CN110483740B

CN110483740B - Polymer, pH-sensitive nano-vesicle, preparation method and application

Info

Publication number: CN110483740B
Application number: CN201910741019.4A
Authority: CN
Inventors: 侯昭升; 滕金伟; 徐钧; 王雪洁
Original assignee: Shandong Normal University
Current assignee: Wuxi Xiangyuan Information Technology Co ltd
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2021-08-27
Anticipated expiration: 2039-08-12
Also published as: CN110483740A

Abstract

The invention provides a polymer, a pH sensitive nano vesicle, a preparation method and an application thereof, wherein the polymer has the following structural formula:

wherein m is₁+n₁＝9～19，m₂+n₂＝9～19，p＝5～10，R₁Is composed of

R₂Is selected from

Vesicles formed from the polymerThe particle size is 100-160 nm, the large-amplitude expansion is generated on a weak acid medium with the pH value of 4.5-6.8, the particle size is increased to 1.4-1.8 times, the multiple increase of the particle size in neutral and alkaline media is less than 1.1 times, the mutation interval is less than 0.25 pH values, and the adjustment can be realized by adjusting the number of pyridine groups in a side chain. The vesicle provided by the disclosure can realize directional administration on diseased cells with weak acidity.

Description

Polymer, pH-sensitive nano-vesicle, preparation method and application

Technical Field

The invention belongs to the technical field of polymer chemistry and biomedical engineering, and relates to a polymer, a pH sensitive nano vesicle, a preparation method and an application thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

The polymersome is a special self-assembly body with a water-containing cavity, which is formed by self-assembling amphiphilic polymers in a certain mode, and has high stability, adjustable membrane property and the capability of simultaneously encapsulating hydrophilic and hydrophobic components. Due to these properties, polymersomes have a wide range of applications in drug delivery, gene therapy and tissue engineering. With the development of nano delivery systems, stimuli-responsive nano-carriers are becoming increasingly popular for cancer therapy due to their ability to release the loaded drug at specific targets and at specific times. The nano-carrier takes a pathological change part as a target spot, responds to local stimulation to release the medicine, greatly increases the concentration of the local treatment medicine, and improves the treatment effect.

It is well known that the special microenvironment of tumor tissue is very different from the pH of normal tissue, wherein the extracellular pH is 7.4, the intracellular pH is 7.2, and the pH is 7.4 during the blood circulation process; the pH value of the inside and outside cells of the tumor tissue is 6.5-7.2, the pH value of an early endosome is 6.0-6.5, the pH value of a late endosome is 5.0-6.0, and the pH value of a lysosome is 4.5-5.0, so that the pH value can be simultaneously used as a trigger factor for designing the fixed-point release of the drug, and the release of the anti-tumor drug in the tumor cells is realized.

Patent CN105997879B discloses a pH and temperature dual-sensitive nano-vesicle, a preparation method and an application thereof, wherein the preparation method comprises the steps of sequentially and gradually synthesizing compounds such as beta-benzyl aspartate, benzyloxycarbonyl aspartic anhydride and the like, finally obtaining polyethylene glycol-poly (aspartic acid-diethylethylenediamine-co-histamine-co-diisopropylethylenediamine), and forming the pH and temperature dual-sensitive nano-vesicle under the action of ultrasound and dialysis. According to the research of the inventor, the nano vesicle has no mutability, and the mutation range is uncontrollable, so that the drug loading has no pertinence.

Patent CN108078924A discloses a preparation method of polyethylene glycol modified nano micelle or vesicle with high drug loading capacity and responsiveness. The method comprises the step of reacting polyethylene glycol with cis-aconitic acid modified doxorubicin to generate an amphiphilic block copolymer, so that a responsive high-drug-loading-rate nano micelle or vesicle with an anticancer drug doxorubicin as a hydrophobic chain segment and polyethylene glycol as a hydrophilic chain segment is formed. The nano micelle or vesicle takes an anticancer drug as a hydrophobic chain segment, so that the nano micelle or vesicle has higher drug loading capacity, and meanwhile, the cis-aconitic acid is sensitive to the acid, so that the prepared nano micelle or vesicle has responsiveness, is suitable for the field of tumor treatment, can improve the chemotherapy efficiency, and reduces the toxic and side effects of the anticancer drug on normal tissues. However, the research of the inventor of the present disclosure finds that the micelle or vesicle prepared by the method has sensitivity under alkaline conditions, has low sensitivity to weak acid, and cannot realize precise release of the drug to the diseased cells.

Patent CN107099007B discloses an amphiphilic block polymer, a light and pH dual-response polymer composite vesicle, and a preparation method and application thereof. The poly (dimethylaminoethyl methacrylate) modified by the azobenzene group is taken as a hydrophilic chain segment, and the polycaprolactone block is taken as a hydrophobic chain segment, and the vesicle is formed through self-assembly. However, the inventor of the present disclosure found that the hydrophobic segment is formed by polymerization of carbon-carbon double bonds, and is difficult to degrade in vivo after releasing the drug.

In general, the inventors of the present disclosure found that the existing pH-sensitive nanovesicles all have the disadvantages of wide mutation range, low sensitivity to medium pH, and difficulty in controlling the mutation range. Particularly, as the medicine carrying vesicle, the pH value of a pathological change region is generally different from that of normal body fluid (or blood), but the difference is small, so that the medicine carrying vesicle which has high pH sensitivity, small mutation region and can accurately control the mutation point has great application value in the aspect of realizing directional administration.

Disclosure of Invention

In order to solve the defects of the prior art, the present disclosure provides a polymer, a pH-sensitive nanovesicle, a preparation method and an application thereof, wherein a side chain of the pH-sensitive nanovesicle formed by the polymer contains a plurality of pH-sensitive groups, and the pH-sensitive nanovesicle can greatly swell (increase the particle size to 1.4-1.8 times) in a weak acid (pH 4.5-6.8) medium, and slightly swell or not swell (increase the particle size to less than 1.1 times) in a neutral or alkaline medium, thereby exhibiting high pH sensitivity. The mutation range is within 0.25 pH value, and the mutation range can be controlled by adjusting the number of pyridine groups in the side chain. Meanwhile, the vesicle can wrap the drug, the drug release rate is high in a weak acidic medium, the drug release amount is higher than 85% in 30-150 hours, the drug release is low in a neutral or alkaline medium, and the drug release amount is less than 15% in 150 hours, so that the drug-loaded vesicle realizes directional drug delivery to diseased cells with weak acidity. Meanwhile, phosphorylcholine groups are arranged on the periphery of the vesicle, and the phospholipid structure similar to the cell wall has high biocompatibility and is easy to approach or penetrate through the cell wall.

In order to achieve the purpose, the technical scheme of the disclosure is as follows:

in a first aspect, the present disclosure provides a polymer having the formula:

wherein m is₁+n₁＝9～19，m₂+n₂＝9～19，p＝5～10，

R₁Is composed of

R₂Is selected from

On the other hand, the disclosure provides a preparation method of the polymer, wherein a single-end dihydroxy pyridine compound PyDH and diisocyanate are subjected to polymerization reaction to prepare polyurethane with isocyanate groups at two ends, the isocyanate groups at two ends of one polyurethane molecule are respectively subjected to addition reaction with amino groups of two chitosan oligosaccharides to obtain a polymer precursor, and a primary amine group in the polymer precursor and an aldehyde group of terminal glyoxal phosphorylcholine are subjected to an aldehyde-amine condensation reaction to obtain the polymer;

the structural formula of the single-end dihydroxypyridine compound PyDH is shown as

The structural formula of the end glyoxal phosphorylcholine is

In a third aspect, the present disclosure provides a pH-sensitive nanovesicle assembled from the above-described polymers.

In a fourth aspect, the present disclosure provides a method for preparing a pH-sensitive nanovesicle, dissolving the above polymer in an organic solvent to obtain a polymer solution, mixing the polymer with water to prepare an emulsion, adding the emulsion into water to prepare a water-oil-water double emulsion, removing the organic solvent, and freeze-drying to obtain the pH-sensitive nanovesicle.

In a fifth aspect, the pH-sensitive nanovesicle is used as a drug carrier.

In a sixth aspect, a targeted drug comprises an active drug and a targeted carrier, wherein the targeted carrier is the pH-sensitive nanovesicle.

In a seventh aspect, a method for preparing a targeted drug comprises dissolving the polymer in an organic solvent to obtain a polymer solution, mixing the polymer with water containing an active drug to prepare an emulsion, adding the emulsion to the water to prepare a water-oil-water double emulsion, removing the organic solvent, and freeze-drying to obtain the targeted drug.

The vesicle disclosed by the invention takes Phosphorylcholine (PC) modified chitosan oligosaccharide as a hydrophilic chain segment, simultaneously contains positive and negative charges, has strong binding capacity with water, is not easy to adsorb and precipitate biological components such as protein on the surface of the material, and shows good biocompatibility. The molecular main chain of the vesicle is a orderly-arranged polyurethane chain segment, and is influenced by the action of hydrogen bonds, so that a three-dimensional network is more stable. The side chain contains a plurality of pyridine groups, the degree of freedom is high, the side chain is sensitive to a medium, the pyridine groups are protonated and converted into quaternary ammonium salts in an acidic medium, the action of hydrogen bonds is weakened, the repulsive force between molecular chains is enhanced, macroscopically, namely, vesicles expand, the degradation rate is accelerated, and the medicine is easy to release. Meanwhile, the orderly-arranged polyurethane can enhance the hydrophobicity of chain segments, the particle size of the formed vesicles is increased, and the drug loading is increased.

The beneficial effect of this disclosure does:

1. the side chain of the nano vesicle provided by the disclosure contains a pyridine group, and the side chain is positioned on the side chain, so that the side chain has higher degree of freedom and high utilization rate, and the vesicle has high pH sensitivity, particularly has high response speed to an acidic medium, can bring obvious change of swelling degree due to small change of external pH, and has the characteristic of high response speed.

2. The periphery of the nano vesicle provided by the disclosure contains a large number of phosphorylcholine groups, so that biological components such as protein, liposome and the like are not easy to adsorb and precipitate, no damage is caused to a human body, and the nano vesicle has good biocompatibility.

3. The main chain of the nano vesicle provided by the disclosure contains a longer polyurethane chain segment, and the hard segment contains a plurality of carbamate groups, so that a compact hydrogen bond can be formed, and the stability of a three-dimensional network is improved. On the other hand, the degradation product is an alkaline substance, and can neutralize and degrade the generated acidic substance, thereby avoiding the generation of acidic inflammation and causing no harm to organisms when being used as a drug carrier.

4. The preparation method disclosed by the invention is simple, has rich principle sources and strong practicability, and is easy to popularize

5. The nano vesicle provided by the disclosure can be used as a drug carrier to realize directional drug delivery to weakly acidic diseased cells.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 shows the NMR spectra of PyDH prepared in the examples of the present disclosure (¹H NMR) pattern;

FIG. 2 shows the NMR spectrum of Polymer A1 in example 1 of the present disclosure (¹H NMR) pattern;

FIG. 3 shows the NMR spectrum of Polymer B1 in example 1 of the present disclosure (¹H NMR) pattern;

FIG. 4 is a plot of particle size curves at different pH for G1 prepared in example 1, G4 prepared in example 4, and G5 prepared in example 5 of the present disclosure

FIG. 5 is a graph of the amount of drug released by G1-Y prepared in example 1 of the present disclosure at various pHs.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the defects that the existing vesicles are low in pH sensitivity, large in mutation area and difficult to achieve directional administration, the disclosure provides a polymer, a pH-sensitive nano vesicle, a preparation method and an application.

In one exemplary embodiment of the present disclosure, a polymer is provided having the following structural formula:

wherein m is₁+n₁＝9～19，m₂+n₂＝9～19，p＝5～10，

R₁Is composed of

R₂Is selected from

Another embodiment of the present disclosure provides a preparation method of the above polymer, the single-end dihydroxy pyridine compound PyDH and diisocyanate are subjected to a polymerization reaction to prepare polyurethane with isocyanate groups at two ends, the isocyanate groups at two ends of one polyurethane molecule are respectively subjected to an addition reaction with amino groups of two chitosan oligosaccharides to obtain a polymer precursor, and a primary amine group in the polymer precursor and an aldehyde group of the terminal glyoxal phosphorylcholine are subjected to an aldehyde-amine condensation reaction to obtain the polymer;

The structural formula of the end glyoxal phosphorylcholine is

In one or more examples of this embodiment, the diisocyanate is an aliphatic diisocyanate having ordered segments, such as hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate, tetramethylene diisocyanate-1, 4-butanediol-tetramethylene diisocyanate, L-lysine diisocyanate-1, 4-butanediol-L-lysine diisocyanate, and the like.

In one or more embodiments of the present disclosure, the molar ratio of the single-ended dihydroxypyridine compound PyDH to the diisocyanate is 1:1.1 to 1: 1.2.

In one or more embodiments of this embodiment, the solvent in the reaction system for preparing the polyurethane is N, N-Dimethylformamide (DMF).

In one or more embodiments of the embodiment, in the polyurethane preparation reaction system, the total concentration of the single-end dihydroxy pyridine compound PyDH and the diisocyanate in the solution is 0.5-1.5 g/mL.

In one or more embodiments of the present disclosure, the polyurethane is prepared at a reaction temperature of 70 to 85 ℃.

In one or more embodiments of this embodiment, the catalyst in the reaction system for preparing the polyurethane is a tin-based catalyst, such as dibutyltin dilaurate, stannous octoate, and the like.

In one or more embodiments of the embodiment, in the polyurethane preparation reaction system, the amount of the catalyst added is 0.5 to 1% of the total mass of the single-ended dihydroxypyridine compound PyDH and the diisocyanate.

In one or more examples of this embodiment, the end point of the reaction of the single-ended dihydroxypyridine compound PyDH with the diisocyanate is determined by the di-n-butylamine method. The time for the reaction to reach the end point is about 3-4 h.

In one or more embodiments of this embodiment, the chitosan oligosaccharide has a number average molecular weight of 1600 to 4000 and a degree of deacetylation of greater than 90%, more preferably greater than 92%.

In one or more embodiments of this embodiment, a chitosan oligosaccharide solution is added to the polymerized material to perform the addition reaction.

In the series of embodiments, the concentration of the chitosan oligosaccharide solution is 1.0-1.5 g/mL.

In one or more embodiments of the present disclosure, the amount of the chitosan oligosaccharide is 4 to 6 times the molar difference between the diisocyanate and the single-terminal dihydroxypyridine compound PyDH. When the chitosan oligosaccharide is in large excess, only one amino group on each chitosan oligosaccharide molecule reacts with-NCO, so that cross-linking is avoided, and vesicles are more easily formed.

In one or more embodiments of this embodiment, the end point of the addition reaction is infrared detection of the-NCO absorption peak (. about.2270 cm)^-1) And (4) disappearing. Namely the time of the addition reaction is 2 to 3.5 hours.

In one or more embodiments of this embodiment, the method of purifying the polymer precursor comprises: diluting the material after the addition reaction, settling by using water, and drying the solid after the precipitation.

In one or more embodiments of this embodiment, the molar amount of the terminal glyoxyl-phosphorylcholine is equal to the molar amount of the primary amine group in the polymer precursor.

In one or more embodiments of the present disclosure, the reaction temperature of the aldehyde-amine condensation reaction is 20 to 30 ℃ and the reaction time is 2 to 3 hours.

In one or more embodiments of the present invention, the material after the aldol condensation reaction is often precipitated with ethanol, and the precipitated solid is washed and dried.

In a third embodiment of the present disclosure, a pH-sensitive nanovesicle is provided, which is assembled from the above-mentioned polymers.

In one or more embodiments of this embodiment, the particle size is 100 to 160 nm.

In one or more embodiments of this embodiment, the particle size of the pH-sensitive nanovesicles is increased to 1.4-1.8 fold in acidic media and to less than 1.1 fold or no increase in neutral or basic media.

In a fourth embodiment of the present disclosure, a method for preparing pH-sensitive nanovesicles is provided, in which the above polymer is dissolved in an organic solvent to obtain a polymer solution, the polymer is mixed with water to prepare an emulsion, the emulsion is added into water to prepare a water-oil-water double emulsion, the organic solvent is removed, and the pH-sensitive nanovesicles are obtained after freeze-drying.

In one or more embodiments of this embodiment, the organic solvent is chloroform.

In one or more embodiments of this embodiment, the polymer solution has a concentration of 0.005 to 0.015 g/mL.

In one or more embodiments of this embodiment, the volume ratio of the polymer solution to water is 4:0.5 to 1.5.

In one or more embodiments of this embodiment, the polymer is mixed with water and sonicated to prepare an emulsion.

In one or more embodiments of the embodiment, the volume ratio of the emulsion to water in the process of preparing the water-oil-water double emulsion is 1: 0.5-1.

In one or more embodiments of this embodiment, the emulsion is added to water and sonicated to prepare a water-oil-water double emulsion.

Diisocyanates having ordered segments (hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate, tetramethylene diisocyanate-1, 4-butanediol-tetramethylene diisocyanate, L-lysine diisocyanate-1, 4-butanediol-L-lysine diisocyanate) used in the present disclosure were prepared according to the method disclosed in patent CN 108976387A.

The preparation method of the single-end dihydroxypyridine compound PyDH disclosed by the invention comprises the following steps: under dry argon, dissolving 4-vinylpyridine and 3-mercapto-1, 2-propylene glycol in DMF, stirring at room temperature for 24h under the action of a catalyst, settling by 12 times of glacial ethyl ether (about 5 ℃), performing suction filtration, and performing vacuum drying at room temperature to constant weight to obtain the single-end dihydroxypyridine compound A with the yield of 92-98%. Preferably, the feeding molar ratio of the 4-vinylpyridine to the 3-mercapto-1, 2-propanediol is 1: 1; the total concentration of the 4-vinylpyridine and the 3-mercapto-1, 2-propanediol in the dimethyl sulfoxide is 0.3-0.6 g/mL; the catalyst is N, N-diisopropylethylamine, and the dosage of the catalyst is 2.5 percent of the mole number of the 4-vinylpyridine. The reaction formula is as follows:

the glyoxal phosphorylcholine is prepared according to a method disclosed in patent CN102070780A, and the glyoxal acetylcholine has a structural formula as follows:

in a fifth embodiment of the present disclosure, there is provided an application of the pH-sensitive nanovesicle as a drug carrier.

In a sixth embodiment of the present disclosure, a targeted drug is provided, which includes an active drug and a targeted carrier, where the targeted carrier is the pH-sensitive nanovesicle described above.

In one or more embodiments of this embodiment, the active agent is an anti-inflammatory agent or an anti-neoplastic agent.

In one or more embodiments of this embodiment, the targeted drug has a particle size of 120 to 190 nm.

In a seventh embodiment of the present disclosure, a method for preparing a targeted drug is provided, in which the above polymer is dissolved in an organic solvent to obtain a polymer solution, the polymer is mixed with water containing an active drug to prepare an emulsion, the emulsion is added into the water to prepare a water-oil-water double emulsion, the organic solvent is removed, and the targeted drug is obtained after freeze-drying.

In one or more embodiments of this embodiment, the volume ratio of the polymer solution to the water containing the active agent is 4:0.5 to 1.5.

In one or more examples of this embodiment, the organic solvent is removed, dialyzed, and then lyophilized. The free active drug is removed.

In this series of examples, dialysis was performed for 2 days using a 10kDa dialysis bag, with deionized water changed every 12 h.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Preparation of PyDH:

dissolving 21.03g of 4-vinylpyridine and 21.63g of 3-mercapto-1, 2-propanediol in 100mL of DMF under dry argon, adding 0.65g N of N-diisopropylethylamine, stirring at room temperature for 24h, settling with 12 times of glacial ethyl ether (about 5 ℃), filtering, drying at room temperature in vacuum to constant weight to obtain a single-end dihydroxypyridine compound PyDH (41.35g, yield 96.9%),¹h NMR is shown in FIG. 1.

Example 1:

(1) preparation of Polymer A1

10.4g of the compound PyDH, 25.59g of hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.2g of dibutyltin dilaurate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3.5 hours at a constant temperature. Then cooled to 22 ℃ and a DMF solution of chitosan oligosaccharide (number average molecular weight 1610, degree of deacetylation 90%) (80.5g chitosan oligosaccharide +100mLDMF) was added and the reaction was continued for 2.5h while maintaining the temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then using deionized water with eight times of volume to settle, repeating twice, filtering, drying in vacuum at 50 ℃ to constant weight to obtain a polymer A1,¹h NMR is shown in FIG. 2.

(2) Preparation of Polymer B1

6.82g of the polymer A1 was dissolved in 50mL of anhydrous chloroform, and 3.6g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 20 ℃ for 2 hours. After the reaction, six times of the volume of the ethylAlcohol sedimentation, suction filtration, ethanol washing for three times, and vacuum drying at normal temperature to constant weight. The polymer B1 was obtained as a result,¹h NMR is shown in FIG. 3.

(3) Preparation of nanovesicles G1

80mL of polymer B1 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 50mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G1.

(4) Preparation of drug-loaded nano-vesicle G1-Y

Mixing 80mL of polymer B1 chloroform solution (0.01g/mL) with 20mL of deionized water (10mg/mL) dissolved with 10-hydroxycamptothecin, performing ultrasonic dispersion to obtain uniform emulsion, slowly adding the emulsion into 50mL of deionized water, and performing ultrasonic dispersion simultaneously; and (3) removing chloroform through reduced pressure distillation, dialyzing the obtained product in deionized water by using a dialysis bag of 10KDa for two days, changing the deionized water every 12 hours, and freeze-drying to obtain the drug-loaded nano vesicle. Denoted as G1-Y, the drug loading encapsulation efficiency was 85%.

Example 2:

(1) preparation of Polymer A2

10.4g of the compound PyDH, 22.5g of tetramethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.18g of dibutyltin dilaurate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was reacted at 75 ℃ for 3 hours in an oil bath. Then cooled to 25 ℃, a DMF solution (80.5g chitosan oligosaccharide +100ml DMF) of chitosan oligosaccharide (number average molecular weight 1610, degree of deacetylation 90%) was added and the reaction was continued for 2.5h maintaining temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A2.

(2) Preparation of Polymer B2

6.51g of the polymer A2 was dissolved in 50mL of anhydrous chloroform, and 3.6g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 20 ℃ for 2 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B2 was obtained.

(3) Preparation of nanovesicles G2

80mL of polymer B2 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 80mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G2.

Example 3:

(1) preparation of Polymer A3

10.4g of the compound PyDH, 32.56g L-lysine diisocyanate-1, 4-butanediol-L-lysine diisocyanate and 0.24g of stannous octoate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3 hours at a constant temperature. Then cooled to 23 ℃ and a DMF solution of chitosan oligosaccharide (number average molecular weight 1610, degree of deacetylation 90%) (96.6g chitosan oligosaccharide +100mLDMF) was added and the reaction was continued for 3h while maintaining the temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A3.

(2) Preparation of Polymer B3

7.52g of the polymer A3 was dissolved in 50mL of anhydrous chloroform, and 3.6g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 25 ℃ for 2 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B3 was obtained.

(3) Preparation of nanovesicles G3

80mL of polymer B3 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 50mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G3.

Example 4:

(1) preparation of Polymer A4

14.56g of the compound PyDH, 34.12g of hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.28g of stannous octoate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3.5 hours at a constant temperature. Then cooled to 22 ℃ and a DMF solution of chitosan oligosaccharide (number average molecular weight 1610, degree of deacetylation 90%) (64.4g chitosan oligosaccharide +100mLDMF) was added and the reaction was continued for 2.5h while maintaining the temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A4.

(2) Preparation of Polymer B4

8.09g of the polymer A4 was dissolved in 50mL of anhydrous chloroform, and 3.6g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 25 ℃ for 2.5 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B4 was obtained.

(3) Preparation of nanovesicles G4

80mL of polymer B4 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 50mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G4.

Example 5:

(1) preparation of Polymer A5

20.8g of the compound PyDH, 46.92g of hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.2g of dibutyltin dilaurate were dissolved in 100mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3.5 hours at a constant temperature. Then cooled to 25 ℃, a DMF solution (80.5g chitosan oligosaccharide +100ml DMF) of chitosan oligosaccharide (number average molecular weight 1610, degree of deacetylation 90%) was added and the reaction was continued for 2.5h maintaining temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A5.

(2) Preparation of Polymer B5

9.99g of the polymer A5 was dissolved in 50mL of anhydrous chloroform, and 3.6g of glyoxyl phosphorylcholine was added thereto to carry out a reaction at a constant temperature of 20 ℃ for 2 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B5 was obtained.

(3) Preparation of nanovesicles G5

80mL of polymer B5 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 60mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G5.

Example 6:

(1) preparation of Polymer A6

10.4g of the compound PyDH, 25.59g of hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.2g of stannous octoate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3.5 hours at a constant temperature. Then cooled to 23 ℃ and a DMF solution of chitosan oligosaccharide (number average molecular weight 2420, degree of deacetylation 93%) (121g chitosan oligosaccharide +100mLDMF) was added and the reaction was continued for 2.5h while maintaining the temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A6.

(2) Preparation of Polymer B6

8.44g of the polymer A6 was dissolved in 50mL of anhydrous chloroform, and 5.6g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 22 ℃ for 2 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B6 was obtained.

(3) Preparation of nanovesicles G6

80mL of polymer B6 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 50mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G6.

Example 7:

(1) preparation of Polymer A7

10.4g of the compound PyDH, 25.59g of hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate and 0.23g of dibutyltin dilaurate were dissolved in 50mL of N, N' -Dimethylformamide (DMF), and the mixture was heated in an oil bath to 80 ℃ and reacted for 3.5 hours at a constant temperature. Then cooled to 25 ℃, a DMF solution (128.8g chitosan oligosaccharide +100ml DMF) of chitosan oligosaccharide (number average molecular weight 3220, degree of deacetylation 95%) was added and the reaction was continued for 2.5h while maintaining the temperature. After the reaction is finished, adding a DMF solution into a reaction bottle to dilute to 0.25g/mL, then settling with eight times of volume of deionized water, repeating twice, filtering, and drying in vacuum at 50 ℃ to constant weight to obtain the polymer A7.

(2) Preparation of Polymer B7

10.04g of the polymer A7 was dissolved in 50mL of anhydrous chloroform, and 8.11g of glyoxyl phosphorylcholine was added thereto to conduct a reaction at a constant temperature of 25 ℃ for 3 hours. After the reaction, the mixture is settled by ethanol with six times of volume, filtered, washed by ethanol for three times, and dried in vacuum at normal temperature to constant weight. Polymer B7 was obtained.

(3) Preparation of nanovesicles G7

80mL of polymer B7 chloroform solution (0.01g/mL) is mixed with 20mL of deionized water, and the mixture is dispersed into uniform emulsion by ultrasound; the emulsion was slowly added to 50mL of deionized water while sonicating, chloroform was removed by distillation under reduced pressure, and lyophilized to give vesicles designated G7.

Analysis and description: the following analytical methods were used for all examples unless otherwise indicated.

Measuring the vesicle particle size: and respectively soaking a certain amount of Nano vesicles in a phosphoric acid buffer solution with the pH value of 4.5-6.5 for 3 hours, and then measuring the particle size of the Nano vesicles by a Nano Zeta Sizer potential particle size analyzer from a certain amount of buffer solution.

The slow release performance of the medicine is as follows: and (3) soaking the nano drug-loaded vesicle in a phosphoric acid buffer solution, measuring the ultraviolet absorption of the nano drug-loaded vesicle by taking the buffer solution at intervals, and calculating the amount of released ceftibuten through an absorbance-concentration absorption curve.

The particle size of the nanovesicles G1, G4, and G5 at different pH was measured to compare the effect of different dosing ratios on the change of the particle size of the nanovesicles, wherein G1, G4, and G5 represent the nanovesicles prepared at different dosing ratios, and the measurement results are shown in fig. 4. The result shows that the nanovesicle prepared by the method has high pH sensitivity, and the mutation range can be controlled within 0.25 pH value. As the number of pyridine groups on the side chain increases, the more sensitive it is to acidic media, the closer the mutation point is to neutrality.

3 parts of nano drug-loaded vesicles G1-Y with equal mass are respectively soaked in solutions with pH 5.1, pH 5.3 and pH 5.5, the ultraviolet absorption of the medium is measured at intervals, and the content of released 10-hydroxycamptothecin is calculated through a standard ultraviolet absorbance-concentration absorption curve. The measurement results are shown in FIG. 5. The result shows that the nano drug-loaded vesicle has pH sensitivity, when the pH value is larger than the mutation range, the drug slow-release rate is relatively gentle, the release time is long, the release amount is extremely low, and the release amount is still less than 15% after 150 hours. When the pH value is smaller than the mutation range, the release rate is fastest, and the release amount can reach more than 90% within 40 h. This shows that the drug-loaded vesicle can realize directional release to weak acidic pathological cells.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A polymer characterized by the structural formula:

wherein m is₁+n₁＝9～19，m₂+n₂＝9～19，p＝5～10，

R₁Is composed of

R₂Is selected from

2. A method for preparing the polymer according to claim 1, wherein the single-end dihydroxy pyridine compound PyDH and diisocyanate are polymerized to prepare polyurethane with two ends containing isocyanate groups, the isocyanate groups at two ends of one polyurethane molecule are respectively added with amino groups of two chitosan oligosaccharides to obtain a polymer precursor, and the primary amine group in the polymer precursor and the aldehyde group of the terminal glyoxal phosphorylcholine are subjected to an aldehyde-amine condensation reaction to obtain the polymer;

The structural formula of the end glyoxal phosphorylcholine is

3. The process for producing a polymer according to claim 2, wherein the diisocyanate is an aliphatic diisocyanate having an ordered segment.

4. A process for preparing a polymer as claimed in claim 3, wherein the diisocyanate is hexamethylene diisocyanate-1, 4-butanediol-hexamethylene diisocyanate, tetramethylene diisocyanate-1, 4-butanediol-tetramethylene diisocyanate or L-lysine diisocyanate-1, 4-butanediol-L-lysine diisocyanate.

5. The method according to claim 2, wherein the molar ratio of the single-terminal dihydroxypyridine compound PyDH to the diisocyanate is 1:1.1 to 1: 1.2.

6. The method for preparing a polymer according to claim 2, wherein the solvent used in the reaction system for preparing the polyurethane is N, N-dimethylformamide.

7. The method according to claim 2, wherein the total concentration of the single-ended dihydroxypyridine compound PyDH and the diisocyanate in the solution in the reaction system for producing polyurethane is 0.5 to 1.5 g/mL.

8. The method for preparing a polymer according to claim 2, wherein the reaction temperature for preparing the polyurethane is 70 to 85 ℃.

9. The method for producing a polymer according to claim 2, wherein the catalyst in the reaction system for producing the polyurethane is a tin-based catalyst.

10. The method for preparing a polymer according to claim 9, wherein the tin catalyst is dibutyltin dilaurate or stannous octoate.

11. The method according to claim 2, wherein the amount of the catalyst added to the reaction system for producing the polyurethane is 0.5 to 1% by mass based on the total mass of the single-terminal dihydroxypyridine compound PyDH and the diisocyanate.

12. The method for preparing a polymer according to claim 2, wherein the chitosan oligosaccharide solution is added to the polymerization reaction mixture to conduct the addition reaction.

13. The method for preparing a polymer according to claim 12, wherein the concentration of the chitosan oligosaccharide solution is 1.0 to 1.5 g/mL.

14. The method according to claim 2, wherein the amount of the chitosan oligosaccharide is 4 to 6 times the molar difference between the diisocyanate and the single-terminal dihydroxypyridine compound PyDH.

15. The method for preparing a polymer according to claim 2, wherein the purification method of the polymer precursor comprises: diluting the material after the addition reaction, settling by using water, and drying the solid after the precipitation.

16. The method for preparing a polymer according to claim 2, wherein the molar amount of the aldehyde-terminated phosphorylcholine is equal to the molar amount of the primary amine group in the polymer precursor.

17. The method for preparing a polymer according to claim 2, wherein the reaction temperature of the aldehyde-amine condensation reaction is 20 to 30 ℃ and the reaction time is 2 to 3 hours.

18. The process for producing a polymer according to claim 2, wherein the material after the aldol condensation is precipitated with ethanol, and the precipitated solid is washed and dried.

19. A pH-sensitive nanovesicle, which is assembled from the polymer according to claim 1.

20. The pH-sensitive nanovesicle of claim 19, wherein the particle size is 100-160 nm.

21. The pH-sensitive nanovesicle of claim 19, wherein the particle size of the pH-sensitive nanovesicle increases 1.4-1.8-fold in an acidic medium and increases less than 1.1-fold or does not increase in a neutral or basic medium.

22. A method for preparing pH-sensitive nanovesicles, comprising the steps of dissolving the polymer of claim 1 in an organic solvent to obtain a polymer solution, mixing the polymer solution with water to prepare an emulsion, adding the emulsion to water to prepare a water-oil-water double emulsion, removing the organic solvent, and freeze-drying to obtain the pH-sensitive nanovesicles.

23. The method for preparing pH-sensitive nanovesicles according to claim 22, wherein the organic solvent is chloroform.

24. The method for preparing pH-sensitive nanovesicles according to claim 22, wherein the concentration of the polymer solution is 0.005-0.015 g/mL.

25. The method for preparing pH-sensitive nanovesicles according to claim 22, wherein a volume ratio of the polymer solution to water is 4: 0.5-1.5.

26. The method for preparing pH-sensitive nanovesicles according to claim 22 wherein the polymer is mixed with water and sonicated to prepare an emulsion.

27. The method for preparing pH-sensitive nanovesicles according to claim 22, wherein in the process of preparing the water-oil-water double emulsion, the volume ratio of the emulsion to water is 1: 0.5-1.

28. The method for preparing pH-sensitive nanovesicles as claimed in claim 22 wherein the emulsion is added to water and sonicated to prepare a water-oil-water double emulsion.

29. Use of the pH-sensitive nanovesicle according to any one of claims 19 to 21 or the pH-sensitive nanovesicle obtained by the preparation method according to any one of claims 22 to 28 as a drug carrier.

30. A targeted drug, which is characterized by comprising an active drug and a targeted carrier, wherein the targeted carrier is the pH-sensitive nanovesicle of any one of claims 19 to 21 or the pH-sensitive nanovesicle obtained by the preparation method of any one of claims 22 to 28.

31. The targeted drug of claim 30, wherein the active drug is an anti-inflammatory drug or an anti-tumor drug.

32. The targeted drug of claim 30, wherein the targeted drug has a particle size of 120 to 190 nm.

33. A process for preparing target medicine includes such steps as dissolving the polymer as claimed in claim 1 in organic solvent to obtain polymer solution, mixing it with water containing active medicine to obtain emulsion, adding the emulsion to water to obtain water-oil-water double emulsion, removing organic solvent, and freeze drying.

34. The method for preparing a targeted drug according to claim 33, wherein the organic solvent is chloroform.

35. The method for preparing the targeted drug according to claim 33, wherein the concentration of the polymer solution is 0.005 to 0.015 g/mL.

36. The method for preparing a targeted drug according to claim 33, wherein the volume ratio of the polymer solution to the water containing the active drug is 4:0.5 to 1.5.

37. The method for preparing a targeted drug according to claim 33, wherein the active drug is an anti-inflammatory drug or an anti-tumor drug.

38. The method for preparing the targeted medicament according to claim 33, wherein in the process of preparing the water-oil-water double emulsion, the volume ratio of the emulsion to the water is 1: 0.5-1.

39. The method for preparing a targeted pharmaceutical according to claim 33, wherein the organic solvent is removed, and after dialysis, lyophilization is performed.

40. The method for preparing the targeted medicament according to claim 39, wherein the dialysis is performed for 2 days by using a 10KDa dialysis bag and the deionized water is changed every 12 hours.