CN113549577B - Method for performing macromolecule functionalization modification on cells - Google Patents

Method for performing macromolecule functionalization modification on cells Download PDF

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CN113549577B
CN113549577B CN202110828968.3A CN202110828968A CN113549577B CN 113549577 B CN113549577 B CN 113549577B CN 202110828968 A CN202110828968 A CN 202110828968A CN 113549577 B CN113549577 B CN 113549577B
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CN113549577A (en
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吴元子
周凯强
毕雪然
舒京晶
李玲
王旭伟
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration

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Abstract

The invention belongs to the field of biological medicine, and provides a method for performing macromolecule functional modification on cells, which is characterized in that polymer macromolecules are connected at the positions of sugar chains of the cells so as to enable the cells to have corresponding functions and physicochemical properties (such as temperature sensitivity) by utilizing the physicochemical characteristics of different types of macromolecules.

Description

Method for performing macromolecule functionalization modification on cells
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a method for performing macromolecule functional modification on cells.
Background
The application of macromolecule functional modification in natural product chemistry is not rare, and the industrial development potential is very great. The plant cells, microorganisms and free enzymes from the nature carry out macromolecule functional modification on natural products, and can realize directional transformation in the aspects of sugar chain positions, sugar group types, sugar group numbers and the like.
The modification of cells by polymer functionalization is currently less studied. The modified cells are endowed with new physicochemical properties, meanwhile, the stability, targeting ability and the like of the cells are enhanced, and the modified cells have potential advantages in the fields of biological medicine, nanotechnology, material science and the like. In order to better promote each performance of the cells, certain special sites on the sugar chains of the cells can be utilized to selectively carry out covalent connection with the high polymer, so that the grafting efficiency is high, the controllability is good, and the like.
The stability of the cells to various denaturing conditions (such as denaturants, heat and the like) can be increased after the cells are subjected to macromolecule functionalization modification, and the cytotoxicity effector function or targeting ability of the cells after partial modification can be enhanced. The research of cell macromolecule functional modification is the research focus of bioconversion in the future, and bioconversion is more tightly combined with a chemical method, plays a more important role in the research and development of natural medicines, and further performs better medical treatment.
Disclosure of Invention
The invention aims to provide a method for performing macromolecule functionalization modification on cells, which can effectively maintain the original structure and activity of the cells while endowing the cells with new functional characteristics.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for modifying the cell by high-molecular functionalization features that the methacrylic acid and acrylic acid polymers are polymerized at the glycosylation site of protein in cell.
The method comprises the following specific steps:
1) Adding the washed cells into phosphate buffer solution, and uniformly mixing to prepare a cell solution with the concentration of 0.1-1 mg/mL; then adding 3- (2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester into the mixture, and carrying out oxidation reaction on the mixture on ice for 30min in a dark place; centrifuging the cell oxide after the reaction is finished, discarding the supernatant, washing with PBS buffer solution, repeating for three times, and re-suspending the precipitate with the PBS buffer solution;
2) Taking out the washed cell oxide, adding an initiator with the concentration of 14mg/mL, performing oximation reaction at 37 ℃, centrifuging the reaction solution after 30min of reaction, discarding the supernatant, washing with PBS buffer solution, and repeating for three times; after the washing is finished, the sediment is resuspended by PBS buffer solution;
3) Transferring the reaction solution into a round-bottom flask A, and connecting a nitrogen purification device to deoxidize 30 min; adding CuBr 2 solution, methanol, monomer and ligand (PMDETA) into a round bottom flask B, connecting a nitrogen purification device to deoxidize for 30min, adding ascorbic acid solution, deoxidizing for 25: 25 min; the reaction in round bottom flask B was forced into round bottom flask a with nitrogen and after keeping the nitrogen deoxygenated for 35min, the mixture was dialyzed overnight in phosphate buffer to give the polymer functionalized modified cell polymer.
The cells in step 1) are eukaryotic cells or prokaryotic cells.
The molar ratio of cells used in step 1) to N-hydroxysuccinimide ester of 3- (2-pyridinedimercapto) propionic acid was 10:1-200:1.
The molar ratio of initiator to cells used in step 2) was 1:10-1:1000; the initiator is bis [2- (2' -bromoisobutyryloxy) ethyl ] disulfide.
The monomer in the step 3) is methacrylic acid or acrylic acid compound.
The molar ratio of ascorbic acid, cuBr 2, ligand (PMDETA), monomer, methanol, water used in step 3) is in the range of 1-100:10-15:13-300:250-6000:5000:175000 to 210000; wherein the concentration of the CuBr 2 solution is 6mg/100 mu L; the concentration of the ascorbic acid solution is 50-90 mg/mL. The phosphate buffer used had a concentration of 0.01M and a pH of 7.4.
The invention has the remarkable advantages that:
(1) According to the method disclosed by the invention, the cell surface is subjected to macromolecule functional modification, so that the cell has corresponding functions and physicochemical properties (such as temperature sensitivity and the like) of the macromolecule, the influence on the surface structure of the cell is low, the original structure and high activity of the cell can be effectively maintained, and the method has considerable application in the aspects of development of biological medicines, slow release of loaded medicines, bioactive materials and the like.
(2) The invention modifies the cells through the cell surface, has instructive significance for identifying and separating cell types, expands the application range of oxime click chemistry method, and provides possibility for related drug development.
Drawings
FIG. 1 is a schematic diagram of the modification of N-hydroxysuccinimide ester (SPDP) of 3- (2-pyridinedimercapto) propionic acid.
FIG. 2 is a graph showing the state of E.coli solution at various temperatures, A is a solution at room temperature, B is a solution at 40 ℃,1 is E.coli protoplast-polymer copolymer, 2 is E.coli protoplast, 3 is GPC experiment group in which the polymer PNIPAm collected on the E.coli surface was modified by DTT and centrifugation, and 4 is GPC experiment control group in which the supernatant collected by DTT and centrifugation was used.
FIG. 3 shows the scanning image of an electron microscope, wherein A is the E.coli protoplast, B is the PNIPAm modified E.coli protoplast, C is the DTT, and the E.coli protoplast is obtained after PNIPAm is removed by centrifugation.
FIG. 4 shows the growth curves of the strains polymerized by ATRP for 15min (a, b) and 30min (c, d) under the culture conditions of 25 ℃ (a, c) and 37 ℃ (b, d). E.coli, proplast E.coli protoplasts; proplast-Polymer E.coli protoplast after chemical treatment; proplast-Polymer+DTT DTT and E.coli protoplasts obtained after removal of PNIPAm by centrifugation.
Fig. 5 fluorescence microscopy, a: fluorescence images without rhodamine monomers added to E.coli under the same conditions; b: and adding rhodamine monomers into escherichia coli to polymerize fluorescence images.
Detailed Description
Example 1
1) Adding the washed escherichia coli protoplast into a phosphate buffer solution, and uniformly mixing to prepare a cell solution with the concentration of 0.5 mg/mL; then adding 3- (2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester into the mixture, and carrying out oxidation reaction on the mixture on ice for 30min in a dark place; centrifuging the cell oxide after the reaction is finished, discarding the supernatant, washing with PBS buffer solution, repeating for three times, and re-suspending the precipitate with the PBS buffer solution;
2) Taking out the washed escherichia coli cell oxide, adding an initiator with the concentration of 14mg/mL, performing oximation reaction at 37 ℃ for 30min, centrifuging the reaction solution, discarding the supernatant, washing with PBS buffer solution, and repeating for three times; after the washing is finished, the sediment is resuspended by PBS buffer solution;
3) Transferring the reaction solution into a round-bottom flask A, and connecting a nitrogen purification device to deoxidize 30min; adding CuBr 2 solution, methanol, monomer and ligand PMDETA into a round bottom flask B, connecting a nitrogen purification device to deoxidize for 30min, adding ascorbic acid solution, deoxidizing 25: 25 min; the reaction product in round bottom flask B was pressed into round bottom flask A with nitrogen, and after keeping the nitrogen deoxidized for 35min, the mixture was dialyzed overnight in phosphate buffer to obtain the polymer of E.coli cell modified by high molecular functionalization.
The molar ratio of cells used in step 1) to N-hydroxysuccinimide ester of 3- (2-pyridinedimercapto) propionic acid was 100:1.
The molar ratio of initiator to cells used in step 2) was 1:500; the initiator is bis [2- (2' -bromoisobutyryloxy) ethyl ] disulfide.
The monomer in the step 3) is NIPAm.
The molar ratio of ascorbic acid, cuBr 2, ligand (PMDETA), monomer, methanol, water used in step 3) was in the range of 1:2:3:50:1000:35000; wherein the concentration of the CuBr 2 solution is 6mg/100 mu L; the concentration of the ascorbic acid solution is 70 mg/mL.
The pH of the phosphate buffer used at a concentration of 0.01M was 7.4.
Experimental results
1. Whether PNIPAm monomer grafting is successful or not
By utilizing reversible rupture of the macromolecule, the disulfide bond between the protein and the polymer biomolecules is reduced by DTT, and further, the polymerization of PNIPAm is verified to occur on the surface of a cytoplasmic membrane. The state of each solution at room temperature and 40C conditions during the experiment is shown in FIG. 2, and when the solution is heated to 40C in a water bath, it is apparent from FIG. 2B that sample No. 1 (E.coli protoplast polymer macromolecule) and sample No. 3 (GPC experiment group: polymer PNIPAm collected by modification of E.coli protoplast surface with DTT and centrifugation) become cloudy and opaque compared with the corresponding sample at room temperature (see FIG. 2A), whereas sample No. 2 (E.coli protoplast) and sample No. 4 (GPC control group: supernatant collected by modification of E.coli protoplast with DTT and centrifugation) remain clear regardless of room temperature (A) or 40C conditions (B). These phenomena are sufficient to demonstrate that PNIPAm was successfully grafted onto E.coli protoplast surface, while PNIPAm can be isolated from lysozyme by heating and simple centrifugation when resuspended pellet samples were treated with reducing agent DTT.
2. Field emission scanning electron microscope characterization
The changes in morphology of E.coli protoplasts and cells before and after chemical treatment were observed by Field Emission Scanning Electron Microscopy (FESEM) and the results are shown in FIG. 3. In FIG. A, B, C, there are single spheroid cells, and also cells that are candid, and also long rod-like cells, which are formed by the fusion of two or more single spheroid cells. The results show that the cell morphology of the E.coli protoplasts is not significantly different before and after the chemical treatment.
3. E.coli viability assay
To further verify whether the polymerization time of PNIPAm and the culture temperature of the strain had an effect on cell viability, experiments were performed on liquid fermentation culture studies of escherichia coli before and after chemical modification (initiator, SPDP access and PNIPAm grafting). In FIG. 4, (a) and (b) are growth curves of samples of different treatment groups at 25℃and 37℃respectively after 15min of ATRP polymerization. From the Proplast-polymer and Protoplast-polymer+DTT experiments in FIG. 4 (a), (b), it can be seen that when the polymerization time was 15min, E.coli protoplasts after chemical treatment (initiator, SPDP grafting and PNIPAm grafting) were almost as viable as strains obtained by disrupting the surface macromolecules of the strain with DTT and removing PNIPAm by centrifugation. After extending the ATRP polymerization time to 30 min, the growth curves of the different treated samples at 25℃and 37℃respectively are shown in FIGS. 4 (c) and (d). From the Proplast-polymer and Protoplast-polymer+DTT experiments in FIG. 4 (c) and (d), it can be seen that when the polymerization time is extended to 30 min, the E.coli protoplasts after chemical modification (initiator, SPDP grafting and PNIPAm grafting) show a difference from the time at which the strain obtained after cleavage of the surface polymer with DTT and removal of PNIPAm by centrifugation starts to grow and propagate at 25℃and 37 ℃.
From a combination of FIG. 4, it was found that, regardless of whether the culture temperature was 25℃or 37℃and the ATRP polymerization time was 15 min or 30min, there was a great possibility that the growth of the E.coli after a series of chemical modifications and removal of the modified E.coli protoplasts was delayed, and that the chemical modifications (initiator, SPDP access and PNIPAm grafting) had adverse effects on the viability of the strain, and that it was a process of strain regeneration. However, when the polymerization time was prolonged to 30min and the PNIPAm-removed E.coli protoplasts were cultured at a temperature higher than the PNIPAm Low Critical Solution Temperature (LCST) and also at the optimal culture temperature for E.coli, the growth retardation was alleviated. All these phenomena above are sufficient to demonstrate that a series of chemical modifications (initiator, SPDP access and PNIPAm grafting) inhibit the viability of the cells, but that the growth and reproduction capacity of the cells is not lost.
Example 2
1) Adding the washed escherichia coli into phosphate buffer solution, and uniformly mixing to prepare a cell solution with the concentration of 0.5 mg/mL; then adding 3- (2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester into the mixture, and carrying out oxidation reaction on the mixture on ice for 30min in a dark place; centrifuging the cell oxide after the reaction is finished, discarding the supernatant, washing with PBS buffer solution, repeating for three times, and re-suspending the precipitate with the PBS buffer solution;
2) Taking out the washed escherichia coli cell oxide, adding an initiator with the concentration of 14mg/mL, performing oximation reaction at 37 ℃ for 30min, centrifuging the reaction solution, discarding the supernatant, washing with PBS buffer solution, and repeating for three times; after the washing is finished, the sediment is resuspended by PBS buffer solution;
3) Transferring the reaction solution into a round-bottom flask A, and connecting a nitrogen purification device to deoxidize 30min; adding CuBr 2 solution, methanol, monomer and ligand PMDETA into a round bottom flask B, connecting a nitrogen purification device to deoxidize for 30min, adding ascorbic acid solution, deoxidizing 25: 25 min; the reaction product in round bottom flask B was pressed into round bottom flask A with nitrogen, and after keeping the nitrogen deoxidized for 35min, the mixture was dialyzed overnight in phosphate buffer to obtain the polymer of E.coli cell modified by high molecular functionalization.
The molar ratio of cells used in step 1) to N-hydroxysuccinimide ester of 3- (2-pyridinedimercapto) propionic acid was 100:1.
The molar ratio of initiator to cells used in step 2) was 1:500; the initiator is bis [2- (2' -bromoisobutyryloxy) ethyl ] disulfide.
The monomer in the step 3) is rhodamine B.
The molar ratio of ascorbic acid, cuBr 2, ligand (PMDETA), monomer, methanol, water used in step 3) was in the range of 1:2:3:80:1000:35000; wherein the concentration of the CuBr 2 solution is 6mg/100 mu L; the concentration of the ascorbic acid solution is 70 mg/mL.
The pH of the phosphate buffer used at a concentration of 0.01M was 7.4.
Experimental results
1. Fluorescence microscope characterization
It was further verified by fluorescence microscopy whether polymerization of rhodamine B occurred on the cell surface. As shown in FIG. 5, the experimental results are shown in FIG. 2A, it is obvious that the E.coli without the fluorescent monomer rhodamine added under the same conditions has no fluorescent cluster and only has sparse fluorescence, while the E.coli with the fluorescent monomer rhodamine polymerized by ATRP in FIG. 2 has obvious fluorescent cluster, which proves that the fluorescent monomer rhodamine is polymerized on the E.coli surface in a large quantity.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (3)

1. A method for performing macromolecule functionalization modification on cells, which is characterized in that: the method comprises the following steps:
1) Adding the washed cells into phosphate buffer solution, and uniformly mixing to prepare a cell solution with the concentration of 0.1-1 mg/mL; then adding 3- (2-pyridine dimercapto) propionic acid N-hydroxysuccinimide ester into the mixture, and carrying out oxidation reaction on the mixture on ice for 30min in a dark place; centrifuging the cell oxide after the reaction is finished, discarding the supernatant, washing with PBS buffer solution, repeating for three times, and re-suspending the precipitate with the PBS buffer solution;
2) Taking out the washed cell oxide, adding an initiator with the concentration of 14mg/mL, performing oximation reaction at 37 ℃, centrifuging the reaction solution after 30min of reaction, discarding the supernatant, washing with PBS buffer solution, and repeating for three times; after the washing is finished, the sediment is resuspended by PBS buffer solution;
3) Transferring the reaction solution into a round-bottom flask A, and connecting a nitrogen purification device to deoxidize for 30min; adding CuBr 2 solution, methanol, monomer and ligand into a round bottom flask B, connecting a nitrogen purification device to deoxidize for 30min, adding ascorbic acid solution, deoxidizing for 25min; pressing the reactant in the round-bottom flask B into the round-bottom flask A by using nitrogen, keeping the nitrogen for deoxidizing for 35min, and dialyzing the mixture in phosphate buffer solution overnight to obtain the high-molecular functional modified cell polymer;
Step 1) the cells are eukaryotic cells or prokaryotic cells;
The molar ratio of cells used in step 1) to N-hydroxysuccinimide ester of 3- (2-pyridinedimercapto) propionic acid was 10:1-200:1, a step of;
The molar ratio of initiator to cells used in step 2) was 1:10-1:1000; the initiator is bis [2- (2' -bromoisobutyryloxy) ethyl ] disulfide;
the monomer in the step 3) is methacrylic acid or acrylic acid compound.
2. The method for performing macromolecule functionalization modification on a cell according to claim 1, wherein: the molar ratio of ascorbic acid, cuBr 2, ligand, monomer, methanol, water used in step 3) is in the range of 1-100:10-15:13-300:250-6000:5000:175000 to 210000; wherein the concentration of the CuBr 2 solution is 6mg/100 mu L; the concentration of the ascorbic acid solution is 50-90mg/mL.
3. The method for performing macromolecule functionalization modification on a cell according to claim 1, wherein: phosphate buffer concentration was 0.01M and pH 7.4.
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CN108531410A (en) * 2018-04-16 2018-09-14 南京工业大学 A method of cell surface is modified based on plant polyphenol tannin acid oxidase auto polymerization
CN108653749A (en) * 2018-07-09 2018-10-16 青岛科技大学 A kind of preparation method of lock nucleic acid nano drug-carrying micella and carrier micelle based on cell-penetrating peptide
WO2019175599A1 (en) * 2018-03-16 2019-09-19 Jones Paul Antonio Surface functionalised materials for sampling biological molecules
CN111848720A (en) * 2020-08-10 2020-10-30 福州大学 Method for performing macromolecule functional modification on glycosylated protein

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2019175599A1 (en) * 2018-03-16 2019-09-19 Jones Paul Antonio Surface functionalised materials for sampling biological molecules
CN108531410A (en) * 2018-04-16 2018-09-14 南京工业大学 A method of cell surface is modified based on plant polyphenol tannin acid oxidase auto polymerization
CN108653749A (en) * 2018-07-09 2018-10-16 青岛科技大学 A kind of preparation method of lock nucleic acid nano drug-carrying micella and carrier micelle based on cell-penetrating peptide
CN111848720A (en) * 2020-08-10 2020-10-30 福州大学 Method for performing macromolecule functional modification on glycosylated protein

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