A tumor cell membrane modified polypeptide nanocarrier, and a preparation method and use thereof

Abstract

The application discloses a tumor cell membrane modified polypeptide nanocarrier, a preparation method and application thereof, and the preparation method of the carrier is that oppositely charged tumor cell membranes and polypeptides are assembled into the tumor cell membrane modified polypeptide nanocarrier through electrostatic adsorption. The tumor cell membrane modified polypeptide nanocarrier prepared by the application can obviously improve the membrane penetration efficiency in tumor cells and immune cells, and can effectively deliver nucleic acid molecules and drug molecules into tumor cells and immune cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tumor cell membrane modified polypeptide nanocarrier, its preparation method, and its uses. Background Technology

[0002] Currently, in the field of anti-tumor drugs, the use of carriers to load tumor drugs to prepare drug delivery systems (DDS) is a topic of much research. DDSs can improve bioavailability by increasing the solubility of hydrophobic drugs. In addition, some nanomaterials have environmentally responsive properties (such as enzyme sensitivity, heat sensitivity, redox sensitivity, and pH sensitivity). The microenvironment of tumor tissue can respond to these properties, thereby increasing the accumulation of drugs at the tumor site and improving the therapeutic effect.

[0003] Currently, numerous researchers are utilizing polymers (peptides and polysaccharides), inorganic substances, polymer-lipid complexes, and liposomes as nanocarriers to construct more efficient drug delivery systems. Among these, peptides have shown great potential in cancer treatment due to their advantages such as small molecular weight, good biocompatibility, and ease of modification. However, research has revealed that traditional peptide nanocarriers still suffer from poor biocompatibility and insufficient targeting, limiting their further clinical application. Summary of the Invention

[0004] The main objective of this invention is to provide a tumor cell membrane modified polypeptide nanocarrier with good biocompatibility, targeting ability and membrane penetration efficiency, as well as its preparation method and uses.

[0005] To achieve the above objectives, the present invention provides a method for preparing a tumor cell membrane modified polypeptide nanocarrier, characterized in that tumor cell membranes and polypeptides with opposite charges are assembled by electrostatic adsorption to form the tumor cell membrane modified polypeptide nanocarrier.

[0006] Furthermore, the polypeptide carries a positive charge, while the tumor cell membrane carries a negative charge. The positively charged polypeptide can load negatively charged nucleic acid molecules, enabling the delivery of nucleic acid molecules.

[0007] Furthermore, the tumor cell membrane is the cell membrane of HeLa cells.

[0008] Furthermore, the cell membrane of the HeLa cells is extracted as follows: HeLa cells are added to a cell membrane extraction buffer, which includes 30 mM Tris-HCl at pH 7.0, 225 mM mannitol and 75 mM sucrose. After sonication and centrifugation, the supernatant is collected and then centrifuged again to obtain the cell membrane of the HeLa cells.

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

[0010]

[0011] This polypeptide incorporates two sulfonium salt centers, which can be reduced by intracellular GSH, thereby releasing the internal nucleic acid drug and greatly improving the delivery efficiency of the carrier for nucleic acid drugs.

[0012] Furthermore, the tumor cell membrane and polypeptide are assembled as follows: the tumor cell membrane and polypeptide are diluted separately in ddH2O, then mixed, and then allowed to stand to obtain the tumor cell membrane modified polypeptide nanocarrier.

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

[0014] The present invention also provides the use of the above-mentioned tumor cell membrane modified polypeptide nanocarriers in the preparation of drug delivery systems.

[0015] The present invention also provides the use of the above-mentioned tumor cell membrane modified polypeptide nanocarriers in the preparation of tumor cell and / or immune cell transfection agents.

[0016] The present invention also provides the use of the above-mentioned tumor cell membrane modified polypeptide nanocarriers in the preparation of antitumor and / or immunotherapy drugs.

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

[0018] This invention combines the biological functions of tumor cell membranes with the designability of peptide carriers to prepare peptide nanocarriers modified with tumor cell membranes. The tumor cell membrane can effectively encapsulate 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 evade the recognition and clearance of the immune system, effectively reducing the immunogenicity of peptide nanocarriers, prolonging the circulation time of peptide carriers in vivo, and improving biocompatibility. In addition, specific receptors or ligands on the tumor cell membrane can achieve targeting of specific cells or tissues, improving the efficiency of drug tumor-targeted delivery.

[0019] The tumor cell membrane-modified polypeptide nanocarrier prepared by this invention can encapsulate various tumor drugs, from small molecule compounds to biomacromolecules, thereby increasing the loading rate of tumor drugs and reducing their toxic side effects in vivo. This provides an option for personalized tumor treatment strategies and is expected to provide new ideas in the future development of drug delivery systems with low toxicity, high delivery efficiency, high release efficiency, and high targeting.

[0020] This invention can prepare relatively stable and uniform nanoparticles by changing the ratio of tumor cell membrane to peptide. Compared with naked cell membrane, the peptide nanocarrier modified with tumor cell membrane prepared by this invention can significantly improve its membrane penetration efficiency in tumor cells and immune cells, and can effectively deliver nucleic acid molecules and drug molecules to tumor cells and immune cells. Attached Figure Description

[0021] Figure 1 Fluorescence images showing the membrane-penetrating effects of tumor cell membranes and tumor cell membrane-modified polypeptide nanocarriers in BMDCs and HeLa cells.

[0022] Figure 2 Figure (a) shows the particle size analysis of polypeptide nanocarriers modified with tumor cell membranes in different ratios; Figure (b) shows the zeta potential analysis of polypeptide nanocarriers modified with tumor cell membranes in different ratios.

[0023] Figure 3 Fluorescence images showing the membrane-penetrating effect of peptide nanocarriers modified with different ratios of tumor cell membranes in HeLa cells. Detailed Implementation

[0024] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0025] Unless otherwise specified, the raw materials, reagents, or apparatus used in the following examples can be obtained from conventional commercial sources or by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods mastered by those skilled in the art. The polypeptides used have the following structural formula: That is, the polypeptide disclosed in Chinese patent ZL201810078986.2.

[0026] Example 1

[0027] Preparation and characterization of tumor cell membrane modified peptide nanocarriers

[0028] (1) Preparation of tumor cell membrane

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

[0030] HeLa cells were collected and resuspended in cell membrane extraction buffer (containing 30 mM Tris-HCl, pH 7.0, 225 mM mannitol, and 75 mM sucrose). The cells were then sonicated (4°C, 1 s on, 4 s off, 270 W) using an ultrasonic cell disruptor. The cells were then centrifuged at 4°C and 10,000 rcf for 25 min. The supernatant was collected and centrifuged again at 4°C and 100,000 rcf for 35 min. The supernatant was discarded, and the residue was resuspended in 0.2 mM EDTA. The resuspension was then centrifuged again at 4°C and 100,000 rcf for 35 min, and the supernatant was discarded. The residue was resuspended in 0.2 mM EDTA to obtain a resuspension containing tumor cell membranes (i.e., HeLa cell membranes). The cell membrane protein concentration in the resuspension was determined using the BCA method. The resuspension was stored at -80°C for later use.

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

[0032] Based on the measured cell membrane protein concentration, a resuspension containing 10 μg of tumor cell membrane was taken, and then 10 μg of peptide was taken. The two were diluted in 100 μL ddH2O. The prepared cell membrane diluent was then added to the peptide diluent, mixed by pipetting, and allowed to stand for 30 min to obtain the peptide nanocarrier modified with tumor cell membrane.

[0033] The particle size and zeta potential of the tumor cell membrane, peptides, and prepared tumor cell membrane-modified peptide nanocarriers used in this embodiment were detected by dynamic light scattering (DLS), and 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 peptides were assembled with the tumor cell membrane, the particle size was significantly increased compared with the cell membrane group. Furthermore, the nanoparticles formed by the peptides with positive surface potential binding with the cell membrane with negative surface potential exhibited a near-neutral potential with a positive charge, thus forming relatively stable cell membrane-peptide nanoparticles.

[0039] Experimental Example 1

[0040] Verification of the membrane penetration efficiency of peptide nanocarriers modified with tumor cell membrane

[0041] Following the same method as in Example 1, before assembling the tumor cell membrane with 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 the DiI-labeled tumor cell membrane modified polypeptide nanocarrier was prepared.

[0042] DiI-labeled tumor cell membranes and the aforementioned DiI-labeled tumor cell membrane-modified polypeptide nanocarriers were added to BMDCs and HeLa cells, respectively, and incubated for 4 hours. Cell smears were collected, and cell nuclei were stained with DAPI dye. The transmembrane penetration of the tumor cell membrane and the tumor cell membrane-modified polypeptide nanocarriers was observed using laser confocal microscopy (Merge figure is a combination of DiI staining and DAPI staining images). Results are as follows: Figure 1 As shown:

[0043] The results showed that no obvious red fluorescence was observed on BMDCs and HeLa cells after incubation with tumor cell membranes, while BMDCs and HeLa cells incubated with the polypeptide nanocarrier modified with tumor cell membranes showed significantly enhanced red fluorescence, indicating that the membrane-penetrating ability of the carrier prepared in this invention is significantly enhanced in tumor cells and immune cells.

[0044] Example 2

[0045] Screening of the ratio of tumor cell membrane to peptides

[0046] Tumor cell membrane-modified polypeptide nanocarriers were prepared according to the same method as in Example 1, with the only difference being the adjustment of the ratio of tumor cell membrane to polypeptide. One group used 5 μg of tumor cell membrane and 10 μg of polypeptide, while the other group used 20 μg of tumor cell membrane and 10 μg of polypeptide. The particle size, polymer dispersion index (PDI), and zeta potential of the final tumor cell membrane-modified polypeptide nanocarriers were then detected using dynamic light scattering (DLS). The results for different tumor cell membrane to polypeptide mass ratios are shown in Tables 3, 4, and 5, and analytical graphs were plotted as follows. Figure 2 As shown ( Figure 2 In section a, the 1:0 part refers to the test data of tumor cell membranes alone.

[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 nanoparticle size distribution of the assembled tumor cell membrane and peptides increased significantly, and the larger the proportion of cell membrane in the carrier, the more uniform the particle size distribution of the carrier and the closer the zeta potential was to electroneutrality. More stable and uniform nanoparticles were obtained when the cell membrane and peptides were assembled at a mass ratio of 2:1.

[0054] Experiment Example 2

[0055] Verification of the membrane penetration efficiency of peptide nanocarriers modified with tumor cell membranes at different ratios

[0056] Following the same methods as in Examples 1 and 2, before assembling the tumor cell membrane with 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 DiI-labeled tumor cell membrane modified polypeptide nanocarriers with cell membrane to polypeptide mass ratios of 1:1 and 2:1 were finally prepared respectively.

[0057] The DiI-labeled tumor cell membrane-modified polypeptide nanocarriers with different ratios and the corresponding DiI-labeled tumor cell membranes were incubated with HeLa cells for 4 hours. Cell smears were collected, and the cell nuclei were stained with DAPI dye. Cell transmembrane penetration was observed using laser confocal microscopy (Merge figure is a combination of DiI staining and DAPI staining images). The results are as follows: Figure 3 As shown:

[0058] The results showed that, compared with a carrier prepared by a cell membrane to polypeptide mass ratio of 1:1, increasing the proportion of cell membrane in the carrier significantly improved the intensity of red fluorescence and increased the membrane penetration rate. This indicates that the tumor cell membrane-modified polypeptide nanocarrier prepared in this invention can indeed enhance its membrane penetration efficiency on tumor cells.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tumor cell membrane-modified polypeptide nanocarrier, characterized by, The tumor cell membrane and the polypeptide are assembled by electrostatic adsorption to form the tumor cell membrane modified polypeptide nanocarrier. The tumor cell membrane is a cell membrane of Hela cells, and the polypeptide has the following structural formula: ； The tumor cell membrane and the polypeptide are assembled by electrostatic adsorption to form the tumor cell membrane modified polypeptide nanocarrier.

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

3. The method for preparing the tumor cell membrane-modified polypeptide nanocarrier as described in claim 1, characterized in that, The cell membrane of the Hela cells is extracted by adding the Hela cells into a cell membrane extraction buffer, ultrasonic crushing, centrifugation, and centrifugal treatment of the supernatant to obtain the cell membrane of the Hela cells.

4. A tumor cell membrane-modified polypeptide nanocarrier, characterized by, Prepared by the method as claimed in any one of claims 1 to 3.

5. Use of the tumor cell membrane modified polypeptide nanocarrier as claimed in claim 4 in the preparation of a drug delivery system.

6. Use of the tumor cell membrane modified polypeptide nanocarrier as claimed in claim 4 in the preparation of a tumor cell and / or immune cell transfection agent.

7. Use of the tumor cell membrane modified polypeptide nanocarrier as claimed in claim 4 in the preparation of an antitumor and / or immunotherapy drug.

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