CN111298116A - Polypeptide drug-loaded thermosensitive liposome and preparation method and application thereof - Google Patents

Abstract

The present invention provides a polypeptide drug-loaded thermosensitive liposome, the drug-loaded thermosensitive liposome is formed by self-assembly of a polypeptide, a chemotherapeutic drug, a photosensitizer and a liposome, wherein the polypeptide is capable of interacting with expression or Cancer cells overexpressing the chemokine receptor protein CXCR4 target the bound polypeptide. Preparation methods and applications thereof are also provided. The drug-loaded thermosensitive liposomes used in combination with the polypeptides, photosensitizers and chemotherapeutic drugs of the present invention have the ability to improve the solubility of the polypeptides in saline solution, improve the binding efficiency of the polypeptides and the chemokine receptor CXCR4 target protein, and improve the The enrichment ability of chemotherapeutic drugs in cancer cells and cancer tissues was demonstrated, and the property of inhibiting the migration of cancer cells was stronger. On the other hand, by using liposomes to improve the drug loading and in vivo bioavailability of chemotherapeutic drugs, and using photosensitizers to achieve precise and controllable release of chemotherapeutic drugs.

Description

Polypeptide drug-loaded thermosensitive liposome and preparation method and application thereof

technical field

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide drug-loaded thermosensitive liposome and a preparation method and application thereof.

Background technique

Cancer is currently the main cause of morbidity and mortality in the world. About 90% of cancer patients die of cancer metastasis and recurrence. Blocking a certain link of cancer metastasis to prevent the spread of cancer is crucial for cancer treatment. Chemokines and chemokine receptors are not only involved in normal physiological activities, but also in pathological processes such as carcinogenesis, invasion and metastasis, among which the CXCR4/SDF-1 (or CXCL12) biochemotactic axis plays a role in cancer migration and invasion. The development of antagonists of the CXCR4 chemokine receptor protein is critical for controlling cancer metastasis and improving cancer cure rates. Because peptides are easy to design and synthesize, easily metabolized in the human body, and do not cause toxic side effects and severe immune responses, the development of peptide antagonists of CXCR4 receptor protein provides a new effective means and strategy for cancer treatment.

Liposomes are a class of biosafety and good drug carriers that have received widespread attention. They are phospholipid bilayers with a diameter of 50-500 nm that are spontaneously formed by phospholipids and cholesterol in a solvent system under appropriate concentration ratios and temperature conditions. system. At present, liposomes have successfully encapsulated a variety of hydrophobic chemotherapeutic drugs, which can not only effectively solve the problems of poor water solubility of chemotherapeutic drugs, rapid degradation in systemic circulation, less drug absorption, and large toxic and side effects, but also enhance the effect of osmotic retention. (Enhanced Permeation Retention Effect, EPR) to achieve its efficient enrichment in the cancer lesion area. However, some liposomes have the defect of slow local drug release, making it difficult for effective drugs to exert their own rapid killing effect, and even cause drug resistance at low drug concentrations that are sustained and sustained. Therefore, the construction of cancer-targeted liposome drug delivery system, reducing the damage of chemotherapy drugs to normal organs, and enhancing the precise and controllable release of chemotherapy drugs in cancer parenchyma have attracted more and more attention of researchers.

Since CXCR4 is highly specifically expressed in various cancer tissues, and most of them are distributed on the surface of cancer cells, it can be used as an active recognition target for nano-drug delivery carriers to develop CXCR4-specific recognition peptides, which can be combined to kill cancer cells. Based on the photothermal effect of the photosensitizer ICG, active targeting thermosensitive liposomes modified with CXCR4-targeting peptides are constructed, which is expected to realize the "drug storm" attack of chemotherapeutic drugs on cancer tissues, thereby significantly enhancing anti-cancer treatment. effect, providing new information and clues for the development of highly effective cancer treatment drugs.

SUMMARY OF THE INVENTION

Therefore, the purpose of the present invention is to overcome the defects in the prior art, and to provide a polypeptide drug-loaded thermosensitive liposome and a preparation method and application thereof.

Before describing the content of the present invention, the terms used herein are defined as follows:

The term "FITC" refers to: fluorescein isothiocyanate.

The term "ICG" refers to: Indocyanine Green.

The term "DPPC" refers to: dipalmitoylphosphatidylcholine.

The term "DSPE" means: distearoylphosphatidylethanolamine.

The term "PEG" means: polyethylene glycol.

The term "MAL" means: 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 thermosensitive liposome, the drug-loaded thermosensitive liposome is formed by self-assembly of a polypeptide, a chemotherapeutic drug, a photosensitizer and a liposome, wherein , the polypeptide is a polypeptide capable of targeted binding with cancer cells expressing or overexpressing the chemokine receptor protein CXCR4;

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

According to the polypeptide drug-loaded thermosensitive liposome according to the first aspect of the present invention, the polypeptide is selected from one or more of the following: polypeptides mainly composed of polar amino acids, polypeptides mainly composed of hydrophobic amino acids, Polypeptides of amino acids and hydrophobic amino acids;

Preferably, the polypeptide consists of 5-100 amino acids, more preferably 10-50 amino acids, more preferably 20-30 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.

According to the polypeptide drug-loaded thermosensitive liposome according to the first aspect of the present invention, 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 drugs are 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, soybean lecithin, lecithin .

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

The photosensitizer is non-covalently bound to the liposome; and/or

The chemotherapeutic drug is combined with the liposome by emulsification.

The second aspect of the present invention provides the preparation method of the polypeptide drug-loaded thermosensitive liposome described in the first aspect, and the preparation method may include the following steps:

(1) prepare a polypeptide-liposome solution;

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

(3) adding ammonium sulfate solution to the product of step (2), then adding chemotherapeutic drug solution, mixing, stirring and standing at room temperature to obtain the polypeptide drug-loaded thermosensitive liposome.

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

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

The incubation temperature is 30-100°C, preferably 50-80°C, and most preferably 60°C.

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

The third aspect of the present invention provides a medicine, which comprises the polypeptide drug-loaded thermosensitive liposome according to the first aspect or the polypeptide drug-loaded thermosensitive liposome prepared according to the preparation method described in the second aspect Liposomes;

Preferably, the drug is a solution or a lyophilized powder;

More preferably, the lyoprotectant is mannitol.

The fourth aspect of the present invention provides that the polypeptide drug-loaded thermosensitive liposome described in the first aspect or the polypeptide drug-loaded thermosensitive liposome prepared according to the preparation method of the second aspect is prepared for the treatment of cancer the application of medicines;

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 CXCR4;

Further preferably, the cancer is selected from one or more of the following: 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 combined with a polypeptide, a chemotherapeutic drug and a photosensitizer, a preparation method and application thereof, and more particularly to a temperature-sensitive dipalmitoyl phosphatidylcholine as a carrier chemical coupler A combined drug-loaded liposome combining a CXCR4 specific targeting polypeptide, a photosensitizer ICG and a chemotherapeutic drug DOX, and a preparation method and application thereof. In the invention, a polypeptide with cancer-targeted therapeutic effect, a chemotherapeutic drug doxorubicin (DOX) and a photosensitizer indocyanine green (ICG) are combined to construct a "polypeptide-chemotherapy drug-photosensitizer". "Thermosensitive PEGylated phospholipid complex"; the cancer-targeted therapeutic polypeptide can specifically bind to the chemokine receptor protein CXCR4 highly expressed by cancer cells, effectively improving the specific selection of drugs for cancer tissues and cancer cells It improves the drug load and biocompatibility of the drug by using the temperature-sensitive PEGylated phospholipid complex, and realizes the controllable release of the drug in the cancer tissue. Toxicity to tissues and normal cells. Compared with the single chemotherapeutic drug doxorubicin, the drug-loaded thermosensitive liposome exerts a stronger advantage of inhibiting the growth of cancer cells and further reduces the toxic and side effects of the chemotherapeutic drugs. The temperature-responsive release drug-loaded liposome of the polypeptide, chemotherapeutic drug and photosensitizer of the present invention provides a feasible method and technology for improving the effect of cancer treatment.

The purpose of the present invention is to provide a preparation method and application of a drug-loaded thermosensitive liposome combining polypeptide, photosensitizer and chemotherapeutic drug for cancer treatment, and to construct a method that can specifically target CXCR4 high-expressing cancer cells. Cell peptides, chemotherapeutic drugs that can kill cancer cells, and thermosensitive nanoliposomes that can improve the precise and controllable release of drugs. The drug-loaded liposome combined with the polypeptide, photosensitizer and chemotherapeutic drug also solves the problems of solubility and biological stability of the cancer-targeting polypeptide with good water solubility but poor salt solubility in salt solution, and improves the peptide and CXCR4 target protein. The binding efficiency of chemotherapeutic drugs enhanced the anti-cancer effect of chemotherapeutic drugs and exhibited excellent inhibition of cancer metastasis.

The present invention adopts following technical scheme:

A polypeptide nanoliposome is formed by chemical bond coupling between distearoyl phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) and cancer-targeting polypeptide P12, the cancer Targeting polypeptides are polypeptides that can target and bind to cancer cells or cancer tissues that express or overexpress the chemokine receptor protein CXCR4.

in:

The distearoyl phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) is polyethylene glycol (hydrophilic block) through covalent bonds and phospholipid molecules (hydrophobic block) A compound formed by the combination of nitrogenous bases on it.

Preferably, the molecular weight of the polyethylene glycol hydrophilic block in the distearoyl phosphatidyl ethanolamine-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 polypeptides mainly consisting of polar amino acids, mainly hydrophobic amino acids, or both polar amino acids and hydrophobic amino acids.

Preferably, the cancer-targeting polypeptide consists of 5-100 amino acids, more preferably 10-50 amino acids, and more preferably 20-30 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. It is found in the present invention that the P12 polypeptide can specifically bind to the surface of cancer cells with high expression of CXCR4 protein, and then play 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 the FITC-labeled P12 polypeptide can be obtained by an existing chemical synthesis method, or a commercial product can be purchased.

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

Preferably, the binding mode of the cancer-targeting polypeptide to DSPE-PEG-MAL is chemical binding.

Preferably, the polypeptide nanoliposomes are in solution form or lyophilized powder form.

The present invention also provides a method for preparing the above-mentioned polypeptide thermosensitive nanoliposome, comprising the following steps:

Prepare DSPE-PEG-2000 molecular solution, DSPE-PEG-P12 molecular solution, cholesterol molecular solution, DPPC molecular solution and ICG solution respectively; mix each component solution, rotate, dry, stand and hydrate to obtain polypeptide Thermosensitive nanoliposome solution.

The preparation method of above-mentioned polypeptide thermosensitive nanoliposome, wherein:

Preferably, the solvent for preparing DSPE-PEG-P12, DSPE-PEG 2000, DPPC and cholesterol solutions is chloroform; the solvent for ICG solution is methanol;

Preferably, the above-mentioned DPPC and cholesterol solutions are prepared into a 2-20 mg/mL solution; the above-mentioned cancer-targeting polypeptides DSPE-PEG-P12, DSPE-PEG2000, and ICG are prepared into a 1-5 mg/mL solution;

Preferably, the mixing is to fully mix the DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12 and ICG solution to obtain a mixed solution, which is rotary evaporated, dried and hydrated;

Preferably, the rotary evaporation time is 1h, the drying time is overnight, the hydration incubation temperature is 60°C, and the incubation time is 30min;

Specifically, the preparation method of the above-mentioned polypeptide thermosensitive nanoliposome comprises the following steps:

(1) Preparation of solution: the above-mentioned DSPE-PEG-P12, DSPE-PEG 2000, DPPC, and cholesterol are prepared into a 2-20 mg/mL solution with a chloroform solution; the above-mentioned ICG molecule is prepared with a methanol solution into a 1-5 mg/mL solution;

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

(3) rotary steam: the obtained mixed solution in step (2) was rotary steamed for 60min in a water bath at 45°C;

Preferably, the incubation temperature is 45°C, and the incubation time is 60min;

(4) Drying: Preferably, the standing drying condition is vacuum drying at 60° C. and standing overnight to obtain a polypeptide thermosensitive liposome membrane.

(5) Hydration: Add 5 mL of phosphate buffer solution (ie, PBS buffer) to the liposome membrane obtained in step (4) in an ultrasonic water bath for 30 minutes, and the incubation temperature is 60°C.

Preferably, the preparation method of the above-mentioned polypeptide thermosensitive nanoliposome further includes the step of sterilizing the obtained polypeptide nanoliposome solution after standing, and further preferably, the sterilizing is after standing in step (4). The obtained polypeptide thermosensitive nanoliposome solution was filtered with a 0.22 μm filter.

According to needs, the preparation method of the above-mentioned polypeptide thermosensitive nanoliposome further includes the step of lyophilizing the sterilized polypeptide thermosensitive nanoliposome solution to prepare the polypeptide thermosensitive nanoliposome freeze-dried powder.

Further preferably, the freeze-drying comprises adding a certain amount of a freeze-drying protection agent to the obtained sterilized polypeptide thermosensitive nanoliposome solution; the freeze-drying protection agent is preferably mannitol, for example, the concentration is 0.01-0.2 g/mL of mannitol.

The polypeptide thermosensitive nanoliposomes of the present invention can be prepared by existing conventional techniques.

In order to further improve the anti-cancer therapeutic effect, the above-mentioned polypeptide nanoliposomes also include chemotherapeutic drugs, and the above-mentioned polypeptide nano-liposomes containing chemotherapeutic drugs are referred to in the present invention as drug-loaded thermosensitive liposomes combined with polypeptides and chemotherapeutic drugs.

In the present invention, the chemotherapeutic drugs may be various chemotherapeutic drugs known to those in the medical field; preferably, the chemotherapeutic drugs are selected from one of doxorubicin, daunorubicin, paclitaxel or docetaxel or several; more preferably doxorubicin and/or paclitaxel; more preferably doxorubicin (DOX).

The drug-loaded thermosensitive liposome combining the above-mentioned polypeptide and a chemotherapeutic drug, wherein the polypeptide nano-liposome and the above-mentioned chemotherapeutic drug are formed by an ammonium sulfate gradient method.

Preferably, the polypeptide nanoliposome and the ammonium sulfate solution are interacted by hydration to obtain the polypeptide nanoliposome ammonium sulfate solution; on this basis, the above chemotherapeutic drugs are added to prepare the polypeptide nanoliposome and chemotherapy Drug-loaded liposomes (ie, drug-loaded thermosensitive liposomes).

Preferably, the DSPE-PEG-MAL is combined with the cancer-targeting polypeptide through chemical coupling to form DSPE-PEG-P12;

Preferably, the ICG is non-covalently bound to the polypeptide nanoliposome.

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

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

Specifically, a drug-loaded thermosensitive liposome combined with a polypeptide and a chemotherapeutic drug is composed of thermosensitive dipalmitoyl phosphatidylcholine (DPPC), cholesterol, an ICG photosensitizer, the above-mentioned cancer-targeting polypeptide and the chemotherapeutic drug Formed by self-assembly, the cancer-targeting polypeptide is a polypeptide capable of targeted binding to cancer cells or cancer tissues that express or overexpress the chemokine receptor CXCR4.

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

The drug-loaded thermosensitive liposome in combination with the above-mentioned polypeptide and a chemotherapeutic drug, wherein:

Preferably, the molar ratio of the thermosensitive dipalmitoyl phosphatidylcholine (DPPC) liposome to the chemotherapeutic drug is 1000:8, 1000:16, 1000:32, more preferably, the mass ratio is 1000:16.

Preferably, the drug-loaded thermosensitive liposome in combination with the above-mentioned polypeptide and a chemotherapeutic drug is in the form of a solution or a lyophilized powder.

The present invention also provides a preparation method of the drug-loaded thermosensitive liposome in combination with the above-mentioned polypeptide, ICG and a chemotherapeutic drug, comprising the following steps:

Prepare DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12, ICG and DOX solutions respectively; mix the DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12, and ICG solutions thoroughly, rotate them, and let them dry , algal bloom, to obtain temperature-sensitive liposomes combined with peptide and photosensitizer ICG.

Preferably, the preparation method of the drug-loaded thermosensitive liposome combined with the above-mentioned thermosensitive liposome and a chemotherapeutic drug comprises the following steps:

DPPC, cholesterol, DSPE-PEG 2000, DSPE-PEG-P12, and ICG solutions were prepared respectively, mixed, rotary evaporated, left to dry, and hydrated to obtain a polypeptide thermosensitive liposome solution;

The polypeptide nano-liposome and the ammonium sulfate solution are interacted by hydration to obtain the polypeptide nano-liposome ammonium sulfate solution; on this basis, the above-mentioned chemotherapeutic drugs are added to prepare the carrier of the combination of the polypeptide nano-liposomes and the chemotherapeutic drugs. Drug-loaded liposomes (ie, drug-loaded thermosensitive liposomes);

The preparation method of the drug-loaded thermosensitive liposome combined with the above-mentioned polypeptide and chemotherapeutic drug, wherein:

Preferably, the solvent for preparing and configuring the thermosensitive nanoliposome solution is any one of phosphate buffered saline (i.e. PBS solution), hydroxyethylpiperazine ethanethiosulfonic acid buffer, physiological saline or sterile ultrapure water; More preferably phosphate buffered saline (ie PBS solution);

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

Preferably, the incubation temperature is 20-30°C, and the incubation time is 20-60 minutes; further preferably, the incubation temperature is 26°C, and the incubation time is 30 minutes.

Preferably, the preparation method of the drug-loaded thermo-sensitive liposome combined with the above-mentioned thermo-sensitive liposome and a chemotherapeutic drug further comprises the step of sterilizing the drug-loaded thermo-sensitive liposome solution obtained after standing, and further preferably, the The sterilization is to filter the drug-loaded thermosensitive liposome solution obtained after standing still with a 0.22 μm filter membrane.

According to needs, the preparation method of the drug-loaded thermosensitive liposome combined with the above-mentioned polypeptide and chemotherapeutic drug further comprises freeze-drying the sterilized solution to prepare the drug-loaded thermosensitive liposome lyophilized powder of the combination of the polypeptide and the chemotherapeutic drug A step of.

Further preferably, the freeze-drying comprises adding a certain amount of a freeze-drying protection agent to the obtained sterilized drug-loaded thermosensitive liposome solution; the freeze-drying protection agent is preferably mannitol, for example, the concentration is 0.01-0.2 g/mL of mannitol.

The drug-loaded thermosensitive liposomes of the present invention can be prepared by existing conventional techniques.

The present invention also includes the above-mentioned polypeptide liposome, the above-mentioned photosensitizer and the temperature-sensitive liposome in combination with the polypeptide liposome, and the above-mentioned preparation method. The application of medicine in the aspect of cancer treatment; preferably, the application in inhibiting cancer metastasis; further preferably, the application in inhibiting cancer metastasis associated with cancer cells or cancer tissues expressing or overexpressing chemokine receptor CXCR4 .

Preferably, the cancer associated with cancer cells or cancer tissues that express or overexpress the chemokine receptor CXCR4 includes any one of breast cancer, leukemia, lymphoma, bladder cancer or liver cancer; further preferably, it is breast cancer cancer.

The polypeptide drug-loaded thermosensitive liposomes of the present invention can have but are not limited to the following beneficial effects:

The drug-loaded thermosensitive liposome used in combination with the polypeptide, the photosensitizer and the chemotherapeutic drug of the present invention has the ability to improve the solubility of the polypeptide in a salt solution, and improves the binding efficiency of the polypeptide and the chemokine receptor CXCR4 target protein. The specific binding of the polypeptide to CXCR4 can not only improve the enrichment ability of chemotherapeutic drugs in cancer cells and cancer tissues, but also the drug-loaded thermosensitive liposome exhibits stronger inhibition of cancer cell migration compared with the single polypeptide. characteristics. On the other hand, by using liposomes to improve the drug loading and in vivo bioavailability of chemotherapeutic drugs, and using photosensitizer ICG to achieve precise and controllable release of chemotherapeutic drugs, compared with the single chemotherapeutic drug doxorubicin, the active Targeted drug-loaded thermosensitive liposomes exhibit stronger cancer growth inhibition properties, providing a feasible method and technology for improving cancer treatment effects.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows the transmission electron microscope picture and dynamic light scattering particle size analysis of DPPC liposomes in PBS solution in Example 1; wherein, Figure 1A shows the transmission electron microscope picture, and Figure 1B shows the dynamic light scattering particle size analyze.

Figure 2 shows the transmission electron microscope picture and dynamic light scattering particle size analysis of the polypeptide P12-Lipo liposome in PBS solution in Example 2; wherein, Figure 2A shows the transmission electron microscope picture; Figure 2B shows the dynamic light Scatter particle size analysis.

Figure 3 shows the transmission electron microscope picture and dynamic light scattering particle size analysis of the polypeptide drug-loaded P12-Lipo-DOX liposome in PBS solution in Example 5; wherein, Figure 3A shows the transmission electron microscope picture; Figure 3B shows Dynamic light scattering particle size analysis.

Figure 4 shows the results of flow cytometry detection of FITC-P12 polypeptides in Examples 6 and 7; wherein, Figure 4A shows the affinity of FITC-P12 polypeptides at different concentrations with CXCR4 low-expressing MCF-7 cells in Example 6 Flow cytometry detection; Figure 4B shows the flow cytometry detection of the affinity of FITC-P12 polypeptide at different concentrations with CXCR4 highly expressing 4T1 cells in Example 7.

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

Figure 6 shows in Example 8, non-targeted thermosensitive liposomes (Lipo-ICG), targeted thermosensitive liposomes (P12-Lipo-ICG) and thermosensitive liposomes in combination with chemotherapeutic drugs (P12 -Lipo-ICG-DOX) on the survival inhibition of breast cancer cells 4T1 results.

Figure 7 shows the evaluation of the anti-cancer therapeutic effect of the polypeptide drug-loaded thermosensitive liposome on tumor-bearing mice in Example 9.

Detailed ways

The present invention is further described below through specific examples, however, it should be understood that these examples are only used for more detailed description, and should not be construed to limit the present invention in any form.

This section provides a general description of the materials and test methods used in the tests of the present invention. While many of the materials and methods of operation used for the purposes of the present invention are known in the art, the present invention is described in as much detail as possible. It is clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods of operation used in the present invention are well known in the art.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

Unless otherwise specified, 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, Chinese Academy of Medical Sciences.

acousticfocusing cytometer，Applied Biosystems，Life Technologies，Carlsbad，CA)。In the embodiment of the present invention, the photosensitizer is indocyanine green (ICG) purchased from thermo Fisher Scientific Company, the chemotherapeutic drug is doxorubicin (Dox) purchased from Shanghai Yuanye Company, dipalmitoyl phosphatidylcholine ( DPPC) was purchased from Avanti Company, and Cell Counting Kit-8 reagent was purchased from Fanbo Biochemical Co., Ltd. The instruments used in the experiment: dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, UK), transmission electron microscopy (transmission electron microscopy, TEM, HT7700, Hitachi), flow cytometer (FCM,

acousticfocusing cytometer, Applied Biosystems, Life Technologies, Carlsbad, CA).

Unless otherwise specified, the solvents of the aqueous solutions used in the following examples are sterile ultrapure aqueous solutions.

Unless otherwise specified, the reagents used in the following examples are of analytical grade.

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

Preparation of 10×PBS solution: NaCl 80.00g, KCl 2g, Na ₂ HPO ₄ 12H ₂ O 35.8g or Na ₂ HPO ₄ 14.2g, KH ₂ PO ₄ 2.7g, make up to 1000mL with ultrapure water, adjust it pH 7.2 to 7.4, autoclaved.

Preparation of 1×PBS solution: Dilute 10×PBS solution 10 times with sterile ultrapure water.

Synthesis of Polypeptide P12

Amino acid sequence of P12 polypeptide: QGSRRRNTVDDWISRRRALC, amino acid sequence of FITC-labeled P12 polypeptide: FITC-QGSRRRNTVDDWISRRRALC. The P12 polypeptide and the FITC-labeled P12 polypeptide (synthesized by Anhui Guoping Pharmaceutical Co., Ltd., with a purity of 98%) were synthesized according to the sequence shown, and the stock solution of appropriate concentration was prepared before the experiment.

Example 1 Preparation of liposome (Lipo) and its dissolution experiment in PBS solution

The DSPE-PEG-MAL solution and the amino group in the P12 polypeptide are mixed and reacted at room temperature to generate DSPE-PEG-P12, which is purified and lyophilized for use. DSPE-PEG-2000 was prepared into a 5 mg/mL solution with chloroform, DSPE-PEG-P12 (where the molecular weight of the PEG segment was 2000) was prepared into a 5 mg/mL solution with chloroform, cholesterol was prepared with chloroform into a 10 mg/mL solution, and DPPC was prepared with chloroform. A 10 mg/mL solution was prepared in chloroform. Mix the above solutions in a molar ratio of 2:1.5:4:15:5. After fully mixing, heat in a water bath at 45°C for 1 hour, and then place in a vacuum drying oven at 60°C overnight. 60 ℃, 45min, make it hydrate into PBS solution of polypeptide nanoliposome. The above prepared solution was placed in a 4°C refrigerator.

The concentration of immobilized nanoliposomes (Lipo) was 100 μM, and the nanoliposomes (Lipo) were sonicated and mixed in a water bath at 45°C for 30 min. Take 10 μL of nanoliposome (Lipo) PBS solution sample and drop it on the surface-activated carbon-coated copper mesh after glow discharge treatment, let it stand for 5 minutes, filter paper to dry the solution, take 5 μL of 2% uranyl acetate or 2% uranyl acetate Tungsten phosphate staining solution (centrifuged at 4000 rpm for 5 min before use, to remove the dyeing solution that could not be completely dissolved) was stained for 60 s, blotted with filter paper, and the samples were observed with a transmission electron microscope (TEM, HT7700, Hitachi). , the sample in Figure 3 was negatively stained with 2% uranyl acetate. Transmission electron microscopy reflects the morphology and particle size of the sample. As shown in Figure 1A, it exists in a spherical structure in PBS solution, and the particle size distribution is relatively uniform; in 1×PBS solution, the particle size is 100-150nm.

The liposome (Lipo) concentration was 100 μM, and the water bath was sonicated at 40° C. for 30 min. The PBS solution of nanoliposomes (Lipo) was shaken, and 1 mL was taken and placed in a 1 cm × 1 cm plastic sample cell for dynamic light scattering test to measure the particle size distribution of the sample. Dynamic light scattering reflects the particle size change of molecules in solution; as shown in Figure 1B, the particle size of nanoliposomes (Lipo) in PBS solution is 100-150 nm.

Example 2 Preparation of polypeptide liposome (P12-Lipo) and its dissolution experiment in PBS solution

DSPE-PEG-2000 was prepared into a 5 mg/mL solution with chloroform, DSPE-PEG-P12 (where the molecular weight of the PEG segment was 2000) was prepared into a 5 mg/mL solution with chloroform, cholesterol was prepared with chloroform into a 10 mg/mL solution, and DPPC was prepared with chloroform. A 10 mg/mL solution was prepared in chloroform. Mix the above solutions according to the mass ratio of 2:1.5:4:15:5. After fully mixing, heat in a water bath at 45°C for 1 h, then place in a vacuum drying oven at 60°C overnight, then add 5 mL of PBS solution, ultrasonically in a water bath At 60°C for 45min, it was hydrated into a PBS solution of polypeptide nano-empty liposomes. The above prepared solution was placed in a 4°C refrigerator.

The concentration of immobilized polypeptide nano-empty liposomes (P12-Lipo) was 100 μM, and the polypeptide nano-empty liposomes (P12-Lipo) were sonicated and mixed in a water bath at 45° C. for 30 min. Take 10 μL of polypeptide nano-empty liposome (P12-Lipo) PBS solution sample and drop it on the surface-activated carbon-coated copper mesh after glow discharge treatment, let it stand for 5 minutes, filter paper to dry the solution, take 5 μL of 2% acetic acid hydrogen peroxide Uranium or 2% tungsten phosphate staining solution (centrifuged at 4000 rpm for 5 min before use, to remove the dyeing solution that was not completely dissolved) was stained for 60s, the filter paper blotted the dyeing solution, and the sample was observed with a transmission electron microscope. The sample in Figure 3 was obtained after 2 % uranyl acetate negative stain. Transmission electron microscopy reflects the morphology and particle size of the sample. As shown in Figure 2A, the polypeptide nano-empty liposome (P12-Lipo) exists as a spherical structure in the PBS solution, and the particle size distribution is relatively uniform, with a particle size of 100 -150nm. When the liposome (Lipo) was loaded with the P12 polypeptide molecule, the polypeptide nanoliposome (P12-Lipo) maintained a spherical structure, and the particle size did not change significantly. It shows that the reason why PEG-PE can increase the solubility of P12 polypeptide molecules in salt solution is that a single P12 polypeptide molecule can be separated and loaded by polypeptide nano-empty liposomes (P12-Lipo), avoiding the separation of P12 polypeptide molecules. aggregation caused by the interaction.

The concentration of polypeptide nano-empty liposomes (P12-Lipo) was 100 μM, and the water bath was ultrasonicated at 40° C. for 30 min. The PBS solution of nanoliposomes (Lipo) was shaken, and 1 mL was placed in a 1cm × 1cm plastic sample cell for dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, UK) test to measure the particle size distribution of the sample. .

Dynamic light scattering reflects the particle size change of molecules in the solution. According to the previous experimental results in the laboratory, P12 polypeptide is insoluble in 1×PBS solution, and white flake precipitates are visible to the naked eye; as shown in Figure 2B, nanometer The particle size of liposome (Lipo) in PBS solution is 100-150nm, which also indicates that P12 polypeptide and DPPC liposome are physically assembled, and liposome can significantly increase the solubility of P12 polypeptide in salt solution.

Example 3 FITC-labeled P12 polypeptide molecules (FITC-P12) and P12-Lipo fluorescent nanoliposomes were dissolved in PBS Solubility test in liquid

The FITC-labeled P12 polypeptide molecule (ie, FITC-P12 polypeptide molecule) was prepared into a 1 mg/mL solution with glucose buffer solution, and the concentration of FITC-P12 polypeptide was 40 μM. In addition, DSPE-PEG-2000 was prepared into a 5 mg/mL solution with chloroform, DSPE-PEG-P12-FITC (wherein the molecular weight of the PEG segment was 2000) was prepared into a 5 mg/mL solution with chloroform, and cholesterol was prepared with chloroform as a 10 mg/mL solution. The solution, DPPC, was prepared as a 10 mg/mL solution in chloroform. Mix the above solutions according to the mass ratio of 2:1.5:4:15:5. After fully mixing, heat in a water bath at 45°C for 1 hour, and then place it in a vacuum drying oven at 60°C overnight. ℃, 45min, make it hydrated to obtain polypeptide nano-fluorescent liposome solution. The above prepared solution was placed in a 4°C refrigerator. The PBS solution of the above-mentioned FITC-P12 and P12-Lipo fluorescent nanoliposomes was detected by a fluorescence microplate reader to detect the relative fluorescence intensity of FITC (excitation emission wavelength 488/535nm) to calculate the dissolved FITC-P12 and P12-Lipo under different conditions Relative concentrations of fluorescent nanoliposomes.

The FITC-P12 polypeptide solution is insoluble in the PBS solution, and precipitates out after standing, the solution becomes cloudy, and the FITC-P12 polypeptide accumulates at the bottom of the test tube. With DSPE-PEG-2000, DSPE-PEG-P12, cholesterol, and DPPC spin membrane hydration to form liposomes, the P12-Lipo fluorescent nanoliposomes containing FITC-P12 polypeptides can be completely dissolved in PBS solution. It shows that the solubility of FITC-P12 polypeptide in saline solution can be significantly increased after nanoliposomeization.

Example 4 Preparation of polypeptide thermosensitive nanoliposomes (P12-Lipo-ICG) and its solubility in PBS solution test

DSPE-PEG-2000 was prepared into a 5 mg/mL solution in chloroform, DSPE-PEG-MAL or DSPE-PEG-P12 (where the molecular weight of the PEG segment was 2000) was prepared into a 5 mg/mL solution in chloroform, and cholesterol was prepared in chloroform. 10 mg/mL solution, DPPC was prepared into 10 mg/mL solution with chloroform, and photosensitizer ICG was prepared as 10 mg/mL solution with methanol solution. Mix the above solutions in a molar ratio of 2:1.5:4:15:5:2. After thorough mixing, heat in a water bath at 45°C for 1 h, then place in a vacuum drying oven at 60°C overnight, and then add 5 mL of PBS solution. Ultrasonic water bath at 60° C. for 45 min to hydrate into a PBS solution of polypeptide thermosensitive nanoliposomes (P12-Lipo-ICG). The above prepared solution was placed in a 4°C refrigerator.

Example 5 Preparation of polypeptide drug-loaded thermosensitive nanoliposomes (P12-Lipo-ICG-DOX) and its application in PBS solution Solubility test

DSPE-PEG-2000 was prepared into a 5 mg/mL solution in chloroform, DSPE-PEG-MAL or DSPE-PEG-P12 (where the molecular weight of the PEG segment was 2000) was prepared into a 5 mg/mL solution in chloroform, and cholesterol was prepared in chloroform. 10 mg/mL solution, DPPC was prepared into 10 mg/mL solution with chloroform, and photosensitizer ICG was prepared as 10 mg/mL solution with methanol solution. Mix the above solutions according to the mass ratio of 2:1.5:4:15:5:2. After fully mixing, heat in a water bath at 45°C, rotate for 1 hour, and then place in a vacuum drying oven at 60°C overnight, and then add ammonium sulfate (250mM ) solution in an ultrasonic water bath at 60 °C for 45 min to hydrate into an ammonium sulfate solution of polypeptide thermosensitive nanoliposomes (P12-Lipo-ICG). Then add 1 mg/mL DOX in PBS solution and stir for 30 min at room temperature at 180 rpm to obtain polypeptide drug-loaded thermosensitive nanoliposomes (P12-Lipo-ICG-DOX). The prepared solution was placed in a 4°C refrigerator.

The carrier concentration of the peptide drug-loaded thermosensitive nanoliposomes (P12-Lipo-ICG-DOX) was 100 μM, and the peptide drug-loaded thermosensitive nanoliposomes were mixed by ultrasonic waves at 45°C for 30min in a water bath. Take 10 μL of the PBS solution sample of polypeptide drug-loaded thermosensitive nanoliposomes and drop it on the surface-activated carbon-coated copper mesh after glow discharge treatment. 2% tungsten phosphate staining solution (centrifuge at 4000 rpm for 5 min before use to remove the dyeing solution that was not completely dissolved) for 60 s, blot the dyeing solution with filter paper, and use transmission electron microscopy samples. The sample in Figure 3 is treated with 2% acetic acid Uranyl negative staining. Transmission electron microscopy reflects the morphology and particle size of the sample. As shown in Figure 3A, the peptide drug-loaded thermosensitive nanoliposome (P12-Lipo-ICG-DOX) exists in a spherical structure in PBS solution, and the particle size distribution More uniform, the particle size is 100-150nm. When the liposome (Lipo) was loaded with P12 polypeptide and chemotherapeutic molecules, the peptide drug-loaded thermosensitive nanoliposome (P12-Lipo-ICG-DOX) maintained a spherical structure, and the particle size did not change significantly.

The carrier concentration of the peptide drug-loaded thermosensitive nanoliposome (P12-Lipo-ICG-DOX) was 100 μM, and the ultrasonic water bath was performed at 40°C for 30 min. The PBS solution of the polypeptide drug-loaded thermosensitive nanoliposome (P12-Lipo-ICG-DOX) was shaken, and 1 mL was placed in a 1 cm × 1 cm plastic sample cell for dynamic light scattering test to measure the particle size distribution of the sample. . As shown in Figure 3B, the particle size of the polypeptide drug-loaded thermosensitive nanoliposomes (P12-Lipo-ICG-DOX) in PBS solution is 100-150 nm, which also indicates that the P12 polypeptide, the photosensitizer ICG, the chemotherapeutic drug DOX and DPPC The liposomes were physically assembled, and the liposomes were able to significantly increase the solubility of the P12 polypeptide in saline solution and successfully load chemotherapeutic drugs.

Example 6 Detection of affinity of FITC-P12 polypeptide or P12-Lipo nanoliposome with CXCR4 receptor in MCF-7 cells Measurement

The MCF-7 cell line was used as a model system for the study of breast cancer cell lines. In a Corning 24-well plate, 1 mL of DMEM medium (containing 10% fetal bovine serum FBS and 1% penicillin streptomycin) was used to culture 2×10 ⁵ cells per well. The cells were pre-incubated for 24 h in a %CO ₂ incubator to make the cells adherent.

The first experimental condition: 10 μL of glucose solutions of different concentrations of FITC-P12 polypeptide were added to each well of Corning 24-well plate, so that the final concentrations of FITC-P12 polypeptide were 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 20 μM , only 10 μL of PBS solution was added to the blank control group, and the 24-well cell culture plate was incubated in an incubator for 1 h.

The second experimental condition: add 10 μL of FITC-P12 polypeptide or P12-Lipo fluorescent nanoliposome (sample of Example 3, 6) PBS solution to Corning 24-well plate, so that the final concentration of FITC-P12 polypeptide is 2 μM and 5μM, P12-Lipo nanoliposomes and 10% P12 polypeptide nano-fluorescent liposomes with P12 polypeptide concentration as the final concentration of 2μM and 5μM, blank control only add 10μL PBS solution, incubate the 24-well cell culture plate in the incubator 1h.

In the above two cases, the flow cytometer was used to set the emission wavelength at 488 nm and the detection wavelength at 535 nm (1 channel). Place the negative control cell sample in the sample rack of the instrument and start the detection. According to the cell size, set the gate on the scatter plot of the forward angle signal (FSC) and the lateral angle signal (SSC), and set it before recording the detection results. Threshold so that the number of fluorescence intensities in the peak graph, the number of cells in the gate whose fluorescence intensity is above the threshold fluorescence intensity is less than 1%, and after the setting is complete, count 10,000 cells. Under the above gate setting and counting conditions, the blank control group and the experimental group samples were detected in turn, and the corresponding detection value, that is, the number percentage of fluorescence intensity higher than the threshold value, was recorded.

In the first case, as shown in Figure 4A, under the same incubation time (1 h), the binding rate of FITC-P12 polypeptide to the CXCR4 receptor of MCF-7 cells (ie, the percentage of positive cells) increased with FITC- increased with increasing concentrations of P12 polypeptides.

In the second case, as shown in Figure 5, the binding rate of FITC-P12 polypeptide alone to the CXCR4 receptor in MCF-7 cells (ie, the percentage of positive cells) increased as the concentration of FITC-P12 polypeptide increased. The binding rate of P12-Lipo fluorescent nanoliposome to CXCR4 receptor of MCF-7 cells (ie the percentage of positive cells) also increased with the increase of the concentration of FITC-P12 polypeptide. However, under the conditions of the same FITC-P12 polypeptide concentration (2 μM and 5 μM) and the same incubation time (1 h), the P12-Lipo fluorescent nanoliposomes (samples of Examples 3 and 6) were not associated with CXCR4 receptors in MCF-7 cells. The binding rate was significantly higher than the binding rate of FITC-P12 polypeptide alone to the CXCR4 receptor of MCF-7 cells.

Example 7 Affinity of FITC-P12 polypeptide or P12-Lipo fluorescent nanoliposome to CXCR4 receptor in 4T1 cells detect

The 4T1 cell line was used as a model system for the study of breast cancer cell lines. In a Corning 24-well plate, culture ² x 105 cells per well using 1 mL of RPM1640 medium (containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin), incubate the 24-well plate at 37°C, 5% _CO2 The cells were pre-cultured for 24 h in a conditioned incubator to make the cells adherent.

The first experimental condition: 10 μL of glucose solutions of different concentrations of FITC-P12 polypeptide were added to each well of Corning 24-well plate, so that the final concentrations of FITC-P12 polypeptide were 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 20 μM , only 10 μL of PBS solution was added to the blank control group, and the 24-well cell culture plate was incubated in an incubator for 1 h.

The second experimental condition: add 10 μL of FITC-P12 polypeptide or P12-Lipo fluorescent nanoliposome (sample of Example 3, 6) PBS solution to Corning 24-well plate, so that the final concentration of FITC-P12 polypeptide is 2 μM and 5μM, P12-Lipo nanoliposomes and 10% P12 polypeptide nano-fluorescent liposomes with P12 polypeptide concentration as the final concentration of 2μM and 5μM, blank control only add 10μL PBS solution, incubate the 24-well cell culture plate in the incubator 1h.

In the above two cases, use flow cytometer to record and detect samples of blank control group and experimental group, and record the corresponding detection value, that is, the number percentage of fluorescence intensity higher than the threshold value.

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

In the second case, as shown in Fig. 5, the binding rate of FITC-P12 polypeptide alone to the CXCR4 receptor of 4T1 cells (ie, the percentage of positive cells) increased with increasing concentration of FITC-P12 polypeptide. The binding rate of P12-Lipo fluorescent nanoliposome to CXCR4 receptor of 4T1 cells (ie the percentage of positive cells) also increased with the increase of the concentration of FITC-P12 polypeptide. However, under the conditions of the same FITC-P12 polypeptide concentration (2 μM and 5 μM) and the same incubation time (1 h), the binding rate of P12-Lipo fluorescent nanoliposomes (samples of Examples 3 and 6) to the CXCR4 receptor in 4T1 cells It was significantly higher than the binding rate of FITC-P12 polypeptide alone to the CXCR4 receptor of 4T1 cells. The above two experimental results all benefit from the fact that liposomeization can significantly increase the solubility of FITC-P12 polypeptide in PBS solution, and promote the binding effect of FITC-P12 polypeptide and CXCR4 receptor.

Example 8 Non-targeted thermosensitive liposomes (Lipo-ICG), targeted thermosensitive liposomes (P12-Lipo-ICG) and chemotherapy Inhibitory effect of drug combined with thermosensitive liposome (P12-Lipo-ICG-DOX) on breast cancer cell 4T1

Using 4T1 tumor cells as a model system for the study of breast cancer cell lines, 4T1 tumor cells were digested with 0.25% trypsin, and 5×10 ³ cells per well were inoculated in a 96-well culture plate, and cultured at 37°C under 5% CO ₂ for 24 h ; Then discard the culture medium, add immobilized non-targeted thermosensitive liposomes (Lipo-ICG), targeted thermosensitive liposomes (P12-Lipo-ICG) and chemotherapeutic drugs combined with thermosensitive liposomes (P12-Lipo -ICG-DOX) in the concentration of ICG (20 μg/mL, samples of Examples 1 and 4), different drug combination solutions were pre-incubated in an ultrasonic water bath at 55 °C for 30 min, then left standing at room temperature for 2 h, added to a 96-well plate, and after 12 h , irradiate each experimental sample with a laser with a wavelength of 808 nm for 3 min or 5 min, then continue to incubate for 2 h, discard the medium, add 100 μL/well of fresh 1640 medium, and then add Cell Counting Kit-8 (10 μL/well), at 37 ℃ , incubate for 2 hours under 5% CO ₂ environment; read the absorbance value at 490nm and calculate the cell growth inhibition rate. , as shown in Figure 6, chemotherapy drugs combined with thermosensitive liposomes (P12-Lipo-ICG-DOX) were significantly better than other groups. As shown in Figure 6, with the concentration of ICG fixed, the targeted thermosensitive liposome (P12-Lipo-ICG) had a higher growth inhibition rate on 4T1 cells than the non-targeted thermosensitive liposome (Lipo-ICG). As shown in Figure 6, as the irradiation time increased, more chemotherapeutic drugs were released, thereby enhancing the inhibitory effect on 4T1 cells.

Example 9 Anti-tumor polypeptide drug-loaded thermosensitive liposome (P12-Lipo-ICG-DOX) therapeutic drug inhibits 4T1 tumor effect of growth

The anti-tumor polypeptide drug-loaded thermosensitive liposomes (P12-Lipo-ICG-DOX) prepared in Examples 4 and 5 were used to determine the effect of anti-4T1 tumor growth, and free chemotherapeutic drug DOX was used as a control sample. BALB/c mice were inoculated with 4T1 breast cancer cells subcutaneously on the back, and when the tumor grew to a volume of 100 mm, saline (Saline) buffer, free chemotherapeutic drug doxorubicin (DOX), drug ^- loaded lipids were injected into the tail vein. Plasmid (Lipo-DOX), polypeptide drug-loaded liposome (P12-Lipo-DOX), polypeptide drug-loaded thermosensitive liposome (P12-Lipo-ICG-DOX), all materials were injected by tail vein every 3 days, The doses were all 3.0 mg/kg DOX, and the peptide drug-loaded thermosensitive liposome plus laser group (P12-Lipo-ICG-DOX+Laser) was irradiated with 808 nm laser 6h after the drug was injected to the tumor site of the mice. Group of 6 mice. After 14 days of treatment, the mice were sacrificed, the tumor tissue was removed, and the tumor weight was weighed. As shown in Figure 7, the peptide drug-loaded thermosensitive liposome plus laser group (P12-Lipo-ICG-DOX+Laser) had the most significant ability to inhibit tumor growth, which was significantly better than that of DOX group, Lipo-DOX group, and P12-Lipo group. -DOX group, P12-Lipo-ICG-DOX without laser group, which shows that the application of peptide drug-loaded thermosensitive liposome (P12-Lipo-ICG-DOX) design has the best therapeutic effect on tumors. The inhibitory effect of P12-Lipo-DOX group on tumor growth was also better than that of Lipo-DOX group, indicating that the targeted drug delivery efficiency of the peptide was higher than that of the non-targeted group. In addition, the rapid release of chemotherapeutic drugs caused by thermal triggering has a significantly better inhibitory effect on tumors than the non-response group (polypeptide drug-loaded thermosensitive liposome, P12-Lipo-ICG-DOX), which also reflects the rapid The anti-tumor nano-drug of the invention can well overcome the defects of low drug enrichment and insufficient bioavailability, and has a good application prospect. Due to space limitations, this article only cites some of the most convincing test cases.

Although this invention has been described to a certain extent, it will be apparent that suitable changes in various conditions may be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the embodiments described, but is to be included within the scope of the claims, which include equivalents for each of the elements described.

Claims (10)

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 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.

2. The polypeptide drug-loaded temperature-sensitive liposome of claim 1, 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 5-100 amino acids, more preferably 10-50 amino acids, and even more preferably 20-30 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.

3. The polypeptide drug-loaded temperature-sensitive liposome of claim 1 or 2, wherein the photosensitizer is selected from one or more of the following: ICG, porphyrin, Fe₃O₄Nanoparticles, 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.

4. The polypeptide drug-loaded temperature-sensitive liposome of claims 1 to 3, wherein the liposome component is selected from one or more of the following: DPPC, DSPE, PEG, MAL, cholesterol, DOPE, soy lecithin, lecithin.

5. The polypeptide drug-loaded temperature-sensitive liposome of claims 1 to 4, wherein the polypeptide is bound to the liposome by 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.

6. The preparation method of the polypeptide drug-loaded temperature-sensitive liposome according to any one of claims 1 to 5, wherein the method comprises 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, incubating and standing to obtain the polypeptide drug-loaded temperature-sensitive liposome.

7. The preparation method according to claim 6, wherein in the step (2), the rotary evaporation temperature is 30-60 ℃, preferably 40-50 ℃, 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 30-100 ℃, preferably 50-80 ℃, and most preferably 60 ℃.

8. The method according to claim 6 or 7, wherein the incubation time in step (3) is 10 to 60min, preferably 30 min.

9. A medicament comprising the polypeptide-carrying thermosensitive liposome according to any one of claims 1 to 5 or the polypeptide-carrying thermosensitive liposome prepared by the preparation method according to any one of claims 6 to 8;

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

more preferably, the lyoprotectant is mannitol.

10. The polypeptide drug-carrying temperature-sensitive liposome of any one of claims 1 to 5 or the polypeptide drug-carrying temperature-sensitive liposome prepared by the preparation method of any one of claims 6 to 8 or the application thereof in preparing drugs for treating cancers;

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.

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