Cell membrane protein fusion liposome as well as preparation method and application thereof
By forming cell membrane protein fusion liposomes, the problem of membrane protein loss during the encapsulation process of cell membrane-coated nanoparticles was solved, significantly improved membrane protein loading, and was successfully applied to tumor-targeted delivery vectors.
Patent Information
- Application Number
- CN202411851438.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-11
AI Technical Summary
Existing cell membrane-coated nanoparticles cause a large amount of membrane protein loss during the encapsulation process, resulting in low membrane protein loading.
By using the lipid solubility of cell membrane proteins and recombination of phospholipid molecules, cholesterol, etc. to form cell membrane protein fusion liposomes, improve the membrane protein load rate, and form a double-layer structure through self-assembly reaction to avoid the loss of membrane proteins caused by mechanical extrusion.
The membrane protein load rate was significantly improved, from 20% to 80%, and was successfully applied to tumor-targeted delivery vehicles, improving the targeting and efficacy of the drug.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a cell membrane protein fusion liposome and a preparation method and application thereof. Background Art
[0002] Cell membrane-coated nanoparticles (CMNPs) have opened up new horizons for the development of drug and gene delivery vectors, with the characteristics of high targeting, precise delivery, controlled drug release, and activation of immune response. CMNPs are extracted from mother cell membrane fragments to preserve the unique membrane surface markers and self-recognition molecules of mother cells. They can target homologous tumor tissues, reduce nonspecific uptake, and reduce the risk of being recognized and cleared by immune tissues, making them good tumor-targeted delivery vectors.
[0003] Although CMNPs relatively completely retain the cell membrane surface structure and can effectively solve the key problem of targeted drug delivery, cell membrane-coated nanoparticles still have obvious limitations. For example, cell membrane-coated nanoparticles are mostly encapsulated by mechanical extrusion, which causes a large amount of membrane protein loss, especially membrane proteins on macromolecular membrane fragments, resulting in a large amount of cell surface receptor loss and low membrane protein loading rate. In summary, the existing delivery carriers have the problem of low membrane protein loading rate. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a cell membrane protein fusion liposome and a preparation method and application thereof.
[0005] A cell membrane protein fusion liposome comprises dioleoyl phosphatidylcholine, 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000], cholesterol, Triton X-100 and cell membrane protein in a mass ratio of 3.17-7.17:0.96-2.87:0.86-2.57:0.5-2.5:0.5-2.5.
[0006] The present invention utilizes the lipid solubility of cell membrane proteins and the recombination of phospholipid molecules, cholesterol and the like to form cell membrane protein fusion liposomes, thereby effectively improving the membrane protein loading rate. The lipid molecules then form a double-layer structure under the action of an aqueous phase to generate a self-assembly reaction. Triton X-100 is added to the synthesis formula to stabilize the membrane protein structure while regulating the flexibility of the cell membrane protein fusion liposomes, thereby avoiding the use of a large number of mechanical extrusion processes during the synthesis process to cause a large amount of membrane protein loss, thereby greatly improving the membrane protein loading rate of the cell membrane protein liposomes.
[0007] Preferably, the cell membrane protein is extracted from neutrophils of bone marrow cells. Compared with cell membrane-coated nanoparticles, the cell membrane protein fusion liposome further processes the cell membrane fragments through a cell membrane protein extraction kit in the synthesis process, so that the cell membrane fragments remove the redundant lipid fragments and form cell membrane proteins.
[0008] The preparation method of the cell membrane lipoprotein fusion liposome comprises the following steps:
[0009] The neutrophils of bone marrow cells are extracted by using lymph separation fluid, and the cell membrane proteins are extracted from the obtained neutrophils by using a cell membrane protein extraction kit;
[0010] Dioleoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000], and cholesterol are dissolved in an alcohol solvent, and then ammonium sulfate and Triton X-100 cell membrane protein are added in sequence to produce a self-assembly reaction, and the cell membrane protein is incubated to maintain the activity, and the cell membrane lipoprotein fusion liposome is obtained by extrusion and dialysis with a molecular weight cutoff of 3kDa-14kDa.
[0011] Preferably, the mixture is incubated at 35° C. to 38° C. for 28 min to 32 min to ensure that the obtained cell membrane protein fusion liposomes have protein biological activity.
[0012] Preferably, during extrusion, the liposomes are passed through 200 nm and 400 nm filter membranes respectively to obtain cell membrane protein fusion liposomes with uniform particle size.
[0013] Preferably, the dialysis is performed for 20 to 30 hours to remove unsynthesized small molecules, and the dialysis at 4° C. ensures that the protein components in the cell membrane protein fusion liposomes do not lose their life activity.
[0014] The cell membrane protein fusion liposome is used as a tumor drug targeted delivery carrier.
[0015] Preferably, the tumor drug is doxorubicin.
[0016] Preferably, the cell membrane protein fusion liposomes and doxorubicin form a tumor-targeted chemotherapy drug;
[0017] The preparation method of the tumor-targeted chemotherapy drug comprises the following steps: incubating the cell membrane protein fusion liposome with doxorubicin at 1° C. to 5° C. for 10 h to 14 h, and dialyzing at 1° C. to 5° C. for 20 h to 30 h, so as to obtain liposomal doxorubicin with tumor cell targeting properties.
[0018] Preferably, the tumor-targeted chemotherapy drug is used to prepare a drug for treating triple-negative breast cancer.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention designs a cell membrane protein-fused liposome (CMPliposome), which significantly improves the membrane protein loading rate from 20% to 80%, providing technical support for the clinical application of cell membrane materials.
[0021] The CMPliposome of the present invention can form a single-layer membrane protein liposome to avoid the obstruction of the membrane protein structure caused by multi-layer packaging, and can also load drugs, thereby being applied to tumor drug targeted delivery carriers.
[0022] The CMPliposome of the present invention is easy to carry out effective load modification of the chemical drug doxorubicin (DOX), providing a favorable tool for tumor-targeted delivery carriers. The present invention successfully prepared DOX@CMPliposome, which can be used to prepare drugs for treating triple-negative breast cancer.
[0023] The CMPliposome of the present invention is a lipid material, which is consistent with the mRNA vaccine vector LNP approved by the FDA and has low toxicity, thus facilitating its clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Showing the physical and chemical properties of DOX@CMPliposome; A is the transmission electron microscopy image of DOX@CMPliposome; B is the hydrated particle size and potential of DOX@CMPliposome; C is the change of hydrated particle size of DOX@CMPliposome over time; D is the UV-visible absorption spectrum of DOX@CMPliposome; E is the absorption peak of DOX@CMPliposome at 500nm; C is the absorption peak of DOX@CMPliposome at 280nm.
[0025] Figure 2 The graph shows the changes in the cell membrane protein loading rate of DOX@CMPliposome as the amount of cell membrane protein added increases.
[0026] Figure 3 Expression profile showing cell membrane protein loading of DOX@CMPliposome.
[0027] Figure 4 The graph shows the change in DOX encapsulation efficiency of DOX@CMPliposome as the amount of DOX added increases.
[0028] Figure 5Showing the targeted endocytosis effect of DOX@CMPliposome; A is a confocal image of mouse breast cancer 4T1 cells, where a, b, c are DAPI, DOX, and Merge cell fluorescence images of the DOX@liposome group, respectively, and d, e, f are DAPI, DOX, and Merge cell fluorescence images of the DOX@CMPliposome group, respectively; B is a comparison of the average fluorescence intensity of DOX between DOX@liposome and DOX@CMPliposome in mouse breast cancer 4T1 cells.
[0029] Figure 6 The results show that DOX@CMPliposome can prevent leukocytes from internalizing. A is a confocal image of peripheral blood neutrophils and peripheral blood mononuclear cells, where a and b are fluorescence images of peripheral blood neutrophils and peripheral blood mononuclear cells in the DOX@liposome group, respectively, and c and d are fluorescence images of peripheral blood neutrophils and peripheral blood mononuclear cells in the DOX@CMPliposome group, respectively. B is a comparison of the average fluorescence intensity of DOX in peripheral blood neutrophils and peripheral blood mononuclear cells between DOX@liposome and DOX@CMPliposome.
[0030] Figure 7 Showing the tumor cell killing effect of DOX@CMPliposome.
[0031] Figure 8 Figure 2 shows the animal treatment effect of DOX@CMPliposome; A is the tumor growth curve of breast cancer in each group of mice; B is the weight change of each group of mice during the treatment; C is the tumor volume change of mice in the DOX@CMPliposome group; D is the tumor volume change of mice in the DOX@liposome group; E is the tumor volume change of mice in the DOX group; F is the tumor volume change of mice in the Control group; G is the tumor weight of mice in each group one week after the end of treatment; H is the in vitro tumor photo of mice in each group one week after the end of treatment.
[0032] Fig. 9 Shows the histochemical staining images of important organs of mice in each group one week after the end of treatment. DETAILED DESCRIPTION
[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.
[0034] The present invention prepares cell membrane protein-fused liposome (CMPliposome) based on CMNPs, which significantly improves the membrane protein loading rate and forms a complete liposome for drug loading. The following problems are effectively solved. 1. Mechanical extrusion can be avoided, and the membrane protein is completely retained and fused and recombined with the liposome, which significantly improves the membrane protein loading rate and avoids the loss of a large number of tumor surface receptors and antigens; 2. The membrane protein-fused liposome is a complete liposome structure, which can be regulated by the inner core drug loading and the outer membrane targeted modification, providing a reliable delivery carrier for the precise targeted delivery of tumor-targeted drugs.
[0035] The present invention uses CMPliposome technology to apply neutrophils to prepare DOX@CMPliposome: 1. DOX@CMPliposome has certain tumor targeting; 2. DOX@CMPliposome avoids macrophage phagocytosis and has long circulation characteristics; 3. It forms a single-layer membrane protein liposome, which can be used for the combined loading of chemotherapy drugs and photodynamic drugs. The purpose of the present invention is to design CMPliposome and prepare the tumor-targeted chemotherapy drug "DOX@CMPliposome" through technology, and treat triple-negative breast cancer through intravenous administration. It is hoped that it can solve the clinical problems of toxic and side effects of existing tumor chemotherapy drugs and enhance tumor efficacy, and provide a safe, reliable and practical delivery tool for the research and development of tumor-targeted drugs.
[0036] Example 1
[0037] The preparation method of cell membrane protein fusion liposomes comprises the following steps:
[0038] (1) Extraction of cell membrane protein
[0039] Mouse bone marrow cells and human whole blood were extracted respectively, and neutrophils were extracted using lymph separation fluid. The obtained neutrophils were subjected to membrane protein extraction using a cell membrane protein extraction kit and stored at 4°C for later use.
[0040] (2) Preparation of cell membrane protein-fused liposome (CMPliposome)
[0041] CMPliposome is synthesized by ethanol injection combined with extrusion. The specific method is as follows:
[0042] Dioleoylphosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000] (DSPE-mPEG2000), and cholesterol were dissolved in 1 mL of anhydrous ethanol, with a mass ratio of DOPC, DSPE-mPEG2000, and cholesterol of 5.17 mg: 1.91 mg: 1.71 mg. 3 mL of 250 mM ammonium sulfate, 0.1 mL of 10 mg / mL Triton X-100 (TritonX-100), and 1 mL of 1 mg / mL cell membrane protein were added in sequence, incubated at 37°C for 30 minutes, and passed through 200 nm and 400 nm filter membrane squeezers for 20 times, respectively. Subsequently, CMPliposome was obtained by dialyzing with 10% sucrose (molecular weight cutoff 14 kDa) at 4°C for 24 hours.
[0043] Preparation of CMPliposome loaded with doxorubicin (DOX@CMPliposome)
[0044] 5 mL of 1 mg / mL CMPliposome synthesized in the previous step was incubated with 1 mL of 2 mg / mL DOX at 37°C for 1 h, followed by dialyzation against 10% sucrose (molecular weight cutoff 14 kDa) at 4°C for 24 h to obtain DOX@CMPliposome.
[0045] Example 2
[0046] The preparation method of cell membrane protein fusion liposomes comprises the following steps:
[0047] (1) Extraction of cell membrane protein
[0048] Mouse bone marrow cells and human whole blood were extracted respectively, and neutrophils were extracted using lymph separation fluid. The obtained neutrophils were subjected to membrane protein extraction using a cell membrane protein extraction kit and stored at 4°C for later use.
[0049] (2) Preparation of cell membrane protein-fused liposome (CMPliposome)
[0050] CMPliposome is synthesized by ethanol injection combined with extrusion. The specific method is as follows:
[0051] Dioleoylphosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000] (DSPE-mPEG2000), and cholesterol were dissolved in 1 mL of anhydrous ethanol. The mass ratio of DOPC, DSPE-mPEG2000, and cholesterol was 3.17 mg: 2.87 mg: 2.57 mg. 3 mL of 250 mM ammonium sulfate, 0.05 mL of 10 mg / mL Triton X-100, and 0.5 mL of 1 mg / mL cell membrane protein were added in sequence. The mixture was incubated at 37°C for 30 minutes, and then filtered through 200 nm and 400 nm filter membrane squeezers for 20 times. Subsequently, CMPliposome was obtained by dialyzing with 10% sucrose at 4°C for 24 hours.
[0052] Preparation of CMPliposome loaded with doxorubicin (DOX@CMPliposome)
[0053] 5 mL of 0.5 mg / mL CMPliposome synthesized in the previous step was incubated with 1 mL of 2.5 mg / mL DOX at 37°C for 2 h, and then dialyzed against 10% sucrose at 4°C for 24 h to obtain DOX@CMPliposome.
[0054] Example 3
[0055] The preparation method of cell membrane protein fusion liposomes comprises the following steps:
[0056] (1) Extraction of cell membrane protein
[0057] Mouse bone marrow cells and human whole blood were extracted respectively, and neutrophils were extracted using lymph separation fluid. The obtained neutrophils were subjected to membrane protein extraction using a cell membrane protein extraction kit and stored at 4°C for later use.
[0058] (2) Preparation of cell membrane protein-fused liposome (CMPliposome)
[0059] CMPliposome is synthesized by ethanol injection combined with extrusion. The specific method is as follows:
[0060] Dioleoylphosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000] (DSPE-mPEG2000), and cholesterol were dissolved in 1 mL of anhydrous ethanol, with a mass ratio of DOPC, DSPE-mPEG2000, and cholesterol of 7.17 mg: 0.96 mg: 0.86 mg. 3 mL of 250 mM ammonium sulfate, 0.25 mL of 10 mg / mL Triton X-100, and 2.5 mL of 1 mg / mL cell membrane protein were added in sequence, incubated at 37°C for 30 minutes, and passed through 200 nm and 400 nm filter membrane squeezers for 20 times, respectively. Subsequently, CMPliposomes were obtained by dialyzing with 10% sucrose at 4°C for 24 hours.
[0061] Preparation of CMPliposome loaded with doxorubicin (DOX@CMPliposome)
[0062] 5 mL of 1.5 mg / mL CMPliposome synthesized in the previous step was incubated with 1 mL of 0.5 mg / mL DOX at 37°C for 1-2 h, and then dialyzed against 10% sucrose at 4°C for 24 h to obtain DOX@CMPliposome.
[0063] Effect verification
[0064] 1. Characterization of the physicochemical properties of DOX@CMPliposome
[0065] (1) The morphology of DOX@CMPliposome was examined by transmission electron microscopy. Figure 1 As shown in A in Figure 1, DOX@CMPliposome is a regular round shape under electron microscope, with a diameter of about 70-80nm; Malvern particle size potential results show that Figure 1 In Figure B, the average hydrated particle size of DOX@CMPliposome is 88.5nm, and its average potential is -16.23, which is much lower than the average potential of DOX@liposome -7.86. Moreover, the average hydrated particle size of DOX@CMPliposome fluctuates less over time, and the average hydrated particle size fluctuates between 88.5-86.8nm within 15 days. Figure 1 C in.
[0066] UV-visible spectrum results, such as Figure 1 As shown in DE in , DOX@CMPliposome has obvious absorption peaks at 280nm and 500nm. The absorption peak at 280nm is the protein absorption peak, and the absorption peak at 500nm is the DOX absorption peak, indicating that DOX@CMPliposome carries membrane protein and DOX.
[0067] 2. Characterization of biological properties of DOX@CMPliposome
[0068] Cell membrane protein determination: 1 mL of cell membrane protein of different concentrations (0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25 mg, 2.5 mg) was used to prepare DOX@CMPliposome, and the cell membrane protein loading rate was detected by BCA protein quantification method.
[0069] like Figure 2 The cell membrane protein loading rate of DOX@CMPliposome varies with the amount of cell membrane protein added, and its cell membrane protein loading rate fluctuates between 52.33% and 82.67%, which is much higher than the cell membrane protein loading rate of cell membrane-wrapped liposomes (CM-liposome), which is 5.33% to 21.67%. At the same time, SDS-PAGE was used to detect the expression spectrum of its cell membrane protein loading, such as Figure 3 The results showed that the protein bands of DOX@CMPliposome were consistent with the cell membrane protein bands, indicating that DOX@CMPliposome can load cell membrane proteins well.
[0070] 3. Exploration of DOX encapsulation efficiency of DOX@CMPliposome
[0071] Determination of DOX encapsulation efficiency: 1 mL of DOX with different concentrations (0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg) was mixed with 5 mL of CMPliposome with a concentration of 1 mg / mL to prepare DOX@CMPliposome, and the DOX encapsulation efficiency was measured by fluorescence spectrophotometry.
[0072] like Figure 4 The DOX encapsulation rate of DOX@CMPliposome varies slightly with the amount of DOX added, and its encapsulation rate fluctuates between 35.27-38.85%, which is lower than the 97.33-98.93% of DOX encapsulation rate of liposome doxorubicin (DOX@liposome). This may be related to the fact that CMPliposome carries a certain amount of cell membrane proteins, which affects the fluidity of its lipid bilayer.
[0073] 4. Cell-targeted endocytosis detection
[0074] 1) Targeted endocytosis experiments in tumor cells
[0075] When the mouse breast cancer cell line 4T1 grows to 75%, it is passaged and inoculated into a confocal dish, 6000 cells per well, 10% FBS DMEM, overnight. The next day, the medium is changed, and 20uL of 5ug / mL DOX@liposome and DOX@CMPliposome are added, respectively, and incubated for 2-4h; DAPI is added after fixation, and cell fluorescence is monitored by laser confocal, parameters: DOX excitation light 488nm, emission light 590nm; DAPI excitation light 405nm, emission light 465nm.
[0076] The results are as follows Figure 5 As shown in A, a, b, c are DAPI, DOX, and Merge cell fluorescence images of the DOX@liposome group, respectively; d, e, f are DAPI, DOX, and Merge cell fluorescence images of the DOX@CMPliposome group, respectively. The results show that the mouse breast cancer cell line 4T1 has a more obvious internalization effect on DOX@CMPliposome, and its average DOX fluorescence intensity is also significantly higher than that of the DOX@liposome group, and the difference is statistically significant, as shown in Figure 1. Figure 5 B in.
[0077] 2) Leukocyte avoidance endocytosis experiment
[0078] Whole blood cells were drawn from mice, and peripheral blood neutrophils and peripheral blood mononuclear cells were extracted using lymph separation fluid, which were then inoculated into confocal dishes with 6000 cells per well. 20uL of 5ug / mL DOX@liposome and DOX@CMPliposome were added respectively and incubated for 2h. Cell fluorescence was monitored by laser confocal microscope with parameters as follows: excitation light 488nm, emission light 590nm.
[0079] like Figure 6 As shown in A, a and b are fluorescence images of peripheral blood neutrophils and peripheral blood mononuclear cells in the DOX@liposome group, respectively; c and d are fluorescence images of peripheral blood neutrophils and peripheral blood mononuclear cells in the DOX@CMPliposome group, respectively. The results show that the red fluorescence of peripheral blood neutrophils and peripheral blood mononuclear cells in the DOX@CMPliposome group was significantly reduced, and the average fluorescence intensity of DOX was also significantly lower than that in the DOX@liposome group, and the difference was statistically significant. Figure 6 B in.
[0080] The above results indicate that these two cell types can reduce the endocytosis of DOX@CMPliposome and avoid damage to blood circulating cells.
[0081] 5. Tumor cell killing experiment
[0082] When the mouse breast cancer cell line 4T1 grows to 75%, it is passaged and inoculated into a 96-well plate, 5000 cells per well, 10% FBS DMEM, overnight. The next day, the medium was changed, and 0, 1.375, 3.75, 7.5, 10ug / mL (calibrated by the concentration of DOX) of DOX, DOX@liposome, and DOX@CMPliposome were added, 3 replicates per group, and incubated for 24h. CCK8 detection reagent was used, 1uL of CCK8 reagent was added to each well, and after incubation for 2h, ABS was detected at 540nm on an ELISA reader.
[0083] like Figure 7 As shown in the figure, at the same DOX content, DOX, DOX@liposome, and DOX@CMPliposome all have an inhibitory effect on the growth of tumors in the mouse breast cancer cell line 4T1 cells, among which DOX@CMPliposome is the most obvious. As the DOX content increases from 1.7ug / mL to 6.9ug / mL, the tumor-killing effect of DOX@CMPliposome on the mouse breast cancer cell line 4T1 cells increases significantly, and the viability of tumor cells in this group is significantly lower than that of DOX@liposome and DOX, and the difference is statistically significant.
[0084] 6. Animal treatment effects
[0085] BALB / c female nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. and raised in an SPF environment. They were tumor-bearing at 6 weeks of age. The mouse breast cancer cell line 4T1 was used, 500,000 / cell / mouse, to prepare a mouse breast cancer model. Drug treatment was performed 1 week after the tumor and divided into a control group (Control), DOX, DOX@liposome group, and DOX@CMPliposome group. Among them, the DOX, DOX@liposome group, and DOX@CMPliposome group were injected with 0.1mL, 1mg / mL (concentration calibrated with the same DOX content) of DOX, DOX@liposome, and DOX@CMPliposome through the tail vein on days 1, 4, and 7, respectively, with 5 mice in each group. During the treatment, the tumor growth and body weight of the mice were monitored, and all mice were killed one week after treatment, and the tumor size and weight of the mice were recorded.
[0086] like Figure 8 As shown in A, DOX, DOX@liposome group, and DOX@CMPliposome all have tumor inhibitory effects on mouse breast cancer, among which the tumor inhibitory effect of DOX@CMPliposome is the most obvious. Figure 8 Result B in the figure shows the weight changes of mice in each group during the treatment period, and the differences were not statistically significant. Figure 8C, D, E, and F show the changes in tumor volume during treatment in the DOX@CMPliposome, DOX@liposome, DOX, and Control groups, respectively. Comparison of the results shows that the tumor volume in the DOX@CMPliposome group grew most slowly. Figure 8 G in the figure shows the tumor weights of the DOX@CMPliposome, DOX@liposome, DOX and Control groups one week after the end of treatment; among them, the DOX@CMPliposome, DOX@liposome and DOX were all lower than those of the Control group, and the tumor weight of DOX@CMPliposome was significantly lower than that of each group, and the difference was statistically significant. Figure 8 .H shows the in vitro tumor photos of DOX@CMPliposome, DOX@liposome, DOX and Control groups one week after the end of treatment, among which the tumor size of DOX@CMPliposome was significantly smaller than that of other groups.
[0087] The biosafety of DOX@CMPliposome was further tested. The results of histochemical (HE) staining of various tissues and organs in mice are as follows: Fig. 9 As shown, DOX@CMPliposome did not cause damage to important organs such as the heart, liver, lungs, and kidneys of mice.
[0088] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.
[0089] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0090] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A cell membrane protein fusion liposome, characterized in that: The cell membrane protein fusion liposome comprises dioleoyl phosphatidylcholine, 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000], cholesterol, Triton X-100 and cell membrane protein in a mass ratio of 3.17-7.17:0.96-2.87:0.86-2.57:0.5-2.5:0.5-2.
5.
2. The cell membrane protein fusion liposome according to claim 1, characterized in that: The cell membrane protein is extracted from neutrophils of bone marrow cells.
3. The method for preparing the cell membrane lipoprotein fusion liposome according to claim 1, characterized in that: The following steps are involved: The neutrophils of bone marrow cells are extracted by using lymph separation fluid, and the cell membrane proteins are extracted from the obtained neutrophils by using a cell membrane protein extraction kit; Dioleoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000], and cholesterol are dissolved in an alcohol solvent, and then ammonium sulfate, Triton X-100, and cell membrane protein are added in sequence to produce a self-assembly reaction, and the cell membrane protein is incubated to maintain the activity, and the cell membrane lipoprotein fusion liposome is obtained by extrusion and dialysis with a molecular weight cutoff of 3kDa-14kDa.
4. The preparation method according to claim 3, characterized in that: Incubate at 35℃~38℃ for 28min~32min.
5. The preparation method according to claim 3, characterized in that: During extrusion, the particles pass through 200nm and 400nm filter membranes respectively.
6. The preparation method according to claim 3, characterized in that: Dialysis lasts 20 to 30 hours.
7. Use of the cell membrane protein fusion liposome according to claim 1 as a tumor drug targeted delivery carrier.
8. The use according to claim 7, characterized in that: The tumor drug is doxorubicin.
9. The use according to claim 8, characterized in that: The cell membrane protein fusion liposome and doxorubicin form a tumor-targeted chemotherapy drug; The preparation method of the tumor-targeted chemotherapy drug comprises the following steps: incubating the cell membrane protein fusion liposome with doxorubicin at 1° C. to 5° C. for 10 h to 14 h, and dialyzing at 1° C. to 5° C. for 20 h to 30 h.
10. The use according to claim 7, characterized in that: The tumor-targeted chemotherapy drug is used to prepare a drug for treating triple-negative breast cancer.
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