CN111621288A

CN111621288A - Self-assembly material capable of forming nano defense network in tumor in situ and preparation method and application thereof

Info

Publication number: CN111621288A
Application number: CN201910147454.4A
Authority: CN
Inventors: 王浩; 王磊; 李秉南
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2019-02-27
Filing date: 2019-02-27
Publication date: 2020-09-04
Anticipated expiration: 2039-02-27
Also published as: CN111621288B

Abstract

The invention relates to a self-assembly material capable of forming a nano defense network in situ on a tumor and a preparation method thereofThe self-assembly material consists of targeting peptide, self-assembly polypeptide and a pyrene fluorescence signal molecule, and the chemical structure of the self-assembly material is shown as the formula (I); r₁From self-assembling polypeptides having multiple hydrogen bonds within the molecule; r₂Derived from a tumor targeting peptide. The preparation method comprises the following steps: the self-assembly material is synthesized by taking amino acid with protected terminal amino group and side chain amino group and a dipyrene fluorescent signal molecule as raw materials through a solid-phase synthesis method. The nano-fiber network can actively target a tumor part, can be induced to deform at the tumor part, and can form a nano-fiber network structure through self-assembly, namely a nano-defense network can be formed in situ in a tumor, so that a vascular endothelial growth factor which induces new blood vessels to generate is released from tumor cells to be captured, the generation of new blood vessels is prevented, the nutrition supply of the tumor is cut off, and the transfer path of the tumor is also blocked.

Description

Self-assembly material capable of forming nano defense network in tumor in situ and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a self-assembly material, a preparation method and application thereof, and particularly relates to a self-assembly material capable of forming a nano defense network in situ on a tumor, and a preparation method and application thereof.

Background

Tumor refers to a new organism formed by local histiocyte hyperplasia under the action of various tumorigenic factors. Tumors are divided into benign tumors and malignant tumors, the malignant tumors seriously harm the health of people, and the reason that the cancer death rate is high is mainly the rapid tumor metastasis, so that the inhibition of tumor metastasis is a key factor for tumor treatment. Tumor metastasis is an extremely complex process, and inhibition of tumor metastasis by inhibiting the activity of metallomatrix proteases is a commonly used strategy, because degradation of extracellular matrix by metallomatrix proteases is a major factor leading to tumor metastasis, but the method has little effect. In addition, there are some reports on tumor metastasis inhibition strategies in the prior art.

CN103520145A discloses the influence of tretinoin and paclitaxel on the in vitro migration ability of tumor cells and the lumen formation ability of vascular endothelial cells, and compares the anti-tumor metastasis abilities of the tretinoin and the paclitaxel, and the experimental results show that: the growth inhibition rate and the lethality of the melanoma cells by the tretinol are lower than those of the taxol, and the inhibition capability of the tretinol on the migration of the melanoma cells and the formation of vascular endothelial cell lumens is slightly lower than that of the taxol. The vitamin A-D-taxol has stronger anti-tumor metastasis activity, and although the anti-metastasis capability of the vitamin A-D-taxol is weaker than that of taxol, the low-cytotoxicity characteristic of the vitamin A-D-taxol is still expected to become a new anti-tumor metastasis medicine.

CN101214342A discloses a rhizoma paridis total saponin extract with anti-tumor metastasis effect and a pharmaceutical preparation thereof, wherein the pharmaceutical preparation consists of the rhizoma paridis total saponin extract as an active ingredient and pharmaceutic adjuvants, wherein the rhizoma paridis total saponin extract accounts for 60-98% of the total weight of the pharmaceutical preparation. The paris polyphylla total saponin extract with the function of resisting tumor metastasis and the pharmaceutical preparation thereof have good inhibiting effect on the metastasis of animal transplanted tumor cells and have no obvious toxicity to main organs.

CN101319008B discloses a polypeptide specifically combined with tumor metastasis cells and application thereof, wherein the amino acid sequence of the polypeptide specifically combined with the surface antigen of the tumor metastasis cells is Leu-Pro-Trp-Lys-Glu-Pro-Tyr-Tyr-Leu-Met-Pro-Pro. The polypeptide has the characteristics of specific binding with tumor metastasis cells and inhibition of tumor metastasis cell movement, so that the polypeptide can be used as a marker for tumor metastasis diagnosis and can be applied to the development of precursors of drugs for inhibiting tumor metastasis.

However, since there are few treatment strategies for inhibiting tumor metastasis in the prior art, it is very important to develop a novel treatment strategy that can inhibit tumor metastasis with a significant therapeutic effect.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a self-assembly material, a preparation method and application thereof, in particular to a self-assembly material capable of forming a nano defense network in situ on a tumor, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

on one hand, the invention provides a self-assembly material capable of forming a nano defense network in situ on a tumor, the self-assembly material consists of targeting peptide, self-assembly polypeptide and a dipyrene fluorescent signal molecule, and the chemical structure of the self-assembly material is shown as a formula (I);

wherein R is₁From self-assembling polypeptides having multiple hydrogen bonds within the moleculeA peptide; r₂Derived from a tumor targeting peptide.

The R is₁Self-assembly polypeptide with multiple hydrogen bonds in molecules, wherein the self-assembly polypeptide is respectively connected with a pyrene fluorescence signal molecule and R in an amido bond connection mode₂Is connected to R₂Derived from a tumor targeting peptide linked to R by an amide bond₁And (4) connecting.

The self-assembly polypeptide in the self-assembly material has good biocompatibility and self-assembly capability, the self-assembly material shown in the formula (I) can be self-assembled to form nano spherical particles through the hydrophilic-hydrophobic water balance of a dipyrene fluorescence signal molecule segment, the nano spherical particles are passively enriched on a tumor part through an EPR effect, and can be induced to deform at the tumor part, and a beta-folded nano fiber mesh structure is formed through self-assembly, so that a nano defense network can be formed in situ of a tumor, so that a tumor cell is released to induce the vascular endothelial growth factor generated by new blood vessels to be captured, the generation of new blood vessels is prevented, the nutrition supply of the tumor is cut off, and the transfer pathway of the tumor is also blocked.

The tumor targeting peptide in the self-assembly material can realize active targeting on a tumor part, and improve the biological safety and bioavailability of the material; the dipyryrene fluorescence signal molecules in the self-assembly material can emit green fluorescence in an aggregation state, so that real-time biological imaging of the self-assembly material can be realized.

Preferably, said R is₁From the polypeptide sequence Leu-Pro-Phe-Phe-Asp.

The structural formula of Leu-Pro-Phe-Phe-Asp is shown as formula (II):

preferably, said R is₂Is derived from the polypeptide sequence Ala-Thr-Trp-Leu-Pro-Pro-Arg, Ala-Ser-Ser-Ser-Tyr-Pro-Leu-Ile-His-Trp-Arg-Pro-Trp-Ala-Arg or Cys-Gly-Leu-Ser-Asp-Ser-Cys.

The structural formula of the Ala-Thr-Trp-Leu-Pro-Pro-Arg is shown as the formula (III):

the structural formula of the Ala-Ser-Ser-Ser-Tyr-Pro-Leu-Ile-His-Trp-Arg-Pro-Trp-Ala-Arg is shown as the formula (IV):

the structural formula of Cys-Gly-Leu-Ser-Asp-Ser-Cys is shown as the formula (V):

preferably, said R is₁Derived from the polypeptide sequence Leu-Pro-Phe-Phe-Asp, said R₂From the polypeptide sequence Ala-Thr-Trp-Leu-Pro-Pro-Arg; wherein R is₁Wherein Asp end is connected with the dipyrene fluorescent signal molecule R₁Leu terminal of (1) and R₂Ala in (b) is terminally attached.

Wherein, the strong hydrogen bonding of the polypeptide sequence Leu-Pro-Phe-Phe-Asp induces the material to be converted into the nano-fiber from the nano-particle. The Ala-Thr-Trp-Leu-Pro-Pro-Arg sequence is capable of targeting nascent vascular endothelial growth factor, preventing binding to its receptor, and inducing R upon binding to endothelial growth factor₁Deformation occurs.

In another aspect, the present invention provides a method for preparing the self-assembly material capable of forming a nano defense network in situ in a tumor, the method comprising:

the self-assembly material capable of forming the nano defense network in situ on the tumor is synthesized by taking amino acid with the terminal amino group and the side chain amino group protected and a dipyrene fluorescence signal molecule as raw materials through a solid phase synthesis method.

The structural formula of the dipyryrene fluorescent signal molecule is shown as a formula (VI).

Preferably, the preparation method specifically comprises the following steps:

(1) swelling the carrier resin;

(2) adopting amino acid with Fmoc protection obtained from terminal amino group, Boc protection obtained from side chain amino group and dipyrene fluorescent signal molecule as raw materials, firstly, according to R₂Amino acid sequence of (1), R₂Adding a first amino acid to the carrier resin, and performing coupling reaction and connection with the carrier resin; removing R₂Fmoc protecting group on the first amino acid, reacting R₂A second amino acid with R₂The first amino acid is coupled and ligated; until R is completed₂Condensation of all amino acids in (1);

(3) removing R₂Fmoc protecting group of the last amino acid, according to R₁Amino acid sequence of (1), R₁First amino acid with R₂The last amino acid is subjected to coupling reaction and ligation; removing R₁Fmoc protecting group on the first amino acid, reacting R₁A second amino acid with R₁The first amino acid is coupled and ligated; until R is completed₁Condensation of all amino acids in (1);

(4) removing R₁Fmoc protecting group of the last amino acid, and the carboxyl end of the dipyrene fluorescent signal molecule and R₁The last amino acid is subjected to coupling reaction and ligation;

(5) and (4) removing the product obtained in the step (4) from the carrier resin, and removing side chain amino protection to obtain the self-assembly material capable of forming the nano defense network in situ on the tumor.

Preferably, the carrier resin of step (1) is Wang resin.

Preferably, the reagent used for swelling in step (1) is N, N-dimethylformamide.

Preferably, the deprotection agent used for removing the Fmoc protecting group is a mixed solvent of N, N-dimethylformamide and piperidine.

Preferably, the volume ratio of the N, N-dimethylformamide to the piperidine is 4: 1.

Preferably, the Fmoc protecting group removal duration is 10-15min, such as 10min, 10.5min, 11min, 11.5min, 12min, 13min, 14min or 15min, and the like.

Preferably, the Fmoc-removal is performed at 20-30 ℃, e.g., 20 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, or 30 ℃.

Preferably, the reaction product is washed after the Fmoc protecting group is removed.

Preferably, the reagents used for the washing are N, N-dimethylformamide and dichloromethane.

Preferably, the coupling agent used in the coupling reaction is a mixed solvent of N, N-dimethylformamide and N-methylmorpholine.

Preferably, the volume ratio of the N, N-dimethylformamide to the N-methylmorpholine is 95: 5.

Preferably, the reaction time of the coupling reaction is 60-90min, such as 60min, 65min, 70min, 72min, 75min, 80min, 85min or 90min, etc.

Preferably, the reaction temperature of the coupling reaction is 20-30 ℃, such as 20 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃ and the like.

Preferably, the reaction product is washed after the coupling reaction is completed.

Preferably, the specific method for removing the product obtained in the step (4) from the carrier resin and removing the side chain amino protection in the step (5) is as follows:

and shrinking the resin, drying in vacuum, cracking the dried resin by using a cracking solution, after cracking, carrying out suction filtration and rotary evaporation on the filtrate, separating out the polypeptide in the rotary-dried product, centrifuging the polypeptide, and drying.

Preferably, the solvent used for the shrink resin is methanol.

Preferably, the duration of the shrinking resin is 20-40min, such as 20min, 23min, 25min, 28min, 30min, 32min, 35min, 37min or 40min, etc.

Preferably, the lysis solution comprises 2.5% of deionized water, 2.5% of triisopropylsilane, 2.5% of 1, 2-ethanedithiol and 92.5% of trifluoroacetic acid by total mass of 100%.

Preferably, the reagent used for precipitating the polypeptide is anhydrous diethyl ether.

In another aspect, the invention provides an application of the self-assembly material capable of forming the nano defense network in situ of a tumor in preparation of an anti-tumor drug.

Compared with the prior art, the invention has the following beneficial effects:

the tumor targeting peptide in the self-assembly material structure can realize active targeting on a tumor part, and improve the biological safety and bioavailability of the material; the dipyryrene fluorescence signal molecules in the structure can spontaneously emit green fluorescence in an aggregation state, so that real-time biological imaging of the self-assembly material is realized; the self-assembled polypeptide in the structure has good biocompatibility and mechanical property, can lead self-assembled materials to form nano spherical particles by self-assembly, is passively enriched on a tumor part through EPR effect, can be induced to deform at the tumor part, and forms a nano fiber net structure by self-assembly, namely a nano defense network can be formed in situ on the tumor, thereby releasing tumor cells to induce the vascular endothelial growth factor generated by new blood vessels to capture, preventing the generation of new blood vessels, cutting off the nutrition supply of the tumor and obstructing the transfer path of the tumor.

Drawings

FIG. 1 is a graph of the results of mass spectrometry characterization of the self-assembled material prepared in example 1;

FIG. 2 is a fluorescence spectrum of self-assembled nanoparticles and self-assembled material monomers prepared in example 2;

FIG. 3 is a transmission electron micrograph of the self-assembled nanoparticles prepared in example 2;

FIG. 4 is a transmission electron micrograph of the nanofiber prepared in example 2;

FIG. 5 is a scanning electron micrograph of example 3;

FIG. 6 is a graph showing the results of wound healing in the control group in example 4;

FIG. 7 is a graph showing the results of wound healing in the experimental group of example 4;

FIG. 8 is a graph showing the results of cell migration in the control group in example 5;

FIG. 9 is a graph showing the results of cell migration in the experimental group in example 5;

FIG. 10 is a sectional view of a tumor in example 6;

FIG. 11 is a lung tissue map of a control group in example 6;

FIG. 12 is a lung tissue map of the experimental group in example 6;

FIG. 13 is a graph showing HE staining of lung tissue in the control group in example 6;

FIG. 14 is a graph showing HE staining of lung tissue in the experimental group of example 6.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The self-assembly material capable of forming a nano defense network in situ on the tumor is constructed in the embodiment, the self-assembly material consists of targeting peptide, self-assembly polypeptide and a pyrene fluorescence signal molecule, and the chemical structure of the self-assembly material is shown as follows;

(1) swelling Wang resin with N, N-dimethylformamide;

(2) adopting amino acid with Fmoc protection obtained from terminal amino group, amino acid with Boc protection obtained from side chain amino group and a dipyrene fluorescent signal molecule as raw materials, firstly, adding Arg into carrier resin according to the amino acid sequence of Ala-Thr-Trp-Leu-Pro-Pro-Arg, and carrying out coupling reaction and connection with Wang resin; removing the Fmoc protecting group on Arg, and performing coupling reaction and connection on a second amino acid Pro and Arg; until the condensation of all amino acids in Ala-Thr-Trp-Leu-Pro-Pro-Arg is completed;

(3) removing the Fmoc protecting group of the last amino acid Ala, and coupling and connecting the first amino acid Leu and Ala according to the amino acid sequence of Leu-Pro-Phe-Phe-Asp; removing the Fmoc protecting group on the Leu, and performing coupling reaction on the second amino acid Pro and the Leu and connecting; until the condensation of all amino acids in Leu-Pro-Phe-Phe-Asp is completed;

(4) removing the Fmoc protecting group of the last amino acid Asp, and performing coupling reaction on the carboxyl end of the pyrene fluorescence signal molecule and Asp and connecting;

(5) and (3) shrinking the resin by using methanol for 30min, carrying out vacuum drying, cracking the dried resin by using a cracking solution, after cracking, carrying out suction filtration and rotary evaporation on the filtrate, separating out the polypeptide in the rotary-dried product by using anhydrous ether, centrifuging the polypeptide, and drying to obtain the self-assembly material capable of forming the nano defense network in situ on the tumor.

The prepared self-assembly material is characterized by mass spectrum, and R is₂The polypeptide sequence of (A) is Ala-Thr-Trp-Leu-Pro-Pro-Arg, and the mass spectrum characterization result is shown in FIG. 1, and it can be seen from FIG. 1 that: the molecular weight of the polypeptide material can be basically consistent with that of the designed polypeptide material as seen by a main peak (m/z-2052.68) of a mass spectrum, thereby deducing that the target molecule is synthesized, and indicating that the self-assembly material with the structure of the formula is successfully synthesized.

Example 2

In this example, a self-assembled nanoparticle solution and a nanofiber dispersion are prepared by the following specific methods:

the self-assembly material prepared in example 1 was dissolved in DMSO solvent (concentration of self-assembly material 1.5 × 10)^-3M), get above-mentioned solution and place in the centrifuging tube, slowly add the centrifuging tube with deionized water again, prepare out the mixed solution of different water contents (0%, 40%, 98%), carry out fluorescence detection with the fluorescence spectrophotometer respectively with self-assembling material monomer solution (water content 0%), self-assembling nanoparticle solution (water content 40%) and self-assembling nanoparticle solution (water content 98%), the fluorescence map is as shown in figure 2, can find out by figure 2: the self-assembly nano-particle solution (with the water content of 40 percent) and the self-assembly nano-particle solution (with the water content of 98 percent) have peak values at the position of 525-550nm,the fluorescence intensity was highest at a water content of 98%. The result shows that the dipyryrene fluorescent molecules gradually form nanoparticles along with the increase of the water content until the nanoparticles are completely aggregated when the water content is more than 98%.

The obtained self-assembled nanoparticles were characterized by transmission electron microscopy, and the results are shown in fig. 3, which is shown in fig. 3: the self-assembled material prepared in example 1 formed self-assembled particles in an aqueous solution.

VEGF solution (vascular endothelial growth factor solution, concentration 3.0 × 10) was added to the self-assembled nanoparticle solution obtained above^-5M, solvent is H₂DMSO 98:2) to a concentration of 1.5 × 10^-3And M, standing for 48 hours to obtain the nanofiber dispersion liquid. The obtained nanofibers were characterized by transmission electron microscopy, and the results are shown in fig. 4, which shows that: the self-assembled polypeptide material becomes short fiber-like.

This phenomenon demonstrates that: the material can be specifically combined with VEGF, and then the nano particles are converted into nano fibers, so that the tumor targeting is realized. Because in the tumor environment, a large amount of VEGF is present, which is not found in normal tissue organs, VEGF is both the target site: the self-assembly material is specifically combined with VEGF, so that the self-assembly material is only gathered at a tumor site and is not other tissues and organs; and also the reason for the deformation of the self-assembly material: the material will specifically bind to VEGF and thereby be converted from nanoparticles to nanofibers.

Example 3

And (3) scanning electron microscope test:

in this example, human breast cancer MDA-MB-231 was used as a cell model, a silicon wafer was placed on the bottom of a culture dish, cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, and 100. mu.g/mL streptomycin for 24 hours, and then 30. mu.M of the self-assembly material prepared in example 1 was added and cultured for 2 hours at 37.0 ℃ under CO₂Was 5.0%, cells were grown on the silicon wafer.

Then the culture medium is discarded, washed with PBS for three times, fixed by adding 4% paraformaldehyde for 1h, washed with PBS for two times, and then the cells are dehydrated in a gradient manner by using 30%, 50%, 70%, 90% and 100% ethanol/PBS solution, each concentration is removed for two times, each time is 10min, and finally the cells are rinsed with tert-butyl alcohol for 30 min. And (4) after the treatment, putting the cells into a vacuum drying oven for drying, and observing and scanning by using a scanning electron microscope after the drying is finished. The test results are shown in fig. 5, from which it can be seen that: the material is subjected to shape transformation near MDA-MB-231 cells to form a network-like nanofiber structure.

Example 4

Cell wound healing assay:

in the embodiment, the human breast cancer cell MDA-MB-231 is taken as a cell model and is divided into a control group and an experimental group. Cells were seeded into 24-well cell culture plates at a concentration of 10 per ml⁴And (3) culturing the cells in a DMEM culture medium for 24 hours, slightly scratching the cells in the monolayer by using a new 200-mu-L gun head without changing the culture medium, wherein the scratch transversely penetrates through the hole, and the gun head is vertical to the bottom of the hole plate. After scratching, the well plate was gently washed with a culture medium 2 times to remove exfoliated cells, and then a fresh culture medium was added to each well, and compared with the control group, 30 μ M of the self-assembly material prepared in example 1 was further added to the culture medium of the experimental group, the cells were cultured for 24 hours, washed with PBS 2 times, then fixed with anhydrous methanol for 30 minutes, stained with 0.1% crystal violet (dissolved in 2% ethanol) for 30 minutes, washed with PBS 3 times, and observed by taking a picture with a microscope. The test results are shown in fig. 6 and 7 (the solid line in the figure indicates the width of the scratch), and it can be seen from the figure that: the blank area in the middle of the experimental group is wider than that of the control group, which shows that the experimental group has stronger capability of inhibiting the lateral migration of the tumor, and proves that the self-assembly material can effectively inhibit the lateral migration of the tumor.

Example 5

Cell migration assay:

in this example, human breast cancer MDA-MB-231 was used as a cell model and divided into a control group and an experimental group. The Transwell chamber was placed in a 24-well plate, 300. mu.L of pre-warmed serum-free medium was added to the upper chamber and allowed to stand at room temperature for 30min to rehydrate the matrigel, and the remaining medium was aspirated. The MDA-MB-231 cells in culture were serum deprived for 12h and further blood was removedEffect of the cleaning, trypsinization of MDA-MB-231 cells to prepare a cell suspension, which is adjusted to 5 × 10/ml with the culture medium⁵The cells were collected and 200. mu.L of each cell was placed in a Transwell chamber, 30. mu.M of the self-assembly material prepared in example 1 was placed in the upper chamber, 500. mu.L of a medium containing 10% fetal bovine serum was placed in the lower chamber, the cells were cultured for 24 hours, the chamber was taken out, the cells on the upper surface of the chamber were gently scraped off with a cotton swab, the cells on the lower surface of the chamber were fixed with absolute methanol for 30 minutes, stained with 0.1% crystal violet (dissolved in 2% ethanol) for 30 minutes, washed with PBS for 3 times, and observed by taking pictures with a microscope. The test results are shown in fig. 8 and 9, from which it can be seen that: the self-assembly material can effectively inhibit the longitudinal migration of tumors.

Example 6

Animal experiments:

the experimental animals selected Balb/c female nude mice 20, were randomly divided into control group and experimental group, 10 animals in each group were pre-fed for 7 days, and injected subcutaneously at the right chest of 20 mice about 5 × 10⁶MDA-MA-231 cells, establishing mouse tumor model. When the tumor volume of the mouse is about 100mm³On the left and right, the administration treatment is started. The mice in the experimental group were intravenously administered 200. mu.L every 72 hours, the self-assembly material prepared in example 1 in a physiological saline solution at a concentration of 100. mu.M for 7 times, and the mice in the control group were intravenously administered 7 times 200. mu.L of physiological saline every 72 hours.

After the end of the experiment, the mice were dissected and the mice tumor sections were performed, as shown in fig. 10 (the arrows in the figure indicate the nanofibrous structure of the material), as can be seen: the material forms a nano-fibrous network structure at the tumor site of the mouse.

The lung tissue of the mouse was taken out and observed, as shown in fig. 11 and 12, fig. 11 is the lung tissue of the control group mouse, fig. 12 is the lung tissue of the experimental group mouse (the arrow in fig. 11 and 12 indicates the position of the lung nodule), and it can be seen that: the number of pulmonary nodules of the mice in the experimental group is remarkably reduced compared with that of the mice in the control group, and the self-assembly material is proved to effectively inhibit the metastasis and invasion of tumor cells.

The results of paraffin section and HE staining of mouse lung tissue are shown in fig. 13 and 14, fig. 13 is lung tissue section of control group mouse, fig. 14 is lung tissue section of experimental group mouse, which can be seen from the figure: the experimental group showed significantly less metastases compared to the control group, further illustrating the ability of the self-assembled material to resist tumor invasion and metastasis, which also corresponds to the above test results for the number of lung nodules.

The applicant states that the self-assembly material capable of forming a nano defense network in situ on a tumor, the preparation method and the application thereof are illustrated by the above embodiments, but the invention is not limited to the above embodiments, i.e. the invention is not meant to be implemented only by relying on the above embodiments. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> national center for Nano science

<120> self-assembly material capable of forming nano defense network in tumor in situ and preparation method and application thereof

<130>2019

<160>4

<170>PatentIn version 3.3

<210>1

<211>5

<212>PRT

<213> artificially synthesized sequence

<400>1

Leu Pro Phe Phe Asp

1 5

<210>2

<211>7

<212>PRT

<213> artificially synthesized sequence

<400>2

Ala Thr Trp Leu Pro Pro Arg

1 5

<210>3

<211>15

<212>PRT

<213> artificially synthesized sequence

<400>3

Ala Ser Ser Ser Tyr Pro Leu Ile His Trp Arg Pro Trp Ala Arg

1 5 10 15

<210>4

<211>7

<212>PRT

<213> artificially synthesized sequence

<400>4

Cys Gly Leu Ser Asp Ser Cys

1 5

Claims

1. A self-assembly material capable of forming a nano defense network in situ on a tumor is characterized by comprising targeting peptide, self-assembly polypeptide and a pyrene fluorescence signal molecule, wherein the chemical structure of the self-assembly material is shown as a formula (I);

wherein R is₁From self-assembling polypeptides having multiple hydrogen bonds within the molecule; r₂Derived from a tumor targeting peptide.

2. The self-assembled material capable of forming nano defense network in tumor in situ according to claim 1, wherein R is₁From the polypeptide sequence Leu-Pro-Phe-Phe-Asp.

3. The self-assembled material capable of forming nano defense network in tumor in situ as claimed in claim 1 or 2, wherein R is₂Is derived from the polypeptide sequence Ala-Thr-Trp-Leu-Pro-Pro-Arg, Ala-Ser-Ser-Ser-Tyr-Pro-Leu-Ile-His-Trp-Arg-Pro-Trp-Ala-Arg or Cys-Gly-Leu-Ser-Asp-Ser-Cys.

4. The self-assembled material capable of forming nano defense network in tumor in situ according to any one of claims 1 to 3, wherein R is₁Derived from the polypeptide sequence Leu-Pro-Phe-Phe-Asp, said R₂From the polypeptide sequence Ala-Thr-Trp-Leu-Pro-Pro-Arg; wherein R is₁Wherein Asp end is connected with the dipyrene fluorescent signal molecule R₁Leu terminal of (1) and R₂Ala in (b) is terminally attached.

5. The method for preparing the self-assembly material capable of forming the nano defense network in the tumor in situ according to any one of claims 1 to 4, wherein the method for preparing the self-assembly material is as follows:

6. The method for preparing the self-assembly material capable of forming the nano defense network in the tumor in situ according to claim 5, wherein the method specifically comprises the following steps:

(1) swelling the carrier resin;

(2) fmoc protection by terminal amino group, Boc protection by side chain amino group, and bispyrene fluorescenceThe signal molecule is used as a starting material, first according to R₂Amino acid sequence of (1), R₂Adding a first amino acid to the carrier resin, and performing coupling reaction and connection with the carrier resin; removing R₂Fmoc protecting group on the first amino acid, reacting R₂A second amino acid with R₂The first amino acid is coupled and ligated; until R is completed₂Condensation of all amino acids in (1);

7. The method for preparing the self-assembly material capable of forming the nano defense network in the tumor in situ according to claim 6, wherein the carrier resin in the step (1) is Wang resin;

preferably, the reagent used for swelling in step (1) is N, N-dimethylformamide;

preferably, the deprotection agent used for removing the Fmoc protecting group is a mixed solvent of N, N-dimethylformamide and piperidine;

preferably, the volume ratio of the N, N-dimethylformamide to the piperidine is 4: 1;

preferably, the duration of the Fmoc protecting group removal is 10-15 min;

preferably, the removal of the Fmoc protecting group is performed at 20-30 ℃;

preferably, after the Fmoc protecting group is removed, the reaction product is washed;

8. The method for preparing the self-assembly material capable of forming the nano defense network in the tumor in situ according to claim 6 or 7, wherein the coupling agent used in the coupling reaction is a mixed solvent of N, N-dimethylformamide and N-methylmorpholine;

preferably, the volume ratio of the N, N-dimethylformamide to the N-methylmorpholine is 95: 5;

preferably, the reaction time of the coupling reaction is 60-90 min;

preferably, the reaction temperature of the coupling reaction is 20-30 ℃;

preferably, the reaction product is washed after the coupling reaction is completed;

preferably, the reagents used for the washing are N, N-dimethylformamide and dichloromethane;

9. The method for preparing the self-assembly material capable of forming the nano defense network in the tumor in situ according to claim 8, wherein the solvent used by the shrinkage resin is methanol;

preferably, the duration of the shrinking resin is 20-40 min;

preferably, the cracking solution comprises 2.5% of deionized water, 2.5% of triisopropylsilane, 2.5% of 1, 2-ethanedithiol and 92.5% of trifluoroacetic acid by total mass of 100%;

10. Use of the self-assembly material capable of forming a nano defense network in tumor in situ according to any one of claims 1 to 4 in preparation of antitumor drugs.