阅读说明：本技术 一种γ-聚谷氨酸-接枝-吲哚结构伯胺及制备方法和应用 (Gamma-polyglutamic acid-graft-indole structure primary amine and preparation method and application thereof ) 是由 袁晓燕 牛情婧 张启发 任丽霞 赵蕴慧 于 2019-10-12 设计创作，主要内容包括：本发明公开一种γ-聚谷氨酸-接枝-吲哚结构伯胺及制备方法和应用。将γ-PGA溶于去离子水中,加入1-羟基苯并三唑,冰浴活化,然后加入溶有吲哚结构伯胺的二甲基亚砜溶液,室温下进行反应。将所得反应液透析、冻干后得到产物γ-聚谷氨酸-接枝-吲哚结构伯胺。用pH值为6.0、6.6、7.0和7.4的磷酸盐缓冲液配制γ-聚谷氨酸-接枝-吲哚结构伯胺的溶液,将细胞在含有海藻糖的γ-聚谷氨酸-接枝-吲哚结构伯胺的磷酸盐缓冲溶液中37℃下孵化3h,在液氮中冻存细胞。γ-聚谷氨酸-接枝-吲哚结构伯胺与海藻糖共同作用,能促进海藻糖的胞内负载,可以使细胞冻存后的存活率达到80％～90％,可运用于细胞冻存领域。(The invention discloses gamma-polyglutamic acid-graft-indole structure primary amine and a preparation method and application thereof. Dissolving gamma-PGA in deionized water, adding 1-hydroxybenzotriazole, activating in ice bath, adding dimethyl sulfoxide solution dissolved with indole structure primary amine, and reacting at room temperature. And dialyzing and freeze-drying the obtained reaction solution to obtain the product gamma-polyglutamic acid-graft-indole structure primary amine. Preparing a solution of gamma-polyglutamic acid-graft-indole structure primary amine by using a phosphate buffer solution with pH values of 6.0, 6.6, 7.0 and 7.4, incubating cells in the phosphate buffer solution of the gamma-polyglutamic acid-graft-indole structure primary amine containing trehalose at 37 ℃ for 3h, and freezing and storing the cells in liquid nitrogen. The gamma-polyglutamic acid-graft-indole structure primary amine and the trehalose act together, so that the intracellular load of the trehalose can be promoted, the survival rate of the frozen cells can reach 80-90%, and the method can be applied to the field of cell freezing.)

1. A gamma-polyglutamic acid-graft-indole structure primary amine is characterized in that the structural formula is as follows:

wherein the molecular weight of the gamma-polyglutamic acid is 1-100 ten thousand; the value of x/n is more than or equal to 0.1 and less than or equal to 0.6, and InR-is indole structural group.

2. The gamma-polyglutamic acid-graft-indole structure primary amine of claim 1, wherein the indole structure group InR-is:

3. the method for preparing gamma-polyglutamic acid-graft-indole structure primary amine according to claim 1, which comprises the steps of:

(1) dissolving gamma-polyglutamic acid in deionized water, adding 1-hydroxybenzotriazole, and activating in ice bath.

(2) Adding dimethyl sulfoxide solution dissolved with primary amine with indole structure, and reacting at room temperature.

(3) And dialyzing and freeze-drying the obtained reaction solution to obtain the product gamma-polyglutamic acid-graft-indole structure primary amine.

4. The method according to claim 3, wherein the molar ratio of the 1-hydroxybenzotriazole to the gamma-polyglutamic acid repeating structural unit in the reaction step (1) is 1-3: 1, and 0.02g of gamma-polyglutamic acid is added to per ml of deionized water.

5. The method according to claim 3, wherein the volume of the dimethyl sulfoxide in the reaction step (2) is the same as that of the deionized water in the reaction step (1), the molar ratio of the indole-structured primary amine in the reaction step (2) to the repeating structural unit of the gamma-polyglutamic acid in the reaction step (1) is 0.1-0.6: 1, and the reaction step (2) is performed by stirring in an ice bath for 10-120 min and then reacting at room temperature for 2-48 h.

6. The method as set forth in claim 3, wherein the reaction solution of gamma-polyglutamic acid and primary amine with indole structure in step (3) is dialyzed in deionized water for 3 days and lyophilized for 1 day to obtain the product of gamma-polyglutamic acid-graft-primary amine with indole structure.

7. The gamma-polyglutamic acid-graft-indole structural primary amine of claim 1, which can facilitate loading of trehalose into cells for cell cryoprotection.

8. The use according to claim 7, wherein the gamma-polyglutamic acid-graft-indole structure primary amine is used for the cryoprotection of stem cells, vascular smooth muscle cells and erythrocytes.

9. The use according to claim 7, wherein the mixed solution of the primary amine with the gamma-polyglutamic acid-graft-indole structure and trehalose is prepared by using a phosphate buffer solution, adding the cell suspension, shaking and incubating for 3 hours in a water bath shaker at 37 ℃, and then freezing and storing the cells in liquid nitrogen.

10. The method according to claim 9, wherein the concentration of the gamma-polyglutamic acid-graft-indole structure primary amine is 1mg/mL, the concentration of trehalose is 0.2-0.4M, the pH of the phosphate buffer is 6.0-7.4, and 80. mu.L of the cell suspension per mL is added.

Technical Field

The invention relates to gamma-polyglutamic acid-graft-indole structure primary amine and a preparation method and application thereof, belonging to the field of biological materials.

Background

Cryoprotectants are chemical substances used to protect cells and biological tissues from freezing injury caused by ice crystal formation. At present, dimethyl sulfoxide or glycerol is usually used as a cryoprotectant for cryopreservation of cells, but the dimethyl sulfoxide and the glycerol influence the activity and the function of the cells, and the cells need a complicated washing process after recovery before use. Trehalose is a natural disaccharide with good biocompatibility and stability, is a good cryoprotectant, and can protect cells and organisms against freezing or dehydration damage. The main methods for loading trehalose in cells comprise chemical methods such as loading and preparing trehalose glycolipid plasmids by using the action of polymers and polypeptides, nanoparticles and cell membranes (S.Stewart, X.He.Intracellularly of trehalose for cell cloning. Langmuir,2019,35(23):7414-7422), gene engineering methods such as trehalose endogenous gene expression or trehalose transporter TRET1 expression, and osmotic methods such as heat, electricity and the like. Apatite nanoparticles stabilized with a positive and negative electrolyte can increase the permeability of trehalose, thereby facilitating the loading of trehalose into erythrocytes (m.stem, k.ward, h.tawfik, r.seemann, v.baulin, y.guo, j.b.fleery, c.drain.application nanoparticles positive protected blood pressure by means of catalytic depletion membrane catalysis, biomaterials,2017,140: 138-. Motta et al, introduced trehalose into cord blood stem cells using trehalose plasmids, improved the freeze-survival of stem cells (J.P.Motta, F.H.Paraguass-Braga, L.F.Bouzas, L.C.Porto.evaluation of intracellular and extracellular trehalases a cryoprotection of stem cells from inorganic cardiac code. Cryobiology,2014,68(3): 343-348). Macromolecular cryoprotectants, such as antifreeze peptide, polyethylene glycol, polyvinylpyrrolidone, albumin, hydroxyethyl starch and the like, can be adsorbed on the surface of ice crystals to reduce the formation rate of the ice crystals and prevent the growth of the ice crystals. In order to enhance the effect of cryoprotectants, it is generally necessary to add two types or more of cryoprotectants during cryopreservation of cells.

Aromatic residues in the polypeptide structure have certain perturbation effect on cell membranes, so that reversible micropores are generated in the cell membranes. In addition, amphiphilic polymers have a disturbing effect on cell membranes and are widely used as carriers for intracellular drug delivery. The Slater group promoted intracellular loading of trehalose by the action of amphiphilic pH-responsive polymers on cell membranes, thereby increasing the freezing survival rate of osteosarcoma cells (S.A. Mercado, N.K.H.Slater.Increase crysuservial of osteoarcoma cells using an amphophilic pH-responsive polymer for trehalose uptake. Cryobiology,2016,73(2): 175-180).

The invention takes the gamma-polyglutamic acid and indole structure primary amine as raw materials and 1-hydroxybenzotriazole as a catalyst to prepare the gamma-polyglutamic acid-graft-indole structure primary amine, and the method has simple and convenient operation and mild reaction conditions. The amphiphilic polymer obtained by grafting the primary amine with the indole structure to the gamma-polyglutamic acid has a disturbing effect on cell membranes, so that trehalose is loaded into cells, and the freezing survival rate of the cells reaches 80-90%. The method of using gamma-polyglutamic acid-graft-indolylamine as cryoprotectant has not been reported.

Disclosure of Invention

The gamma-polyglutamic acid-graft-indole structure primary amine prepared by the invention can be used for improving the freezing survival rate of cells under the combined action of trehalose. The hydrophobic modification of gamma-polyglutamic acid is carried out by using primary amines with indole structures such as tryptamine, 4-aminomethyl indole, indole-3-methylamine and the like, the obtained primary amines with the gamma-polyglutamic acid-graft-indole structures have interaction with cell membranes, and can be used for intracellular loading of non-membrane permeability protective agents such as trehalose and the like so as to be applied to the fields of cell cryopreservation, drug carriers and the like.

The technical scheme of the invention is as follows:

the structural formula of gamma-polyglutamic acid-graft-indole structure primary amine is as follows:

wherein InR-is an indole structure-containing group, such as:

and the like.

In the formula, n represents the number of the repeating units of the polymer, and x/n represents the grafting rate of the gamma-polyglutamic acid, and the value range of x/n is more than or equal to 0.10 and less than or equal to 0.60.

The reaction equation for preparing gamma-polyglutamic acid-graft-indole structure primary amine is as follows:

wherein HOBt is 1-hydroxybenzotriazole.

The preparation method of the gamma-polyglutamic acid-graft-indole structure primary amine comprises the following steps:

(1) dissolving gamma-polyglutamic acid in deionized water, adding 1-hydroxybenzotriazole, and activating in ice bath.

(2) Adding dimethyl sulfoxide solution dissolved with primary amine with indole structure, and reacting at room temperature.

(3) And dialyzing and freeze-drying the obtained reaction solution to obtain the product gamma-polyglutamic acid-graft-indole structure primary amine.

In the step (1), the molecular weight of the gamma-polyglutamic acid is 1-100 ten thousand, the molar ratio of the 1-hydroxybenzotriazole to the gamma-polyglutamic acid structural unit is 1-3: 1, and 0.02g of gamma-polyglutamic acid is added into per milliliter of deionized water.

The volume of the dimethyl sulfoxide in the step (2) is equal to that of the deionized water in the step (1), the molar ratio of the indole-structure primary amine to the gamma-polyglutamic acid structural unit is 0.1-0.6: 1, the ice bath time is 10-120 min, and the room-temperature reaction time is 2-48 h.

And (3) dialyzing the reaction solution obtained by room-temperature reaction in deionized water for more than 3 days, and freeze-drying for 1 day to obtain the gamma-polyglutamic acid-graft-indole structure primary amine.

The gamma-polyglutamic acid-graft-indole structure primary amine can promote trehalose to be loaded into cells and is used for cell freezing protection, and the application method comprises the following steps:

preparing a mixed solution of gamma-polyglutamic acid-graft-indole structure primary amine and trehalose by using a phosphate buffer solution, adding the cell suspension, shaking uniformly, incubating in a water bath shaker at 37 ℃, and then freezing and storing the cells in liquid nitrogen.

The pH value of the phosphate buffer solution is 6.0, 6.4, 7.0 and 7.4, the concentration of gamma-polyglutamic acid-graft-indole structure primary amine is 1mg/mL, the concentration of trehalose is 0.2-0.4M, the used cells comprise hematopoietic stem cells, vascular smooth muscle cells and red blood cells, and the amount of the added cells per milliliter is 80 mu L.

The invention discloses a gamma-polyglutamic acid-graft-indole structure primary amine and a preparation method and application thereof. Preparing a 1mg/mL gamma-polyglutamic acid-graft-indole structure primary amine solution by using a phosphate buffer solution with a pH value of 6.0-7.4, and incubating cells in the phosphate buffer solution containing 0.2-0.4M trehalose gamma-polyglutamic acid-graft-indole structure primary amine at 37 ℃ for 3 h. The gamma-polyglutamic acid-graft-indole structure primary amine and the trehalose act together to promote the intracellular load of the trehalose and ensure that the survival rate of the cells after cryopreservation reaches 80-90 percent. The gamma-polyglutamic acid-graft-indole structure primary amine cryoprotectant is used together with dimethyl sulfoxide and glycerol, so that the dosage of the dimethyl sulfoxide and the glycerol can be reduced to reduce the damage to cells.

Detailed description of the invention

The technical solution of the present invention is further described below by the following embodiments, which are further illustrative of the present invention and do not limit the applicable scope of the present invention.

(1) Preparation of gamma-polyglutamic acid-graft-indole structure primary amine

Dissolving 1-hydroxybenzotriazole/gamma-polyglutamic acid structural unit in a molar ratio of 1-3: 1 in deionized water, and adding 0.02g of gamma-polyglutamic acid in each milliliter of deionized water. Adding dimethyl sulfoxide dissolved with indole structure primary amine with the same volume, wherein the molar weight of the indole structure primary amine/gamma-polyglutamic acid structural unit is 0.1-0.6: 1, firstly stirring in an ice bath for 10-120 min, and then reacting at room temperature for 2-48 h. Dialyzing in deionized water for 3 days and then freeze-drying for 1 day to obtain the gamma-polyglutamic acid-graft-indole structure primary amine.

(2) Gamma-polyglutamic acid-graft-indole structure primary amine for cell cryopreservation

1mL of polymer trehalose mixed solution is prepared by using phosphate buffer solution with the pH value of 6.0, 6.6, 7.0 or 7.4, wherein the concentration of the polymer is 1mg/mL, and the concentration of trehalose is 0.2-0.4M. Add 80. mu.L of cell suspension, shake and incubate in 37 ℃ water bath shaker for 3h, then freeze in liquid nitrogen for more than 2 h. After being frozen and taken out, the cells were immediately thawed in a water bath at 37 ℃ and the freezing survival rate was calculated. Wherein the negative control group is phosphate buffer solution of trehalose with corresponding pH value of each sample, and the positive control group is added with deionized water to ensure that cells are fully hemolyzed.