CN111298116B

CN111298116B - Polypeptide drug-loaded temperature-sensitive liposome and preparation method and application thereof

Info

Publication number: CN111298116B
Application number: CN202010234616.0A
Authority: CN
Inventors: 王琛; 张凯悦; 方小翠; 游青; 杨延莲
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-12-13
Anticipated expiration: 2040-03-30
Also published as: CN111298116A

Abstract

The invention provides a polypeptide drug-loaded temperature-sensitive liposome which is formed by self-assembling a polypeptide, a chemotherapeutic drug, a photosensitizer and a liposome, wherein the polypeptide can be combined with cancer cells expressing or over-expressing a chemokine receptor protein CXCR4 in a targeting manner. Also provides a preparation method and application thereof. The drug-loaded temperature-sensitive liposome combining the polypeptide, the photosensitizer and the chemotherapeutic drug has the capability of improving the solubility of the polypeptide in a salt solution, improves the binding efficiency of the polypeptide and a chemokine receptor CXCR4 target protein, improves the enrichment capability of the chemotherapeutic drug in cancer cells and cancer tissues, and shows stronger characteristic of inhibiting the migration of the cancer cells. On the other hand, the drug-loading rate and in-vivo bioavailability of the chemotherapeutic drug are improved by utilizing the liposome, and the precise controllable release of the chemotherapeutic drug is realized by utilizing the photosensitizer.

Description

Polypeptide drug-loaded temperature-sensitive liposome and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a polypeptide drug-loaded temperature-sensitive liposome, and a preparation method and application thereof.

Background

Cancer is currently the leading cause of morbidity and mortality worldwide, with about 90% of cancer patients dying from cancer metastasis and relapse, and blocking a certain segment of cancer metastasis for the purpose of suppressing cancer spread is crucial for cancer treatment. Chemokines and chemokine receptors are involved in not only normal physiological activities but also pathological processes such as cancer occurrence, infiltration and metastasis, wherein the biological chemotactic axis of CXCR4/SDF-1 (or CXCL 12) plays an important role in cancer migration and invasion, and the development of antagonists of CXCR4 chemokine receptor proteins is very critical for controlling cancer metastasis and improving the cancer cure rate. The development of polypeptide antagonists of CXCR4 receptor proteins provides new and effective means and strategies for cancer therapy because of the ease of design and synthesis of polypeptides, their metabolism in humans and their lack of toxic side effects and severe immune responses.

The liposome is a biological safe and good drug carrier which is widely concerned, and is a phospholipid bilayer structure system with the diameter of 50-500 nm, which is spontaneously formed by phospholipid and cholesterol in a solvent system under the conditions of proper concentration ratio and temperature. At present, the liposome successfully realizes the entrapment of various hydrophobic chemotherapeutic drugs, can effectively solve the problems of poor water solubility, quick degradation in systemic circulation, less drug absorption, large toxic and side effects and the like of the chemotherapeutic drugs, and can realize the high-efficiency enrichment of the chemotherapeutic drugs in a cancer focus area through an Enhanced Retention Effect (EPR). However, part of the liposome has the defect of slow local drug release, so that the effective drug hardly exerts the fast killing effect per se, and the drug resistance is generated even under the low drug concentration of continuous slow release. Therefore, the construction of cancer-targeted liposome drug delivery systems, the reduction of the damage of chemotherapeutic drugs to normal organs, and the enhancement of the precise and controllable release of chemotherapeutic drugs in the cancer parenchyma are attracting more and more attention of researchers.

Because CXCR4 is specifically and highly expressed in various cancer tissues and is mostly distributed on the surface of cancer cells, the CXCR4 can be used as an active recognition target of a nano drug delivery carrier to develop CXCR4 specific recognition polypeptide, and combined with a chemotherapeutic drug with a killing effect on the cancer cells, the CXCR4 targeted polypeptide modified active targeting type temperature-sensitive liposome is constructed based on the photothermal effect of a photosensitizer ICG, so that the drug storm attack of the chemotherapeutic drug on the cancer tissues is expected to be realized, the anti-cancer treatment effect is further remarkably enhanced, and new information and clues are provided for developing efficient cancer treatment drugs.

Disclosure of Invention

Therefore, the invention aims to overcome the defects in the prior art and provides a polypeptide drug-loaded temperature-sensitive liposome and a preparation method and application thereof.

Before setting forth the context of the present invention, the terms used herein are defined as follows:

the term "FITC" refers to: fluorescein isothiocyanate (fluorescein isothiocyanate).

The term "ICG" refers to: indocyanine Green (Indocyanine Green).

The term "DPPC" refers to: dipalmitoylphosphatidylcholine.

The term "DSPE" refers to: distearoyl phosphatidyl ethanolamine.

The term "PEG" refers to: polyethylene glycol.

The term "MAL" refers to: a maleimide.

The term "DOPE" refers to dioleoylphosphatidylethanolamine.

In order to achieve the above object, the first aspect of the present invention provides a polypeptide drug-loaded thermo-sensitive liposome, which is formed by self-assembly of a polypeptide, a chemotherapeutic drug, a photosensitizer and a liposome, wherein the polypeptide is a polypeptide capable of being combined with a cancer cell expressing or overexpressing a chemokine receptor protein CXCR4 in a targeted manner;

preferably, the particle size of the polypeptide drug-loaded temperature-sensitive liposome is 50-500 nm, preferably 50-200 nm, and more preferably 100-150 nm.

The polypeptide drug-loaded thermosensitive liposome according to the first aspect of the invention, wherein the polypeptide is selected from one or more of the following: polar amino acid-based polypeptides, hydrophobic amino acid-based polypeptides, and polypeptides having both polar amino acids and hydrophobic amino acids;

preferably, the polypeptide consists of 5 to 100 amino acids, more preferably 10 to 50 amino acids, and even more preferably 20 to 30 amino acids;

more preferably, the cancer targeting polypeptide is a P12 polypeptide or a FITC-labeled P12 polypeptide; wherein the amino acid sequence of the P12 polypeptide is QGSRRRNTVDDWISRRRALC; the amino acid sequence of the FITC-labeled P12 polypeptide is FITC-QGSRRRNTVDDWISRRRALC.

The polypeptide drug-loaded thermosensitive liposome according to the first aspect of the present invention, wherein the photosensitizer is selected from one or more of the following: ICG, porphyrin, fe ₃ O ₄ Nanoparticles, gold nanoparticles; preferably ICG and/or porphyrin; more preferably ICG.

The chemotherapeutic drug is selected from one or more of the following: doxorubicin, daunorubicin, paclitaxel, docetaxel; preferably doxorubicin and/or paclitaxel; more preferably doxorubicin.

The polypeptide drug-loaded thermosensitive liposome according to the first aspect of the present invention, wherein the liposome component is selected from one or more of the following: DPPC, DSPE, PEG, MAL, cholesterol, DOPE, soy lecithin, lecithin.

The polypeptide drug-loaded thermosensitive liposome according to the first aspect of the invention, wherein the polypeptide is bound to the liposome through chemical coupling;

the photosensitizer is bound to the liposome by non-covalent interactions; and/or

The chemotherapeutic agent is combined with the liposome by emulsification.

The second aspect of the present invention provides a preparation method of the polypeptide drug-loaded temperature-sensitive liposome of the first aspect, which may include the following steps:

(1) Preparing a polypeptide-liposome solution;

(2) Uniformly mixing the liposome solution, the polypeptide-liposome solution prepared in the step (1) and the photosensitizer solution, performing rotary evaporation, incubating and standing;

(3) And (3) adding an ammonium sulfate solution into the product obtained in the step (2), adding a chemotherapeutic drug solution, uniformly mixing at room temperature, stirring, and standing to obtain the polypeptide drug-loaded temperature-sensitive liposome.

The preparation method according to the second aspect of the present invention, wherein, in the step (2), the rotary evaporation temperature is 30 to 60 ℃, preferably 40 to 50 ℃, and most preferably 45 ℃;

the rotary evaporation time is 10-90 minutes, preferably 20-70 minutes, and more preferably 30-60 minutes; and/or

The incubation temperature is between 30 and 100 ℃, preferably between 50 and 80 ℃, most preferably 60 ℃.

The production method according to the second aspect of the present invention, wherein, in the step (3), the stirring time is 10 to 60min, preferably 30min.

The third aspect of the invention provides a medicament, which comprises the polypeptide drug-carrying temperature-sensitive liposome of the first aspect or the polypeptide drug-carrying temperature-sensitive liposome prepared by the preparation method of the second aspect;

preferably, the medicament is a solution or a lyophilized powder;

more preferably, the lyoprotectant is mannitol.

The fourth aspect of the invention provides the application of the polypeptide drug-carrying temperature-sensitive liposome of the first aspect or the polypeptide drug-carrying temperature-sensitive liposome prepared by the preparation method of the second aspect in preparing a medicament for treating cancer;

preferably, the drug is a drug that inhibits cancer metastasis;

more preferably, the cancer is a cancer associated with cancer cells or cancer tissues that express or overexpress the chemokine receptor protein CXCR 4;

further preferably, the cancer is selected from one or more of: breast cancer, leukemia, lymphoma, bladder cancer, liver cancer, preferably breast cancer or liver cancer, most preferably breast cancer.

The invention relates to a drug-loaded temperature-sensitive liposome combining polypeptide, chemotherapeutic drugs and photosensitizers, and a preparation method and application thereof, and in particular relates to a drug-loaded liposome chemically coupled with CXCR4 specific targeting polypeptide, photosensitizers ICG and chemotherapeutic drugs DOX by taking temperature-sensitive dipalmitoyl phosphatidylcholine as a carrier, and a preparation method and application thereof. The invention combines polypeptide with cancer targeting treatment effect, chemotherapy drug adriamycin (DOX) and photosensitizer Indocyanine Green (ICG) to construct and obtain a temperature-sensitive pegylated phospholipid compound of polypeptide-chemotherapy drug-photosensitizer; the polypeptide with the cancer targeting treatment effect can be specifically combined with a chemokine receptor protein CXCR4 highly expressed by cancer cells, so that the specific selectivity and the targeted permeability of the medicament to cancer tissues and cancer cells are effectively improved, the medicament loading rate and the biocompatibility of the medicament are improved by utilizing the temperature-sensitive pegylated phospholipid compound, the controllable release of the medicament to the cancer tissues is realized, the anti-cancer synergistic effect is improved, and meanwhile, the toxicity of the medicament to normal tissues and normal cells is reduced. Compared with the independent chemotherapeutic drug adriamycin, the drug-loaded temperature-sensitive liposome exerts the advantage of stronger inhibition of the growth of cancer cells and further reduces the toxic and side effects of the chemotherapeutic drug. The temperature response released drug-loaded liposome combined by the polypeptide, the chemotherapeutic drug and the photosensitizer provides a feasible method and technology for improving the treatment effect of cancer.

The invention aims to provide a preparation method and application of a drug-loaded temperature-sensitive liposome for treating cancer, which integrates polypeptide, photosensitizer and chemotherapeutic drug to combine, and constructs the polypeptide capable of specifically targeting CXCR4 high-expression cancer cells, the chemotherapeutic drug capable of killing the cancer cells and the temperature-sensitive nanoliposome capable of improving the accurate and controllable release capacity of the drug. The drug-loaded liposome combining the polypeptide, the photosensitizer and the chemotherapeutic drug also solves the problems of solubility and biological stability of the cancer targeting polypeptide with good water solubility and poor salt solubility in a salt solution, improves the combination efficiency of the polypeptide and CXCR4 target protein, enhances the anti-cancer effect of the chemotherapeutic drug and shows excellent effect of inhibiting cancer metastasis.

The invention adopts the following technical scheme:

a polypeptide nanoliposome is formed by coupling distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and a cancer targeting polypeptide P12 through a chemical bond, wherein the cancer targeting polypeptide is a polypeptide capable of being combined with cancer cells or cancer tissues expressing or over-expressing a chemokine receptor protein CXCR4 in a targeting way.

Wherein:

the distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) is a compound formed by combining polyethylene glycol (hydrophilic block) with nitrogenous bases on phospholipid molecules (hydrophobic block) through covalent bonds.

Preferably, the molecular weight of the polyethylene glycol hydrophilic block in the distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) molecule is 2000.

Preferably, the particle size of the polypeptide nanoliposome is 100-150 nm.

Preferably, the cancer targeting polypeptide is selected from one or more of polar amino acid-based polypeptides, hydrophobic amino acid-based polypeptides or polypeptides both containing polar amino acid and hydrophobic amino acid.

Preferably, the cancer targeting polypeptide consists of 5 to 100 amino acids, more preferably 10 to 50 amino acids, and even more preferably 20 to 30 amino acids.

Most preferably, the cancer targeting polypeptide is a P12 polypeptide or a FITC-labeled P12 polypeptide.

The P12 polypeptide consists of 20 amino acids. The invention discovers that the P12 polypeptide can be specifically bound to the surface of a cancer cell with high expression of CXCR4 protein, and further plays a role.

Specifically, the amino acid sequence of the P12 polypeptide: QGSRRRNTVDDWISRRRALC; the amino acid sequence of the FITC-labeled P12 polypeptide: FITC-QGSRRRNTVDDWISRRRALC.

The P12 polypeptide or FITC labeled P12 polypeptide can be obtained by the existing chemical synthesis method, and can also be purchased as a commercial product.

The P12 polypeptide or FITC marked P12 polypeptide has the characteristics of good water solubility and poor salt solubility.

Preferably, the cancer-targeting polypeptide is chemically conjugated to the DSPE-PEG-MAL.

Preferably, the polypeptide nanoliposome is in the form of a solution or a lyophilized powder.

The invention also provides a preparation method of the polypeptide temperature-sensitive nano-liposome, which comprises the following steps:

respectively preparing DSPE-PEG-2000 molecular solution, DSPE-PEG-P12 molecular solution, cholesterol molecular solution, DPPC molecular solution and ICG solution; and uniformly mixing the component solutions, performing rotary evaporation, drying, standing and hydrating to obtain the polypeptide temperature-sensitive nano liposome solution.

The preparation method of the polypeptide temperature-sensitive nano-liposome comprises the following steps:

preferably, the solvent for preparing the DSPE-PEG-P12, DSPE-PEG2000, DPPC and cholesterol solution is chloroform; the solvent of the ICG solution is methanol;

preferably, the DPPC and the cholesterol solution are prepared into a solution of 2-20 mg/mL; preparing the cancer targeting polypeptides DSPE-PEG-P12, DSPE-PEG2000 and ICG into 1-5 mg/mL solution;

preferably, the mixing step is to fully mix the DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG-P12 and ICG solution to obtain a mixed solution, and then spin-steaming, drying and hydrating the mixed solution;

preferably, the rotary steaming time is 1h, the drying time is overnight, the hydration incubation temperature is 60 ℃, and the incubation time is 30min;

Specifically, the preparation method of the polypeptide temperature-sensitive nano liposome comprises the following steps:

(1) Preparing a solution: preparing the DSPE-PEG-P12, the DSPE-PEG 2000, the DPPC and the cholesterol into a solution of 2 to 20mg/mL by using a chloroform solution; preparing the ICG molecule into a solution of 1-5 mg/mL by using a methanol solution;

(2) Uniformly mixing: fully and uniformly mixing the DSPE-PEG-P12 solution, the DSPE-PEG 2000 solution, the DPPC solution, the cholesterol solution and the ICG methanol solution in a 100mL flask to obtain a mixed solution;

(3) And (3) rotary steaming: carrying out water bath rotary steaming on the mixed solution obtained in the step (2) at 45 ℃ for 60min;

preferably, the incubation temperature is 45 ℃ and the incubation time is 60min;

(4) And (3) drying: preferably, the standing and drying condition is vacuum drying at 60 ℃ for standing overnight; obtaining the polypeptide temperature-sensitive liposome membrane.

(5) Hydration: and (3) adding 5mL of phosphate buffer solution (namely PBS buffer solution) into the liposome membrane obtained in the step (4) for 30min in an ultrasonic water bath, and incubating at the temperature of 60 ℃.

Preferably, the preparation method of the polypeptide temperature-sensitive nanoliposome further comprises a step of sterilizing the polypeptide nanoliposome solution obtained after standing, and further preferably, the sterilization is to filter the polypeptide temperature-sensitive nanoliposome solution obtained after standing in the step (4) by using a 0.22-micrometer filter membrane.

According to the requirement, the preparation method of the polypeptide temperature-sensitive nano liposome further comprises the step of freeze-drying the sterilized polypeptide temperature-sensitive nano liposome solution to prepare the polypeptide temperature-sensitive nano liposome freeze-dried powder.

Further preferably, the freeze-drying comprises adding a certain amount of freeze-drying protective agent into the sterilized polypeptide temperature-sensitive nano liposome solution; the lyoprotectant is preferably mannitol, for example mannitol at a concentration of 0.01 to 0.2 g/mL.

The polypeptide temperature-sensitive nano liposome can be prepared by the prior conventional technology.

In order to further improve the anti-cancer treatment effect, the polypeptide nano-liposome also comprises a chemotherapeutic drug, and the polypeptide nano-liposome containing the chemotherapeutic drug is called a drug-loaded temperature-sensitive liposome combining the polypeptide and the chemotherapeutic drug in the invention.

In the present invention, the chemotherapeutic agent may be any of a variety of chemotherapeutic agents well known to those in the medical arts; preferably, the chemotherapeutic drug is selected from one or more of adriamycin, daunorubicin, paclitaxel or docetaxel; further preferably doxorubicin and/or paclitaxel; more preferably Doxorubicin (DOX).

The polypeptide and chemotherapeutic drug combined drug-loaded temperature-sensitive liposome is formed by an ammonium sulfate gradient method.

Preferably, the polypeptide nanoliposome and an ammonium sulfate solution are subjected to hydration interaction to obtain the polypeptide nanoliposome ammonium sulfate solution; on the basis, the chemotherapeutic drug is added to prepare the drug-loaded liposome (namely the drug-loaded temperature-sensitive liposome) combining the polypeptide nano-liposome and the chemotherapeutic drug.

Preferably, the DSPE-PEG-MAL is conjugated to the cancer targeting polypeptide by chemical coupling to form DSPE-PEG-P12;

preferably, the ICG is associated with the polypeptide nanoliposome by non-covalent interaction.

Preferably, the polypeptide nanoliposome is combined with the chemotherapeutic drug through emulsification.

Preferably, the particle size of the drug-loaded liposome of the combination of the polypeptide and the chemotherapeutic drug is 100-150 nm.

The drug-loaded temperature-sensitive liposome combined by the polypeptide and the chemotherapeutic drug is formed by self-assembling temperature-sensitive dipalmitoyl phosphatidylcholine (DPPC), cholesterol, an ICG photosensitizer and the cancer targeting polypeptide and the chemotherapeutic drug, wherein the cancer targeting polypeptide can be combined with cancer cells or cancer tissues expressing or over-expressing chemokine receptor CXCR4 in a targeting manner.

Preferably, the chemotherapeutic drug is selected from one or more of adriamycin, daunorubicin, paclitaxel or docetaxel; further preferably doxorubicin and/or paclitaxel; more preferably doxorubicin.

The drug-loaded temperature-sensitive liposome combined by the polypeptide and the chemotherapeutic drug comprises the following components:

preferably, the molar ratio of the thermosensitive Dipalmitoylphosphatidylcholine (DPPC) liposome to the chemotherapeutic drug is 1000.

Preferably, the drug-loaded temperature-sensitive liposome combined by the polypeptide and the chemotherapeutic drug is in a solution form or a freeze-dried powder form.

The invention also provides a preparation method of the drug-loaded temperature-sensitive liposome combined by the polypeptide, the ICG and the chemotherapeutic drug, which comprises the following steps:

respectively preparing DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12, ICG and DOX solution; fully and uniformly mixing DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12 and ICG solution, performing rotary evaporation, standing, drying and water bloom to obtain the thermosensitive liposome combining polypeptide and photosensitizer ICG.

Preferably, the preparation method of the drug-loaded temperature-sensitive liposome combining the temperature-sensitive liposome and the chemotherapeutic drug comprises the following steps:

respectively preparing DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12 and ICG solution, mixing, rotary steaming, standing, drying and hydrating to obtain polypeptide temperature sensitive liposome solution;

carrying out hydration interaction on the polypeptide nanoliposome and an ammonium sulfate solution to obtain a polypeptide nanoliposome ammonium sulfate solution; on the basis, the chemotherapeutic drug is added to prepare a drug-loaded liposome (namely a drug-loaded temperature-sensitive liposome) combining the polypeptide nano-liposome and the chemotherapeutic drug;

The preparation method of the drug-loaded temperature-sensitive liposome combining the polypeptide and the chemotherapeutic drug comprises the following steps:

preferably, the solvent for preparing the temperature-sensitive nano liposome solution is any one of phosphate buffer solution (namely PBS solution), hydroxyethyl piperazine ethanethiosulfonic acid buffer solution, normal saline or sterile ultrapure water; more preferably phosphate buffered saline (i.e., PBS solution);

preferably, the liposome solution is prepared into 2-20 mg/mL solution; the chemotherapeutic drug DOX is prepared into a 1mg/mL solution

Preferably, the incubation temperature is 20-30 ℃, and the incubation time is 20-60 min; further preferably, the incubation temperature is 26 ℃ and the incubation time is 30min.

Preferably, the preparation method of the drug-loaded thermo-sensitive liposome combining the thermo-sensitive liposome and the chemotherapeutic drug further comprises the step of sterilizing the drug-loaded thermo-sensitive liposome solution obtained after standing, and further preferably, the sterilization is to filter the drug-loaded thermo-sensitive liposome solution obtained after standing by a 0.22 μm filter membrane.

According to the requirement, the preparation method of the drug-loaded temperature-sensitive liposome combined with the polypeptide and the chemotherapeutic drug further comprises the step of freeze-drying the sterilized solution to prepare the drug-loaded temperature-sensitive liposome freeze-dried powder combined with the polypeptide and the chemotherapeutic drug.

Further preferably, the freeze-drying comprises adding a certain amount of freeze-drying protective agent into the sterilized drug-loaded temperature-sensitive liposome solution; the lyoprotectant is preferably mannitol, for example mannitol at a concentration of 0.01 to 0.2 g/mL.

The drug-loaded temperature-sensitive liposome can be prepared by the conventional technology.

The invention also comprises the polypeptide liposome, the temperature-sensitive liposome combined by the photosensitizer and the polypeptide liposome, and the application of the temperature-sensitive liposome prepared by the preparation method and the drug-carrying temperature-sensitive liposome of chemotherapeutic drugs as candidate drugs in the aspect of cancer treatment; preferably, for use in inhibiting cancer metastasis; further preferred is the use in inhibiting cancer metastasis associated with cancer cells or cancer tissues expressing or overexpressing the chemokine receptor CXCR 4.

Preferably, the cancer associated with cancer cells or cancer tissues expressing or overexpressing chemokine receptor CXCR4 comprises any one of breast cancer, leukemia, lymphoma, bladder cancer or liver cancer; further preferably, it is breast cancer.

The polypeptide drug-loaded temperature-sensitive liposome disclosed by the invention can have the following beneficial effects but not limited to:

The drug-loaded temperature-sensitive liposome combining the polypeptide, the photosensitizer and the chemotherapeutic drug has the capability of improving the solubility of the polypeptide in a salt solution, and improves the combination efficiency of the polypeptide and a chemokine receptor CXCR4 target protein. The polypeptide is specifically combined with CXCR4, so that the enrichment capacity of chemotherapeutic drugs in cancer cells and cancer tissues can be improved, and compared with the single polypeptide, the drug-loaded temperature-sensitive liposome has the characteristic of stronger inhibition of cancer cell migration. On the other hand, the drug-loading rate and in-vivo bioavailability of the chemotherapeutic drug are improved by using the liposome, and the accurate and controllable release of the chemotherapeutic drug is realized by using the photosensitizer ICG.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows transmission electron microscopy images and dynamic light scattering particle size analysis of DPPC liposomes in PBS solution in example 1; wherein, fig. 1A shows a transmission electron microscope picture, and fig. 1B shows a dynamic light scattering particle size analysis.

FIG. 2 shows transmission electron microscopy pictures and dynamic light scattering particle size analysis of the polypeptide P12-Lipo liposomes in PBS solution in example 2; wherein, fig. 2A shows a transmission electron microscope picture; fig. 2B shows dynamic light scattering particle size analysis.

FIG. 3 shows transmission electron microscopy pictures and dynamic light scattering particle size analysis of the polypeptide drug-loaded P12-Lipo-DOX liposomes in PBS solution in example 5; wherein, fig. 3A shows a transmission electron microscope picture; fig. 3B shows dynamic light scattering particle size analysis.

FIG. 4 shows the results of flow cytometry detection of FITC-P12 polypeptide in examples 6 and 7; wherein, figure 4A shows flow cytometry detection of affinity of different concentrations of FITC-P12 polypeptide to CXCR4 under-expressed MCF-7 cells in example 6; figure 4B shows flow cytometry assays of affinity of FITC-P12 polypeptides for CXCR4 high expressing 4T1 cells at different concentrations in example 7.

FIG. 5 shows the FITC-P12 polypeptide positive binding rate and relative mean fluorescence intensity in examples 6 and 7; wherein, FIG. 5A shows the positive binding rate of FITC-P12 polypeptide to MCF-7 cells and 4T1 cells at various concentrations; FIG. 5B shows the relative mean fluorescence intensity of FITC-P12 polypeptide and FITC-P12-Lipo liposomes versus MCF-7 cells and 4T1 cells at various concentrations.

FIG. 6 is a graph showing the results of the survival inhibitory effect of non-targeted temperature sensitive liposomes (Lipo-ICG), targeted temperature sensitive liposomes (P12-Lipo-ICG) and temperature sensitive liposomes (P12-Lipo-ICG-DOX) combined with chemotherapeutic drugs on 4T1 of breast cancer cells in example 8.

Fig. 7 shows the evaluation of the anti-cancer therapeutic effect of the polypeptide drug-loaded temperature-sensitive liposome on tumor-bearing mice in example 9.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purposes of this invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Unless otherwise indicated, the human breast cancer cell line MCF-7 and the murine breast cancer cell line 4T1 used in the following examples were purchased from the cell center of the institute of basic medicine of the Chinese academy of medical sciences.

In the examples of the present invention, the photosensitizer is indocyanine green (ICG) available from thermo Fisher Scientific, the chemotherapeutic agent is doxorubicin (Dox) available from Shanghai Yuye, dipalmitoylphosphatidylcholine (DPPC) available from Avanti, and Cell Counting Kit-8 reagent available from Panbobiochemistry, inc. The apparatus used for the experiment: dynamic light scattering (DLS, zetasizer Nano ZS, malvern, uk), transmission electron microscopy (TEM, HT7700, hitachi), flow cytometry (FCM,

acoustic focusing cytometer，Applied Biosystems，Life Technologies，Carlsbad，CA)。

the solvents of the aqueous solutions used in the examples below were all sterile ultrapure aqueous solutions unless otherwise specified.

Unless otherwise indicated, all reagents used in the following examples were analytical reagents.

Unless otherwise specified, all PBS solutions used in the following examples are 1 × PBS solutions.

Preparation of 10 × PBS solution: 80.00g of NaCl, 2g of KCl, na ₂ HPO ₄ ·12H ₂ O35.8 g or Na ₂ HPO ₄ 14.2g，KH ₂ PO ₄ 2.7g, diluting to 1000mL with ultrapure water, adjusting the pH value to 7.2-7.4, and autoclaving.

Preparation of 1 × PBS solution: the 10 XPBS solution was diluted 10-fold with sterile ultrapure water.

Synthesis of polypeptide P12

Amino acid sequence of P12 polypeptide: qgsrrrntvddwissrrralc, amino acid sequence of FITC-labeled P12 polypeptide: FITC-QGSRRRNTVDDWISRRRALC. P12 polypeptide and FITC labeled P12 polypeptide (synthesized by Anhui national pharmaceutical Co., ltd., purity 98%) were synthesized according to the indicated sequences, respectively, and a mother solution of appropriate concentration was prepared before the experiment.

Example 1 preparation of liposomes (Lipo) and experiments of their dissolution in PBS solution

The DSPE-PEG-MAL solution and amino groups in the P12 polypeptide are mixed at normal temperature to react to generate the DSPE-PEG-P12 which is purified and then freeze-dried for later use. Respectively preparing 5mg/mL solution of DSPE-PEG-2000 with chloroform, 5mg/mL solution of DSPE-PEG-P12 (wherein the molecular weight of PEG segment is 2000) with chloroform, 10mg/mL solution of cholesterol with chloroform, and 10mg/mL solution of DPPC with chloroform. Mixing the above solutions according to a molar ratio of 2. The prepared solution is placed in a refrigerator at 4 ℃.

Fixing the concentration of the nano liposome (Lipo) at 100 mu M, and uniformly mixing the nano liposome (Lipo) in a water bath at 45 ℃ by ultrasound for 30min. A10 mu L sample of a nano liposome (Lipo) PBS solution is dropped on a carbon-coated copper net with the surface activated after glow discharge treatment, the mixture is kept stand for 5min, filter paper is used for sucking the solution, 5 mu L of 2% uranyl acetate or 2% tungsten phosphate staining solution (the staining solution which is not completely dissolved is removed by centrifugation at 4000rpm before use) is used for staining for 60s, the staining solution is sucked by the filter paper, a transmission electron microscope (transmission electron microscopy, TEM, HT7700, hitachi) is used for observing the sample, and the sample in figure 3 is negatively stained by 2% uranyl acetate. The transmission electron microscope reflects the appearance and the particle size of the sample, and as shown in FIG. 1A, the particle size distribution is uniform due to the existence of a spherical structure in the PBS solution; the particle size in 1 XPBS solution is 100-150nm.

The liposome (Lipo) concentration was 100. Mu.M, and the mixture was subjected to ultrasonic water bath at 40 ℃ for 30min. Shaking the PBS solution of the nanoliposome (Lipo) uniformly, placing 1mL of the solution into a plastic sample cell with the size of 1cm multiplied by 1cm, and carrying out dynamic light scattering test to measure the particle size distribution condition of the sample. Dynamic light scattering reflects the particle size change of molecules in solution; as shown in FIG. 1B, the particle size of nanoliposome (Lipo) in PBS solution is 100-150nm.

Example 2 preparation of polypeptide liposomes (P12-Lipo) and experiments on their solubilization in PBS solution

Respectively preparing 5mg/mL solution of DSPE-PEG-2000 with chloroform, 5mg/mL solution of DSPE-PEG-P12 (wherein the molecular weight of PEG segment is 2000) with chloroform, 10mg/mL solution of cholesterol with chloroform, and 10mg/mL solution of DPPC with chloroform. Mixing the above solutions according to a mass ratio of 2. The prepared solution is placed in a refrigerator at 4 ℃.

Fixing the concentration of the polypeptide nano empty liposome (P12-Lipo) to be 100 mu M, and uniformly mixing the polypeptide nano empty liposome (P12-Lipo) with ultrasonic water bath at 45 ℃ for 30 min. Dripping 10 mu L of a PBS solution sample of the polypeptide nanometer empty liposome (P12-Lipo) on a carbon-coated film copper net with activated surface after glow discharge treatment, standing for 5min, sucking the solution by using filter paper, taking 5 mu L of 2% uranyl acetate or 2% tungsten phosphate staining solution (centrifuging at 4000rpm for 5min before use, removing the staining solution which is not completely dissolved) for staining for 60s, sucking the staining solution by using the filter paper, observing the sample by using a transmission electron microscope, wherein the sample in figure 3 is negatively stained by 2% uranyl acetate. The transmission electron microscope reflects the morphology and the particle size of the sample, and as shown in fig. 2A, the polypeptide nano hollow liposome (P12-Lipo) exists in a spherical structure in the PBS solution, the particle size distribution is uniform, and the particle size is 100-150nm. After the liposome (Lipo) is loaded with P12 polypeptide molecules, the polypeptide nanoliposome (P12-Lipo) keeps a spherical structure, and the particle size is not obviously changed. The PEG-PE can increase the solubility of the P12 polypeptide molecules in a saline solution, because a single P12 polypeptide molecule can be loaded by the separation of the polypeptide nanometer empty liposome (P12-Lipo), and the aggregation caused by the interaction between the P12 polypeptide molecules is avoided.

The concentration of the polypeptide nanometer empty liposome (P12-Lipo) is 100 MuM, and the ultrasonic water bath is carried out for 30min at the temperature of 40 ℃. After shaking the nanoliposome (Lipo) in PBS, 1mL of the nanoliposome was placed in a 1cm X1 cm plastic sample cell and subjected to dynamic light scattering (DLS, zetasizer Nano ZS, malvern, UK) test to measure the particle size distribution of the sample.

The dynamic light scattering reflects the particle size change of molecules in the solution, and according to the early-stage experimental result in a laboratory, the P12 polypeptide is insoluble in a 1 XPBS solution, and white precipitate visible to the naked eye appears; as shown in fig. 2B, the particle size of the nanoliposome (Lipo) in the PBS solution is 100-150nm, which also indicates that the P12 polypeptide and DPPC liposome are physically assembled, and the liposome can significantly increase the solubility of the P12 polypeptide in the saline solution.

Example 3 FITC-labeled P12 polypeptide molecule (FITC-P12) and P12-Lipo fluorescent nanoliposome dissolved in PBS Solubility in liquid test

FITC-labeled P12 polypeptide molecules (namely FITC-P12 polypeptide molecules) are prepared into a 1mg/mL solution by using a glucose buffer solution, and the concentration of the FITC-P12 polypeptide is 40 mu M. In addition, DSPE-PEG-2000 was prepared as a 5mg/mL solution with chloroform, DSPE-PEG-P12-FITC (in which the molecular weight of the PEG moiety was 2000) was prepared as a 5mg/mL solution with chloroform, cholesterol was prepared as a 10mg/mL solution with chloroform, and DPPC was prepared as a 10mg/mL solution with chloroform. The preparation method comprises the following steps of mixing the above solutions according to the mass ratio of 2. The prepared solution was placed in a refrigerator at 4 ℃. The PBS solution of FITC-P12 and P12-Lipo fluorescent nanoliposome is used for detecting the relative fluorescence intensity (excitation emission wavelength 488/535 nm) of FITC by a fluorescence microplate reader to calculate the relative concentration of the dissolved FITC-P12 and P12-Lipo fluorescent nanoliposome under different conditions.

The FITC-P12 polypeptide solution is insoluble in the PBS solution, precipitates after standing, becomes turbid, and the FITC-P12 polypeptide aggregates at the bottom of the test tube. After the DSPE-PEG-2000, DSPE-PEG-P12, cholesterol and DPPC are hydrated to form liposome, the P12-Lipo fluorescent nano liposome containing FITC-P12 polypeptide can be completely dissolved in PBS solution. The nano lipidization can obviously increase the solubility of the FITC-P12 polypeptide in a salt solution.

Example 4 preparation of polypeptide temperature sensitive nanoliposome (P12-Lipo-ICG) and its solubility in PBS solution Test (experiment)

Respectively preparing 5mg/mL solution of DSPE-PEG-2000 with chloroform, 5mg/mL solution of DSPE-PEG-MAL or DSPE-PEG-P12 (wherein the molecular weight of PEG segment is 2000), 10mg/mL solution of cholesterol with chloroform, 10mg/mL solution of DPPC with chloroform, and 10mg/mL solution of photosensitizer ICG with methanol. The molar ratio is 2: 2, mixing the solutions, fully and uniformly mixing, heating in a water bath at 45 ℃, carrying out rotary evaporation for 1h, then placing in a vacuum drying oven at 60 ℃ overnight, then adding 5mL of PBS solution, carrying out ultrasonic water bath at 60 ℃ for 45min, and hydrating the PBS solution into a PBS solution of polypeptide temperature-sensitive nano liposomes (P12-Lipo-ICG). The prepared solution was placed in a refrigerator at 4 ℃.

Example 5 preparation of polypeptide drug-loaded temperature-sensitive nanoliposome (P12-Lipo-ICG-DOX) and its use in PBS solution Solubility test

Respectively preparing 5mg/mL solution of DSPE-PEG-2000 with chloroform, 5mg/mL solution of DSPE-PEG-MAL or DSPE-PEG-P12 (wherein the molecular weight of PEG segment is 2000), 10mg/mL solution of cholesterol with chloroform, 10mg/mL solution of DPPC with chloroform, and 10mg/mL solution of photosensitizer ICG with methanol. According to the mass ratio of 2: 2, mixing the above solutions, fully and uniformly mixing, heating in a water bath at 45 ℃, carrying out rotary evaporation for 1h, then placing in a vacuum drying oven at 60 ℃ overnight, then adding an ammonium sulfate (250 mM) solution, carrying out ultrasonic water bath for 60 ℃ and 45min, and hydrating the ammonium sulfate solution into an ammonium sulfate solution of the polypeptide temperature-sensitive nano liposome (P12-Lipo-ICG). Then adding 1mg/mL DOX PBS solution, stirring at room temperature of 180rpm for 30min to obtain polypeptide drug-loaded temperature-sensitive nano liposome (P12-Lipo-ICG-DOX), and placing the prepared solution in a refrigerator at 4 ℃.

The carrier concentration of the polypeptide drug-loaded temperature-sensitive nano liposome (P12-Lipo-ICG-DOX) is 100 mu M, and the polypeptide drug-loaded temperature-sensitive nano liposome is evenly mixed in a water bath at the temperature of 45 ℃ by ultrasound for 30 min. Dropping 10 mu L of PBS solution sample of the polypeptide drug-loaded temperature-sensitive nano liposome on a carbon-coated film copper net with activated surface after glow discharge treatment, standing for 5min, sucking dry the solution by using filter paper, dyeing for 60s by using 5 mu L of 2% uranyl acetate or 2% tungsten phosphate dyeing solution (centrifuging at 4000rpm for 5min before use, removing the dyeing solution which is not completely dissolved), sucking dry the dyeing solution by using the filter paper, and carrying out negative dyeing by using a transmission electron microscope sample, wherein the sample in figure 3 is 2% uranyl acetate. The transmission electron microscope reflects the morphology and particle size of the sample, as shown in fig. 3A, the polypeptide drug-loaded temperature-sensitive nanoliposome (P12-Lipo-ICG-DOX) exists in a spherical structure in the PBS solution, the particle size distribution is uniform, and the particle size is 100-150nm. After the liposome (Lipo) is loaded with P12 polypeptide and chemotherapeutic drug molecules, the polypeptide drug-loaded temperature-sensitive nano-liposome (P12-Lipo-ICG-DOX) keeps a spherical structure, and the particle size is not obviously changed.

The carrier concentration of the polypeptide drug-loaded temperature-sensitive nano liposome (P12-Lipo-ICG-DOX) is 100 mu M, and the ultrasonic water bath is carried out for 30min at 40 ℃. The method comprises the steps of shaking up a PBS solution of the polypeptide drug-loaded temperature-sensitive nano liposome (P12-Lipo-ICG-DOX), placing 1mL of the solution into a plastic sample cell with the size of 1cm multiplied by 1cm, and carrying out dynamic light scattering test to measure the particle size distribution condition of the sample. As shown in FIG. 3B, the particle size of the polypeptide drug-loaded temperature-sensitive nanoliposome (P12-Lipo-ICG-DOX) in PBS solution is 100-150nm, which also indicates that the P12 polypeptide, photosensitizer ICG, chemotherapeutic drug DOX and DPPC liposome are physically assembled, and the liposome can significantly increase the solubility of the P12 polypeptide in the saline solution and successfully load chemotherapeutic drugs.

Example 6 detection of affinity of FITC-P12 polypeptide or P12-Lipo nanoliposome to CXCR4 receptor of MCF-7 cells Measuring

MCF-7 cell line is used as a model system for researching breast cancer cell line. 2X 10 cultures of 2 mL DMEM medium (containing 10% fetal bovine serum FBS and 1% streptomycin) per well in Corning (Corning) 24-well plates ⁵ The cells were treated with 24-well plates at 37 ℃ and 5% CO ₂ And pre-culturing for 24h in an incubator under the condition to allow the cells to adhere to the wall.

First experimental conditions: mu.L of glucose solutions of FITC-P12 polypeptide at various concentrations were added to each well of a Corning 24-well plate to give final concentrations of FITC-P12 polypeptide of 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M and 20. Mu.M, and only 10. Mu.L of PBS solution was added to a blank control group, and 24-well cell culture plates were incubated in an incubator for 1 hour.

Second experimental conditions: to Corning 24-well plates were added 10. Mu.L of FITC-P12 polypeptide or P12-Lipo fluorescent nanoliposomes (samples of examples 3, 6) PBS solution to make the final concentrations of FITC-P12 polypeptide 2. Mu.M and 5. Mu.M, P12-Lipo nanoliposomes and 10% of the nanofluorescent liposomes of P12 polypeptide 2. Mu.M and 5. Mu.M at the final concentration of P12 polypeptide, and the blank control was added only 10. Mu.L of PBS solution, and 24-well cell culture plates were incubated for 1h in an incubator.

In both cases, the emission wavelength was set to 488nm using a flow cytometer and the detection wavelength was 535nm (1 channel). The negative control group cell sample was placed on the instrument sample holder and the test was started, and according to the cell size, a gate was set in the scattergram of the forward angle signal (FSC), the lateral angle signal (SSC), and a threshold value was set before the test result was recorded so that the number of fluorescence intensities in the gate, which was higher than the threshold fluorescence intensity, was less than 1% in the number statistical peak chart of the fluorescence intensities, and after the setting was completed, 10,000 cells were counted. Under the above gate settings and counting conditions, the blank control and experimental group samples were sequentially tested and the corresponding test values, i.e. the percentage of the number above the threshold fluorescence intensity, were recorded.

In the first case, as shown in FIG. 4A, at the same incubation time (1 h), the binding rate of FITC-P12 polypeptide to CXCR4 receptor of MCF-7 cells (% of positive cells) increased with increasing concentration of FITC-P12 polypeptide.

In the second case, as shown in FIG. 5, the binding rate of FITC-P12 polypeptide alone to CXCR4 receptor of MCF-7 cells (i.e.,% of positive cells) increased with increasing concentration of FITC-P12 polypeptide. The binding rate (namely the percentage of positive cells) of the P12-Lipo fluorescent nanoliposome to the CXCR4 receptor of MCF-7 cells also increases along with the increase of the concentration of the FITC-P12 polypeptide. However, under the same FITC-P12 polypeptide concentration (2. Mu.M and 5. Mu.M) and the same incubation time (1 h), the binding rate of the P12-Lipo fluorescent nanoliposomes (samples of examples 3 and 6) to MCF-7 cellular CXCR4 receptor is obviously higher than that of the FITC-P12 polypeptide alone to the MCF-7 cellular CXCR4 receptor.

Example 7 affinity of FITC-P12 Polypeptides or P12-Lipo fluorescent Naniposomes with 4T1 cells CXCR4 receptor Detection

The 4T1 cell line was used as a model system for the study of breast cancer cell lines. In Corning 24-well plates, 2X 10 wells were cultured in 1mL of RPM1640 medium (containing 10% fetal bovine serum FBS and 1% streptomycin) ⁵ The cells were treated with 24-well plates at 37 ℃ and 5% CO ₂ And pre-culturing for 24h in an incubator under the condition to allow the cells to adhere to the wall.

First experimental conditions: mu.L of glucose solutions of FITC-P12 polypeptide at different concentrations were added to each well of Corning 24-well plates to give final concentrations of FITC-P12 polypeptide of 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M and 20. Mu.M, only 10. Mu.L of PBS solution was added to the blank control group, and 24-well cell culture plates were incubated in an incubator for 1 hour.

In both cases, flow cytometry was used to record the samples of the control and experimental groups and the corresponding measurements, i.e., the percentage of the amount above the threshold fluorescence intensity, were recorded.

In the first case, as shown in FIG. 4A, at the same incubation time (1 h), the binding rate of FITC-P12 polypeptide to CXCR4 receptor of 4T1 cells (% of positive cells) increased with increasing concentration of FITC-P12 polypeptide.

In the second case, as shown in FIG. 5, the binding rate of FITC-P12 polypeptide alone to CXCR4 receptor of 4T1 cells (% of positive cells) increased with increasing concentration of FITC-P12 polypeptide. The binding rate (namely, the percentage of positive cells) of the P12-Lipo fluorescent nano liposome to the CXCR4 receptor of the 4T1 cells is increased along with the increase of the concentration of the FITC-P12 polypeptide. However, under the same FITC-P12 polypeptide concentration (2 μ M and 5 μ M) and the same incubation time (1 h), the binding rate of the P12-Lipo fluorescent nanoliposome (samples of examples 3 and 6) to 4T1 cell CXCR4 receptor is obviously higher than that of the FITC-P12 polypeptide alone to 4T1 cell CXCR4 receptor. The two experimental results both benefit from the fact that the solubility of the FITC-P12 polypeptide in the PBS solution can be obviously increased through lipidization, and the binding effect of the FITC-P12 polypeptide and the CXCR4 receptor is promoted.

Example 8 non-targeted temperature sensitive liposomes (Lipo-ICG), targeted temperature sensitive liposomes (P12-Lipo-ICG) and chemotherapy Inhibition effect of drug-combined temperature-sensitive liposome (P12-Lipo-ICG-DOX) on breast cancer cell 4T1

4T1 tumor cells were used as a model system for studying breast cancer cell lines, and 4T1 tumor cells were digested with 0.25% trypsin at 5X 10 cells per well ³ Cells were seeded in 96-well plates at 37 ℃ and 5% CO ₂ Culturing for 24h under the condition; then removing culture solution, adding fixed non-target temperature-sensitive liposome (Lipo-ICG) and target temperature-sensitive lipidICG concentration (20. Mu.g/mL, sample of example 1, 4) in plastid (P12-Lipo-ICG) and chemotherapeutic drug in combination with temperature sensitive liposomes (P12-Lipo-ICG-DOX), different drug combination solutions were incubated in an ultrasonic water bath at 55 ℃ for 30min in advance, then left to stand at room temperature for 2h, added to a 96-well plate, 12h later, each experimental sample was irradiated with a laser at wavelength 808nm for 3min or 5min, then continued incubation for 2h, the medium was discarded, fresh 1640 medium was added at 100. Mu.L/well, cell Counting Kit-8 (10. Mu.L/well) was added at 37 ℃,5 CO was added ₂ Incubating for 2 hours; the 490nm absorbance value was read and the cell growth inhibition was calculated. As shown in FIG. 6, the chemotherapeutic drug combined with the temperature sensitive liposome (P12-Lipo-ICG-DOX) is significantly superior to other groups. As shown in FIG. 6, the growth inhibition rate of the targeted temperature-sensitive liposome (P12-Lipo-ICG) on 4T1 cells is higher than that of the non-targeted temperature-sensitive liposome (Lipo-ICG) when the concentration of ICG is fixed. As shown in fig. 6, the release of more chemotherapeutic agent was allowed with increasing irradiation time, thereby enhancing the inhibitory effect on 4T1 cells.

Example 9 anti-tumor polypeptide drug-loaded thermo-sensitive liposomes (P12-Lipo-ICG-DOX) therapeutic drugs inhibit 4T1 tumors Effect of growth

The anti-tumor polypeptide drug-loaded temperature-sensitive liposome (P12-Lipo-ICG-DOX) prepared in examples 4 and 5 was used to determine the effect of resisting the growth of 4T1 tumor, and free chemotherapeutic drug DOX was used as a control sample. Inoculating BALB/c white mouse with 4T1 breast cancer cells at the subcutaneous part of the back until the tumor grows to be 100mm in volume ³ When in use, physiological Saline (Saline) buffer solution, free chemotherapeutic adriamycin (DOX), drug-loaded liposome (Lipo-DOX), polypeptide drug-loaded liposome (P12-Lipo-DOX) and polypeptide drug-loaded thermo-sensitive liposome (P12-Lipo-ICG-DOX) are injected into tail vein once every 3 days, the dose is 3.0mg/kg DOX, wherein after the polypeptide drug-loaded thermo-sensitive liposome and a Laser group (P12-Lipo-ICG-DOX + Laser) are injected into the drug for 6 hours, a 808nm Laser is utilized to irradiate the tumor part of a mouse respectively, and 6 mice in each group are obtained. After 14 days of treatment, the mice were sacrificed, tumor tissue was removed, and the tumors were weighed. As shown in figure 7, the polypeptide drug-loaded temperature-sensitive liposome plus Laser group (P12-Lipo-ICG-DOX + Laser) has the most remarkable ability of inhibiting tumor growth and is obviously superior to that of the polypeptide drug-loaded temperature-sensitive liposome plus Laser groupThe DOX group, the Lipo-DOX group, the P12-Lipo-DOX group and the P12-Lipo-ICG-DOX group are not added with laser, which shows that the design of the polypeptide drug-loaded temperature-sensitive liposome (P12-Lipo-ICG-DOX) has the best treatment effect on tumors. The growth inhibition effect of the P12-Lipo-DOX group on the tumor is better than that of the Lipo-DOX group, which indicates that the targeted drug delivery efficiency of the polypeptide is higher than that of the non-targeted group. In addition, the rapid chemotherapy drug release caused by thermal triggering is obviously superior to a non-response group (polypeptide drug-loaded temperature-sensitive liposome, P12-Lipo-ICG-DOX) in the tumor inhibition effect, so that the rapid drug release can cause more effective killing effect on the tumor, and the utilization rate of the drug is improved. Only some of the most compelling test cases are exemplified herein, to the extent of space.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be accorded the scope of the appended claims, including equivalents of each element described.

Claims

1. The polypeptide drug-loaded temperature-sensitive liposome is characterized by being formed by self-assembling a polypeptide, a chemotherapeutic drug, a photosensitizer and a liposome, wherein the polypeptide can be combined with cancer cells expressing or over-expressing a chemokine receptor protein CXCR4 in a targeting manner;

the particle size of the polypeptide drug-loaded temperature-sensitive liposome is 50-500 nm;

the cancer cell targeted binding polypeptide is P12 polypeptide or FITC labeled P12 polypeptide; wherein the amino acid sequence of the P12 polypeptide is QGSRRRNTVDDWISRRRALC; the amino acid sequence of the FITC-labeled P12 polypeptide is FITC-QGSRRRNTVDDWISRRRALC;

the liposome comprises DPPC, DSPE-PEG-MAL, DSPE-PEG and cholesterol.

2. The polypeptide drug-loaded thermosensitive liposome according to claim 1, wherein the particle size of the polypeptide drug-loaded thermosensitive liposome is 50-200 nm.

3. The polypeptide drug-loaded temperature-sensitive liposome of claim 2, wherein the particle size of the polypeptide drug-loaded temperature-sensitive liposome is 100-150 nm.

4. The polypeptide drug-loaded temperature-sensitive liposome of claim 1, wherein the photosensitizer is selected from one or more of the following: ICG, porphyrin, fe ₃ O ₄ Nanoparticles, gold nanoparticles;

the chemotherapeutic drug is selected from one or more of the following: adriamycin, daunorubicin, paclitaxel and docetaxel.

5. The polypeptide drug-loaded thermosensitive liposome according to claim 4, wherein the photosensitizer is ICG and/or porphyrin;

the chemotherapy medicine is adriamycin and/or paclitaxel.

6. The polypeptide drug-loaded thermosensitive liposome according to claim 5, wherein the photosensitizer is ICG;

the chemotherapy drug is adriamycin.

7. The polypeptide drug-loaded thermosensitive liposome according to any one of claims 1 to 6, wherein the polypeptide is bound to the liposome through chemical coupling;

The chemotherapeutic agent is combined with the liposomes by emulsification.

8. The preparation method of the polypeptide drug-loaded temperature-sensitive liposome according to any one of claims 1 to 7, wherein the method comprises the following steps:

(1) Preparing a polypeptide-liposome solution;

(3) And (3) adding an ammonium sulfate solution into the product obtained in the step (2), adding a chemotherapeutic drug solution, uniformly mixing, incubating and standing to obtain the polypeptide drug-loaded temperature-sensitive liposome.

9. The preparation method according to claim 8, wherein in the step (2), the rotary evaporation temperature is 30-60 ℃;

the rotary steaming time is 10-90 minutes; and/or

The temperature of the incubation is 30-100 ℃.

10. The method according to claim 9, wherein, in the step (2),

the rotary evaporation temperature is 40-50 ℃;

the rotary evaporation time is 20-70 minutes; and/or

The incubation temperature is 50-80 ℃.

11. The method according to claim 10, wherein in the step (2),

the rotary evaporation temperature is 45 ℃;

the rotary steaming time is 30-60 minutes; and/or

The incubation temperature was 60 ℃.

12. The method according to claim 8, wherein the incubation time in the step (3) is 10 to 60min.

13. The method according to claim 12, wherein the incubation time in the step (3) is 30min.

14. A medicament comprising the polypeptide-carrying thermosensitive liposome according to any one of claims 1 to 7 or the polypeptide-carrying thermosensitive liposome prepared by the preparation method according to any one of claims 8 to 13.

15. The medicament of claim 14, wherein the medicament is a solution or a lyophilized powder.

16. The medicament of claim 15, wherein the lyoprotectant of the lyophilized powder is mannitol.

17. Use of the polypeptide drug-loaded thermo-sensitive liposome of any one of claims 1 to 7 or the polypeptide drug-loaded thermo-sensitive liposome prepared by the preparation method of any one of claims 8 to 13 in the preparation of a medicament for treating cancer.

18. The use of claim 17, wherein the medicament is a medicament for inhibiting cancer metastasis.

19. The use according to claim 18, wherein the cancer is a cancer associated with cancer cells or cancer tissues expressing or overexpressing the chemokine receptor protein CXCR 4.

20. The use according to claim 19, wherein the cancer is selected from one or more of: breast cancer, leukemia, lymphoma, bladder cancer, and liver cancer.

21. The use of claim 20, wherein the cancer is breast cancer or liver cancer.

22. The use of claim 21, wherein the cancer is breast cancer.