CN113754730B

CN113754730B - Polypeptide capable of self-assembling to form PH-responsive drug-loaded hydrogel, preparation method and application thereof

Info

Publication number: CN113754730B
Application number: CN202111096041.1A
Authority: CN
Inventors: 葛亮; 朱颖; 沈娟
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2022-11-08
Anticipated expiration: 2041-09-17
Also published as: CN113754730A

Abstract

The invention discloses a polypeptide capable of self-assembling to form PH response drug-loaded hydrogel, a preparation method and application thereof, wherein the polypeptide sequence is as follows: FOVVVEF, where F = phenylalanine, O = ornithine, V = valine, E = glutamic acid. The polypeptide with seven amino acids is synthesized by a solid phase synthesis method, and the polypeptide self-assembles to form polypeptide hydrogel wrapping the doxorubicin hydrochloride. The polypeptide has good biocompatibility and biodegradability and good mechanical property, can achieve a slow release effect after being wrapped by a medicament, has higher sensitivity to PH, and can realize targeted slow release administration of an anti-tumor medicament under the stimulation of an acid tumor microenvironment, improve the chemotherapy effect, improve the anti-tumor effect and reduce toxic and side effects by injecting the medicament-carrying gel around a tumor in an in-situ injection mode at a tumor part.

Description

Polypeptide capable of self-assembling to form PH-responsive drug-loaded hydrogel, preparation method and application thereof

Technical Field

The invention relates to a biomedical material, a preparation method and application thereof, in particular to polypeptide and hydrogel capable of forming PH response drug-loaded hydrogel through self-assembly, and a preparation method and application thereof.

Background

At present, the cancer is mainly treated by operation treatment, chemotherapy, radiotherapy and the like. The traditional chemical drug therapy has many problems and defects, such as high metabolism speed, poor bioavailability, unsatisfactory drug absorption and distribution, poor selectivity, large toxic and side effects and the like, and is always the limitation of cancer combined therapy. In order to overcome the above disadvantages, more and more novel drug-loaded materials have been developed. The polypeptide hydrogel is used as a special drug carrier, and has attracted great attention due to excellent biocompatibility, controllable degradability and slow control property. The injectable polypeptide hydrogel can be directly injected into a human body through an injector or a catheter, and is used as a carrier for local and continuous administration, so that the injectable polypeptide hydrogel has good injectability, can delay the release of a medicament, and has the effect of improving the treatment effect.

With the continuous and deep research and exploration, the intelligent response type polypeptide hydrogel is very popular with people. The intelligent response type polypeptide hydrogel can generate sol-gel transformation under the external physical or chemical stimulation (such as PH, temperature, solvent, pressure, illumination and ionic strength). Therefore, according to the characteristics of the tumor microenvironment (such as low PH and high glutathione content), the polypeptide hydrogel carrier capable of intelligently responding in the tumor microenvironment is designed, and the polypeptide hydrogel carrier has wide application prospect in the aspect of the controlled release of anti-cancer chemical drugs.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide polypeptide and hydrogel which have better biocompatibility, degradability, mechanical properties and the like and can be self-assembled to form PH-responsive drug-loaded hydrogel.

The invention also aims to provide a preparation method and application of the polypeptide and hydrogel which have better biocompatibility, degradability, mechanical properties and the like and can be self-assembled to form the PH-responsive drug-loaded hydrogel.

The technical scheme is as follows: the invention provides a polypeptide capable of forming a PH response drug-loaded hydrogel through self-assembly, which has the polypeptide sequence as follows: FOVVVEF, where F = phenylalanine, O = ornithine, V = valine, E = glutamic acid.

A method for preparing a PH-responsive self-assembled drug-loaded polypeptide hydrogel, which comprises the steps of dissolving the polypeptide in the claim 1 in deionized water, whirling until the polypeptide is fully dissolved, adjusting the PH, and standing at room temperature to form gel.

Further, the gelling concentration is 15-20mg/ml. The drug loading is doxorubicin hydrochloride. The standing time at room temperature is 20min-1day.

The preparation method of the polypeptide capable of self-assembling to form the PH response drug-loaded hydrogel comprises the following steps:

(1) Covalently attaching a first amino acid to the resin:

weighing the first amino acid (4 mmol) at the C terminal into a 50mL centrifuge tube, adding 20mL DMF to dissolve the first amino acid, then adding 2mL DIEA (12 mmol), shaking and shaking for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ for reaction. Then washed four times with 3 resin volumes of DMF and drained for use.

(2) Deprotection:

the resin with the first amino acid attached synthesized above was placed in a reactor, 30mL of 20% piperidine/DMF solution was added to immerse the resin, and the resin was shaken on a decolorization shaker for 20min to remove the Fmoc protecting group from the resin. After the protection is removed, washing with DMF for four times and draining. And (3) detecting a small amount of resin by an indantrione method, wherein the resin is developed (blue or bluish-purple), which indicates that the deprotection is successful.

(3) Activation and crosslinking:

weighing the second amino acid (4 mmol) and 0.541g (4 mmol) of HOBt in a 50mL centrifuge tube, adding 25mL of DMF to dissolve, then adding 0.622mL (4 mmol) of DIC, shaking for 1min, adding the solution into a reactor after the solution is clear, and placing the reactor in a shaker at 30 ℃ for reaction. After 1h, taking a small amount of resin, and detecting by an indantrione method, wherein if the resin is colorless, the reaction is complete; if the resin is developed, it is not condensed completely, and the reaction is continued. Activating carboxyl on the next amino by using activators HBTU and HOBt, and crosslinking with amino on the resin to form peptide bonds;

(4) Repeating the steps (2) and (3), repeatedly and circularly adding the monomer amino acid, and synthesizing from right to left according to the sequence of the FOVVEF sequence until the synthesis is finished;

(5) Acetylation:

fmoc on the amino group was removed with 20% piperidine in alkaline solvent to expose the amino group, 2ml of 20% acetic anhydride and 200. Mu.l DIEA were added and reacted for 30min.

(6) Elution and deprotection:

adding 95 cutting fluid (TFA/TIS/EDT/H2O = 95/2/2/1) into a reactor (10 mL of cutting fluid is added for every 1g of resin), oscillating and reacting at 0 ℃ for 0.5H, gradually heating to room temperature, reacting at room temperature for 2H, removing resin by suction filtration, washing the resin by using a small amount of lysis fluid, combining washing fluid and the lysis fluid, pouring the mixture into a centrifuge tube filled with glacial ethyl ether, centrifuging at 4 ℃ and 7000rmp for 5min after precipitation, removing supernatant, repeating the steps, and centrifuging and settling for four times to obtain a crude polypeptide. The crude product was purified by HPLC analysis, lyophilized and stored.

The hydrogel capable of self-assembling to form PH response drug-loaded hydrogel can be used for preparing anticancer drug-loaded hydrogel.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the polypeptide has short polypeptide sequence length, simple synthesis and convenient preparation of polypeptide hydrogel.

2. The polypeptide of the invention has good biocompatibility and biodegradability and good mechanical property, can achieve a slow release effect after being wrapped by a medicament, and is a potential medicament delivery carrier.

3. The PH response polypeptide hydrogel provided by the invention has higher sensitivity to PH, the drug-loaded gel is injected around the tumor by a tumor site in-situ injection mode, and the drug-loaded polypeptide hydrogel can realize targeted sustained release administration of the anti-tumor drug under the stimulation of an acid tumor microenvironment, improve the chemotherapy effect, prolong the treatment time and reduce the toxic and side effect of the anti-tumor drug on normal tissues.

Drawings

FIG. 1 is an accumulative release diagram of drug-loaded hydrogel at different drug-loaded concentrations and different pH values;

FIG. 2 is a FOVVEF sequence peptide hydrogel CD spectrum;

fig. 3 is a transmission electron micrograph of PH =5.8 FOVVVEF;

fig. 4 is a transmission electron micrograph of PH =7.4 FOVVVEF.

Detailed Description

Example 1 (please supplement the specific amounts of the following raw and auxiliary materials)

The polypeptide synthesis method of the present invention is a conventional solid phase synthesis method, that is, a method of fixing the C-terminal of a polypeptide chain on a resin by using an insoluble resin as a solid phase carrier, and sequentially condensing amino acids from the C-terminal to the N-terminal to extend the polypeptide chain.

1. Step of polypeptide Synthesis

In the first step, the first amino acid is covalently attached to the resin

Weighing 2g 2-CTC resin (with substitution degree of 0.9 mmol/g) into a 150mL reactor, soaking with 50mL EDCM for 2h to swell, then washing the resin with 3 times of DMF volume, draining, repeating the above steps for four times, and draining the resin for standby. Weighing the first amino acid (4 mmol) at the C terminal into a 50mL centrifuge tube, adding 20mL DMF to dissolve the first amino acid, then adding 2mL DIEA (12 mmol), shaking and shaking for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ for reaction. Adding 2mL of methanol after 2h, continuing to react for 0.5h for end sealing, pumping out reaction liquid, washing with DMF (dimethyl formamide) with the volume of 3 times of that of the resin for four times, and pumping out for later use

Adding condensing agents HBTU and HOBt to ensure that the carboxyl terminal of the protected amino acid and resin form common ester to complete the fixation of the amino acid;

second, deprotection

Removing Fmoc on the amino group by using 20% piperidine in an alkaline solvent to expose the amino group;

third step, activation and crosslinking

Activating carboxyl on the next amino by using activators HBTU and HOBt, and crosslinking with amino on the resin to form peptide bonds;

and fourthly, repeating the second step and the third step, repeatedly and circularly adding the monomer amino acid, and synthesizing from right to left according to the sequence of the FOVVEF sequence until the synthesis is finished.

The fifth step of acetylation

Removing Fmoc on amino by using an alkaline solvent of 20% piperidine to expose the amino, adding 2ml of 20% acetic anhydride and 200 microliters of DIEA, and reacting for 30min;

sixth step, elution and deprotection

And (3) eluting the peptide chain from the resin by using a deprotection agent, namely trifluoroacetic acid (TFA), removing a protecting group, analyzing and purifying by HPLC, and freeze-drying and storing.

2. Preparation method of self-assembled polypeptide hydrogel

And dissolving the synthesized polypeptide in deionized water, vortexing until the polypeptide is sufficiently dissolved to form a polypeptide solution with the concentration of 20mg/ml, adjusting the pH with a 0.5M NaOH solution, and standing at room temperature for 20min to form the pH-responsive self-assembled polypeptide hydrogel. Example 2

The synthesis of the polypeptide was performed as in example 1. Dissolving the synthesized polypeptide in deionized water, vortexing to fully dissolve the polypeptide to obtain a polypeptide solution with a polypeptide concentration of 10mg/ml, adjusting the pH of the polypeptide solution to 7.4 by using a 0.5M NaOH solution, and standing for 20min at room temperature to form the gel.

Example 3

The synthesis of the polypeptide was performed as in example 1. And dissolving the synthesized polypeptide in deionized water, vortexing until the polypeptide is sufficiently dissolved to obtain a polypeptide solution with the polypeptide concentration of 15mg/ml, adjusting the pH of the polypeptide solution to 7.4 by using a 0.5M NaOH solution, and standing for 20min at room temperature to form the gel.

Example 4

The synthesis of the polypeptide was performed as in example 1. Dissolving the synthesized polypeptide in deionized water, vortexing to fully dissolve the polypeptide to obtain a polypeptide solution with the polypeptide concentration of 20mg/ml, adjusting the pH of the polypeptide solution to 7.4 by using a 0.5M NaOH solution, and standing for 20min at room temperature to form the gel.

Comparative examples

Comparative examples 2-4 PH-responsive polypeptide hydrogels prepared at different polypeptide concentrations are shown in table 1.

TABLE 1 PH-responsive polypeptide hydrogels prepared at different polypeptide concentrations

Table 1 shows that the concentration of the polypeptide has a large influence on the gelling properties. The concentration range of the polypeptide gel forming is 15-30mg/ml, in the concentration range, the higher the concentration of the polypeptide solution is, the shorter the gel forming time is, but when the concentration of the polypeptide is as high as 30mg/ml, the formed hydrogel is in a turbid state, which indicates that at the concentration, the concentration of the polypeptide is too high, and partial polypeptide powder cannot be completely dissolved. Therefore, considering the combination of gelling time and gelling effect, we chose a polypeptide concentration of 20mg/ml, at which gelling time is shorter and a clear gel is formed.

Example 5

The polypeptide synthesis method is shown as an example 1, the polypeptide concentration is shown as an example 4, doxorubicin hydrochloride is selected as a loading drug of the polypeptide hydrogel, the synthesized polypeptides are respectively dissolved in 1mg/ml doxorubicin hydrochloride solution, the solution is swirled to be fully dissolved to obtain a drug-loaded polypeptide solution with the polypeptide concentration of 20mg/ml, the pH of the polypeptide solution is adjusted to 7.4 by using 0.5M NaOH, and the solution is kept stand at room temperature for 20min to form the pH response drug-loaded polypeptide hydrogel.

To the DOX-loaded hydrogel was added 1ml of 10mM PBS (pH 7.4 and 5.8, respectively) in a shaker at 37 deg.C, at 120rpm, and the release medium was removed at a specific time point for drug content testing and supplemented with the same volume of fresh release medium.

Example 6

The polypeptide synthesis method is shown as an example 1, the polypeptide concentration is shown as an example 4, doxorubicin hydrochloride is selected as a loading drug of the polypeptide hydrogel, the synthesized polypeptides are respectively dissolved in 2mg/ml doxorubicin hydrochloride solution, the solution is swirled to be fully dissolved to obtain a drug-loaded polypeptide solution with the polypeptide concentration of 20mg/ml, the pH of the polypeptide solution is adjusted to 7.4 by using 0.5M NaOH, and the solution is kept stand at room temperature for 20min to form the pH response drug-loaded polypeptide hydrogel.

To the DOX-loaded hydrogel was added 1ml of 10mM PBS (pH 7.4 and 5.8, respectively) in a shaker at 37 deg.C, at 120rpm, and the release medium was removed at specific time points for drug content testing and supplemented with the same volume of fresh release medium.

Comparative examples

Comparative examples 5-6, the release behavior of different drug concentrations in a PH-responsive self-assembled hydrogel was compared, as shown in figure 1.

When the PH is 5.8, the drug-loaded hydrogel with the adriamycin concentration of 2mg/ml has far greater drug release amount than the drug-loaded hydrogel with the adriamycin concentration of 1 mg/ml; at the pH of 7.4, the drug-loaded hydrogel with the adriamycin concentration of 2mg/ml and the drug-loaded hydrogel with the adriamycin concentration of 1mg/ml have no obvious difference in drug release amount. It can be seen that when the PH is 5.8, the gel structure of the polypeptide hydrogel is destroyed and is converted into a sol state, so that the originally encapsulated drug is more easily released, and therefore, the higher the doxorubicin concentration of the drug-loaded hydrogel, the higher the drug release amount. And when the pH =7.4, the polypeptide hydrogel is in a gel state, has a better encapsulation effect on the drug, and has no obvious difference in drug release amount even if the drug-loading concentration of the hydrogel is different. The result shows that the self-assembled drug-loaded polypeptide hydrogel has good PH responsiveness, is used as a carrier of an anti-tumor drug, and has a wide application prospect.

Example 7

The secondary structure of the polypeptide synthesized in example 1 was characterized by circular dichroism, with the following experimental procedures:

(1) Preparing a polypeptide solution with the concentration of 0.1 mg/ml;

(2) Respectively adjusting the pH values of the polypeptide solutions to be 7.4 and 5.8;

(3) Putting a 15uL sample into a 0.1cm quartz cuvette;

(4) Placing the quartz cuvette in a circular dichroism chromatograph, setting the scanning wavelength range to be 190-260 nm, the bandwidth to be 1nm, the response time to be 1s, the scanning speed to be 100nm/min and the temperature to be 25 ℃;

(5) After scanning, the data were rolled out and plotted.

Wherein the detection result of the circular dichroism chart is shown in figure 2. Under neutral conditions, the polypeptide presents a single negative peak shape which is specific to a B-fold structure; under the acidic condition, the polypeptide is in a chromatographic characteristic of a random coil conformation, namely, a negative peak is generated at a position with a shorter wavelength, which shows that the polypeptide can generate beta-folding under the neutral condition to form stable hydrogel, the secondary structure of the polypeptide is changed under the acidic condition, the beta-folding and the random coil structure coexist, and the short peptide is changed from a fiber network hydrogel state to a solution state in a macroscopic view. The polypeptide is proved to have good PH responsiveness.

Example 8

The microscopic morphology characterization of the polypeptide in example 1 was performed by transmission electron microscopy, and the specific experimental procedures were as follows:

(1) Preparing polypeptide hydrogel with the concentration of 20mg/ml, and respectively adjusting the pH values to 7.4 and 5.8;

(2) Diluting the polypeptide hydrogel by 10 times with ultrapure water with different pH values;

(3) Sucking a drop of polypeptide solution, gently dropping the polypeptide solution on the surface of the carbon-coated copper mesh, removing the redundant liquid by using filter paper after 1min, and drying at room temperature for 3-5min;

(4) Dropwise adding a proper amount of phosphotungstic acid solution for dyeing for 3-5min, and transferring the copper mesh to a position below an infrared lamp for drying;

(5) The microscopic morphology of the polypeptide under different pH conditions was observed by transmission electron microscopy.

The transmission electron microscope detection result is shown in fig. 3. At pH 5.8, transmission electron microscopy images of the diluted peptide hydrogels showed a loose distribution of peptide fragments in the form of long, narrow stripes, slightly coiled, within about 50nm in diameter, and between 0.5 μm and 1.5 μm in length (FIG. 3). At pH7.4, the diluted peptide hydrogel structure was in the form of an aggregated straight rod, interleaved stacked. The diameter of the band is 200nm-600nm, and the length is 2 μm-7 μm (FIG. 4). The polypeptide hydrogel has obvious microscopic morphological difference under different pH values, and shows obvious pH sensitivity. The FOVVEF peptide fragment is primarily judged to be mainly of a beta sheet structure according to an image obtained by TEM.

Example 9

The synthesis of the polypeptide was performed as in example 1. Doxorubicin hydrochloride is selected as a loading drug of the polypeptide hydrogel, the synthesized polypeptide is dissolved in 1mg/ml doxorubicin hydrochloride solution, the polypeptide solution with the concentration of 20mg/ml is formed after the polypeptide solution is dissolved by vortex, the pH value of the polypeptide solution is adjusted to 7.4 by 0.5M NaOH, and the polypeptide hydrogel is kept stand at room temperature for 20min to form the pH response polypeptide hydrogel.

Gently rinsing the surface of the gel with PBS (phosphate buffered saline) at pH7.4 for 2 times, collecting the rinsing solution, and testing the content of the drug by using an ultraviolet spectrophotometer to calculate the Entrapment Efficiency (EE) of the drug-loaded gel, wherein the calculation formula is as follows:

TABLE 3-2 encapsulation efficiency determination

(Biqu: A =0.0178X + 0.0047)

When the drug loading concentration is 1mg/ml and 2mg/ml, the encapsulation efficiency of the polypeptide hydrogel is more than 98%, which shows that the polypeptide hydrogel has good encapsulation performance.

Claims

1. A polypeptide capable of self-assembling to form a PH response drug-loaded hydrogel is characterized in that: the polypeptide sequence is: FOVVVEF, where F = phenylalanine, O = ornithine, V = valine, E = glutamic acid.

2. A preparation method of a PH response self-assembly drug-loaded polypeptide hydrogel is characterized by comprising the following steps: dissolving the polypeptide of claim 1 in deionized water, vortexing to dissolve sufficiently, adjusting the pH, and standing at room temperature to gel.

3. The method for preparing the PH-responsive self-assembled drug-loaded polypeptide hydrogel according to claim 2, wherein the method comprises the following steps: the gelling concentration is 15-20mg/ml.

4. The method for preparing the PH-responsive self-assembled drug-loaded polypeptide hydrogel according to claim 2, wherein the method comprises the following steps: the drug-loading agent is doxorubicin hydrochloride.

5. The preparation method of the PH-responsive self-assembled drug-loaded polypeptide hydrogel according to claim 2, wherein the preparation method comprises the following steps: the standing time at room temperature is 20min-1day.

6. The method for preparing the polypeptide capable of self-assembling to form the PH-responsive drug-loaded hydrogel according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(1) Covalently attaching a first amino acid to the resin:

adding condensing agents HBTU and HOBt to enable the carboxyl terminal of the protected amino acid and resin to form co-ester so as to complete the fixation of the amino acid;

(2) Deprotection:

removing Fmoc on the amino group by adopting an alkaline solvent to expose the amino group;

(3) Activation and crosslinking:

(5) Acetylation:

removing Fmoc on amino by adopting an alkaline solvent to expose the amino, adding acetic anhydride and DIEA (diethylhexyl Ether-CoA) for reaction;

(6) Elution and deprotection:

7. The method for preparing the polypeptide capable of self-assembling to form the PH-responsive drug-loaded hydrogel according to claim 6, wherein the method comprises the following steps: the alkaline solvent is 20% piperidine.

8. Use of the polypeptide of claim 1 that can self-assemble to form a PH-responsive drug-loaded hydrogel in the preparation of an anticancer drug-loaded hydrogel.