CN112210027B

CN112210027B - Precursor polymer of cleavable material, cleavable material and preparation method thereof

Info

Publication number: CN112210027B
Application number: CN202011089781.8A
Authority: CN
Inventors: 高庆; 王鹏; 简宇航
Original assignee: Suzhou Yongqinquan Intelligent Equipment Co ltd
Current assignee: Suzhou Yongqinquan Intelligent Equipment Co ltd
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2023-10-20
Anticipated expiration: 2040-10-13
Also published as: CN112210027A

Abstract

The invention discloses a precursor polymer of a cleavable material, the cleavable material and a preparation method thereof. The prepared precursor polymer of the cleavable material is prepared by introducing a cleavable chemical bond coupled olefin functional group on a precursor polymer, so that the photo-curing property and the controllable cleavage property of the precursor material are endowed. The cleavable material prepared by the invention comprises hydrogel and a freeze-dried porous bracket thereof, and is rapidly cleaved into a solution under the action of a cleavage reagent. The cleavable material prepared by the invention has wide application prospect in the field of cell culture and tissue engineering.

Description

Precursor polymer of cleavable material, cleavable material and preparation method thereof

Technical Field

The invention relates to the field of biological materials, in particular to a precursor polymer of a cleavable material, the cleavable material and a preparation method thereof.

Background

Compared with the traditional 2D cell culture, the 3D cell culture in the scaffold materials such as hydrogel, porous scaffold, microsphere and the like can better simulate the in vivo environment. Among them, hydrogel, which is a high-water-content material, has been widely used in the fields of drug carriers, cell culture, tissue regeneration, etc., due to its structural similarity with extracellular matrix.

The ideal 3D cell carrier is capable of being rapidly disrupted after the culture is completed. However, due to the uncontrollable degradation of hydrogels, how to quickly disrupt the gel and extract the cells after 3D culture and expansion of the cells in hydrogels is a challenge to be solved.

Literature: sun M, wong JY, nugraha B, et al clear cellulosic sponge for functional hepatic cell culture and retrieval [ J ]. Biomaterials,2019,201:16-32. A material based on hydroxypropyl cellulose is described, wherein hydroxypropyl cellulose is first carboxyl functionalized with disulfide-coupled dicarboxylic acids, and then carboxyl modified double bonds are used. The gel precursor is crosslinked and solidified through gamma ray irradiation, and is freeze-dried to form the cleavable porous sponge material. The material is subjected to esterification reaction between carboxyl and hydroxyl on a polymer in the first step of carboxyl functionalization in the synthesis process, the reaction efficiency is low, and the material needs to be carried out in an organic solvent. The subsequent material curing and crosslinking adopts a gamma ray irradiation mode, the material preparation mode is complex, and the requirements of quick curing and in-situ cell culture of the hydrogel cannot be met.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a precursor polymer capable of preparing a cleavable material, wherein a precursor polymer solution can be cured in situ through light irradiation, and the cleavable chemical bond in the cleavable material is utilized to rapidly cleave the material by adding a cleavage reagent, so that cells cultured in the material can be easily recovered.

The invention also provides a cleavable material obtained from the precursor polymer.

The invention also provides a preparation method of the precursor polymer or the cleavable material.

In order to achieve the above purpose, the technical scheme provided is as follows:

a method for preparing a cleavable material precursor polymer comprising the steps of:

1-1: modifying the functional group of the polymer I to obtain a functional group modified polymer with a cleavable bond;

1-2: and (3) performing double bond modification on the functional group modified polymer with the cleavable bond prepared in the step (1-1) to obtain a double bond modified polymer with the cleavable bond.

Preferably, in step 1-1, the functional group is modified to an amino group. The cleavable bond is-S-. By the step 1-1, the amination modification of the polymer I to obtain the disulfide bond coupled amino modified polymerPolymer-ss-NH ₂ . Polymer-ss-NH prepared by step 1-2, for 1-1 ₂ Double bond modification is carried out to obtain the Polymer-ss-ene of the disulfide bond coupled olefin modified Polymer.

Preferably, the polymer I is a hydrophilic polymer with carboxyl functionality; the modification of the functional groups is more easily achieved with carboxyl functional groups.

Preferably, the polymer I may be selected from polymers having carboxyl groups in themselves; for example, polyglutamic acid or its derivatives, polyacrylic acid or its derivatives, alginic acid or its derivatives, hyaluronic acid or its derivatives, and the like can be selected. The polymer I may be a carboxyl-containing derivative of another polymer, for example, one or more of a carboxyl-containing derivative of chitosan, a carboxyl-containing derivative of gelatin, a carboxyl-containing derivative of polylysine, a carboxyl-containing derivative of silk fibroin, and the like may be selected. Further preferably, the polymer I is one or more selected from polyglutamic acid, polyacrylic acid, alginic acid, hyaluronic acid, carboxymethyl chitosan, and the like. The invention has low requirement on the molecular weight of the polymer I, and can select proper polymer I according to actual needs.

The amination modification of the polymer I described in step 1-1 is accomplished by amidation reaction between the carboxyl groups on the polymer and the amino groups of the diamine molecule (diamine compound) under the action of a coupling agent. Preferably, in step 1-1, the functional group modification of polymer I is performed as follows: under the action of a coupling agent, the carboxyl on the polymer I and one amino group of a diamine compound with a cleavable bond are subjected to amidation reaction to obtain the functional group modified polymer with the amino group on the surface.

Preferably, the diamine compound having a cleavable bond used for the amination modification of the polymer in step 1-1 is a diamine compound having-S-or a disulfide bond coupled diamine compound. As a further preference, the disulfide-coupled diamine molecule used is preferably cystamine or a derivative thereof.

Preferably, the amination modification of the polymer described in step 1-1 can be carried out by amidation reaction with a coupling agent such as a common amide reaction coupling agent, e.g., 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), NN' -Carbonyldiimidazole (CDI), etc., with the EDC+NHS combination being preferred.

Preferably, the solvent used in the amidation reaction is water; of course, other solvents that dissolve the starting materials may be used.

Preferably, the molar ratio of the polymer I (calculated by the molar amount of carboxyl groups), the coupling agent and the diamine compound is 0.5-3:1:0.5-3. Further preferably, the molar ratio of the polymer I (calculated as the molar amount of carboxyl groups), the coupling agent, and the diamine compound is 0.8 to 1:1:0.8 to 1.2.

Preferably, the amination modification of the polymer in step 1-1 is performed in an aqueous solvent at a pH of 4 to 6 (which may be adjusted by an acid-base regulator, such as dilute hydrochloric acid or dilute sulfuric acid, and an alkaline regulator, such as sodium hydroxide, sodium carbonate or sodium bicarbonate).

Preferably, the amination modification reaction in the step 1-1 is carried out for 18-30 hours at the temperature of 25-50 ℃; more preferably 18 to 24 hours.

Preferably, after the amination modification reaction in the step 1-1 is finished, the reaction solution is dialyzed by deionized water to remove impurities and then freeze-dried.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in step 1-1 ₂ Double bond modification is carried out by using Polymer-ss-NH ₂ The amidation reaction of the amino group on the molecule can be carried out by using the carboxyl-containing olefin through a coupling agent, or can be carried out by directly using an acylating agent containing double bonds.

As a further preference, in step 1-2, the double bond modification may be carried out by one of the following methods:

(A) Under the action of a coupling agent, the functional group modified polymer with a cleavable bond and the carboxyl-containing olefin compound are subjected to amidation reaction to realize the double bond modification;

(B) And carrying out amidation reaction on the functional group modified polymer with the cleavable bond and an acylating agent containing double bonds to realize the double bond modification.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in said step 1-1 ₂ When a carboxyl group-containing olefin is used, the carboxyl group-containing olefin may be one or more of acrylic acid, methacrylic acid, etc., and methacrylic acid is preferable.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in step 1-1 ₂ Double bond modification is carried out, when using carboxyl-containing olefins, polymer-ss-NH ₂ The mass ratio of the carboxyl-containing olefin to the carboxyl-containing olefin is 100:10-200 (Polymer-ss-NH is adopted ₂ The molar amount of the amino groups in the Polymer-ss-NH is calculated ₂ The molar ratio of the carboxyl-containing olefin to the EDC, HCl and NHS is 1:1-2:1-2, and the preferable molar ratio is 1:1:1.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in step 1-1 ₂ When a double bond-containing acylating agent is used, the double bond-containing acylating agent may be one or more of acryloyl chloride, methacryloyl chloride, acrylic anhydride, methacrylic anhydride, glycidyl methacrylate, etc., and among these, methacrylic anhydride is preferable.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in step 1-1 ₂ Double bond modification is carried out, when double bond-containing acylating agents are used, polymer-ss-NH ₂ The mass ratio of the double bond-containing acylating agent to the double bond-containing acylating agent is 100:10-200 (Polymer-ss-NH is adopted) ₂ The molar amount of the amino groups in the Polymer-ss-NH is calculated ₂ The molar ratio of the double bond-containing acylating agent to the double bond-containing acylating agent is 1:0.5-2).

Preferably, in the step 1-2, the double bond modification reaction is carried out for 1-18 h at the temperature of 25-50 ℃; when the carboxyl group-containing olefin is used for double bond modification, it is more preferably 4 to 8 hours; when double bond modification is performed using a double bond-containing acylating agent, it is more preferable to use the double bond-containing acylating agent for 0.5 to 2 hours.

Preferably, in the step 1-2, after the double-bonding modification reaction is finished, the reaction solution is dialyzed by deionized water to remove impurities and then freeze-dried, so that a final product Polymer-ss-ene is obtained.

A cleavable material precursor polymer prepared by the preparation method of any one of the above technical schemes.

A cleavable material obtained by curing a cleavable material precursor polymer prepared according to any one of the above-described aspects under curing conditions; or further freeze-drying the solidified product.

Preferably, the curing conditions are photo-curing. In actual curing, the corresponding photoinitiator and the light irradiation condition are required to be matched.

The cleavable material may be a hydrogel material obtainable by irradiating a cleavable material precursor polymer in a solvent, in the presence of a photoinitiator; the cleavable material can also be a freeze-dried hydrogel scaffold, and can be obtained from the corresponding hydrogel through a freeze-drying process.

Preferably, a cleavable material comprising the steps of:

2-1: the cleavable material precursor Polymer-ss-ene and the photoinitiator are dissolved in deionized water, PBS solution or cell culture medium solution.

2-2: and (3) irradiating the hydrogel precursor solution prepared in the step (2-1) by a light source to cure the hydrogel precursor solution into hydrogel. The hydrogel product can be obtained at this time.

2-3: freezing the hydrogel prepared in the step 2-2 in an environment below 0 ℃.

2-4: the frozen hydrogel prepared in step 2-3 is placed in a freeze drying agent for freeze drying.

2-5: cutting the freeze-dried hydrogel prepared in the step 2-4 into a sheet-shaped freeze-dried hydrogel bracket. The lyophilized hydrogel scaffold material can be obtained at this time.

Preferably, in the step 2-1, the mass ratio of the cleavable material precursor Polymer-ss-ene to the solvent (ion water, PBS solution or cell culture medium solution) is 0.5-10:100.

In step 2-1, preferably, the photoinitiator is one or more of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropaneketone (I2959), phenyl-2, 4, 6-trimethylbenzoylphosphine (LAP), preferably LAP.

Preferably, in the step 2-1, the mass ratio of the photoinitiator to the solvent is 0.1-1:100, and more preferably 0.2-0.5:100; still more preferably 0.25:100.

Preferably, in the step 2-2, the wavelength of the irradiation light source is 365-450 nm, and more preferably 400-420 nm; even more preferably 405nm.

Preferably, in the step 2-3, the freezing temperature is-5 to-200 ℃, and more preferably-10 to-50 ℃; still more preferably at-20 ℃.

When the hydrogel is used as a carrier for biological applications, the hydrogel precursor solution may be sterilized by filtration or the like before step 2-2.

When the hydrogel freeze-dried scaffold is used as a carrier for biological application, the prepared sheet-shaped freeze-dried hydrogel scaffold can be subjected to sterilization treatment such as ultraviolet irradiation or gamma ray irradiation after the step 2-5.

The cleavable material obtained by the invention can realize rapid cleavage under the corresponding cleavage conditions, and the cleavage process of the hydrogel or the freeze-dried porous scaffold is as follows:

3-1 after the biological application is completed, soaking the hydrogel or the freeze-dried bracket carrier thereof in PBS buffer solution containing a lysis reagent, and standing or standing on a shaking table for a period of time to realize the complete disintegration of the hydrogel or the bracket thereof.

Preferably, in step 3-1, the cleavage reagent may be one or more of Glutathione (GSH), dithiothreitol (DTT), β -thioethanol (β -ME), tris (hydroxymethyl) aminomethane phosphine (TCEP).

Preferably, in step 3-1, the lysis reagent is present in the PBS buffer at a molar concentration of 5 to 50mM.

The prepared precursor polymer of the cleavable material is prepared by introducing a cleavable chemical bond coupled olefin functional group on a precursor polymer, so that the photo-curing property and the controllable cleavage property of the precursor material are endowed. The cleavable material prepared by the invention comprises hydrogel and a freeze-dried porous bracket thereof, and is rapidly cleaved into a solution under the action of a cleavage reagent. The cleavable material prepared by the invention has wide application prospect in the field of cell culture and tissue engineering.

Drawings

FIG. 1 is a schematic diagram of a cleavable material precursor polymer synthesis process;

FIG. 2 is a schematic diagram of a curing and cracking process of a cleavable material;

FIG. 3 is a digital photograph of the hydrogel prepared in example 1 and its freeze-dried scaffold cleavage process;

fig. 4 is a freeze-dried stent SEM picture.

Detailed Description

In order that the invention may be more readily understood, the invention is further described in connection with the following drawings and examples:

example 1: see fig. 1 and 2

1. Preparation of cleavable precursor polymers:

1) 0.5g (6.579 mmol in terms of-COOH) of a polyacrylic acid as a starting polymer (average molecular weight Mw of polyacrylic acid: 4,000,000) was dissolved in 100mL of deionized water to prepare a 0.5% (w/v) aqueous solution;

2) 1.482g (6.579 mmol) of cystamine dihydrochloride is added for full dissolution, and then 0.1M sodium hydroxide is used for adjusting the pH value to 4-6;

3) NHS 0.757g (6.579 mmol) and EDC.HCl 1.261g (6.579 mmol) were added in sequence to dissolve well;

4) Stirring at room temperature for reaction for 24 hours;

5) Dialyzing the reaction solution with deionized water for 2 days;

6) Lyophilizing to obtain Polymer-ss-NH ₂ 。

7) Taking the above lyophilized product Polymer-ss-NH ₂ 0.25g was dissolved in 10mL deionized water.

8) 0.05g (0.58 mmol) of methacrylic acid was added to dissolve thoroughly

9) 0.067g (0.58 mmol) of NHS and 0.111g (0.58 mmol) of EDC.HCl equimolar to methacrylic acid were added in order and fully dissolved;

10 Stirring at room temperature for reaction for 5h.

11 Dialyzing the reaction solution with deionized water for 2 days;

12 Freeze-drying to obtain Polymer-ss-ene.

2. Preparation of cleavable hydrogel and freeze-dried bracket thereof

1) The precursor Polymer ss-ene 0.1g and LAP 0.025g prepared above were added to 10mL of PBS buffer, and dissolved in the dark to prepare a hydrogel precursor solution (Polymer ss-ene concentration 1% w/w).

2) The precursor solution was properly taken and put into a cylindrical mold having a diameter of 8mm and a height of 4mm, and was irradiated with blue light of 405nm for 30 seconds to be cured into a hydrogel.

3) The hydrogels were frozen at-20 ℃ for 3h, and then the frozen samples were transferred to a lyophilizer for lyophilization.

4) The upper and lower surfaces of the lyophilized hydrogel were cut off to obtain cylindrical porous lyophilized gel scaffold sheets having a height of about 2mm, and fig. 4 is an SEM photograph of the lyophilized scaffold.

3. Cleavage of hydrogels and their freeze-dried scaffolds

The hydrogel and the freeze-dried scaffold prepared according to the preparation method are placed in a 24-well plate, and 1mL of PBS solution of a lysis reagent is added. The lysis reagent was DTT at a concentration of 25mM. The hydrogel and its scaffold were observed for cleavage at room temperature, and PBS solution without reagent was used as a control group. The results are shown in FIG. 3 and Table 2.

Examples 2 to 9 have the same main procedure as in example 1, except that some experimental parameters were changed, the specific experimental parameters are shown in Table 1

TABLE 1 list of parameters for examples 2-9

TABLE 2 time required for complete cleavage of hydrogels and lyophilized scaffolds prepared in examples 1 to 9

Preparation of the cleavable precursor polymers, cleavable hydrogels and their lyophilized scaffolds of examples 2-10 were accomplished according to the parameters of table 1 and the methods provided in example 1, while full cleavage time detection of hydrogels and their lyophilized scaffolds was performed according to the steps provided in example 1. The results are shown in Table 2, and it is clear from Table 2 that the cleavable materials prepared by the method of the present invention can achieve rapid cleavage, and particularly the cleavage time of the obtained freeze-dried gel scaffold is within half an hour.

Example 10

1. Preparation of cleavable precursor polymers:

1) 0.5g (2.381 mmol as-COOH) of hyaluronic acid, an initial polymer, was dissolved in 100mL of deionized water to prepare a 0.5% (w/w) aqueous solution;

2) Adding 0.536g (2.381 mmol) of cystamine dihydrochloride, fully dissolving, and then adjusting the pH to 4-6 by 0.1M sodium hydroxide;

3) NHS 0.548g (4.762 mmol) and EDC.HCl 0.912g (4.762 mmol) were added in sequence to dissolve well;

4) Stirring at room temperature for reaction for 18h;

5) Dialyzing the reaction solution with deionized water for 2 days;

6) Lyophilizing to obtain Polymer-ss-NH ₂ 。

8) 0.05g (0.58 mmol) of methacrylic anhydride was added to dissolve the mixture sufficiently

9) The reaction was stirred at room temperature for 1h.

10 Dialyzing the reaction solution with deionized water for 2 days;

11 Freeze-drying to obtain Polymer-ss-ene.

2. Preparation of cleavable hydrogel and freeze-dried bracket thereof

1) The prepared precursor Polymer ss-ene 0.1g and LAP 0.025g were added to 10mL PBS buffer, and dissolved in the dark to prepare a hydrogel precursor solution.

2) The precursor solution was properly taken and put into a cylindrical mold having a diameter of 8mm and a height of 4mm, and was cured into a hydrogel by irradiation with blue light of 405nm for 30 seconds.

4) The upper and lower surfaces of the lyophilized hydrogel were cut off to obtain cylindrical porous lyophilized gel scaffold sheets having a height of about 2 mm.

3. Cleavage of hydrogels and their freeze-dried scaffolds

The hydrogel and lyophilized scaffold prepared according to the above method were placed in a 24-well plate, and 1mL of a PBS solution of a lysis reagent was added. The lysis reagent was DTT at a concentration of 25mM. Standing at room temperature, and observing the cracking condition of the hydrogel and the stent.

TABLE 3 list of parameters for examples 2-9

TABLE 4 hydrogels prepared in examples 10 to 16 and the time required for complete cleavage of the lyophilized porous scaffold thereof

Preparation of the cleavable precursor polymers, cleavable hydrogels and their lyophilized scaffolds of examples 11-16 were accomplished according to the parameters of table 3 and the methods provided in example 10, while full cleavage time detection of hydrogels and their lyophilized scaffolds was performed according to the procedure provided in example 10. The results are shown in Table 4, and it can be seen from Table 4 that the cleavable materials prepared by the method of the present invention can achieve rapid cleavage, and in particular, the cleavage time of the obtained lyophilized gel scaffold is within half an hour. Meanwhile, the double bond modification is performed by adopting an acylating reagent, the reaction time is fast, and the cracking speed of the hydrogel or the freeze-dried bracket is also fast.

Claims

1. A method for preparing a precursor polymer of a cleavable material, comprising the steps of:

1-2: double bond modification is carried out on the functional group modified polymer with the cleavable bond prepared in the step 1-1 to obtain a double bond modified polymer with the cleavable bond;

the polymer I is a hydrophilic polymer containing carboxyl functional groups; the cleavable bond is-S-S-; the functional group is modified into amino;

in step 1-1, the method for modifying the functional group of the polymer I is as follows: under the action of a coupling agent, carrying out amidation reaction on carboxyl on the polymer I and one amino group of a diamine compound with a cleavable bond to obtain a functional group modified polymer with the amino group on the surface; the diamine compound with a cleavable bond is a diamine compound containing-S-S-; the amination modification reaction in the step 1-1 is carried out for 18-24 hours at the temperature of 25-50 ℃;

in step 1-2, the double bond modification may be performed by one of the following methods:

(B) Carrying out amidation reaction on the functional group modified polymer with the cleavable bond and an acylating agent containing double bonds to realize double bond modification;

in the step 1-2, when the carboxyl-containing olefin is used for double bond modification, the double bond modification reaction is carried out for 4-8 hours at the temperature of 25-50 ℃; when double bond modification is carried out by using an acylating agent containing double bonds, the double bond modification reaction is carried out for 0.5-2 hours at the temperature of 25-50 ℃;

the polymer I is one or more selected from polyglutamic acid or derivatives thereof, polyacrylic acid or derivatives thereof, alginic acid or derivatives thereof, hyaluronic acid or derivatives thereof, carboxyl-containing derivatives of chitosan, carboxyl-containing derivatives of gelatin, carboxyl-containing derivatives of polylysine and carboxyl-containing derivatives of silk fibroin;

the diamine compound is cystamine or a derivative thereof; the solvent adopted in the amidation reaction is water; the coupling agent is selected from the group consisting of 1-ethyl- (3-dimethylaminopropyl) carbodiimide in combination with N-hydroxysuccinimide, NN' -carbonyldiimidazole;

the molar ratio of the polymer I to the coupling agent to the diamine compound is 0.8-1:1:0.8-1.2, wherein the amount of the polymer I is calculated by the molar amount of carboxyl;

the carboxyl-containing olefin compound comprises one or more of acrylic acid and methacrylic acid; the double bond-containing acylating reagent comprises one or more of acryloyl chloride, methacryloyl chloride, acrylic anhydride and methacrylic anhydride.

2. A cleavable material precursor polymer prepared by the method of claim 1.

3. A cleavable material obtained by curing the cleavable material precursor polymer of claim 2 under curing conditions; or further freeze-drying the solidified product.

4. A cleavable material according to claim 3, wherein the curing conditions employ photocuring; the photoinitiator used for photocuring is one or more of 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone (I2959) and phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate; the wavelength of the irradiation light source is 365-450 nm.