CN109369913B - PH-responsive polyaspartic acid grafted with hydrophobic amino acid and preparation method thereof - Google Patents
PH-responsive polyaspartic acid grafted with hydrophobic amino acid and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a pH responsive polyaspartic acid grafted with hydrophobic amino acid and a preparation method thereof, wherein polyaspartic acid-grafted-phenylalanine is obtained by taking polyaspartic acid and L-phenylalanine methyl ester hydrochloride as raw materials through 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide coupling reaction and alkali washing; the method comprises the following steps of taking polyaspartic acid-graft-phenylalanine and hydrophobic amino acid hydrochloride as raw materials, and carrying out coupling reaction and alkali washing to obtain a product. The hydrochloride salt of a hydrophobic amino acid comprises Nε-benzyloxycarbonyl lysine benzyl ester hydrochloride, tryptophan methyl ester hydrochloride, valine methyl ester hydrochloride and isoleucine methyl ester hydrochloride; two amino acids are continuously grafted on the side group, the polymer contains anionic carboxyl with pH responsiveness, the biocompatibility is good, and the survival rate of smooth muscle cells is over 80% when the concentration of the polymer is less than 0.1 mg/mL. Simple operation and low cost. Is applicable to the field of biomedical drug carrier materials.
Description
Technical Field
The invention relates to pH responsive polyaspartic acid grafted with hydrophobic amino acid and a preparation method thereof, in particular to preparation and application of the pH responsive polyaspartic acid grafted with hydrophobic amino acid, belonging to the field of biomedical drug carrier materials.
Background
As the pH of the solution changes, the conformation of the pH responsive polymer changes accordingly. Currently, there are a number of reports on the study of pH-responsive polymers (Kanamala M, Wilson WR, Yang M, Palmer BD, Wu Z. mechanisms and biological materials in pH-responsive structured drug delivery: A review. biological materials,2016,85, 152-. The pH-responsive polymer may be classified into an anionic polymer and a cationic polymer according to ionic properties in its structure. Wherein, the pH responsive anionic polymer comprises polyaspartic acid, polyacrylic acid, polyethyl acrylic acid, polymethacrylic acid, polysulfonamide and the like; and the pH responsive cationic polymer includes polylysine, polyhistidine, etc. In addition, pH-responsive block copolymers such as polyethylene glycol-b-polyglutamic acid-b-polymyosine are reported, and the pH-responsive polymers have important application values in the biomedical field, such as delivery of tumor-targeted drugs.
In recent years, the research reports on the preparation of amphiphilic polyamino acid grafted hydrophobic compounds and nanoparticles thereof and the application of the amphiphilic polyamino acid grafted hydrophobic compounds and nanoparticles thereof as drug carriers are increasing. Chinese patent (CN 105492494B) discloses a graft copolymer of polyamino acid and hydrophobic primary amine compound, and the obtained nanoparticles of the graft product of poly (gamma-glutamic acid) and ethyl phenylalanine are used as vaccine adjuvants. Chinese patent (CN 102875818B) discloses a graft copolymer of polyamino acid and hydrophilic and hydrophobic compounds, such as polyglutamic acid, polyaspartic acid grafted fatty alcohol of C8-C30, which can encapsulate drugs in an aqueous medium, suitable for intravenous delivery of drugs. In addition, chinese patent (CN 102369242B) discloses a polyion complex containing hydrophobic acidic polyamino acid and basic polypeptide, which can be combined with antigen into nanoparticles for use as an immunological agent.
Therefore, as a pH-responsive polymer for drug delivery, polyaspartic acid grafted with hydrophobic amino acid has biodegradability and biocompatibility, and amphiphilicity provided by hydrophilic and hydrophobic groups and pH-responsiveness provided by anionic carboxyl groups, and the anionic carboxyl group polymer is relatively less cytotoxic than cationic amino polymers. Most of the anionic polyamino acids reported at present are single hydrophobic amino acid esters grafted on the side chain, and two kinds of hydrophobic amino acids are not reported to be grafted continuously.
Disclosure of Invention
The invention aims to provide pH responsive polyaspartic acid grafted with hydrophobic amino acid and a preparation method thereof. The polyaspartic acid grafted with the hydrophobic amino acid has good biocompatibility and pH responsiveness, and is suitable for the field of biomedical drug carrier materials.
The technical scheme of the invention is as follows:
a pH-responsive polyaspartic acid grafted with a hydrophobic amino acid, having the structural formula:
in the formula (I), R is Nε-benzyloxycarbonyl lysine or tryptophan or valine or isoleucine; n is 39-800;
x is more than or equal to 0, y is more than or equal to 0, z is more than or equal to 0, and x, y and z cannot be 0 at the same time; n > x + y >0, n > x + y + z > 0.
The preparation method of the polyaspartic acid grafted with the hydrophobic amino acid takes the polyaspartic acid as a raw material, and obtains the polyaspartic acid-grafted-phenylalanine by alkali washing after grafting L-phenylalanine methyl ester hydrochloride through 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide coupling reaction;
further using polyaspartic acid-graft-phenylalanine as raw material, and making 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide coupling reaction to graft hydrophobic amino acid methyl ester hydrochloride (N)εBenzyloxycarbonyl lysine methyl ester hydrochloride, tryptophan methyl ester hydrochloride, valine methyl ester hydrochloride or isoleucine methyl ester hydrochloride), and alkaline washing to obtain a polyaspartic acid product grafted with the hydrophobic amino acid.
Wherein the molar ratio of the carboxyl of the polyaspartic acid to the L-phenylalanine methyl ester is 1: 0.6-1.2; the molar ratio of the carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid methyl ester is 1: 0.5-1.2; the molar ratio of the polyaspartic acid carboxyl to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1: 0.5-1.0.
Among them, several kinds of hydrophobic amino acid methyl ester hydrochloride (N)εBenzyloxycarbonyl lysine methyl ester hydrochloride, tryptophan methyl esterHydrochloride, valine methyl ester hydrochloride or isoleucine methyl ester hydrochloride) respectively react with polyaspartic acid-graft-phenylalanine to respectively obtain polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine, polyaspartic acid-graft-phenylalanine-graft-tryptophan, polyaspartic acid-graft-phenylalanine-graft-valine and polyaspartic acid-graft-phenylalanine-graft-isoleucine; the amino groups of the four hydrophobic amino acids are respectively subjected to random coupling reaction with carboxyl groups of the polyaspartic acid-graft-phenylalanine molecular chain side groups, and after the reaction is finished, the positions of the hydrophobic amino acids on the polymer side groups are random, and the specific structural formula is shown as (I).
The preparation method of the pH responsive polyaspartic acid grafted with the hydrophobic amino acid comprises the following steps:
1) preparing a sodium bicarbonate aqueous solution with the mass concentration of 0.21-0.63 wt% by taking water as a solvent, dissolving the carboxyl content of polyaspartic acid and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide into the sodium bicarbonate aqueous solution according to the molar ratio of 1: 0.5-1.0 to obtain a solution with the polyaspartic acid concentration of 1-2 wt%, and stirring for 30-60 min;
2) adding an L-phenylalanine methyl ester hydrochloride aqueous solution into the polyaspartic acid carboxyl and L-phenylalanine methyl ester according to the molar ratio of 1: 0.6-1.2, and reacting for 48-72h at room temperature;
3) adding 2.0-5.0 wt% of sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight, wherein the volume fraction of the sodium hydroxide ethanol solution to the reaction system is 50-80%;
4) and (3) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5-1 kDa, dialyzing for 3-5 days by using deionized water, and freeze-drying to obtain the polyaspartic acid-grafted-phenylalanine as shown in the formula (II).
In the formula (II), t is the number of polyaspartic acid-graft-phenylalanine repeating units, and x + y is t; n > t > 0; n is 39-800.
5) Taking the polyaspartic acid-graft-phenylalanine (II) obtained in the step 4) as a raw material, and enabling the carboxyl content of the polyaspartic acid and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to be in a molar ratio of 1: 0.5-1.0; dissolving the mixture in a sodium bicarbonate water solution with the mass concentration of sodium bicarbonate of 0.21-0.63 wt% to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 0.5-1.0 wt%; activating for 30-60 min at room temperature;
6) adding hydrophobic amino acid methyl ester hydrochloride (N) according to the molar ratio of carboxyl of polyaspartic acid-graft-phenylalanine to hydrophobic amino acid of 1: 0.1-1.2εBenzyloxycarbonyl lysine methyl ester hydrochloride, tryptophan methyl ester hydrochloride, valine methyl ester hydrochloride or isoleucine methyl ester hydrochloride) aqueous solution, and continuously reacting at room temperature for 48-72 h;
7) adding 2.0-5.0 wt% of sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight, wherein the volume fraction of the sodium hydroxide ethanol solution to the reaction system is 50-80%;
8) dialyzing the solution with deionized water for 3-5 days, and freeze-drying for 24-48 h to obtain the polyaspartic acid-grafted-hydrophobic amino acid shown in the formula (I).
The polyaspartic acid-graft-hydrophobic amino acid is suitable for the field of biomedical drug carrier materials.
The method comprises the steps of taking polyaspartic acid and L-phenylalanine methyl ester hydrochloride as raw materials, and obtaining polyaspartic acid-graft-phenylalanine through 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide coupling reaction and alkali washing; and then, the polyaspartic acid-graft-phenylalanine and hydrophobic amino acid are used as raw materials, and a product is obtained through coupling reaction and alkali washing. The method has the advantages of simple operation, low cost, mild reaction conditions and easily obtained experimental raw materials. The polyaspartic acid-graft-hydrophobic amino acid has adjustable hydrophilicity and hydrophobicity, pH responsiveness and good cell compatibility. The pH-responsive polyaspartic acid-graft-hydrophobic amino acid is suitable for the field of biomedical drug carrier materials.
Detailed Description
The technical solution of the present invention is further described below by the following embodiments, which are further illustrative of the present invention and do not limit the applicable scope of the present invention.
Example 1:
(1) preparation of polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine
1) Polyaspartic acid (molecular weight M)w4500) (3.00g, amount of carboxyl substance 0.026mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 4.98g,0.026mol) were dissolved in 150mL of an aqueous solution of sodium bicarbonate with a mass concentration of 0.63 wt% to obtain a solution of polyaspartic acid with a concentration of 2.0 wt%, and stirred for 30 min;
2) adding an aqueous solution of L-phenylalanine methyl ester hydrochloride (6.69g,0.031mol) according to the molar ratio of polyaspartic acid carboxyl to L-phenylalanine methyl ester hydrochloride of 1:1.2, and reacting at room temperature for 72 h;
3) adding 75mL of 5.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5kDa, dialyzing with deionized water for 3 days, purifying, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine, wherein the grafting rate of the phenylalanine is 71.8 percent, and the structural formula is as follows:
5) adding polyaspartic acid-graft-phenylalanine of the step 4) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, and adding polyaspartic acid-graft-phenylalanine (0.5g, the amount of carboxyl substances is 0.0023mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.44g,0.0023mol) and 100mL of an aqueous sodium bicarbonate solution (0.42 wt%) to obtain a solution with a polyaspartic acid-graft-phenylalanine concentration of 0.5 wt%; activating at room temperature for 60 min;
6) adding aqueous solution of carbobenzoxy lysine methyl ester hydrochloride (1.14g,0.0028mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:1.2, and continuously reacting for 56h at room temperature;
7) adding 50mL of 3.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
8) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing the solution with deionized water for 4 days, and freeze-drying for 48 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 79 percent and the grafting rate of the benzyloxycarbonyl lysine of 41.1 percent, and the structural formula is as follows:
wherein x + y is 28; x + z is 16.
(2) Preparation of polyaspartic acid-graft-phenylalanine-graft-tryptophan
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask provided with a magnetic stirring bar, wherein the polyaspartic acid-graft-phenylalanine (1.0g, the amount of carboxyl substances is 0.0046mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.44g,0.0023mol) and 100mL of sodium bicarbonate aqueous solution (0.21 wt%) are added to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 1 wt%; activating at room temperature for 30 min;
2) adding an aqueous solution of tryptophan methyl ester hydrochloride (0.59g,0.0023mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.5, and continuously reacting for 48h at room temperature;
3) adding 80mL of 2.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.8kDa, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine-graft-tryptophan with the yield of about 75 percent and the tryptophan grafting rate of 28.2 percent, and the structural formula is as follows:
wherein x + y is 28; x + z is 11.
(3) Preparation of polyaspartic acid-graft-phenylalanine-graft-valine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask provided with a magnetic stirring bar, wherein the polyaspartic acid-graft-phenylalanine (0.75g, the amount of carboxyl substances is 0.0031mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.45g,0.0024mol) and 100mL of sodium bicarbonate aqueous solution (0.45 wt%) are added to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 0.75 wt%; activating at room temperature for 45 min;
2) adding an aqueous solution of valine methyl ester hydrochloride (0.31g,0.0019mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.6, and continuously reacting for 56h at room temperature;
3) adding 80mL of 4.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, and freeze-drying for 48 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-valine with the yield of about 78 percent and the valine grafting rate of 35.9 percent, and the structural formula is as follows:
x+y＝28；x+z＝14
(4) preparation of polyaspartic acid-graft-phenylalanine-graft-isoleucine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, wherein the polyaspartic acid-graft-phenylalanine (0.80g, the amount of carboxyl substances is 0.0036mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.70g,0.0036mol) and 100mL of an aqueous sodium bicarbonate solution (0.50 wt%) are added to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 0.8 wt%; activating at room temperature for 50 min;
2) adding an aqueous solution of isoleucine methyl ester hydrochloride (0.53g,0.0029mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.8, and continuously reacting for 54h at room temperature;
3) adding 60mL of 4.5 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.8kDa, and freeze-drying for 36 hours to obtain polyaspartic acid-graft-phenylalanine-graft-isoleucine with the yield of about 79 percent and the isoleucine grafting rate of 30.8 percent, wherein the structural formula is as follows:
x+y＝28；x+z＝12
(5) performance testing
The prepared polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has pH responsiveness, is a transparent solution at a pH of 7.4, and becomes an emulsion when the pH is less than 5.8, and the polymer is aggregated to form particles; cell toxicity experiments are carried out by adopting an Alamar Blue kit, and when the concentration of the polymer is less than 0.1mg/mL, the survival rate of smooth muscle cells is over 80 percent, which shows that the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has good cell compatibility.
The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan is self-assembled at a concentration of 1.0mg/mL and at a pH of 4.8-7.4, and when the pH value is more than 5.5, the solution is changed from emulsion to transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out cytotoxicity detection experiments, and when the concentration is less than 0.2mg/mL, the survival rate of smooth muscle cells is over 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-valine self-assembled at a concentration of 1.0mg/mL and at a pH of 4.4-7.4, the solution changed from emulsion to transparent solution at a pH of greater than 4.4. The prepared polyaspartic acid-graft-phenylalanine-graft-valine adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out a cytotoxicity detection experiment, and when the concentration is less than 1.2mg/mL, the survival rate of smooth muscle cells is more than 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine self-assembles at a concentration of 1.0mg/mL at a pH of between 4.6 and 7.4, and the solution changes from an emulsion to a clear solution at a pH of greater than 4.6. The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is subjected to a cytotoxicity detection experiment by adopting an AB reagent, and when the concentration is less than 1.5mg/mL, the survival rate of smooth muscle cells is over 80%.
Example 2:
(1) preparation of polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine
1) Polyaspartic acid (molecular weight M)w46100) (1.50g, the amount of the carboxyl substance was 0.013mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 4.98g,0.026mol) were dissolved in 150mL of an aqueous solution of sodium bicarbonate having a mass concentration of 0.21 wt% to obtain a solution having a polyaspartic acid concentration of 1.0 wt%, and stirred for 60 min;
2) adding an aqueous solution of L-phenylalanine methyl ester hydrochloride (1.68g,0.0078mol) according to the molar ratio of polyaspartic acid carboxyl to L-phenylalanine methyl ester hydrochloride of 1:0.6, and reacting at room temperature for 72 h;
3) adding 80mL of 2.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing with deionized water for 3 days, purifying, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine, wherein the grafting rate of the phenylalanine is 31.5 percent, and the structural formula is as follows:
5) adding polyaspartic acid-graft-phenylalanine of the step 4) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, 1.0g of polyaspartic acid-graft-phenylalanine (amount of carboxyl substance: 0.0062mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (1.19g,0.0062mol), and 100mL of an aqueous sodium bicarbonate solution (0.42 wt%), to obtain a solution with a polyaspartic acid-graft-phenylalanine concentration of 1.0 wt%; activating at room temperature for 45 min;
6) adding aqueous solution of carbobenzoxy lysine methyl ester hydrochloride (3.01g,0.0074mol) according to the molar ratio of polyaspartic acid-graft-phenylalanine carboxyl to hydrophobic amino acid of 1:1.2, and continuously reacting for 60h at room temperature;
7) adding 50mL of 5.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
8) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5kDa, dialyzing for 5 days, and freeze-drying for 48 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 78 percent and the grafting rate of the benzyloxycarbonyl lysine of 27.5 percent, and the structural formula is as follows:
wherein x + y is 126; x + z is 110.
(2) Preparation of polyaspartic acid-graft-phenylalanine-graft-tryptophan
1) Adding polyaspartic acid-graft-phenylalanine of the step 4) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, 1.0g of polyaspartic acid-graft-phenylalanine (amount of carboxyl substance: 0.0062mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (1.19g,0.0062mol), and 100mL of an aqueous sodium bicarbonate solution (0.42 wt%), to obtain a solution with a polyaspartic acid-graft-phenylalanine concentration of 1.0 wt%; activating at room temperature for 45 min;
2) adding aqueous solution of tryptophan methyl ester hydrochloride (1.89g,0.0074mol) according to the molar ratio of polyaspartic acid-graft-phenylalanine carboxyl to hydrophobic amino acid of 1:1.2, and continuing to react for 60h at room temperature;
3) adding 50mL of 5.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5kDa, dialyzing for 3 days, and freeze-drying for 24 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 73 percent and the grafting rate of the benzyloxycarbonyl lysine of 31.0 percent, and the structural formula is as follows:
wherein x + y is 126; and x + z 124.
(3) Preparation of polyaspartic acid-graft-phenylalanine-graft-valine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask provided with a magnetic stirring bar, wherein the polyaspartic acid-graft-phenylalanine (0.60g, the amount of carboxyl substances is 0.0037mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.36g,0.0019mol) and 100mL of sodium bicarbonate aqueous solution (0.32 wt%) are added to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 0.6 wt%; activating at room temperature for 30 min;
2) adding an aqueous solution of valine methyl ester hydrochloride (0.31g,0.0015mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.4, and continuously reacting for 48 hours at room temperature;
3) adding 60mL of 3.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) loading the solution into a dialysis bag with a cut-off molecular weight of 0.5kDa, dialyzing for 4 days, and freeze-drying to obtain polyaspartic acid-graft-phenylalanine-graft-valine with a yield of about 65% and a valine grafting rate of 37.1%, wherein the structural formula is as follows:
x+y＝126；x+z＝148
(4) preparation of polyaspartic acid-graft-phenylalanine-graft-isoleucine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in (1) as a raw material to a 250mL single-neck flask equipped with a magnetic stirrer, polyaspartic acid-graft-phenylalanine (0.90g, amount of carboxyl substance 0.0056mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.96g,0.0050mol), 100mL of an aqueous sodium bicarbonate solution (0.40 wt%), to obtain a solution having a concentration of polyaspartic acid-graft-phenylalanine of 0.9 wt%; activating at room temperature for 50 min;
2) adding an aqueous solution of isoleucine methyl ester hydrochloride (0.71g,0.0039mol) according to the molar ratio of polyaspartic acid-graft-phenylalanine carboxyl to hydrophobic amino acid of 1:0.7, and continuously reacting for 48 hours at room temperature;
3) adding 50mL of 4.8 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5kDa, dialyzing for 5 days, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine-graft-isoleucine with the yield of about 77 percent and the grafting rate of isoleucine of 30.8 percent, and the structural formula is as follows:
x+y＝126；x+z＝123
(5) performance testing
The prepared polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has pH responsiveness, is a transparent solution at the pH value of 7.4, has a polymer molecular chain in an extended state, and is changed into an emulsion when the pH value is less than 5.9; cell toxicity experiments are carried out by adopting an Alamar Blue kit, and when the concentration of the polymer is less than 0.1mg/mL, the survival rate of smooth muscle cells is over 80 percent, which shows that the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has good cell compatibility.
The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan self-assembles at a concentration of 1.0mg/mL and at a pH of between 4.9 and 7.4, and when the pH is greater than 5.4, the solution changes from an emulsion to a transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out cytotoxicity detection experiments, and when the concentration is less than 0.3mg/mL, the survival rate of smooth muscle cells is over 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-valine self-assembled at a concentration of 1.0mg/mL and at a pH of 4.8-7.4, the solution changed from emulsion to transparent solution at a pH of greater than 5.0. The prepared polyaspartic acid-graft-phenylalanine-graft-valine adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out a cytotoxicity detection experiment, and when the concentration is less than 1.3mg/mL, the survival rate of smooth muscle cells is more than 80%.
The concentration of the prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is 1.0mg/mL, when the solution is self-assembled at the pH value of 4.6-7.4, and when the pH value is more than 5.2, the solution is changed from emulsion to transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is subjected to a cytotoxicity detection experiment by adopting an AB reagent, and when the concentration is less than 1.7mg/mL, the survival rate of smooth muscle cells is over 80%.
Example 3:
(1) preparation of polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine
1) Polyaspartic acid (molecular weight M)w16800) (2.0g, the amount of the carboxyl group substance was 0.017mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 2.3g,0.012mol) were dissolved in 100mL of an aqueous sodium bicarbonate solution having a mass concentration of 0.54 wt% to obtain a solution having a polyaspartic acid concentration of 2.0 wt%, followed by stirring for 50 min;
2) adding an aqueous solution of L-phenylalanine methyl ester hydrochloride (3.33g,0.015mol) according to the molar ratio of polyaspartic acid carboxyl to L-phenylalanine methyl ester hydrochloride of 1:0.9, and reacting for 72h at room temperature;
3) adding 50mL of 4.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 3 days, purifying, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine, wherein the grafting rate of the phenylalanine is 43.1 percent, and the structural formula is as follows:
5) adding polyaspartic acid-graft-phenylalanine of step 4) as a raw material to a 250mL single-neck flask equipped with a magnetic stirrer, polyaspartic acid-graft-phenylalanine (1.0g, amount of carboxyl substance 0.0056mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.86g,0.0045mol), 100mL of an aqueous sodium bicarbonate solution (0.63 wt%), to obtain a solution having a polyaspartic acid-graft-phenylalanine concentration of 1.0 wt%; activating at room temperature for 54 min;
6) adding aqueous solution of benzyloxycarbonylamino acid methyl ester hydrochloride (2.28g,0.0056mol) according to the molar ratio of polyaspartic acid-graft-phenylalanine carboxyl to hydrophobic amino acid of 1:1, and continuing the reaction at room temperature for 72 hours;
7) adding 50mL of 4.2 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
8) dialyzing the solution with deionized water for three days, and freeze-drying to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 69 percent and the grafting rate of the benzyloxycarbonyl lysine of 23.6 percent, and the structural formula is as follows:
wherein x + y is 62; x + z is 34.
(2) Preparation of polyaspartic acid-graft-phenylalanine-graft-tryptophan
1) Adding polyaspartic acid-graft-phenylalanine of the step 4) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, and adding polyaspartic acid-graft-phenylalanine (0.8g, the amount of carboxyl substances is 0.0045mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.52g,0.0027mol) and 100mL of an aqueous sodium bicarbonate solution (0.36 wt%) to obtain a solution with a polyaspartic acid-graft-phenylalanine concentration of 0.8 wt%; activating at room temperature for 30 min;
2) adding aqueous solution of tryptophan methyl ester hydrochloride (0.80g,0.0032mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.7, and continuously reacting for 48h at room temperature;
3) adding 50mL of 4.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 3 days, and freeze-drying for 48 hours to obtain the polyaspartic acid-graft-phenylalanine-benzyloxycarbonyl lysine with the yield of about 71 percent and the benzyloxycarbonyl lysine grafting rate of 37.5 percent, and the structural formula is as follows:
wherein x + y is 62; x + z 54.
(3) Preparation of polyaspartic acid-graft-phenylalanine-graft-valine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, wherein the polyaspartic acid-graft-phenylalanine (0.60g, the amount of a carboxyl substance is 0.0037mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.71g,0.0037mol) and 100mL of an aqueous sodium bicarbonate solution (0.41 wt%) are added to obtain a solution with the concentration of polyaspartic acid-graft-phenylalanine of 0.6 wt%; activating at room temperature for 45 min;
2) adding an aqueous solution of valine methyl ester hydrochloride (0.19g,0.0011mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.3, and continuously reacting for 54h at room temperature;
3) adding 60mL of 2.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 3 days, and freeze-drying for 24 hours to obtain poly-graft-phenylalanine-graft-valine with the yield of about 68 percent and the valine grafting rate of 25.0 percent, and the structural formula is as follows:
x+y＝62；x+z＝36
(4) preparation of polyaspartic acid-graft-phenylalanine-graft-isoleucine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in (1) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, and adding polyaspartic acid-graft-phenylalanine (0.90g, the amount of a carboxyl substance is 0.0056mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.75g,0.0039mol) and 100mL of an aqueous sodium bicarbonate solution (0.35 wt%) to obtain a solution with a concentration of polyaspartic acid-graft-phenylalanine of 0.9 wt%; activating at room temperature for 60 min;
2) adding an aqueous solution of isoleucine methyl ester hydrochloride (0.81g,0.0045mol) according to the molar ratio of carboxyl to hydrophobic amino acid of 1:0.8, and continuing to react for 56h at room temperature;
3) adding 50mL of 5.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 4 days, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine-graft-isoleucine with the yield of about 74 percent and the isoleucine grafting rate of 28.5 percent, wherein the structural formula is as follows:
x+y＝62；x+z＝41
(5) performance testing
The prepared polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has pH responsiveness, is a transparent solution at a pH of 7.4, and becomes an emulsion when the pH is less than 5.8; cell toxicity experiments are carried out by adopting an Alamar Blue kit, and when the concentration of the polymer is less than 0.1mg/mL, the survival rate of smooth muscle cells is over 80 percent, which shows that the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has good cell compatibility.
The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan self-assembles at a concentration of 1.0mg/mL and at a pH of 4.8-7.4, and when the pH is more than 5.1, the solution changes from emulsion to transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out cytotoxicity detection experiments, and when the concentration is less than 0.3mg/mL, the survival rate of smooth muscle cells is over 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-valine self-assembled at a concentration of 1.0mg/mL and at a pH of 4.5-7.4, the solution changed from emulsion to transparent solution at a pH of greater than 4.9. The prepared polyaspartic acid-graft-phenylalanine-graft-valine adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out a cytotoxicity detection experiment, and when the concentration is less than 1.3mg/mL, the survival rate of smooth muscle cells is more than 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine self-assembles at a concentration of 1.0mg/mL at a pH between 4.6 and 7.4, and the solution changes from an emulsion to a clear solution at a pH greater than 4.8. The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is subjected to a cytotoxicity detection experiment by adopting an AB reagent, and when the concentration is less than 1.7mg/mL, the survival rate of smooth muscle cells is over 80%.
Example 4:
(1) preparation of polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine
1) Polyaspartic acid (molecular weight M)w92300) (3.0g, amount of carboxyl substance 0.026mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 3.49g,0.018mol) were dissolved in 200mL of an aqueous solution of sodium bicarbonate with a mass concentration of 0.54 wt% to give a solution of polyaspartic acid with a concentration of 1.5 wt%, followed by stirring for 60 min;
2) adding an aqueous solution of L-phenylalanine methyl ester hydrochloride (6.73g,0.031mol) according to the molar ratio of polyaspartic acid carboxyl to L-phenylalanine methyl ester hydrochloride of 1:1.2, and reacting at room temperature for 72 h;
3) adding 100mL of 4.5 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 4 days, purifying, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine, wherein the grafting rate of the phenylalanine is 51.5 percent, and the structural formula is as follows:
5) using the polyaspartic acid-graft-phenylalanine of step 4) as a raw material, adding polyaspartic acid-graft-phenylalanine (1.5g, amount of carboxyl substance 0.0079mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (1.5g,0.0079mol), 150mL of aqueous sodium bicarbonate solution (0.53 wt%) in a 250mL single-neck flask equipped with a magnetic stirrer to obtain a solution having a polyaspartic acid-graft-phenylalanine concentration of 1.0 wt%; activating at room temperature for 60 min;
6) adding aqueous solution of carbobenzoxy amino acid methyl ester hydrochloride (3.86g,0.0095mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:1.2, and continuing to react for 60 hours at room temperature;
7) adding 80mL of 4.5 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
8) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 4 days, and freeze-drying for 24 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 64 percent and the grafting rate of the benzyloxycarbonyl lysine of 14.5 percent, and the structural formula is as follows:
wherein x + y is 412; x + z 116.
(2) Preparation of polyaspartic acid-graft-phenylalanine-graft-tryptophan
1) Using the polyaspartic acid-graft-phenylalanine of step 4) as a raw material, adding polyaspartic acid-graft-phenylalanine (1.5g, amount of carboxyl substance 0.0079mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (1.36g,0.0071mol), 100mL of aqueous sodium bicarbonate solution (0.40 wt%) in a 250mL single-neck flask equipped with a magnetic stirrer to obtain a solution with polyaspartic acid-graft-phenylalanine concentration of 0.65 wt%; activating at room temperature for 45 min;
2) adding aqueous solution of tryptophan methyl ester hydrochloride (1.21g,0.0047mol) according to the molar ratio of carboxyl to hydrophobic amino acid of 1:0.6, and continuing the reaction at room temperature for 56 h;
3) adding 60mL of 4.8 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 4 days, and freeze-drying for 24 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine with the yield of about 64 percent and the grafting rate of the benzyloxycarbonyl lysine of 27.1 percent, and the structural formula is as follows:
wherein x + y is 412; x + z 217.
(3) Preparation of polyaspartic acid-graft-phenylalanine-graft-valine
1) Adding the product of step 4) in (1) of polyaspartic acid-graft-phenylalanine (II) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, wherein the raw material comprises polyaspartic acid-graft-phenylalanine (1.5g, the amount of carboxyl substances is 0.0079mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (1.51g,0.0079mol) and 100mL of an aqueous solution of sodium bicarbonate (0.50 wt%), thereby obtaining a solution with a concentration of polyaspartic acid-graft-phenylalanine of 1.0 wt%; activating at room temperature for 54 min;
2) adding an aqueous solution of valine methyl ester hydrochloride (0.79g,0.0047mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.3, and continuously reacting for 60h at room temperature;
3) adding 50mL of 3.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 3 days, and freeze-drying for 24 hours to obtain the polyaspartic acid-graft-phenylalanine-graft-valine with the yield of about 71 percent and the valine grafting rate of 26.1 percent, and the structural formula is as follows:
x+y＝412；x+z＝209
(4) preparation of polyaspartic acid-graft-phenylalanine-graft-isoleucine
1) Adding polyaspartic acid-graft-phenylalanine (II) which is the product of the step 4) in the step (1) as a raw material into a 250mL single-neck flask equipped with a magnetic stirrer, wherein the polyaspartic acid-graft-phenylalanine (0.90g, the amount of a carboxyl substance is 0.0056mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (0.91g,0.0048mol) and 100mL of an aqueous sodium bicarbonate solution (0.35 wt%) are added to obtain a solution with a concentration of polyaspartic acid-graft-phenylalanine of 0.9 wt%; activating at room temperature for 60 min;
2) adding an aqueous solution of isoleucine methyl ester hydrochloride (0.92g,0.0050mol) according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1:0.9, and continuously reacting for 72 hours at room temperature;
3) adding 80mL of 4.0 wt% sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 1kDa, dialyzing for 3 days, and freeze-drying for 24 hours to obtain polyaspartic acid-graft-phenylalanine-graft-isoleucine with the yield of about 76 percent and the isoleucine grafting rate of 29.5 percent, wherein the structural formula is as follows:
x+y＝412；x+z＝236
(5) performance testing
The prepared polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has pH responsiveness, is a transparent solution when the pH is 7.4, has a polymer molecular chain in an extended state, and is changed into an emulsion when the pH is less than 5.7, and the polymer is aggregated to form particles; cell toxicity experiments are carried out by adopting an Alamar Blue kit, and when the concentration of the polymer is less than 0.1mg/mL, the survival rate of smooth muscle cells is over 80 percent, which shows that the polyaspartic acid-graft-phenylalanine-graft-benzyloxycarbonyl lysine has good cell compatibility.
The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan self-assembles at a concentration of 1.0mg/mL and at a pH of 4.5-7.4, and when the pH is more than 5.1, the solution changes from emulsion to transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-tryptophan adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out cytotoxicity detection experiments, and when the concentration is less than 0.4mg/mL, the survival rate of smooth muscle cells is over 80%.
The prepared polyaspartic acid-graft-phenylalanine-graft-valine self-assembled at a concentration of 1.0mg/mL and at a pH of 4.4-7.4, the solution changed from emulsion to transparent solution at a pH of greater than 4.5. The prepared polyaspartic acid-graft-phenylalanine-graft-valine adopts an Alamar Blue cell proliferation/cytotoxicity detection kit to carry out a cytotoxicity detection experiment, and when the concentration is less than 1.6mg/mL, the survival rate of smooth muscle cells is more than 80%.
The concentration of the prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is 1.0mg/mL, when the solution is self-assembled at the pH value of 4.6-7.4, and when the pH value is more than 4.8, the solution is changed from emulsion to transparent solution. The prepared polyaspartic acid-graft-phenylalanine-graft-isoleucine is subjected to a cytotoxicity detection experiment by adopting an AB reagent, and when the concentration is less than 2.1mg/mL, the survival rate of smooth muscle cells is over 80%.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention will fall within the scope of the invention.
Claims (5)
1. A pH-responsive polyaspartic acid grafted with a hydrophobic amino acid, characterized by the structural formula:
in the formula (I), R is Nε-benzyloxycarbonyl lysine or tryptophan or valine or isoleucine; n is 39-800; x is the number of>0, y is more than or equal to 0, z is more than or equal to 0, and y and z can not be 0 at the same time; n is>x+y>0,n≥x+y+z>0。
2. The method for preparing polyaspartic acid with pH responsiveness of grafting hydrophobic amino acid according to claim 1, wherein polyaspartic acid-graft-phenylalanine is prepared by using polyaspartic acid and L-phenylalanine methyl ester hydrochloride as raw materials, performing a catalytic reaction of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and then performing alkali washing; and then, the polyaspartic acid-graft-phenylalanine and hydrophobic amino acid methyl ester hydrochloride are used as raw materials, and a product is obtained through coupling reaction and alkali washing.
3. The method for preparing pH-responsive polyaspartic acid grafted with hydrophobic amino acid according to claim 2, wherein the molar ratio of the carboxyl group of polyaspartic acid to the charged L-phenylalanine methyl ester is 1: 0.6-1.2; the molar ratio of polyaspartic acid carboxyl to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1: 0.5-1.0, and the molar ratio of polyaspartic acid-graft-phenylalanine carboxyl to hydrophobic amino acid methyl ester hydrochloride is 1: 0.5-1.2; wherein the hydrophobic amino acid methyl ester hydrochloride reacts with the polyaspartic acid-graft-phenylalanine to obtain the structure shown in the formula (I).
4. The method for preparing pH-responsive polyaspartic acid grafted with hydrophobic amino acid according to claim 2, which comprises the steps of:
1) preparing a sodium bicarbonate aqueous solution with the mass concentration of 0.21-0.63 wt% by taking water as a solvent, dissolving the carboxyl content of polyaspartic acid and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide into the sodium bicarbonate aqueous solution according to the molar ratio of 1: 0.5-1.0 to obtain a solution with the polyaspartic acid concentration of 1-2 wt%, and stirring for 30-60 min;
2) adding an L-phenylalanine methyl ester hydrochloride aqueous solution into the polyaspartic acid carboxyl and L-phenylalanine methyl ester hydrochloride according to the molar ratio of 1: 0.6-1.2, and reacting for 48-72h at room temperature;
3) adding 2.0-5.0 wt% of sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight, wherein the volume fraction of the sodium hydroxide ethanol solution to the reaction system is 50-80%;
4) putting the solution into a dialysis bag with the molecular weight cutoff of 0.5-1 kDa, dialyzing with deionized water for 3-5 days, purifying, freeze-drying to obtain polyaspartic acid-graft-phenylalanine as shown in formula (II),
in the formula (II), t is the number of polyaspartic acid-graft-phenylalanine repeating units, and n is more than t and is more than 0; n is 39-800;
5) taking the polyaspartic acid-graft-phenylalanine (II) in the step 4) as a raw material, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide according to the molar ratio of the carboxyl content of the polyaspartic acid-graft-phenylalanine (II) to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide of 1: 0.5-1.0; dissolving the poly (aspartic acid) grafted phenylalanine copolymer in a sodium bicarbonate water solution with the mass concentration of 0.21-0.63 wt% of sodium bicarbonate to obtain a solution with the concentration of 0.5-1.0 wt% of the poly (aspartic acid) grafted phenylalanine copolymer; activating for 30-60 min at room temperature;
6) adding hydrophobic amino acid methyl ester hydrochloride aqueous solution according to the molar ratio of carboxyl of the polyaspartic acid-graft-phenylalanine to the hydrophobic amino acid of 1: 0.5-1.2, and continuously reacting for 48-72h at room temperature;
7) adding 2.0-5.0 wt% of sodium hydroxide ethanol solution for alkali washing, and stirring at room temperature overnight, wherein the volume fraction of the sodium hydroxide ethanol solution to the reaction system is 50-80%;
8) dialyzing the solution with deionized water for 3-5 days, and freeze-drying for 24-48 h to obtain the polyaspartic acid-graft-phenylalanine-graft-hydrophobic amino acid shown in the formula (I).
5. Use of the pH-responsive polyaspartic acid grafted with a hydrophobic amino acid according to claim 1 for the preparation of a pharmaceutical carrier material.
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