CN106349331B

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Publication number: CN106349331B
Application number: CN201610709616.5A
Authority: CN
Inventors: 王浩; 罗强; 杨培培; 王磊; 齐国斌
Original assignee: Beijing Institute of Nanoenergy and Nanosystems
Current assignee: Beijing Institute of Nanoenergy and Nanosystems
Priority date: 2016-08-23
Filing date: 2016-08-23
Publication date: 2020-05-15
Anticipated expiration: 2036-08-23
Also published as: CN106349331A

Abstract

Translated fromChinese

本发明提供了一种基于双芘的pH响应自组装的多肽纳米材料及其制备方法和应用，所述多肽纳米材料具有式I‑IV所示结构，本发明的基于双芘的多肽纳米材料具有pH响应性，在1.0≤pH＜8.0的环境下自组装形成纳米纤维，在pH≥8.0的环境下组装成纳米球，可以招募疏水的小分子成像剂和药物，大大提高小分子成像剂和治疗药物的利用率，该纳米材料可在疾病组织或细胞部位原位转化为纤维，可以大大延长滞留时间，可用于生物成像和治疗，为疾病治疗提供可靠诊断以及靶向治疗方法，具有广泛的应用前景。The present invention provides a bipyrene-based pH-responsive self-assembled polypeptide nanomaterial and a preparation method and application thereof, wherein the polypeptide nanomaterial has a structure shown in formula I-IV, and the bipyrene-based polypeptide nanomaterial of the present invention has pH-responsive, self-assembled to form nanofibers in an environment of 1.0≤pH<8.0, and assembled into nanospheres in an environment of pH≥8.0, which can recruit hydrophobic small-molecule imaging agents and drugs, greatly improving small-molecule imaging agents and therapeutics The utilization rate of drugs, the nanomaterials can be converted into fibers in situ in diseased tissues or cells, which can greatly prolong the residence time, can be used for biological imaging and therapy, provide reliable diagnosis and targeted therapy for disease treatment, and have a wide range of applications prospect.

Description

Bipyrene-based pH response self-assembly polypeptide nano material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano materials, relates to a polypeptide nano material and a preparation method and application thereof, and particularly relates to a pH response self-assembly polypeptide nano material based on bispyrene and a preparation method and application thereof.

Background

In recent years, the application of nano materials in the diagnosis and treatment of clinical diseases has received great attention. Polypeptide self-assembled nanomaterials are widely used in biological imaging and therapy due to their good biocompatibility and chemical diversity. And nano-materials of different sizes and morphologies have great influence on biological imaging and therapy. Such as nanoparticles, related studies have shown that when the size of the nanoparticles is in the range of 10-100nm, the circulation time of the nanoparticles in vivo is increased; when the nanometer size is 15-350nm, the nanometer particles can be targeted and gathered at the myocardial infarction, tumor and other inflammation parts; but such nanoparticles will be rapidly cleared by the targeted tissue. Moreover, the structure of the nanoparticles is a metastable state, and the morphology of the nanoparticles is transformed along with the change of time and other factors (enzyme, temperature, pH and the like). Secondly, compared with nanoparticles, nanofibers have better stability and are not easily removed in vivo, which can prolong the residence time of the material. By utilizing the property of the nano fiber, the nano fiber can be used for long-acting biological imaging and treatment. On the other hand, small molecule imaging agents and therapeutic drugs have certain hydrophobicity, which limits their biological applications. The current method of use for small molecule imaging agents and therapeutic drugs is to encapsulate them with polymeric nanomaterials, which also presents certain challenges for the controlled release of small molecule imaging agents and drugs. Therefore, it is necessary to develop a new strategy for tumor detection and treatment.

Therefore, in the field, it is expected that a polypeptide nano-material capable of being transformed in situ to form a fiber can be designed by combining the stability of the nano-fiber and the hydrophobicity of the small molecule drug, so as to be used for recruiting the hydrophobic small molecule material and be used for biological imaging and treatment, and the novel diagnosis and treatment strategy with potential has wide research significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a polypeptide nano material based on bispyrene and a preparation method and application thereof, in particular to a polypeptide nano material based on pH response self-assembly of the bispyrene and a preparation method and application thereof.

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

in one aspect, the invention provides a polypeptide nanomaterial based on pyrene, wherein the nanomaterial has a structure shown in formulas I-IV as follows:

wherein R is₁Is a polypeptide sequence with multiple hydrogen bonds in the molecule, R₂A polypeptide sequence consisting of histidine, R₃Polyethylene glycol chain being hydrophilic, R₁、R₂And R₃Are connected by amido bond.

The polypeptide nano material with the structure has pH responsiveness, can be converted in situ to form fibers, is used for recruiting hydrophobic small molecule imaging agents and medicines, and greatly improves the utilization rate of the small molecule imaging agents and the medicines for treatment.

Preferably, the donor of the bispyrene compound group in the polypeptide nanomaterial based on bispyrene is a bispyrene compound with a structure shown in a formula V:

dipyrene compound group and R in polypeptide nano material based on bispyrene₁Or R₂Form amide bonds to be linked together.

Preferably, R₁Comprises the following steps:

wherein

Represents the site of attachment of the group. That is, in the present invention, R₁The donor structure of (a) is:

(may be represented by KLVFF) or

(may be denoted by LPFFD).

Preferably, R₂The donor is a polypeptide consisting of 4-10 histidines, e.g. R₂The donor of (a) is a polypeptide consisting of 4, 5, 6, 7, 8, 9 or 10 histidines, preferably a hexapeptide consisting of 6 histidines.

Preferably, R₃The donor of (a) is a carboxyl-terminated polyethylene glycol having a weight average molecular weight of 300-2000, such as 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 or 2000.

On the other hand, the invention provides a preparation method of the polypeptide nano material based on the dipyryrene, which comprises the steps of taking resin as a carrier, taking a dipyryrene compound shown as a formula V and amino acid as raw materials, and preparing the polypeptide nano material based on the dipyryrene by using a solid-phase synthesis method.

In another aspect, the invention provides a pH-responsive self-assembly method of the polypeptide nanomaterial based on bispyrene, which comprises the following steps:

(1) dissolving a polypeptide nano material based on the dipyrene in an organic solvent to obtain a polypeptide nano material solution;

(2) adding the polypeptide nano material solution obtained in the step (1) into a buffer solution, and self-assembling the polypeptide nano material into nano fibers in the buffer solution with the pH value of more than or equal to 1.0 and less than 8.0; in the buffer solution with the pH value more than or equal to 8.0, the polypeptide nano material is self-assembled into nanospheres.

Preferably, the organic solvent in step (1) is any one or a combination of at least two of DMSO, DMF or 1, 4-dioxane, preferably DMSO.

Preferably, the concentration of the polypeptide nano material solution in the step (1) is 10^-4-10^-2M, e.g. 1X 10^-4M、3×10^-4M、5×10^-4M、8×10^-4M、1×10^-3M、3×10^-3M、5×10^-3M、8×10^-3M or 1X 10^-2M。

Preferably, the self-assembly of step (2) is performed under ultrasound for a period of 1-10min, such as 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10 min.

Preferably, the sonication in step (2) is maintained for 0.5-12h (e.g., 0.8h, 1h, 3h, 5h, 8h, 10h, 11h or 12h) at 20-40 ℃ (e.g., 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃ or 38 ℃).

The polypeptide nano material based on the bispyrene has pH responsiveness, can be self-assembled into different forms under different pH values, and compared with nano particles, the nano fiber or the nano sphere prepared by the invention has better stability, is not easy to be eliminated in organisms, has longer detention time in the organisms, and has good biocompatibility, EPR effect, good pH sensitivity and the capacity of recruiting hydrophobic small molecule drugs.

On the other hand, the invention provides application of the polypeptide nano material based on the dipyryrene in preparation of cancer treatment drugs and biological imaging agents.

The pH responsive polypeptide nano material provided by the invention can be converted in situ to form fibers based on the good sensitivity to pH, is used for recruiting hydrophobic small molecule imaging agents and medicines, greatly improves the utilization rate of the small molecule imaging agents and the medicines for treatment, and has wide application prospect.

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

the polypeptid nano material based on the bispyrene has pH responsiveness, forms nano fibers by self-assembly in the environment of pH being more than or equal to 1.0 and less than 8.0, is assembled into nanospheres in the environment of pH being more than or equal to 8.0, can recruit hydrophobic micromolecule imaging agents and medicines, greatly improves the utilization rate of the micromolecule imaging agents and the treatment medicines, can be converted into fibers in situ at diseased tissues or cell parts, can greatly prolong the retention time, can be used for biological imaging and treatment, provides a reliable diagnosis and targeted treatment method for disease (especially cancer) treatment, and has wide application prospect.

Drawings

FIG. 1 is a mass spectrum of a polypeptide nanomaterial based on bispyrene prepared in example 1;

FIG. 2 is a fluorescence intensity curve obtained by a self-assembly capability test of the polypeptide nanomaterial based on bispyrene prepared in example 1;

FIG. 3 is a transmission electron microscope image of the polypepyrene-based polypeptide nanomaterial prepared in example 1 after self-assembly in ultrapure water (panel A) and a buffer solution (panel B) with pH of 6.0, wherein the scales in the panels A and B are both 100 nm;

FIG. 4 is a single photon laser confocal micrograph of cells incubated with Nile Red by the polyp-based polypeptide nanomaterial prepared in example 1, wherein A is a bright field confocal result of a cytosphere under a 405nm laser excitation condition, B is a green result of the polyp when 450-;

FIG. 5 is a scanning electron micrograph of the bispyrene-based polypeptide nanomaterial prepared in example 1 showing in-situ morphology transformation on the surface of cancer cells, wherein a B picture is an enlarged view of selected positions in a picture A, a scale of the picture A is 50 μm, and a scale of the picture B is 5 μm;

FIG. 6 is a graph showing the results of a small animal imaging experiment in which the polypeptidic nanomaterial based on bispyrene prepared in example 1 was used to recruit the fluorescent dye Nile Red; wherein, the A picture is an imaging result picture after intravenous injection of the polypeptide nano material based on the dipyryrene and the Nile red for 4h, and the B picture is an imaging result picture after intravenous injection of the polypeptide nano material based on the dipyryrene and the Nile red for 96 h;

FIG. 7 is a mass spectrum of the polypeptidic nanomaterial based on bispyrene prepared in example 6;

FIG. 8 is a fluorescence intensity profile obtained by a self-assembly capability test of the polypeptide nanomaterial based on bispyrene prepared in example 6;

fig. 9 is a transmission electron microscope image of the polypepyrene-based polypeptide nanomaterial prepared in example 6 after self-assembly in a buffer (panel a) and a buffer (panel B) with a pH of 6.0, wherein scales in the panels a and B are both 100 nm;

FIG. 10 is a single photon laser confocal micrograph of the double pyrene-based polypeptide nanomaterial prepared in example 6 after co-incubation with Nile Red on cells, wherein A is a bright field confocal result of a cytosphere under a 405nm laser excitation condition, B is a result of double pyrene green light when 450-550nm and C is a result of Nile Red light when 550-700nm are collected under the 405nm laser excitation condition;

FIG. 11 is a scanning electron micrograph of the bispyrene-based polypeptide nanomaterial of example 6 showing in situ morphological transformation on the surface of cancer cells, wherein panel B is an enlarged view of selected sites in panel A, the scale of panel A is 50 μm, and the scale of panel B is 5 μm;

fig. 12 is a graph showing the result of an imaging experiment on a small animal in which the polypeptidic nanophase material based on bispyrene prepared in example 6 was used to recruit the fluorescent dye nile red, in which a is a graph showing the imaging result after 4 hours of intravenous injection of the polypeptidic nanophase material based on bispyrene and nile red, and B is a graph showing the imaging result after 96 hours of intravenous injection of the polypeptidic nanophase material based on bispyrene and nile red.

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

In this example, the structure of the polypeptidic nanomaterial based on bispyrene is shown below:

that is, in the formula II provided by the present invention, R₁The donor of (A) is KLVFF, R₂The donor of (A) is a hexapeptide consisting of 6 histidines, R₃The donor of (A) is a carboxyl-terminated polyethylene glycol having a molecular weight of 1000g mol^-1。

The polypepyrene-based polypeptide nano material is synthesized by a solid-phase synthesis method of polypeptide, Wang resin is adopted, coupling is carried out by a coupling agent (azomethylmorpholine: DMF: 5: 95, volume ratio) according to the sequence of the polypeptide, wherein PEG with carboxyl and a polypepyrene compound are also reacted according to the coupling method of amino acid, and finally, the polypepyrene-based polypeptide nano material is obtained by trifluoroacetic acid cracking, rotary evaporation and ether purification.

The results of mass spectrometry characterization of the polypeptide nanomaterial based on bispyrene synthesized in this example are shown in fig. 1, from which it can be seen that the ion peak appearing in the figure satisfies the characteristic peak of the molecular weight of the material molecule, and the peak shape completely conforms to the characteristic peak of the PEG polymeric chain.

Example 2

In this example, the polypeptidic nanomaterial based on bispyrene prepared in example 1 was self-assembled, and the pH responsiveness of the material was examined.

Firstly, the self-assembly ability of the polypeptidic nanomaterial based on the bispyrene is tested, the polypeptidic nanomaterial based on the bispyrene prepared in the embodiment 1 is dissolved in DMSO to test the fluorescence intensity, then water is added for group assembly, the water and the organic phase are fully mixed after adding a certain amount of water, then the mixture is kept stand for one minute to test the fluorescence intensity, and the test result is shown in figure 2.

As can be seen from FIG. 2, H was added to the DMSO solution of the polypeptidic nanomaterial based on bispyrene₂The fluorescence increases at O, indicating that the material changes from a monodisperse to an aggregate state.

The pH responsive self-assembly method of the polypeptide nano material based on the dipyryrene comprises the following steps:

weighing 5mg of polypeptid nano material based on the dipyrene, dissolving the polypeptid nano material in 10mL of DMSO solvent, respectively and quickly injecting the solution into 90mL of ultrapure water and buffer solution with the pH value of 6.0 by using an injector, carrying out ultrasonic treatment for 30 minutes, and standing for two hours to respectively obtain fluorescent nano particles and nano fiber dispersion liquid.

Fig. 3 is a transmission electron microscope image of the polypeptidic nanomaterial based on bispyrene prepared in example 1 after self-assembly in ultrapure water and a buffer solution with pH of 6.0, and it can be seen from fig. 3 that the polypeptidic nanomaterial based on bispyrene has pH responsiveness, and the morphology changes from nanoparticles to fibers under the condition that pH gradually changes to acidity.

Example 3

The polypeptide nano-material based on the bispyrene prepared in the example 1 is subjected to a cell confocal test to examine the recruitment condition of the polypeptide nano-material to drugs or imaging agents, and the method comprises the following steps:

cell suspensions were prepared, 1mL per confocal dish, and incubated overnight. Taking out the culture medium, adding 1mL of the culture medium with the polypeptide nano material concentration of 20 mu M based on the dipyrene, and standing overnight; the drug is replaced by Nile Red (Nile Red) with 20 mu M, the culture is carried out for 2 hours, PBS is used for cleaning for 3 times, the confocal imaging experiment is carried out, a 405nm laser channel is adopted, and the green light wave band of 450-.

The result is shown in FIG. 4, the cytosphere is incubated with the material and Nile red, and in the single-photon co-focusing experiment, when the cell is excited at 405nm, the cell is in a green light of the bispyrene when the cell is collected in the range of 450-550 nm; when the collection is carried out in the range of 550-700nm, the red light of the Nile red is displayed, namely FRET (fluorescence resonance energy transfer) occurs, which proves that the material can be greatly accumulated on the cell surface and effectively recruit the fluorescent dye of the Nile red.

Example 4

In this example, the transformation of the polypeptide nanomaterial based on bispyrene prepared in example 1 on the surface of cancer cells was examined by the following method:

a1% agarose gel solution (0.1g agarose in 10mL water) was prepared, heated to boiling, transferred quickly to a clean bench, and 100. mu.L agarose solution was added to each well of a 96-well plate. Then the ultraviolet lamp is turned on to irradiate for 30 min. MCF-7 cell suspensions were prepared by adding 2000 cells per well, 200. mu.L per well. Culturing for 7 days, taking out the culture medium, adding the polypeptide nano-material based on the dipyrene into each hole, wherein the concentration of the polypeptide nano-material is 20 mu M, the volume is 200 mu L, and culturing for 24 h. The cell spheres were removed, solidified for 2-3 hours with 20% glutaraldehyde solution (glutaraldehyde: PBS buffer 1:4), and dehydrated with 30%, 50%, 70%, 90%, 100% ethanol solution diluted with PBS sequentially for 10 minutes three times at each concentration. Then, water and ethanol were replaced with a tert-butanol solution 3 times for 10 minutes each. And finally, dropwise adding the treated cell mass experiment to a silicon wafer, drying, and scanning and observing.

The results are shown in fig. 5, and it can be seen that the polypeptide nanomaterial based on bispyrene prepared in example 1 undergoes in-situ transformation on the surface of cancer cells to form fibers.

Example 5

In this example, the imaging situation of the polypeptide nanomaterial based on bispyrene prepared in example 1 in the small animal body is examined, and the method is as follows:

mice were injected subcutaneously with about 10⁶MCF-7 cells, used for establishing a mouse model for tumor growth. When the mouse tumor grew to around 5.0mm in diameter, it was started for the imaging experiment. Firstly, 200 mu L of medicine is injected into the vein, and the concentration is 200 mu M; after 8 hours, 200. mu.L of 20. mu.M Nile Red solution was injected intravenously, and immediately a small animal imaging experiment was performed, collecting the band 550-600 nm.

The results are shown in FIG. 6, which shows that: injecting Nile red after intravenous injection of the medicine, wherein a Nile red signal can be detected within 4h, which indicates that the material has the capability of recruiting fluorescent molecules; after 96h, signals of nile red can be detected at the tumor site of the mouse, which indicates that the material can be retained at the tumor site for a very long time and can recruit fluorescent molecules for cancer detection.

Example 6

that is, in formula IV provided by the present invention, R₁The donor of (A) is KLVFF, R₂The donor of (A) is a hexapeptide consisting of 6 histidines, R₃The donor of (A) is a carboxyl-terminated polyethylene glycol having a molecular weight of 1000g mol^-1。

The polypepyrene-based polypeptide nanomaterial was synthesized using the solid phase synthesis method for the polypeptide as described in example 1.

The results of mass spectrometry characterization of the polypeptide nanomaterial based on bispyrene synthesized in this example are shown in fig. 7, from which it can be seen that the ion peak appearing in the figure satisfies the characteristic peak of the molecular weight of the material molecule, and the peak shape completely conforms to the characteristic peak of the PEG polymeric chain.

Example 7

In this example, the polypeptide nanomaterial based on bispyrene prepared in example 6 was self-assembled, and the self-assembly ability and pH responsiveness of the material were examined.

Firstly, the self-assembly ability of the polypeptidic nanomaterial based on the bispyrene is tested, the polypeptidic nanomaterial based on the bispyrene prepared in the embodiment 6 is dissolved in DMSO to test the fluorescence intensity, then water is added for group assembly, the water and the organic phase are fully mixed after adding a certain amount of water, then the mixture is kept stand for one minute to test the fluorescence intensity, and the test result is shown in FIG. 8.

As can be seen from FIG. 8, H was added to the DMSO solution of the polypeptidic nanomaterial based on bispyrene₂The fluorescence increases at O, indicating that the material changes from a monodisperse to an aggregate state.

compound 5mg was weighed and dissolved in 10mL DMSO solvent, and the solution was rapidly injected into 90mL ultrapure water and pH 6.0 buffer solution by syringe, sonicated for 30 minutes, and left for two hours to obtain fluorescent nanoparticle and nanofiber dispersions, respectively.

Fig. 9 is a transmission electron microscope image of the polypeptidic nanomaterial based on bispyrene prepared in example 6 after self-assembly in ultrapure water and a buffer solution with pH of 6.0, and it can be seen from fig. 9 that the polypeptidic nanomaterial based on bispyrene has pH responsiveness, and the morphology changes from nanoparticles to fibers under the condition that pH gradually changes to acidity.

Example 8

The polypeptide nano-material based on the bispyrene prepared in the example 6 is subjected to a cell confocal test to examine the recruitment condition of the polypeptide nano-material to drugs or imaging agents, and the method comprises the following steps:

The result is shown in FIG. 10, the cytosphere is incubated with the material and Nile red, and in the single-photon confocal experiment, when the cell is excited at 405nm, the cell is in a green light of the bispyrene when the cell is collected in the range of 450-550 nm; when the collection is carried out in the range of 550-700nm, the red light of the Nile red is displayed, namely FRET (fluorescence resonance energy transfer) occurs, which proves that the material can be greatly accumulated on the cell surface and effectively recruit the fluorescent dye of the Nile red.

Example 9

In this example, the transformation of the polypeptide nanomaterial based on bispyrene prepared in example 6 on the surface of cancer cells was examined by the following method:

a1% agarose gel solution (0.1g agarose in 10mL water) was prepared, heated to boiling, transferred quickly to a clean bench, and 100. mu.L agarose solution was added to each well of a 96-well plate. Then the ultraviolet lamp is turned on to irradiate for 30 min. MCF-7 cell suspensions were prepared by adding 2000 cells per well, 200. mu.L per well. After 7 days of culture, the medium was removed and a volume of 200. mu.L of medium with a drug concentration of 20. mu.M was added to each well and cultured for 24 hours. The cell spheres were removed, solidified for 2-3 hours with 20% glutaraldehyde solution (glutaraldehyde: PBS buffer 1:4), and dehydrated with 30%, 50%, 70%, 90%, 100% ethanol solution diluted with PBS sequentially for 10 minutes three times at each concentration. Then, water and ethanol were replaced with a tert-butanol solution 3 times for 10 minutes each. And finally, dropwise adding the treated cell mass experiment to a silicon wafer, drying, and scanning and observing.

The result is shown in fig. 11, which shows that the polypeptide nanomaterial based on bispyrene prepared in example 6 is transformed in situ on the surface of cancer cells to form fibers.

Example 10

In this example, the imaging situation of the polypeptide nanomaterial based on bispyrene prepared in example 6 in the small animal body is examined, and the method is as follows:

The results are shown in FIG. 12, which shows that: injecting Nile red after intravenous injection of the medicine, wherein a Nile red signal can be detected within 4h, which indicates that the material has the capability of recruiting fluorescent molecules; after 96h, signals of nile red can be detected at the tumor site of the mouse, which indicates that the material can be retained at the tumor site for a very long time and can recruit fluorescent molecules for cancer detection.

The applicant states that the present invention is illustrated by the above examples to the polypeptide nanomaterial based on pH-responsive self-assembly of bispyrene and the preparation method and application thereof, but the present invention is not limited by the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

Translated fromChinese

1.一种基于双芘的多肽纳米材料，其特征在于，所述多肽纳米材料具有如下式II所示结构：1. a polypeptide nanomaterial based on bipyrene, is characterized in that, described polypeptide nanomaterial has structure shown in following formula II:

R₁的供体为KLVFF；The donor of R₁ is KLVFF;

R₂的供体为由6个组氨酸构成的六肽；The donor of R₂ is a hexapeptide consisting of 6 histidines;

R₃的供体为羧基封端的聚乙二醇，其分子量为1000g mol^-1；The donor of R₃ is a carboxyl-terminated polyethylene glycol with a molecular weight of 1000 g mol⁻¹ ;

所述多肽纳米材料的结构式具体如下所示：The structural formula of the polypeptide nanomaterial is as follows:

所述基于双芘的多肽纳米材料具有pH响应性，在pH＝6.0的环境下自组装形成纳米纤维，能够招募疏水的小分子成像剂和药物。The bipyrene-based polypeptide nanomaterial is pH-responsive, self-assembles to form nanofibers in an environment of pH=6.0, and can recruit hydrophobic small molecule imaging agents and drugs.

2.根据权利要求1所述的基于双芘的多肽纳米材料的制备方法，其特征在于，以树脂为载体，以如下式V所示双芘化合物和氨基酸作为原料，利用固相合成方法制备得到所述基于双芘的多肽纳米材料；2. the preparation method of the polypeptide nanomaterial based on bipyrene according to claim 1, is characterized in that, with resin as carrier, with bipyrene compound shown in following formula V and amino acid as raw material, utilize solid-phase synthesis method to prepare The bipyrene-based polypeptide nanomaterial;

3.根据权利要求1所述的基于双芘的多肽纳米材料的pH响应性自组装方法，所述方法包括以下步骤：3. The pH-responsive self-assembly method of bipyrene-based polypeptide nanomaterials according to claim 1, the method comprising the following steps:

(1)将基于双芘的多肽纳米材料溶于DMSO中，得到多肽纳米材料溶液；步骤(1)所述多肽纳米材料溶液的浓度为1.6×10^-4M；(1) dissolving the polypeptide nanomaterial based on bipyrene in DMSO to obtain a polypeptide nanomaterial solution; the concentration of the polypeptide nanomaterial solution in step (1) is 1.6×10^-4 M;

(2)将步骤(1)得到的多肽纳米材料溶液加入缓冲溶液中，在pH＝6.0的缓冲液中，所述多肽纳米材料自组装为纳米纤维；在超纯水的缓冲液中，所述多肽纳米材料自组装为纳米球；(2) adding the polypeptide nanomaterial solution obtained in step (1) into the buffer solution, in the buffer solution of pH=6.0, the polypeptide nanomaterial self-assembles into nanofibers; in the buffer solution of ultrapure water, the polypeptide nanomaterials self-assemble into nanofibers; Polypeptide nanomaterials self-assemble into nanospheres;

步骤(2)所述自组装在超声下进行，所述超声的时间为30分钟；超声后静置两小时。In step (2), the self-assembly is carried out under ultrasound, and the duration of the ultrasound is 30 minutes; after the ultrasound, it is allowed to stand for two hours.

4.根据权利要求1所述的基于双芘的多肽纳米材料在制备癌症治疗药物以及生物成像剂中的应用。4. The application of the bipyrene-based polypeptide nanomaterial according to claim 1 in the preparation of cancer therapeutic drugs and biological imaging agents.