阅读说明：本技术 磷酸化蛋白质、基于磷酸化蛋白质的胞内递送体系及制备方法与应用 (Phosphorylated protein, intracellular delivery system based on phosphorylated protein, preparation method and application ) 是由 殷黎晨 刘勇 吴宇辰 于 2020-06-27 设计创作，主要内容包括：本发明公开了磷酸化蛋白质、基于磷酸化蛋白质的胞内递送体系及制备方法与应用；提供了一种蛋白质磷酸化修饰方法,所述磷酸化蛋白质具有蛋白质结构、通过氨基接于所述蛋白质表面的苯硼酸基团和通过硼酸与邻二醇反应接于所述蛋白质表面的三磷酸腺苷；将阳离子多肽溶液和磷酸化蛋白质在碱液中混合,震荡后静置,得到基于磷酸化蛋白质的胞内递送体系。在肿瘤细胞内低pH值和高浓度的活性氧环境下,苯硼酸和三磷酸腺苷从蛋白质表面脱落,蛋白质与阳离子聚多肽的作用力减弱,从复合物中释放出来,同时恢复活性,杀死肿瘤细胞。(The invention discloses a phosphorylated protein, an intracellular delivery system based on the phosphorylated protein, a preparation method and application thereof; a method for modifying protein phosphorylation is provided, wherein the protein phosphorylation has a protein structure, a phenylboronic acid group attached to the protein surface through an amino group, and adenosine triphosphate attached to the protein surface through a reaction between boric acid and vicinal diol; and mixing the cationic polypeptide solution and the phosphorylated protein in an alkaline solution, shaking and standing to obtain the intracellular delivery system based on the phosphorylated protein. In the low pH value and high concentration active oxygen environment in tumor cells, the phenylboronic acid and the adenosine triphosphate fall off from the surface of the protein, the acting force of the protein and the cationic polypeptide is weakened, the protein and the cationic polypeptide are released from the compound, and the activity is recovered to kill the tumor cells.)

1. Phosphorylated proteins are prepared from proteins, phenylboronic acid molecules and adenosine phosphate.

2. The phosphorylated protein according to claim 1, wherein the molar ratio of the amine group to the phenylboronic acid molecule to the adenosine phosphate on the protein is 1: 1.3 to 1.8: 1.2 to 1.8.

3. The phosphorylated protein of claim 1, wherein the phenylboronic acid molecule is 4-hydroxymethylphenylboronic acid pinacol carbonyl imidazole; adenosine phosphate is adenosine triphosphate.

4. The phosphorylated protein according to claim 1, wherein a solution of phenylboronic acid molecules is added to a protein phosphate buffer solution and reacted to obtain a solution of R-PBA; then adding adenosine phosphate solution to react to obtain phosphorylated protein.

5. A phosphorylated protein-based intracellular delivery system comprising a phosphorylated protein according to claim 1 and a cationic polypeptide.

6. The intracellular delivery system based on the phosphorylated protein, according to claim 5, wherein the mass ratio of the phosphorylated protein to the cationic polypeptide is 1: (0.5-10); the cationic polypeptide has the following chemical structural formula:

。

7. the phosphorylated protein-based intracellular delivery system of claim 5, wherein the phosphorylated protein-based intracellular delivery system is obtained by mixing the cationic polypeptide solution and the phosphorylated protein in an alkaline solution, shaking the mixture, and standing the mixture.

8. The phosphorylated protein-based intracellular delivery system of claim 7, wherein the alkaline solution is an aqueous sodium bicarbonate solution.

9. Use of the phosphorylated protein-based intracellular delivery system according to claim 5 or of the phosphorylated protein according to claim 1 for the preparation of a nano-drug.

10. Use of a phosphorylated protein according to claim 1 for the preparation of a phosphorylated protein based intracellular delivery system according to claim 5.

Technical Field

The present application relates to phosphorylated proteins, and more particularly to phosphorylated proteins for use in intracellular protein delivery systems.

Background

All protein drugs currently on the market are developed based on extracellular targets, such as cell membrane proteins (programmed cell death receptor 1, PD-1; human epidermal growth factor receptor 2, HER 2; insulin receptor, etc.) and secreted proteins (tumor necrosis factor alpha, TNF alpha; interleukin 12, IL 12; vascular endothelial growth factor, VEGF, etc.). This is mainly due to the large molecular nature and hydrophilicity of the protein, which makes it incapable of penetrating cell membranes. However, more than about 70% of the proteins encoded by the genome are located intracellularly. These proteins are difficult to "druggy" due to the inability to penetrate the cell membrane. Therefore, in recent years, the development of a simple and efficient intracellular protein delivery system has been increasingly emphasized.

Covalent attachment of components with membrane-penetrating functions to proteins is a common intracellular delivery of proteins. Common components with membrane penetration include protein transduction domains, cell penetrating peptides, cationic polymers, and some small amphiphilic molecules. The protein after functional modification has more excellent membrane penetrating capacity, but such chemical modification strategy is difficult to modify chemical substances with membrane penetrating function to specific sites of the protein, and the biological activity of the modified protein may be irreversibly reduced or changed. Thus, various dynamic covalent bonds have been used to link proteins to functional groups. The protein modified by the dynamic covalent bond can release the loading protein under the stimulation of endogenous or exogenous, and the effect is achieved. Another commonly used intracellular delivery strategy for proteins is the use of delivery vehicles such as liposomes, cell-derived vesicles, inorganic nanoparticles, nanogels and polymers. These carriers are typically associated with proteins by non-covalent interactions. Proteins are biomolecules with uncertain charge properties and large volumes, and the number of sites on the surface of the protein, which can be combined with a carrier, is limited. In addition, the properties such as isoelectric point, hydrophilicity and hydrophobicity and the like of different proteins are greatly different, so that it is difficult to design a protein delivery system with universality.

Disclosure of Invention

The invention solves the problems by carrying out specific phosphorylation modification on protein and assisting with cationic polypeptide, can effectively penetrate cell membranes, and the obtained nano-medicament has small particle size.

The invention adopts the following technical scheme:

phosphorylated proteins are prepared from proteins, phenylboronic acid molecules and adenosine phosphate.

An intracellular delivery system based on phosphorylated proteins, comprising the phosphorylated proteins and a cationic polypeptide.

In the invention, the molar ratio of amino groups on the protein to phenylboronic acid molecules to adenosine phosphate is 1: 1.3-1.8: 1.2-1.8, and after the obtained phosphorylated protein is compounded with cationic polypeptide, the obtained nano-drug has small particle size and good stability.

In the invention, the mass ratio of the phosphorylated protein to the cationic polypeptide is 1: 0.5-10.

In the invention, the phenylboronic acid molecule is 4-hydroxymethylphenylboronic acid pinacol carbonyl imidazole; adenosine phosphate is adenosine triphosphate.

In the present invention, the cationic polypeptide has the following chemical structural formula:

the preparation method of the phosphorylated protein comprises the steps of adding a phenylboronic acid molecular solution into a protein phosphate buffer solution, and reacting to obtain an R-PBA solution; then adding adenosine phosphate solution to react to obtain phosphorylated protein.

In the above technical scheme, the reaction is a room temperature reaction.

The preparation method of the intracellular delivery system based on the phosphorylated protein comprises the steps of mixing a cationic polypeptide solution and the phosphorylated protein in an alkaline solution, shaking and standing to obtain the intracellular delivery system based on the phosphorylated protein. The alkali solution is sodium bicarbonate water solution.

The invention further discloses the application of the phosphorylated protein or the intracellular delivery system based on the phosphorylated protein in the preparation of nano-drugs, in particular anti-tumor protein drugs.

Based on the findings, the invention provides a protein intracellular delivery system assisted by cationic polypeptide based on protein phosphorylation modification; the phosphorylated protein has a protein structure, phenylboronic acid groups attached to the protein surface through amino groups, and adenosine triphosphate attached to the protein surface through the reaction of boric acid with vicinal diols.

The invention has the advantages that the phosphorylated protein has higher negative charge density, so that the electrostatic binding force between the modified protein and the cationic polypeptide is enhanced, and a stable nano-composite is formed. Meanwhile, the salt bridge function between the phosphate group and the guanidyl group is also beneficial to enhancing the interaction force of the cationic polypeptide and the phosphorylated protein, and the nano compound is further stabilized. In the low pH value and high concentration active oxygen environment in tumor cells, the phenylboronic acid and the adenosine triphosphate fall off from the surface of the protein, the acting force of the protein and the cationic polypeptide is weakened, the protein and the cationic polypeptide are released from the compound, and the activity is recovered to kill the tumor cells.

Drawings

FIG. 1 is a mass spectrum of macromolecules before and after RNase A phosphorylation modification in example 1;

FIG. 2 is the cytotoxicity of LPP in example 2; (a) relative survival rates (n = 3) after 10 hours of incubation of MCF-7, (B) B16F10, (c) HeLa cells with different concentrations of LPP and 38 hours of continued incubation in fresh medium;

FIG. 3 is a CLSM picture of phosphorylated proteins and nanocomplexes of example 2, after MCF-7 cells were incubated with FITC labeled free RNase A, free R-P-ATP and LPP/R-P-ATP complex under serum-free conditions for 4 hours, nuclei and lysosomes were stained with Hoechst and Lysotracker Deep Red (LDR), respectively, on a scale of 100 μm;

FIG. 4 is a graph of the flow cytometric analysis of the phosphorylated proteins and nanocomplexes of example 2, MCF-7 cells incubated with free RNaseA, free R-P-ATP and LPP/R-P-ATP complexes under serum-free conditions for 4 hours, and the mean fluorescence intensity of MCF-7 cells calculated from the profiles (n = 3);

FIG. 5 is the mechanism of cellular uptake of the nanocomplexes of example 2;

FIG. 6 is a graph showing the in vitro tumor suppression effect of the nanocomposite in example 2;

FIG. 7 is a graph showing the in vivo tumor suppression effect of the nanocomposite in example 2; (a) mouse tumor growth curves over 12 days observation period (n = 10), arrows indicate intratumoral injection of different drug formulations: PBS, free RNase A (1 mg/kg RNase A), LPP/B-P-ATP (1 mg/kg BSA), LPP/R-P-ATP (1 mg/kg RNase A); (b) body weight change in tumor-bearing mice over a 12-day observation period (n = 10), (c) survival rate;

FIG. 8 shows LPP in TFA-dIn (1)¹H NMR spectrum;

FIG. 9 is a representation of the unmodified RNase A and R-P-ATP (a) RNase A, R-P-ATP and acid and H₂O₂MALDI-TOF spectrum of the treated R-P-ATP; particle size (b) and zeta potential (c) of RNase A and R-P-ATP;

FIG. 10 shows the particle size change of LPP/RNase A nanocomposite in PBS.

FIG. 11 is a representation of LPP/R-P-ATP nanocomplex. Particle size of the complexes prepared at different LPP/R-P-ATP mass ratios (a), zeta potential (b) and particle size change in PBS (c);

FIG. 12 is an enzyme activity assay by the EtBr method for different proteins or nanocomplexes under different conditions. (a) The fluorescence intensity change of the RNA-EtBr complex under different protein or nano-composite treatments; (b) RNase A, R-P-ATP, LPP/R-P-ATP complex and acid and H₂O₂Relative enzymatic activity of LPP/R-P-ATP complex after treatment (n = 3);

FIG. 13 shows phosphorylation modifications of BSA and Cyt C and endocytosis studies after complexing with LPP. (a) Particle size, potential of LPP/BSA-P-ATP and (b) LPP/Cyt C-P-ATP; (c) uptake of LPP/FITC-BSA-P-ATP and (d) LPP/FITC-Cyt C-P-ATP complexes on MCF-7 cells, scale 100 μm.

Detailed Description

The invention provides a protein phosphorylation modification method, which prepares phosphorylated protein from protein, phenylboronic acid molecules and adenosine triphosphate, and is schematically shown as follows:

the phosphorylated protein of the invention can be compounded with cationic polypeptide through charge interaction to form a nano-composite. Therefore, the invention provides a phosphorylated protein-based intracellular delivery system which is composed of phosphorylated protein and cationic polypeptide, and the preparation method comprises the steps of dissolving the water-soluble phosphorylated protein in sodium bicarbonate buffer solution to form solution, and then mixing the solution with the cationic polypeptide solution to obtain the phosphorylated protein-based intracellular delivery system which is nano-drug.