CN114507326A - pH response crosslinking bond-based hydrophilic side chain-adjustable triblock polymer and preparation and application thereof - Google Patents

Abstract

The invention discloses a pH response crosslinking bond-based hydrophilic side chain-adjustable triblock polymer, and preparation and application thereof. The polymer is poly (2-phenoxyethyl methacrylate) -b-poly (2-oxopropyl methacrylate) -b-poly (monomethoxy polyethylene glycol) methacrylate, and the hydrophilic side chain of the polymer is easy to regulate and control and can be self-assembled to form micelles with different stabilities. The pH response cross-linked micelle of the polymer can effectively load the water-insoluble drug. The micelle disclosed by the invention enhances the stability of the micelle in a physiological environment through hydrophilic side chain regulation and cross-linking strategies, has a better pH response performance, can quickly release a drug in a tumor microenvironment, and improves the drug delivery efficiency.

Description

A triblock polymer with tunable hydrophilic side chain based on pH-responsive crosslinks and its preparation and application

technical field

The invention belongs to the technical field of biological macromolecular polymer materials, in particular to a triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds, and preparation and application thereof.

Background technique

In recent years, more and more attention has been paid to the harm of cancer to human health. Chemotherapy is a mainstream treatment method among many cancer treatment strategies. However, traditional chemotherapeutic drugs have disadvantages such as poor water solubility and strong toxic and side effects. The use of drug carrier materials to deliver drugs can effectively remedy these deficiencies. Among the currently developed drug-carrying materials, polymer micelles are widely used for the encapsulation and delivery of poorly water-soluble drugs due to their excellent biocompatibility and easy chemical modification.

Polymeric micelles are dynamic equilibrium aggregates formed by the self-assembly of amphiphilic block polymers in aqueous media. In the process of drug delivery in vivo, micelles are often destabilized due to the adsorption of proteins, lipids and other substances in the blood, which eventually leads to the disintegration of the structure. At the same time, foreign substances such as proteins adsorbed on the surface of micelles may promote the aggregation and sedimentation of micelles through bridging. In addition, there is a large amount of fluid in the blood circulation, which reduces the concentration of micelles to below the critical micelle concentration (CMC) through dilution, resulting in the destabilization and disintegration of micelles, which seriously affects the drug delivery effect. Aiming at the above problems, it is mainly achieved by improving the structural stability and colloidal stability. Crosslinking by chemical covalent bonds is one of the effective strategies to improve the stability of micelle structure. Zhang et al. prepared a shell-layer reversible cross-linked micelle based on a four-arm star polymer, forming a cross-linked structure with reversible hydrazone bonds and disulfide bonds in the shell layer. After 1000-fold dilution with deionized water, the cross-linked The micelles still maintain the intact structure and have good anti-dilution ability and stability. For colloidal stability, changing the properties of the surface micelle hydrophilic block (such as adjusting its length, grafting density and chemical structure, etc.) can enhance its colloidal stability (Polymer 2017, 114, 161-172). Shoichet et al. studied the effect of the grafting density of hydrophilic polyethylene glycol (PEG) on the stability of nanoparticles. With the increase of PEG grafting density, the stability of nanoparticles in physiological environment was enhanced (Chem.Mater. 2014, 26, 2847-2855).

In the process of drug delivery, polymer micelles should not only maintain a high physiological environment stability, but also achieve accurate and rapid drug release at the tumor site. Selecting a reversible cross-linked bond structure that responds to breaking in the tumor microenvironment can achieve the combination of the above two and maximize the effect of drug delivery. Patent CN111978553B discloses a triple stimuli-responsive interface cross-linked polymer micelle, in which the disulfide cross-links are broken under reducing conditions, which effectively increases the amount of drug release. However, the regulation of stability by polymer structure and the combination of stability and rapid drug release have not been systematically studied.

To sum up, how to realize the above requirements such as micelle stability and drug controlled release is a technical problem that needs to be solved at present.

SUMMARY OF THE INVENTION

In order to overcome the shortcomings of the prior art such as complex polymer structure, difficult stability control, and poor controlled release performance, the primary purpose of the present invention is to provide a pH-responsive crosslink-based triad with adjustable hydrophilic side chains. segment polymer.

The structure of the polymer of the present invention is: polymethacrylate-2-phenoxyethyl ester-b-polymethacrylate-2-oxopropyl ester-b-polymethacrylate monomethoxypolyethylene glycol ( PPOEMA-b-POPMA-b-PPEGMA).

The polymer preparation process of the invention is simple and convenient, and the length of the hydrophilic side chain of the amphiphilic block polymer can be accurately and quickly regulated, thereby enhancing the stability and drug delivery performance of the micelle formed by the polymer.

Another object of the present invention is to provide a method for preparing the above-mentioned triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds. This method first oxidizes 2-hydroxypropyl methacrylate (HPMA) to the keto-functionalized monomer 2-oxopropyl methacrylate (OPMA) with pyridine chlorochromate; then uses electron transfer to activate regenerated atoms Radical transfer polymerization (ARGET ATRP) method, using a small molecular initiator to combine the hydrophobic monomer-2-phenoxyethyl methacrylate (POEMA) and the keto-functional monomer-2-oxopropyl methacrylate (OPMA) and the hydrophilic monomer monomethoxypolyethylene glycol methacrylate (PEGMA) were polymerized in turn to obtain the triblock polymer PPEEMA-b-POPMA-b-PPEGMA. Among them, triblock polymers with different hydrophilic side chains can be obtained by adding equimolar amounts of PEGMA with different relative molecular weights.

Another object of the present invention is to provide the application of the above-mentioned triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds in loading poorly water-soluble drugs, especially poorly water-soluble anticancer drugs (such as doxorubicin). The triblock polymer of the present invention is dissolved in a solvent and then dialyzed to obtain a polymer micelle in which the core layer is a hydrophobic block, the middle layer contains ketone groups that can be used for reversible covalent cross-linking, and the shell layer is a hydrophilic block , in the environment of pH 6.5 and under the action of the catalyst 2-amino-5-methoxybenzoic acid, it reacts with the hydrazide bond in the cross-linking agent adipic acid dihydrazide to generate an acylhydrazone that can be reversibly broken under acidic conditions bond cross-linking structure. Under normal physiological environment, doxorubicin is stably loaded in the micellar core through the π-π stacking interaction with the benzene ring of the micellar core layer, and at the same time, due to the effect of the cross-linked structure and long hydrophilic side chains, the loading is further enhanced. stability. When the micelles were in the acidic microenvironment of the tumor, the acylhydrazone cross-links in the middle layer of the micelles were broken, resulting in the disintegration of the micelle structure and the release of doxorubicin from the inner core of the micelles. At the same time, the protonation of the amino group of doxorubicin under acidity improves the water solubility of the drug and further accelerates the release of the drug.

The object of the present invention is achieved through the following technical solutions:

A triblock polymer with tunable hydrophilic side chains based on pH-responsive cross-linking bonds, the structural formula of which is as follows:

Wherein, x=15-35, y=5-20, z=15-35, and the number of structural units n of the side chain ethylene glycol group of the hydrophilic block monomer PEGMA is 5-20.

The triblock polymer with adjustable hydrophilic side chain based on pH-responsive cross-linking bond of the present invention is named PPEEMA-b-POPMA-b-PPEGMA.

The number-average molecular weight of the triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds is 8499-43503 g/mol.

The above-mentioned preparation method of a triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds, comprising the following steps:

(1) Preparation of keto-functional monomer (OPMA): Dissolve pyridine chlorochromate (PCC) and silica gel powder in a solvent, add 2-hydroxypropyl methacrylate (HPMA) under ice bath conditions, and go to Continue the reaction at room temperature to obtain keto-functional monomer 2-oxopropyl methacrylate (OPMA);

(2) Preparation of amphiphilic triblock polymer (PPOEMA-b-POPMA-b-PPEGMA): catalyst, monomer 2-phenoxyethyl methacrylate (POEMA), ligand 1,1,4 ,7,10,10-Hexamethyltriethylenetetramine (HMTETA) is dissolved in the solvent, fully stirred, and then add the reducing agent, stir evenly, add the small molecule initiator and heat the reaction, after the monomer conversion is complete, add the step ( 1) The prepared keto-functional monomer methacrylate-2-oxopropyl methacrylate (OPMA) continues to react, and after the monomer conversion is complete, the monomer methacrylate monomethoxy polyethylene glycol ester ( PEGMA) to continue the reaction to obtain an amphiphilic triblock polymer (PPOEMA-b-POPMA-b-PPEGMA).

Preferably, the molar ratio of 2-hydroxypropyl methacrylate and pyridine chlorochromate in step (1) is 1:1.2-2.

Preferably, the silica gel powder in step (1) is used for dispersing the reactants and improving the reaction yield, and the amount thereof is the same as the quality of PCC.

Preferably, the solvent in step (1) is dichloromethane; the molar volume ratio of the pyridine chlorochromate to the solvent is 0.3-0.5 mmol/mL.

Preferably, the reaction time at room temperature in step (1) is 12-18 h.

Preferably, the catalyst in step (2) is at least one of copper bromide (CuBr ₂ ) and copper chloride; the reducing agent is at least one of stannous octoate (Sn(Oct) ₂ ) and ascorbic acid ; The small molecule initiator is ethyl bromoisobutyrate (EBriB).

Preferably, the catalyst in step (2), 2-phenoxyethyl methacrylate, 1,1,4,7,10,10-hexamethyltriethylenetetramine, reducing agent, small molecule initiator, The molar ratio of 2-oxopropyl methacrylate to monomethoxy polyethylene glycol methacrylate is 0.04-0.06: 15-35: 0.4-0.6: 0.4-0.6: 1: 5-20: 15 ~35.

Preferably, the solvent in step (2) is at least one of anisole and N,N-dimethylformamide (DMF); The molar volume ratio is 0.52-2.09 mmol/mL.

Preferably, the temperature of the monomer 2-phenoxyethyl methacrylate heating reaction in step (2) is 40 to 50° C., and the time is 4 to 10 h; the temperature of the reaction of 2-oxopropyl methacrylate is The reaction temperature is 40-50 DEG C, and the time is 12-18 hours; the reaction temperature of the monomethoxy polyethylene glycol methacrylate is 60-70 DEG C, and the time is 36-72 hours.

Preferably, after the reaction in step (1) is completed, the reaction product system is purified and concentrated to obtain a purified product. The purification refers to first adding the reaction solution to diatomaceous earth for suction filtration under reduced pressure, and rotating the concentrated filtrate to obtain a crude product, and then eluting the product through silica gel column chromatography, and finally rotating the eluate to concentrate to obtain the purified product. .

Preferably, after the reaction of the monomer monomethoxy polyethylene glycol methacrylate in step (2) is completed, the reaction product system is cooled, purified and dried to obtain a purified product. The purification refers to adding tetrahydrofuran (THF) to the reaction product system after cooling to terminate the reaction, then passing through a neutral alumina chromatography column to remove the catalyst, and then adding dropwise to 10 times of ice-n-hexane to precipitate after rotary evaporation and concentration, and the rotary evaporation-precipitation repeats. Three times, the purified product was obtained after vacuum drying.

Preferably, the reactions in steps (1) to (2) are carried out under inert gas protection and anhydrous conditions.

Application of a triblock polymer with tunable hydrophilic side chains based on pH-responsive crosslinks in loading poorly water-soluble drugs.

Preferably, the application is as follows: dissolving the above-mentioned triblock polymer with adjustable hydrophilic side chain based on pH-responsive cross-linking bonds and a poorly water-soluble drug in a solvent, after mixing uniformly, dialyzing in a phosphate buffered saline solution , then add catalyst and cross-linking agent, stir the reaction at room temperature, and dialyze with deionized water to obtain a pH-responsive cross-linked micelle system loaded with poorly water-soluble drugs.

More preferably, the solvent is dimethyl sulfoxide (DMSO); the mass-volume ratio of the triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds to the solvent is 1-5 mg/mL .

More preferably, the mass ratio of the triblock polymer with adjustable hydrophilic side chain based on the pH-responsive cross-linking bond to the poorly water-soluble drug is 2-10:1.

More preferably, the poorly water-soluble drug is a poorly water-soluble anticancer drug, such as deacidified doxorubicin (DOX) and paclitaxel; the poorly water-soluble drug refers to a drug with a solubility of less than or equal to 1 g in 1 L of water.

More preferably, the mixing time is 3-5 hours.

More preferably, the catalyst is 2-amino-5-methoxybenzoic acid; the cross-linking agent is adipic acid dihydrazide (ADH); the hydrophilic side chain based on the pH-responsive cross-linking bond can be The molar ratio of the adjusted triblock polymer and the cross-linking agent is 2:1; the concentration of the catalyst in the micelle solution obtained after dialysis in a phosphate buffered saline solution is 8-12 mmol/L.

More preferably, the stirring reaction time at room temperature is 20-30 h.

More preferably, the pH of the phosphate buffered saline solution is 6.5; the time of the dialysis in the phosphate buffered saline solution is 18-30 hours, and the dialysate is replaced every 2-6 hours.

More preferably, the time of the deionized water dialysis is 10-24 hours, and the dialysate is replaced every 2 hours.

The drug loaded in the pH-responsive cross-linked micelle system loaded with poorly water-soluble drugs is released slowly in a normal physiological environment (pH 7.4), and the pH-responsive cross-linked bonds are broken in an acidic tumor microenvironment (pH 5.0), promoting the drug Quick release.

The mechanism of the present invention is:

The present invention utilizes the oxidant pyridine chlorochromate (PCC) to oxidize 2-hydroxypropyl methacrylate (HPMA) to keto-functional monomer 2-oxopropyl methacrylate (OPMA), and then pass through ARGET ATRP The triblock polymer was obtained by polymerizing 2-phenoxyethyl methacrylate (POEMA), OPMA and monomethoxy polyethylene glycol methacrylate (PEGMA) in turn, which was dissolved in a solvent and then dialyzed. After the cross-linking reaction at room temperature, a cross-linked polymer micelle with hydrophobic PPEEMA as the core, POPMA with acylhydrazone bond cross-linked structure as the middle layer, and hydrophilic layer PPEGMA as the outer shell was obtained. POEMA has a benzene ring, which can efficiently load the hydrophobic anticancer drug doxorubicin (DOX) through π-π stacking. The ketone group in OPMA can form a pH-responsive acylhydrazone bond cross-linked structure under mild conditions at room temperature. Good water and biocompatibility, excellent anti-protein adsorption performance, can better stabilize the micellar structure, and the length of the hydrophilic side chain of the micellar can be precisely regulated by the different PEGMA structural units added to ARGET ATRP, and then Enhances the stability of polymer micelles. The broad-spectrum anticancer drug DOX is loaded in the inner core of the micelle, and can be stably encapsulated by the micelle at pH 7.4 (normal physiological environment). The cleavage of acid-sensitive acylhydrazone bonds leads to the disintegration of the micellar structure, achieving the goal of rapid drug release and precise delivery.

The invention provides a triblock polymer with adjustable hydrophilic side chains based on pH-responsive cross-linking bonds and a micelle thereof. By combining the regulation of the hydrophilic side chains and the cross-linking strategy, the colloid and the colloid of the micelle are enhanced at the same time. Structural stability, pH-responsive bond cleavage in the acidic microenvironment of tumors, and rapid drug release, is a potential drug delivery material.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The preparation process of the present invention is simple, the conditions are mild, the block composition and polymerization degree of the triblock polymer can be precisely regulated, and the hydrophilic side chain of the polymer is changed by inputting PEGMA monomers with different ethylene glycol structural units. length, and accurately regulate the stability of the formed micelles.

(2) The present invention adopts the strategy of regulating the length of the hydrophilic side chain and the reversible cross-linking at the same time to maximize the stability of the polymer micelle and improve the drug delivery efficiency of the micelle.

(3) The cross-linking bond constructed in the present invention is an acylhydrazone bond structure that responds to pH. When the drug-loaded micelle reaches the tumor cell, it can be broken in response to the acidic environment in the microenvironment and rapidly release the anticancer drug doxorubicin. Therapeutic effect of chemotherapeutic drugs.

Description of drawings

FIG. 1 is the hydrogen nuclear magnetic spectrum of the keto-functional monomer OPMA in Example 1. FIG.

FIG. 2 is the hydrogen nuclear magnetic spectrum of the triblock polymer PPEEMA-b-POPMA-b-PPEGMA in Example 2. FIG.

3 is the GPC elution curve of the triblock polymer PPEEMA-b-POPMA-b-PPEGMA in Example 2.

4 is a DLS graph of the pH-responsive cross-linked polymer micelles in Example 5. FIG.

FIG. 5 is a graph of the stability test of the cross-linked polymer micelles in Example 6 under a physiological environment.

FIG. 6 is a TEM image of the drug-loaded cross-linked polymer micelles in Example 7. FIG.

FIG. 7 is the in vitro release curve of the drug-loaded cross-linked micelles in Example 8. FIG.

Figure 8 is a graph of the cytotoxicity experiment of drug-loaded micelles and free doxorubicin in Example 9.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

If the specific conditions are not indicated in the examples of the present invention, the conventional conditions or the conditions suggested by the manufacturer are used. The raw materials, reagents, etc., which are not specified by the manufacturer, are all conventional products that can be purchased from the market.

Example 1: Keto-functionalized monomer (OPMA)

A stirring bar, PCC (4.85g, 22.50mmol) and 4.85g of silica gel were added to a 50mL dry eggplant-shaped bottle, argon was passed through for 10min, then the eggplant-shaped bottle was sealed, 50mL of dichloromethane was injected into the eggplant-shaped bottle with a syringe, and the eggplant The flask was placed in an ice bath, and then a solution of HPMA (2.16 mL, 15 mmol) in dichloroethane was added dropwise (HPMA was dissolved in 10 mL of dichloromethane in advance), and the reaction was carried out at room temperature for 12 h. 50 mL of ether was added to the reaction solution, diatomaceous earth was spread on the Buchner funnel, and then filtered under reduced pressure and the filtrate was collected. The solvent was removed by rotary evaporation, and the obtained liquid was eluted with silica gel column chromatography (mobile phase: n-hexane:ethyl acetate 15:1), and rotary evaporated again to obtain a yellow oily liquid. The synthetic reaction formula is shown in formula (1). The structure of the product was characterized and analyzed by hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR), and the results are shown in Figure 1 .

Example 2: Preparation of triblock polymer (PPOEMA-b-POPMA-b-PPEGMA) (x:y:z=35:15:25, n=20)

Take a 50mL dry eggplant-shaped bottle, add stirring bar and catalyst CuBr ₂ (5.01mg, 0.022mmol) to it, seal the reaction bottle and evacuate - pass argon 3 times, add solvent anhydrous anisole (8mL) successively with a syringe , monomer POEMA (3 mL, 15.75 mmol), ligand HMTETA (0.06 mL, 0.22 mmol), fully stirred for 10 min to form a catalyst complex. Then, the reducing agent Sn(Oct) ₂ (0.07 mL, 0.22 mmol) pre-dissolved in 2 mL of anhydrous anisole was added and stirred for 10 min. After adding the small-molecule initiator EBriB (0.06 mL, 0.45 mmol) with a micro syringe, transfer React in an oil bath at 40°C. After the POEMA reaction was completed, the monomer OPMA (0.96g, 6.75mmol) was added to continue the reaction for 15h, and then the monomer PEGMA (Mn=950g/mol, 10.66g, 11.25mmol) pre-dissolved in 1mL of anhydrous anisole was added, The temperature was adjusted to 60°C and the reaction was continued for 72h. After the reaction was completed, the eggplant-shaped flask was cooled to room temperature and THF was added to terminate the reaction, then passed through a neutral alumina column (THF as eluent), concentrated by rotary evaporation, and slowly added dropwise to ten times the amount of n-hexane for precipitation at 45°C. 24h under vacuum drying at 35mbar to obtain the product. The synthetic reaction formula is shown in formula (2). The composition and structure of the product were analyzed by ¹ H NMR and GPC, the results are shown in Figure 2 and Figure 3, Mn=17.0kDa,

Example 3: Preparation of triblock polymer (PPOEMA-b-POPMA-b-PPEGMA) (x:y:z=35:15:25, n=9)

Take a 50mL dry eggplant-shaped bottle, add stirring bar and catalyst CuBr ₂ (5.01mg, 0.022mmol) to it, seal the reaction bottle and evacuate - pass argon 3 times, add solvent anhydrous anisole (8mL) successively with a syringe , monomer POEMA (3 mL, 15.75 mmol), ligand HMTETA (0.06 mL, 0.22 mmol), fully stirred for 10 min to form a catalyst complex. Then, the reducing agent Sn(Oct) ₂ (0.07 mL, 0.22 mmol) pre-dissolved in 2 mL of anhydrous anisole was added and stirred for 10 min. After adding the small-molecule initiator EBriB (0.06 mL, 0.45 mmol) with a micro syringe, transfer React in an oil bath at 40°C. After the POEMA reaction was completed, the monomer OPMA (0.96 g, 6.75 mmol) was added to continue the reaction for 15 h, and then the monomer PEGMA (Mn=475 g/mol, 5.33 g, 11.25 mmol) was added, and the temperature was adjusted to 60 °C to continue the reaction for 48 h. After the reaction was completed, the eggplant-shaped flask was cooled to room temperature and THF was added to terminate the reaction, then passed through a neutral alumina column (THF as eluent), concentrated by rotary evaporation, and slowly added dropwise to ten times the amount of n-hexane for precipitation at 45°C. 24h under vacuum drying at 35mbar to obtain the product. The synthetic reaction formula is shown in formula (2). Mn=12.7kDa,

Example 4: Preparation of triblock polymer (PPOEMA-b-POPMA-b-PPEGMA) (x:y:z=35:15:25, n=5)

Take a 50mL dry eggplant-shaped bottle, add stirring bar and catalyst CuBr ₂ (5.01mg, 0.022mmol) to it, seal the reaction bottle and evacuate - pass argon 3 times, add solvent anhydrous anisole (8mL) successively with a syringe , monomer POEMA (3 mL, 15.75 mmol), ligand HMTETA (0.06 mL, 0.22 mmol), fully stirred for 10 min to form a catalyst complex. Then, the reducing agent Sn(Oct) ₂ (0.07 mL, 0.22 mmol) pre-dissolved in 2 mL of anhydrous anisole was added and stirred for 5 min. After adding the small-molecule initiator EBriB (0.06 mL, 0.45 mmol) with a micro syringe, transfer React in an oil bath at 40°C. After the POEMA reaction was completed, the monomer OPMA (0.96g, 6.75mmol) was added to continue the reaction for 15h, and then the monomer PEGMA (Mn=300g/mol, 3.37g, 11.25mmol) was added, and the temperature was adjusted to 55°C to continue the reaction for 48h. After the reaction was completed, the eggplant-shaped flask was cooled to room temperature and THF was added to terminate the reaction, then passed through a neutral alumina column (THF as eluent), concentrated by rotary evaporation, and slowly added dropwise to ten times the amount of n-hexane for precipitation at 45°C. 24h under vacuum drying at 35mbar to obtain the product. The synthetic reaction formula is shown in formula (2). Mn=11.1kDa,

Example 5: Preparation of pH-responsive cross-linked polymeric micelles

The pH-responsive cross-linked polymer micelles were prepared by dialysis: the polymer (120 mg) of Example 2 was dissolved in 30 mL of DMSO and transferred to a dialysis bag (MWCO=3.5 kDa), and PBS solution (50 mmol/L, pH 6.5) was used to prepare it. Dialysis for 24h. The dialysate was changed every two hours for the first 12h, then every 6h. Subsequently, the micelle solution was transferred to the flask, the catalyst 2-amino-5-methoxybenzoic acid (the concentration in the micelle solution was 10 mmol/L) was added, and the cross-linking agent adipic acid dihydrazide (ADH) was added. A solution with a concentration of 50 mg/mL was prepared and dropped into the flask until the molar ratio of polymer and ADH was 2/1. After stirring the reaction for one day, the micelle solution was dialyzed with deionized water for another 12 h, and the dialysate was replaced every two hours. Finally, the cross-linked micelle solution was filtered through a 0.45 μm aqueous microporous membrane and lyophilized to obtain cross-linked polymer micelles.

The particle size and polydispersity (PDI) of the crosslinked micelles were characterized by dynamic light scattering (DLS). The hydrodynamic diameter (Dh) of the cross-linked micelles was 43.71 nm and the PDI was 0.12 (Figure 4).

Example 6: Stability test of cross-linked polymer micelles in physiological environment

Take the polymer (120 mg) of Example 2, fluorescent dyes DiO (150 μg) and DiI (150 μg) and dissolve them in 30 mL of DMSO and stir for 4 h, then transfer them to a dialysis bag (MWCO=3.5 kDa) with PBS solution (50 mmol/L , pH 6.5) dialysis for 24h. The dialysate was changed every two hours for the first 12h, then every 6h. Subsequently, the micelle solution was transferred to the flask, the catalyst 2-amino-5-methoxybenzoic acid (the concentration in the micelle solution was 10 mmol/L) was added, and the cross-linking agent ADH was made into a concentration of 50 mg/mL. solution, dropwise into the flask until the molar ratio of polymer and ADH is 2/1. After stirring the reaction for one day, the micelle solution was dialyzed with deionized water for another 12 h, and the dialysate was replaced every two hours. Finally, the cross-linked micelle solution was filtered with a 0.45 μm aqueous microporous membrane to obtain cross-linked polymer micelles loaded with two fluorescent dyes. Then, the filtered micelle solution was prepared into 1 mL of a micelle solution containing 10% fetal bovine serum (FBS), and incubated at 37°C with shaking. Molecular fluorometer was used to measure the fluorescence emission intensity of micelle solution containing 10% fetal bovine serum (FBS) at 490-600 nm at different times, and the ratio of the sum of the intensity values at 505 nm and 505 nm to 572 nm was calculated to obtain the FRET efficiency.

As shown in Figure 5, the FRET efficiency of cross-linked polymer micelles did not change significantly within 72 h of incubation with fetal bovine serum, indicating that the micelles can still maintain good structural stability for a long time under the action of physiological substances such as proteins. , so as to stably encapsulate the fluorescent dye.

Example 7: Preparation of cross-linked polymer micelles loaded with anticancer drug DOX

The drug-loaded cross-linked micelles were prepared by dialysis method: dissolve 100 mg DOX HCl in 10 mL borax buffered saline solution (pH 9.0), stir at room temperature for 12 h, centrifuge at 13000 rpm for 15 min, take the precipitate and freeze-dry to obtain deacidified DOX. Then, the deacidified DOX (36 mg) and the polymer (120 mg) in Example 2 were dissolved in 30 mL of DMSO and stirred for 4 h, and then transferred to a dialysis bag (MWCO=3.5 kDa) with PBS solution (50 mmol/L, pH 6.5) Dialysis for 24h. The dialysate was changed every two hours for the first 12h and then every 6h. Subsequently, the micelle solution was transferred to the flask, the catalyst 2-amino-5-methoxybenzoic acid (the concentration in the micelle solution was 10 mmol/L) was added, and the cross-linking agent ADH was made into a concentration of 50 mg/mL. The solution was dropped into the flask until the polymer to ADH molar ratio was 2/1. After stirring the reaction for one day, the micelle solution was dialyzed with deionized water for another 12 h, and the dialysate was replaced every two hours. Finally, the cross-linked micelle solution was filtered with a 0.45 μm aqueous microporous membrane and lyophilized to obtain DOX-loaded cross-linked polymer micelles.

Dissolve 1 mg of drug-loaded micelles in 10 mL of DMSO, measure the absorption value of the micelle solution at a wavelength of 480 nm with UV-Vis spectrophotometer, calculate the drug loading (LC) and encapsulation efficiency (EE), LC≈11.32%, EE ≈37.73%.

The morphology and particle size of the drug-loaded cross-linked micelles were characterized by transmission electron microscopy (TEM). The drug-loaded cross-linked micelles showed a relatively uniform spherical morphology with a particle size of 61.11 nm (Figure 6).

Example 8: In vitro release of drug-loaded cross-linked micelles

The drug-loaded cross-linked micelles (3 mg) in Example 7 were dispersed in 3 mL of PBS solution (pH 7.4) and acetate buffer (pH 5.0), respectively, and then transferred to a dialysis bag (MWCO=3.5 kDa) and immersed in the corresponding In a buffer solution (47 mL), dialysis was performed at 37°C and 100 rpm. Select a specific time point to take the extradialysis fluid (4 mL) and add an equal amount of fresh corresponding buffer (4 mL). The absorbance at 480 nm of the dialysate external fluid at different times was measured with a UV-visible spectrophotometer, and the in vitro release curve was drawn, as shown in FIG. 7 .

Example 9: Cytotoxicity assay

HepG2 cells were subcultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin (the raw material is penicillin-streptomycin solution (100X), Biyuntian CO222) at 37°C under 5% _CO concentration. . Then, cells were plated in 96-well plates at a density of 5000 cells per well and incubated for 24 h. After the old medium was aspirated and washed, 150 μL of medium containing 10 μL of drug-loaded micelles or free DOX was added to the well plate and incubated for 24 h. Then aspirated and washed, added 150 μL of medium containing 10 μL of LCCCK-8 reagent for 3 h, tested the UV absorption value of the well plate at a wavelength of 450 nm, and calculated the cell viability under different material concentrations, as shown in Figure 8.

Comparative Example 1:

Preparation of triblock polymer (PPOEMA-b-POPMA-b-PPEGMA) using toluene as solvent (x:y:z=35:15:25, n=9)

且洗脱曲线出现拖尾峰，表明聚合物的均一性不佳。Take a 50mL dry eggplant-shaped bottle, add a stirrer and catalyst CuBr ₂ (4.01mg, 0.018mmol) to it, seal the reaction bottle and evacuate - pass argon 3 times, add solvent anhydrous toluene (8mL), mono The body POEMA (3 mL, 15.75 mmol) and the ligand HMTETA (0.05 mL, 0.18 mmol) were stirred thoroughly for 10 min to form the catalyst complex. Then, the reducing agent Sn(Oct) ₂ (0.06 mL, 0.18 mmol) pre-dissolved in 2 mL of anhydrous toluene was added and stirred for 10 min. After adding the small molecule initiator EBriB (0.06 mL, 0.45 mmol) with a micro syringe, it was transferred to 80 ℃ oil bath for 6h. Then, the monomer OPMA (0.96 g, 6.75 mmol) pre-dissolved in 1 mL of anhydrous toluene was added, the temperature was adjusted to 70 °C and the reaction was continued for 12 h, and then the monomer PEGMA ( _Mn = 475 g/mol) dissolved in 1 mL of anhydrous toluene was added. , 5.33g, 11.25mmol) continued to react for 48h. After the reaction was completed, the eggplant-shaped flask was cooled to room temperature and THF was added to terminate the reaction, then passed through a neutral alumina column (THF as eluent), concentrated by rotary evaporation, and slowly added dropwise to ten times the amount of n-hexane for precipitation at 45°C. 24h under vacuum drying at 35mbar to obtain the product. The composition and structure of the product were analyzed by GPC, _Mn = 1.77kDa,

And the elution curve appeared tailing peak, indicating that the homogeneity of the polymer is not good.

Comparative example 2: Preparation of DOX-loaded cross-linked polymer micelles

Dissolve 45 mg of DOX·HCl in 15 mL of DMSO, add triethylamine (TEA, 1.08 mL), and stir for 24 h in the dark. Then, the polymer (120 mg) in Example 2 was dissolved in 15 mL of DMSO, mixed with the above DOX solution and stirred for 4 h, then transferred to a dialysis bag (MWCO=3.5 kDa) and dialyzed with a PBS solution (50 mmol/L, pH 6.5). 24h. The dialysate was changed every two hours for the first 12h, then every 6h. Subsequently, the micelle solution was transferred to the flask, the catalyst 2-amino-5-methoxybenzoic acid (the concentration in the micelle solution was 10 mmol/L) was added, and the cross-linking agent ADH was made into a concentration of 50 mg/mL. solution, dropwise into the flask until the molar ratio of polymer and ADH is 2/1. After stirring the reaction for one day, the micelle solution was dialyzed with deionized water for another 12 h, and the dialysate was replaced every two hours. Finally, the cross-linked micelle solution was filtered through a 0.45 μm aqueous microporous membrane and lyophilized to obtain DOX-loaded cross-linked polymer micelles.

Dissolve 1 mg of drug-loaded micelles in 10 mL of DMSO, measure the absorption value of the micelle solution at a wavelength of 480 nm with a UV-Vis spectrophotometer, and calculate the drug loading (LC) and encapsulation efficiency (EE). LC≈3.87%, EE≈10.34%.

Comparative Example 3:

Preparation of triblock polymer (PPOEMA-b-POPMA-b-PPEGMA) using toluene as solvent (x:y:z=35:15:25, n=9)

Take a 50mL dry eggplant-shaped bottle, add a stirrer and catalyst CuBr ₂ (5.01mg, 0.022mmol) to it, seal the reaction bottle and evacuate - pass argon for 3 times, and add solvent anhydrous toluene (8mL), single The body POEMA (3 mL, 15.75 mmol) and the ligand HMTETA (0.06 mL, 0.22 mmol) were stirred thoroughly for 10 min to form the catalyst complex. Then, the reducing agent Sn(Oct) ₂ (0.07 mL, 0.22 mmol) pre-dissolved in 2 mL of anhydrous toluene was added and stirred for 10 min. After adding the small molecule initiator EBriB (0.06 mL, 0.45 mmol) with a micro syringe, it was transferred to 40 °C in an oil bath. After the POEMA reaction was completed, monomer OPMA (0.96g, 6.75mmol) was added to continue the reaction for 15h, and then monomer PEGMA ( _Mn =475g/mol, 5.33g, 11.25mmol) was added to continue the reaction for 48h. After the reaction was completed, the eggplant flask was cooled to room temperature and THF was added to terminate the reaction, then passed through a neutral alumina column (THF as eluent), concentrated by rotary evaporation, and slowly added dropwise to ten times the amount of n-hexane for precipitation.

During the reaction, the apparent viscosity did not change significantly, and no precipitation occurred after being added dropwise to n-hexane, indicating that no polymer was formed. The structure of the product was characterized by ¹ H NMR, and a peak appeared at a chemical shift of 5-6 ppm, indicating that there were still a large number of monomers containing carbon-carbon double bonds, and the polymerization reaction did not occur or the degree of polymerization was very low.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. A pH-responsive cross-linked bond-based hydrophilic side chain-tunable triblock polymer characterized by the following structural formula:

wherein x is 15-35, y is 5-20, z is 15-35, and n is 5-20.

2. The method for preparing the triblock polymer with adjustable hydrophilic side chains based on the pH response crosslinking bond as claimed in claim 1 is characterized by comprising the following steps:

(1) dissolving pyridine chlorochromate and silica gel powder in a solvent, adding 2-hydroxypropyl methacrylate under an ice bath condition, and continuing to react at room temperature to obtain a keto-functionalized monomer 2-oxopropyl methacrylate;

(2) dissolving a catalyst, monomer methacrylic acid-2-phenoxyethyl ester and ligand 1,1,4,7,10, 10-hexamethyl triethylene tetramine in a solvent, fully stirring, adding a reducing agent, uniformly stirring, adding a small molecular initiator, heating for reaction, adding the ketone group functionalized monomer methacrylic acid-2-oxopropyl ester prepared in the step (1) for continuous reaction after the monomer is completely converted, and adding monomer monomethoxy polyethylene glycol methacrylate for continuous reaction after the monomer is completely converted to obtain the amphiphilic triblock polymer.

3. The method for preparing a triblock polymer with tunable hydrophilic side chains based on pH-responsive cross-links according to claim 2, wherein the solvent in the step (2) is at least one of anisole and N, N-dimethylformamide; the molar volume ratio of the monomer methacrylic acid-2-phenoxyethyl ester to the solvent is 0.52-2.09 mmol/mL.

4. The method for preparing a triblock polymer with adjustable hydrophilic side chains based on pH response cross-linking bonds according to claim 2, wherein the molar ratio of 2-hydroxypropyl methacrylate to pyridinium chlorochromate in the step (1) is 1: 1.2-2;

the molar ratio of the catalyst in the step (2), 2-phenoxyethyl methacrylate, 1,4,7,10, 10-hexamethyltriethylene tetramine, a reducing agent, a small molecular initiator, 2-oxopropyl methacrylate and monomethoxy polyethylene glycol methacrylate is 0.04-0.06: 15-35: 0.4-0.6: 0.4-0.6: 1: 5-20: 15 to 35.

5. The preparation method of the triblock polymer with the adjustable hydrophilic side chains based on the pH response cross-links as claimed in claim 2, wherein the reaction time of the step (1) at room temperature is 12-18 h;

heating the monomer 2-phenoxyethyl methacrylate in the step (2) to react at 40-50 ℃ for 4-10 h; the reaction temperature of the 2-oxopropyl methacrylate is 40-50 ℃, and the reaction time is 12-18 h; the reaction temperature of the monomethoxy polyethylene glycol methacrylate is 60-70 ℃, and the reaction time is 36-72 hours.

6. The method for preparing a triblock polymer with tunable hydrophilic side chains based on pH response cross-linking bonds according to claim 2, wherein the solvent in the step (1) is dichloromethane; the molar volume ratio of the pyridinium chlorochromate to the solvent is 0.3-0.5 mmol/mL;

the mass ratio of the silica gel powder to the pyridinium chlorochromate in the step (1) is 1: 1;

the catalyst in the step (2) is at least one of copper bromide and copper chloride; the reducing agent is at least one of stannous octoate and ascorbic acid; the micromolecular initiator is ethyl bromoisobutyrate;

the reactions in the steps (1) to (2) are carried out under the protection of inert gas and under anhydrous conditions.

7. The application of the triblock polymer with the adjustable hydrophilic side chain based on the pH-responsive cross-linked bond as claimed in claim 1 in loading of a poorly water-soluble drug is characterized in that the triblock polymer with the adjustable hydrophilic side chain based on the pH-responsive cross-linked bond as claimed in claim 1 and the poorly water-soluble drug are dissolved in a solvent, after uniform mixing, the mixture is dialyzed in a phosphate buffer solution, then a catalyst and a cross-linking agent are added, stirring reaction is carried out at room temperature, and dialysis is carried out with deionized water, so that a pH-responsive cross-linked micelle system loading the poorly water-soluble drug is obtained.

8. The application of the pH-responsive cross-linked hydrophilic side chain-tunable triblock polymer in loading of a poorly water-soluble drug is characterized in that the mass ratio of the pH-responsive cross-linked hydrophilic side chain-tunable triblock polymer to the poorly water-soluble drug is 2-10: 1;

the water-insoluble medicine is a water-insoluble anticancer medicine.

9. The preparation method of the triblock polymer with adjustable hydrophilic side chain based on the pH response cross-linking bond as claimed in claim 2, wherein the catalyst is 2-amino-5-methoxybenzoic acid; the cross-linking agent is adipic acid dihydrazide; the molar ratio of the pH response crosslinking bond-based hydrophilic side chain-adjustable triblock polymer to the crosslinking agent is 2: 1; the concentration of the catalyst in a micelle solution obtained after dialysis in a phosphate buffer salt solution is 8-12 mmol/L;

the stirring reaction at room temperature is carried out for 20-30 h;

the water-insoluble drug is at least one of deacidified adriamycin and paclitaxel.

10. The use of the triblock polymer with tunable hydrophilic side chains based on pH-responsive crosslinks for loading poorly water soluble drugs as claimed in claim 7, wherein the phosphate buffered saline solution has a pH of 6.5; the dialysis time in phosphate buffer saline solution is 18-30 h, and the dialysate is replaced once every 2-6 h;

the time of dialysis by deionized water is 10-24 h, and the dialysate is replaced once every 2 h;

the solvent is dimethyl sulfoxide; the mass-to-volume ratio of the hydrophilic side chain-tunable triblock polymer based on pH response cross-links to the solvent is 4 mg/mL.

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