阅读说明：本技术 一种无载体型超支化大分子聚合物及制备方法 (Carrier-free hyperbranched macromolecular polymer and preparation method thereof ) 是由 田威 刘程飞 庞俊 李鹏翔 李慧鑫 于 2019-10-28 设计创作，主要内容包括：本发明涉及一种无载体型超支化大分子聚合物及制备方法,以两种常见的疏水性药物甲氨蝶呤和苯丁酸氮芥为基元,对这两种疏水性药物进行结构修饰改性,使其结构中分别含有动态共价键“二硫键”和“酯键”,然后以“A<Sub>2</Sub>+B<Sub>3</Sub>”法聚合成为超支化大分子聚合物,最后自组装成为超支化大分子药物型的无载体纳米药物自递送系统,从而为无载体纳米药物递送系统的构筑提供了新的思路,并且解决了现有超支化聚合物在药物包载方面所存在的载药量低,细胞毒性大,对于肾脏等器官毒副作用大以及响应性不足等问题。(The invention relates to a carrier-free hyperbranched macromolecular polymer and a preparation method thereof, which takes two common hydrophobic drugs of methotrexate and chlorambucil as basic elements, modifies the structures of the two hydrophobic drugs to ensure that the structures respectively contain dynamic covalent bonds of disulfide bond and ester bond, and then uses A 2 +B 3 The method is polymerized into hyperbranched macromolecular polymer and finally self-assembled into the hyperbranched macromolecular drug type carrier-free nano-drug self-delivery system, thereby providing a new idea for the construction of the carrier-free nano-drug delivery system andsolves the problems of low drug loading, high cytotoxicity, high toxic and side effects on organs such as kidney and the like, insufficient responsiveness and the like of the existing hyperbranched polymer in the aspect of drug loading.)

1. A non-carrier type hyperbranched macromolecular polymer is characterized in that the structural formula is as follows:

2. a method for preparing the non-carrier hyperbranched macromolecular polymer of claim 1, which is characterized by comprising the following steps:

step 1: mixing cystamine dihydrochloride and triethylamine in a molar ratio of 1: 2.5-3.5 under an ice bath condition, and stirring for 20-40 min by using methanol as a solvent; weighing di-tert-butyl dicarbonate with a molar ratio of 1: 0.3-0.35 to cystamine dihydrochloride, dissolving the di-tert-butyl dicarbonate in methanol, dropwise adding the solution into a reaction system, and reacting for 4-8 hours after dropwise adding; adding deionized water with the volume equal to that of the reaction solvent, evaporating the solvent by using a rotary evaporator, dissolving the solvent by using dichloromethane as the solvent, adding a saturated sodium chloride solution for extraction, drying a lower organic phase by using anhydrous sodium sulfate, concentrating a filtrate by rotary evaporation, passing the filtrate through a silica gel column, taking the filtrate, spin-drying the solvent by using the rotary evaporator, and drying the filtrate in a vacuum drying oven for 36-60 hours to obtain a light yellow oily liquid which is cystamine with a single-end tert-butoxycarbonyl protected amino group;

step 2: mixing methotrexate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1: 1.8-2.2 under an ice bath condition, adding N, N-dimethylformamide as a solvent, and stirring for 25-35 min; then dissolving cystamine with a single-ended tert-butoxycarbonyl protected amino group and methotrexate in a molar weight ratio of 1: 2.3-2.7 in a solvent N, N-dimethylformamide, dropwise adding the mixture into a reaction system, and reacting for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator to obtain a brown yellow oily liquid, adding trichloromethane as a solvent for dissolving, adding a saturated sodium bicarbonate solution for extracting, drying a lower organic phase by using anhydrous sodium sulfate, then, rotatably evaporating the filtrate, adding dichloromethane as a solvent for dissolving, concentrating the liquid by rotary evaporation, passing through a silica gel column, taking the filtrate, and evaporating the solvent by using the rotary evaporator to obtain yellow solid powder, namely methotrexate protected by tert-butyloxycarbonyl at two ends;

and step 3: adding methotrexate and trifluoroacetic acid with both ends protected by tert-butyloxycarbonyl according to a molar ratio of 1: 0.4-0.6, taking dichloromethane as a solvent, and stirring for reaction for 12-16 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, and then adding 1mol/L sodium hydroxide solution with the volume ratio of the solvent to dichloromethane being 1: 2-2.5; finally, spin-drying the solvent by using a rotary evaporator, and drying the solvent in a vacuum drying oven for 46-50 hours to obtain a yellow solid, namely the methotrexate monomer with two amino-modified ends;

and 4, step 4: adding chlorambucil, trihydroxymethyl aminomethane and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline according to a molar ratio of 1: 1.1-1.2, taking ethanol as a solvent, refluxing, heating and stirring for reaction at 55-65 ℃ for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, passing through a silica gel column, taking the filtrate, and evaporating the solvent by using the rotary evaporator to obtain white solid powder, namely chlorambucil with the tail end modified by three hydroxyl groups;

and 5: under the ice bath condition, mixing chlorambucil with three hydroxyl-modified terminals and triethylamine according to the molar ratio of 1: 8-12, and stirring for 30-40 min by taking tetrahydrofuran as a solvent; dissolving succinic anhydride with the molar weight ratio of the succinic anhydride to trihydroxy phenylbutyric acid chlorambucil being 1: 3.5-4.0 in tetrahydrofuran, dropwise adding the mixture into a reaction system, and stirring and reacting for 10-14 hours after dropwise adding; after the reaction is finished, evaporating the solvent by using a rotary evaporator, adding dichloromethane as the solvent for dissolution, and extracting by using a saturated ammonium chloride solution; concentrating the liquid by rotary evaporation, passing through a silica gel column, taking filtrate, and evaporating the solvent by using a rotary evaporator to obtain white solid powder, namely three carboxybenzene butyric acid nitrogen mustard monomers at the tail end;

step 6: under ice bath condition, the molar ratio of the mixture is 1: 2.0-2.2: 22-27, stirring for 25-35 min by taking N, N-dimethylformamide as a solvent, wherein three carboxychlorambucil monomers, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine are mixed at the tail end; dissolving a methotrexate monomer with the molar weight ratio of three carboxybenzene butyric acid nitrogen mustard monomers at the tail end of 1: 2.8-3.2 and two amino-modified terminals in a solvent N, N-dimethylformamide, dropwise adding the methotrexate monomer into a reaction system, and reacting for 70-75 h; after the reaction is finished, adding deionized water with the same volume as that of the reaction liquid, concentrating the liquid through rotary evaporation, and then transferring the liquid to a dialysis bag with the molecular weight cutoff of 5000 to dialyze in N, N-dimethylformamide for 2-3 d; and then, further concentrating the liquid by rotary evaporation, and adding ice tetrahydrofuran with the volume of 25-35 times that of the concentrated reaction solution for precipitation to obtain a brown yellow solid, namely the carrier-free hyperbranched macromolecular polymer.

3. The method of claim 2, wherein: the extraction operation in the steps 1,2, 3 and 5 refers to collecting the organic phase and repeating the organic phase three times after fully mixing the organic phase with the corresponding aqueous solution in a separating funnel.

4. The method of claim 2, wherein: and 4, the reflux heating in the step 4 is to add a condensation reflux pipe on the mouth of the single-mouth bottle and introduce condensed water under the condition of constant-temperature oil bath.

5. The method of claim 2, wherein: and in the step 6, the time for dripping the methotrexate monomer with two amino-modified ends lasts for 15-20 min.

6. The method of claim 2, wherein: the glacial tetrahydrofuran in the step 6 refers to: placing tetrahydrofuran in a freezer at a temperature of between 18 ℃ below zero and 22 ℃ below zero for 30min to 1 h.

Technical Field

The invention belongs to the field of synthesis of high polymer materials and a preparation method of functional drugs, and relates to a carrier-free hyperbranched macromolecular polymer and a preparation method thereof.

Background

The carrier-free nano-drug delivery system has the advantages of high drug loading, simple preparation strategy, accurate structure, stable drug release and the like, and has become a main trend of nano-drug development. The complete removal of inert excipient not only can make the drug-loading rate reach 100%, greatly improve the treatment effect, but also can effectively avoid toxicity and immune reaction caused by the carrier.

Document 1 "Xiujuan Xi, Shiqi Hu, Zhuxian Zhou and Youqing Shen et al. Dedrimers with the protocatechuic acid building block for anti-drug delivery [ J ]. Journal of Materials Chemistry B,2016,4, 5236-.

Document 2, "Yajun Huang, Xiaokang Ding, Yu Qi, Bingran Yu and Fu-Jian xu. reduction-responsive polyfunctional polysaccharides with the use of xcellent anti-activity, biocompatibilities and gene transduction capabilities [ J ] Biomaterials, 2016,106: 134-.

Document 3 "Xiaoopin Duan, Jiansing Xiao, Qi Yin and Yang Li et al Smart pH-Sensitive and Temporal-Controlled Polymeric drugs for effective combination Therapy of Doxorubicin and Disulorfiram [ J ]. ACS Nano,2013,7(7): 5858-5869" discloses a strategy for anticancer by using a combination of two drugs, namely, adriamycin and dithiolan, but the traditional method of physical coating with a carrier leads to the disadvantages of low drug loading rate of the system, unstable drug delivery and the like.

Disclosure of Invention

Technical problem to be solved

In order to avoid the defects of the prior art, the invention provides a carrier-free hyperbranched macromolecular polymer and a preparation method thereof, and the carrier-free hyperbranched macromolecular polymer is synthesized by a two-monomer method by using a methotrexate monomer which takes hydrophobic methotrexate as a basic unit and contains a disulfide bond structure and two amino groups at two ends and a chlorambucil monomer which takes a hydrophobic drug chlorambucil as a basic unit and contains an ester bond and three carboxyl groups at the tail end.

Technical scheme

A non-carrier type hyperbranched macromolecular polymer is characterized in that the structural formula is as follows:

a preparation method of the carrier-free hyperbranched macromolecular polymer is characterized by comprising the following steps:

step 1: mixing cystamine dihydrochloride and triethylamine in a molar ratio of 1: 2.5-3.5 under an ice bath condition, and stirring for 20-40 min by using methanol as a solvent; weighing di-tert-butyl dicarbonate with a molar ratio of 1: 0.3-0.35 to cystamine dihydrochloride, dissolving the di-tert-butyl dicarbonate in methanol, dropwise adding the solution into a reaction system, and reacting for 4-8 hours after dropwise adding; adding deionized water with the volume equal to that of the reaction solvent, evaporating the solvent by using a rotary evaporator, dissolving the solvent by using dichloromethane as the solvent, adding a saturated sodium chloride solution for extraction, drying a lower-layer organic phase by using anhydrous sodium sulfate, concentrating a filtrate by rotary evaporation, passing the filtrate through a silica gel column, taking the filtrate, spin-drying the solvent by using the rotary evaporator, and drying the filtrate in a vacuum drying oven for 36-60 hours to obtain a light yellow oil liquid which is cystamine with single-ended tert-butoxycarbonyl protected amino;

step 2: mixing methotrexate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1: 1.8-2.2 under an ice bath condition, adding N, N-dimethylformamide as a solvent, and stirring for 25-35 min; then dissolving cystamine with a single-ended tert-butoxycarbonyl protected amino group and methotrexate in a molar weight ratio of 1: 2.3-2.7 in a solvent N, N-dimethylformamide, dropwise adding the mixture into a reaction system, and reacting for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator to obtain a brown yellow oily liquid, adding trichloromethane as a solvent for dissolution, adding a saturated sodium bicarbonate solution for extraction, drying a lower organic phase by using anhydrous sodium sulfate, then, rotatably evaporating the filtrate, adding dichloromethane as a solvent for dissolution, concentrating the liquid by rotary evaporation, passing through a silica gel column, taking the filtrate, and evaporating the solvent by using the rotary evaporator to obtain yellow solid powder, namely the methotrexate protected by the tert-butoxycarbonyl at two ends;

and step 3: adding methotrexate and trifluoroacetic acid with both ends protected by tert-butyloxycarbonyl according to a molar ratio of 1: 0.4-0.6, taking dichloromethane as a solvent, and stirring for reaction for 12-16 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, and then adding 1mol/L sodium hydroxide solution with the volume ratio of the solvent to dichloromethane being 1: 2-2.5; finally, spin-drying the solvent by using a rotary evaporator, and drying the solvent in a vacuum drying oven for 46-50 hours to obtain a yellow solid, namely the methotrexate monomer with two amino-modified ends;

and 4, step 4: adding chlorambucil, trihydroxymethyl aminomethane and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline according to a molar ratio of 1: 1.1-1.2, taking ethanol as a solvent, refluxing, heating and stirring for reaction at 55-65 ℃ for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, passing through a silica gel column, taking filtrate, and evaporating the solvent by using the rotary evaporator to obtain white solid powder, namely chlorambucil with the tail end modified by three hydroxyl groups;

and 5: under the ice bath condition, mixing chlorambucil with three hydroxyl-modified tail ends and triethylamine according to the molar ratio of 1: 8-12, and stirring for 30-40 min by taking tetrahydrofuran as a solvent; dissolving succinic anhydride with the molar weight ratio of the succinic anhydride to trihydroxy phenylbutyric acid chlorambucil being 1: 3.5-4.0 in tetrahydrofuran, dropwise adding the mixture into a reaction system, and stirring and reacting for 10-14 hours after dropwise adding; after the reaction is finished, evaporating the solvent by using a rotary evaporator, adding dichloromethane as the solvent for dissolution, and extracting by using a saturated ammonium chloride solution; concentrating the liquid by rotary evaporation, passing through a silica gel column, taking filtrate, and evaporating the solvent by using a rotary evaporator to obtain white solid powder, namely three carboxybenzene butyric acid nitrogen mustard monomers at the tail end;

step 6: under ice bath condition, the molar ratio of the mixture is 1: 2.0-2.2: 22-27, stirring for 25-35 min by taking N, N-dimethylformamide as a solvent, wherein three carboxychlorambucil monomers, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine are mixed at the tail end; dissolving a methotrexate monomer with two amino-modified ends and three carboxybenzene butyric acid nitrogen mustard monomers at the tail end in a molar weight ratio of 1: 2.8-3.2 in a solvent N, N-dimethylformamide, dropwise adding the methotrexate monomer into a reaction system, and reacting for 70-75 h; after the reaction is finished, adding deionized water with the same volume as that of the reaction liquid, concentrating the liquid through rotary evaporation, and then transferring the liquid to a dialysis bag with the molecular weight cutoff of 5000 to dialyze in N, N-dimethylformamide for 2-3 d; and then, further concentrating the liquid by rotary evaporation, adding ice tetrahydrofuran with the volume of 25-35 times that of the concentrated reaction solution for precipitation, and obtaining a brown yellow solid, namely the unsupported hyperbranched macromolecular polymer.

The extraction operation in the steps 1,2, 3 and 5 refers to collecting the organic phase and repeating the organic phase three times after fully mixing the organic phase with the corresponding aqueous solution in a separating funnel.

And 4, the reflux heating in the step 4 is to add a condensation reflux pipe on the mouth of the single-mouth bottle and introduce condensed water under the condition of constant-temperature oil bath.

And in the step 6, the time for dripping the methotrexate monomer with two amino-modified ends lasts for 15-20 min.

The glacial tetrahydrofuran in the step 6 refers to: placing tetrahydrofuran in a freezer at a temperature of between 18 ℃ below zero and 22 ℃ below zero for 30min to 1 h.

Advantageous effects

The invention provides a carrier-free hyperbranched macromolecular polymer and a preparation method thereof, which takes two common hydrophobic drugs of methotrexate and chlorambucil as basic elements to carry out structural modification on the two hydrophobic drugsModifying to make its structure respectively contain dynamic covalent bond "disulfide bond" and "ester bond", then using "A" to make₂+B₃The method is polymerized into hyperbranched macromolecular polymers, and finally the hyperbranched macromolecular drug type carrier-free nano-drug self-delivery system is self-assembled, so that a new thought is provided for the construction of the carrier-free nano-drug delivery system, and the problems of low drug loading, high cytotoxicity, high toxic and side effects on organs such as kidney and the like, insufficient responsiveness and the like of the existing hyperbranched polymers in the aspect of drug loading are solved.

The invention has the beneficial effects that: the hyperbranched macromolecular drug is obtained through the amidation polymerization between the methotrexate monomer with two amino-modified ends and the nitrogen mustard monomer with three carboxyl phenylbutyrate at the tail end, and the self-assembly is carried out to form the carrier-free nano-drug self-delivery system. The system solves the problems of low drug loading, high cytotoxicity and the like of the existing hyperbranched polymer self-assembly in the aspect of drug loading; meanwhile, the method provides ideas and methods for the construction of a novel carrier-free nano-drug delivery system.

Drawings

FIG. 1: nuclear magnetic resonance hydrogen spectrogram of carrier-free hyperbranched macromolecular drug

Detailed Description

The invention will now be further described with reference to the following examples and drawings:

the invention relates to a carrier-free hyperbranched macromolecular polymer, which comprises two monomers:

one of the monomer structural formulas:

a second monomer formula:

with "A₂+B₃The hyperbranched macromolecular polymer is synthesized by the method, and the structural formula is as follows:

the preparation method comprises the following steps:

step 1: under the ice bath condition, adding cystamine dihydrochloride and triethylamine into a dry single-neck flask according to the molar ratio of 1: 2.5-3.5, taking methanol as a solvent, and stirring for 20-40 min; weighing di-tert-butyl dicarbonate with a molar ratio of 1: 0.3-0.35 to cystamine dihydrochloride, dissolving the di-tert-butyl dicarbonate in methanol, dropwise adding the solution into a reaction system, and reacting for 4-8 hours after dropwise adding; adding deionized water with the volume equal to that of the reaction solvent, evaporating the solvent by using a rotary evaporator, dissolving the solvent by using dichloromethane as the solvent, adding saturated sodium chloride solution for extraction, drying a lower organic phase by using anhydrous sodium sulfate, concentrating filtrate by rotary evaporation, passing the filtrate through a silica gel column, taking the filtrate, spin-drying the solvent by using the rotary evaporator, and drying the solvent in a vacuum drying oven for 36-60 hours to obtain a light yellow oily liquid which is cystamine with a single-ended tert-butoxycarbonyl protected amino group;

step 2: under ice-bath conditions, adding a mixture of a dry single-neck flask and a water-soluble organic solvent in a molar ratio of 1: 1.8-2.2: 1.8-2.2 adding methotrexate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole, adding N, N-dimethylformamide as a solvent, and stirring for 25-35 min; then the molar weight ratio of the methotrexate to the methotrexate is 1: 2.3-2.7, dissolving cystamine with amino protected by single-ended tert-butyloxycarbonyl in a solvent N, N-dimethylformamide, dropwise adding the mixture into a reaction system, and reacting for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator to obtain brown yellow oily liquid, adding chloroform as a solvent for dissolving, adding a saturated sodium bicarbonate solution for extraction, drying a lower organic phase by using anhydrous sodium sulfate, then, rotatably drying filtrate by rotary evaporation, adding dichloromethane as a solvent for dissolving, concentrating the liquid by rotary evaporation, passing through a silica gel column, taking the filtrate, and evaporating the solvent by using the rotary evaporator to obtain yellow solid powder, namely the methotrexate protected by the tert-butyloxycarbonyl group at two ends;

and step 3: into a dry single-neck flask, the molar ratio of 1: 0.4-0.6, adding methotrexate and trifluoroacetic acid with two ends protected by tert-butyloxycarbonyl, taking dichloromethane as a solvent, and stirring for reaction for 12-16 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, and then adding 1mol/L sodium hydroxide solution with the volume ratio of the solvent to dichloromethane being 1: 2-2.5; finally, spin-drying the solvent by using a rotary evaporator, and drying the solvent in a vacuum drying oven for 46-50 hours to obtain a yellow solid, namely the methotrexate monomer with two amino-modified ends;

and 4, step 4: into a dry single-neck flask, the molar ratio of 1: 1.1-1.2: 1.1-1.2 adding chlorambucil, tris (hydroxymethyl) aminomethane and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, taking ethanol as a solvent, refluxing, heating and stirring at 55-65 ℃, and reacting for 10-14 h; after the reaction is finished, evaporating the solvent by using a rotary evaporator, passing through a silica gel column, taking the filtrate, evaporating the solvent by using the rotary evaporator to obtain white solid powder, namely chlorambucil with the tail end modified by three hydroxyl groups;

and 5: under ice-bath conditions, adding a mixture of a dry single-neck flask and a water-soluble organic solvent in a molar ratio of 1: 8-12, adding chlorambucil and triethylamine of which the tail ends are modified by three hydroxyl groups, and stirring for 30-40 min by taking tetrahydrofuran as a solvent; and then mixing the mixture with CB-TRIS in a molar ratio of 1: 3.5-4.0 parts of succinic anhydride is dissolved in tetrahydrofuran and is added into the reaction system drop by drop, and stirring and reacting are carried out for 10-14 hours after the dropwise addition is finished. After the reaction is finished, evaporating the solvent by using a rotary evaporator, adding dichloromethane as the solvent for dissolution, and extracting by using a saturated ammonium chloride solution; concentrating the liquid by rotary evaporation, passing through a silica gel column, taking filtrate, and evaporating the solvent by using a rotary evaporator to obtain white solid powder, namely three carboxybenzene butyric acid nitrogen mustard monomers at the tail end;

step 6: under ice-bath conditions, adding a mixture of a dry single-neck flask and a water-soluble organic solvent in a molar ratio of 1: 2.0-2.2: 2.0-2.2: 22-27, adding three carboxyl phenylbutyric acid chlorambucil monomers at the tail end, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine, taking N, N-dimethylformamide as a solvent, and stirring for 25-35 min; then the molar weight ratio of the monomer to the monomer which is three carboxyl phenylbutyric acid nitrogen mustard at the end is 1: 2.8-3.2, dissolving the methotrexate monomer with two amino-modified amino groups at two ends in a solvent N, N-dimethylformamide, dropwise adding the mixture into a reaction system, and reacting for 70-75 h; and after the reaction is finished, adding deionized water with the same volume as that of the reaction liquid, concentrating the liquid by rotary evaporation, and then transferring the liquid to a dialysis bag with the molecular weight cutoff of 5000 for dialysis in N, N-dimethylformamide for 2-3 d. And then, further concentrating the liquid by rotary evaporation, and adding ice tetrahydrofuran with the volume of 25-35 times that of the concentrated reaction solution for precipitation to obtain a brown yellow solid, namely the carrier-free hyperbranched macromolecular drug.