A multifunctional nano-therapeutic agent modified with a biomimetic biofilm, and its preparation method and application

Abstract

The present invention discloses a multifunctional nano-therapeutic reagent modified with a biomimetic biofilm, which is a multifunctional nano-therapeutic agent modified with tumor cell membranes. The multifunctional therapeutic agent is a BPP nanoparticle self-assembled from 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, protocatechuic acid, and distearoylphosphatidylethanolamine-polyethylene glycol. The present invention discloses the application of the multifunctional nano-therapeutic agent modified with a biomimetic biofilm in the preparation of drugs for the synergistic treatment of tumors by phototherapy, chemotherapy, and anti-angiogenesis. The multifunctional nano-therapeutic agent modified with a biomimetic biofilm in the present invention has the special abilities of immune escape and homologous targeting of cancer cell membranes, enabling the nano-therapeutic agent to be efficiently enriched in tumor sites, exerting the ability of multi-modal tumor synergistic treatment, and realizing multi-modal tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of nano-medicine, and relates to a multifunctional nano-therapeutic agent modified with a biomimetic biofilm, a preparation method thereof, and an application of the multifunctional nano-therapeutic agent in the synergistic treatment of tumors. Background Art

[0002] Phototherapy has received extensive attention due to its non-invasive, highly selective, and negligible systemic cytotoxicity. However, the problem of tumor hypoxia has always restricted phototherapy. The oxygen partial pressure in solid tumors is only about 10 mmHg, far lower than that in normal peripheral tissues (40 - 60 mmHg), which seriously hinders the effect of oxygen-dependent photodynamic therapy (PDT). In addition, photothermal therapy (PTT) often induces the up-regulation of heat shock proteins in tumor tissues, leading to rapid tumor tolerance and ultimately affecting the treatment effect. Although the synergistic effect of PDT and PTT can improve the oxygen supply of tumor tissues by increasing blood flow velocity, thereby improving the efficiency of PDT, and PDT can also in turn induce the heat-resistant death of tumor cells, the unsatisfactory excitation light penetration depth and the low specificity of photothermal reagents still hinder the treatment effect.

[0003] The occurrence of tumors is often accompanied by the formation of new blood vessels, which is not only a prerequisite for tumorigenesis but also the basis for tumor growth, invasion, and metastasis. Therefore, when treating cancer, targeting tumor blood vessels is as crucial as targeting tumor cells. Anti-angiogenic therapy inhibits the formation of new blood vessels by neutralizing or blocking the expression of vascular endothelial growth factor (VEGF), thereby reducing the nutrient supply of tumor tissues and ultimately leading to apoptosis or necrosis of tumor tissues from the inside out. Therefore, combining phototherapy with different anti-tumor mechanisms and anti-angiogenic therapy is considered a breakthrough strategy to achieve the effect of "1 + 2 > 3" and improve the treatment effect. On the one hand, phototherapy can eradicate tumor cells nourished by marginal normal blood vessels, and such tumor cells still exist after anti-angiogenic therapy. On the other hand, anti-angiogenic therapy can destroy deep tumor cells that are difficult for light to completely penetrate. At the same time, tumor angiogenesis inhibitors can neutralize or block the action of VEGF, thereby "normalizing" tumor blood vessels for a certain period. Therefore, the combination of phototherapy and anti-angiogenic therapy can relieve local tumor hypoxia, improve local transport, and the accumulation of therapeutic drugs in tumors, thereby maximizing the treatment effect of phototherapy.

[0004] Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a widely distributed natural phenolic compound with a simple structure and high biological activity. In addition, PCA has been identified as a multi-target bioactive compound. It can not only block the VEGFR2-dependent Akt / MMP2 and ERK pathways in vascular endothelial cells to inhibit angiogenesis, but also has good killing effects on various tumor cells with obvious chemotoxicity. However, it is difficult to kill all tumor cells in actual treatment, resulting in tumor recurrence and metastasis.

[0005] Most inorganic nanomaterials have disadvantages such as poor biocompatibility and difficulty in metabolism. The organic small molecule pyrrolopyrrole dione (DPP) derivatives are organic small molecule dyes that are easy to modify, have high biocompatibility and low toxicity, and have extensive applications in the fields of organic electronic devices and biological probes. However, in the field of biomedicine, the water solubility and targeting of DPP are still a huge challenge. At the same time, in actual research, it has also been found that the simple phototherapy generated by DPP, like the chemotherapy and anti-angiogenic therapy mediated by PCA, cannot completely ablate tumor cells. To overcome these shortcomings, combining phototherapy, chemotherapy and anti-angiogenic therapy with different anti-tumor mechanisms is considered a breakthrough strategy to achieve the effect of "1+2>3" and improve the treatment effect.

[0006] However, due to the biological barriers in the body, nanoparticles (NPs), as exogenous materials, may be recognized by the early immune system and then cleared by the liver and kidneys, which limits the further development of nanoparticles.

[0007] Therefore, designing and preparing multifunctional nano-drugs modified with biomimetic biofilms, which have the abilities of phototherapy, chemotherapy and anti-angiogenesis, has broad prospects in the field of tumor treatment. Summary of the Invention

[0008] The purpose of the present invention is to prepare multifunctional nano-therapeutics modified with biomimetic biofilms for anti-tumor at the nanoscale. By modifying the nanoparticles with biomimetic biofilms, the accumulation at the tumor site can be improved and the retention in healthy tissues can be reduced, which is beneficial to drug delivery and local treatment of tumors.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A phototherapeutic agent 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (BPA) with the structure shown in Formula I:

[0011]

[0012] Another object of the present invention is to provide the use of 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione in the preparation of tumor phototherapeutic drugs.

[0013] Another object of the present invention is to provide a biomimetic biofilm-modified multifunctional nanotherapeutic agent, which is a multifunctional nanotherapeutic agent modified with tumor cell membranes; the multifunctional therapeutic agent is a BPP nanoparticle (BPP NPs) self-assembled from the phototherapeutic agent 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (BPA), a chemotherapeutic agent, and protocatechuic acid (PCA) as an anti-angiogenic agent under the action of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000).

[0014] The tumor cell membrane is a HeLa cell membrane.

[0015] The tumor cell membrane is prepared by those skilled in the art through conventional methods. Specifically, the following method can be adopted: suspend the tumor cells in membrane protein extraction buffer A (Beyotime cell membrane protein and cytoplasmic protein extraction kit) containing phenylmethylsulfonyl fluoride (1 mM), incubate the mixture in an ice bath for 15 min to lyse the cells; then, freeze the cell suspension in liquid nitrogen and thaw it at room temperature; repeat the freeze-thaw cycle 2 times; centrifuge at 700 g for 10 min at 4 °C to remove the precipitate, and further centrifuge the supernatant at high speed for 30 min at 4 °C to collect the precipitate, which is the tumor cell membrane fragment.

[0016] Another object of the present invention is to provide a preparation method of the biomimetic biofilm-modified multifunctional nanotherapeutic agent, including:

[0017] Step (1), under a nitrogen atmosphere, add 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid, bis(3-5-bromofuran-2-yl)-5-di(6-4-bromohexyl)pyrrolopyrrole(2,5)-1,4-dione (DPP-1), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), and tripotassium phosphate into 1,4-dioxane, heat and stir; remove the solvent to obtain a crude product, and separate the crude product to obtain 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (BPA);

[0018]

[0019] Step (2): Dissolve 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (BPA) and protocatechuic acid (PCA) in tetrahydrofuran to obtain a mixed solution; under stirring, slowly drop the mixed solution into deionized water containing distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000). After the dropping is completed, continue stirring for 5 - 10 minutes, fully blow nitrogen to remove tetrahydrofuran in the reaction solution, centrifuge, and take the supernatant to obtain a BPP nanoparticle (BPP NPs) aqueous dispersion with chemotherapy, phototherapy, and anti-angiogenic functions.

[0020] Step (3): Dissolve the tumor cell membrane in deionized water and ultrasonically disrupt it to obtain a tumor cell membrane solution; under stirring, slowly drop the tumor cell membrane solution into the BPP nanoparticle (BPP NPs) aqueous dispersion obtained in step (2), incubate in the dark, centrifuge, and take the supernatant to obtain a biomimetic biofilm-modified multifunctional nano-therapeutic agent, mBPP nanoparticle (mBPP NPs).

[0021] In step (1), the molar ratio of DPP-1 to 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid is 1:2 - 1:10, preferably 1:5; the molar ratio of DPP-1 to tetrakis(triphenylphosphine)palladium is 1:0.01 - 1:0.1, preferably 1:0.05 - 1:0.06; the molar ratio of DPP-1 to tripotassium phosphate is 1:2 - 1:10, preferably 1:4.

[0022] The temperature of the heating and stirring is 25 - 120 °C, and the time of the heating and stirring is 8 - 36 hours; preferably, the temperature of the heating and stirring is 90 - 100 °C, and the time of the heating and stirring is 12 - 24 hours.

[0023] The crude product is separated and purified by a silica gel chromatographic column to obtain BPA, and the eluent is petroleum ether:dichloromethane = 1:2 V / V.

[0024] In step (2), it is sufficient to dissolve BPA and PCA in tetrahydrofuran. Generally, the concentration of BPA in tetrahydrofuran is 1 - 5 mg / mL, and the concentration of PCA in tetrahydrofuran is 1 - 5 mg / mL.

[0025] The mass ratio of BPA, PCA, and DSPE-mPEG2000 is 1:1:0.5.

[0026] The concentration of DSPE-mPEG2000 in deionized water is 0.1 - 0.5 mg / mL.

[0027] The dropping rate of the mixed solution is 1 to 30 drops per minute; the rotation speed of stirring is 500 to 2000 revolutions per minute.

[0028] The concentration of BPP nanoparticles in the aqueous dispersion of BPP nanoparticles (BPP NPs) is 100 to 200 μg / mL.

[0029] In step (3), the time of ultrasonic fragmentation is 2 to 10 minutes, and the power of ultrasonic is 400 W.

[0030] The cell membrane concentration in the described tumor cell membrane solution is 0.1 to 1 mg / mL.

[0031] The mass ratio of the described tumor cell membrane to BPP nanoparticles is 1:1.

[0032] At a stirring rate of 200 to 500 revolutions per minute and a temperature of 4 to 8 °C, slowly drop the tumor cell membrane solution into the aqueous dispersion of BPP nanoparticles.

[0033] The described dark incubation is carried out under stirring, the stirring rate of incubation is 200 to 500 revolutions per minute, the incubation temperature is 4 to 8 °C, and the incubation time is 8 to 24 hours.

[0034] Another object of the present invention is to provide the application of the multifunctional nano-therapeutic agent modified with the bionic biomembrane in the preparation of drugs for the combined treatment of tumors by phototherapy, chemotherapy, and anti-angiogenesis.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] The present invention obtains a multifunctional nano-therapeutic agent modified with a bionic biomembrane through the methods of self-assembly and co-incubation. This multifunctional nano-therapeutic agent has the homologous targeting and immune escape functions modified by cancer cell membranes, and can cooperate with various tumor treatment methods such as phototherapy, chemotherapy, and anti-angiogenesis therapy. Specifically, it is manifested as:

[0037] (1) The synthesis process of the multifunctional nano-therapeutic agent modified with the bionic biomembrane of the present invention is simple and has a high yield;

[0038] (2) The multifunctional nano-therapeutic agent modified with the bionic biomembrane of the present invention has accurate active and passive tumor targeting properties. It can not only achieve homologous targeting of tumor cells through the modification of the bionic biomembrane, but also passively target the tumor site through the enhanced permeability and retention effect of nanoparticles. At the same time, the modification of the bionic biomembrane also endows the nanoparticles with immune escape ability, avoiding their being phagocytosed by the immune system. Eventually, the nano-therapeutic agent is more enriched around the tumor, improving the therapeutic effect;

[0039] (3) The multifunctional nano-therapeutic agent modified with the biomimetic biofilm of the present invention can synergistically kill tumor cells, inhibit the regeneration and metastasis of tumor cells, and achieve multi-modal tumor treatment through multiple methods such as phototherapy, chemotherapy, and anti-angiogenesis therapy, with broad application prospects. Description of the Drawings

[0040] Figure 1 1H-NMR of BPA 1 1H-NMR spectrum (400 MHz, CDCl3), the abscissa is the chemical shift and the ordinate is the intensity.

[0041] Figure 2 Transmission electron microscopy image of the multifunctional nano-therapeutic agent BPP nanoparticles.

[0042] Figure 3 Detail transmission electron microscopy image of the multifunctional nano-therapeutic agent BPP nanoparticles.

[0043] Figure 4 Transmission electron microscopy image of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm.

[0044] Figure 5 Detail transmission electron microscopy image of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm.

[0045] Figure 6 Detection results of the photothermal conversion efficiency of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm in water. Among them, curve (a) is the heating and cooling curve of mBPP nanoparticles, curve (b) is the heating and cooling curve of deionized water, and the regression line (c) is the relationship between the -ln value of the temperature drop of mBPP nanoparticles and time.

[0046] Figure 7 Mechanism diagram of apoptosis of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm analyzed by flow cytometry at different concentrations (0 μM, 10 μM, 20 μM, 30 μM).

[0047] Figure 8 Endocytosis of HeLa cells observed by fluorescence confocal microscopy for the multifunctional therapeutic agent BPP nanoparticles and the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm.

[0048] Figure 9 Immune escape of endocytosis of RAW264.7 cells observed by fluorescence confocal microscopy for the multifunctional therapeutic agent BPP nanoparticles and the multifunctional nano-therapeutic agent mBPP nanoparticles modified with the biomimetic biofilm.

[0049] Figure 10Inhibitory angiogenesis experiment of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with biomimetic biofilm detected by human umbilical vein endothelial cells.

[0050] Figure 11 Change process of the tumor volume of nude mice treated with the multifunctional nano-therapeutic agent mBPP nanoparticles modified with biomimetic biofilm, where the abscissa represents time and the ordinate represents tumor volume.

[0051] Figure 12 Anatomical tumor size of nude mice after treatment with the multifunctional nano-therapeutic agent mBPP nanoparticles modified with biomimetic biofilm. Detailed implementation mode

[0052] The technical solutions of the present invention will be further described below through examples to better understand the content of the present invention.

[0053] Example 1

[0054] Synthesis of BPA

[0055] Under a nitrogen atmosphere, 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid (0.68 g, 2.00 mmol), DPP-1 (0.30 g, 0.40 mmol), tetrakis(triphenylphosphine)palladium (0.026 g, 0.0225 mmol) and tripotassium phosphate (0.34 g, 1.60 mmol) were added to 1,4-dioxane (80 mL) and refluxed at 100 °C for 24 h; after removing the solvent, the crude product was obtained. The crude product was separated and purified by a silica gel chromatographic column (the eluent was petroleum ether:dichloromethane = 1:2 V / V) to obtain 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (BPA) with a yield of 96%.

[0056] NMR of BPA 1 The 1H-NMR spectrum (400 MHz, CDCl3) is shown in Figure 1 . 11H NMR (400 MHz, CDCl3, ppm): δ 8.22 (d, J = 3.8 Hz, 2H), 7.84 (d, J = 8.4 Hz, 4H), 7.75 (d, J = 8.2 Hz, 2H), 7.50 - 7.36 (m, 8H), 7.34 - 7.25 (m, 4H), 7.22 - 7.14 (m, 4H), 7.10 - 7.03 (m, 4H), 6.99 - 6.92 (m, 2H), 6.91 - 6.85 (m, 4H), 6.69 (d, J = 3.8 Hz, 2H), 4.05 (t, J = 7.3 Hz, 4H), 3.21 (t, J = 6.8 Hz, 4H), 1.68 (t, J = 6.9 Hz, 8H), 1.39 - 1.26 (m, 8H) 13 13C NMR (100 MHz, CDCl3, ppm): 148.1, 146.2, 142.2, 141.6, 134.3, 130.9, 130.02, 128.3, 128.0, 127.85, 127.5, 127.3, 126.4, 126.0, 125.7, 125.4, 125.4, 125.1, 125.0, 124.4, 122.9, 122.2, 122.1, 120.8, 120.6, 119.2, 115.1, 106.7, 41.3, 32.7, 31.6, 29.1, 26.9, 25.2.

[0057] Preparation of BPP nanoparticles (BPP NPs)

[0058] 1 mg of DSPE - mPEG2000 was ultrasonically dissolved in 5 mL of deionized water to obtain a DSPE - mPEG2000 solution; 2 mg of BPA and 2 mg of PCA were dissolved in 500 μL of THF to obtain a mixed solution; while maintaining the DSPE - mPEG2000 solution in a state of rapid stirring (rotation speed of 1000 revolutions per minute), the mixed solution was slowly added dropwise to the DSPE - mPEG2000 solution at a dropping rate of 20 drops per minute; after the addition was completed, stirring was continued for 5 minutes, and nitrogen was bubbled thoroughly to remove tetrahydrofuran in the solution, and then centrifuged, and the supernatant was taken to obtain an aqueous dispersion of multifunctional nano - therapeutic agent BPP nanoparticles (BPP NPs), and the concentration of BPP nanoparticles was 200 μg / mL.

[0059] Preparation of mBPP nanoparticles (mBPP NPs)

[0060] 5×10 collected by culturing 7One HeLa cell was suspended in 1 mL of membrane protein extraction buffer A (Beyotime cell membrane and cytoplasmic protein extraction kit) containing phenylmethylsulfonyl fluoride (1 mM). The mixture was incubated in an ice bath for 15 min to lyse the cells. Subsequently, the cell suspension was frozen in liquid nitrogen and thawed at room temperature. The freeze-thaw cycle was repeated twice. At 4 °C, the cell suspension was centrifuged at 700 g for 10 min to remove the precipitate. The resulting supernatant was further centrifuged at high speed at 4 °C for 30 min, and the precipitate was collected, which was the tumor cell membrane fragments.

[0061] The HeLa cancer cell membrane was dissolved in deionized water (concentration: 200 μg / mL), and ultrasonicated at 400 W for 4 min to obtain the HeLa cancer cell membrane solution. At 4 °C, the stirring rate of the maintained HeLa cancer cell membrane solution was 200 revolutions per minute. According to the mass ratio of tumor cell membrane to BPP nanoparticles of 1:1, the HeLa cancer cell membrane solution was slowly added dropwise to the stirred (rotation speed: 200 revolutions per minute) BPP nanoparticle aqueous dispersion (concentration: 200 μg / mL) at a dropping rate of 20 drops per minute. After the dropping was completed, it was incubated in the dark at a stirring rate of 200 revolutions per minute and a temperature of 4 °C for 12 h, centrifuged, and the supernatant was taken to obtain the multifunctional nano-therapeutic agent mBPP nanoparticles (mBPP NPs) modified with biomimetic biomembrane.

[0062] The transmission electron micrograph of the multifunctional nano-therapeutic agent BPP nanoparticles is shown in Figure 2 and Figure 3 , with an average size of 90 nm; the transmission electron micrograph of the multifunctional nano-therapeutic agent mBPP nanoparticles modified with biomimetic biomembrane is shown in Figure 4 and Figure 5 , with an average size of 101 nm. It can be clearly found through the detailed comparison of the transmission electron microscopy that the multifunctional nano-therapeutic agent mBPP nanoparticles modified with biomimetic biomembrane have an obvious core-shell structure, and the average size increases by about 10 nm.

[0063] Example 2

[0064] Detection of singlet oxygen and photothermal conversion efficiency of mBPP nanoparticles

[0065] The mBPP nanoparticle aqueous dispersion was irradiated with a 660 nm laser (1.3 W / cm 2 ) for 10 minutes, and deionized water was used as a blank control under the same conditions. The results are shown in Figure 6 . When the temperature reached equilibrium, it was cooled at room temperature. The significant increase in temperature indicated that the mBPP nanoparticles had extremely strong photothermal conversion ability.

[0066] Example 3

[0067] In vitro cytotoxicity detection of mBPP nanoparticles

[0068] HeLa tumor cells (purchased from GIBCO) were selected for in vitro quantitative toxicity experiments to test the laser toxicity of mBPP nanoparticles.

[0069] The specific experimental steps were as follows: mBPP nanoparticles were diluted with DMEM medium to different concentrations (0 μM, 10 μM, 20 μM, 30 μM). HeLa tumor cells were inoculated in a 6-well culture plate and cultured at 37 °C for 24 hours to allow them to adhere and grow. The drug was added under light protection (500 μL), and the cells were cultured for 24 hours under light protection. Each well was irradiated with a 660 nm laser for 5 minutes (1.3 W / cm 2 ), and the cells were cultured for another 12 hours. Then, the cells were stained with Annexin V-FITC and propidium iodide (PI). An absolute cell survival rate was calculated using a flow cytometer. As Figure 7 shown, the cell survival rate showed an inverse concentration dependence, and the higher the drug concentration, the lower the cell survival rate. The IC 50 value was approximately 23 μM.

[0070] Example 4

[0071] In vitro cell uptake experiment and immune escape experiment of mBPP nanoparticles

[0072] HeLa tumor cells (purchased from GIBCO) were selected for in vitro cell uptake experiments. The experimental steps were as follows: HeLa tumor cells were inoculated into two confocal dishes. After the cells adhered, the culture medium was changed to DMEM medium containing BPP nanoparticles (concentration of 23 μM) and DMEM medium containing mBPP nanoparticles (concentration of IC 50 concentration 23 μM), respectively. After incubation for 4 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, and stained with DAPI. After thorough rinsing with PBS, the cell uptake was observed under a confocal microscope. As Figure 8 shown, compared with HeLa cells treated with BPP nanoparticles, HeLa cells treated with mBPP nanoparticles showed more obvious red fluorescence, indicating that mBPP nanoparticles modified with cancer cell membranes had stronger targeting to the same type of cells.

[0073] Mouse monocyte-macrophage leukemia cells (RAW264.7) (purchased from GIBCO) were selected for in vitro immune escape experiments. The experimental steps were as follows: RAW264.7 cells were seeded into two confocal dishes. After the cells adhered, the DMEM medium was changed to DMEM medium containing BPP nanoparticles (concentration: 30 μM) and DMEM medium containing mBPP nanoparticles (concentration: 30 μM), respectively. After incubation for 12 hours, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, stained with DAPI, and thoroughly rinsed with PBS. Then, the uptake of immune cells was observed under a confocal microscope. As Figure 9 shown, RAW264.7 immune cells treated with mBPP nanoparticles showed a weaker red fluorescence signal, indicating that mBPP nanoparticles could be less taken up by immune cells, and the immune escape ability of the cancer cell membrane was fully demonstrated.

[0074] Example 5

[0075] Experiment for detecting the inhibition of mBPP nanoparticles on angiogenesis

[0076] Matrigel was thawed on ice and dropped onto pre-cooled angiogenesis slides (10 μL / well), and incubated at 37 °C for 1 h. Then, human umbilical vein endothelial cells (HUVECs, 2×10 5 cells / well) were mixed with mBPP nanoparticles at different concentrations (0, 10, 20, 30 μM) prepared with DMEM medium and then seeded. The cells were cultured in a carbon dioxide incubator, and the vascular structure of endothelial cells was observed and photographed under an inverted fluorescence microscope. As Figure 10 shown, in the blank group (0 μM), blood vessels gradually formed a dense vascular network over time, while the formation of the vascular network in the drug-administered groups was significantly inhibited and showed a concentration-dependent manner.

[0077] Example 6

[0078] In vivo tumor therapeutic performance of mBPP nanoparticles

[0079] Nude mice with HeLa tumor cells injected into the armpit were selected as tumor models. Twenty-four nude mice were randomly divided into 6 groups. When the tumor volume was approximately 100 mm 3At that time, the first group (Control) was intravenously injected with normal saline through the tail vein, the second group (BPA NPs) of mice was intravenously injected with BPA nanoparticles (100 μg / mL, 100 μL, prepared with normal saline), the third group (BPA nanoparticle laser irradiation group, BPA NPs+L) of mice was intravenously injected with BPA nanoparticles (100 μg / mL, 100 μL), the fourth group (PCA group) of mice was intravenously injected with PCA solution (100 μg / mL, 100 μL, prepared with normal saline), the fifth group (mBPP NPs) of mice was intravenously injected with mBPP nanoparticles (100 μg / mL, 100 μL, prepared with normal saline), and the sixth group (mBPP nanoparticle laser irradiation group, mBPP NPs+L) of mice was intravenously injected with mBPP nanoparticles (100 μg / mL, 100 μL). 12 hours later, the tumors of the mice in the third and sixth groups were irradiated with a 660 nm (1 W / cm 2 ) laser for 5 minutes each, and the other groups were not irradiated. The tumor size was measured every 2 days, and the results are as Figure 11 shown. The tumor tissue was basically eliminated in the sixth group on the 4th day. The mice were sacrificed after 14 days, and the tumor tissue was observed and measured. As Figure 12 shown, the sixth group could effectively and completely eliminate the tumor, and there were residues to varying degrees in the other groups.

Claims

1. A multifunctional nano-therapeutic reagent modified with a biomimetic biofilm, characterized in that: It is a multifunctional nano-therapeutic agent for modifying the cell membrane of tumor cells; the multifunctional nano-therapeutic agent is a BPP nanoparticle self-assembled from 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione and protocatechuic acid under the action of distearoyl phosphatidylethanolamine-polyethylene glycol, with a structure as shown in Formula I; 2. A method for preparing the multifunctional nano-therapeutic reagent modified with the bionic biofilm according to claim 1, characterized in that: Including: Step (1): Under a nitrogen atmosphere, 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid, bis(3-(5-bromofuran-2-yl))-5-bis(6-(4-bromohexyl))pyrrolopyrrole(2,5)-1,4-dione, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) and tripotassium phosphate are added to 1,4-dioxane, heated and stirred; after removing the solvent, a crude product is obtained, and the crude product is separated to obtain 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione; Step (2): 2,5-bis(6-bromohexyl)-3,6-bis(5-(4-(naphthalen-1-yl(phenyl)amino)phenyl)furan-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione and protocatechuic acid are dissolved in tetrahydrofuran to obtain a mixed solution; under stirring, the mixed solution is added dropwise to deionized water containing distearoyl phosphatidylethanolamine-polyethylene glycol. After the addition is complete, stirring is continued for 5-10 minutes, and nitrogen is bubbled sufficiently to remove tetrahydrofuran. After centrifugation, the supernatant is taken to obtain a BPP nanoparticle aqueous dispersion; Step (3): The tumor cell membrane is dissolved in deionized water and ultrasonically broken to obtain a tumor cell membrane solution; under stirring, the tumor cell membrane solution is added dropwise to the BPP nanoparticle aqueous dispersion, incubated in the dark, centrifuged, and the supernatant is taken to obtain a multifunctional nano-therapeutic agent mBPP nanoparticle modified with a biomimetic biological membrane.

3. The preparation method of the multifunctional nano-therapeutic reagent modified with the biomimetic biofilm according to claim 2, wherein: In step (1), the molar ratio of bis(3-(5-bromofuran-2-yl))-5-bis(6-(4-bromohexyl))pyrrolopyrrole(2,5)-1,4-dione to 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid is 1:2 - 1:10; the molar ratio of bis(3-(5-bromofuran-2-yl))-5-bis(6-(4-bromohexyl))pyrrolopyrrole(2,5)-1,4-dione to tetrakis(triphenylphosphine)palladium is 1:0.01 - 1:0.1; the molar ratio of bis(3-(5-bromofuran-2-yl))-5-bis(6-(4-bromohexyl))pyrrolopyrrole(2,5)-1,4-dione to tripotassium phosphate is 1:2 - 1:

10.

4. The preparation method of the multifunctional nano-therapeutic reagent modified with the bionic biofilm according to claim 3, characterized in that: In step (1), the molar ratio of bis(3-5-bromofuran-2-yl)-5-bis(6-4-bromohexyl)pyrrolopyrrole(2,5)-1,4-dione to 4-(N-(naphthalen-1-yl)-N-phenylamino)phenylboronic acid is 1:5; the molar ratio of bis(3-5-bromofuran-2-yl)-5-bis(6-4-bromohexyl)pyrrolopyrrole(2,5)-1,4-dione to tetrakis(triphenylphosphine)palladium is 1:0.05 - 1:0.06; the molar ratio of bis(3-5-bromofuran-2-yl)-5-bis(6-4-bromohexyl)pyrrolopyrrole(2,5)-1,4-dione to tripotassium phosphate is 1:

4.

5. The preparation method of the multifunctional nano-therapeutic reagent modified with the bionic biofilm according to claim 2, characterized in that: In step (1), the temperature of the heating and stirring is 25 - 120 °C, and the time of the heating and stirring is 8 - 36 hours.

6. The preparation method of the multifunctional nano-therapeutic reagent modified with the biomimetic biofilm according to claim 5, characterized in that: In step (1), the temperature of the heating and stirring is 90 - 100 °C, and the time of the heating and stirring is 12 - 24 hours.

7. The preparation method of the multifunctional nano-therapeutic reagent modified with the bionic biofilm according to claim 2, characterized in that: In step (2), the mass ratio of BPA, PCA and DSPE-mPEG2000 is 1:1:0.5; the concentration of DSPE-mPEG2000 in deionized water is 0.1 - 0.5 mg / mL.

8. The preparation method of the multifunctional nano-therapeutic reagent modified with the biomimetic biofilm according to claim 2, wherein: In step (3), the cell membrane concentration in the tumor cell membrane solution is 0.1 - 1 mg / mL; the mass ratio of the tumor cell membrane to the BPP nanoparticles is 1:1; The light-shielded incubation is carried out under stirring, the stirring rate of the incubation is 200 - 500 revolutions per minute, the incubation temperature is 4 - 8 °C, and the incubation time is 8 - 24 hours.

9. Use of the multifunctional nano-therapeutic agent modified with the biomimetic biofilm according to claim 1 in the preparation of a drug for synergistic treatment of tumors by phototherapy, chemotherapy and anti-angiogenesis.

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