CN113274353A - Drug-loaded nano micelle preparation, and preparation method and application thereof - Google Patents
Drug-loaded nano micelle preparation, and preparation method and application thereof Download PDFInfo
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- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/605—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the macromolecule containing phosphorus in the main chain, e.g. poly-phosphazene
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- A61P35/04—Antineoplastic agents specific for metastasis
Abstract
The invention provides a drug-loaded nano micelle preparation, and a preparation method and application thereof. Also provides a preparation method of the polypeptide drug and a nano micelle preparation thereof. Wherein, the drug-loaded nano micelle preparation is formed by assembling polyethylene glycol phospholipid and cancer therapeutic polypeptide. The polypeptide drug can be specifically bound to a chemokine receptor CXCR4, and can inhibit the migration of pancreatic cancer cells induced by a chemotactic axis CXCL12/CXCR 4. The polypeptide medicament and the micelle preparation thereof provide feasible methods and technologies for treating pancreatic cancer.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a drug-loaded nano micelle preparation, and a preparation method and application thereof.
Background
Pancreatic cancer is the digestive tract tumor with the highest malignancy, the fatality rate is extremely high, and the five-year survival rate is lower than 10%. Chemotherapy is the most effective treatment for pancreatic cancer treatment in addition to surgical treatment, however even the first-line chemotherapeutic drug gemcitabine is far less effective than clinically expected.
The chemokine CXCL12 and its receptor CXCR4 play an important role in the development of many carcinogenesis. CXCR4 is expressed at high levels in pancreatic, breast, non-small cell lung, and esophageal cancers, and is associated with lymph node metastasis and poor prognosis. Among them, CXCR4 is highly expressed in pancreatic cancer tissues, and is closely related to differentiation, metastasis, growth, and prognosis of pancreatic cancer. Immunohistochemical analysis is carried out on normal pancreatic tissues, pancreatic cancer tissues, paracancerous tissues and peripancreatic lymph node tissues, and the CXCR4 protein is found to be low expressed in the normal pancreatic tissues and high expressed in the pancreatic cancer tissues, the paracancerous tissues and the peripancreatic lymph node tissues, so that the CXCL12/CXCR4 chemotactic axis is possibly involved in the process of pancreatic cancer development, and meanwhile, the over-expression of CXCR4 at the lymph node position also indicates that the CXCR4 is possibly related to lymph node metastasis and distant metastasis of pancreatic cancer. Thus, CXCR4 may serve as a potential marker protein for the treatment of pancreatic cancer.
There are 4 main CXCR4 antagonists, small molecule antagonists, polypeptide antagonists, CXCR4 antibodies, and CXCL12 derivatives. Compared with the other three types, the polypeptide has the characteristics of small molecular weight, simple structure, easy synthesis, strong specificity, high activity, low toxicity and low immunogenicity, and has strong advantages in the aspect of disease treatment. Meanwhile, the polypeptide drug also has the problems of low solubility, poor stability, short circulation time and the like, and the drug effect and bioavailability of the polypeptide drug are hindered. With the development of nanotechnology, nano-drug carriers can overcome these problems to some extent. The nano-drug carrier is a drug delivery system with the size in the nano-scale range, can improve the bioavailability of the drug, for example, can control the release process of the drug in the body, promote the crossing of biological barriers, and have longer half-life period and different biological distribution characteristics. In addition, according to the high-permeability long-retention effect (EPR effect), the nano-carrier can be preferentially gathered in tumor tissues, so that the toxicity to normal cells is reduced, and the tumor can be better treated. Common nanocarriers for drug delivery are generally classified into two types, organic nanocarriers and inorganic nanocarriers. The organic nano-carrier mainly comprises liposome, dendritic macromolecule, polymer nano-particle, polymer micelle and the like, and the inorganic nano-carrier comprises metal nano-particle, carbon nano-tube, super-paramagnetic iron oxide nano-particle and the like.
The polymer nano-micelle is used as one of nano drug-carrying systems, can improve the solubility of E5 polypeptide in a salt solution, protects E5 polypeptide, and is a good choice for the drug-carrying systems. However, until now, no polypeptide drug or micelle preparation thereof has been found to be useful for the treatment of pancreatic cancer.
Disclosure of Invention
Before setting forth the context of the present invention, the terms used herein are defined as follows:
the term "E5 polypeptide" refers to: a polypeptide having the amino acid sequence of GGRSFFLLRRIQGCRFRNTVDD (N-terminal to C-terminal).
The term "CXCR 4" refers to: the 7-transmembrane G protein-coupled receptor, a receptor specific for chemokine stromal cell derived factor-1 (CXCL 12).
The term "FITC" refers to: fluorescein isothiocyanate.
The term "CXCL 12" refers to: a chemokine, stromal cell derived factor-1 (SDF-1), a class of small cytokines or signaling proteins secreted by cells.
The term "PANC-1" refers to: human pancreatic cancer cells; PANC-1 was derived from pancreatic cancer ductal cells with a doubling time of 52 hours. The term "Capan-1" means: the human pancreatic cancer tumor cell line, Capan-1.
The term "BxPC-3" means: human in situ pancreatic adenocarcinoma cell line BxPC-3.
The term "MiaPaCa-2" means: human pancreatic cancer cell line MiaPaCa-2.
The term "T3M 4" refers to: human pancreatic adenocarcinoma cell line T3M 4.
The term "Transwell experiment" means: cell migration assay.
Therefore, the invention aims to overcome the defects in the prior art and provide a drug-loaded nano-micelle preparation, a preparation method thereof and application thereof in pancreatic cancer treatment.
In order to achieve the above object, the first aspect of the present invention provides a drug-loaded nanomicelle preparation, which is formed by assembling pegylated phospholipid and cancer therapeutic polypeptide; wherein:
the polyethylene glycol phospholipid is formed by combining polyethylene glycol serving as a hydrophilic block with nitrogenous bases on phospholipid molecules serving as a hydrophobic block through covalent bonds; and is
The cancer therapeutic polypeptide is a polypeptide capable of binding to chemokine receptor CXCR4, thereby inhibiting pancreatic cancer cell migration;
preferably, the drug-loaded nanomicelle formulation is in the form of a solution or lyophilized.
The drug-loaded nanomicelle formulation according to the first aspect of the invention, wherein the cancer therapeutic polypeptide is selected from one or more of the following: polypeptides mainly comprising polar amino acids, polypeptides mainly comprising hydrophobic amino acids, and polypeptides having both polar amino acids and hydrophobic amino acids;
preferably, the cancer therapeutic polypeptide consists of 5-50 amino acids, more preferably 10-30 amino acids, even more preferably 15-25 amino acids.
According to the drug-loaded nano-micelle preparation of the first aspect of the invention, the cancer therapeutic polypeptide is E5 polypeptide or FITC-labeled E5 polypeptide;
preferably, the E5 polypeptide is probe-labeled or nanoparticle-entrapped;
more preferably, wherein:
the probe is selected from one or more of the following: fluorescent molecules, quantum dots, radioactive elements, horseradish peroxidase and alkaline phosphatase;
the nanoparticles are selected from one or more of the following: liposome, polymer micelle, dendritic macromolecule and gold nanoparticle.
According to the drug-loaded nano-micelle preparation of the first aspect of the invention, the amino acid sequence of the E5 polypeptide is as follows: GGRSFFLLRRIQGCRFRNTVDD, the amino acid sequence of the fluorescent probe-labeled E5 polypeptide is: FITC-GGRSFFLLRRIQGCRFRNTVDD;
preferably, the drug-loaded nano-micelle preparation is a nano-micelle embedded with E5 polypeptide, which is formed by assembling polyethylene glycol phospholipid molecules and E5 polypeptide through electrostatic interaction.
According to the drug-carrying nano-micelle preparation of the first aspect of the invention, the molecular weight of the polyethylene glycol hydrophilic block in the polyethylene glycol phospholipid molecule is 500-10000, preferably 1000-5000, and further preferably 2000-4000;
the particle size of the drug-loaded nano micelle is 10-100nm, preferably 10-50nm, and more preferably 10-30 nm;
the molar ratio of the pegylated phospholipid to the cancer therapeutic polypeptide is 2-10: 1, preferably 4 to 5: 1.
the second aspect of the present invention provides a method for preparing the drug-loaded nanomicelle preparation of the first aspect, wherein the method comprises the following steps:
(1) respectively preparing a polyethylene glycol phospholipid molecular solution and an E5 polypeptide molecular solution, uniformly mixing, incubating in a water bath, and standing to obtain a drug-loaded nano micelle solution of the polypeptide;
(2) preferably, the drug-loaded nano-micelle solution obtained after the standing in the step (1) is sterilized and filtered.
The production method according to the second aspect of the present invention, wherein, in step (1):
the solvent is one or more of the following: phosphate buffer, ultrapure water, physiological saline, preferably phosphate buffer and ultrapure water, most preferably ultrapure water;
the concentration of the PEGylated phospholipid solution is 1-50mg/mL, preferably 1-30mg/mL, and more preferably 2-20 mg/mL;
the concentration of the E5 polypeptide solution is 0.1-15mg/mL, preferably 0.1-10mg/mL, and more preferably 0.5-5 mg/mL;
the water bath incubation temperature is 30-65 ℃, preferably 40-60 ℃, and further preferably 40-55 ℃;
the water bath incubation time is 20-60min, preferably 20-50min, and more preferably 25-40 min;
the standing temperature is 4-30 ℃, preferably 10-30 ℃, and more preferably 20-25 ℃;
the standing time is 2 to 48 hours, preferably 2 to 36 hours, and further preferably 4 to 24 hours;
the molar ratio of the pegylated phospholipid to the E5 polypeptide is: 40-50: 1-20, preferably 2-10: 1, more preferably 4 to 5: 1. .
The production method according to the second aspect of the present invention, wherein, in step (2): the pore size of the filtration membrane is 0.1 to 0.8 μm, preferably 0.1 to 0.6. mu.m, more preferably 0.1 to 0.4. mu.m, and most preferably 0.22. mu.m.
The third aspect of the present invention provides the use of the drug-loaded nanomicelle formulation of the first aspect or the drug-loaded nanomicelle formulation prepared according to the method of the second aspect for the preparation of a medicament for the treatment of cancer:
preferably, the cancer is pancreatic cancer;
more preferably, the pancreatic cancer characterizes pancreatic cancer cells or pancreatic cancer tissue having expressed or overexpressed CXCR 4.
According to the use of the third aspect of the invention, the pancreatic cancer cell species is selected from one or more of: PANC-1, Capan-1, BxPC-3, MiaPaCa-2, T3M4, preferably PANC-1 or MiaPaCa-2.
According to a particularly preferred embodiment of the invention, the polypeptide E5 and the drug-loaded nano-micelle thereof, which can be used for targeting a CXCR4 target, are provided, and can inhibit the migration of pancreatic cancer cells by combining with CXCR4, so that the purpose of treating pancreatic cancer is achieved.
The polypeptide medicament and the micelle preparation thereof can have the following beneficial effects:
1. the drug-loaded nano-micelle improves the solubility and the biological stability of the E5 polypeptide in a physiological solution, and improves the binding capacity of the E5 polypeptide and a CXCR4 target, thereby improving the inhibiting capacity on pancreatic cancer cell migration.
2. The polypeptide E5 has higher affinity with pancreatic cancer cells expressing CXCR4, which indicates that the polypeptide E5 can be used as a CXCR4 antagonist for treating pancreatic cancer (figure 2). The E5 polypeptide has good safety and no obvious cytotoxicity in the concentration range of 60 micromolar. (FIG. 3)
3. The polypeptide drug and the micelle preparation thereof are shown by a transmission electron microscope, the micelle has a spherical structure and uniform particle size, and the particle size of the prepared micelle is distributed between 10 and 30nm (figure 4).
4. The polypeptide E5 drug-loaded nano-micelle can inhibit cell migration of pancreatic cancer cell lines PANC-1 and MiaPaCa-2, and can obviously inhibit migration of pancreatic cancer cells when the concentration of the pegylated phospholipid molecules is 200 micromoles per liter (figure 6).
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the measurement of the expression level of CXCR4 of the surface chemokine receptor of three different cells (MS-5, PANC-1 and MiaPaCa-2) in example 2.
FIG. 2 shows the binding experiments of the polypeptide E5 with cells expressing different amounts of CXCR4 (MS-5, PANC-1 and MiaPaCa-2) in example 3.
FIG. 3 shows the cytotoxicity (MS-5, PANC-1 and MiaPaCa-2) experiments of the polypeptide E5 in test example 1. Specifically, FIG. 3A, FIG. 3B and FIG. 3C show the toxicity assessment of the E5 polypeptide on MS-5 cells, the E5 polypeptide on PANC-1 cells and the E5 polypeptide on MiaPaCa-2 cells, respectively.
FIG. 4 shows the morphology analysis of the E5 polypeptide drug-loaded nano-micelle (M-E5) in example 4.
FIG. 5 shows the inhibitory effect of the E5 polypeptide on the migration of different pancreatic cancer cells (PANC-1 and MiaPaCa-2) in Experimental example 2. Specifically, FIG. 5A and FIG. 5B show the inhibitory effect of the E5 polypeptide on migration of pancreatic cancer cells MiaPaCa-2 and the inhibitory effect of the E5 polypeptide on migration of pancreatic cancer cells PANC-1, respectively.
FIG. 6 shows the inhibitory effect of E5 polypeptide drug-loaded nanomicelles (M-E5) on migration of different pancreatic cancer cells (PANC-1 and MiaPaCa-2) in Experimental example 3. Specifically, FIG. 6A and FIG. 6B show the inhibition effect of M-E5 on migration of pancreatic cancer cells MiaPaCa-2 and M-E5 on migration of different pancreatic cancer cells PANC-1, respectively.
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 purpose of carrying out the 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 materials, reagents and instruments used in the following examples are as follows:
materials:
human pancreatic cancer cell line PANC-1 was purchased from: cell resource center of the institute of basic medicine of the Chinese academy of medical sciences.
Human pancreatic cancer cell line MiaPaCa-2 was purchased from: china Center for Type Culture Collection (CCTCC) cell bank.
Mouse derived bone marrow stromal cells were purchased from: qingqi (shanghai) biotechnology development limited.
Reagent:
the E5 polypeptide was purchased from national drug industry Co., Ltd, Anhui province, and had a purity of 98%.
Pegylated phospholipids were purchased from Avanti Polar Lipids (Alabama) Inc., USA.
Both the E5 polypeptide and the PEGylated phospholipid molecules used were prepared as a mother liquor with ultrapure water to the appropriate concentration prior to the experiment.
A sterile ultrapure aqueous solution having a water quality parameter of 18.2M Ω. cm @25 ℃.
PBS buffer, DMEM high-sugar medium, 1640 medium, pancreatin and fetal bovine serum were purchased from Gibco, USA.
The CCK-8 kit was purchased from DOJINDO, Japan.
The crystal violet solution was purchased from Solebao.
The instrument comprises the following steps:
transwell cells were purchased from R & D, usa.
Continuous spectrum multifunctional microplate readers are available from Molecular Devices, USA, under the model of SpectraMax i 3.
Flow cytometry was purchased from BD, usa under model C6.
Transmission Electron Microscopy (TEM) was purchased from Hitachi, Japan, model HT 7700.
Example 1:
this example illustrates the preparation of the polypeptide drug of the present invention and its micelle preparation.
(1) The preparation method of the PEGylated phospholipid molecule solution comprises the following steps: dissolving the polyethylene glycol phospholipid powder in a proper amount of ultrapure water, and completely dissolving to obtain a 1mM polyethylene glycol phospholipid molecular solution.
(2) E5 polypeptide molecule solution: the powder of E5 polypeptide was dissolved in an appropriate amount of ultrapure water and sonicated for 5 minutes to give a 500. mu.M solution of E5 polypeptide molecules.
(3) And (3) uniformly mixing the two molecular solutions in the step (1) and the step (2), incubating in a water bath, and standing to obtain the drug-loaded nano-micelle solution of the polypeptide.
(4) And (4) standing the drug-loaded nano micelle solution obtained in the step (3), sterilizing, and filtering with a 0.22-micron filter membrane.
Example 2: determination of expression level of CXCR4 on cell surface
When the expression level of CXCR4 on the cell surface is determined by flow cytometry, the cells are divided into 1.5mL centrifuge tubes, about 50 ten thousand cells are put in each tube, an experimental group incubates antibodies (primary antibody and mouse anti-human) of a chemokine receptor CXCR4, the cells are blown uniformly and incubated at room temperature for 1 hour, then the cell suspension is centrifuged, the supernatant is discarded, the cells are resuspended by PBS and centrifuged again, the operation is repeated for three times, the non-specifically bound antibodies are washed off, then 200 microliter of prepared goat anti-mouse secondary antibody solution with AF488 markers is added, the same secondary antibody solution is also added into a control group, the cells are blown uniformly and incubated together at room temperature for 30min, then the cell suspension is centrifuged, the supernatant is discarded, the cell suspension is resuspended by PBS and centrifuged again, the operation is repeated for three times, the non-specifically bound antibodies are washed off, and finally the cells are resuspended by 200 microliter of PBS buffer. As shown in figure 1, a CXCR4 antibody is utilized to screen out a cell strain MS-5 with low expression of CXCR4, two cell strains PANC-1 and MiaPaCa-2 with high expression of CXCR 4. The expression levels of CXCR4 of MS-5, PANC-1 and MiaPaCa-2 are respectively as follows: 4.0%, 51.1% and 39.7%. MS-5 cells were CXCR4 negative cells, PANC-1 and MiaPaCa-2 were CXCR4 positive cells.
Example 3: affinity assay for E5 polypeptide and CXCR4
Flow cytometry was used to examine the binding capacity of the E5 polypeptide to cell lines expressing different chemokine receptors CXCR 4. The cells were dispensed into 1.5mL centrifuge tubes, and about 50 ten thousand cells per tube were centrifuged to discard the supernatant. Preparing FITC-E5 polypeptide solutions with the concentrations of 2, 4, 6, 8 and 10 mu M respectively, adding 50 mu L of the prepared FITC-E5 polypeptide solutions into a centrifuge tube in which cells are collected respectively, adding 200 mu L of PBS buffer solution into a control group without adding the polypeptide, gently blowing and beating the cells by using a pipette until the cells are uniformly suspended, and incubating the cells for 2 hours at 37 ℃. Adding a proper amount of PBS buffer solution into the incubated cells for resuspension, centrifuging, discarding the supernatant, resuspending the cells by using a proper amount of PBS buffer solution, centrifuging again, repeating for 3 times, washing off non-specifically bound polypeptide, and finally resuspending by using 200 mu L of PBS buffer solution to obtain a test sample. As shown in figure 2, the binding capacity of the cell lines PANC-1 and MiaPaCa-2 which over-express CXCR4 to the E5 polypeptide is strong, and the binding capacity of the CXCR4 negative cell line MS-5 to the E5 polypeptide is basically negligible, which indicates that the E5 polypeptide has higher binding capacity to the CXCR 4.
Example 4: morphology analysis of E5 polypeptide drug-loaded nano-micelle
The morphology of the E5 polypeptide drug-loaded nano-micelle (M-E5) is characterized by using a transmission electron microscope. Diluting the micelle solution to 20 mu M, dropping 10 mu L of the liquid on a copper net, standing overnight, dropping 2 wt% uranyl acetate solution, dyeing for 1min, sucking the liquid by using filter paper, naturally drying, and observing and photographing under a transmission electron microscope. As shown in FIG. 4, the size of the drug-loaded nano-micelle is about 20 nm.
Test example 1: cytotoxicity assay for E5 Polypeptides
The CCK-8 method was used to examine the effect of E5 polypeptide on cell viability of different cell lines. Cells, PANC-1 and MS-5 cells 4X 103 per well and MiaPaCa-2 cells 6X 103 per well were cultured in 96-well plates (Corning) using the corresponding media, and the 96-well plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 h. E5 polypeptide solutions with different concentrations are prepared by corresponding culture media, the cells in a 96-well plate are changed, after 48 hours of culture, CCK-8 solution with the volume of 10 mu L is added into each well, the incubation is carried out for 1 hour at 37 ℃, the absorbance (OD) value at the wavelength of 450nm is measured by a continuous spectrum multifunctional microplate reader (SpectraMax i3, USA), and the survival rate of the cells is calculated. Three parallel multiple-hole hairs are arranged on each sample, and the experimental result is the average value of three experiments. As shown in FIG. 3, the E5 polypeptide was itself safer and showed no significant cytotoxicity at 60. mu. molar concentration.
Test example 2: evaluation of inhibition ability of free E5 polypeptide on pancreatic cancer cell migration
The inhibition ability of free E5 polypeptide on pancreatic cancer cell migration was explored using a Transwell assay. Complete medium without or with CXCL12 protein was added to 24-well plates. Serum-free media containing different concentrations of the E5 polypeptide were prepared. The cells were collected in a 1.5mL centrifuge tube, the prepared medium containing E5 polypeptide was added, incubated at 37 ℃ for 1h, the cells were resuspended again, 100. mu.L of cell suspension was added to a Transwell chamber having a pore size of 8 μm, suspended in the corresponding lower chamber, and incubated in an incubator at 37 ℃ under 5% CO2 for 24 h.
Placing the cell into 0.1% crystal violet solution, dyeing for 10min, washing with clear water, wiping the cells on the inner side of the cell filter membrane with a cotton swab, drying the cell in the air, and taking a picture under a microscope. After photographing, adding glacial acetic acid for decoloring for 10min, sucking 200 mu L of each sample into a 96-well plate, and measuring the absorbance (OD) value at the wavelength of 530nm by using a continuous spectrum multifunctional microplate reader so as to calculate the cell migration rate. As shown in FIG. 5, after adding E5 polypeptide at 1. mu.M, 10. mu.M and 50. mu.M, it can be seen that the amount of migration was significantly reduced at a concentration of 50. mu.M of E5 polypeptide. Indicating that the E5 polypeptide can inhibit the migration of pancreatic cancer cells.
Test example 3: evaluation of inhibition capability of E5 polypeptide drug-loaded nano-micelle (M-E5) on pancreatic cancer cell migration
The ability of M-E5 to inhibit pancreatic cancer cell migration was explored using the Transwell assay. Complete medium without or with CXCL12 protein was added to 24-well plates. Serum-free media containing different concentrations of M-E5 were prepared. The cells were collected in a 1.5mL centrifuge tube, the prepared medium containing M-E5 was added, incubated at 37 ℃ for 1h, the cells were resuspended again, 100. mu.L of the cell suspension was added to a Transwell chamber having a pore size of 8 μ M, suspended in the corresponding lower chamber, and incubated in an incubator at 37 ℃ under 5% CO2 for 24 h.
Placing the cell into 0.1% crystal violet solution, dyeing for 10min, washing with clear water, wiping the cells on the inner side of the cell filter membrane with a cotton swab, drying the cell in the air, and taking a picture under a microscope. After photographing, adding glacial acetic acid for decoloring for 10min, sucking 200 mu L of each sample into a 96-well plate, and measuring the absorbance (OD) value at the wavelength of 530nm by using a continuous spectrum multifunctional microplate reader so as to calculate the cell migration rate. As shown in FIG. 6, the amount of cell migration was significantly reduced when the concentration of M-E5 was 200. mu.M, i.e., the concentration of E5 polypeptide was 50. mu.M, based on the concentration of PEG-PE molecules in the system. The E5 polypeptide drug-loaded nano-micelle can inhibit the migration of pancreatic cancer cells.
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 limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (10)
1. The drug-carrying nano micelle preparation is characterized by being formed by assembling polyethylene glycol phospholipid and cancer therapeutic polypeptide; wherein:
the polyethylene glycol phospholipid is formed by combining polyethylene glycol serving as a hydrophilic block with nitrogenous bases on phospholipid molecules serving as a hydrophobic block through covalent bonds; and is
The cancer therapeutic polypeptide is a polypeptide capable of binding to chemokine receptor CXCR4, thereby inhibiting pancreatic cancer cell migration;
preferably, the drug-loaded nanomicelle formulation is in the form of a solution or lyophilized.
2. The drug-loaded nanomicelle formulation of claim 1, wherein the cancer therapeutic polypeptide is selected from one or more of the group consisting of: polypeptides mainly comprising polar amino acids, polypeptides mainly comprising hydrophobic amino acids, and polypeptides having both polar amino acids and hydrophobic amino acids;
preferably, the cancer therapeutic polypeptide consists of 5-50 amino acids, more preferably 10-30 amino acids, even more preferably 15-25 amino acids.
3. The drug-loaded nanomicelle formulation of claim 1 or 2, wherein the cancer therapeutic polypeptide is E5 polypeptide or FITC-labeled E5 polypeptide;
preferably, the E5 polypeptide is probe-labeled or nanoparticle-entrapped;
more preferably, wherein:
the probe is selected from one or more of the following: fluorescent molecules, quantum dots, radioactive elements, horseradish peroxidase and alkaline phosphatase;
the nanoparticles are selected from one or more of the following: liposome, polymer micelle, dendritic macromolecule and gold nanoparticle.
4. The drug-loaded nanomicelle formulation of claim 3, wherein:
the amino acid sequence of the E5 polypeptide is: GGRSFFLLRRIQGCRFRNTVDD, the amino acid sequence of the fluorescent probe-labeled E5 polypeptide is: FITC-GGRSFFLLRRIQGCRFRNTVDD;
preferably, the drug-loaded nano-micelle preparation is a nano-micelle embedded with E5 polypeptide, which is formed by assembling polyethylene glycol phospholipid molecules and E5 polypeptide through electrostatic interaction.
5. The drug-loaded nanomicelle formulation according to any one of claims 1 to 4, characterized in that:
the molecular weight of the polyethylene glycol hydrophilic block in the polyethylene glycol phospholipid molecule is 500-10000, preferably 1000-5000, and more preferably 2000-4000;
the particle size of the drug-loaded nano micelle is 10-100nm, preferably 10-50nm, and more preferably 10-30 nm;
the molar ratio of the pegylated phospholipid to the cancer therapeutic polypeptide is 2-10: 1, preferably 4 to 5: 1.
6. the method for preparing a drug-loaded nanomicelle formulation according to any one of claims 1 to 5, characterized in that the method comprises the following steps:
(1) respectively preparing a polyethylene glycol phospholipid molecular solution and an E5 polypeptide molecular solution, uniformly mixing, incubating in a water bath, and standing to obtain a drug-loaded nano micelle solution of the polypeptide;
(2) preferably, the drug-loaded nano-micelle solution obtained after the standing in the step (1) is sterilized and filtered.
7. The method for preparing a drug-loaded nanomicelle formulation according to claim 6, wherein in step (1):
the solvent is one or more of the following: phosphate buffer, ultrapure water, physiological saline, preferably phosphate buffer and ultrapure water, most preferably ultrapure water;
the concentration of the PEGylated phospholipid solution is 1-50mg/mL, preferably 1-30mg/mL, and more preferably 2-20 mg/mL;
the concentration of the E5 polypeptide solution is 0.1-15mg/mL, preferably 0.1-10mg/mL, and more preferably 0.5-5 mg/mL;
the water bath incubation temperature is 30-65 ℃, preferably 40-60 ℃, and further preferably 40-55 ℃;
the water bath incubation time is 20-60min, preferably 20-50min, and more preferably 25-40 min;
the standing temperature is 4-30 ℃, preferably 10-30 ℃, and more preferably 20-25 ℃;
the standing time is 2 to 48 hours, preferably 2 to 36 hours, and further preferably 4 to 24 hours;
the molar ratio of the pegylated phospholipid to the E5 polypeptide is: 40-50: 1-20, preferably 2-10: 1, more preferably 4 to 5: 1.
8. the method for preparing a drug-loaded nanomicelle formulation according to claim 6, wherein in step (2): the pore size of the filtration membrane is 0.1 to 0.8 μm, preferably 0.1 to 0.6. mu.m, more preferably 0.1 to 0.4. mu.m, and most preferably 0.22. mu.m.
9. Use of a polypeptide agent of any one of claims 1 to 5 and a micelle formulation thereof or a polypeptide agent prepared by a method of any one of claims 6 to 8 and a micelle formulation thereof for the manufacture of a medicament for the treatment of cancer;
preferably, the cancer is pancreatic cancer;
more preferably, the pancreatic cancer characterizes pancreatic cancer cells or pancreatic cancer tissue having expressed or overexpressed CXCR 4.
10. The use of claim 9, wherein the pancreatic cancer cells are of a species selected from one or more of: PANC-1, Capan-1, BxPC-3, MiaPaCa-2, T3M4, preferably PANC-1 or MiaPaCa-2.
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