Tumor cell membrane modified polypeptide nano-carrier as well as preparation method and application thereof

Abstract

The invention discloses a tumor cell membrane modified polypeptide nano-carrier as well as a preparation method and application thereof. The preparation method of the carrier comprises the step of assembling a tumor cell membrane and polypeptide with opposite charges through an electrostatic adsorption effect to form the tumor cell membrane modified polypeptide nano-carrier. According to the tumor cell membrane modified polypeptide nano-carrier prepared by the invention, the membrane penetrating efficiency in tumor cells and immune cells can be obviously improved, and nucleic acid molecules and drug molecules can be effectively delivered to the tumor cells and the immune cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a polypeptide nanocarrier modified with tumor cell membranes, a preparation method thereof, and uses thereof. Background Art

[0002] Currently, in the field of anti-tumor drugs, using carriers to load anti-tumor drugs to prepare drug delivery systems (DDSs) is a topic that has been studied extensively. DDSs can improve the bioavailability by increasing the solubility of hydrophobic drugs. In addition, some nanomaterials have environmental response characteristics (such as enzyme sensitivity, thermal sensitivity, redox sensitivity, and pH sensitivity), and the microenvironment of tumor tissues can respond to these characteristics, thereby increasing the accumulation of drugs at the tumor site and improving the therapeutic effect.

[0003] Currently, a large number of researchers have used polymers (peptides and polysaccharides), inorganic substances, polymer-lipid complexes, liposomes, etc. as nanocarriers to construct more efficient drug delivery systems. Among them, polypeptides have shown great application potential in tumor treatment due to their advantages such as small molecular weight, good biocompatibility, and easy modification. However, it has been found in the research that traditional polypeptide nanocarriers still have problems such as poor biocompatibility and insufficient targeting, which limit their further clinical applications. Summary of the Invention

[0004] The main object of the present invention is to provide a polypeptide nanocarrier modified with tumor cell membranes, a preparation method thereof, and uses thereof, which have good biocompatibility, targeting, and membrane penetration efficiency.

[0005] To achieve the above object, the present invention provides a preparation method of a polypeptide nanocarrier modified with tumor cell membranes, which is characterized in that the tumor cell membranes and polypeptides with opposite charges are assembled through electrostatic adsorption to form the polypeptide nanocarrier modified with tumor cell membranes.

[0006] Further, the polypeptide is positively charged, and the tumor cell membrane is negatively charged. The positively charged polypeptide can load negatively charged nucleic acid molecules to achieve the delivery of nucleic acid molecules.

[0007] Further, the tumor cell membrane is the cell membrane of Hela cells.

[0008] Further, the extraction method of the cell membrane of Hela cells is as follows: Hela cells are added to a cell membrane extraction buffer, and the cell membrane extraction buffer includes 30 mM Tris-HCl with pH = 7.0, 225 mM mannitol, and 75 mM sucrose. After ultrasonic disruption, the supernatant is taken by centrifugation, and then the supernatant is centrifuged to obtain the cell membrane of Hela cells.

[0009] Furthermore, the structural formula of the polypeptide is as follows:

[0010]

[0011] Two sulfonium salt centers are introduced into the polypeptide, which can be reduced by intracellular GSH, thereby releasing the internal nucleic acid drug, and can greatly improve the delivery efficiency of the carrier for nucleic acid drugs.

[0012] Furthermore, the assembly method of the tumor cell membrane and the polypeptide is as follows: Dilute the tumor cell membrane and the polypeptide in ddH 2 O respectively, then mix them, and then let them stand for treatment to obtain the polypeptide nanocarrier modified with the tumor cell membrane.

[0013] The present invention also provides a polypeptide nanocarrier modified with a tumor cell membrane, which is prepared by the above method.

[0014] The present invention also provides the use of the polypeptide nanocarrier modified with the tumor cell membrane in the preparation of a drug delivery system.

[0015] The present invention also provides the use of the polypeptide nanocarrier modified with the tumor cell membrane in the preparation of a tumor cell and / or immune cell transfection agent.

[0016] The present invention also provides the use of the polypeptide nanocarrier modified with the tumor cell membrane in the preparation of an anti-tumor and / or immunotherapy drug.

[0017] The beneficial effects of the present invention are reflected in:

[0018] The present invention combines the biological function of the tumor cell membrane with the designability of the polypeptide carrier to prepare a polypeptide nanocarrier modified with the tumor cell membrane. The tumor cell membrane can effectively coat drug molecules, effectively enhancing the tumor targeting effect of the carrier. As a natural barrier between cells and the external environment, the cell membrane has good biocompatibility and complex biological functions. Cell membrane surface markers can avoid the recognition and clearance of the immune system, effectively reducing the immunogenicity of the polypeptide nanocarrier and prolonging the circulation time of the polypeptide carrier in vivo, improving biocompatibility; in addition, specific receptors or ligands on the tumor cell membrane can achieve targeting to specific cells or tissues, improving the tumor-targeted delivery efficiency of drugs.

[0019] The polypeptide nanocarrier modified with the tumor cell membrane prepared by the present invention can encapsulate various tumor drugs from small molecule compounds to biological macromolecules, improve the loading rate of tumor drugs, and reduce their toxic and side effects in the body, providing a choice for personalized tumor treatment strategies. In the future, it is expected to provide new ideas in the development of drug delivery systems with low toxicity, high delivery efficiency, high release efficiency, and high targeting.

[0020] The present invention can prepare relatively stable and uniform nanoparticles by changing the ratio of tumor cell membrane to polypeptide. Compared with the naked cell membrane, the polypeptide nanocarrier modified with the tumor cell membrane prepared by the present invention can significantly improve its transmembrane efficiency in tumor cells and immune cells, and can effectively deliver nucleic acid molecules and drug molecules into tumor cells and immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a fluorescence image of the transmembrane effect of tumor cell membrane and polypeptide nanocarrier modified with tumor cell membrane in BMDCs and Hela cells.

[0022] Figure 2 In Figure (a), it is a particle size analysis diagram of polypeptide nanocarriers modified with tumor cell membranes with different ratios; in Figure (b), it is a Zeta potential analysis diagram of polypeptide nanocarriers modified with tumor cell membranes with different ratios.

[0023] Figure 3 It is a fluorescence image of the transmembrane effect of polypeptide nanocarriers modified with tumor cell membranes with different ratios in Hela cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] In order to make those skilled in the art more clearly understand the technical solutions described in the present invention, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0025] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods; unless otherwise specified, the methods used in the examples of the present invention are all methods mastered by those skilled in the art. Among them, the polypeptide used has the structural formula That is, the polypeptide disclosed in Chinese Patent ZL201810078986.2.

[0026] Example 1

[0027] Preparation and Characterization of Polypeptide Nanocarrier Modified with Tumor Cell Membrane

[0028] (1) Preparation of Tumor Cell Membrane

[0029] Hela cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody.

[0030] Hela cells were collected and resuspended in cell membrane extraction buffer (including 30 mM Tris-HCl with pH = 7.0, 225 mM mannitol, and 75 mM sucrose). After that, they were placed in an ultrasonic cell disruptor for ultrasonic disruption (at 4 °C, 1 s on, 4 s off, power 270 W). Then, they were centrifuged at 4 °C and 10,000 rcf for 25 min. The supernatant was taken and ultracentrifuged at 4 °C and 100,000 rcf for 35 min. The supernatant was discarded. The residue was resuspended in 0.2 mM EDTA and ultracentrifuged again at 4 °C and 100,000 rcf for 35 min. Then, the supernatant was discarded. The residue was resuspended in 0.2 mM EDTA to obtain a resuspension containing tumor cell membranes (i.e., the cell membranes of Hela cells). The concentration of cell membrane proteins in the resuspension was detected by the BCA method and stored at -80 °C for later use;

[0031] (2) Assembly and characterization of the carrier

[0032] According to the measured concentration of cell membrane proteins, a resuspension containing 10 μg of tumor cell membranes was taken, and 10 μg of polypeptide was also taken. They were respectively diluted in 100 μL of ddH 2 O. Then, the prepared cell membrane dilution was added to the polypeptide dilution, and they were pipetted and mixed well and left standing for 30 min to obtain a polypeptide nanocarrier modified with tumor cell membranes.

[0033] The particle size and Zeta potential of the tumor cell membranes, polypeptides, and the prepared polypeptide nanocarriers modified with tumor cell membranes used in this example were detected by a dynamic light scattering instrument (DLS). The results are shown in Tables 1 and 2:

[0034] Table 1 Particle size data

[0035]

[0036] Table 2 Potential data

[0037]

[0038] The results showed that after the polypeptide was assembled with the tumor cell membranes, the particle size increased significantly compared with the cell membrane group, and the potential of the nanoparticles formed after the polypeptide with a positive surface potential was combined with the cell membrane with a negative surface potential showed a nearly electro-neutral positive charge, forming relatively stable cell membrane-polypeptide nanoparticles.

[0039] Experimental Example 1

[0040] Verification of the transmembrane efficiency of the polypeptide nanocarrier modified with tumor cell membranes

[0041] According to the method of Example 1, before the assembly of the tumor cell membrane and the polypeptide, the tumor cell membrane was first labeled with DiI, that is, the prepared tumor cell membrane and the cell membrane fluorescent probe DiI were incubated at 37 °C for 20 min, and finally a polypeptide nanocarrier modified with DiI-labeled tumor cell membrane was prepared;

[0042] The DiI-labeled tumor cell membrane and the polypeptide nanocarrier modified with the DiI-labeled tumor cell membrane were respectively added to BMDCs and Hela cells, incubated for 4 h, and cell slides were collected. In addition, the cell nuclei were stained with DAPI dye, and the penetration of the tumor cell membrane and the polypeptide nanocarrier modified with the tumor cell membrane was observed by a laser confocal microscope (the Merge figure is the combination of the DiI staining and DAPI staining figures). The results are as Figure 1 shown:

[0043] The results showed that no obvious red fluorescence was observed on BMDCs and HeLa cells after incubation with the tumor cell membrane, while obvious enhanced red fluorescence was presented on BMDCs and HeLa cells after incubation with the polypeptide nanocarrier modified with the tumor cell membrane, indicating that the prepared carrier in the present invention has significantly enhanced membrane penetration in tumor cells and immune cells.

[0044] Example 2

[0045] Ratio screening of tumor cell membrane and polypeptide

[0046] The polypeptide nanocarrier modified with the tumor cell membrane was prepared according to the method of Example 1, except that the dosage ratio of the tumor cell membrane and the polypeptide was adjusted. One group used 5 μg of tumor cell membrane and 10 μg of polypeptide, and the other group used 20 μg of tumor cell membrane and 10 μg of polypeptide. Then, the particle size, polymer dispersion index (PDI) and Zeta potential of the finally prepared polypeptide nanocarrier modified with the tumor cell membrane were detected by a dynamic light scattering instrument (DLS). The results of different mass ratios of the tumor cell membrane and the polypeptide are shown in Tables 3, 4 and 5, and an analysis diagram is made as Figure 2 shown ( Figure 2 the part of 1:0 in a refers to the test data of the pure tumor cell membrane):

[0047] Table 3 Particle size data

[0048]

[0049] Table 4 PDI data

[0050]

[0051] Table 5 Potential data

[0052]

[0053] The results showed that the uniformity of the nanoparticles assembled from tumor cell membranes and polypeptides increased significantly. Moreover, the larger the proportion of cell membranes in the carrier, the more uniform the particle size distribution of the carrier and the closer the Zeta potential was to electrical neutrality. When the cell membrane and polypeptide were assembled at a mass ratio of 2:1, more stable and uniform nanoparticles could be obtained.

[0054] Experimental Example 2

[0055] Verification of the transmembrane efficiency of polypeptide nanocarriers modified with tumor cell membranes at different ratios

[0056] According to the methods of Examples 1 and 2, before the assembly of the tumor cell membrane and polypeptide, the tumor cell membrane was first labeled with DiI, that is, the prepared tumor cell membrane was incubated with the cell membrane fluorescent probe DiI at 37 °C for 20 min, and polypeptide nanocarriers modified with DiI-labeled tumor cell membranes with mass ratios of cell membrane to polypeptide = 1:1 and 2:1 were finally prepared respectively;

[0057] The polypeptide nanocarriers modified with DiI-labeled tumor cell membranes at the above different ratios and the DiI-labeled tumor cell membranes in the corresponding preparation process were incubated with Hela cells for 4 h, and cell slides were collected. In addition, the cell nuclei were stained with DAPI dye, and their transmembrane conditions were observed by laser confocal microscopy (the Merge figure is the combination of the DiI staining and DAPI staining figures). The results are as Figure 3 shown:

[0058] The results showed that compared with the carrier prepared with a mass ratio of cell membrane to polypeptide = 1:1, increasing the proportion of cell membrane in the carrier could significantly increase the intensity of red fluorescence and the transmembrane rate. It was shown that the polypeptide nanocarriers modified with tumor cell membranes prepared by the present invention could indeed enhance their transmembrane efficiency on tumor cells.

[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a tumor cell membrane-modified polypeptide nanocarrier, characterized in that: The tumor cell membrane and polypeptide with opposite charges are assembled through electrostatic adsorption to form the tumor cell membrane modified polypeptide nanocarrier.

2. The method for preparing a tumor cell membrane-modified polypeptide nanocarrier according to claim 1, characterized in that: The polypeptide is positively charged and the tumor cell membrane is negatively charged.

3. The method for preparing a tumor cell membrane-modified polypeptide nanocarrier according to claim 2, characterized in that: The tumor cell membrane is the cell membrane of Hela cells.

4. The method for preparing a tumor cell membrane-modified polypeptide nanocarrier according to claim 3, characterized in that: The cell membrane of the Hela cells is extracted as follows: Hela cells are added to a cell membrane extraction buffer, wherein the cell membrane extraction buffer includes 30mM Tris-HCl with a pH of 7.0, 225mM mannitol and 75mM sucrose, ultrasonically disrupted, centrifuged to obtain a supernatant, and then the supernatant is centrifuged to obtain the cell membrane of the Hela cells.

5. The method for preparing a tumor cell membrane-modified polypeptide nanocarrier according to any one of claims 1 to 4, characterized in that: The structural formula of the polypeptide is as follows:

6. The method for preparing a tumor cell membrane-modified polypeptide nanocarrier according to any one of claims 1 to 4, characterized in that: The tumor cell membrane and the polypeptide are assembled as follows: the tumor cell membrane and the polypeptide are diluted in ddH2O respectively, then mixed, and then allowed to stand to obtain the tumor cell membrane-modified polypeptide nanocarrier.

7. A tumor cell membrane-modified polypeptide nanocarrier, characterized in that: Prepared according to the method according to any one of claims 1 to 4.

8. Use of the tumor cell membrane-modified polypeptide nanocarrier as claimed in claim 7 in preparing a drug delivery system.

9. Use of the tumor cell membrane-modified polypeptide nanocarrier according to claim 7 in the preparation of a tumor cell and / or immune cell transfection agent.

10. Use of the tumor cell membrane-modified polypeptide nanocarrier according to claim 7 in the preparation of anti-tumor and / or immunotherapy drugs.

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