CN111621024B - Preparation method of block copolymer containing double selenium bonds with rapid oxidation/reduction dual responsiveness - Google Patents
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Abstract
The invention discloses a preparation method and application of a quick oxidation/reduction dual-responsiveness block copolymer containing diselenide. The specific preparation method comprises the steps of firstly respectively preparing the bis-selenium micromolecules (N) blocked by the diazide groups3‑SeSe‑N3) And dipropargyl-terminated polyethylene glycol (PA-PEG-PA). The azide group and the alkynyl group in the structure are utilized to react to obtain the diselenide-containing polyethylene glycol alternating copolymer (PA-PEG-alt-SeSe-PA). Then, polycaprolactone (PCL-N) mono-terminated with azido3) And reacting to obtain the double-selenium-containing block copolymer (PCL-PEGSeSe-PCL) with quick oxidation/reduction dual responsiveness. The amphiphilic copolymer is self-assembled into nano particles in aqueous solution, and has good biocompatibility and biodegradability. The hydrophobic anticancer drug is entrapped, so that the polymer nanoparticles can be destroyed in the tumor cell environment to quickly release the entrapped drug, and the preparation method has potential application prospect in the aspect of cancer treatment.
Description
The invention belongs to a preparation method of a block copolymer containing a double selenium bond with quick oxidation/reduction dual responsiveness and application thereof, and divisional application of invention application with application number of 2017110594936 and application date of 2017, 11 and 1, and belongs to a part of a product preparation method.
Technical Field
The invention belongs to the field of biomedical high polymer materials, and particularly relates to a preparation method and application of a rapid oxidation/reduction dual-responsiveness block copolymer containing a double selenium bond.
Background
Traditional nano-drug carriers are often prepared from non-degradable polymers such as polyacrylics, polyethylenes, polystyrenes, and the like. These polymeric carriers are not easily degraded after entering the body, are not easily excreted by the kidney, and finally remain and accumulate in the body, which in the past causes many side effects for patients.
To date, polylactic acid (PLA), polyethylene glycol (PEG), Polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), etc. have been approved by the Food and Drug Administration (FDA) for use in medical materials. Many nano-drug carriers based on such polymers have been developed and used. The polymer material has good biocompatibility and biodegradability, and can be hydrolyzed or enzymolyzed under physiological conditions to form molecules which can be absorbed by organisms or excreted by kidneys. However, they are still insufficient as nano drug carriers, i.e. the polymer itself degrades slowly, and the hydrolysis or enzymolysis rate cannot meet the controllable release of the drug, which may result in insufficient release of the drug at the tumor focus site, and affect the therapeutic effect to some extent. Therefore, in recent years, the synthesis of various biodegradable 'intelligent' nano-drug carriers with good biocompatibility has been proposed.
Compared with the traditional nano-drug carrier, the intelligent nano-drug carrier can be dissociated under the stimulation of in-vivo microenvironment (pH value, redox, enzyme and the like) or external (temperature, light, ultrasonic and the like) conditions, so that the controlled release of the drug is realized. The redox responsiveness of the support is currently a more studied class. The stimulation mainly comes from the reducing or oxidizing environment in the tumor cells, so that chemical bonds with redox responsiveness are broken, the hydrophilic and hydrophobic properties of the polymer are further influenced, finally, the drug-loaded nanoparticles are dissociated, and the drug is rapidly released.
In the prior art, some reports have been made on fast oxidation/reduction sensitive polymers containing diselenide bonds, however, in these structures, the limited diselenide bonds are only linked between longer different polymer blocks, and as drug carriers, the fast response and dissociation degree of copolymer carriers in vivo is very limited. Therefore, there is a need to develop a new polymer, in which the double selenium bonds are alternately bonded to the blocks in the main chain of the polymer, so that the drug-loaded nanoparticles formed from the polymer have fast oxidation/reduction responsiveness in the environment of tumor cells, thereby releasing the drug rapidly.
Disclosure of Invention
The invention aims to provide a block copolymer containing double selenium bonds with quick oxidation/reduction dual responsiveness based on polyethylene glycol and polycaprolactone and a preparation method thereof; in the copolymer, selenium is used as a basic trace element in a human body and has an indispensable effect on the health of the human body, the selenium can clear free radicals in the human body, effectively inhibit the generation of lipid peroxide, stimulate the immune response of the human body, improve the protective capability of the immune system and prevent diseases, and more importantly, the selenium is closely and indiscriminately connected with cancer prevention; selenium mainly exists in the form of selenase and selenoprotein in a human body, the invention creatively designs the block copolymer containing double selenium bonds, has the effect of antioxidation, can well protect cell membranes from being damaged by peroxide, prevents the accumulation of the peroxide in the body, reduces the damage of the peroxide to DNA, prevents mutation and achieves the effect of preventing canceration; further, selenium and an element in the same main group as chalcogen are distinguished, however, in that selenium has a larger atomic radius than sulfur and is less electronegative than sulfur, so that the bond energy of the diselenide bond (172 kJ/mol) is lower than the bond energy of the disulfide bond (240 kJ/mol). Thus, the diselenide bond can be oxidized in addition to being reduced, and even has a radiation response characteristic. Therefore, the chemical bond energy of selenium is smaller, the chemical bond is more sensitive, and the application of the double selenium bond in the field of stimulus-responsive drug carriers is greatly promoted.
The specific technical scheme of the invention is as follows: a fast oxidation/reduction dual-responsive double selenium bond-containing block copolymer expressed by the following chemical structural formula:
wherein m is 5-15, n is 4-114, and x is 15-45.
In the technical scheme, the rapid oxidation/reduction dual-responsiveness block copolymer containing the double selenium bonds has the double selenium bonds in the structure, the alternating copolymer of the double selenium component and PEG is a hydrophilic section, and PCL is a hydrophobic section; the formed nano particles have good stability under normal physiological conditions, and under the oxidation and reduction conditions, the double selenium bond is broken, so that the nano particles are destroyed, and the hydrophobic anticancer drugs gathered in the nano particles are rapidly released.
In the preferred technical scheme, the number average molecular weight of the block copolymer containing the quick oxidation/reduction dual-responsiveness double selenium bond is 7220-98250 g multiplied by mol-1(ii) a Wherein the polyethylene glycol repeating chain segment is 15-45, and the polycaprolactone repeating chain segment is 5-15.
The invention adopts a structure containing diselenide and polyethylene glycol (PA-PAG) with the end blocked by propynylaltSeSe-PA) as basic raw material reacts with azido single-terminated polycaprolactone (PCL-N) under the catalysis of copper salt and ligand3) System ofPreparing a rapid oxidation/reduction dual-responsiveness block copolymer PCL-PEGSeSee-PCL containing diselenide based on polyethylene glycol and polycaprolactone.
The preparation method of the rapid oxidation/reduction dual-responsiveness block copolymer containing diselenide comprises the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) and (3) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing the diselenide bond at two ends prepared in the step (3) with the azido-single-terminated polycaprolactone prepared in the step (4) to prepare the rapid oxidation/reduction dual-responsiveness block copolymer containing the diselenide bond.
In the technical scheme, in the step (1), a reaction product of selenocysteine hydrochloride and an acyl chloride compound is reacted with an azide compound to prepare an azide-terminated diselenide micromolecule; in the step (2), reacting a reaction product of polyethylene glycol and potassium hydride with a propine compound to prepare polyethylene glycol with propargyl groups at two ends blocked; in the step (3), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand; in the step (4), initiating the polymerization of e-caprolactone by using small molecular alcohol in the presence of an organic catalyst; in step (5), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand. In the step (1), the acyl chloride compound is chloracetyl chloride, and the azide compound is sodium azide; in the step (2), the propyne compound is 3-bromopropyne; in the step (3), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine; in the step (4), the catalyst is selected from stannous octoate or 1, 8-diazabicycloundecen-7-ene; in the step (5), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine.
In the technical scheme, in the step (1), the reaction temperature is 25-60 ℃, and the reaction time is 12-40 h; in the step (2), the reaction temperature is 0-50 ℃, and the reaction time is 1-40 h; in the step (3), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h; in the step (4), the reaction temperature is 60-90 ℃, and the reaction time is 4-24 h; in the step (5), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h.
In the step (1), selenocysteine hydrochloride is used as a raw material to react with chloroacetyl chloride with high activity, and dichloro-terminated diselenide micromolecules (Cl-SeSe-Cl) are prepared in the presence of a catalyst; further reacting with sodium azide to obtain double selenium micromolecules blocked by azide groups; in the step (2), the KH with high activity is reacted with polyethylene glycol to prepare an oxyanion initiator; further carrying out nucleophilic reaction with 3-bromopropyne to prepare polyethylene glycol (PA-PEG-PA) with propargyl end caps at two ends; in the step (3), in the presence of a copper salt catalyst and a ligand, the bis-selenium micromolecule (N) blocked by azide group3-SeSe-N3) Reacting with polyethylene glycol (PA-PEG-PA) with propargyl end caps at two ends to obtain the polyethylene glycol alternating copolymer (PA-PAG) containing diselenide with alkynyl end caps at two endsalt-SeSe-PA); in the step (4), 2-bromoethanol is used as an initiator, and Sn (Oct)2Initiating the polymerization of e-caprolactone (e-CL) by using ring-opening polymerization as a catalyst to prepare the mono-bromo-terminated polycaprolactone (PCL-Br); further modifying the end group of PCL-Br by sodium azide to prepare azido single-ended polycaprolactone (PCL-N)3) (ii) a In the step (5), alkyne at two ends is carried out in the presence of copper salt catalyst and ligandThe base-terminated polyethylene glycol alternating copolymer containing double selenium bonds reacts with the azido single-terminated polycaprolactone to obtain the rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds. In the step (1), the molar ratio of the selenocysteine hydrochloride to the chloracetyl chloride is 1: (2-10); the molar ratio of dichloro-terminated diselenide small molecules (Cl-SeSe-Cl) to sodium azide is 1: (2-10); in the step (2), the molar ratio of the polyethylene glycol to the potassium hydride to the 3-bromopropyne is 1: (2-6): (2-10); in the step (3), the double selenium micromolecules (N) blocked by the azide group3-SeSe-N3) The molar ratio of the catalyst to polyethylene glycol and copper salt catalyst with propargyl end capping at two ends is 1 to (1.05-1.3) to (0.5-1.5); the molar ratio of the copper salt catalyst to the ligand is 1: 1-2; in the step (4), the molar ratio of the 2-bromoethanol to the e-caprolactone is 1: (15-45); in the step (5), the molar ratio of the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end caps to the azido single end capped polycaprolactone is 1: (2-4).
The invention also discloses the application of the rapid oxidation/reduction dual-responsiveness block copolymer containing the double selenium bond in the preparation of nano-drugs; or the block copolymer with quick oxidation/reduction dual responsiveness and containing the double selenium bonds is used as a nano-drug carrier.
The invention also discloses a fast oxidation/reduction dual-responsiveness block copolymer nano particle containing a double selenium bond and a preparation method thereof, and the fast oxidation/reduction dual-responsiveness block copolymer nano particle comprises the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;
(6) and (3) self-assembling and dialyzing the block copolymer containing the double selenium bonds with the fast oxidation/reduction dual responsiveness prepared in the step (5) to prepare the block copolymer nano particles containing the double selenium with the fast oxidation/reduction dual responsiveness. For example, the rapid oxidation/reduction dual-responsive diselenide-containing block copolymer is self-assembled in an aqueous solution to form nanoparticles; the fast oxidation/reduction dual-responsiveness double-selenium-containing segmented copolymer nanoparticles are prepared by a good solvent dialysis method.
The invention also discloses the application of the block copolymer nano particle containing double selenium bonds with quick oxidation/reduction dual responsiveness in preparing nano medicaments; or as a nano-drug carrier.
The invention also discloses a rapid oxidation/reduction dual-responsiveness anticancer nano-drug system and a preparation method thereof, and the preparation method comprises the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;
(6) and (3) mixing the block copolymer with the rapid oxidation/reduction dual responsiveness and containing the double selenium bonds and the anticancer drug, and then carrying out self-assembly and dialysis to prepare the rapid oxidation/reduction dual responsiveness anticancer nano-drug system. For example, the anti-cancer nano drug-carrying system with the rapid oxidation/reduction dual-responsiveness and containing the double selenium bonds is prepared by mixing the block copolymer with the rapid oxidation/reduction dual-responsiveness and the anti-cancer drug, dissolving the block copolymer with the rapid oxidation/reduction dual-responsiveness and containing the double selenium bonds in a good solvent and dialyzing the mixture in a water phase.
The invention also discloses application of the rapid oxidation/reduction dual-responsiveness anticancer nano drug system in preparation of anticancer drugs, in particular application in preparation of stimulus-responsiveness anticancer nano drug-loaded systems.
In the invention, the anti-cancer drug is selected from one of adriamycin, paclitaxel, camptothecin and curcumin.
Specifically, the following scheme can be adopted by the invention as an example:
(1) synthesizing a dichloro-terminated diselenide micromolecule by taking selenocysteine hydrochloride and chloroacetyl chloride as raw materials, taking dichloromethane as a solvent and triethylamine, pyridine or ethylenediamine as an acid-binding agent through an amide-type reaction; further reacts with sodium azide to prepare the bis-selenium micromolecule (N) blocked by diazide3-SeSe-N3）；
Wherein the molar ratio of the selenocysteine hydrochloride to the chloroacetyl chloride is 1: (2-10);
the chemical structural formula of the dichloro-terminated diselenide micromolecule is as follows:
the chemical structural formula of the diazido-terminated diselenide micromolecule is as follows:
(2) polyethylene glycol is used as a raw material, tetrahydrofuran is used as a solvent, and under the anhydrous and oxygen-free conditions, terminal hydroxyl reacts with potassium hydride (KH) to form oxyanions; further reacting with 3-bromopropyne to prepare dialkynyl terminated polyethylene glycol (PA-PEG-PA);
wherein the molar ratio of the reaction of the polyethylene glycol, the potassium hydride and the 3-bromopropyne is 1: (2-6): (2-10);
the chemical structural formula of the oxyanion is as follows:
the chemical structural formula of the dipropargyl-terminated polyethylene glycol is as follows:
n is 4 to 114;
under the condition of inert gas atmosphere, under the existence of copper salt catalyst and ligand, propargyl-terminated polyethylene glycol and diazido-terminated diselenide micromolecules are used as raw materials
As a ligand, toN, N,Dimethyl formamide as solvent, and preparing polyethylene glycol alternating copolymer (PA-PAG) with two end alkynyl end caps through reactionalt-SeSe-PA)；
Wherein the molar ratio of the propargyl terminated polyethylene glycol to the diazido terminated diselenide micromolecule to the copper salt catalyst is 1: (1.05-1.3): (0.5 to 1.5); the mole ratio of copper salt catalyst and ligand is 1: (1-2);
the chemical structural formula of the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end closures is as follows:
m is 5 to 15, n is 4 to 114;
(4) 2-bromoethanol is used as an initiator to initiate
Ring-opening polymerization is carried out, and modification is carried out to obtain azido single-end-capped polycaprolactone (PCL-N)3)；
Wherein the content of the first and second substances,
and sodium azide at a 1: (15-45): (2-4);
the chemical structural formula of the azido single-terminated polycaprolactone is as follows:
x is 15-45;
(5) under the condition of inert gas atmosphere, in the presence of a copper salt catalyst and a ligand, taking a polyethylene glycol alternating copolymer with two end alkynyl end caps and azido group single end capped polycaprolactone as raw materials
As a ligand, toN,N,Dimethyl formamide is used as a solvent, and a rapid oxidation/reduction dual-responsiveness block copolymer (PCL-PEGSeSE-PCL) containing a dual selenium bond is prepared through reaction;
wherein, the mol ratio of the selenium-containing polyethylene glycol alternating copolymer with two end alkynyl end-capped ends, the azido single end-capped polycaprolactone and the copper salt is 1: (2-4): (0.5 to 1.5); the mole ratio of copper salt catalyst and ligand is 1: (1-2);
the structural formula of the rapid oxidation/reduction dual-responsiveness double-selenium-containing block copolymer is as follows:
m is 5 to 15, n is 4 to 114, and x is 15 to 45.
According to the invention, the selenium-containing polyethylene glycol alternating copolymer raw material with two end alkynyl end closures and the quick oxidation/reduction dual-responsiveness double-selenium-containing block copolymer are prepared by limiting the raw material and parameters in the presence of a copper salt catalyst and a ligand for the first time, so that the problem that the existing reaction system cannot prepare the copolymer with the main chain containing the double-selenium alternating is solved, and the method is a novel, efficient and quick synthesis method.
According to a further technical scheme, after the steps (1) to (5) are finished, products are respectively purified, and the purification process comprises the following steps:
(i) purifying the bisazido-terminated diselenide micromolecules: after the reaction was completed, the crude product was filtered to remove unreacted sodium azide. The DMF solvent was removed under reduced pressure using an oil pump. Adding CH2Cl2The concentrated product was dissolved sufficiently and extracted. Collecting organic phase, drying with anhydrous sodium sulfate, filtering, and rotary evaporating to remove CH2Cl2A solvent. Putting the obtained product into a vacuum drying oven to be dried to constant weight to obtain a dark yellow solid product;
(ii) purification of propargyl terminated polyethylene glycol: after the reaction was complete, the crude product was filtered and the THF solvent was removed by rotary evaporation. By CH2Cl2Extracting with solvent, collecting organic phase, drying with anhydrous sodium sulfate, filtering, and rotary evaporating to remove most CH2Cl2A solvent. Precipitating in n-hexane for three times, and drying the product in a vacuum drying oven to constant weight to obtain a light yellow viscous liquid product;
(iii) purifying the diselenide-containing polyethylene glycol alternating copolymer with two end alkynyl end closures: after the reaction is finished, the crude product is passed through neutral Al2O3Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48-72 h, periodically changing water, and freeze drying to obtain light yellow solid product;
(iv) purification of azido single-terminated polycaprolactone: after the reaction was completed, the crude product was filtered to remove unreacted sodium azide. Concentrating the reaction solution under reduced pressure with an oil pump, and using CH2Cl2And (4) solvent extraction. Harvesting machineThe organic layer was collected, and dried with anhydrous sodium sulfate. Then filtered and rotary evaporated to remove CH2Cl2A solvent. Putting the obtained product into a vacuum drying oven to be dried to constant weight to obtain a white solid product;
(v) purification of fast oxidation/reduction dual-responsive diselenide-containing block copolymers: after the reaction is finished, the crude product is passed through neutral Al2O3Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48-72 h, periodically changing water, and freeze drying to obtain light yellow solid product.
The invention discloses a rapid oxidation/reduction dual-responsiveness block copolymer PCL-PEGSeSee-PCL containing diselenide, which can be self-assembled in an aqueous solution to form nanoparticles. The hydrophobic polycaprolactone block forms the core of the nanoparticle; the hydrophilic polyethylene glycol chain segment forms the shell of the nano particle, and plays a role in stabilizing the nano particle. The double selenium bond is easy to break under the condition of rapid oxidation/reduction, and the nano particles are destroyed, thereby rapidly releasing the encapsulated hydrophobic anticancer drug. Therefore, the invention requests to protect the application of the rapid oxidation/reduction dual-responsive diselenide-containing block copolymer in the preparation of a stimulus-responsive anticancer nano-drug system.
Due to the implementation of the scheme, compared with the prior art, the invention has the following advantages:
1. according to the invention, hydrophilic polyethylene glycol and hydrophobic polycaprolactone with good biocompatibility are used as raw materials, a double selenium bond is introduced into a main chain of a block copolymer for the first time through a chemical reaction, and the fast response and the dissociation degree of a copolymer carrier in vivo are very excellent; in aqueous solution, the block copolymer can self-assemble to form nano particles and is used as a carrier of a hydrophobic anticancer drug;
2. in the polymer disclosed by the invention, the double selenium bond can be broken under the action of a reducing agent and even under the action of an oxidizing agent, so that the drug-loaded nano particle is endowed with a rapid oxidation/reduction double stimulation response behavior;under normal physiological conditions, the drug-loaded nanoparticles can exist stably and in an oxidizing environment (such as H)2O2) Or under reducing conditions (such as glutathione), the double selenium bond can be rapidly broken, and the polymer is dissociated, so that the polymer nanoparticles are rapidly destroyed, and the anticancer drug is rapidly released; in addition, selenium is used as an anticancer element, can be synergistically acted with the medicament, and can well play a role in preventing cancers, so that the medicament-carrying nano particles have potential application value in the aspect of cancer treatment;
3. the quick oxidation/reduction dual-responsiveness block copolymer containing diselenide provided by the invention has a definite structure and mild synthesis conditions, and has the following remarkable characteristics: (1) the raw materials and reagents are easy to obtain; (2) the reaction condition is simple and mild; (3) the yield is high; (4) the product is simple and convenient to separate; (5) the product has good stability and convenient purification.
Drawings
FIG. 1 shows an example of a bisazido-terminated diselenide small molecule (N)3-SeSe-N3) And nuclear magnetic resonance hydrogen spectra of the intermediate: (A) NH (NH)2-SeSe-NH2；(B) Cl-SeSe-Cl；(C) N3-SeSe-N3(ii) a The solvent is deuterated dimethyl sulfoxide;
FIG. 2 shows the bisazido-terminated diselenide small molecule (N) in example one3-SeSe-N3) And the infrared spectrogram of the intermediate: (A) NH (NH)2-SeSe-NH2; (B) Cl-SeSe-Cl; (C) N3-SeSe-N3；
FIG. 3 is the NMR spectrum of diynyl terminated polyethylene glycol (PA-PEG-PA) in example II with deuterated dimethyl sulfoxide as the solvent; (A) HO-PEG-OH; (B) PA-PEG-PA;
FIG. 4 shows two alkynyl-terminated diselenide-containing polyethylene glycol alternating copolymers (PA-PEG-alt-se-PA) nuclear magnetic resonance hydrogen spectrum;
FIG. 5 is a NMR chart of the azido single-capped polycaprolactone and the intermediate of example IV, with deuterated chloroform as the solvent; (A) PCL-Br; (B) PCL-N3；
FIG. 6 shows a fourth embodiment of a sandwichGel permeation chromatography outflow curves of the nitrogen-based single-end-capped polycaprolactone and the intermediate, (A) PCL-Br; (B) PCL-N3；
FIG. 7 is the IR spectrum of the azido single-terminated polycaprolactone and the intermediate of example IV, (A) PCL-Br; (B) PCL-N3；
FIG. 8 is the NMR spectrum of the fast oxidation/reduction dual-responsive diselenide-containing block copolymer (PCL-PEGSeSE-PCL) synthesized in example five, with deuterated dimethyl sulfoxide as the solvent;
FIG. 9 is a gel permeation chromatography elution profile of the azido single-capped polycaprolactone, dialkynyl-capped polyethylene glycol, alkynyl-capped alternating copolymers of diselenide-containing polyethylene glycol, and diselenide-containing triblock copolymers of example V, (A) PA-PEG-PA; (B) PCL-N3；(C) PA-PEG-alt-SeSe-PA ；(D) PCL-PEGSeSe-PCL；
FIG. 10 is the particle size distribution curve and nanoparticles formed by the fast oxidation/reduction dual-responsive diselenide-containing block copolymer (PCL-PEGSeSE-PCL) in the sixth embodiment in the aqueous solution;
FIG. 11 is a graph showing the particle size change of drug-loaded nanoparticles formed by the fast oxidation/reduction dual-responsive diselenide-containing block copolymer and doxorubicin under different conditions in example seven;
FIG. 12 is an in vitro drug release profile of the drug-loaded nanoparticles of example eight at different Glutathione (GSH) concentrations;
FIG. 13 shows the cell survival rates of the L929 cell and the HeLa cell in the ninth embodiment after culturing with different concentrations of diselenide polymer nanoparticles and drug-loaded nanoparticle solution thereof for 72 h, respectively;
FIG. 14 shows the results of endocytosis observed using the living cell workstation in example ten: (A) fluorography of Doxorubicin (DOX) -entrapped nanoparticles with (B) free DOX into HeLa cells (DOX concentration of 1)
) (ii) a The fluorescence signals from left to right are blue fluorescence of nuclei stained with the blue dye Hoechst 33342, and doxorubicin-spontaneous red, respectivelyColor fluorescence and overlapping fluorescence of the two fluorescence signals;
FIG. 15 is a flow cytometric curve of the loaded nanoparticles and free DOX of example ten.
Detailed Description
The invention is further described below with reference to examples and figures:
the first embodiment is as follows: bis-selenium small molecule (N) terminated with diazido groups3-SeSe-N3) Synthesis of (2)
Bis-selenium small molecule (N) terminated with diazido groups3-SeSe-N3) The preparation method mainly comprises two steps, wherein in the first step, dichloroyl-terminated diselenide micromolecules (Cl-SeSe-Cl) are prepared; second, preparing the bis-selenium micromolecules (N) blocked by diazide groups3-SeSe-N3) The specific synthesis method comprises the following steps:
synthesis of dichloro-terminated bis-selenium Small molecule (Cl-SeSe-Cl): and (3) putting a 250 mL three-neck flask with a stirrer, a bent pipe, a constant-pressure dropping funnel and a piston into a 120 ℃ oven for drying for 12 h, taking out the flask from the oven before use, putting the flask into a dryer, and cooling the flask to room temperature for use. The apparatus was mounted, nitrogen was introduced, and 70 mL of anhydrous CH was added to a three-necked flask2Cl2Selenocysteine hydrochloride (1 g, 3.12 mmol) is weighed and added into a three-neck flask, and anhydrous triethylamine (2.536 g, 25 mmol) is extracted by using a syringe and added to be fully and uniformly mixed. 30 mL of anhydrous CH was added to a constant pressure dropping funnel2Cl2Chloroacetyl chloride (1.416 g, 12.5 mmol) was added, and the mixture was sealed after three times of gassing. And (3) placing the reaction bottle in an ice-water bath, opening the constant-pressure dropping funnel after the temperature of the reaction liquid is reduced, and slowly dropping the liquid at the speed of 1 drop/2 second. After the solution was completely dripped, it was transferred from the ice water bath to a 25 ℃ oil bath and reacted for 24 h. After the reaction was complete, the crude product was transferred to a 250 mL round bottom flask and concentrated. 150 mL of THF was added and the salt was removed by filtration. THF was removed by rotary evaporation and 100 mL CH was added2Cl2And fully dissolved. Then, extraction treatment is carried out: 50 mL of saturated saline was added, followed by shaking and standing, and the organic phase was collected. Then 30 mL CH2Cl2The aqueous phase was extracted, the organic phases combined and the operation repeated three times. Harvesting machineCollecting organic phase, adding appropriate amount of anhydrous sodium sulfate, drying to remove water, filtering, and rotary evaporating to remove solvent. The resulting product was dried in a vacuum oven to constant weight to give the product as a dark yellow solid, i.e., Cl-SeSe-Cl (0.93 g, 38.4% yield). The nuclear magnetic resonance spectrum and the infrared spectrum of the product are respectively shown in FIG. 1(B) and FIG. 2 (B).
Synthesis of the Diazido-end-capped diselenide Small molecule (N)3-SeSe-N3): adding Cl-SeSe-Cl (0.4 g, 1 mmol) and NaN3(0.65 g, 10 mmol) and 10 mL of DMF were added sequentially to a 50 mL round bottom flask. The round bottom flask was transferred to a 60 ℃ oil bath and reacted for 40 h. After the reaction was completed, the crude product was filtered to remove unreacted sodium azide. The DMF solvent was removed under reduced pressure using an oil pump. 100 mL of CH was added2Cl2The concentrated product was dissolved sufficiently and extracted. Collecting organic phase, adding anhydrous sodium sulfate for drying, filtering, and rotary evaporating to remove CH2Cl2A solvent. Drying the obtained product in a vacuum drying oven to constant weight to obtain a dark yellow solid product, namely N3-SeSe-N3(0.38 g, yield 36.2%). The nuclear magnetic resonance spectrum and the infrared spectrum of the product are respectively shown in FIG. 1(C) and FIG. 2 (C).
Example two: synthesis of Dipropargyl terminated polyethylene glycol (PA-PEG-PA)
The dipropargyl-terminated polyethylene glycol (PA-PEG-PA) is mainly prepared by a one-pot method. The first step is as follows: preparing an initiator of oxyanion by reacting high-activity KH with terminal hydroxyl of ethylene glycol; secondly, reacting an oxyanion initiator with 3-bromopropyne to prepare the dipropargyl-terminated polyethylene glycol (PA-PEG-PA), wherein the specific synthetic method comprises the following steps:
putting a 100 mL branch flask with a stirrer, a constant-pressure dropping funnel and a piston into a 120 ℃ oven for drying for 12 h, taking out the flask from the oven before use, putting the flask into a dryer, and cooling to room temperature for further use. HO-PEG-OH (5 g, 5 mmol) was first weighed into a branched flask, followed by azeotropic removal of water by addition of 50 mL of toluene and repeated distillation twice. The manifold flask was then charged three times with double exhaust tubing and charged with 50 mL of anhydrous THF using a syringe and appropriately heated until HO-PEG-OH was completely dissolved. KH encapsulated in mineral oil was aspirated into a small saline bottle and washed three times with anhydrous THF. After the mixture was drained, KH (1.2 g, 30 mmol) was weighed out from the mixture and charged into a branched flask, and stirred for 1 hour, and then a constant pressure dropping funnel was placed, 20 mL of anhydrous THF was added, and 3-bromopropyne (5.9 g, 50 mmol) was taken out using a syringe and charged into the constant pressure dropping funnel. And (5) inflating and deflating the reaction device for three times and then sealing. The constant pressure dropping funnel was opened and the liquid was slowly dropped at a rate of 1 drop/2 sec. The reaction flask was transferred to a 45 ℃ oil bath and reacted for 40 h.
After the reaction was complete, the crude product was filtered and the THF solvent was removed by rotary evaporation. Using CH2Cl2And (5) carrying out extraction treatment. Collecting organic phase, adding anhydrous sodium sulfate for drying, filtering, and rotary evaporating to remove most CH2Cl2A solvent. Precipitated three times in n-hexane and the product was dried in a vacuum oven to constant weight to give a pale yellow viscous liquid, PA-PEG-PA (4.19 g, 38.4% yield). The NMR spectrum of the product is shown in FIG. 3 (B).
Example three: bi-selenium-containing polyethylene glycol alternating copolymer (PA-PEG-alt-SeSe-PA) Synthesis
And (3) putting the 50 mL branch flask with the stirrer into a 120 ℃ oven for drying for 12 h, taking out the flask from the oven before use, cooling by using a double-row pipe, and cooling to room temperature. CuBr (71.73 mg, 0.5 mmol), PMDETA (86.65 mg, 0.5 mmol) and 5 mL anhydrous DMF were added and stirred well. Weighing PA-PEG-PA (281.5 mg, 0.25 mmol) and N3-SeSe-N3(103.5 mg, 0.25 mmol) was put into a centrifuge tube, 5 mL of anhydrous DMF was added to completely dissolve the two materials, the mixture was put into a branched flask, the flask was sealed after being charged and discharged three times, the branched flask was transferred to a 35 ℃ oil bath, and the reaction was carried out for 24 hours. To ensure that the polymer was end-capped with propargyl, excess PA-PEG-PA (140.8 mg, 0.125 mmol) was added and the reaction was continued for 24 h.
After the reaction is finished, the crude product is filtered by a neutral Al2O3Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48 hr, periodically changing water, freeze drying,obtaining a light yellow solid product, namely PA-PEG-altSeSe-PA (0.25 g, 67% yield). The NMR spectrum of the product is shown in FIG. 4.
Example four: azido single-terminated polycaprolactone (PCL-N)3) Synthesis of (2)
The azido-terminated polycaprolactone is prepared mainly by two steps. The first step, 2-bromoethanol is used as an initiator, Sn (Oct)2Initiation of the ring-opening polymerization as a catalyst
Polymerizing to prepare mono-bromine terminated polycaprolactone (PCL-Br); secondly, modifying the end group of PCL-Br by using sodium azide to prepare azido-terminated polycaprolactone (PCL-N)3) The specific synthesis method comprises the following steps:
in the first step, PCL-Br is synthesized. A50 mL branched flask equipped with a stirrer was placed in a 120 ℃ oven and dried for 12 h, removed from the oven before use and cooled to room temperature using a double calandria pump. 2-bromoethanol (0.23 g, 1.84 mmol),
(3.58 g, 31.28 mmol) and 10 mL of anhydrous toluene were added to the flask in a branched flask in this order, and the mixture was stirred sufficiently to mix them uniformly. Adding Sn (Oct)2(0.037 g, 0.092 mmol), the flask was sealed after being charged and discharged three times, and the branched flask was transferred to a 90 ℃ oil bath to react for 4 hours. After the reaction is finished, most of toluene is removed by rotary evaporation. Precipitated three times in ether, and the product was dried in a vacuum oven to constant weight to give PCL-Br (2.84 g, 74.5% yield) as a white solid. The NMR spectrum of the product is shown in FIG. 5 (A).
Second, synthesizing PCL-N3. PCL-Br (1.08 g, 0.44 mmol) synthesized in the first step and NaN3(0.14 g, 2.2 mmol) and 10 mL of DMF were added sequentially to a 50 mL round bottom flask. The round bottom flask was transferred to a 60 ℃ oil bath and reacted for 24 h. After the reaction was completed, the crude product was filtered to remove unreacted sodium azide. The reaction solution was concentrated under reduced pressure using an oil pump. Using CH2Cl2And (5) carrying out extraction treatment. The organic layer was collected, and dried by adding anhydrous sodium sulfate. Then filtered and rotary evaporated to remove CH2Cl2A solvent. Drying the obtained product in a vacuum drying oven to constant weight to obtain white solid, namely PCL-N3(0.89 g, 73.0% yield). The NMR spectrum of the product is shown in FIG. 5(B), the gel permeation chromatography outflow curve is shown in FIG. 6, and the infrared spectrum characterization is shown in FIG. 7.
Example five: synthesis of ABA type triblock copolymer PCL-PEGSeSE-PCL with double response of quick oxidation/reduction of azide
A50 mL branched flask equipped with a stirrer was placed in a 120 ℃ oven and dried for 12 h, removed from the oven before use and cooled down by double calandria, and after cooling to room temperature CuBr (27.7 mg, 0.193 mmol), PMDETA (66.8 mg, 0.386 mmol) and 5 mL DMF were added and stirred well. Weighing PA-PEG-alt-SeSe-PA（206.8 mg, 0.016 mmol）、PCL-N3(176.0 mg, 0.064 mmol) was placed in a centrifuge tube, 3 mL of DMF was added to completely dissolve the two materials, the mixture was placed in a branched flask, and the flask was sealed after three times of aeration. The branched flask was transferred to a 45 ℃ oil bath and reacted for 24 hours. After the reaction is finished, alkynyl modified polystyrene resin is added to continue the reaction for 24 hours so as to react and adsorb residual PCL-N3. After the reaction is finished, the crude product is filtered by a neutral Al2O3Short chromatographic column of (4). Transferring the solution into dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 48 h, periodically changing water, and freeze-drying to obtain light yellow solid product, i.e. PCL-PEGSeSE-PCL (197.6 mg, 51.7% yield). The NMR spectrum of the product is shown in FIG. 8, and the gel permeation chromatography outflow curve is shown in FIG. 9.
Example six: preparation of PCL-PEGSeSe-PCL polymer nano-particle by dialysis method
Firstly, accurately weighing 7.93 mg of pyrene to be dissolved in acetone solution, and fixing the volume to 50 mL to obtain 0.78 mM pyrene/acetone solution. 50 microliter of pyrene/acetone solution was added to a number of small saline bottles with a stirrer and the acetone was suction dried using reduced pressure suction. 5 mL of PCL-PEGSeSE-PCL polymer (product of example five) with different concentrations were added to dissolveLiquid (A), (B)
) Vigorously stir for 48 h. And detecting the fluorescence signals of the solutions in the saline bottles by adopting a fluorescence spectrophotometer, wherein the excitation wavelength is set to be 335 nm, the emission wavelength scanning range is 350-550 nm, the slit width is set to be 2.5 nm, and the voltage is set to be 800V. Analyzing the fluorescence spectrum, takingI 3/I 1And (3) drawing with Log C, wherein the intersection point of the linear fitting curves of the high concentration and the low concentration is the critical aggregation concentration of the PCL-PEGSeSee-PCL polymer.
The PCL-PEGSeSE-PCL polymer nano particles are prepared by a good solvent dialysis method. 10 mg of PCL-PEGSeSE-PCL polymer was weighed out accurately, dissolved in 1.5 mL of dimethyl sulfoxide, and stirred for 4 hours to dissolve completely. Followed by stirring while using a micro-pump (WZS-50F) at 1.5 mL h-1Slowly add 6 mL of deionized water. After the dropwise addition, stirring is continued for 6 h, then the solution is transferred to a dialysis bag (MWCO 7000 Da), placed in secondary water for dialysis for 18 h, water is periodically changed, finally the dialysate is made to be 10 mL by using the secondary water, and the nanoparticles formed in the aqueous solution and the particle size distribution curve thereof are shown in FIG. 10.
Example seven: the drug-loaded nano-particles formed by the rapid oxidation/reduction dual-responsiveness bi-selenium-containing block copolymer and the adriamycin are adopted to change the particle size under different conditions
50 mg of PCL-PEGSeSE-PCL polymer (product of example five) was dissolved in 4 mL of Dimethylsulfoxide (DMSO) and stirred for 4 h to dissolve completely. 4 mL DOX/DMSO solution (1.25 mg mL) was added-1) After stirring for 4 hours, the mixture was stirred with a micro-pump for 3 mL hours -125 mL of deionized water was added dropwise. Stirring for 4 h after the dropwise addition is completed, transferring the solution into a dialysis bag (MWCO 7000 Da), dialyzing in secondary water for 24 h, periodically changing water, and fixing the volume of the dialysate to 50 mL by using the secondary water.
DLS is used for observing the particle size change of the drug-loaded nanoparticles along with time under different conditions. The specific operation is as follows: taking two 5 mL medicine-carrying nano particle solutions, and respectively adding 0.1mL 30% H2O2Solution and 16 mg Glutathione (GSH), configured to 0.5 wt% H2O2The solution and 10 mM GSH solution were stirred at room temperature, the particle size of the drug-loaded nanoparticles was measured at intervals, and the change in particle size was observed, and the results are shown in FIG. 11.
Example eight: in vitro drug release testing of drug-loaded nanoparticles at different Glutathione (GSH) concentrations
5 mL of DOX-loaded nano solution of PCL-PEGSeSE-PCL polymer (example seven) was transferred to a dialysis bag (MWCO 7000 Da), sealed and immersed in a centrifuge tube containing 20 mL of buffer solution with different conditions. The tube was transferred to a constant temperature water bath and shaken at constant temperature (37 ℃). 5 mL of release solution is taken out from the centrifuge tube at regular intervals for monitoring the drug release behavior of the centrifuge tube, and 5 mL of corresponding buffer solution is added to keep the total volume of the release solution unchanged. And detecting the fluorescence intensity of DOX in the release solution by using a fluorescence spectrophotometer, wherein the excitation wavelength is 480 nm, the scanning range of the emission wavelength is 520-620 nm, the slit width is 10 nm, the voltage is 700V, and the concentration of DOX in the release solution is obtained according to a DOX fluorescence emission concentration standard curve. When no GSH is present or the GSH concentration is very low (2 μ M), the drug release rate is slow, releasing only about 23% or 28% of DOX over 60 hours; under the reducing condition of simulating the intracellular GSH concentration, the drug release rate of DOX is obviously accelerated, and nearly 72 percent of DOX can be released in 60 hours, as shown in figure 12. The drug release result shows that the block copolymer containing the diselenide (PCL-PEGSeSee-PCL) nano particle has obvious quick oxidation/reduction dual responsiveness, and can achieve the purpose of controlling and releasing the anti-cancer drug.
Example nine: study of in vitro toxicity of nanoparticles
The biocompatibility of the blank nano-particles and the capacity of the drug-loaded nano-particles loaded with the adriamycin for inhibiting the proliferation of tumor cells are detected by adopting a conventional tetramethyl azoazolate trace enzyme reaction colorimetric method (MTT method). The specific operation is as follows: mouse fibroblasts (L929 cells) and cervical cancer cells (HeLa cells) were inoculated onto 96-well culture dishes containing a culture medium (DMEM) containing 10% heat-inactivated fetal bovine bloodSerum (FBS), 1% penicillin (penicillin) and streptomycin (streptomycin). It was placed at 37 ℃ in 5% CO2After culturing for 12 h under the condition, adding nanoparticle solutions with different concentrations to continue culturing for 72 h. Add 25. mu.l of MTT solution (5 mg mL) to each well of the dish-1) After culturing for 4 hours, the supernatant was aspirated, and 150. mu.l of dimethyl sulfoxide was added to dissolve formazan crystals formed. The absorbance (OD) at 570 nm of each well was measured using a microplate reader (Bio-Rad 680). The relative cell viability was calculated according to the following formula: relative cell survival (%) = (OD)Test/ODControl)
100. In the formula, ODTestIs the absorbance measurement, OD, of the solution in the well of the sample to be testedControlThere is no absorbance measurement of the solution in the sample well to be tested. Five replicates of each concentration sample were run, each sample tested in triplicate and averaged. FIG. 13 shows the cell survival rates of L929 cells and HeLa cells of human cervical cancer respectively cultured with diselenide polymer (product of example five) nanoparticles of different concentrations and drug-loaded nanoparticle solution thereof for 72 h. The results show that the blank nanoparticles are almost non-cytotoxic, even though the nanoparticle concentration reaches 0.2 mg mL-1After the copolymer is cultured with L929 cells and HeLa cells for 72 hours, the survival rate of the cells is still over 90 percent, and the synthesized copolymer containing the double selenium bonds is proved to have excellent biocompatibility. In addition, the drug-loaded nanoparticles show certain capacity of inhibiting tumor cell proliferation, and show the potential of the drug-loaded nanoparticles as drug carriers.
Example ten: endocytosis assay for drug-loaded nanoparticles
The endocytosis of the drug-loaded nanoparticles in HeLa cells was observed using a Live Cell imaging system (Cell' R, Olympus, Japan). The specific operation is as follows: HeLa cells were seeded on 6-well culture dishes containing DMEM medium and placed at 37 ℃ with 5% CO2Culturing for 12 h under the condition to make the culture grow adherently. The supernatant was aspirated and washed 3 times with PBS buffer, added Hoechst 33342 and washed againCulturing for 30 min to stain cell nucleus. The culture dish is placed on the objective table, and the sampling tube is installed at the same time. Appropriate cell regions were selected for observation from an inverted microscope. The medium in the dish was replaced with an equal volume of medium containing DOX/Polymer drug loaded nanoparticles (example seven) or free DOX (DOX concentration 1 mg L) using a sample tube-1) And observing for 6 h in real time at 40 times of focal length. And (4) taking a fluorescence imaging picture of the cells in the selected area every half hour, and tracking and recording the change of the fluorescence intensity in the HeLa cells in real time. As a result, as shown in FIG. 14, (A) nanoparticles carrying Doxorubicin (DOX) and (B) free DOX were taken as a fluorescence photograph of HeLa cells (the concentration of DOX was 1 mg L)-1). The fluorescence signals from left to right are the blue fluorescence of nuclei stained with the blue dye Hoechst 33342, the red fluorescence spontaneously emitted by doxorubicin, and the overlapping fluorescence of the two fluorescence signals, respectively.
The fluorescence intensity of the polymer nanoparticles coated with DOX and free DOX entering the cells was measured by flow cytometry, and the experimental results are shown in fig. 15. With the prolonged culture time of the polymer drug-loaded nanoparticle solution, the fluorescence intensity in HeLa cells is gradually increased, which indicates that more and more DOX/polymer drug-loaded nanoparticles enter the cells through endocytosis and release DOX in the cells. Fluorescence of DOX was also present in HeLa cells after 5 h incubation with free DOX. However, the average fluorescence intensity was much weaker than that of DOX/polymer drug-loaded nanoparticles, and the experimental results were consistent with those obtained by a living cell workstation.
The results show that the DOX/polymer drug-loaded nanoparticles can be efficiently endocytosed into HeLa cells, and the effective release of the drug is realized under the action of high-concentration GSH or active oxygen of tumor cells, so that the rapid drug release capability is shown. FIG. 15 is a flow cytometric curve showing different time periods for DOX/polymer drug-loaded nanoparticles and free DOX in HeLa cell culture (concentration of DOX is 1 mg L)-1）。
Replacing other catalysts and ligands defined by the invention can also obtain products; the molecular weight of the polyethylene glycol and the raw material proportion limited by the invention are changed, and products with different m values and n values can be obtained. The diselenide-containing block copolymer (PCL-PEGSeSE-PCL) with rapid oxidation/reduction dual responsiveness can be assembled into polymer nanoparticle nanoparticles in aqueous solution and stably exist. Under the oxidation or reduction condition, the diselenide bond is broken to destroy the nano particles, so that the encapsulated anticancer drug can be quickly released for treatment, and the technical effects as described in the embodiments are achieved. Therefore, the polymer containing the diselenide bond disclosed by the invention has very quick oxidation and reduction stimulation response characteristics, can realize quick cracking in tumor cells, so that the drug is released and cannot be enriched in the cells, and in addition, as a carrier of an anti-tumor drug, the polymer containing the diselenide bond with good biocompatibility and biodegradability, even nanoparticles have the capacity of inhibiting the proliferation of the tumor cells.
Claims (6)
1. A preparation method of a block copolymer containing a double selenium bond with rapid oxidation/reduction dual responsiveness is characterized by comprising the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing the diselenide bond at two ends prepared in the step (3) with the azido-single-terminated polycaprolactone prepared in the step (4) to prepare the rapid oxidation/reduction dual-responsiveness block copolymer containing the diselenide bond;
the fast oxidation/reduction dual-responsive double-selenium bond-containing block copolymer is expressed by the following chemical structural formula:
wherein m is 5-15, n is 4-114, and x is 15-45.
2. The method for preparing a rapid oxidation/reduction dual-responsive diselenide bond-containing block copolymer according to claim 1, wherein in step (1), the reaction product of cysteamine seleno hydrochloride and an acid chloride compound is reacted with an azide compound to prepare an azide-terminated diselenide small molecule; in the step (2), reacting a reaction product of polyethylene glycol and potassium hydride with a propine compound to prepare polyethylene glycol with propargyl groups at two ends blocked; in the step (3), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand; in the step (4), initiating the polymerization of e-caprolactone by using small molecular alcohol in the presence of an organic catalyst; in step (5), the reaction is carried out in the presence of a copper salt catalyst and a catalyst ligand.
3. The method for preparing a fast oxidation/reduction dual-responsive block copolymer containing a diselenide bond as set forth in claim 2, wherein: in the step (1), the acyl chloride compound is chloracetyl chloride, and the azide compound is sodium azide; in the step (2), the propyne compound is 3-bromopropyne; in the step (3), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine; in the step (4), the catalyst is selected from stannous octoate or 1, 8-diazabicycloundecen-7-ene; in the step (5), the copper salt catalyst is selected from copper sulfate pentahydrate, cuprous chloride or cuprous bromide, and the catalyst ligand is selected from one of sodium ascorbate, bipyridyl, pentamethyldiethylenetriamine, tetramethylethylenediamine or hexamethyltriethylenetetramine.
4. The method of claim 1, wherein the fast oxidation/reduction dual-responsive block copolymer containing diselenide linkages comprises: in the step (1), the reaction temperature is 25-60 ℃, and the reaction time is 12-40 h; in the step (2), the reaction temperature is 0-50 ℃, and the reaction time is 1-40 h; in the step (3), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h; in the step (4), the reaction temperature is 60-90 ℃, and the reaction time is 4-24 h; in the step (5), the reaction temperature is 35-45 ℃, and the reaction time is 24-48 h.
5. A preparation method of a block copolymer nanoparticle containing a double selenium bond with rapid oxidation/reduction dual responsiveness is characterized by comprising the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;
(6) and (3) self-assembling and dialyzing the block copolymer containing the double selenium bonds with the fast oxidation/reduction dual responsiveness prepared in the step (5) to prepare the block copolymer nano particles containing the double selenium with the fast oxidation/reduction dual responsiveness.
6. A preparation method of a rapid oxidation/reduction dual-responsiveness anticancer nano-drug system is characterized by comprising the following steps:
(1) reacting and preparing the azido-terminated diselenide micromolecule by taking selenocysteine hydrochloride, acyl chloride compound and azido compound as raw materials;
(2) polyethylene glycol, potassium hydride and a propine compound are used as raw materials to prepare polyethylene glycol with propargyl end capping at two ends through reaction;
(3) reacting the azido-terminated diselenide micromolecules prepared in the step (1) with the two-end propargyl-terminated polyethylene glycol prepared in the step (2) to prepare two-end propargyl-terminated diselenide bond-containing polyethylene glycol alternating copolymers;
(4) polymerizing e-caprolactone, and reacting with azide to prepare azide single-ended polycaprolactone;
(5) reacting the propargyl-terminated polyethylene glycol alternating copolymer containing double selenium bonds at two ends prepared in the step (3) with the azido-singly-terminated polycaprolactone prepared in the step (4) to prepare a rapid oxidation/reduction dual-responsiveness block copolymer containing double selenium bonds;
(6) and (3) mixing the block copolymer with the rapid oxidation/reduction dual responsiveness and containing the double selenium bonds and the anticancer drug, and then carrying out self-assembly and dialysis to prepare the rapid oxidation/reduction dual responsiveness anticancer nano-drug system.
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| KR101992236B1 (en) * | 2018-05-16 | 2019-06-24 | 가톨릭대학교 산학협력단 | Reductive Degradable Poly(ε-caprolactone) Polymer Having Covalent Bond Between Diselenodipropionate and Poly(ε-caprolactone) Polymer, Anticancer Composition and Drug Delivery Composition Comprising the Same |
| CN110801432A (en) * | 2019-11-12 | 2020-02-18 | 重庆医科大学 | Multifunctional amphiphilic camptothecin-fluorouracil-loaded targeting mixed micelle drug delivery system and preparation method thereof |
| CN111789964B (en) * | 2020-08-28 | 2022-05-27 | 南开大学 | Selenium polymer prodrug micelle with reduction responsiveness, preparation method and application |
| CN112341607B (en) * | 2020-10-13 | 2021-11-19 | 中国科学院化学研究所 | Degradable copolymer and preparation method and application thereof |
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