CN112029091A - PH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA - Google Patents
PH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA Download PDFInfo
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Abstract
The invention discloses a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA, a nano micelle, a preparation method and application thereof, and belongs to the technical field of polymer synthesis and biomedical materials. The invention uses polyethylene glycol as an initiator to initiate beta-benzyl-L-aspartic acid-NRing-opening polymerization of the carboxylactam and subsequent debenzylationThen obtaining a polyethylene glycol-polyaspartic acid block copolymer, and modifying a small molecule with pH/reduction dual responsiveness on a branched chain of polyaspartic acidNAnd (2- ((2-aminoethyl) disulfanyl) ethyl) -3- (piperidin-1-yl) propanamide (CPA) is purified to obtain the pH/reduction dual-responsiveness block copolymer. The formed nano micelle has pH/reduction dual responsiveness and good biocompatibility, can load anticancer drugs and realizes controlled release at a tumor target position.
Description
Technical Field
The invention belongs to the technical field of polymer synthesis and biomedical materials, and relates to a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA, a nano micelle thereof, a preparation method and application thereof.
Background
Cancer is considered the first leading killer to death in humans. The current important method for treating cancer is chemotherapy, but in the whole chemotherapy treatment process, because the treatment drug cannot be delivered to a pathological change part with high selectivity, the drug effect is reduced, the toxic and side effects are increased, and the drug is easy to be eliminated by a reticuloendothelial system, so that the absorption of tumor cells is poor. The nano-drug delivery system can enhance the drug permeability and retention effect, avoid the recognition and capture of a reticuloendothelial system of a human body, prolong the circulation time of a drug-carrying system in blood and improve the bioavailability of the drug.
In certain pathological conditions, specific physicochemical properties at the systemic, tissue and cellular level have been determined. For example, the extracellular environment of tumor tissue is significantly lower than the pH of normal tissue and systemic blood. This is because when tumor cells proliferate at an abnormally high rate, the lack of nutrients results in a high rate of glycolysis and accumulation of lactate, thereby lowering the pH of the environment. The acidic environment of tumor tissues (around pH 6.5) and intracellular inclusion bodies and lysosomes (pH 4.0-6.0) is widely used for designing pH stimuli-responsive nanocarriers. Polymers containing tertiary amine groups have been widely explored and used to prepare pH-responsive nanomaterials, where protonation of the nitrogen atom increases the hydrophilicity of the polymer. The amine groups can also effectively neutralize the acidic endosome environment by protonation, which increases the pH of the endosome, triggering the transport of endosome ions into the lumen, which eventually swell and burst, releasing the internalized nanocarriers (proton sponge effect). In addition, tumors develop to the extent that they formAn anoxic zone. Reduced Glutathione (GSH) concentration is abnormal, and GSH concentration in tumor cytoplasm (2-10mmol L)-1) Much higher than the extracellular GSH concentration (2-20. mu. mol. L)-1) And is 7-10 times the concentration of GSH in the normal cytoplasm. The disulfide bond is the most widely applied chemical bond in a redox system, can be introduced into a skeleton and a side chain of a carrier or used as a cross-linking agent, is stable outside cells, enters into tumor cytoplasm and is reduced into sulfydryl by GSH, so that the nanoparticles are disintegrated, and then the loaded drug is quickly released.
The polyethylene glycol-polyaspartic acid copolymer (PEG-b-PASp) has good biocompatibility and biodegradability, and the PEG block has good water solubility, so that the solubility of the whole drug-loading system can be increased. Aspartic acid is an essential amino acid of human body, has good compatibility with human body, and metabolite can be discharged out of body through metabolism without toxic and side effects. On the other hand, the aspartic acid has a large number of carboxyl terminals, and can be modified by sensitive groups to achieve the effect of targeted release. Therefore, the synthesis of the block copolymer with polyaspartic acid as the functional carboxyl chain end has great significance.
Disclosure of Invention
The purpose of the invention is as follows: the first purpose of the invention is to provide a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA.
The second purpose of the invention is to provide a preparation method of the PEG-b-PASp-g-CPA.
The third purpose of the invention is to provide the application of the PEG-b-PASp-g-CPA as an anti-tumor nano-carrier.
The fourth purpose of the invention is to provide a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA nano micelle.
The fifth object of the present invention is to provide a method for preparing the above nanomicelle.
A sixth object of the present invention is to provide the use of the above nanomicelle.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA has the following structural formula:
the PASp end of the polyethylene glycol-polyaspartic acid (PEG-b-PASp) is modified with different groups, so as to respond to different tumor microenvironments.
Wherein the CPA is N- (2- ((2-aminoethyl) disulfanyl) ethyl) -3- (piperidin-1-yl) propionamide.
As a preferred embodiment of the present invention, the polymerization degree of the polyethylene glycol-polyaspartic acid is 20 to 60.
More preferably, the polymerization degree of the polyethylene glycol-polyaspartic acid is 30 to 60.
The invention also discloses a preparation method of the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA, firstly an amphiphilic copolymer PEG-b-PASp main chain is obtained through ring-opening polymerization reaction, then the pH/reduction dual-responsiveness micromolecule CPA is connected to the PASp end of the copolymer through side chain group reaction, and finally the PEG-b-PASp-g-CPA is obtained.
As a preferred technical scheme of the application, the synthesis method of the amphiphilic block copolymer PEG-b-PASp comprises the following steps: adding PEG-NH2Adding L-aspartic acid-beta-benzyl ester-N-carboxyl lactam (BLA-NCA) into a sealed tube, and adding an anhydrous DMF/DCM mixed solution; stirring and reacting at room temperature; after the reaction is stopped, adding anhydrous ether to generate white precipitate, and performing vacuum filtration to obtain the block copolymer PEG-b-PASp.
More preferably, the PEG-NH2And BLA-NCA in a molar ratio of 1: 80-320 parts.
More preferably, the volume ratio of the anhydrous DMF/DCM mixed solution is 1: 1.5 to 3.
More preferably, the PEG-NH2And BLA-NCA reaction time of 5-7 days.
As a preferred embodiment of the present application, the small molecule CPA is prepared as follows: firstly, dissolving cystamine dihydrochloride in methanol, adding triethylamine to adjust the pH value to be alkalescent, and stirring at room temperature for 0.5 h; dissolving di-tert-butyl dicarbonate in methylAdding alcohol into the mixed solution drop by drop; after stirring at room temperature for 12h, the mixture was evaporated under reduced pressure and the residue was poured into saturated NaH2PO4In solution; extracting with diethyl ether for three times to remove double protection byproducts, adjusting the pH of the water phase to be alkalescent with 1M NaOH, extracting with ethyl acetate for three times, combining the organic phases, and carrying out rotary evaporation under reduced pressure to obtain a crude product; purifying by column chromatography to obtain compound 1; dissolving 1, 1-piperidine propionic acid and HATU in DMF in proportion, and adding triethylamine to adjust pH to alkalescence; reacting at room temperature for 5h, after TLC detection reaction is completed, pouring the reaction liquid into five times of equivalent of distilled water, extracting with ethyl acetate for three times, combining organic phases, and carrying out reduced pressure rotary evaporation to obtain a crude product; and dissolving the obtained crude product in a mixed solution of trifluoroacetic acid/dichloromethane for deprotection, reacting at room temperature for 1h, carrying out reduced pressure rotary evaporation, and purifying by column chromatography to obtain CPA.
More preferably, the molar ratio of cystamine dihydrochloride to di-tert-butyl dicarbonate is 1: 1 to 2.
More preferably, the volume ratio of the trifluoroacetic acid/dichloromethane mixed solution is 1: 3 to 5.
As a preferred technical scheme of the application, the PEG-b-PASp-g-CPA is synthesized as follows: dissolving PEG-b-PASp and excessive CPA in DMF, adding condensing agents HATU and triethylamine, and reacting at room temperature; after the reaction is stopped, filling the reaction solution into a dialysis bag, and dialyzing to remove small molecular impurities in the reaction solution; and finally, freeze-drying to obtain PEG-b-PASp-g-CPA.
More preferably, the molar ratio of the PEG-b-PASp to the CPA is determined by the mole number of carboxyl groups on the PASp branch chain, and the mole number of the CPA is twice of the mole number of the carboxyl groups.
More preferably, the reaction time of the PEG-b-PASp and the CPA is two days.
The invention also provides a pH/reduction dual-responsiveness nano micelle which mainly comprises PEG-b-PASp or the PEG-b-PASp-g-CPA.
As a preferred technical scheme of the application, the particle size of the PEG-b-PASp and PEG-b-PASp-g-CPA micelle is 100-250 nm.
A preparation method of pH/reduction dual-responsiveness nano-micelle comprises the following steps: dissolving PEG-b-PASp or PEG-b-PASp-g-CPA polymer in DMF respectively, slowly dropwise adding deionized water, and stirring; and transferring the solution to a 3500DA dialysis bag with the molecular weight cutoff after the dropwise addition is finished, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample.
Preferably, the PEG-b-PASp and the PEG-b-PASp-g-CPA are prepared by the method of the invention.
The invention also protects a carrier material of the nano-micelle for drug delivery.
Advantageous effects
The invention has the following advantages:
(1) the amino group in the amino polyethylene glycol is used as a nucleophilic initiation group to react with the active monomer BLA-NCA, so that the polymerization monomer can be initiated in one step conveniently, and the synthesis reaction condition is simple and the yield is high.
(2) The nano micelle is prepared by a dialysis method, the particle size can be controlled and distributed uniformly, and the repeatability is good.
(3) The pH/reduction dual-responsive group CPA is connected, so that the carrier material can sensitively respond to a tumor microenvironment and has good biocompatibility.
The amphiphilic block copolymer and the nano micelle thereof have good biodegradability, biocompatibility and tumor microenvironment responsiveness. The nano-micelle can be used for targeted delivery of in-vivo tumor drugs, and when the drug-loaded nano-micelle reaches a tumor part through blood circulation, the drug-loaded nano-micelle firstly responds to the low pH value of a tumor microenvironment, so that the piperidine group in the CPA is protonated to further realize the conversion of the surface charge of the nano-micelle from negative to positive, and the nano-micelle with positive charge is easier to be taken up by tumor cells. After the nano-micelle enters the tumor cell, the disulfide bond in the CPA can respond to high-concentration reductive glutathione in the tumor cell to break, so that the nano-micelle is disintegrated, the loaded drug is fully released, and the anti-tumor effect is further realized.
Drawings
FIG. 1 is a nuclear magnetic spectrum of PEG-B-PASp of example 1, FIG. 1B is a nuclear magnetic spectrum of PEG-B-PASp of example 2, FIG. 1C is a nuclear magnetic spectrum of PEG-B-PASp of example 3, FIG. 1D is a nuclear magnetic spectrum of PEG-B-PASp of example 4, FIG. 1E is a nuclear magnetic spectrum of PEG-B-PASp of comparative example 1, FIG. 1F is a nuclear magnetic spectrum of CPA of example 5, and FIG. 1G is a nuclear magnetic spectrum of PEG-B-PASp-G-CPA of example 6;
FIG. 2 shows PEG-b-PASp58-g-CPA49A particle size change diagram of the micelle under different pH conditions and in the presence or absence of GSH;
FIG. 3 shows PEG-b-PASp58-g-CPA49Electron microscopy of nanoparticles;
FIG. 4 is data of cytotoxicity experiments for copolymers; wherein, FIG. 4A is the cytotoxicity test data of PEG-B-PASp micelle with different block ratios, and FIG. 4B is the PEG-B-PASp micelle58-g-CPA49Cytotoxicity experimental data of micelles.
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.
A preparation method of a pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA comprises the following steps:
(1) synthesis of amphiphilic Block copolymer PEG-b-PASp (reaction formula 1)
Adding PEG-NH2And L-aspartic acid-beta-benzyl ester-N-carboxylactam (BLA-NCA) was added to the block and the dry DMF/DCM mixture was added. The reaction was stirred at room temperature for 5-7 days. After the reaction was stopped, anhydrous ether was added to obtain a white precipitate. Dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2h at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp.
(2) The preparation process of the pH/reduction dual-responsiveness small molecule CPA is as follows (reaction formula 2)
Firstly, cystamine dihydrochloride is dissolved in methanol, triethylamine is added to adjust the pH value to be alkalescent, and the mixture is stirred for 0.5h at room temperature. Di-tert-butyl dicarbonate is dissolved in methanol and then added dropwise to the above mixed solution. After stirring at room temperature for 12h, the mixture was evaporated under reduced pressure and the residue was poured into saturated NaH2PO4In solution. Extraction with diethyl ether three times to remove bisProtecting the by-products, adjusting the pH of the water phase to 9 by using 1M NaOH, extracting by using ethyl acetate for three times, combining organic phases, and carrying out reduced pressure rotary evaporation to obtain a crude product. Purifying by column chromatography (mobile phase methanol/dichloromethane, volume ratio 1/60) to obtain compound 1; the compound 1, 1-piperidine propionic acid and HATU are dissolved in DMF according to the proportion, and triethylamine is added to adjust the pH value to be alkalescent. Reacting for 5 hours at room temperature, after TLC detection reaction is completed, pouring the reaction liquid into five times of equivalent of distilled water, extracting for three times by ethyl acetate, combining organic phases, and carrying out rotary evaporation under reduced pressure to obtain a crude product. And dissolving the obtained crude product in a mixed solution of trifluoroacetic acid and dichloromethane for deprotection, reacting at room temperature for 1h, performing reduced pressure rotary evaporation, and purifying by column chromatography (mobile phase methanol/dichloromethane, volume ratio 1/40) to obtain CPA.
(3) Synthesis of pH/reduction Dual-responsive Block copolymer PEG-b-PASp-g-CPA (reaction formula 3)
PEG-b-PASp and excess CPA were dissolved in DMF and the condensing agents HATU and triethylamine were added to react at room temperature for two days. After the reaction was stopped, the reaction solution was filled into a dialysis bag (MWCO ═ 3500Da), and small-molecule impurities in the reaction solution were removed by dialysis. And finally, freeze-drying to obtain PEG-b-PASp-g-CPA.
As a preferred example, in the step (1), the initiator PEG-NH2Molar ratio to BLA-NCA 1: 80-320 parts; the reaction solvent is an anhydrous DMF/DCM mixed solution (volume ratio is 1: 1.5-3).
As a preferred example, in the step (2), the mole ratio of cystamine dihydrochloride to di-tert-butyl dicarbonate is 1: 1-2; the volume ratio of the trifluoroacetic acid/dichloromethane mixed solution is 1: 3-5; the pH value of the reaction solution is about 9.
As a preferred example, in the step (3), the molar ratio of PEG-b-PAsp to CPA is determined by the number of moles of carboxyl groups on the PAsp branches, and the number of moles of CPA is twice as large as the number of moles of carboxyl groups; the reaction time was two days.
EXAMPLE 1 preparation of amphiphilic Block copolymer PEG-b-PASp
Adding PEG-NH2(0.1g, 0.05mmol) and L-aspartic acid- β -benzyl ester-N-carboxylactam (BLA-NCA) (1g, 4mmol) were added to a 250mL block tube, followed by 30mL of dry DMF/DCM mixture (volume ratio 1/2). The reaction was stirred at room temperature for 5-7 days. And after the reaction is stopped, adding anhydrous ether to generate white precipitate, dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2 hours at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp. .
Nuclear magnetic resonance (1H-NMR) (DMSO) as shown in fig. 1A indicates 3.51ppm (-CH)2CH2O-) is the characteristic peak of PEG, 8.2ppm (-CONH-), 4.52ppm (-COCH-), 2.7ppm (-COCH-)2-) belong to the characteristic peaks of PAsp. The Degree of Polymerization (DP) of PAsp was 28 and the molecular weight was 5220 as calculated by integrating the peak area at 3.51ppm of PEG main peak and the peak area at 8.2ppm of PAsp.
Taking a small amount of PEG-b-PASp28The polymer is dissolved in a small amount of DMF, and deionized water is slowly added dropwise and stirred. And after the dropwise addition is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. The particle size distribution of DLS detection is 0.172 when the particle size is 98 nm.
EXAMPLE 2 preparation of amphiphilic Block copolymer PEG-b-PASp
Adding PEG-NH2(0.1g, 0.05mmol) and L-aspartic acid- β -benzyl ester-N-carboxylactam (BLA-NCA) (2g, 8mmol) were added to a 250mL block tube, followed by 30mL of dry DMF/DCM mixture (volume ratio 1/2). The reaction was stirred at room temperature for 5-7 days. And after the reaction is stopped, adding anhydrous ether to generate white precipitate, dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2 hours at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp. Nuclear magnetic (1H-NMR) (DMSO) as shown in fig. 1B shows 3.51ppm (-CH)2CH2O-) is the characteristic peak of PEG, 8.2ppm (-CONH-), 4.52ppm (-COCH-), 2.7ppm (-COCH-)2-) belong to the characteristic peaks of PAsp. The Degree of Polymerization (DP) of PAsp was 35 and the molecular weight was 6025 as calculated from the integration ratio of the peak area at 3.51ppm of PEG main peak to the peak area at 8.2ppm of PAsp.
Taking a small amount of PEG-b-PASp35The polymer was dissolved in a small amount of DMFSlowly dropping deionized water and stirring. Transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA after the addition is finished, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. DLS detection particle size 108nm, particle size distribution 0.208.
Example 3 preparation of amphiphilic Block copolymer PEG-b-PASp
Adding PEG-NH2(0.1g, 0.05mmol) and L-aspartic acid- β -benzyl ester-N-carboxylactam (BLA-NCA) (3g, 12mmol) were added to a 250mL block tube, followed by 30mL of dry DMF/DCM mixture (volume ratio 1/2). The reaction was stirred at room temperature for 5-7 days. And after the reaction is stopped, adding anhydrous ether to generate white precipitate, dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2 hours at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp. Nuclear magnetic resonance (1H-NMR) (DMSO) as shown in fig. 1C indicates 3.51ppm (-CH)2CH2O-) is the characteristic peak of PEG, 8.2ppm (-CONH-), 4.52ppm (-COCH-), 2.7ppm (-COCH-)2-) belong to the characteristic peaks of PAsp. The Degree of Polymerization (DP) of PAsp was 46 and the molecular weight was 7290 as calculated by integrating the peak area at 3.51ppm of PEG main peak and the peak area at 8.2ppm on PAsp.
Taking a small amount of PEG-b-PASp46The polymer is dissolved in a small amount of DMF, and deionized water is slowly added dropwise and stirred. And after the dropwise addition is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. DLS detection particle size 122nm, particle size distribution 0.154.
Example 4 preparation of amphiphilic Block copolymer PEG-b-PASp
Adding PEG-NH2(0.1g, 0.05mmol) and L-aspartic acid- β -benzyl ester-N-carboxylactam (BLA-NCA) (4g, 16mmol) were added to a 250mL block tube, followed by 30mL of dry DMF/DCM mixture (volume ratio 1/2). The reaction was stirred at room temperature for 5-7 days. And after the reaction is stopped, adding anhydrous ether to generate white precipitate, dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2 hours at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp. Nuclear magnetic resonance (1H-NMR) (DMSO) as shown in fig. 1D indicates 3.51ppm (-CH)2CH2O-) is the characteristic peak of PEG, 8.2ppm (-CONH-), 4.52ppm (-CONH-)COCH-)，2.7ppm(-COCH2-) belong to the characteristic peaks of PAsp. The Degree of Polymerization (DP) of PAsp was 58 and the molecular weight was 8670 as calculated from the integral ratio of the peak area at 3.51ppm for the PEG main peak to the peak area at 8.2ppm for PAsp.
Taking a small amount of PEG-b-PASp58The polymer is dissolved in a small amount of DMF, and deionized water is slowly added dropwise and stirred. And after the dropwise addition is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. DLS detection particle size 138nm, particle size distribution 0.255.
Comparative example 1 preparation of amphiphilic Block copolymer PEG-b-PASp
Adding PEG-NH2(0.1g, 0.05mmol) and L-aspartic acid- β -benzyl ester-N-carboxylactam (BLA-NCA) (5g, 20mmol) were added to a 250mL block tube, followed by 30mL of dry DMF/DCM mixture (volume ratio 1/2). The reaction was stirred at room temperature for 5-7 days. And after the reaction is stopped, adding anhydrous ether to generate white precipitate, dissolving the white precipitate in a hydrobromic acid/acetic acid mixed solution, reacting for 2 hours at room temperature, removing benzyl, and dialyzing to obtain the block copolymer PEG-b-PASp. Nuclear magnetic (1H-NMR) (DMSO) as shown in figure 1E indicates 3.51ppm (-CH)2CH2O-) is the characteristic peak of PEG, 8.2ppm (-CONH-), 4.52ppm (-COCH-), 2.7ppm (-COCH-)2-) belong to the characteristic peaks of PAsp. The Degree of Polymerization (DP) of PAsp was 72 and the molecular weight was 10762, calculated from the integral ratio of the peak area at 3.51ppm for the PEG main peak to the peak area at 8.2ppm for PAsp.
Taking a small amount of PEG-b-PASp72The polymer is dissolved in a small amount of DMF, and deionized water is slowly added dropwise and stirred. And after the dropwise addition is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. The particle size distribution of the particle size distribution is 0.340 when the particle size distribution is 237nm by DLS detection. The particle size is too large and is not suitable for preparing the drug-loaded micelle.
Example 5 preparation of pH/reduction Dual responsive Small molecule CPA
Cystamine dihydrochloride (1.0g, 4.44mmol) was first dissolved in 20mL of methanol, adjusted to pH 9 with triethylamine and stirred at room temperature for 0.5 h. Di-tert-butyl dicarbonate (0.97g, 4.44mmol) was dissolved in 5mL of methanolAnd dropwise adding the mixture into the mixed solution. After stirring at room temperature for 12h, the mixture was evaporated under reduced pressure and the residue was poured into 20mL of saturated NaH2PO4In solution. And (3) carrying out ether extraction for three times to remove double-protection byproducts, adjusting the pH of the water phase to 9 by using 1M NaOH, extracting for three times by using ethyl acetate, combining organic phases, and carrying out rotary evaporation under reduced pressure to obtain a crude product. Purification by column chromatography (mobile phase methanol/dichloromethane, vol 1/60) gave 0.9g of Compound 1; compound 1(0.9g, 3.57mmol), 1-piperidinepropionic acid (1.13g, 7.14mmol) and HATU (2.75g, 7.14mmol) were dissolved in 20mL DMF and adjusted to pH 9 with triethylamine. Reacting for 5 hours at room temperature, after TLC detection reaction is completed, pouring the reaction liquid into five times of equivalent of distilled water, extracting for three times by ethyl acetate, combining organic phases, and carrying out rotary evaporation under reduced pressure to obtain a crude product. The crude product was dissolved in 20mL of a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio: 1/4) to carry out deprotection, reacted at room temperature for 1 hour, then rotary evaporated under reduced pressure, and purified by column chromatography (mobile phase methanol/dichloromethane, volume ratio: 1/40) to obtain 0.2g of CPA pure product. Nuclear magnetism (1H-NMR) (CDCl3) It was shown (see FIG. 1F) that the characteristic peaks at chemical shifts of 1.42ppm and 1.55ppm were assigned to the piperidine ring and that the characteristic peaks at chemical shifts of 3.30 and 3.10ppm were assigned to cystamine. And the peak area ratio is 1: 2: 1: 1, demonstrating an equivalent ligation of the two. In addition, the chemical shift of 8.26ppm peak is the characteristic peak of amide bond, which also proves the successful connection of the two.
Example 6 preparation of pH/reduction Dual-responsive Block copolymer PEG-b-PASp-g-CPA
Mixing PEG-b-PASp58(0.1g, carboxyl group-containing 0.67mmol) and CPA (0.4g, 1.34mmol) were dissolved in 30mL of DMF, and the mixture was reacted with 1mL of HATU (0.5g, 1.34mmol) and triethylamine at room temperature for two days. After the reaction was stopped, the reaction solution was filled into a dialysis bag (MWCO ═ 3500Da), and small-molecule impurities in the reaction solution were removed by dialysis. Finally freeze-drying to obtain PEG-b-PASp58-g-CPA. Nuclear magnetism (1H-NMR) (DMSO) as FIG. 1G shows that the pH/reduction dual-responsive block copolymer was successfully synthesized. The grafting degree of CPA was 49 and the molecular weight was 22047, calculated from the ratio of the peak area of the PEG main peak 3.51ppm to the peak areas of CPA of 1.42ppm and 1.55 ppm.
Taking a small amount of PEG-b-PASp58-g-CPA49The polymer was dissolved in a small amount of DMFSlowly dropping deionized water and stirring. And after the dropwise addition is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 3500DA, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample. The DLS detection particle size is 240nm, and the particle size distribution is 0.216.
Example 7 PEG-b-PASp58-g-CPA49Evaluation of micelle responsiveness to pH/reduction
PEG-b-PAsp58-g-CPA49Ultrasonic treating micelle water solution (1mg/mL) for 30min, respectively incubating under different pH conditions and in the presence or absence of GSH, and detecting PEG-b-PASp by DLS58-g-CPA49The size of the micelle varies. The results show (as figure 2) that the micelle particle size gradually increases with the decrease of the pH value and the addition of GSH, the micelle particle size reaches 1034nm under the condition of the existence of the pH value of 5.0 and 10mM GSH, the particle size distribution is 1.0, and the phenomena of micelle swelling and disintegration are most obvious. The DLS result proves that the PEG-b-PASp is more58-g-CPA49Micelles have a sensitive response to pH/reduction.
Example 8 examination of PEG-b-PASp by MTT method58-g-CPA49Biocompatibility of micelle and PEG-b-PASp micelle with different block ratios
Cell culture: HFL-1 cells, all cultured in 10% FBS-containing high-glucose DMEM medium at 37 deg.C and 5% CO2In the incubator, when the cells grow to be full of 80% under the field of view, the cells are digested and passaged by 0.25% trypsin, and the cells in logarithmic phase are taken for subsequent experiments. HFL-1 cells were seeded in 96-well plates at a density of 800 cells per well, at 100. mu.L per well. At 5% CO2And culturing in an incubator at 37 ℃ for 48 hours. Removing the culture solution, washing with precooled PBS, adding PEG-b-PASp polymers with certain concentration and different block ratios, continuing to culture for 24h under 5 the same environment, and testing six duplicate wells in parallel at the same concentration. Incubation was continued for 4h by adding 20. mu.L of MTT solution before the end of the incubation. At the end of incubation, the solution was aspirated and dissolved with 150 μ L of DMSO solution. After standing for 10min, absorbance was measured at 490nm and cell viability was calculated. Survival rate (dose-blank OD value)/(negative control OD value-blank OD value) × 100%.
The results show (see FIG. 4) that PEG-b-PASP58-g-CPA49The micelle and the PEG-b-PASp micelle with different block ratios have no obvious toxicity to HFL-1 cells and have good biocompatibility.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.
Claims (10)
1. A pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA is characterized in that the structural formula is as follows:
different groups are modified at the PASp end of the PEG-b-PASp so as to respond to different tumor microenvironments.
2. The pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 1, wherein the polymerization degree of the polyethylene glycol-polyaspartic acid is 20 to 60.
3. The preparation method of the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA as claimed in claim 1, wherein the amphiphilic block copolymer PEG-b-PASp main chain is obtained by ring-opening polymerization reaction, and then the pH/reduction dual-responsiveness small molecule CPA is connected to the PASp end of the copolymer by side chain group reaction, and finally the PEG-b-PASp-g-CPA is obtained.
4. The method for preparing the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 3, wherein the amphiphilic block copolymer PEG-b-PASp is synthesized by the following steps: adding PEG-NH2Adding L-aspartic acid-beta-benzyl ester-N-carboxyl lactam (BLA-NCA) into a sealed tube, and adding an anhydrous DMF/DCM mixed solution; stirring and reacting at room temperature; after the reaction is stoppedAdding anhydrous ether to generate white precipitate, and vacuum filtering to obtain block copolymer PEG-b-PASp.
5. The method for preparing the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 4, wherein the PEG-NH is added2And BLA-NCA in a molar ratio of 1: 80-320 parts; preferably, the volume ratio of the anhydrous DMF/DCM mixed solution is 1: 1.5 to 3; preferably, the PEG-NH2And BLA-NCA reaction time of 5-7 days.
6. The method for preparing the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 3, wherein the small molecule CPA is prepared as follows: firstly, dissolving cystamine dihydrochloride in methanol, adding triethylamine to adjust the pH value to be alkalescent, and stirring at room temperature; dissolving di-tert-butyl dicarbonate in methanol and then dropwise adding the solution into the mixed solution; after stirring at room temperature, the mixture was rotary-evaporated under reduced pressure, and the residue was poured into saturated NaH2PO4In solution; extracting with diethyl ether for three times to remove double protection byproducts, adjusting the pH of the water phase to be alkalescent with 1M NaOH, extracting with ethyl acetate for three times, combining the organic phases, and carrying out rotary evaporation under reduced pressure to obtain a crude product; purifying by column chromatography to obtain compound 1; dissolving 1, 1-piperidine propionic acid and HATU in DMF in proportion, and adding triethylamine to adjust pH to alkalescence; reacting at room temperature, after TLC detection reaction is completed, pouring the reaction solution into five times of equivalent of distilled water, extracting with ethyl acetate for three times, combining organic phases, and carrying out reduced pressure rotary evaporation to obtain a crude product; and dissolving the obtained crude product in a mixed solution of trifluoroacetic acid/dichloromethane for deprotection, reacting at room temperature for 1h, carrying out reduced pressure rotary evaporation, and purifying by column chromatography to obtain CPA.
7. The method for preparing the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 6, wherein the molar ratio of the cystamine dihydrochloride to the di-tert-butyl dicarbonate is 1: 1-2; preferably, the volume ratio of the trifluoroacetic acid/dichloromethane mixed solution is 1: 3 to 5.
8. The method for preparing the pH/reduction dual-responsiveness block copolymer PEG-b-PASp-g-CPA according to claim 3, wherein the PEG-b-PASp-g-CPA is synthesized as follows: dissolving PEG-b-PASp and excessive CPA in DMF, adding condensing agents HATU and triethylamine, and reacting at room temperature; after the reaction is stopped, filling the reaction solution into a dialysis bag, and dialyzing to remove small molecular impurities in the reaction solution; finally, freeze-drying to obtain PEG-b-PASp-g-CPA; preferably, the molar ratio of the PEG-b-PASp to the CPA is determined by the mole number of carboxyl on a PASp branched chain, and the mole number of the CPA is twice of the mole number of the carboxyl; preferably, the reaction time of the PEG-b-PASp and the CPA is two days.
9. The pH/reduction dual-responsiveness segmented copolymer nano micelle is characterized in that the main component is PEG-b-PASp or PEG-b-PASp-g-CPA; preferably, the particle size of the PEG-b-PASp and PEG-b-PASp-g-CPA micelle is 100-250 nm; preferably, the preparation method of the nano-micelle comprises the following steps: dissolving the PEG-b-PASp of claim 4 or the PEG-b-PASp-g-CPA polymer of claim 1 in DMF, slowly adding deionized water dropwise, and stirring; and transferring the solution to a 3500DA dialysis bag with the molecular weight cutoff after the dropwise addition is finished, removing the organic solvent to obtain a micelle aqueous solution, and freeze-drying to obtain a micelle sample.
10. A carrier material for drug delivery of the nanomicelle of claim 9.
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