A pH and reduction dual-responsive polymer cross-linked micelle and a preparation method thereof

Abstract

The present application relates to the technical field of polymer micelles, and particularly relates to a pH and reduction dual-responsive star polymer cross-linked micelle and a preparation method thereof.The pH and reduction dual-responsive star polymer cross-linked micelle takes pentaerythritol as a core, and other block structures include polyethylene glycol monomethyl ether, a dynamic imine bond, gamma-amino-epsilon-caprolactone, epsilon-caprolactone and a disulfide bond.After loading an anticancer drug doxorubicin, the cross-linked micelle can stably exist under a neutral condition of a human body normal tissue, and can slowly release the drug under a micro-acidic and high glutathione environment of a tumor cell.Compared with a non-cross-linked micelle, the stability of the cross-linked micelle under a neutral condition is obviously improved, and the cross-linked micelle can slowly release the drug under a tumor environment.

Description

Technical Field

[0001] This invention relates to the field of polymer micelle technology, and in particular to a pH- and reduction-responsive star-shaped polymer crosslinked micelle and its preparation method. Background Technology

[0002] Cancer poses a serious threat to human health and is one of the leading causes of death. Studies have found that the microenvironment of tumor cells differs significantly from that of normal cells. For example, the pH of the tumor cell microenvironment is lower than that of normal cells; specifically, the pH of the tumor cell microenvironment is 5.0–6.0, while the pH of normal cells is 7.4. Furthermore, research has shown that the concentration of glutathione in tumor cells is several times higher than in normal tissue cells, resulting in a higher reduction response in tumor cells compared to normal cells. Therefore, the characteristics that distinguish tumor cells from normal cells—high reduction responsiveness and slightly acidic environment—provide a theoretical basis for researchers to construct drug carriers for cancer treatment.

[0003] Micelles possess inherent advantages in loading anticancer drugs. Composed of amphiphilic polymers, polymer micelles spontaneously assemble into a shell-core structure with a hydrophilic exterior and a hydrophobic interior in an aqueous environment. The hydrophobic blocks encapsulate poorly soluble drugs, releasing them at the target site; while the hydrophilic blocks encapsulate the hydrophobic core, reducing the overall interaction between the drug-loaded micelle and the bloodstream, thus improving drug targeting and in vivo bioavailability. By leveraging the characteristics of these two blocks, responsive polymer micelles can be designed, such as by incorporating pH-responsive blocks and introducing cross-linking sites. This allows for targeted delivery of anticancer drugs while ensuring the stability of the cross-linked micelles in the bloodstream, releasing drugs at tumor cells. Although the successful market launch of various polymer micelles indicates a significant milestone in anticancer drug delivery research, improvements in the stability and controlled drug release performance of drug-loaded micelles are still needed. Summary of the Invention

[0004] One of the objectives of this invention is to overcome the shortcomings of the prior art by providing a pH- and reduction-responsive star polymer crosslinked micelle and its preparation method, which can produce drug-loaded micelles with good stability and precise controlled drug release.

[0005] The second objective of this invention is to provide a pH- and reduction-responsive star-shaped polymer crosslinked micelle.

[0006] To achieve one of the above objectives, the present invention provides the following technical solution:

[0007] The first aspect of this invention provides a pH- and reduction-responsive star-shaped polymer crosslinked micelle, wherein the polymer in the crosslinked micelle has the following structural formula:

[0008]

[0009] Where x = 5 to 30, y = 1 to 10, and m = 1 to 2.

[0010] Preferably, the number-average molecular weight of the pH- and reduction-responsive star-shaped polymer crosslinked micelles is 22288–71648 g / mol.

[0011] This invention provides a beneficial effect of pH- and reduction-responsive star-shaped polymer crosslinked micelles:

[0012] The pH- and reduction-responsive star-shaped polymer crosslinked micelles of the present invention include pentaerythritol, polyethylene glycol monomethyl ether, γ-amino-ε-caprolactone, ε-caprolactone, dynamic imine bonds, and disulfide bonds in their structure. Polyethylene glycol monomethyl ether has good water solubility, which can prolong the circulation time of micelles in a normal blood environment; ε-caprolactone is a hydrophobic block, which can improve the drug loading capacity of micelles for hydrophobic drugs; the amino group in γ-amino-ε-caprolactone acts as a crosslinking group, which can undergo a crosslinking reaction with a small molecule crosslinking agent containing disulfide bonds under slightly alkaline conditions to form a stable chemical crosslinking bond, improving the stability of the micelles; the disulfide bond acts as a reduction-responsive group, which breaks through a thiol-disulfide bond exchange reaction under the high glutathione reduction conditions of tumor tissue, thereby de-crosslinking the crosslinked micelles and allowing anticancer drugs to be released smoothly from the hydrophobic core; the dynamic imine bond and the primary amino group in γ-amino-ε-caprolactone act as pH-responsive groups, and the imine bond can be hydrolyzed and broken under the slightly acidic conditions of tumor tissue. Polyethylene glycol monomethyl ether (PEG) is hydrophilic and can serve as a hydrophilic shell for micelles, encapsulating the hydrophobic core and drug within. The removal of the hydrophilic block exposes the hydrophobic core, releasing some of the drug. By gradually removing the PEG PEG hydrophilic protective shell, the micelles can more easily enter tumor cells and partially release the drug. Meanwhile, the primary amino group in the uncrosslinked γ-amino-ε-caprolactone undergoes protonation, gradually changing from completely hydrophobic to partially hydrophilic, causing the hydrophobic core to swell and further releasing the drug loaded within it. Therefore, the pH- and reduction-responsive star-shaped polymer crosslinked micelles provided by this invention are stable in the microenvironment of normal cells, while in the acidic environment of tumor cells, as well as in environments with high glutathione concentrations or other high reduction responses and slightly acidic conditions, they can release the drug in a controlled manner. Compared with non-crosslinked micelles, the drug-loaded crosslinked micelles prepared in this invention, after being loaded with anticancer drugs, are stable under the neutral conditions of normal human tissues, and release drugs in a controlled manner under the acidic and high-glutathione environment of tumor cells. That is, the stability of crosslinked micelles under neutral conditions is significantly improved, and the characteristic of controlled drug release in the tumor environment is further enhanced. This avoids unnecessary drug release in non-tumor environments, but allows for controlled drug release in the tumor environment, thereby improving the drug utilization efficiency of drug-loaded micelles.

[0013] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0014] A method for preparing the above-mentioned pH- and reduction-responsive star-shaped polymer crosslinked micelles is provided, the method comprising the following steps:

[0015] After mixing polyethylene glycol monomethyl ether, 4-carboxybenzaldehyde, dehydrating agent, solvent and first catalyst, the mixture was evacuated and purged with nitrogen three times, and stirred at room temperature to carry out esterification reaction to obtain aldehyde-modified polyethylene glycol.

[0016] Under an argon atmosphere, pentaerythritol, ε-caprolactone, γ-(tert-butyl carbamate)-ε-caprolactone, solvent, and a second catalyst were mixed and subjected to a ring-opening polymerization reaction to obtain star-shaped polymer molecules.

[0017] The star-shaped polymer molecules were dissolved in dichloromethane with trifluoroacetic acid and then stirred to obtain a pH-responsive star-shaped polymer.

[0018] Under a nitrogen atmosphere, the pH-responsive star polymer and the aldehyde-modified polyethylene glycol were dissolved in dimethylformamide and subjected to a Schiff base reaction to obtain a pH-responsive star amphiphilic polymer.

[0019] pH-responsive star-shaped amphiphilic polymers, disulfide-containing small molecule crosslinking agents, and anticancer drugs were subjected to dialysis to obtain pH- and reduction-responsive star-shaped polymer drug-loaded crosslinked micelles.

[0020] The dehydrating agent is dicyclohexylcarbodiimide, the first catalyst is dimethylaminopyridine, the solvent is dichloromethane or dimethylformamide, and the esterification reaction time is 24 h;

[0021] In the preparation of aldehyde-modified polyethylene glycol, the solvent is toluene, the second catalyst is stannous isooctanoate, and the ring-opening polymerization reaction is carried out at a temperature of 130°C for 24 hours.

[0022] The Schiff base reaction time was 48 hours.

[0023] Preferably, the preparation of the pH- and reduction-responsive star-shaped polymer drug-loaded crosslinked micelles includes: weighing a pH-responsive star-shaped amphiphilic polymer, a small molecule crosslinking agent containing disulfide bonds, and a hydrophobic anticancer drug, dissolving them in an organic solvent at a ratio of 30-40:5-2:20-15, stirring for 4-48 hours, and then dialyzing with deionized water for 24-72 hours, changing the deionized water every 3-6 hours during this period.

[0024] Preferably, the hydrophobic anticancer drug is doxorubicin.

[0025] It should be noted that the pH- and reduction-responsive star-shaped polymer drug-loaded crosslinked micelles provided by the present invention include hydrophobic blocks, which are suitable for loading hydrophobic drugs and can solubilize hydrophobic anticancer drugs, thereby improving the drug loading capacity of the polymer crosslinked micelles.

[0026] Preferably, the Schiele reaction further includes: removing anhydrous sodium sulfate from the reaction solution through a needle filter and then adding it dropwise to anhydrous diethyl ether to precipitate the solution.

[0027] Preferably, the raw materials for preparing the aldehyde-modified polyethylene glycol, calculated by mass, include: 2-4 parts by mass of polyethylene glycol monomethyl ether, 0.55-1.22 parts by mass of 4-carboxybenzaldehyde, 0.22-0.41 parts by mass of dicyclohexylcarbodiimide, and 0.15-0.27 parts by mass of dimethylaminopyridine.

[0028] Preferably, before obtaining aldehyde-modified polyethylene glycol, the process further includes: concentration, precipitation, filtration, and drying. The precipitation is achieved by dissolving the concentrated product in a small amount of dichloromethane and then transferring this solution into 10 times its volume of cold diethyl ether for precipitation.

[0029] Preferably, the pH-responsive star polymer comprises the following raw materials in parts by weight: 0.056–0.0681 parts pentaerythritol, 3.56–6.84 parts ε-caprolactone, 0.78–4.6 parts γ-(tert-butyl carbamate)-ε-caprolactone, 0.00025–0.00087 parts stannous isooctanoate, and 6.55–7.68 parts trifluoroacetic acid.

[0030] Preferably, the pH-responsive star-shaped amphiphilic polymer comprises the following parts by weight of raw materials: 0.08 to 0.12 parts by weight of pH-responsive star-shaped polymer and 0.03 to 0.16 parts by weight of aldehyde-modified polyethylene glycol.

[0031] Preferably, the pH and reduction dual-responsive star polymer micelles comprise the following raw materials in parts by weight: 0.03 to 0.06 parts by weight of pH-responsive star amphiphilic polymer, and 0.002 to 0.005 parts by weight of small molecule crosslinking agent containing disulfide bonds.

[0032] Before obtaining star-shaped polymer molecules, the process also includes: concentration, precipitation, filtration, and drying;

[0033] The precipitation is achieved by dissolving the concentrated product in a small amount of dichloromethane, and then transferring this solution into 10 times its volume of cold diethyl ether for precipitation.

[0034] Before obtaining the pH-responsive star polymer, the process also includes: concentration, precipitation, filtration, and drying;

[0035] The precipitation is achieved by dissolving the concentrated product in a small amount of dichloromethane, and then transferring this solution into 10 times its volume of cold diethyl ether for precipitation.

[0036] Preferably, the preparation method of γ-(tert-butyl carbamate)-ε-caprolactone includes: dissolving 4-(tert-butyloxycarbonylamino)cyclohexanone and 3-chloroperoxybenzoic acid in dichloromethane and reacting at 50°C for 15 h to obtain γ-(tert-butyl carbamate)-ε-caprolactone.

[0037] Preferably, the γ-(tert-butyl carbamate)-ε-caprolactone comprises the following raw materials in parts by weight: 20.8 to 21.3 parts by weight of 4-(tert-butyloxycarbonylamino)cyclohexanone, and 19.7 to 20.6 parts by weight of 3-chloroperoxybenzoic acid.

[0038] Preferably, the preparation method of the disulfide-containing small molecule crosslinking agent includes: dissolving 3,3-dithiodipropionic acid in dichloromethane, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and stirring at 25°C for 2 hours. After concentration, purification is performed by silica gel column chromatography.

[0039] Preferably, the eluent for the silica gel chromatography column is ethyl acetate / n-hexane = 1:5.

[0040] Preferably, the disulfide-containing small molecule crosslinking agent comprises the following raw materials in parts by weight: 1 to 3 parts by weight of 3,3-dithiodipropionic acid, 2.16 to 3.44 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1.21 to 3.68 parts by weight of N-hydroxysuccinimide.

[0041] Preferably, after the reaction is complete, the process further includes concentrating and purifying small molecule crosslinking agents containing disulfide bonds;

[0042] Preferably, the concentration includes: subjecting the reaction solution to rotary evaporation under reduced pressure to remove the organic solvent;

[0043] The purification includes: purification by silica gel column chromatography with ethyl acetate / n-hexane as the eluent in a ratio of 1:5.

[0044] The present invention provides a method for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles with the following advantages:

[0045] The preparation method of this invention makes full use of the chemical properties of the reactants, effectively preparing pH and reduction dual-responsive star polymer crosslinked micelles. It has strong operational repeatability and is suitable for large-scale production applications. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 The synthetic route diagram for γ-(tert-butyl carbamate)-ε-caprolactone;

[0048] Figure 2 A flowchart illustrating the synthetic process of small molecule crosslinking agents containing disulfide bonds;

[0049] Figure 3 A flowchart illustrating the synthetic process of terminally aldehyde-modified polyethylene glycol;

[0050] Figure 4 Synthesis process route diagram for pH and reduction dual-responsive star polymer crosslinked micelles;

[0051] Figure 5 The 1H NMR spectrum of γ-(tert-butyl carbamate)-ε-caprolactone in Example 1;

[0052] Figure 6 The 1H NMR spectrum of the terminal aldehyde-terminated polyethylene glycol in Example 2;

[0053] Figure 7 The 1H NMR spectrum of the disulfide-containing small molecule crosslinking agent in Example 3;

[0054] Figure 8 The image shows the hydrogen nuclear magnetic resonance spectrum of the star-shaped polymer molecule in step 1 of Example 4;

[0055] Figure 9 The 1H NMR spectrum of the pH-responsive star-shaped amphiphilic polymer in step 3 of Example 4;

[0056] Figure 10 The above is the 1H NMR spectrum of the pH- and reduction-responsive star polymer crosslinked micelles in step 4 of Example 4.

[0057] Figure 11 The graph shows the particle size variation over time of the pH and reduction dual-response blank cross-linked micelles and the pH-response blank non-cross-linked micelles in Example 7.

[0058] Figure 12 Transmission electron microscopy images of pH- and reduction-responsive drug-loaded crosslinked micelles and pH-responsive non-crosslinked drug-loaded micelles in Example 8;

[0059] Figure 13This is an in vitro release curve of pH- and reduction-responsive drug-loaded cross-linked micelles and pH-responsive non-cross-linked drug-loaded micelles in Example 9. Detailed Implementation

[0060] To make the objectives, features, and advantages of this invention more apparent and understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the embodiments described below are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0061] Example 1

[0062] Example 1 provides a preparation process for γ-(tert-butyl carbamate)-ε-caprolactone. Figure 1 As shown, the steps include:

[0063] 4-(tert-butyloxycarbonylamino)cyclohexanone (21.3 g, 0.1 mol) and 3-chloroperoxybenzoic acid (20.6 g, 0.12 mol) were dissolved in anhydrous dichloromethane (100 mL), and the mixture was refluxed at 50 °C for 15 h. After the reaction was completed, the dichloromethane was rotary evaporated, and the concentrated product was added dropwise to a volume of ten times its volume of cold diethyl ether to precipitate. The product was filtered and dried under vacuum at 30 °C for 24 h to obtain γ-(tert-butyl carbamate)-ε-caprolactone.

[0064] The 1H NMR spectrum of γ-(tert-butyl carbamate)-ε-caprolactone was used for characterization. Figure 5 This indicates that γ-(tert-butyl carbamate)-ε-caprolactone can be prepared.

[0065] Example 2

[0066] Example 2 provides a process for preparing aldehyde-modified polyethylene glycol. Figure 2 As shown, the steps include:

[0067] Polyethylene glycol monomethyl ether (4 g, 2 mmol) was dissolved in anhydrous dichloromethane (40 mL), and dicyclohexylcarbodiimide (0.41 g, 2 mmol) and dimethylaminopyridine (0.27 g, 2 mmol) were added to the above solution. 4-Carboxybenzaldehyde (1.22 g, 10 mmol) was dissolved in dimethylformamide (5 mL), and after complete dissolution, it was added to the above solution. The mixture was evacuated and purged with nitrogen three times, then sealed in a container and stirred at room temperature for 48 hours. The solution was concentrated to 10 mL by rotary evaporation, stored at low temperature, filtered, and the filtrate was added dropwise to 500 mL of diethyl ether solution. After stirring for 15 minutes, the residue was filtered and dissolved in 10 mL of dimethylformamide. The residue was then poured into a 1000 Da dialysis bag and dialyzed for three days. The dialyzed liquid was freeze-dried to obtain aldehyde-modified polyethylene glycol.

[0068] The aldehyde-modified polyethylene glycol was characterized by proton NMR spectroscopy. Figure 6 This indicates that aldehyde-modified polyethylene glycol can be prepared.

[0069] Example 3

[0070] Example 3 provides a preparation process for a small molecule crosslinking agent containing disulfide bonds. Figure 3 As shown, the steps include:

[0071] 3,3-Dithiodipropionic acid (1 g, 4.75 mmol) was added to 20 mL of dichloromethane and stirred vigorously until completely dissolved. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (2.16 g, 10.5 mmol) and N-hydroxysuccinimide (1.21 g, 10.5 mmol) were added to the solution. The mixture was stirred at 25 °C for 2 hours, concentrated by rotary evaporation, and then purified by silica gel column chromatography with ethyl acetate / n-hexane as the eluent at a ratio of 1:5. After complete separation, a small molecule crosslinking agent containing disulfide bonds was obtained.

[0072] The small molecule crosslinking agents containing disulfide bonds were characterized by proton NMR spectroscopy. Figure 7 This indicates that small molecule crosslinking agents containing disulfide bonds can be prepared.

[0073] Example 4

[0074] Example 4 provides a process for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles. Figure 4 As shown, the steps include:

[0075] Step 1: Under an argon atmosphere, pentaerythritol (0.0681 g, 1 mmol), ε-caprolactone (4.55 g, 40 mmol), and γ-(tert-butyl carbamate)-ε-caprolactone (0.92 g, 4 mmol) were added to a flask. The flask was placed in a 50°C oil bath, stirred, and evacuated for 2 hours. 1 mL of toluene containing stannous isooctanoate (0.1 wt% of the monomer) was added using a syringe. Toluene was removed by repeated purging and degassing. The flask was then vacuum-sealed and magnetically stirred in a 130°C oil bath for 24 hours. The flask was rapidly cooled to room temperature to terminate the polymerization. The copolymer was dissolved in 10 mL of dichloromethane and added dropwise to 500 mL of cold diethyl ether to precipitate the copolymer. The precipitate was filtered and dried to constant weight in an empty drying oven to obtain star-shaped polymer molecules.

[0076] The star-shaped polymer molecules were characterized by proton nuclear magnetic resonance spectroscopy. Figure 8 This indicates that star-shaped polymer molecules can be prepared.

[0077] Step 2: Dissolve the star-shaped polymer prepared in Step 1 in 5 mL of dichloromethane and stir for 15 minutes until fully dissolved. Add 5 mL of trifluoroacetic acid and stir vigorously at 0 °C for 2 hours. Terminate the reaction when no bubbles are generated. After removing the solvent by rotary evaporation, add 5 mL of dichloromethane and 1 mL of triethylamine and stir vigorously for 1 hour. After the solution becomes clear, precipitate the mixture dropwise into cold diethyl ether, filter, and vacuum dry for 24 hours to obtain the pH-responsive star-shaped polymer.

[0078] Step 3: Add the pH-responsive star polymer (0.12 g) and the aldehyde-modified polyethylene glycol (0.08 g) prepared in Example 2 to a nitrogen-filled Stokes flask. Add 5 mL of anhydrous dimethylformamide to dissolve the polymer completely. Add a small amount of anhydrous sodium sulfate to absorb the trace amounts of water generated during the reaction. Seal the container and react for 48 h. After removing the anhydrous sodium sulfate through a needle filter, add the polymer dropwise to anhydrous diethyl ether to precipitate the product. Vacuum dry the precipitate for 24 h to obtain the pH-responsive star amphiphilic polymer. Characterize the pH-responsive star amphiphilic polymer by proton NMR spectroscopy. Figure 9 This indicates that pH-responsive star-shaped amphiphilic polymers can be prepared.

[0079] Step 4: Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 3 in 1 mL of dimethyl sulfoxide, sonicate for 10 min to ensure complete dissolution, and slowly add it dropwise to 10 mL of distilled water at a rate of 10 seconds / drop. At this point, the solution will exhibit an opalescent appearance. Add another 20 mL of distilled water to stabilize the micelles, transfer the solution to a 1.5 kDa dialysis bag, and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, for a total of three changes, then change the dialysis medium every 12 h. After dialysis, transfer the solution from the dialysis bag to a beaker and adjust the pH to 9 with 0.1 mol / L NaOH solution. Weigh out 0.002 g (0.01 mmol) of the small molecule crosslinking agent and add it to a beaker. Maintain the pH at 9.0 and stir at room temperature for 48 h. Then, transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 volumes of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution through a 0.22 μm needle filter to obtain a crosslinked micelle solution. Freeze-dry the solution to obtain a crosslinked micelle powder.

[0080] Hydrogen nuclear magnetic resonance (HMR) spectroscopy was used to characterize the pH- and reduction-responsive star-shaped polymer crosslinked micelles. Figure 10 This indicates that pH-responsive star-shaped amphiphilic polymers can be prepared.

[0081] Example 5

[0082] Example 5 provides a process for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles, including the following steps:

[0083] Step 1: Under an argon atmosphere, pentaerythritol (0.0681 g, 1 mmol), ε-caprolactone (4.55 g, 40 mmol), and γ-(tert-butyl carbamate)-ε-caprolactone (2.76 g, 12 mmol) were added to a flask. The flask was placed in a 50°C oil bath, stirred, and evacuated for 2 hours. 1 mL of toluene containing stannous isooctanoate (0.1 wt% of the monomer) was added using a syringe. Toluene was removed by repeated purging and degassing. The flask was then vacuum-sealed and magnetically stirred in a 130°C oil bath for 24 hours. The flask was rapidly cooled to room temperature to terminate the polymerization. The copolymer was dissolved in 10 mL of dichloromethane and added dropwise to 500 mL of cold diethyl ether to precipitate the copolymer. The precipitate was filtered and dried to constant weight in an empty drying oven to obtain star-shaped polymer molecules.

[0084] Step 2: Dissolve the star-shaped polymer prepared in Step 1 in 5 mL of dichloromethane and stir for 15 minutes until fully dissolved. Add 5 mL of trifluoroacetic acid and stir vigorously at 0 °C for 2 hours. Terminate the reaction when no bubbles are generated. After removing the solvent by rotary evaporation, add 5 mL of dichloromethane and 1 mL of triethylamine and stir vigorously for 1 hour. After the solution becomes clear, precipitate the mixture dropwise into cold diethyl ether, filter, and vacuum dry for 24 hours to obtain the pH-responsive star-shaped polymer.

[0085] Step 3: Add the pH-responsive star polymer (0.12 g) and the aldehyde-modified polyethylene glycol (0.08 g) prepared in Example 2 to a nitrogen-filled Stock flask, add 5 mL of anhydrous dimethylformamide to dissolve it completely, add a small amount of anhydrous sodium sulfate to absorb the trace amount of water generated in the reaction, seal the container and react for 48 h, remove the anhydrous sodium sulfate through a needle filter and then add it dropwise to anhydrous diethyl ether to precipitate, and vacuum dry the precipitate for 24 h to obtain the pH-responsive star amphiphilic polymer;

[0086] Step 4: Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 3 in 1 mL of dimethyl sulfoxide, sonicate for 10 min to ensure complete dissolution, and slowly add it dropwise to 10 mL of distilled water at a rate of 10 seconds / drop. At this point, the solution will exhibit an opalescent appearance. Add another 20 mL of distilled water to stabilize the micelles, transfer the solution to a 1.5 kDa dialysis bag, and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, for a total of three changes, then change the dialysis medium every 12 h. After dialysis, transfer the solution from the dialysis bag to a beaker and adjust the pH to 9 with 0.1 mol / L NaOH solution. Weigh out 0.002 g (0.01 mmol) of the small molecule crosslinking agent and add it to a beaker. Maintain the pH at 9.0 and stir at room temperature for 48 h. Then, transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 volumes of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution through a 0.22 μm needle filter to obtain a crosslinked micelle solution. Freeze-dry the solution to obtain a crosslinked micelle powder.

[0087] Example 6

[0088] Example 6 provides a process for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles, including the following steps:

[0089] Step 1: Under an argon atmosphere, pentaerythritol (0.0681 g, 1 mmol), ε-caprolactone (6.84 g, 4 mmol), and γ-(tert-butyl carbamate)-ε-caprolactone (4.6 g, 20 mmol) were added to a flask. The flask was placed in a 50°C oil bath, stirred, and evacuated for 2 hours. 1 mL of toluene containing stannous isooctanoate (0.1 wt% of the monomer) was added using a syringe. Toluene was removed by repeated purging and venting. The flask was then vacuum-sealed and magnetically stirred in a 130°C oil bath for 24 hours. The flask was rapidly cooled to room temperature to terminate the polymerization. The copolymer was dissolved in 10 mL of dichloromethane and added dropwise to 500 mL of cold diethyl ether to precipitate the copolymer. The precipitate was filtered and dried to constant weight in an empty drying oven to obtain star-shaped polymer molecules.

[0090] Step 2: Dissolve the star-shaped polymer prepared in Step 1 in 5 mL of dichloromethane and stir for 15 minutes until fully dissolved. Add 5 mL of trifluoroacetic acid and stir vigorously at 0 °C for 2 hours. Terminate the reaction when no bubbles are generated. After removing the solvent by rotary evaporation, add 5 mL of dichloromethane and 1 mL of triethylamine and stir vigorously for 1 hour. After the solution becomes clear, precipitate the mixture dropwise into cold diethyl ether, filter, and vacuum dry for 24 hours to obtain the pH-responsive star-shaped polymer.

[0091] Step 3: Add the pH-responsive star polymer (0.12 g) and the aldehyde-modified polyethylene glycol (0.16 g) prepared in Example 2 to a nitrogen-filled Stock flask, add 5 mL of anhydrous dimethylformamide to dissolve it completely, add a small amount of anhydrous sodium sulfate to absorb the trace amount of water generated in the reaction, seal the container and react for 48 h, remove the anhydrous sodium sulfate through a needle filter and then add it dropwise to anhydrous diethyl ether to precipitate, and vacuum dry the precipitate for 24 h to obtain the pH-responsive star amphiphilic polymer;

[0092] Step 4: Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 3 in 1 mL of dimethyl sulfoxide, sonicate for 10 min to ensure complete dissolution, and slowly add it dropwise to 10 mL of distilled water at a rate of 10 seconds / drop. At this point, the solution will exhibit an opalescent appearance. Add another 20 mL of distilled water to stabilize the micelles, transfer the solution to a 1.5 kDa dialysis bag, and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, for a total of three changes, then change the dialysis medium every 12 h. After dialysis, transfer the solution from the dialysis bag to a beaker and adjust the pH to 9 with 0.1 mol / L NaOH solution. Weigh out 0.002 g (0.01 mmol) of the small molecule crosslinking agent and add it to a beaker. Maintain the pH at 9.0 and stir at room temperature for 48 h. Then, transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 volumes of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution through a 0.22 μm needle filter to obtain a crosslinked micelle solution. Freeze-dry the solution to obtain a crosslinked micelle powder.

[0093] Example 7

[0094] Example 7 provides a process for preparing pH-responsive star-shaped amphiphilic polymer non-crosslinked blank micelles and pH / reduction dual-responsive star-shaped polymer crosslinked blank micelles, including the following steps:

[0095] Step 1: Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 2 of Example 4 in 1 mL of dimethyl sulfoxide and sonicate for 10 min until completely dissolved. Then, slowly add the solution dropwise to 10 mL of distilled water at a rate of 10 seconds / drop. At this point, the solution will exhibit an opalescent effect. Add another 20 mL of distilled water to stabilize the micelles. The mixed solution is then transferred to a 1.5 kDa dialysis bag and dialyzed with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution using a 0.22 μm syringe filter to obtain a blank micelle solution SM with a concentration of approximately 1 mg / mL.

[0096] Step 2: Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 2 of Example 4 in 1 mL of dimethyl sulfoxide. Sonicate for 10 min to ensure complete dissolution. Slowly add the solution dropwise to 10 mL of distilled water at a rate of 10 drops per second. The solution will exhibit an opalescent appearance. Add another 20 mL of distilled water to stabilize the micelles. Transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, repeating this process three times. After three changes, change the dialysis medium every 12 h. After dialysis, transfer the solution from the dialysis bag to a beaker and adjust the pH to 9 with 0.1 mol / L NaOH solution. Weigh out 0.002 g (0.01 mmol) of the small molecule crosslinking agent and add it to a beaker. Maintain the pH at 9.0 and stir at room temperature for 48 h. Then, transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 volumes of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution using a 0.22 μm needle filter to obtain the crosslinked micelle solution (CSM).

[0097] Step 3: The stability of the micelle solutions prepared in Step 1 and Step 2 is tested. The test steps include: using dynamic light scattering method to determine the particle size change of non-crosslinked blank micelles SM and crosslinked blank micelles CSM after 120 h of incubation in PBS buffer solution at pH 7.4.

[0098] The result is as follows Figure 11 As shown, the stability of pH and reduction dual-responsive star polymer micelles is improved after cross-linking, and the cross-linking method is beneficial for the stable existence of micelles in normal cells.

[0099] Example 8

[0100] Example 8 provides a process for preparing pH-responsive non-crosslinked drug-loaded micelles and pH and reduction-responsive crosslinked drug-loaded micelles.

[0101] Step 1: Add 15 mg of doxorubicin hydrochloride to 0.5 mL of dimethyl sulfoxide, followed by 0.1 mL of triethylamine and stirring in the dark for 1 h. Dissolve 30 mg of the pH-responsive star-shaped amphiphilic polymer prepared in Step 2 of Example 4 in 1 mL of dimethyl sulfoxide. After complete dissolution, mix with the doxorubicin solution. Stir in the dark for 12 h, then slowly add the mixture dropwise to 10 mL of distilled water at a rate of 10 seconds / drop, followed by adding 20 mL of distilled water to stabilize the micelles. Transfer the mixture to a 1.5 kDa dialysis bag and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, repeating this process three times, and then every 12 h. After dialysis, filter the solution using a 0.22 μm needle filter and freeze-dry to obtain non-crosslinked drug-loaded micelles.

[0102] Step 2: Dissolve 30 mg of the cross-linked micelle powder prepared in Example 4 in 1 mL of dimethyl sulfoxide to obtain a cross-linked micelle solution. Add 15 mg of doxorubicin hydrochloride to 1 mL of dimethyl sulfoxide, add 0.1 mL of triethylamine and stir in the dark for 1 h. Slowly add the triethylamine to the cross-linked micelle solution at a rate of 30 seconds / drop and stir in the dark for 12 h. Transfer the solution to a 1.5 kDa dialysis bag and dialyze with 100 times the volume of PBS solution for 48 h. Change the dialysis medium every 4 h, and after three changes, change the dialysis medium every 12 h. After dialysis, filter the solution using a 0.22 μm needle filter and freeze-dry to obtain cross-linked drug-loaded micelles.

[0103] Step 3: Morphology test of the micelle solution prepared in Step 1 and Step 2. The test steps include: adding one drop of micelle solution to the front side of a normal carbon film using a 10 μL pipette, letting it stand for 5 min, then using a pipette to remove the remaining liquid on the surface of the carbon film, letting it stand overnight at room temperature to fully evaporate the solvent, and using TEM to observe the micromorphology and particle size of the micelles in the dry state.

[0104] Morphological characteristics such as Figure 12 As shown, the particle sizes of the drug-loaded micelles before and after crosslinking are 90.4 nm and 80.2 nm, respectively, and both have a spherical morphology.

[0105] Example 9

[0106] Example 9 describes a drug release test on the pH- and reduction-responsive cross-linked drug-loaded micelles and the pH-responsive non-cross-linked drug-loaded micelles prepared in Example 8. The test includes the following steps:

[0107] Weigh out three 5 mg portions of non-crosslinked drug-loaded micelle powder and four 5 mg portions of crosslinked drug-loaded micelle powder. Dissolve the non-crosslinked samples in 5 mL of buffer solutions at pH 7.4, pH 7.4 / 10 mM DTT, and pH 5.0 / 10 mM DTT, respectively. Dissolve the crosslinked samples in 5 mL of buffer solutions at pH 7.4, pH 5.0, pH 7.4 / 10 mM DTT, and pH 5.0 / 10 mM DTT, respectively. After complete dissolution, transfer the solutions to a 3.5 kDa dialysis bag and immerse it in 95 mL of the corresponding buffer solution for drug release. Set the temperature and rotation speed to 37 °C and 100 rpm, respectively. At the set time intervals (0.5, 1, 2, 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 h), remove 5 mL of solution from the beaker and simultaneously add 5 mL of the corresponding buffer solution. The absorbance of the solution at 498 nm was determined by ultraviolet spectrophotometry, and the in vitro release curve was plotted.

[0108] The results can be found in [link to results]. Figure 13 ,from Figure 13It can be seen that in a normal blood environment (pH 7.4 / 0mM DTT), the drug release rate of both types of drug-loaded micelles is slow. However, the cumulative drug release of non-crosslinked micelles (SM) reaches 26.3% within 120 hours, while that of crosslinked micelles (CSM) is 17.3% within 120 hours. At pH 7.4 / 10mM DTT, the release curve of non-crosslinked micelles (SM) is similar to that under normal physiological conditions, with a cumulative drug release of 25.6% within 120 hours. The reduction response of crosslinked micelles (CSM) results in a faster initial drug release rate, which plateaus after 24 hours, with a cumulative drug release of 36.6% within 120 hours. At pH 5.0 / 0mM DTT, the crosslinked micelles (CSM) show a cumulative drug release of 58.2% within 120 hours because the internal disulfide bonds are not broken, hindering micelle swelling. At pH 5.0 / 10mM DTT... Under DTT conditions, both types of drug-loaded micelles exhibited rapid drug release rates. The non-crosslinked micelle (SM) showed a cumulative drug release of 82.6% within 120 hours, while the crosslinked micelle (CSM) showed a cumulative release of 76.1% within the same timeframe. These results are quite similar. This indicates that reversible nuclear crosslinking of drug-loaded micelles can improve drug leakage under normal physiological conditions, while not significantly hindering drug release in a tumor environment.

[0109] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles, characterized in that, Includes the following steps: After mixing polyethylene glycol monomethyl ether, 4-carboxybenzaldehyde, dehydrating agent, solvent and first catalyst, the mixture was evacuated and purged with nitrogen three times, and stirred at room temperature to carry out esterification reaction to obtain aldehyde-modified polyethylene glycol. Under an argon atmosphere, pentaerythritol, ε-caprolactone, γ-(tert-butyl carbamate)-ε-caprolactone, solvent, and a second catalyst were mixed and subjected to a ring-opening polymerization reaction to obtain star-shaped polymer molecules. The star-shaped polymer molecules were dissolved in dichloromethane with trifluoroacetic acid and then stirred to obtain a pH-responsive star-shaped polymer. Under a nitrogen atmosphere, the pH-responsive star polymer and the aldehyde-modified polyethylene glycol were dissolved in dimethylformamide and subjected to a Schiff base reaction to obtain a pH-responsive star amphiphilic polymer. pH-responsive star-shaped amphiphilic polymers, disulfide-containing small molecule crosslinking agents, and anticancer drugs were subjected to dialysis to obtain pH- and reduction-responsive star-shaped polymer drug-loaded crosslinked micelles. The dehydrating agent is dicyclohexylcarbodiimide, the first catalyst is dimethylaminopyridine, the solvent is dichloromethane or dimethylformamide, and the esterification reaction time is 24 h. In the preparation of aldehyde-modified polyethylene glycol, the solvent is toluene, the second catalyst is stannous isooctanoate, and the ring-opening polymerization reaction is carried out at a temperature of 130°C for 24 hours. The Schiff base reaction time was 48 hours. The preparation method of γ-(tert-butyl carbamate)-ε-caprolactone includes the following steps: dissolving 4-(tert-butyloxycarbonylamino)cyclohexanone and 3-chloroperoxybenzoic acid in dichloromethane and reacting at 50°C for 15 h to obtain γ-(tert-butyl carbamate)-ε-caprolactone. The preparation method of the disulfide-containing small molecule crosslinking agent includes the following steps: adding 3,3-dithiodipropionic acid to dichloromethane and stirring until dissolved; adding N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution and stirring at 25°C for 2 hours to obtain the disulfide-containing small molecule crosslinking agent. The aldehyde-modified polyethylene glycol comprises the following raw materials: 4g polyethylene glycol monomethyl ether, 1.22g 4-carboxybenzaldehyde, 0.41g dicyclohexylcarbodiimide, and 0.27g dimethylaminopyridine; The pH-responsive star polymer comprises the following raw materials: 0.0681g pentaerythritol, 6.84g ε-caprolactone, 4.6g γ-(tert-butyl carbamate)-ε-caprolactone, 1ml stannous isooctanoate, and 5ml trifluoroacetic acid. The pH-responsive star-shaped amphiphilic polymer comprises the following raw materials: 0.12 g pH-responsive star-shaped polymer, 0.16 g aldehyde-modified polyethylene glycol; the pH and reduction dual-responsive star-shaped polymer micelles comprise the following raw materials: 0.03 g pH-responsive star-shaped amphiphilic polymer, 0.002 g disulfide-containing small molecule crosslinking agent.

2. The method for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles according to claim 1, characterized in that, The anticancer drug is a hydrophobic anticancer drug; the hydrophobic anticancer drug is doxorubicin.

3. The method for preparing pH- and reduction-responsive star-shaped polymer crosslinked micelles according to claim 1, characterized in that, The number-average molecular weight of the pH- and reduction-responsive star polymer is 22288–71648 g / mol.

4. A pH- and reduction-responsive star-shaped polymer crosslinked micelle, characterized in that, It is prepared by cross-linking micelles of pH- and reduction-responsive star polymers as described in any one of claims 1-3.

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