR strain mice, male, 20±2g (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.).

[0150] (2) Mouse grouping and drug dosage

[0151] The dosage of the nanoparticles of the present invention is 4 mg / kg, the dosage of the Norb-allyl mPEG5000 and Norb-mPEG2000-tBA mixed polymer is 10 mg / kg, the dosage of the positive control doxorubicin hydrochloride is 2 μmol / kg, and the negative control (NS) is normal saline; the administration is continued for 10 days, for a total of 10 times.

[0152] (3) Experimental operation

[0153] Under sterile conditions, extract S180 ascites tumor fluid for inoculation, dilute the cell fluid with physiological saline at a ratio of 1:2 and mix thoroughly, dye the tumor cell suspension with freshly prepared 0.2% trypan blue, mix well and count according to the white blood cell counting method, blue-stained cells are dead cells, unstained cells are live cells, and the cell concentration and survival rate are calculated according to the following formula:

[0154] Cell concentration = number of live cells in 4 large squares / 4×10 4 × dilution factor = number of cells / mL

[0155] Cell survival rate = number of live cells / (number of live cells + number of dead cells) × 100%

[0156] The cell suspension with a survival rate greater than 90% was prepared into 2.0×10 7 / mL, inoculated subcutaneously in the armpit of mice, 0.3mL / mouse, to produce S180 tumor mice. Three days after tumor inoculation, mice in the test compound group were intravenously injected with nanoparticles NPs, Norb-allyl mPEG5000, Norb-mPEG2000-tBA mixed polymers at a given dose, and doxorubicin was intraperitoneally injected at a given dose. The administration was continued for 10 days, for a total of 10 times. On the 11th day, blood was collected from the eyeballs after ether anesthesia, and the mice were killed by cervical dislocation. The tumors were then bluntly dissected, and the organs of the mice were weighed. The weights of tumors and organs were expressed as mean ± SD g. The experimental data were statistically analyzed using t-test and variance analysis.

[0157] (4) Experimental results

[0158] The experimental results of each compound are shown in Table 2. It can be seen from the results that the nanoparticles of the present invention showed good anti-tumor activity at a dose of 4 mg / mL / day, while the polymer did not show obvious anti-tumor activity, indicating that it has good bioavailability and can be metabolized in the body as a drug carrier, and has good application prospects. Compared with NS, P < 0.01.

[0159] Table 2 Effects of nanoparticles and polymers on tumor growth

[0160]

[0161] Note: n = 6. a: Compared with NS, P < 0.01.

[0162] The nanoparticles provided by the present invention use linear polymers that can be metabolized in vivo, have good biocompatibility, and are highly safe for the human body. In the subsequent application process, positive drugs, fluorescent groups, targeting groups, etc. can be introduced into the double-bond or triple-bond side chains of the nanoparticles, so that the nanoparticles become drug-carrying molecules, break through the biological barrier in a targeted manner, and deliver the drugs to the lesion site, so as to achieve the purpose of drug concentration accumulation and treat the disease. The nanomaterials provided by the present invention have broad application prospects as drug-carrying materials in the treatment of tumors and other diseases.

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing magnetic nanoparticles, characterized in that: The following steps are involved: (1) under anhydrous and oxygen-free condition, polymer monomer A and polymer monomer B are added to a solvent, a catalyst is added to cause a ring-opening metathesis polymerization reaction for 2-3 hours, after the reaction is completed, vinyl ethyl ether is added dropwise to terminate the reaction, and the obtained reaction solution is dissolved, centrifuged, and dried to obtain a chain polymer I; (2) dissolving the chain polymer I in a dichloromethane solvent, then adding trifluoroacetic acid to react for 40-50 hours, draining the solvent, then adding dichloromethane to dissolve again, and draining twice; finally, adding a small amount of ether, and draining with a water pump in a warm water bath at 30-60° C. to obtain a chain polymer II; (3) dissolving the chain polymer II in a mixed solvent I of dichloromethane and methanol under an inert environment to obtain a reactant a; Anhydrous ferric chloride and anhydrous ferrous chloride are dissolved in a mixed solvent II of dichloromethane and methanol to obtain reactant b; then reactant a and reactant b are added to a mixed solvent III of dichloromethane and methanol, after the dropwise addition is completed, ammonia water is added and stirred to react for 3-5 hours, and the solvent is removed by reduced pressure distillation to obtain the magnetic nanoparticles.

2. The method for preparing magnetic nanoparticles according to claim 1, characterized in that: The polymer monomer A in step (1) is composed of an intermediate monomer C and a cycloolefin group; one end of the intermediate monomer C is connected to the cycloolefin group, and the other end is connected to the terminal group E; The intermediate monomer C is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl and their derivatives; the terminal group E is any one of methoxy, phenyl, vinyl, ester, amide, alkyl, benzyl, acyl halide, pyridine, ketone, naphthyl or substituted naphthyl.

3. The method for preparing magnetic nanoparticles according to claim 2, characterized in that: The molecular weight of the polymer monomer A is 500-50000.

4. The method for preparing magnetic nanoparticles according to claim 2, characterized in that: The polymer monomer A in step (1) is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol.

5. The method for preparing magnetic nanoparticles according to claim 1, characterized in that: The polymer monomer B in step (1) comprises an intermediate monomer D, a cycloolefin group and a tert-butyl ester group; one end of the intermediate monomer D is connected to the cycloolefin group, and the other end is connected to the tert-butyl ester group; The intermediate monomer D is any one of polyethylene glycol, polycarbonate lactone, polyvinyl alcohol, polycaprolactam, polylactic acid, polymethyl methacrylate, and polycaprolactone; the cycloolefin group is any one of norbornene, cyclobutenyl, cyclopentenyl, and their derivatives.

6. The method for preparing magnetic nanoparticles according to claim 5, characterized in that: The molecular weight of the polymer monomer B is 500-50000.

7. The method for preparing magnetic nanoparticles according to claim 5, characterized in that: The polymer monomer B is cis-5-norbornene-2,3-dicarboxylic anhydride-polyethylene glycol-tert-butyl ester.

8. The method for preparing magnetic nanoparticles according to claim 1, characterized in that: The mass ratio of the polymer monomer A to the polymer monomer B in step (1) is 1:100-100:1; The solvent in step (1) is dichloromethane, and the catalyst is Grubbs third generation catalyst; In the removal reaction of step (2), the ratio of chain polymer I, dichloromethane and trifluoroacetic acid is 50-55 mg: 5-6 mL: 300-350 μL; in the re-dissolving of step (2), the ratio of dichloromethane to chain polymer I is 1: 1-1: 5; the ratio of ether to chain polymer I is 100 μL: 50 mg; Step (3) The ratio of dichloromethane to methanol in the mixed solvent I is 1:1-1:5; the ratio of dichloromethane to methanol in the mixed solvent II is 1:1-1:5; the ratio of dichloromethane to methanol in the mixed solvent III is 1:1-1:5; In step (3), the ratio of the chain polymer I to the mixed solvent I is 5-50 mg: 3-5 ml; the ratio of the anhydrous ferric chloride, anhydrous ferrous chloride and the mixed solvent II is 0.1-3 mg: 0.1-1.5 mg: 3-5 ml; In step (3), the volume ratio of the mixed solvent I, the mixed solvent II, the mixed solvent III and the ammonia water is 3-5 ml: 3-5 ml: 40-50 ml: 1-2 ml; the concentration of the ammonia water is 20-30%.

9. A magnetic nanoparticle, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. The use of magnetic nanoparticles according to claim 9, characterized in that: The magnetic nanoparticles are used for preparing anti-tumor drugs.

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