CN112210027A

CN112210027A - Cleavable material precursor polymer, cleavable material and preparation method thereof

Info

Publication number: CN112210027A
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: 2021-01-12
Anticipated expiration: 2040-10-13
Also published as: CN112210027B

Abstract

The invention discloses a cleavable material precursor polymer, a cleavable material and a preparation method thereof. The precursor polymer of the cleavable material prepared by the invention is endowed with photocuring performance and controllable cracking performance by introducing the olefin functional group coupled with the cleavable chemical bond on the precursor polymer. The cleavable material prepared by the invention comprises hydrogel and a freeze-dried porous scaffold thereof, and the cleavable material is quickly cracked into solution under the action of a cracking reagent. The cleavable material prepared by the invention has wide application prospect in the fields of cell culture and tissue engineering.

Description

Cleavable material precursor polymer, cleavable material and preparation method thereof

Technical Field

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

Background

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

The ideal 3D cell carrier is capable of being rapidly disrupted after culture is complete. However, due to the uncontrollable degradation of the hydrogel, how to rapidly break the gel and extract the cells after 3D culture and amplification of the cells in the hydrogel is a difficult problem to be solved.

The literature: sun M, Wong JY, Nugraha B, et al, clean cellular space for functional cellulosic culture and recovery [ J ] Biomaterials,2019,201: 16-32, describes a material based on hydroxypropyl cellulose, which is first carboxyl functionalized by a dicarboxylic acid coupled by a disulfide bond, and then the double bond is modified by the carboxyl group. The gel precursor is irradiated by gamma rays for crosslinking and curing, and is freeze-dried to form a cleavable porous sponge material. The material is subjected to esterification reaction by carboxyl and hydroxyl on a polymer during the first step of carboxyl functionalization in the synthesis process, the reaction efficiency is low, and the esterification reaction needs to be carried out in an organic solvent. The subsequent material adopts gamma ray irradiation mode for curing and crosslinking, the material preparation mode is complex, and the requirements of hydrogel rapid curing and in-situ cell culture cannot be met.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a precursor polymer capable of preparing a cleavable material, the precursor polymer solution can be cured in situ through light irradiation, and the material can be rapidly cleaved by adding a cleavage reagent by utilizing cleavable chemical bonds in the cleavable material, 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 realize the purpose, the technical scheme is as follows:

a preparation method of a precursor polymer of a cleavable material comprises the following steps:

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

1-2: and (2) carrying out double bond modification on the functional group modified polymer with the cleavable bond prepared in the step (1-1) to obtain the 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-S-. Through the step 1-1, the Polymer I is aminated and modified to obtain the amino modified Polymer-ss-NH coupled with the disulfide bond₂. Polymer-ss-NH prepared by step 1-2, for 1-1₂Double bond modification is carried out to obtain the olefin modified Polymer-ss-ene coupled by the disulfide bond.

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

Preferably, the polymer I can be selected from polymers which have carboxyl groups per se; for example, polyglutamic acid or a derivative thereof, polyacrylic acid or a derivative thereof, alginic acid or a derivative thereof, hyaluronic acid or a derivative thereof, and the like can be selected. The polymer I can also be carboxyl-containing derivatives of other polymers, such as one or more of carboxyl-containing derivatives of chitosan, carboxyl-containing derivatives of gelatin, carboxyl-containing derivatives of polylysine, carboxyl-containing derivatives of silk fibroin and the like can be selected. More 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 the proper polymer I according to the actual requirement.

The amination modification of the polymer I in the step 1-1 is completed by performing amidation reaction between carboxyl groups on the polymer and amino groups of diamine molecules (diamine compounds) under the action of a coupling agent. Preferably, in step 1-1, the method for modifying the functional group of the polymer I is as follows: and carrying out amidation reaction on carboxyl on the polymer I and one amino of a diamine compound with a cleavable bond under the action of a coupling agent to obtain a functional group modified polymer with amino 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 containing-S-or a diamine compound coupled by a disulfide bond. Further preferably, the disulfide-bond-coupled diamine molecule used is preferably cystamine or a derivative thereof.

Preferably, the polymer is subjected to amination modification in step 1-1, and amidation reaction is carried out with a coupling agent, and the coupling agent used is a common amide reaction coupling agent, such as 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine (EDC) in combination with N-hydroxysuccinimide (NHS), NN' -Carbonyldiimidazole (CDI), and the like, and the combination of EDC + NHS is 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 weight of carboxyl groups), the coupling agent and the diamine compound is 0.5-3: 1: 0.5-3. More preferably, the molar ratio of the polymer I (calculated by the molar amount of carboxyl groups), the coupling agent and the diamine compound is 0.8-1: 1: 0.8-1.2.

Preferably, the polymer is subjected to amination modification in the step 1-1, and when the polymer is in an aqueous phase solvent, the pH value of the solvent is 4-6 (the pH value can be adjusted by an acid-base regulator, the acid regulator can be diluted hydrochloric acid, diluted sulfuric acid and the like generally, and the alkaline regulator can be sodium hydroxide, sodium carbonate, sodium bicarbonate and the like).

Preferably, the amination modification reaction in the step 1-1 is carried out for 18-30 h 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, namely deionized water, is dialyzed to remove impurities and then is lyophilized.

Preferably, the Polymer-ss-NH prepared in step 1-1 is subjected to step 1-2₂Double bond modification is carried out by using Polymer-ss-NH₂Amidation of amino groups on the molecule, using carboxyl-containing olefins via coupling agentsBy carrying out the amidation reaction, the amidation reaction can also be carried out by using an acylating agent containing a double bond as it is.

Further preferably, 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 the cleavable bond and a 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 acylation reagent containing a double bond to realize the double bond modification.

Preferably, step 1-2 is performed on Polymer-ss-NH prepared in said step 1-1₂The double bond modification is carried out, and when a carboxyl group-containing olefin is used, the carboxyl group-containing olefin may be one or more of acrylic acid, methacrylic acid, and the like, and is preferably methacrylic acid.

Preferably, step 1-2 is performed on the Polymer-ss-NH prepared in step 1-1₂Double bond modification, Polymer-ss-NH when using carboxyl group-containing olefins₂The mass ratio of the carboxyl-containing olefin to the carboxyl-containing olefin is 100: 10-200 (by Polymer-ss-NH)₂Calculated by the molar amount of the amino groups in the Polymer-ss-NH₂The molar ratio of the carboxyl-containing olefin to the carboxyl-containing olefin is 1: 0.5-2), and the molar ratio of the carboxyl-containing olefin to EDC.HCl and NHS is 1: 1-2, preferably 1:1: 1.

Preferably, the Polymer-ss-NH prepared in step 1-1 is subjected to step 1-2₂The double bond modification is carried out, and when a double bond-containing acylating agent is used, the double bond-containing acylating agent can be one or more of acryloyl chloride, methacryloyl chloride, acrylic anhydride, methacrylic anhydride, glycidyl methacrylate and the like, wherein methacrylic anhydride is preferred.

Preferably, the Polymer-ss-NH prepared in step 1-1 is subjected to step 1-2₂Double bond modification, Polymer-ss-NH when using double bond-containing acylating agents₂The mass ratio of the double bond-containing acylating agent to the double bond-containing acylating agent is 100: 10-200 (by Polymer-ss-NH)₂Calculated by the molar amount of the amino groups in the Polymer-ss-NH₂The mol ratio of the double bond acylation reagent to the double bond acylation reagent 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 double bond modification is carried out by using carboxyl-containing olefin, the preferable time is 4 to 8 hours; when double bond modification is performed using a double bond-containing acylating agent, the double bond modification is more preferably performed 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 is lyophilized, so that the final product Polymer-ss-ene is obtained.

A precursor polymer of a cleavable material is prepared by the preparation method in any technical scheme.

A cleavable material is obtained by curing a precursor polymer of the cleavable material prepared by any one of the technical schemes under a curing condition; or further lyophilizing the solidified product.

Preferably, the curing conditions are light curing. When the curing is actually carried out, the corresponding photoinitiator and light irradiation conditions are matched.

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

Preferably, a cleavable material, comprising the steps of:

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

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

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

2-4: and (3) freeze-drying the frozen hydrogel prepared in the step 2-3 in a freeze-drying agent.

2-5: and (4) cutting the freeze-dried hydrogel prepared in the step (2-4) into a sheet-shaped freeze-dried hydrogel support. The lyophilized hydrogel scaffold material can now be obtained.

Preferably, in the step 2-1, the mass ratio of the cleavable material precursor Polymer-ss-ene to the solvent (ionic 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-methyl propiophenone (I2959) and lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate (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; still more preferably 405 nm.

Preferably, in step 2-3, the freezing temperature is-5 to-200 ℃, and more preferably-10 to-50 ℃; still more preferably-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 applications, the prepared sheet-like freeze-dried hydrogel scaffold may be subjected to sterilization treatment such as ultraviolet irradiation or gamma ray irradiation after steps 2 to 5.

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

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

Preferably, in step 3-1, the lysis reagent may be one or more of Glutathione (GSH), Dithiothreitol (DTT), β -mercaptoethanol (β -ME), tris phosphine (TCEP).

Preferably, in step 3-1, the lysis reagent has a molarity of 5-50 mM in PBS buffer.

The precursor polymer of the cleavable material prepared by the invention is endowed with photocuring performance and controllable cracking performance by introducing the olefin functional group coupled with the cleavable chemical bond on the precursor polymer. The cleavable material prepared by the invention comprises hydrogel and a freeze-dried porous scaffold thereof, and the cleavable material is quickly cracked into solution under the action of a cracking reagent. The cleavable material prepared by the invention has wide application prospect in the fields 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 process for curing and cracking a cleavable material;

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

fig. 4 is a SEM picture of the lyophilized scaffold.

Detailed Description

In order that the invention may be more readily understood, it will now be further described with reference to the following drawings and examples:

example 1: see fig. 1 and 2

1. Preparation of cleavable precursor polymer:

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

2) adding 1.482g (6.579mmol) of cystamine dihydrochloride to dissolve fully, and then adjusting the pH value to 4-6 by 0.1M sodium hydroxide;

3) 0.757g (6.579mmol) of NHS and 1.261g (6.579mmol) of EDC.HCl are added successively and dissolved thoroughly;

4) stirring and reacting for 24 hours at room temperature;

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

6) freeze-drying to obtain Polymer-ss-NH₂。

7) Taking the freeze-dried product Polymer-ss-NH₂0.25g was dissolved in 10mL of deionized water.

8) 0.05g (0.58mmol) of methacrylic acid was added thereto and sufficiently dissolved

9) 0.067g (0.58mmol) of NHS and 0.111g (0.58mmol) of EDC.HCl which are equimolar with methacrylic acid are added in sequence to be fully dissolved;

10) the reaction was stirred at room temperature for 5 h.

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

12) and freeze-drying to obtain the Polymer-ss-ene.

2. Cleavable hydrogel and preparation of freeze-dried scaffold thereof

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

2) And (3) taking a proper amount of the precursor solution, adding the precursor solution into a cylindrical mold with the diameter of 8mm and the height of 4mm, and irradiating the cylindrical mold with 405nm blue light for 30s to solidify the precursor solution into hydrogel.

3) The hydrogel was frozen at-20 ℃ for 3h, and the frozen sample was then transferred to a lyophilizer for lyophilization.

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

3. Lysis of hydrogels and their lyophilized scaffolds

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

Examples 2-9 the main steps were the same as in example 1, except that some experimental parameters were changed, and the specific experimental parameters are shown in Table 1

TABLE 1 parameter lists of examples 2-9

Table 2 time required for complete lysis of the hydrogels prepared in examples 1 to 9 and the freeze-dried scaffolds thereof

The preparation of the cleavable precursor polymer, the preparation of the cleavable hydrogel and the freeze-drying scaffold thereof in the embodiments 2 to 10 are realized according to the parameters in table 1 and the method provided in the embodiment 1, and the complete lysis time detection of the hydrogel and the freeze-drying scaffold thereof is performed according to the steps provided in the embodiment 1. The results are shown in table 2, and it can be seen from table 2 that the cleavable material 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 polymer:

1) taking 0.5g (2.381mmol calculated by-COOH) of initial polymer hyaluronic acid, dissolving in 100mL deionized water, and preparing 0.5% (w/w) water solution;

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

3) NHS 0.548g (4.762mmol) and EDC.HCl 0.912g (4.762mmol) were added successively and dissolved thoroughly;

4) stirring and reacting for 18h at room temperature;

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

6) freeze-drying to obtain Polymer-ss-NH₂。

8) 0.05g (0.58mmol) of methacrylic anhydride was added thereto and sufficiently dissolved

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

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

11) and freeze-drying to obtain the Polymer-ss-ene.

2. Cleavable hydrogel and preparation of freeze-dried scaffold thereof

1) And adding 0.1g of the prepared precursor Polymer-ss-ene and 0.025g of LAP into 10mL of PBS buffer solution, and dissolving in the dark to prepare a hydrogel precursor solution.

2) And (3) taking a proper amount of the precursor solution, adding the precursor solution into a cylindrical mold with the diameter of 8mm and the height of 4mm, and irradiating the cylindrical mold for 30s by using 405nm blue light to solidify the precursor solution into hydrogel.

4) Cutting the upper and lower surfaces of the freeze-dried hydrogel to obtain a cylindrical porous freeze-dried gel support sheet with the height of about 2 mm.

3. Lysis of hydrogels and their lyophilized scaffolds

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

TABLE 3 parameter lists of examples 2 to 9

Table 4 time required for complete lysis of the hydrogels and lyophilized porous scaffolds prepared in examples 10-16

The preparation of the cleavable precursor polymers, the preparation of the cleavable hydrogel and the freeze-dried scaffold thereof in the embodiments 11 to 16 are realized according to the parameters in table 3 and the method provided in the embodiment 10, and the detection of the complete lysis time of the hydrogel and the freeze-dried scaffold thereof is performed according to the steps provided in the embodiment 10. The results are shown in table 4, and it can be seen from table 4 that the cleavable material 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. Meanwhile, the double bond modification is carried out by adopting the acylation reagent, the reaction time is short, and the cracking speed of the hydrogel or the freeze-dried scaffold is high.

Claims

1. A preparation method of a precursor polymer of a cleavable material is characterized by comprising the following steps:

2. The method for preparing a cleavable material precursor polymer according to claim 1, characterized in that said polymer I is a hydrophilic polymer containing carboxyl functional groups; the cleavable bond is-S-; the functional group modification is amino modification.

3. The method for preparing a cleavable material precursor polymer according to claim 1, characterized in that the polymer I is selected from one or more of polyglutamic acid or its derivatives, polyacrylic acid or its derivatives, alginic acid or its derivatives, hyaluronic acid or its derivatives, carboxyl-containing derivatives of chitosan, carboxyl-containing derivatives of gelatin, carboxyl-containing derivatives of polylysine, carboxyl-containing derivatives of silk fibroin.

4. The method for preparing the cleavable material precursor polymer according to claim 2, characterized in that in step 1-1, the functional group modification of polymer I is performed as follows: performing amidation reaction on carboxyl on the polymer I and one amino of a diamine compound with a cleavable bond under the action of a coupling agent to obtain a functional group modified polymer with amino on the surface; the diamine compound with the cleavable bond is a diamine compound containing-S-S-.

5. The method for preparing a cleavable material precursor polymer according to claim 4, characterized in that the diamine compound is cystamine or a derivative thereof; the solvent adopted in the amidation reaction is water; the coupling agent is selected from 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide combination, and NN' -carbonyldiimidazole; the molar ratio of the polymer I to the coupling agent to the diamine compound is 0.5-3: 1: 0.5-3.

6. The method for preparing the precursor polymer of the cleavable material according to claim 2, wherein in the step 1-2, the double bond modification is performed by one of the following methods:

7. The method of preparing a cleavable material precursor polymer according to claim 6, characterized in that the carboxyl-containing olefinic compound comprises one or more of acrylic acid, methacrylic acid; the double-bond-containing acylating reagent comprises one or more of acryloyl chloride, methacryloyl chloride, acrylic anhydride, methacrylic anhydride and glycidyl methacrylate.

8. A precursor polymer of a cleavable material, which is prepared by the preparation method of any one of claims 1 to 7.

9. A cleavable material, characterized in that it is obtained by curing the cleavable material precursor polymer according to claim 8 under curing conditions; or further lyophilizing the solidified product.

10. The cleavable material according to claim 9, characterized in that the curing conditions are light curing; the photoinitiator used for photocuring is one or more of 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone (I2959) and lithium phenyl-2, 4, 6-trimethyl benzoyl phosphonate; the wavelength of the irradiation light source is 365-450 nm.