Folic acid modified pH / reduction dual-response selenium-containing mixed polymer cross-linked micelle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polymer micelles, and discloses a folic acid modified pH / reduction dual-response selenium-containing mixed polymer cross-linked micelle as well as a preparation method and application thereof. The selenium-containing mixed polymer cross-linked micelle takes polycaprolactone (PCL) and methacrylic acid N, N-diethylaminoethyl ester (DEAEMA) as a core, and other block structures comprise polyethylene glycol (PEG), polyethylene glycol monomethyl ether monomethacrylate (PEGMA), hydroxyethyl methylacrylate (PHEM), folic acid (FA) and selenium caprylic acid (SA). After an anti-cancer drug adriamycin is loaded, the selenium-containing mixed polymer cross-linked micelle can stably exist in a neutral environment of normal tissues of a human body, has less drug leakage, can be actively recognized by tumor cells at a tumor part, is accumulated and endocytosed, and smoothly releases the drug; the technical problems that in the prior art, the stability, the drug controlled release and the targeting performance of polymer drug-loaded micelles still need to be improved are solved to a certain extent.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer micelles, and particularly relates to a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle, and a preparation method and application thereof. Background Art

[0002] Cancer is a serious threat to human health and is one of the main causes of human death. However, traditional chemotherapy drugs have always had limitations in clinical application and efficacy, such as poor solubility, significant toxic side effects, easy degradation, poor pharmacokinetic properties, and lack of targeting selectivity for specific tissues during drug transport in the body, which leads to the need for high-dose, continuous infusion administration, which kills cancer cells and a large number of normal cells at the same time. To solve the above limitations, drug delivery systems (DDS) can select appropriate carrier materials and dosage forms based on the properties of drug molecules, and achieve accurate and convenient drug delivery technology through reasonable design to deliver drugs to the body to achieve optimal bioavailability and therapeutic effects. In order to improve the utilization of drugs in the body and reduce toxic side effects, the development of excellent DDS has become a research hotspot in medicine and pharmacy.

[0003] Smart responsive nano polymer drug-loaded micelles can flexibly carry anti-tumor drugs to overcome physiological and pathological barriers, complete continuous targeted delivery and controlled release, and achieve the effect of reducing toxicity and enhancing the efficacy of anti-tumor drugs. It is one of the key research and development directions of the current new drug delivery system. It uses amphiphilic polymers to self-assemble in solution to form core / shell structured nanoparticles. The hydrophobic core can encapsulate hydrophobic drugs and improve drug solubility; the hydrophilic shell stabilizes and protects the micelles, which can reduce the toxic and side effects of drugs in normal human tissues and avoid phagocytosis of the reticuloendothelial system or absorption and clearance by tissues such as the liver, spleen, and kidneys. Functional modification of polymer micelles, such as introducing stimulus-responsive groups or modifying targeting ligands, can be used to make use of the differences between the normal human cell microenvironment (pH≈7.4, 36-37℃, low GSH) and the tumor tissue microenvironment (pH≈5.0-6.5, 37-42℃, high GSH, overexpression of proteins / enzymes / receptors, etc.), which can give them the characteristics of smart response and active targeting, and achieve effective targeted delivery and controlled release of anti-cancer drugs.

[0004] As a hydrophobic drug delivery system with excellent application prospects, smart responsive polymer micelles are easy to functionalize on their surfaces, which can effectively improve the bioavailability of hydrophobic drugs and reduce drug toxicity and side effects. However, despite the great progress made in the delivery of anticancer drugs by various polymer micelles, this smart drug delivery system still has the following shortcomings: (1) In terms of preparation technology, the single molecule structure type design, multifunctional blocks lead to long polymer chains, and the synthetic route is complicated; (2) In terms of controlled release performance, the carrier is not stable in the blood circulation, the efficiency of targeted precise release in the tumor environment is low, and the target environment response is slow. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies in the above-mentioned prior art, the primary purpose of the present invention is to provide a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles.

[0007] Another object of the present invention is to provide the application of the above-mentioned folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles. The selenium-containing mixed polymer cross-linked micelles are used as carriers of hydrophobic anticancer drugs, which solves the technical problems in the prior art that the stability of polymer drug-loaded micelles, drug controlled release, targeting performance and preparation technology still need to be improved.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle, wherein the mixed polymer cross-linked micelle is formed by mixed cross-linking self-assembly of a folic acid-modified polymer P1-SA and a polymer P2-SA with polycaprolactone as the core; the polymer P1-SA is FA-PEG-(PHEMA-g-SA)-PDEAEMA, and its structural formula is shown in formula (1), wherein X is a positive integer of 30 to 40, Y is a positive integer of 10 to 20, and Z is a positive integer of 30 to 40; the polymer P2-SA is PCL-(PHEMA-g-SA)-PEGMA, and its structural formula is shown in formula (2), wherein L is a positive integer of 50 to 70, and M and N are positive integers of 10 to 20:

[0010]

[0011] The number average molecular weight of the polymer P1-SA is 13500 to 20000 g / mol; the number average molecular weight of the polymer P2-SA is 13600 to 22500 g / mol.

[0012] The method for preparing the above-mentioned folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles comprises the following steps:

[0013] (1) In an argon atmosphere, polyethylene glycol, a solvent, and an acid-binding agent are mixed and then placed in an ice bath, 2-bromoisobutyryl bromide is added, and the mixture is stirred at low temperature to perform a bromination reaction to obtain brominated polyethylene glycol (PEG-iBuBr);

[0014] (2) mixing the brominated polyethylene glycol (PEG-iBuBr) obtained in step (1), folic acid (FA), a dehydrating agent, a solvent and a first catalyst, evacuating and filling with nitrogen three times, stirring at room temperature, performing an esterification reaction, and dialyzing against deionized water to obtain polyethylene glycol grafted with folic acid (FA-PEG-iBuBr);

[0015] (3) After mixing the polyethylene glycol grafted with folic acid (FA-PEG-iBuBr) obtained in step (2) and the second catalyst, evacuating and filling with nitrogen three times, adding a solvent, hydroxyethyl methacrylate, a ligand and a reducing agent, and performing an ARGET ATRP (electron transfer activation regeneration atom transfer radical polymerization) primary reaction, and then adding diethylaminoethyl methacrylate to perform an ARGET ATRP secondary reaction to obtain a pH-responsive polymer FA-PHEMA-PDEAEMA (P1);

[0016] (4) mixing the pH-responsive polymer FA-PHEMA-PDEAEMA obtained in step (3), a dehydrating agent, a solvent and a first catalyst, evacuating and filling with nitrogen three times, stirring at room temperature, adding selenoic acid (SA) for esterification, dialyzing with deionized water, filtering and freeze-drying to obtain a polymer FA-PEG-(PHEMA-g-SA)-PDEAEMA, namely, polymer P1-SA;

[0017] (5) Under an argon atmosphere, 1-butanol, ε-caprolactone, a solvent, and a catalyst were mixed and stirred at 130° C. for 24 h to perform a ROP ring-opening polymerization reaction to obtain polycaprolactone (PCL);

[0018] (6) Under an argon atmosphere, the polycaprolactone obtained in step (5), the solvent and the acid binding agent are mixed and then placed in an ice bath, 2-bromoisobutyryl bromide is added, and the mixture is stirred at low temperature to carry out a bromination reaction to obtain brominated polycaprolactone (PCL-iBuBr);

[0019] (7) After mixing the brominated polycaprolactone (PCL-iBuBr) obtained in step (6) and the second catalyst, evacuating and filling with nitrogen three times, adding a solvent, hydroxyethyl methacrylate, a ligand and a reducing agent, performing an ARGET ATRP primary reaction, and then adding polyethylene glycol monomethyl ether monomethacrylate to perform an ARGET ATRP secondary reaction to obtain a polymer PCL-PHEMA-PPEGMA (P2);

[0020] (8) mixing the polymer PCL-PHEMA-PPEGMA obtained in step (7), a dehydrating agent, a solvent and a first catalyst, evacuating and filling with nitrogen three times, stirring at room temperature, adding selenoic acid (SA) to carry out esterification reaction, dialyzing with deionized water, filtering and freeze-drying to obtain a polymer PCL-(PHEMA-g-SA)-PEGMA, i.e., a polymer P2-SA;

[0021] (9) dissolving the polymer P1-SA obtained in step (4) and the polymer P2-SA obtained in step (8) in a mass ratio of 1:1 in DMSO, and dialyzing in deionized water to obtain a blank mixed non-cross-linked micelle dialysate; filtering and freeze-drying the blank mixed non-cross-linked micelle dialysate to obtain a blank mixed non-cross-linked micelle powder;

[0022] (10) The blank mixed non-cross-linked micelle dialysate obtained in step (9) is adjusted to pH 8.5, and a reducing agent is added to cause the micelles to undergo self-cross-linking reaction, followed by continued dialysis to obtain a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle dialysate. The dialysate is filtered and freeze-dried to obtain a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle powder.

[0023] In step (1) and step (6), the acid binding agent is triethylamine, the solvent is tetrahydrofuran, the temperature of the bromination reaction is 0° C., and the time of the bromination reaction is 3 h;

[0024] In step (2), step (4) and step (8), the dehydrating agent is dicyclohexylcarbodiimide, the first catalyst is dimethylaminopyridine, the solvent is dimethyl sulfoxide, the esterification reaction time is 24 hours, and the deionized water dialysis time is 48 hours;

[0025] In step (3) and step (7), the second catalyst is copper bromide, the ligand is pentamethyldiethylenetriamine, the reducing agent is stannous isooctanoate, the time of the ARGET ATRP primary reaction is 6 hours, and the time of the ARGET ATRP secondary reaction is 8 hours; the temperature of the ARGET ATRP primary reaction and the ARGET ATRP secondary reaction is 70° C.;

[0026] The dialysis medium in step (10) is PBS solution, and the dialysis time is 48 hours;

[0027] In step (10), the reducing agent is dithiothreitol (DTT), the self-crosslinking reaction time is 24 hours, and the dialysis time is 48 hours.

[0028] After the ARGET ATRP secondary reaction in step (3) and the bromination reaction in step (6), concentration, precipitation, filtration and drying are performed to finally obtain the product; the precipitation is performed by dissolving the concentrated sample in a small amount of tetrahydrofuran, and then transferring the solution into 10 times the volume of ice n-hexane for precipitation.

[0029] The solvent in step (5) is tetrahydrofuran, and the catalyst is stannous isooctanoate.

[0030] The raw materials for preparing the polymer P1-SA in step (4) include: 5 parts by mass of pH-responsive polymer FA-PHEMA-PDEAEMA, 0.44 parts by mass of selenoic acid (SA), 0.41 parts by mass of a dehydrating agent, and 0.024 parts by mass of a first catalyst.

[0031] The raw materials for preparing the polymer P2-SA in step (8) include: 5 parts by mass of polymer PCL-PHEMA-PPEGMA, 0.44 parts by mass of selenoic acid (SA), 0.41 parts by mass of a dehydrating agent, and 0.024 parts by mass of a first catalyst.

[0032] The amount of the reducing agent used in step (10) is 10% of the total molar number of the polymer P1-SA obtained in step (4) and the polymer P2-SA obtained in step (8).

[0033] Preferably, the number average molecular weight of the polymer P1-SA is 14785 g / mol; the number average molecular weight of the polymer P2-SA is 18055 g / mol.

[0034] Preferably, in the step (1), after the bromination reaction, the step further comprises: washing with dilute hydrochloric acid, sodium bicarbonate and water in sequence, and adding dropwise into n-hexane for precipitation, to finally obtain brominated polyethylene glycol (PEG-iBuBr).

[0035] Preferably, in the step (1), after the bromination reaction, the steps further include: concentration, precipitation, filtration, and drying to finally obtain brominated polyethylene glycol (PEG-iBuBr); the precipitation is to dissolve the concentrated product in a small amount of dichloromethane, and then transfer the solution into 10 times the volume of n-hexane for precipitation.

[0036] The raw materials for preparing the brominated polyethylene glycol in step (1) include: 10 parts by mass of polyethylene glycol, 0.46 parts by mass of 2-bromoisobutyryl bromide, and 0.40 parts by mass of triethylamine.

[0037] The raw materials for preparing the folic acid-grafted polyethylene glycol (FA-PEG-iBuBr) in step (2) include: 5 parts by mass of PEG-iBuBr, 0.44 parts by mass of folic acid (Folic Acid), 0.41 parts by mass of a dehydrating agent, and 0.024 parts by mass of a first catalyst.

[0038] The raw materials for preparing the pH responsive polymer FA-PHEMA-PDEAEMA (P1) in step (3) include: 2.40 parts by mass of FA-PEG-iBuBr, 0.012 parts by mass of a second catalyst, 1.30 parts by mass of hydroxyethyl methacrylate, 0.086 parts by mass of pentamethyldiethylenetriamine, 0.20 parts by mass of a reducing agent, and 3.70 parts by mass of diethylaminoethyl methacrylate.

[0039] The raw materials for preparing the polycaprolactone (PCL) in step (5) include: 7.99 parts by mass of ε-caprolactone, 0.12 parts by mass of 1-butanol, and 0.08 parts by mass of a catalyst stannous isooctanoate.

[0040] The raw materials for preparing the brominated polycaprolactone (PCL-iBuBr) in step (6) include: 8 parts by mass of polycaprolactone, 0.23 parts by mass of 2-bromoisobutyryl bromide, and 0.20 parts by mass of an acid binding agent.

[0041] The raw materials for preparing the polymer PCL-PHEMA-PPEGMA (P2) in step (7) include: 8 parts by mass of brominated polycaprolactone (PCL-iBuBr), 0.012 parts by mass of a second catalyst, 1.30 parts by mass of hydroxyethyl methacrylate, 0.086 parts by mass of pentamethyldiethylenetriamine, 0.20 parts by mass of stannous isooctanoate, and 3.70 parts by mass of polyethylene glycol monomethyl ether monomethacrylate.

[0042] The above-mentioned folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles are used as drug carriers in the delivery of anticancer drugs.

[0043] It should be noted that the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles provided by the present invention have a low critical micelle concentration, can self-assemble to form polymer micelles in aqueous solution, and are used for encapsulating anticancer drugs.

[0044] Preferably, the anticancer drug is a hydrophobic anticancer drug.

[0045] The folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles provided by the present invention include hydrophobic blocks, are suitable for loading hydrophobic drugs, can solubilize hydrophobic anticancer drugs, and improve the drug loading capacity of polymer micelles.

[0046] The folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked drug-loaded micelles are prepared according to the following method: the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles and the hydrophobic anticancer drug are dissolved in an organic solvent at a mass ratio of 2:1, stirred for 4 to 12 hours, dialyzed with deionized water for 48 hours, replaced the deionized water every 4 hours, and replaced the deionized water every 12 hours after three changes, and then freeze-dried to obtain the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked drug-loaded micelles.

[0047] It should be noted that when the ratio of selenium-containing mixed polymer cross-linked micelles to hydrophobic anticancer drugs is 2:1, polymer drug-loaded micelles with uniform particle size distribution and capable of maintaining a high drug loading amount can be obtained.

[0048] In summary, the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles of the present invention are formed by self-assembly of polymers P1-SA and P2-SA, and the structure thereof includes polycaprolactone (PCL), N,N-diethylaminoethyl methacrylate (DEAEMA), polyethylene glycol (PEG), polyethylene glycol monomethyl ether monomethacrylate (PEGMA), hydroxyethyl methacrylate (PHEM), folic acid (FA) and selenoic acid (SA). Among them, PEG and PEGMA are hydrophilic blocks with good water solubility, which can prolong the circulation time of micelles in a normal blood environment; PCL and DEAEMA are hydrophobic blocks, which can improve the drug loading of micelles for hydrophobic drugs; the tertiary amino group in DEAEMA acts as a pH-responsive group, and under the slightly acidic conditions of tumor tissue, the tertiary amino group of DEAEMA is protonated, gradually changing from completely hydrophobic to partially hydrophilic, causing the hydrophobic core to swell, thereby releasing the drug loaded inside the hydrophobic core. The PHEM structure contains terminal hydroxyl groups, which can modify selenoic acid. The diselenide bond in the five-membered ring of selenooctanoic acid can be self-crosslinked by opening the ring using a trace amount of DTT; at the same time, the diselenide bond has reduction responsiveness and can be reduced and broken in the high GSH concentration environment of tumor tissue, thereby accelerating drug release. FA can be actively recognized by the folate receptors on the surface of tumor cells, and can provide active targeting of micelles. Therefore, the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles provided by the present invention application have active targeting and pH / reduction dual responsiveness at the same time, and its polymer preparation route is short and the synthesis difficulty is relatively low. The drug-loaded micelles prepared by the present invention application can be stably present and leak less drugs in normal intracellular microenvironment conditions, and can be actively recognized, accumulated and internalized by tumor cells at the tumor site, and smoothly release drugs, and have the advantages of non-immunity, non-antigenicity, non-toxicity and no pollution to most proteins, which can solve the technical problems that the stability of polymer drug-loaded micelles, drug controlled release, targeting performance and preparation technology in the prior art still need to be improved.

[0049] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0050] The present invention designs and synthesizes a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle, which has a uniform particle size distribution and a high drug loading capacity. In order to reduce the difficulty of synthesizing the multifunctional polymer carrier, the mixed micelle strategy is adopted to prepare the micelle. After the self-crosslinking is initiated by trace dithiothreitol to prepare the drug-loaded cross-linked micelle, the stability is significantly improved compared with the non-cross-linked drug-loaded micelle, and it also has active targeting and pH / reduction dual responsiveness. The drug-loaded cross-linked micelle can exist stably for a long time in a neutral environment with little drug leakage; and can be actively recognized, accumulated and internalized by tumor cells at the tumor site, and the drug is released smoothly. Therefore, the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle prepared by the present invention can solve the technical problems of poor stability, weak targeting and low drug controlled release ability of the current intelligent response polymer drug-loaded micelle, and reduce the difficulty of synthesizing the multifunctional carrier material. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The synthetic process route of polymer P1-SA is shown in FIG.

[0052] Figure 2 The synthetic process route of polymer P2-SA is shown in FIG.

[0053] Figure 3 are hydrogen NMR spectra of polymer P1 and polymer P1-SA in Example 3 and Example 4, wherein (a) is a hydrogen NMR spectrum of polymer P1, and (b) is a hydrogen NMR spectrum of polymer P1-SA;

[0054] Figure 4 is the gel permeation chromatogram of polymer P1 and polymer P1-SA in Example 3 and Example 4;

[0055] Figure 5 are hydrogen NMR spectra of polymer P2 and polymer P2-SA in Example 7 and Example 8, wherein (a) is a hydrogen NMR spectrum of polymer P2, and (b) is a hydrogen NMR spectrum of polymer P2-SA;

[0056] Figure 6 is the gel permeation chromatogram of polymer P2 and polymer P2-SA in Example 7 and Example 8;

[0057] Figure 7 is a graph showing the critical micelle concentration of the polymer in Example 9;

[0058] Figure 8 This is a graph showing the change in particle size over time of the blank micelles of the pH / reduction dual-responsive selenium-containing mixed polymer modified with folic acid before and after cross-linking in Example 10;

[0059] Fig. 9 is an electron micrograph of the drug-loaded cross-linked micelles in Example 11;

[0060] Fig.10 This is the drug release diagram of the drug-loaded cross-linked micelles in Example 12;

[0061] Fig.11 These are the toxicity graphs of the polymers and drug-loaded cross-linked micelles in Example 13, where (a) is the toxicity graph of blank micelles of each polymer, and (b) is the toxicity graph of polymer drug-loaded cross-linked micelles. DETAILED DESCRIPTION

[0062] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0063] Example 1

[0064] This embodiment 1 provides a preparation process of brominated polyethylene glycol (PEG-iBuBr), comprising the steps of:

[0065] Dissolve polyethylene glycol (5 g, 1 mmol) in anhydrous dichloromethane (30 mL), add triethylamine (0.1 g, 1 mmol) and stir the mixture at 0°C, add 2-bromoisobutyryl bromide (0.29 g, 1 mmol) and react for 3 h. After reaction, wash the mixture three times with dilute hydrochloric acid, sodium bicarbonate and water. Add the concentrated product dropwise into cold n-hexane with a volume ten times that of the product to precipitate, filter and dry in vacuo at 30°C for 24 h to obtain brominated polyethylene glycol (PEG-iBuBr).

[0066] Example 2

[0067] This Example 2 provides a preparation process of polyethylene glycol grafted with folic acid (FA-PEG-iBuBr), comprising the steps of:

[0068] Folic acid (0.4 g, 1 mmol) and dicyclohexylcarbodiimide (0.41 g, 2 mmol) were dissolved in dimethyl sulfoxide (30 mL). After activation for 24 h, PEG-iBuBr (5.1 g, 1 mmol) and dimethylaminopyridine (0.27 g, 2 mmol) obtained in Example 1 were added to the activated folic acid. The mixture was stirred at room temperature for esterification for 24 h, and dialyzed with deionized water for 48 h. The dialyzed liquid was freeze-dried to obtain the product, folic acid-grafted polyethylene glycol (FA-PEG-iBuBr).

[0069] Example 3

[0070] This Example 3 provides a preparation process of a pH responsive polymer P1, comprising the steps of:

[0071] Under argon atmosphere, polyethylene glycol grafted with folic acid (FA-PEG-iBuBr) (1.4 g, 1 mmol), copper bromide (4.55 g, 40 mmol) and toluene (30 mL) obtained in Example 2 were added to the bottle for dissolution. Hydroxyethyl methacrylate (2.6 g, 20 mmol) filtered by alumina and ligand pentamethyldiethyltriamine (0.17 g, 1 mmol) were added and stirred for 15 minutes, and then the reducing agent stannous isooctanoate (0.4 g, 1 mmol) was added with a syringe, and stirred in a 70°C oil bath for 6 hours, and then diethylaminoethyl methacrylate (7.4 g, 40 mmol) filtered by alumina was added, and stirred in a 70°C oil bath for 8 hours. The flask was quickly cooled to room temperature to terminate the polymerization. The copolymer was dissolved in 10 mL of tetrahydrofuran, passed through a 5 cm thick neutral alumina column, concentrated by rotary evaporation, and added dropwise to 500 ml of cold n-hexane for precipitation, suction filtered, and freeze-dried to obtain pH-responsive polymer P1 molecules. The molecular weight Mn of polymer P1 was 12497 by gel permeation chromatography.

[0072] Example 4

[0073] This embodiment 4 provides a preparation process of polymer P1-SA, comprising the steps of:

[0074] SA (1mmol, 0.3g), 40mL DCM were added to the dry reaction bottle, and after sealing, evacuated and argon was passed for 5min, and DCC (1.46mmol, 0.3g) pre-dissolved in DCM was added, and activated at room temperature in the dark for 24 hours, and the excess DCM was evaporated. 20mL DMSO, polymer P1 (0.1mmol, 1.4g) obtained in Example 3, and DMAP (0.02mmol, 0.0024g) were added to continue the reaction for 48 hours. After the reaction was completed, the dialysis bag was used and the deionized water was dialyzed for 48h. After the dialysis was completed, the dialyzate was passed through a 0.45μm aqueous phase filter membrane, and freeze-dried to obtain a selenoic acid polymer P1-SA. The molecular weight Mn of the polymer P1-SA characterized by gel permeation chromatography was 14785.

[0075] The synthetic route of polymer P1-SA is as follows Figure 1 The NMR and GPC characterizations of polymers P1 and P1-SA are shown in Figure 3 and Figure 4 As shown, it shows that polymers P1 and P1-SA were successfully synthesized.

[0076] Example 5

[0077] This embodiment 5 provides a preparation process of polycaprolactone (PCL), comprising the steps of:

[0078] Caprolactone (7.99 g, 70 mmol) and 1-butanol (0.114 g, 1 mmol) were dissolved in anhydrous tetrahydrofuran (30 mL), stannous isooctanoate (0.08 g, 0.1 mmol) was added and the mixture was stirred at 130 °C for 24 h. After the reaction, 20 mL of anhydrous tetrahydrofuran was added, and the mixture was cooled to room temperature. The white solid was dissolved by ultrasonication, and the product was added dropwise into cold n-hexane with a volume ten times that of the product to precipitate, filtered, and dried in vacuo at 30 °C for 24 h to obtain polycaprolactone (PCL).

[0079] Example 6

[0080] This Example 6 provides a preparation process of brominated polycaprolactone (PCL-iBuBr), comprising the steps of:

[0081] The polycaprolactone (7 g, 1 mmol) obtained in Example 5 was dissolved in anhydrous tetrahydrofuran (30 mL), triethylamine (0.1 g, 1 mmol) was added and the mixture was stirred at 0°C, 2-bromoisobutyryl bromide (0.29 g, 1 mmol) was added and the reaction was carried out for 3 h. After the reaction was completed, the product was passed through a 5 cm thick neutral alumina column, and the concentrated product was added dropwise into cold n-hexane with a volume ten times that of the product to precipitate, filtered, and vacuum dried at 30°C for 24 h to obtain brominated polycaprolactone (PCL-iBuBr).

[0082] Example 7

[0083] This embodiment 7 provides a preparation process of polymer P2, comprising the steps of:

[0084] Under argon atmosphere, the brominated polycaprolactone (PCL-iBuBr) (0.0681 g, 1 mmol), copper bromide (4.55 g, 40 mmol) and toluene (30 mL) obtained in Example 6 were added to a bottle and dissolved. Hydroxyethyl methacrylate (2.6 g, 20 mmol) filtered through alkaline alumina and ligand pentamethyldiethyltriamine (0.17 g, 1 mmol) were added and stirred for 15 minutes, and then the reducing agent stannous isooctanoate (0.4 g, 1 mmol) was added with a syringe, and stirred in a 70°C oil bath for 6 hours, and then polyethylene glycol monomethyl ether monomethacrylate (7.12 g, 15 mmol) filtered through alumina was added, and stirred in a 70°C oil bath for 8 hours, and the flask was quickly cooled to room temperature to terminate the polymerization. The copolymer was dissolved in 10 mL of tetrahydrofuran, passed through a 5 cm thick neutral alumina column, concentrated by rotary evaporation, and added dropwise to 500 ml of cold n-hexane for precipitation, suction filtered, and freeze-dried to obtain polymer P2 molecules. The molecular weight Mn of polymer P2 was 16053 as characterized by gel permeation chromatography.

[0085] Example 8

[0086] This Example 8 provides a preparation process of polymer P2-SA, comprising the steps of:

[0087] SA (1mmol, 0.3g) and 40mL DCM were added to the dry reaction bottle, and after sealing, evacuated and argon was passed for 5min, and DCC (1.46mmol, 0.3g) pre-dissolved in DCM was added, and activated at room temperature in the dark for 24 hours, and the excess DCM was evaporated. 20mL DMSO, P2 (0.1mmol, 1.7g) obtained in Example 7 and DMAP (0.02mmol, 0.0024g) were added and the reaction was continued for 48 hours. After the reaction was completed, the dialysis bag was used and dialyzed with deionized water for 72 hours. After the dialysis was completed, the dialyzate was passed through a 0.45μm aqueous phase filter membrane and freeze-dried to obtain the selenoctanoic acid polymer P2-SA. The molecular weight Mn of the polymer P2-SA characterized by gel permeation chromatography was 18055.

[0088] The synthetic route of polymer P2-SA is as follows Figure 2 The NMR and GPC characterizations of polymers P2 and P2-SA are shown in Figure 5 and Figure 6 As shown, it shows that polymers P1 and P1-SA were successfully synthesized.

[0089] Example 9

[0090] In this Example 9, the critical micelle concentration of the polymer P1-SA, polymer P2-SA and the mixture of P1-SA and P2-SA prepared in Example 4 and Example 8 was tested. The testing method was a fluorescent probe method, and the testing steps included:

[0091] Step 1: Prepare pyrene solution by dissolving pyrene in acetone to a concentration of 6×10 -5 M containing pyrene acetone solution;

[0092] Step 2: Accurately weigh 10 mg of polymer P1-SA, polymer P2-SA and a mixture of P1-SA and P2-SA (5 mg / 5 mg) prepared in Example 3 and Example 5, respectively, and dissolve them in 5 mL of acetone, and add them dropwise into 100 mL of deionized water. After evaporating the acetone, a 0.1 mg / mL solution is obtained, which is then diluted with deionized water to a series of concentrations (0.0001 to 0.1 mg / ml). Take 18 10 mL brown volumetric flasks, add 0.1 mL of pyrene solution to each, and then add the above polymer solutions of different concentrations to prepare sample solutions. The concentration of pyrene in the sample solution is 6×10 -7 M;

[0093] Step 3, fluorescence spectrum test: take 373nm as the emission wavelength, test the excitation spectrum of the sample solution in the range of 300-350nm, and take I 337 / I331 The ratio is plotted against the logarithm of the concentration, log C ( Figure 7 ), the obtained curve was segmentedly fitted, and the abscissa corresponding to the intersection of the two fitting trend lines was the critical micelle concentration value. The critical micelle concentrations of polymer P1-SA, polymer P2-SA and the mixture of P1-SA and P2-SA were measured to be 3.31 mg / L, 1.41 mg / L and 1.95 mg / L, respectively.

[0094] Example 10

[0095] This Example 10 provides a preparation process of pH / reduction dual-responsive selenium-containing mixed polymer cross-linked blank micelles modified with folic acid, comprising the steps of:

[0096] Step 1, weigh 15 mg of polymers P1-SA and P2-SA prepared in Example 4 and Example 8 respectively, mix and dissolve in 1 mL DMSO, ultrasonicate for 10 minutes to help dissolution, transfer to dialysis bag (MWCO1500) after complete dissolution, dialyze in 1 L deionized water for 48 hours, replace the dialysis medium every four hours, and after dialysis, filter the resulting solution through a water filter with a pore size of 0.22 μm to obtain blank mixed non-cross-linked micelles Mixed Micelle-SA (MMSA). Adjust the pH of the blank mixed non-cross-linked micelles Mixed Micelle-SA (MMSA) to 8.5, add 10% DTT of the molar number of diselenide bonds in the micelles to achieve cross-linking, and react at room temperature for 24 hours under a nitrogen atmosphere. After the reaction is completed, the cross-linked mixed micelle solution is placed back into the dialysis bag and dialyzed for 48 hours. After dialysis, filter with a 0.45 μm filter membrane to obtain blank mixed cross-linked micelles Mixed cross-linked micelles (MCLM).

[0097] Step 2, performing a stability test on the micelle solution obtained in step 1, the test steps comprising: using dynamic light scattering (DLS) to measure the particle size change of blank mixed non-cross-linked micelles MMSA and blank mixed cross-linked micelles MCLM after incubation in a neutral environment for 120 hours.

[0098] The results are as follows Figure 8 As shown, the stability of the pH / reduction dual-responsive selenium-containing polymer micelles after cross-linking is significantly improved, which is conducive to the stable existence of the micelles in normal cells.

[0099] Embodiment 11

[0100] This Example 11 is to load the MCLM prepared in Example 10 with anticancer drugs, and characterize the particle size distribution and morphology.

[0101] Anticancer drug loading includes: adding 15mg of doxorubicin hydrochloride to 0.5ml dimethyl sulfoxide, then adding 0.1ml TEA and stirring in dark for 1h. Weigh 30mg of MCLM micelles prepared in Example 10 respectively and dissolve in 0.5mL dimethyl sulfoxide, and mix with doxorubicin solution after complete dissolution. After stirring in dark for 12h, slowly drip into 10ml distilled water at a speed of 10 seconds / drop, and then pour 20ml distilled water to stabilize micelles. The mixed solution is then transferred to a 1500Da dialysis bag, dialyzed with 100 times the volume of PBS solution for 48h, and the dialysis medium is replaced every 4h. After changing three times, the dialysis medium is changed every 12h. After dialysis, the solution is filtered using a needle filter with a pore size of 0.22μm, and a drug-loaded micelle solution with a concentration of about 1mg / mL is finally obtained. After freeze drying, a mixed drug-loaded cross-linked micelle powder is obtained, which is a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle powder loaded with anticancer drugs.

[0102] Morphological characterization Fig. 9 As shown, the particle size and particle size distribution were measured by dynamic light scattering, and the average particle size of the pH / reduction dual-responsive mixed polymer cross-linked micelles modified with folic acid loaded with anticancer drugs was 117 nm by laser light scattering, and the particle size distribution was 0.327; TEM observation showed that the morphology was spherical.

[0103] Example 12

[0104] This Example 12 is to conduct a drug release test on the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles loaded with anticancer drugs prepared in Example 11. The test includes the following steps:

[0105] Accurately weigh four portions of 5mg of the folic acid-modified pH / reduction dual-response selenium-containing mixed polymer cross-linked micelle powder of the load anticancer drug prepared in Example 11, and dissolve them in 5mL of pH 7.4 / 0mM DTT, pH 5.0 / 0mM DTT, pH 7.4 / 10mM DTT, pH 5.0 / 10mM DTT buffer solution. After fully dissolving, the solution is transferred to a 3500Da dialysis bag and immersed in 95mL of the corresponding buffer solution for drug release, and the temperature and speed are set to be 37°C and 100rpm respectively. At the time interval set (0.5, 1, 2, 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120h), take out the solution in the 5mL beaker and supplement 5mL of the corresponding buffer solution. The absorbance of the solution taken at 503nm is measured using ultraviolet spectrophotometry to draw an in vitro release curve.

[0106] The results can be found in Fig.10 ,from Fig.10It can be seen that in the normal blood pH 7.4 / 0mM DTT environment, the drug release rate of the drug-loaded micelles is slow, with a cumulative release of 10% in 24 hours and 16.2% in 120 hours; while in the pH 5.0 / 10mM DTT environment, MCLM shows excellent pH and reduction response performance, with a cumulative drug release of 75.8% within 120 hours. The above experimental results show that the pH / reduction dual-responsive selenium-containing mixed polymer cross-linked drug-loaded micelles modified with folic acid have good stability and pH / reduction dual responsiveness, without burst release, and meet the requirements of sustained release.

[0107] Embodiment 13

[0108] This Example 13 is to test the cytotoxicity of the pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles modified with folic acid and loaded with anticancer drugs prepared in Example 11 and the cross-linked micelles modified with non-folic acid, and the test includes the following steps:

[0109] HepG2 cells were seeded into 96-well plates (1×10 4 / well), and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. Free DOX (0.1, 1, 2, 5, 10, 20 mg / L), polymer P1-SA obtained in Example 4, P2-SA obtained in Example 8, and blank mixed cross-linked micelles MCLM obtained in Example 10 (1, 5, 10, 100, 200, 300 mg / L), folic acid-loaded micelles MCLM (i.e., mixed drug-loaded cross-linked micelles obtained in Example 11), and folic acid-free drug-loaded micelles NMCLM (the preparation method of NMCLM is similar to that of folic acid-loaded micelles MCLM, except that folic acid is not modified when preparing the intermediate polymer P1-SA) (0.5, 5, 10, 25, 50, 100 mg / L, and its DOX content is equivalent to free DOX) were added and cultured for 48 hours. After removing the original culture medium, wash twice with PBS solution, and then replace the culture medium in the 96-well plate with 100 μL of fresh endothelial cell-specific culture medium. CCK-8 solution (10 μL) was added to each well. The well plate was then placed in an environment set at 37°C and containing 5% CO2 for 2 hours. After the incubation was completed, the absorbance of each well at a wavelength of 450 nm was measured, and the cell survival rate of each sample was calculated.

[0110] The results can be found in Fig.11 ,from Fig.11It can be seen that the cell survival rate of the blank micelles of pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles modified with folic acid is greater than 80%, which means that the polymer micelles are non-toxic drug carrier materials. The tumor cell inhibition rate of mixed drug-loaded cross-linked micelles (MCLM) containing folic acid is significantly higher than that of non-folic acid-modified drug-loaded cross-linked micelles (NMCLM), indicating that the pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles modified with folic acid are more easily actively recognized and internalized by tumor cells, showing obvious active targeting.

[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle, characterized in that: The mixed polymer cross-linked micelle is formed by mixed cross-linking self-assembly of a folic acid-modified polymer P1-SA and a polymer P2-SA with polycaprolactone as the core; the polymer P1-SA is FA-PEG-(PHEMA-g-SA)-PDEAEMA, and its structural formula is shown in formula (1), wherein X is a positive integer of 30 to 40, Y is a positive integer of 10 to 20, and Z is a positive integer of 30 to 40; the polymer P2-SA is PCL-(PHEMA-g-SA)-PEGMA, and its structural formula is shown in formula (2), wherein L is a positive integer of 50 to 70, and M and N are positive integers of 10 to 20:

2. The folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 1, characterized in that: The number average molecular weight of the polymer P1-SA is 13500 to 20000 g / mol; the number average molecular weight of the polymer P2-SA is 13600 to 22500 g / mol.

3. The method for preparing a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 1, characterized in that The following steps are involved: (1) In an argon atmosphere, polyethylene glycol, a solvent, and an acid-binding agent are mixed and then placed in an ice bath, 2-bromoisobutyryl bromide is added, and the mixture is stirred at low temperature to carry out a bromination reaction to obtain brominated polyethylene glycol; (2) mixing the brominated polyethylene glycol obtained in step (1), folic acid, a dehydrating agent, a solvent and a first catalyst, evacuating and filling with nitrogen three times, stirring at room temperature, performing an esterification reaction, and dialyzing against deionized water to obtain polyethylene glycol grafted with folic acid; (3) After mixing the polyethylene glycol grafted with folic acid obtained in step (2) and the second catalyst, evacuating and filling with nitrogen three times, adding a solvent, hydroxyethyl methacrylate, a ligand and a reducing agent, performing an ARGET ATRP primary reaction, and then adding diethylaminoethyl methacrylate to perform an ARGET ATRP secondary reaction to obtain a pH-responsive polymer FA-PHEMA-PDEAEMA; (4) mixing the pH-responsive polymer FA-PHEMA-PDEAEMA obtained in step (3), a dehydrating agent, a solvent and a first catalyst, evacuating and filling with nitrogen three times, stirring at room temperature, adding selenoic acid to carry out esterification reaction, dialyzing with deionized water, filtering and freeze-drying to obtain a polymer FA-PEG-(PHEMA-g-SA)-PDEAEMA, namely, polymer P1-SA; (5) Under an argon atmosphere, 1-butanol, ε-caprolactone, a solvent, and a catalyst were mixed, and stirred at 130° C. for 24 h to perform a ROP ring-opening polymerization reaction to obtain polycaprolactone; (6) Under an argon atmosphere, the polycaprolactone obtained in step (5), the solvent and the acid binding agent are mixed and then placed in an ice bath, 2-bromoisobutyryl bromide is added, and the mixture is stirred at low temperature to carry out a bromination reaction to obtain brominated polycaprolactone; (7) After mixing the brominated polycaprolactone obtained in step (6) and the second catalyst, evacuating and filling with nitrogen three times, adding a solvent, hydroxyethyl methacrylate, a ligand and a reducing agent, performing an ARGET ATRP primary reaction, and then adding polyethylene glycol monomethyl ether monomethacrylate to perform an ARGET ATRP secondary reaction to obtain a polymer PCL-PHEMA-PPEGMA; (8) The polymer PCL-PHEMA-PPEGMA obtained in step (7), a dehydrating agent, a solvent and a first catalyst are mixed, evacuated and filled with nitrogen three times, stirred at room temperature, selenoic acid is added for esterification reaction, dialyzed with deionized water, filtered and freeze-dried to obtain a polymer PCL-(PHEMA-g-SA)-PEGMA, i.e., polymer P2-SA; (9) dissolving the polymer P1-SA obtained in step (4) and the polymer P2-SA obtained in step (8) in a mass ratio of 1:1 in DMSO, and dialyzing in deionized water to obtain a blank mixed non-cross-linked micelle dialysate; filtering and freeze-drying the blank mixed non-cross-linked micelle dialysate to obtain a blank mixed non-cross-linked micelle powder; (10) adjusting the pH of the blank mixed non-cross-linked micelle dialysate obtained in step (9) to 8.5, adding a reducing agent to cause the micelles to undergo a self-cross-linking reaction, and then continuing the dialysis to obtain a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle dialysate, and filtering and freeze-drying the dialysate to obtain a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle powder; In step (1) and step (6), the acid binding agent is triethylamine, the solvent is tetrahydrofuran, the temperature of the bromination reaction is 0° C., and the time of the bromination reaction is 3 h; In step (2), step (4) and step (8), the dehydrating agent is dicyclohexylcarbodiimide, the first catalyst is dimethylaminopyridine, the solvent is dimethyl sulfoxide, the esterification reaction time is 24 hours, and the deionized water dialysis time is 48 hours; In step (3) and step (7), the second catalyst is copper bromide, the ligand is pentamethyldiethylenetriamine, the reducing agent is stannous isooctanoate, the time of the ARGET ATRP primary reaction is 6 hours, and the time of the ARGET ATRP secondary reaction is 8 hours; the temperature of the ARGET ATRP primary reaction and the ATRP ROP secondary reaction is 70°C; The dialysis medium in step (10) is PBS solution, and the dialysis time is 48 hours; In step (10), the reducing agent is dithiothreitol, the self-crosslinking reaction time is 24 hours, and the dialysis time is 48 hours.

4. The method for preparing a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 3, characterized in that: The raw materials for preparing the polymer P1-SA in step (4) include: 5 parts by mass of pH-responsive polymer FA-PHEMA-PDEAEMA, 0.44 parts by mass of selenoic acid (SA), 0.41 parts by mass of a dehydrating agent, and 0.024 parts by mass of a first catalyst.

5. The method for preparing a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 3, characterized in that: The raw materials for preparing the polymer P2-SA in step (8) include: 5 parts by mass of polymer PCL-PHEMA-PPEGMA, 0.44 parts by mass of selenoic acid, 0.41 parts by mass of a dehydrating agent, and 0.024 parts by mass of a first catalyst.

6. The method for preparing a folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 3, characterized in that: The amount of the reducing agent used in step (10) is 10% of the total molar number of the polymer P1-SA obtained in step (4) and the polymer P2-SA obtained in step (8).

7. Use of the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelle according to claim 1 or 2 as a drug carrier in the delivery of anticancer drugs.

8. The use according to claim 7, characterized in that: The folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles are used as drug carriers. The folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked micelles and hydrophobic anticancer drugs are dissolved in an organic solvent at a mass ratio of 2:1, stirred for 4 to 12 hours, dialyzed with deionized water for 48 hours, and the deionized water is replaced every 4 hours. After three replacements, the deionized water is replaced every 12 hours, and then freeze-dried to obtain the folic acid-modified pH / reduction dual-responsive selenium-containing mixed polymer cross-linked drug-loaded micelles.

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