阅读说明：本技术 一种多肽纳米载体及其制备方法和应用 (Polypeptide nano-carrier and preparation method and application thereof ) 是由 管斐 李国瑞 田泾 宫春爱 韩治敏 陈继源 武鑫 顾永卫 傅志勤 许幼发 张元声 于 2019-10-10 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及一种多肽纳米载体及其制备方法和应用。本发明提供一种多肽构建的纳米载体,由丙氨酸,甘氨酸,等组成的短肽及胆固醇组成。本发明的所形成的多肽可在细胞自然降解,多肽中的多种氨基酸均为体内存在的氨基酸,对细胞及人体无毒副作用,CCK-8法细胞增殖试验表明,制备的纳米载体具有很低的细胞毒性,同时又有较好的共载基因与不溶性化合物的能力,本发明的纳米载体能在乳腺癌免疫治疗中能特异性靶向肿瘤细胞,递送免疫检查点阻断剂,促进自身抗肿瘤免疫,促使乳腺癌细胞的凋亡,从而成为乳腺癌免疫治疗的一种靶向、高效、低毒的纳米级递送系统。(The invention relates to the technical field of medicines, in particular to a polypeptide nano-carrier and a preparation method and application thereof. The invention provides a nano-carrier constructed by polypeptide, which consists of short peptide consisting of alanine, glycine and the like and cholesterol. The polypeptide formed by the invention can be naturally degraded in cells, various amino acids in the polypeptide are all amino acids existing in vivo, and have no toxic or side effect on cells and human bodies, and CCK-8 cell proliferation tests show that the prepared nano carrier has very low cytotoxicity and better capability of co-loading genes and insoluble compounds.)

1. A polypeptide, characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A cholesterol-modified polypeptide according to claim 1, wherein the cholesterol-modified polypeptide is a cholesterol-modified polypeptide, and the cholesterol modification is that an amino group of the polypeptide is connected with a carboxyl group of cholesterol by an amido bond, and the chemical structure is shown as formula (I):

3. the cholesterol-modified polypeptide of claim 2, wherein the molecular weight of the polypeptide nanocarrier is 2289-2300 Da.

4. A polypeptide nano-carrier is characterized in that the preparation method comprises the following steps:

(A) synthesis of the cholesterol-modified polypeptide of claim 2;

(B) dissolving the synthesized cholesterol modified polypeptide in DMSO, adding into a dialysis bag with molecular cut-off of 1000, dialyzing in water for 12h, changing water every 2h, concentrating, lyophilizing, and redissolving.

5. Use of the polypeptide of claim 1, the cholesterol-modified polypeptide of claim 2, the polypeptide nanocarrier of claim 4 in the preparation of chemotherapeutic or genetic drugs.

6. Use of the polypeptide of claim 1, the cholesterol-modified polypeptide of claim 2, the polypeptide nanocarrier of claim 4 for the preparation of a combination chemotherapeutic and genetic drug.

7. Use of the polypeptide of claim 1, the cholesterol-modified polypeptide of claim 2, the polypeptide nanocarrier of claim 4 in the preparation of a medicament for treating breast cancer.

8. The use of the polypeptide nanocarrier of claim 4 for co-loading a gene and a chemotherapeutic agent in the preparation of a medicament for treating breast cancer.

9. The use of claims 5-8, wherein said gene is siRNA; the chemotherapeutic drug is water-insoluble 1-methyltryptophan.

10. The application of claim 9, wherein the application:

preparing nano micelle by a dialysis method for the nano carrier and the chemotherapeutic drug, and mixing the nano micelle with siRNA to prepare a co-loading system;

the encapsulation rate of the chemotherapeutic drug is 75-80%, and the nitrogen-phosphorus ratio of the nano carrier to the siRNA is 5:1-40: 1.

Technical Field

The invention relates to the technical field of medicines, in particular to a polypeptide nano-carrier and a preparation method and application thereof.

Background

Breast cancer is one of the most common malignant cancers in women worldwide. Currently, the breast cancer treatment methods in clinical use include radiotherapy, chemotherapy, targeted therapy, endocrine therapy, and the like, and chemotherapy is considered as an important means for treating breast cancer. However, during chemotherapy, drug resistance often develops, reducing the cure rate of breast cancer and often leading to patient death. Breakthrough of new treatment modes is urgently needed.

With the intensive research on tumor immunology, cancer immunotherapy is becoming the standard method for treating many cancers, especially with the drug of anti PD-1/PD-L1 (immune checkpoint blocker), with beneficial clinical results and a multi billion dollar market, further promoting its research and development. A series of clinical trials involving PD-1/PD-L1 breast cancer have also published promising results. However, only a small fraction of patients benefit from anti PD-1/PD-L1 treatment. anti PD-1/PD-L1 enhances T cell responses by blocking PD-1 function, but immune escape from tumors also involves other immunosuppressive molecules such as indoleamine 2,3-dioxygenase (IDO). IDO (indoleamine 2, 3-dioxygenase), which is the rate-limiting enzyme for the metabolic breakdown of tryptophan along the kynurenine pathway, leads to the depletion of tryptophan, and has been shown in studies that the metabolite kynurenine of tryptophan inactivates effector T cells and inhibits dendritic cell immunosuppression. A series of studies suggest that blocking the PD-1 and IDO pathways may be a potential therapeutic strategy for breast cancer treatment.

At present, there is no nanoparticle of polypeptide, and a carrier capable of carrying out targeted immunotherapy for treating breast cancer.

Disclosure of Invention

In the traditional administration mode, the off-target effect of the antibody and normal tissues and the reason that the antibody cannot be effectively transmitted in vivo can bring a series of immune-related side effects, and the expected therapeutic effect cannot be achieved, so that the invention considers introducing siRNA interference and realizing a precisely positioned mode to interfere the PD-1/PD-L1 pathway. 1-methyl-DL-tryptophan (1-MT) has pharmacological inhibiting effect of IDO, and can enhance T cell dependent antitumor immunity. The invention assumes that the combination of 1MT and anti-PD-L1siRNA has good prospect in treating breast cancer.

The nano-carrier with a core-shell nano-structure represented by the polymer micelle can effectively encapsulate genes and chemotherapeutic drugs. Based on the amphiphilic structure of the peptide, a hydrophobic core can be coated with a water-insoluble drug, and a cationic peptide segment can compress a negatively charged siRNA. Linear polypeptide (Lin TT1) (SEQ ID NO: AKRGARSTA) can effectively bind to p32/gC1qR on the surface of tumor cells and endothelial cells, achieving effective breast cancer tumor homing and penetration. In addition, histidine has been shown to be effective in achieving endosomal escape in a number of studies, since arginine residues can be protonated and deprotonated in different pH environments, which is based on the proton sponge hypothesis to achieve endosomal escape.

According to the invention, by constructing cholesterol modified polypeptide Chol-HHHHHHHAKRGARSTA (CHL for short), siRNA and chemical drug 1MT can be entrapped in micelle in a self-assembly manner. In physiological pH environment, 1MT is coated in a hydrophobic core through hydrophobic acting force and pi-pi conjugation, and meanwhile, siRNA is combined on a cation shell formed by Lin TT 1. After intravenous administration, because the targeting property of Lin TT1 can be accurately targeted to a tumor site, after cell internalization, the micelle complex can escape from the cell due to protonation of histidine structure in CHL under low pH environment, thereby causing swelling of the complex, accelerating drug release and delivering therapeutic genes to cell nucleus.

The invention aims to provide a safe and low-toxicity polypeptide nano-carrier which wraps up immunotherapy chemical drugs and has high gene transfection efficiency of external wrapped genes. The invention also aims to provide a preparation method of the polypeptide nano-carrier; the third purpose of the invention is to provide the combined application of the polypeptide nano-carrier in immunotherapy chemical drugs and gene therapy drugs.

The invention aims to solve the main technical problems that: how to improve the capacity of the polypeptide nano-carrier for carrying siRNA gene segments into cells and effectively transfecting the cells and how to improve the capacity of the polypeptide nano-carrier for carrying immunotherapy chemicals,

the invention designs a cholesterol-modified polypeptide nano-carrier, Lin TT1 has positive charges, can be combined with gene segments with negative charges, has the functions of breast cancer membrane penetration and positioning, and histidine promotes the escape of endosomes of the gene segments due to the proton sponge effect of histidine, so that the purposes of endosome escape and drug release can be achieved after the histidine enters cells.

In a first aspect of the present invention, there is provided a polypeptide, the amino acid sequence of which is as follows:

HHHHHHHAKRGARSTA (SEQ ID NO. 1). The amino acids are connected by peptide bonds. The polypeptide part is abbreviated in English to HL.

In a second aspect of the present invention, there is provided a cholesterol-modified polypeptide as described above, wherein the cholesterol modification is a linkage of an amino group of the polypeptide to a carboxyl group of cholesterol via an amide bond. The english abbreviation of cholesterol-modified polypeptide is CHL.

Further, the chemical structure of the cholesterol-modified polypeptide is shown as the formula (I):

further, the molecular weight of the cholesterol modified polypeptide is 2289-2300 Da. Preferably 2289.65 Da.

In a third aspect of the present invention, there is provided a polypeptide nanocarrier, wherein the preparation method comprises the following steps:

(A) synthesis of cholesterol-modified polypeptides as described above: synthesizing Chol-HHHHHHHAKRGARSTA (CHL);

(B) dissolving the synthesized CHL in DMSO, adding into a dialysis bag with molecular interception of 1000, dialyzing in water for 12h, changing water every 2h, concentrating, lyophilizing, and redissolving.

Further, the solution reacted in the step (B) is concentrated and then is freeze-dried by a freeze dryer.

Further, in the dialysis bag in the step (B), the dialysate is distilled water, and the dialysis is carried out for 12 hours.

In an embodiment of the present invention, step (B) is specifically: and dissolving the synthesized CHL in DMSO, adding into a dialysis bag with the molecular retention of 1000, dialyzing in water for 12h, changing water every 2h, concentrating, freeze-drying, and re-dissolving to obtain Blank CHL nano micelle (Blank-CHL).

In order to maintain the higher activity of the polypeptide nano carrier material, the dialyzed solution is freeze-dried and stored at-20 ℃, and the nano material can be stored for a long time at 4 ℃ after being redissolved.

The fourth aspect of the invention provides an application of the polypeptide, the cholesterol modified polypeptide and the polypeptide nano-carrier in preparing chemotherapeutic drugs or gene drugs.

In the fifth aspect of the invention, the application of the polypeptide, the cholesterol modified polypeptide and the polypeptide nano-carrier in preparing combined chemotherapeutic drugs and gene drugs is provided.

Furthermore, the invention provides application of the polypeptide, the cholesterol modified polypeptide and the polypeptide nano-carrier in preparation of a breast cancer treatment drug.

Furthermore, the invention also provides application of the polypeptide nano-carrier co-carried gene and chemotherapeutic drugs in preparation of breast cancer treatment drugs.

The application refers to that arginine in the polypeptide nano-carrier is positively charged and can be combined with negatively charged genes.

The application refers to that the fat solubility of cholesterol in the polypeptide nano-carrier can encapsulate fat-soluble chemical drugs.

The gene is siRNA.

Preferably, the gene is siPD-L1, and the sequence is shown as follows:

sense strand: 5'-AGAcGuAAGcAGuGuuGAA-3' (SEQ ID NO. 2);

antisense strand: 5'-UUcAAcACUGCUuACGUCU-3' (SEQ ID NO. 3).

The sense strand and the antisense strand can be added with TT at the 3' end to increase the stability of the siRNA.

The chemotherapeutic drug is water-insoluble 1-methyltryptophan (1 MT).

Further, the application comprises the following steps:

preparing nano micelle by a dialysis method for the nano carrier and the chemotherapeutic drug, and mixing the nano micelle with siRNA to prepare a co-loading system;

the encapsulation rate of the chemotherapeutic drug is 75-80%, and the nitrogen-phosphorus ratio of the nano-carrier to RNA is 5:1-40: 1.

The polypeptide nano-carrier has the capability of loading 1MT, and has better drug loading rate and encapsulation efficiency when the feeding ratio CHL:1MT is 3: 1.

Further, the polypeptide nano-carrier is mixed with siPD-L1 to prepare a gene transfection system.

The N/P ratio of CHL to sipD-L1 is 5:1-40:1, in the proportion range, the nano carrier material can guide the siPD-L1 into cells, and has higher transfection efficiency. Preferably, the N/P ratio is 20: 1.

in one embodiment of the invention, CHL and 1-methyltryptophan (1MT) are respectively dissolved in DMSO to prepare a solution, the solution is added into a dialysis bag with the molecular interception amount of 1000, dialysis is carried out in water for 12h, water is changed once every 2h, micelle 1MT-CHL is prepared after dialysis, freeze-drying is carried out at low temperature, then preservation is carried out at-20 ℃, and after 1MT-CHL is redissolved, the ratio of nitrogen to phosphorus (N/P) of CHL to sipD-L1 is 5:1-40:1 and siPD-L1, whirling for 10s, and standing for 30min to obtain Co-CHL (Co-CHL nano micelle loaded with immune checkpoint blockers siPD-L1 and 1 MT).

Preferably, the CHL and the siPD-L1 are mixed in a buffer solution, the pH value of the buffer solution is 5.0-7.0, the incubation is carried out for 20-30 minutes at room temperature, and the reasonable pH value and the incubation time ensure the formation of a gene transfection system.

The polypeptide nano-carrier provided by the invention is suitable for therapeutic siRNA and chemotherapeutic drugs required by experiments.

The invention has the advantages that:

1. the polypeptide nano-carrier of the invention is composed of a plurality of amino acids and cholesterol, the formed cholesterol modified polypeptide has no toxic or side effect on cells and human bodies, and CCK-8 cell proliferation experiments show that the prepared nano-carrier has very low cytotoxicity and better capability of co-loading genes and chemical drugs, thus being very suitable for in-vivo and in-vitro chemotherapy and gene therapy research and application.

2. The preparation method disclosed by the invention is simple to operate, the reaction reagent and the obtained product are non-toxic, the environment cannot be polluted, the reaction condition is mild, the polypeptide nano-carrier obtained after the reaction is simple to purify, the cost is low, and the preparation method is beneficial to large-scale popularization in the research and application fields.

3. The polypeptide nano-carrier can successfully deliver siRNA to inhibit autophagy specificity caused by chemotherapeutic drugs in breast cancer treatment, enhance the sensitivity of tumor cells to the chemotherapeutic drugs and promote the apoptosis of breast cancer cells, thereby becoming a targeted, efficient and low-toxicity nano-scale delivery system for breast cancer treatment.

Drawings

FIG. 1 nuclear magnetic resonance hydrogen spectrum of CHL;

FIG. 2 HPLC plot of CHL synthetic purity;

FIG. 3 is a particle size diagram of Co-CHL nanomicelle when N/P is 20;

FIG. 4 is a potential diagram of Co-CHL nano-micelle with N/P of 20;

FIG. 5 is a perspective electron microscope image of Co-CHL nano-micelle;

FIG. 6 examination of the release of 1MT under different pH conditions;

FIG. 7 shows the uptake of Nile Red-CHL by 4T1 cells at different time points;

FIG. 8 uptake of FAM-siPD-L1 by different N/P4T 1 cells;

FIG. 9 intracellular distribution of Co-CHL in 4T1 cells;

FIG. 10 shows the cytotoxicity of the vector on cells under different concentrations;

FIG. 11.1 MT, 1MT-CHL, Co-CHL cytotoxicity Studies;

FIG. 12 transfection effects of siPD-L1 at different concentrations;

FIG. 13 inhibition of kynurenine production;

FIG. 14 drug-stimulated apoptosis of tumor cells in tumor-lymphocyte co-culture environment.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.