A bionic nanocarrier system responsive to active oxygen, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to an active oxygen responsive bionic nanocarrier system, a preparation method and an application thereof. The active oxygen responsive bionic nanocarrier system provided by the present invention is in a core-shell structure, wherein the core is a nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6, and the shell is a cell membrane. After entering the body, the active oxygen responsive bionic nanocarrier of the present invention has a red-cancer cell fusion membrane coating on the surface, so that its circulation time in the body can be prolonged and homologously targeted to the tumor site, and further endocytosed and taken up by the tumor cells. Under the irradiation of 660nm infrared light, active oxygen is generated by the photosensitizer Ce6 to kill tumor cells, while triggering the core to disintegrate and quickly release Oxa, further killing tumor cells, improving the anti-tumor effect of Oxa, reducing toxic side effects, and having an application prospect of chemotherapy-photodynamic combined treatment of colon cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an active oxygen responsive bionic nanocarrier system, a preparation method and an application thereof. Background Art

[0002] Colon cancer is one of the common malignant tumors of the digestive system, which seriously endangers human health. At present, chemotherapy is still one of the main methods for treating patients with colon cancer. Commonly used first-line chemotherapy drugs include oxaliplatin, 5-fluorouracil, irinotecan, etc. Since chemotherapy drugs are not very selective for tumors, while killing tumor cells, they will also attack other normal cells in the body, making chemotherapy drugs unable to exert the best anti-tumor effect and causing serious toxic side effects. Therefore, it is necessary to develop a drug for the treatment of colon cancer with strong targeting and few side effects. Summary of the invention

[0003] In view of the problems existing in the prior art, the purpose of the present invention is to provide an active oxygen responsive bionic nanocarrier system, a preparation method and an application. The active oxygen responsive bionic nanocarrier system provided by the present invention can achieve long-term circulation of nanodrug particles in the body and homologously target the tumor site, enhance the enrichment and retention of nanodrug particles in the tumor site, and has a high application prospect for chemotherapy-photodynamic synergistic treatment of colon cancer.

[0004] Based on the above purpose, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides an active oxygen responsive bionic nanocarrier system, which is a core-shell structure, wherein the core is a nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6, and the shell is a cell membrane.

[0006] The active oxygen-responsive erythrocyte membrane co-loaded with oxaliplatin and dihydrochlorin e6 of the present invention is fused with the homologous HCT116 cell membrane. Under the irradiation of 660nm infrared light, the photosensitizer dihydrochlorin e6 generates active oxygen to kill tumor cells, while triggering the inner core to disintegrate and quickly release oxaliplatin, further killing tumor cells, thereby improving the anti-tumor effect of oxaliplatin and reducing toxic side effects.

[0007] Preferably, the cell membrane is one of a red blood cell membrane, a HCT116 cell membrane, a CT26 cell membrane, a fusion membrane formed by a red blood cell membrane and a HCT116 cell membrane, and a fusion membrane formed by a red blood cell membrane and a CT26 cell membrane.

[0008] The reactive oxygen-responsive cell membrane biomimetic nanocarriers co-loaded with oxaliplatin (Oxa) and dihydrochlorin e6 (Ce6) can achieve long-term circulation of nanodrug particles in vivo and homologously target the tumor site, enhance the enrichment and retention of nanodrug particles in the tumor site, inhibit the occurrence and development of the tumor, and have low biological toxicity.

[0009] In a second aspect, the present invention provides a method for preparing an active oxygen responsive biomimetic nanocarrier system, comprising the following steps:

[0010] (1) mixing hydrophobic oxaliplatin and / or dihydrochlorin e6 with TK-PPE, and preparing a nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6 by a nanoprecipitation method;

[0011] (2) The nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6 is mixed with the cell membrane, and the active oxygen responsive biomimetic nanocarrier system with the cell membrane encapsulating the nanocore is prepared by a membrane extrusion method.

[0012] Preferably, the nanoprecipitation method comprises the following steps: uniformly dispersing hydrophobic oxaliplatin and / or dihydrochlorin e6 and TK-PPE in dimethyl sulfoxide to prepare a mixed solution, and then adding the mixed solution dropwise into water to form a nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6.

[0013] Preferably, the membrane extrusion method comprises the following steps: uniformly mixing the nanocore loaded with hydrophobic oxaliplatin and / or dihydrochlorin e6 with the cell membrane, extruding through a polycarbonate membrane with a pore size of 200nm to 400nm, and then centrifuging to obtain an active oxygen responsive biomimetic nanocarrier system with the cell membrane encapsulating the nanocore.

[0014] Preferably, the hydrophobic oxaliplatin is prepared by the following method, which comprises the following steps:

[0015] (1) dispersing oxaliplatin in hydrogen peroxide, oxidizing and drying to obtain oxidized oxaliplatin;

[0016] (2) The oxidized oxaliplatin and lauric anhydride are dissolved in N,N-dimethylformamide, stirred and reacted at 100° C. to 120° C., and then recrystallized to obtain hydrophobic oxaliplatin.

[0017] The encapsulation of the drug is through the hydrophilic-hydrophobic interaction. If it is a hydrophilic drug, it is directly dissolved in water and is not a nano preparation. Therefore, oxaliplatin must be hydrophobized before it can form a nano preparation. The present invention adopts the hydrophobic treatment of grafting alkyl groups on both sides of oxaliplatin. This is because the valence of the platinum element of oxaliplatin is divalent. If alkyl groups are connected to both sides, platinum needs to be oxidized to tetravalent, so it must be oxidized first, and then esterification reaction is carried out to connect alkyl groups to complete hydrophobic treatment. In addition, if oxaliplatin that is not completely hydrophobized is contained, the drug loading rate of the drug will be reduced, and it is best that the hydrophobic reaction is complete to achieve optimal drug loading efficiency.

[0018] The present invention adopts a method of subjecting oxaliplatin to hydrophobic treatment after oxidation, thereby achieving cell membrane coating of oxaliplatin while maintaining the efficacy of oxaliplatin, which helps the coated oxaliplatin nanoparticles to circulate for a long time in the body and homologously target the tumor site, thereby enhancing the enrichment and retention of the nanoparticles at the tumor site.

[0019] In a third aspect, the present invention provides the use of an active oxygen responsive bionic nanocarrier system in the preparation of a drug for treating colon cancer.

[0020] In a fourth aspect, the present invention provides a drug for treating colon cancer, the drug comprising an active oxygen responsive bionic nanocarrier system.

[0021] Preferably, the active oxygen responsive bionic nanocarrier system is a fusion membrane formed by an erythrocyte membrane and an HCT116 cell membrane, which encapsulates and simultaneously loads a hydrophobic oxaliplatin and a dihydrochlorin e6 nanocore to form a nanocarrier system;

[0022] Or for,

[0023] The fusion membrane formed by the red blood cell membrane and the HCT116 cell membrane encapsulates the hydrophobic oxaliplatin-loaded nanocore to form a nanocarrier system;

[0024] Or for,

[0025] The fusion membrane formed by the red blood cell membrane and the HCT116 cell membrane encapsulates the dihydrochlorin e6 nanocore to form a nanocarrier system;

[0026] Or for,

[0027] The red blood cell membrane wraps and loads the hydrophobic oxaliplatin and dihydrochlorin e6 nanocore to form a nanocarrier system;

[0028] Or for,

[0029] The HCT116 cell membrane is wrapped and loaded with hydrophobic oxaliplatin and dihydrochlorin e6 nanocore to form a nanocarrier system;

[0030] Or for,

[0031] The fusion membrane formed by the red blood cell membrane and the CT26 cell membrane encapsulates and simultaneously loads the hydrophobic oxaliplatin and dihydrochlorin e6 nanocore to form a nanocarrier system;

[0032] Or for,

[0033] The fusion membrane formed by the red blood cell membrane and the CT26 cell membrane encapsulates the hydrophobic oxaliplatin-loaded nanocore to form a nanocarrier system;

[0034] Or for,

[0035] The fusion membrane formed by the red blood cell membrane and the CT26 cell membrane encapsulates the loaded dihydrochlorin e6 nanocore to form a nanocarrier system;

[0036] Or for,

[0037] A mixture of two nanocarrier systems, wherein the two nanocarrier systems are: a) a nanocarrier system formed by a fusion membrane formed by an erythrocyte membrane and a CT26 cell membrane, encapsulating a hydrophobic oxaliplatin nanocore; b) a nanocarrier system formed by a fusion membrane formed by an erythrocyte membrane and a CT26 cell membrane, encapsulating a dihydrochlorin e6 nanocore;

[0038] Or for,

[0039] A mixture of two nanocarrier systems, wherein the two nanocarrier systems are: a) a nanocarrier system formed by a fusion membrane formed by an erythrocyte membrane and an HCT116 cell membrane, which wraps a hydrophobic oxaliplatin nanocore; b) a nanocarrier system formed by a fusion membrane formed by an erythrocyte membrane and an HCT116 cell membrane, which wraps a dihydrochlorin e6 nanocore.

[0040] Anti-tumor experiments on tumor-bearing mice revealed that the above-mentioned nanocarrier system has an inhibitory effect on the growth of colon cancer tumors.

[0041] Preferably, the active oxygen responsive bionic nanocarrier system is a fusion membrane formed by an erythrocyte membrane and an HCT116 cell membrane, which encapsulates and simultaneously loads a hydrophobic oxaliplatin and a dihydrochlorin e6 nanocore to form a nanocarrier system.

[0042] Anti-tumor experiments on tumor-bearing mice revealed that this nanocarrier system has the best inhibitory effect on colon cancer tumor growth and has good advantages in achieving chemotherapy-photodynamic combined treatment of colon cancer.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The active oxygen-responsive erythrocyte membrane co-loaded with oxaliplatin (Oxa) and dihydrochlorin e6 (Ce6) and the homologous HCT116 cell membrane fusion membrane biomimetic nanocarrier hNPOC of the present invention can achieve long-term circulation of nanoparticles in vivo and homologously target to tumor sites, thereby enhancing the enrichment and retention of nanocarriers at tumor sites.

[0045] After entering the body, the active oxygen responsive biomimetic nanocarrier of the present invention has a red-cancer cell fusion membrane coating on its surface, which can prolong its circulation time in the body and homologously target the tumor site, and further be internalized and taken up by tumor cells. Under the irradiation of 660nm infrared light, while the photosensitizer Ce6 generates active oxygen to kill tumor cells, it triggers the inner core to disintegrate and quickly release Oxa, further killing tumor cells, improving the anti-tumor effect of Oxa, and reducing toxic side effects. It has an application prospect of chemotherapy-photodynamic combined treatment of colon cancer, and provides a nanomedicine reference for the treatment of colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 for Figure 1 The particle size distribution and transmission electron microscopy images of NPOC and hNPOC nanoparticles;

[0047] Figure 2 Surface potential diagrams of NPOC, hNPOC and hM;

[0048] Figure 3 is the micellar stability diagram of hNPOC;

[0049] Figure 4 This is a diagram of membrane protein analysis of hNPOC;

[0050] Figure 5 is the UV absorption spectra of NPOC and hNPOC;

[0051] Figure 6 is the drug release curve of hNPOC under illumination or non-illumination conditions;

[0052] Figure 7 The uptake graphs of rNPOC or hNPOC by HCT116 tumor cells after incubation for different time periods;

[0053] Figure 8 Figure 2 is the uptake graph of hNPOC by syngeneic HCT116 tumor cells or heterogeneous CT26 tumor cells after incubation for different time periods;

[0054] Fig. 9 Confocal fluorescence imaging of homologous HCT116 cells or heterologous CT26 cells incubated with hNPOC for 4 hours, compared with HCT116 cells incubated with rNPOC for 4 hours as a control group;

[0055] Fig.10 is the uptake graph of NPOC, rNPOC, cNPOC or hNPOC by RAW264.7 cells after 4 h of incubation;

[0056] Fig.11 Fluorescence imaging of hNPOC enrichment and retention in the tumor sites of mice bearing syngeneic HCT116 cells or heterogeneous CT26 cells;

[0057] Fig.12 The pharmacokinetic curves of free Ce6, rNPOC, cNPOC and hNPOC in vivo;

[0058] Fig.13 It is a tumor growth curve of hNPOC anti-tumor treatment;

[0059] Fig.14 This is a curve chart of body weight changes of mice in each experimental group during the in vivo treatment experiment. DETAILED DESCRIPTION

[0060] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0061] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0062] Example 1 Extraction and fusion of erythrocyte membrane and cancer cell membrane

[0063] 1. Extraction of red blood cell membrane (hypotonic lysis method)

[0064] (1) Collect blood and wash red blood cells

[0065] Collect the blood sample into a heparinized container, centrifuge at 2000rpm for 10 minutes at 4℃, and remove the supernatant and white villi (leukocytes, etc.) with a pipette. Suspend the packed red blood cells with 10 times the volume of 4℃ isotonic buffer, and repeat the above centrifugation steps 2-3 times until the supernatant is colorless and there is no white villi layer.

[0066] (2) Preparation of cell membrane

[0067] Add 10 times the volume of hypotonic buffer to the above packed red blood cells, mix well, and place at 4°C for 20 minutes until the cell suspension changes from bright red to dark red and is transparent under fluorescent light. Centrifuge at 12000rpm for 30 minutes at 4°C, remove the dark red supernatant, add 10 times the volume of hypotonic buffer to the precipitate, mix well, and repeat the above centrifugation steps 2-3 times until the precipitate is milky white. Discard the supernatant and the brown fibrin-like precipitate at the bottom of the centrifuge tube, and collect the white or pink blood film.

[0068] (3) Cell membrane preservation and identification

[0069] Suspend the blood ghost membrane with a certain volume of hypotonic buffer, mix well, and determine the protein content with a BCA kit. Dilute it into a suspension with a certain protein content as needed, quantitatively dispense it into small containers, and store it at -80°C for later use, thereby obtaining the red blood cell membrane (rM).

[0070] 2. Extraction of cancer cell membrane (homogenization, differential centrifugation)

[0071] (1) Collect cells

[0072] Take the cultured HCT116 cells, discard the old culture medium, wash once with PBS, treat with 3 mL EDTA (0.05%) digestion solution to make the cells no longer adhere to the wall tightly, and blow the cells off with a pipette. Centrifuge (800 rpm, 5 min) the cell suspension, discard the supernatant and keep the cell pellet.

[0073] (2) Washing cells

[0074] Resuspend the cells in ice-cold PBS, take a small amount of cells for counting, and centrifuge the remaining cells at 4°C, 600g for 5 minutes to precipitate the cells. Discard the supernatant, then centrifuge at 4°C, 600g for 1 minute to further precipitate the cells, and aspirate all the residual liquid.

[0075] (3) Cell pretreatment

[0076] Take 1 mL of membrane protein extraction reagent A with PMSF added to the cell pellet, gently and fully suspend the cells, and place on ice for 10-15 minutes.

[0077] (4) Cell sample disruption and disruption effect identification

[0078] Transfer the cell suspension to a homogenizer of appropriate size and homogenize for 20 minutes (6000rpm, 10 cycles×20s, pause 10s). Usually, you can take about 2-3 microliters of cell homogenate after homogenization and drop it on the cover glass and observe it under a microscope. If you see a perinuclear halo or complete cell morphology, it means that the cells are still intact. If 70%-80% of the cells have no perinuclear halo and complete cell morphology, it means that the cells have been fully broken and the next step of the experiment can be carried out. Otherwise, re-homogenize 10-30 times until at least 70% of the cells have been broken.

[0079] (5) Removal of cell nuclei and unbroken cells

[0080] Centrifuge at 4°C, 700g for 10 minutes, and carefully collect the supernatant into a new 2mL centrifuge tube (do not touch the precipitate when aspirating the supernatant, and do not aspirate about 30-50μL of supernatant remaining to ensure that the aspirated supernatant has a higher purity).

[0081] (6) Precipitate cell membrane fragments

[0082] Centrifuge at 4°C, 14000g for 30 minutes to precipitate cell membrane fragments, mix the cell membrane with a certain volume of water, and determine the protein content using a BCA kit. Dilute to a suspension with a certain protein content as needed, quantitatively dispense into small containers, and store at -80°C for later use.

[0083] HCT116 cell membrane (cM) was obtained by the method in steps (1) to (6) above.

[0084] The heterologous colon cancer cell CT26 cell membrane (CT26-M) was obtained from colon cancer cell CT26 cells by referring to the method of steps (1) to (6) above.

[0085] 3. Fusion of red blood cell membrane and cancer cell membrane

[0086] The red blood cell membrane and the cancer cell membrane, i.e., HCT116 cell membrane, were mixed at a mass ratio of 1:1, and then ultrasonicated (130 W, 10 min) and sequentially extruded through 800 nm and 400 nm polycarbonate membranes to obtain a fusion membrane (hM).

[0087] The red blood cell membrane and the cancer cell membrane, i.e., CT26 cell membrane, were mixed at a mass ratio of 1:1, and then extruded through 800 nm and 400 nm polycarbonate membranes in sequence after ultrasound (130 W, 10 min) to prepare a fusion membrane of heterologous CT26 cell membrane and red blood cell membrane (CT26-hM).

[0088] The protein content of the prepared fusion membrane was determined using a BCA kit.

[0089] Example 2 Preparation of Oxa-la

[0090] 1. Oxidation of Oxa

[0091] 0.5 g, i.e., 1.26 mmol, of oxaliplatin (Oxa) was weighed and dissolved in 30% hydrogen peroxide solution (20 mL), stirred at 50° C. for overnight reaction, and freeze-dried to obtain oxidized oxaliplatin (Oxa-OH).

[0092] 2. Preparation of Oxa-la

[0093] Weigh the oxidized oxaliplatin (0.245 g, 0.57 mmol) and lauric anhydride (0.445 g, 1.16 mmol) and dissolve them in 10 mL of anhydrous N, N-dimethylformamide, and stir the reaction at 100-120° C., preferably 110° C., for 4-6 hours, preferably 4 hours. After the reaction is completed, the solution is recrystallized, preferably at -20° C., and the precipitate is collected, washed with deionized water 3 times, and freeze-dried to obtain the product hydrophobic oxaliplatin (Oxa-la, 0.394 g, 57%).

[0094] Example 3 Preparation of NPOC, hNPOC

[0095] 1. Preparation of NPOC (nanoprecipitation method)

[0096] Weigh the active oxygen responsive polyphosphate material TK-PPE (2.5 mg), Oxa-la (wherein the metal platinum is 0.25 mg) and dihydrochlorin e6, i.e. Ce6 (0.25 mg) and dissolve them in 0.6 mL of DMSO. After mixing, add them dropwise to 2.5 mL of deionized water under stirring conditions and stir for 2 hours. The particle solution is placed in a MWCO = 3500Da dialysis bag and dialyzed overnight to remove DMSO. Finally, the excess drug is filtered out through a 0.45 μm filter to obtain a nano-core NPOC.

[0097] As a control, Oxa-la and Ce6 were loaded into the active oxygen responsive polyphosphate material TK-PPE according to the above method to obtain the Oxa-la loaded nanocore NPO and the Ce6 loaded nanocore NPC, respectively.

[0098] 2. Preparation of hNPOC nanocarriers

[0099] hNPOC was prepared by extrusion method, the specific method is as follows:

[0100] The NPOC particle solution (1 ml, 1 mg / mL) was mixed with the hM fusion membrane solution (1 ml, 1 mg / mL), sonicated for 2 min (130 W) in an ice bath, and then sequentially extruded through 400 nm and 200 nm polycarbonate membranes, and finally centrifuged at high speed (18000 g, 10 min) to obtain the reactive oxygen species responsive biomimetic nanocarrier (hNPOC). The excess membrane was removed by washing with PBS three times, and the nanocarrier hNPOC was resuspended in deionized water for later use.

[0101] Referring to the preparation method of hNPOC nanocarrier, the hM fusion membrane was replaced with rM, cM, and CT26-hM in sequence, and the prepared nanocarriers were recorded as rNPOC, cNPOC or CT26-hNPOC in sequence.

[0102] Referring to the preparation method of hNPOC nanocarrier, NPO particle solution (1 ml, 1 mg / mL) was mixed with hM fusion membrane solution (1 ml, 1 mg / mL), and hM fusion membrane-wrapped NPO nanocarrier was prepared according to the above method, which was recorded as hNPO.

[0103] Referring to the preparation method of hNPOC nanocarrier, NPC particle solution (1 ml, 1 mg / mL) was mixed with hM fusion membrane solution (1 ml, 1 mg / mL), and hM fusion membrane-wrapped NPC nanocarrier was prepared according to the above method, which was recorded as hNPC.

[0104] The mixture formed by mixing two nanocarriers hNPO and hNPC at the same drug concentration was recorded as hNPO / hNPC.

[0105] Example 4 Application of hNPOC

[0106] 1. Characteristics of hNPOC nanocarriers

[0107] (1) Cell membrane successfully coated on the surface of nanocarrier

[0108] Two nanoparticles, NPOC and hNPOC, were obtained by nanoprecipitation, and the particle size, potential, and morphology of the nanoparticles were observed by dynamic light scattering (DLS) and transmission electron microscope (TEM). Furthermore, cell membrane proteins were characterized by polyacrylamide gel electrophoresis and protein blotting.

[0109] like Figure 1As shown, the particle size of NPOC is about 96.9±0.8nm, and the particle size of hNPOC after cell membrane coating is about 120.4±3.6nm, which is consistent with the TEM detection results. In addition, the thickness of the cell membrane coating detected by TEM is about 16.5nm.

[0110] like Figure 2 As shown, after cell membrane coating, the surface potential of hNPOC changed from -22.5 mV to -33.9 mV, while the surface potential of hM was -35.9 mV, and the results of the two were consistent.

[0111] like Figure 3 As shown in the figure, hNPOC has a good stability. In the co-culture of 1×PBS and 1×PBS solution containing 10% fetal bovine serum for 48 hours, the particle size of hNPOC did not change significantly.

[0112] like Figure 4 As shown, hNPOC, like hM, successfully inherited most of the membrane proteins of rM and cM; Western blotting analysis showed that membrane proteins related to homologous targeting in cM (E-cadherin and Galectin-3) and membrane proteins related to the ability to reduce macrophage uptake in rM (CD47) were highly expressed in hNPOC.

[0113] (2) Drug loading and reactive oxygen species response performance of hNPOC

[0114] After ultraviolet spectral scanning, the absorption peaks of NPOC and hNPOC were compared with the absorption peak of free Ce6. 100 μL of hNPOC solution was taken and freeze-dried and then redissolved in 1 mL of DMSO to prepare the Ce6 standard curve solution. The absorbance of each particle sample of the standard curve was detected at 405 nm, and the drug concentration was calculated according to the standard curve. At the same time, a platinum (Pt) standard curve solution was prepared, and the concentration of Pt was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES). The loading content (LC) of nanoparticles encapsulating Pt or Ce6 was calculated by the following formula:

[0115] Drug loading (LC%) = (mass of Pt or Ce6 / total mass of nanocarrier) × 100%;

[0116] like Figure 5 As shown, the absorption peaks of hNPOC and NPOC were similar to that of free Ce6, and the drug loadings of Pt and Ce6 were 3.9±0.3% and 5.1±0.2%, respectively.

[0117] The hNPOC solution ([Pt] = 160.92 μg / mL, 1 mL) was irradiated with a 660 nm laser (0.3 W / cm2) for 1 h, and the same hNPOC solution was taken as a control group without illumination, and three parallel experiments were performed. The solution was then transferred to a dialysis bag (MWCO 14000 Da) soaked in 15 mL of phosphate buffer (PB buffer, 0.02 M, pH 7.4) and placed in a water bath shaker for shaking (37 ° C, 100 rpm). The PB buffer was collected at different time intervals and replaced with an equal amount of fresh PB buffer. The collected PB buffer was analyzed for platinum concentration using an inductively coupled plasma optical emission spectrometer (ICP-OES).

[0118] like Figure 6 As shown, Oxa can be rapidly released in hNPOC after light exposure.

[0119] 2. hNPOC uptake by cells

[0120] (1) Behavior of rNPOC and hNPOC uptake by HCT116 cells

[0121] Syngeneic HCT116 cells were seeded at a number of 8 × 10 4 Cells / well were added to a 24-well plate, 0.5 mL of DMEM (containing 10% FBS) was added, and the cells were incubated at 37°C, 5% CO2 for 12 h. The old culture medium was replaced with fresh DMEM medium (containing 10% FBS) containing rNPOC or hNPOC ([Ce6] = 5 μg / mL), and after 4 h of co-incubation, the cells were washed with PBS to remove extracellular particles, and the cell suspension obtained by digestion of the cells was then used to detect the Ce6 fluorescence intensity in the tumor cells by flow cytometry.

[0122] like Figure 7 As shown, compared with rNPOC, hNPOC showed more obvious cellular uptake at various time points of HCT116 cell incubation, proving that membrane protein molecules inherited from homologous cancer cells can enhance the homologous targeted uptake ability of hNPOC.

[0123] (2) hNPOC uptake by HCT116 cells or CT26 cells

[0124] Syngeneic HCT116 cells and heterogeneous CT26 cells were seeded at a number of 8×10 4Cells / well were added to a 24-well plate, 0.5 mL DMEM (containing 10% FBS) was added, and incubated for 12 h at 37°C and 5% CO2. The old culture medium was replaced with fresh DMEM culture medium (containing 10% FBS) containing hNPOC ([Ce6] = 5 μg / mL). After 4 h of co-incubation, the cells were washed with PBS to remove extracellular particles, and the cell suspension obtained by digestion of the cells was then used to detect the Ce6 fluorescence intensity in the tumor cells by flow cytometry.

[0125] like Figure 8 As shown, the intracellular fluorescence level of hNPOC cells co-incubated with heterologous CT26 was lower than that of syngeneic HCT116 cells, further confirming this homologous targeting effect.

[0126] Syngeneic HCT116 cells and heterogeneous CT26 cells were co-incubated with hNPOC for 4 h, and HCT116 cells co-incubated with rNPOC were used as a control group. Then, the intracellular fluorescence of Ce6 was observed using a confocal laser scanning microscope.

[0127] like Fig. 9 As shown, confocal laser scanning microscopy imaging also confirmed that hNPOC exhibited the best internalization efficiency when incubated with syngeneic HCT116 cells, which was consistent with the results obtained by flow cytometry.

[0128] (3) Uptake of NPOC, rNPOC, cNPOC, and hNPOC by RAW264.7 cells

[0129] Mouse macrophage RAW264.7 cells were seeded at a number of 1×10 5 Cells / well were added to a 24-well plate, 0.5 mL DMEM (containing 10% FBS) was added, and incubated for 12 h at 37°C and 5% CO2. The old culture medium was replaced with fresh DMEM culture medium (containing 10% FBS) containing NPOC or rNPOC or cNPOC or hNPOC ([Ce6] = 5 μg / mL). After 4 h of co-incubation, the cells were washed with PBS to remove extracellular particles, and the cell suspension obtained by digestion of the cells was then used to detect the fluorescence intensity of Ce6 in tumor cells by flow cytometry.

[0130] like Fig.10 As shown, RAW264.7 cells have a strong internalization efficiency of NPOC and cNPOC, while rNPOC and hNPOC can reduce the uptake of nanoparticles by RAW264.7 cells. In summary, hNPOC can acquire homologous self-targeting ability while avoiding clearance by macrophages.

[0131] 3. hNPOC enhances tumor accumulation and retention

[0132] HCT116 cell tumor-bearing mice and CT26 cell tumor-bearing mice were randomly divided into two groups, 3 in each group. The hNPOC particle solution was injected into the tail vein, and the Ce6 dosage was 2 mg / kg per mouse. The fluorescence of the tumor site was observed using a small animal in vivo imaging device at 2h, 4h, 8h, 12h, 24h, and 36h after administration (Ex: 660nm, Em: 710nm).

[0133] like Fig.11 As shown, hNPOC retained longer in the tumor site of HCT116 cell-bearing mice compared with the CT26 cell-bearing mice group.

[0134] 6. In vivo pharmacokinetic study of hNPOC and its anti-tumor efficacy in vivo

[0135] (1) Pharmacokinetic experiments

[0136] Twelve ICR mice were randomly divided into four groups and injected with NPOC, rNPOC, cNPOC or hNPOC ([Ce6] = 5 mg / kg) through the tail vein. Blood was collected from the mouse eye sockets at a pre-set time point, and the upper serum was collected after centrifugation. The collected serum was mixed with 1 mL of tetrahydrofuran, centrifuged at 10000g for 10 min, and the supernatant was removed. After natural air drying, 100 μL of dimethyl sulfoxide was added. A standard curve solution of Ce6 was prepared, and the Ce6 content in each serum sample was detected using a multifunctional well plate detection system (Biotek Cytation5, BioTek, United States, 405 nm).

[0137] like Fig.12 As shown in Figure 3, hNPOC and rNPOC had significantly longer systemic circulation times than cNPOC and free Ce6, which was attributed to the effective reduction of clearance by the reticuloendothelial system (RES) by rM.

[0138] (2) In vivo anti-tumor therapy test

[0139] 40 tumor-bearing mice (tumor size ∼100 mm) were randomly selected. 3) were divided into 8 groups. On the 1st, 4th and 7th days, the mice were injected with PBS, free Oxa-la and Ce6 (2% dimethyl sulfoxide solubilization) plus light (L+), hNPO (L+), hNPC (L+), hNPO / hNPC (L+), CT26-hNPOC (L+) and hNPOC (L+) or (L-) through the tail vein, respectively. The dosage of Pt was 3.2 mg / kg of mouse body weight, and the dosage of Ce6 was 2.8 mg / kg of mouse body weight. The weight of the mice and the length and width of the tumor mass were measured every three days. After 18 days of treatment, the mice were killed and the intact tumor tissue was removed. The tumor tissue was photographed and weighed. The weight changes of mice in each experimental group were also detected. The formula for calculating the tumor volume is as follows: Volume (mm 3 )=0.5×length(mm)×width(mm) 2 .

[0140] like Fig.13 As shown, compared with the PBS group, free Oxa-la and Ce6 (L+), hNPO (L+) or hNPOC (L-) had no obvious inhibitory effect on HCT116 tumor; hNPC (L+), hNPO / hNPC (L+) or CT26-hNPOC (L+) had a slight inhibitory effect on tumor growth; hNPOC (L+) had the best inhibitory effect on HCT116 tumor growth, showing the good advantages of this red cancer cell membrane bionic nanocarrier in achieving chemotherapy-photodynamic combined therapy.

[0141] like Fig.14 As shown, there was no significant change in the body weight of mice during the entire treatment period, indicating that the bionic nanocarriers would not cause serious toxic side effects to mice and cause a sharp drop in body weight.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An active oxygen responsive bionic nanocarrier system, characterized in that: The active oxygen responsive biomimetic nanocarrier system is a core-shell structure, wherein the core is a nanocore loaded with hydrophobic oxaliplatin and dihydrochlorin e6, and the shell is a cell membrane; The cell membrane is a fusion membrane formed by the red blood cell membrane and the HCT116 cell membrane; The carrier of the nano core loaded with hydrophobic oxaliplatin and dihydrochlorin e6 is a polyphosphate material TK-PPE; The preparation method of hydrophobic oxaliplatin comprises the following steps: (1) dispersing oxaliplatin in hydrogen peroxide, oxidizing and drying to obtain oxidized oxaliplatin; (2) The oxidized oxaliplatin and lauric anhydride are dissolved in N,N-dimethylformamide, stirred and reacted at 100° C. to 120° C., and then recrystallized to obtain hydrophobic oxaliplatin.

2. A method for preparing the active oxygen responsive biomimetic nanocarrier system according to claim 1, characterized in that: The steps include: (1) mixing hydrophobic oxaliplatin and dihydrochlorin e6 with TK-PPE, and preparing a nanocore loaded with hydrophobic oxaliplatin and dihydrochlorin e6 by a nanoprecipitation method; (2) mixing the nanocore loaded with hydrophobic oxaliplatin and dihydrochlorin e6 with the cell membrane, and preparing the active oxygen responsive biomimetic nanocarrier system with the cell membrane encapsulating the nanocore by membrane extrusion method; Wherein, the preparation method of the hydrophobic oxaliplatin comprises the following steps: (1) dispersing oxaliplatin in hydrogen peroxide, oxidizing and drying to obtain oxidized oxaliplatin; (2) The oxidized oxaliplatin and lauric anhydride are dissolved in N,N-dimethylformamide, stirred and reacted at 100° C. to 120° C., and then recrystallized to obtain hydrophobic oxaliplatin.

3. The method for preparing an active oxygen responsive biomimetic nanocarrier system according to claim 2, characterized in that: The nanoprecipitation method comprises the following steps: uniformly dispersing hydrophobic oxaliplatin, dihydrochlorin e6 and TK-PPE in dimethyl sulfoxide to prepare a mixed solution, and then dropwise adding the mixed solution into water to form a nanocore loaded with hydrophobic oxaliplatin and dihydrochlorin e6.

4. The method for preparing an active oxygen responsive biomimetic nanocarrier system according to claim 2, characterized in that: The membrane extrusion method comprises the following steps: uniformly mixing the nanocore loaded with hydrophobic oxaliplatin and dihydrochlorin e6 with the cell membrane, extruding through a polycarbonate membrane with a pore size of 200nm to 400nm, and then centrifuging to obtain an active oxygen responsive biomimetic nanocarrier system with the cell membrane encapsulating the nanocore.

5. Use of the active oxygen responsive bionic nanocarrier system according to claim 1 in the preparation of drugs for treating colon cancer.

6. A drug for treating colon cancer, characterized in that: The drug comprises the active oxygen responsive bionic nanocarrier system according to claim 1.

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