X-ray activated bionic neutrophil based on conjugated polymer and preparation method and application thereof

Abstract

The invention discloses an X-ray activated bionic neutrophil based on a conjugated polymer, a preparation method and an application thereof, belonging to the field of nanomedicine. Bovine serum albumin BSA is used to encapsulate a radiotherapy sensitizer PFODBT and an elastase PPE, and nanoparticles are formed with a polymer shell DSPE-TK-PEG, HSPC and cholesterol. A layer of neutrophil membrane extracted from mice is coated on the outer surface to obtain the final product. The nano immune drug has high safety, can enhance the effect of immunotherapy for treating brain glioma, and is fruitful in anti-tumor application.

Description

Technical Field

[0001] The invention belongs to the technical field of conjugated polymer nanomaterials, and in particular relates to an X-ray activated bionic neutrophil based on a conjugated polymer, and a preparation method and application thereof. Background Art

[0002] Tumors are caused by the loss of normal regulation of the growth of a cell in a local tissue at the genetic level under the influence of various carcinogenic factors, leading to abnormal clonal proliferation and formation of new organisms. Malignant tumors are generally referred to as cancer, and people have never stopped exploring tumor treatment.

[0003] Glioblastoma is the most aggressive brain tumor, characterized by the highest mortality, short survival, poor prognosis, and high tendency to relapse. Surgery can remove the tumor, but due to damage to the healthy brain, tumor cells infiltrating the normal brain parenchyma cannot be completely removed. These residual tumor cells are protected by the blood-brain barrier (BBB), which hinders the delivery of chemotherapeutic drugs. Therefore, therapeutic agents using endogenous cells have been considered as a powerful potential brain-targeted drug delivery strategy.

[0004] Studies have found that neutrophils (NE) are the most abundant type of immune cells. NE have the natural ability to cross the brain barrier and penetrate glioma sites, and release catalytically active neutrophil elastase (ELANE) to kill many types of cancer cells while sparing non-cancerous cells. ELANE proteolytically releases the CD95 death domain, which interacts with the histone H1 isoform to selectively eradicate cancer cells. Porcine pancreatic elastase (PPE) is a homolog of ELANE, resistant to tumor-derived protease inhibitors, and exhibits significantly improved therapeutic effects.

[0005] The application of nanotechnology in medicine aims to develop precise theranostics of diseases by exploiting the extraordinary effects of engineered nanomedicines, materials, and biomaterials. However, finely controlling the biological behavior of nanomaterials in complex living systems remains a great challenge in terms of accuracy and intelligence. Inspired by nature, biomimeticism has become an attractive strategy for developing clinically valuable materials and technologies based on highly ordered natural biological structures and producing excellent bioavailability. The rational combination of nanomaterials and biomimetic strategies can enable a variety of nanomaterials to transform from a “distinguishable state” to a “camouflaged state”, which can be achieved by transplanting target recognition molecules (such as aptamers, peptides, and antibodies) onto the surface of nanoparticles to evade host immune responses. Biomembranes, as nanodrug carriers, not only maintain the inherent physicochemical properties of nanomaterials, but also give them unique biological properties. Nanoparticles wrapped with neutrophil membranes, which mimic neutrophil killing of tumors, are a potential brain-targeted drug system with strong potential.

[0006] Currently available nanoparticle responsive carriers mainly include: based on tumor microenvironment, near-infrared light responsive, and ultrasound responsive. However, the specificity and controllability of tumor microenvironment responsive drugs are limited, and there is still a risk of causing toxic side effects. The tissue penetration ability of light is weak, and light-responsive drug delivery systems can often only be used to treat shallow tumors. They are not suitable for most tumors in deep tissues in clinical practice, which greatly restricts their clinical transformation and application. Excessive power in ultrasound treatment can easily cause muscle lysis of the host and then induce tumor metastasis. Therefore, how to achieve safe and efficient immunotherapy for tumors in deep tissues is a key scientific problem that needs to be solved urgently. X-ray induced dynamic therapy (XDT) is a new and precise tumor treatment method that uses the strong penetrating power of X-rays to effectively kill cancer cells deep in tissues, and its application in cancer treatment is becoming more and more promising.

[0007] X-ray induced dynamic therapy can achieve considerable precision regulation by consuming oxygen to produce highly toxic singlet oxygen through the radiosensitizer PFODBT. The singlet oxygen-responsive shell is used as a carrier to encapsulate the drug and the nano-delivery system constructed by the neutrophil membrane, which is enriched in the tumor area. Under X-ray irradiation, the singlet oxygen produced can cleave the liposome shell to release porcine pancreatic elastase (PPE). PPE can hydrolyze the transmembrane protein CD95 receptor on the cell membrane, and the released CD95 death domain can bind to the H1 histone and co-localize to the mitochondria to produce ROS and cause cell apoptosis. At the same time, it can also produce a series of immune responses to further enhance the effect of killing tumors.

[0008] After searching domestic and foreign literature, no relevant reports were found on the nanodelivery system constructed by bionic neutrophils using singlet oxygen-responsive shells as carriers to encapsulate drugs. Summary of the invention

[0009] The present invention discloses a conjugated polymer-based X-ray activated bionic neutrophil, a nano delivery system constructed by coating PPE with a singlet oxygen responsive shell as a carrier and outer layer of neutrophil membrane; the singlet oxygen responsive shell is DSPE-TK-PEG; and the system also includes a radiotherapy sensitizer composed of PFODBT, PPE and neutrophil membrane.

[0010] A method for preparing X-ray activated bionic neutrophils based on conjugated polymers comprises the following steps:

[0011] Step (1) dissolving the radiosensitizer PFODBT in tetrahydrofuran;

[0012] Step (2) dissolving BSA in water;

[0013] Step (3) adding the solutions of the above steps (1) and (2) into the aqueous solution under ultrasonic conditions, and ultrasonicating;

[0014] Step (4) using a shaking table to fully volatilize tetrahydrofuran;

[0015] Step (5) dissolving PPE in water and mixing it with the volatilized BSA@PFODBT;

[0016] Step (6) dissolving DSPE-TK-PEG, HSPC and cholesterol in chloroform;

[0017] Step (7) volatilizes using a rotary evaporator;

[0018] After volatilization in step (8), water is added and ultrasonication is performed;

[0019] Step (9) adding water to hydrate the product of step (8) in a 60° C. water bath;

[0020] Step (10) mixing the products of step (5) and step (9) under ultrasonic conditions;

[0021] Step (11) extruding using a liposome extruder;

[0022] Step (12) extracting neutrophils from BALB / c mice using a Percoll gradient method, and activating the neutrophils using a medium containing lipopolysaccharide (LPS);

[0023] Step (13) using an ultrasonic disruptor to disrupt the activated neutrophils;

[0024] Step (14) The products of step (11) and step (13) are mixed, extruded using a liposome extruder, and then passed through a membrane and ultrafiltered.

[0025] A nanomedicine derived from X-ray activated bionic neutrophils based on conjugated polymers, which can be used to cross the blood-brain barrier and treat brain gliomas.

[0026] The present invention adopts the above technical solution and has the following advantages compared with the prior art:

[0027] (1) The low-dose X-rays used in the present invention can provide precise control externally, thereby improving the therapeutic and diagnostic effects;

[0028] (2) The present invention uses novel materials and achieves targeted delivery of nanomedicine by wrapping the bionic neutrophil membrane to penetrate the blood-brain barrier;

[0029] (3) The drug encapsulated by the present invention has a precise controlled release effect, which can improve the safety, effectiveness and utilization rate of the drug.

[0030] (4) The materials used in the present invention can significantly enhance the production of singlet oxygen, cause DNA damage, and enhance immune cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation of SPNPs for the present invention NE Schematic diagram of the synthesis of

[0032] Figure 2 SPN, SPNP, SPN prepared in Example 1 of the present invention NE and SPNP NE UV spectrum of

[0033] Figure 3 SPN, SPNP, SPN prepared in Example 1 of the present invention NE 、SPNP NE Surface potential diagram of NE (a) and SPN, SPNP, SPN NE and SPNP NE Particle size distribution (b);

[0034] Figure 4 SPN prepared in Example 1 of the present invention NE (a) SPNP NE Transmission electron microscopy images of (b) SPN (c) SPNP (d).

[0035] Figure 5 SPNP prepared in Example 1 of the present invention NE The release percentage of PPE in the nanoplatform under X-ray irradiation;

[0036] Figure 6 SPN, SPNP, SPN prepared in Example 1 of the present invention NE 、SPNP NE and polyacrylamide gel electrophoresis of NE;

[0037] Figure 7 SPN, SPNP, SPN prepared in Example 1 of the present invention NE 、SPNP NE and Western blot of NE;

[0038] Figure 8 Gl261-luc cells tested by CCK-8 method were incubated with PBS buffer (control) and SPN prepared by the present invention. NE and SPNP NE (PFODBT concentration ranged from 6.25 to 100 μg / mL) Cell viability after 24 h of treatment;

[0039] Fig. 9SPN prepared in Example 1 of the present invention NE and SPNP NE penetration in bEnd.3 cells;

[0040] Fig.10 SPN prepared in Example 1 of the present invention NE and SPNP NE Evaluation of singlet oxygen generation capacity in vitro;

[0041] Fig.11 SPNP prepared in Example 1 of the present invention NE Ability to generate CD95 death domain in vitro;

[0042] Fig.12 SPN prepared in Example 1 of the present invention NE and SPNP NE Evaluation of in vivo fluorescence imaging capabilities;

[0043] Fig.13 SPN of the present invention NE and SPNP NE Mice were injected via tail vein and irradiated with 6 Gy using an X-ray irradiator. The body weight changes during the treatment (a) and the survival rate of the mice at 65 days (b) were recorded. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0045] like Figure 1 As shown, a method for preparing X-ray activated bionic neutrophils based on conjugated polymers comprises:

[0046] (1) Dissolve the radiosensitizer PFODBT in tetrahydrofuran. After it is fully dissolved, add the above solution to the aqueous solution containing BSA protein under ultrasonic conditions and sonicate; put the above solution in a constant temperature shaker, fully volatilize the tetrahydrofuran, and then add the PPE protein aqueous solution; dissolve DSPE-TK-PEG, HSPC, and cholesterol in chloroform, transfer the above solution to a rotary evaporator, and after the chloroform is completely volatilized, add water to a water bath to hydrate the solution; mix the above two solutions together under ultrasonic conditions, extrude them with a liposome extruder, and pass them through a PES membrane to obtain DSPE-TK-PEG@BSA@PFODBT@PPE.

[0047] The mass ratio of DSPE-TK-PEG: HSPC: cholesterol: BSA: radiosensitizer PFODBT: PPE is 10:10:3:1:0.5:0.5; the volume of tetrahydrofuran is 1 mL; the total volume of the aqueous solution containing BSA is 9 mL; the aqueous solutions for dissolving BSA and PPE proteins are 1 mL respectively; the ultrasonic time is 30 min; the constant temperature shaker evaporation time is 12 h; the setting parameters of the rotary evaporator are: 60°C, 20 rpm, 20 min; the molecular retention capacity of the PES membrane filter device used is 0.22 μm; the volume of the aqueous solution added for hydration is 3 mL; the temperature of the constant temperature water bath is 60°C, 30 min; the liposome extruder needs to squeeze back and forth 10 times.

[0048] (2) Extract bone marrow from the hind legs of BALB / c mice; transfer the cell suspension to a nylon mesh filter centrifuge tube and centrifuge. Prepare four concentration gradients of 100% Percoll, 80% Percoll, 65% Percoll, and 55% Percoll. Add 80% Percoll, 65% Percoll, and 55% Percoll to the centrifuge tube from bottom to top, let it stand; add the bone marrow cell suspension to the top layer and centrifuge. Collect the cells between the 80% and 65% Percoll layers in a new centrifuge tube, add 1×PBS to 14mL, mix thoroughly, and centrifuge. Add pre-cooled red blood cell lysis buffer, add 1×PBS to 14mL after lysis, centrifuge; discard the supernatant. Add LPS to the separated neutrophil culture medium, and further culture the neutrophils in an incubator to obtain activated neutrophils, then wash with 1×PBS, centrifuge, and finally obtain neutrophils.

[0049] The nylon mesh is 200 mesh; the centrifuge tube is 15mL; the centrifugation parameters are 500g / 1200g, 4℃, 5min / 30min; the configuration of 100% Percoll is 10×PBS and Percoll stock solution in a ratio of 1:9; 100% Percoll is diluted with 1×PBS to three concentration gradients of 80% Percoll, 65% Percoll, and 55% Percoll, with volumes of 2mL, 1.5mL, and 1.5mL respectively; standing for 5min; the volume of bone marrow cell suspension is 1mL; the volume of red blood cell lysate is 3mL, and the lysis time is 5min; the concentration of LPS is 100ng / mL; and the culture is incubated in an incubator for 4h.

[0050] (3) Using an ultrasonic disruptor to disrupt the neutrophils in step 2; adding the DSPE-TK-PEG@BSA@PFODBT@PPE obtained in step 1 to the disrupted neutrophil cell membrane; repeatedly extruding and ultrafiltration using a liposome extruder to obtain the final material DSPE-TK-PEG@BSA@PFODBT@PPE@NM.

[0051] The power for crushing neutrophils is 40W, 20min; the liposome extruder needs to squeeze back and forth 20 times; the molecular weight cutoff of the ultrafiltration tube is 50000; the process parameters of ultrafiltration are: 4℃, 2000rpm, 40min, repeated times: 3-5 times.

[0052] The prepared nanoimmunotherapy drugs (SPNs) were characterized by transmission electron microscopy (TEM), ultraviolet-visible absorption spectroscopy (UV-Vis), Zeta potential and dynamic light scattering analysis (DLS). NE and SPNP NE ); then by cell viability analysis (CCK-8 test), simulated penetration of the blood-brain barrier (Transwell), immunofluorescence test of CD95 death domain, in vivo fluorescence imaging, mouse survival rate and body weight monitoring.

[0053] Example 1

[0054] Preparation of a conjugated polymer-based X-ray activated biomimetic neutrophil (SPNP) NE ) specific steps:

[0055] (1) Dissolve the radiosensitizer PFODBT (0.5 mg) in tetrahydrofuran (1 mL). After fully dissolved, add the above solution to an aqueous solution (9 mL) containing BSA protein (1 mg) under ultrasonic conditions and ultrasonicate for 30 min. Place the above solution in a constant temperature shaker to evaporate for 12 h. After the tetrahydrofuran is fully evaporated, add PPE (0.5 mg) protein aqueous solution (1 mL). Add DSPE-TK-PEG (10 mg), HSPC (10 mg), and bile Sterol (3 mg) was dissolved in chloroform (3 mL), and the solution was transferred to a rotary evaporator at 60 ° C, 20 rpm, 20 min. After the chloroform was completely volatilized, an aqueous solution (3 mL) was added to a water bath at 60 ° C for 30 min. After hydration, the two solutions were mixed together under ultrasonic conditions, repeatedly extruded 10 times with a liposome extruder, and passed through a 220 nm PES membrane to obtain DSPE-TK-PEG@BSA@PFODBT@PPE.

[0056] (2) Extract bone marrow from the hind legs of BALB / c mice; transfer the cell suspension to a nylon mesh (200 mesh) filtered centrifuge tube (15 mL) and centrifuge at 500 g, 4°C, for 5 min. Prepare a 100% Percoll solution (10×PBS and Percoll stock solution in a ratio of 1:9), and dilute 100% Percoll with 1×PBS to three concentration gradients of 80% Percoll, 65% Percoll, and 55% Percoll. Add 80% Percoll (2 mL), 65% Percoll (1.5 mL), and 55% Percoll (1.5 mL) from bottom to top in a 15 mL centrifuge tube and let stand for 5 min; add 1 mL of bone marrow cell suspension resuspended in 1×PBS to the top layer and centrifuge at 1200 g, 4°C, for 30 min. Collect the cells between the 80% and 65% Percoll layers in a new 15 mL centrifuge tube, add 1×PBS to the liquid to 14 mL, mix thoroughly, and centrifuge at 500 g, 4°C, for 5 min. Add 3 mL of pre-cooled red blood cell lysis buffer, and after 5 minutes, add 1×PBS to 14 mL of the liquid, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Add LPS (100 ng / mL) to the separated neutrophil culture medium, and further culture the neutrophils in the incubator for 4 hours to obtain activated neutrophils, then wash with 1×PBS, centrifuge at 500g, 4°C for 5 minutes, and collect the neutrophils;

[0057] (3) The neutrophils in step 2 were disrupted by ultrasonic disruption (40W, 20min); the DSPE-TK-PEG@BSA@PFODBT@PPE obtained in step 1 was added to the disrupted neutrophil membrane; the liposome extruder was used to repeatedly extrude 20 times, and ultrafiltration was performed 3-5 times with an ultrafiltration tube with a molecular cutoff of 50,000 (centrifuge parameters: 4°C, 2000rpm, 40min) to obtain the final material DSPE-TK-PEG@BSA@PFODBT@PPE@NM. (SPN NE and SPNP NE The synthetic steps are similar to the above steps.)

[0058] Example 2

[0059] UV-Visible Spectrophotometer Test:

[0060] Take SPN, SPNP, SPN prepared in Example 1 NE and SPNP NE The PFODBT concentration was 10 μg / mL and the volume was 3 mL. The UV absorption spectrum was measured using a UV spectrophotometer. Figure 2 As shown, SPN, SPNP, SPN NE and SPNPNE There is a peak of PFODBT at 540nm, SPN NE and SPNP NE There is a peak of NE at 280 nm because SPNP and SPNP NE The peak of PPE is included at 280nm, so SPNP and SPNP NE The peak at 280nm is significantly higher than that of SPN and SPN NE .

[0061] UV-Vis Test Results

[0062] Reference Manual Attached Figure 2 The SPN, SPNP and SPN prepared by the present invention NE and SPNP NE There is a surface plasmon resonance peak of the radiosensitizer PFODBT at 540nm. NE and SPNP NE There is a peak of NE at 280 nm because SPNP and SPNP NE The peak of PPE is included at 280nm, so SPNP and SPNP NE The peak at 280nm is significantly higher than that of SPN and SPN NE It can be seen that PFODBT, PPE and NE have been successfully encapsulated in nanoparticles.

[0063] Example 3

[0064] Particle size measurement and Zeta potential testing:

[0065] Take SPN, SPNP, SPN prepared in Example 1 NE and SPNP NE A solution with a PFODBT concentration of 10 μg / mL was prepared and the size and Zeta potential were measured using a Zetasizer Nano ZS nanoparticle size potential analyzer. Figure 3 As shown, dynamic light scattering measurements show that SPN, SPNP, SPN NE and SPNP NE The hydrodynamic sizes of SPN, SPNP, SPN are 23.88nm, 27.77nm, 68.69nm and 79.88nm respectively; NE 、SPNP NE The Zeta potentials of NE and NE are -38.91mV, -43.23mV, -29.6mV, -27.0mV and -21.0mV, respectively.

[0066] Hydration kinetic diameter and Zeta potential test results

[0067] Reference Manual Attached Figure 3 a-3b, respectively NE, SPN, SPNP, SPN NE 、SPNP NE Surface potential and SPN, SPNP, SPN NE 、SPNP NE The hydrodynamic particle size of the prepared SPN, SPNP, and SPN NE and SPNP NE The hydrodynamic particle sizes were 23.88 nm, 27.77 nm, 68.69 nm, and 79.88 nm, respectively, showing an increasing trend. SPN and SPNP exhibited similar negative surfaces, with zeta potentials of -38.91 mV and -43.23 mV, respectively. NE 、SPNP NE and NE exhibited a similar negative surface with a zeta potential ranging from −21.0 to −30.0 mV.

[0068] Example 4

[0069] Transmission electron microscopy test:

[0070] Take SPN, SPNP, SPN prepared in Example 1 NE and SPNP NE The nanoparticles were prepared into 500 μL of a solution with a PFODBT concentration of 15 μg / mL using ultrapure water. 5 μL of the nanoparticle suspension was dropped on the surface of the copper mesh. After it was allowed to dry naturally, it was photographed using a JEM-2100 transmission electron microscope.

[0071] Transmission electron microscopy test results

[0072] Reference Manual Attached Figure 4 , SPN, SPNP, SPN NE and SPNP NE All of them are spherical in shape with uniform size and good dispersion. NE and SPNP NE It is covered with neutrophil membrane and has a larger particle size than SPN and SPNP.

[0073] Example 5

[0074] PPE Release Kinetics:

[0075] Take 5.6 mg of Na2CO3 and 80.0 mg of NaHCO3 and add 10 mL of H2O to prepare a 0.1 mg / mL Na2CO3 solution with a pH of 9.0. Take 1 mg of FITC powder and dissolve it in anhydrous DMSO to prepare a 1 mg / mL FITC solution. Add 1 mg / mL of PPE protein solution to 200 μL of FITC solution and stir for 12 hours at 4°C in the dark. After stirring, dialyze for 72 hours at 4°C in the dark. After dialysis, synthesize SPNP with FITC according to the method in Example 1 NE Take the SPNP with FITC prepared in Example 1 NE The nanoparticles were prepared into 6 mL of a solution with a PFODBT concentration of 25 μg / mL using ultrapure water and divided into SPNP NE – FITC – X-ray and SPNP NE –FITC+X-ray groups, using fluorescence chromatograph to detect the release of PPE-FITC. Under X-ray (8Gy) irradiation, a strong fluorescence signal was detected, SPNP NE –The fluorescence signal intensity of FITC+X-ray group is higher than that of SPNP NE The –FITC–X-ray group was 12.8 times higher.

[0076] PPE release test results of nanoimmunotherapy

[0077] Reference Manual Attached Figure 5 , the drug release percentages under 8Gy dose of X-ray irradiation were 92.7%, indicating that the nanomaterial has an excellent controlled release effect.

[0078] Example 6

[0079] Polyacrylamide gel electrophoresis:

[0080] Prepare a 10% mini gel using a one-step PAGE gel rapid preparation kit, prepare the separation gel and the concentration gel according to the instructions, install the prepared gel in the electrophoresis tank, and add the electrophoresis solution to the assembled electrophoresis instrument. NE and SPNP NE Nanoparticles were prepared with ultrapure water to make a 10 μL solution with a PFODBT concentration of 15 μg / mL. NE was taken with 10 μL, and 1 μL of loading buffer was added to each. Mixed with a mixer. 10 μL of samples were taken respectively according to Marker, SPN, SPNP, NE, SPN NE and SPNP NEAdd to the sample tank in sequence, add 5μL of Marker. Cover the electrophoresis tank lid, turn on the power, 200V, 60min. After the electrophoresis is completed, turn off the power, take out the gel and put it in a dish, pour in the Coomassie Brilliant Blue dye solution, and shake it for 30min. After the dyeing is completed, recover the Coomassie Brilliant Blue dye solution, add deionized water and shake it repeatedly to wash and decolorize. After the decolorization is completed, take pictures with a developer and observe the experimental results.

[0081] Polyacrylamide gel electrophoresis results

[0082] Reference Manual Attached Figure 6 , SPN NE and SPNP NE The band of is consistent with the position of NE, while SPN and SPNP do not have bands consistent with NE, indicating that the nanoparticles are wrapped with the neutrophil membrane.

[0083] Example 7

[0084] Western Blot:

[0085] Take SPN, SPNP, SPN prepared in Example 1 NE and SPNP NE The nanoparticles were prepared into 200 μL of a solution with a PFODBT concentration of 600 μg / mL using ultrapure water and 1 mL of a NE solution, which was refrigerated at -80°C for 12 h and subjected to Western blotting experiments.

[0086] Western blot results

[0087] Reference Manual Attached Figure 7 , SPN NE and SPNP NE The bands of and NE both contain Ly6G protein, a marker of neutrophil membrane, while SPN and SPNP do not, further demonstrating that the nanoparticles are coated with the neutrophil membrane.

[0088] Example 8

[0089] Cytotoxicity results:

[0090] Evaluation of SPN prepared by the present invention using Gl261-luc cells as model cells NE and SPNP NE Effects of two nanoparticles on cell proliferation. SPNs of different concentrations were prepared with sterile PBS. NE and SPNP NE Nanoparticle solution was sterilized by UV irradiation overnight. NE and SPNP NENanoparticles (PFODBT concentrations of 6.25, 12.5, 25, 50, 100 μg / mL) solution, 5 replicate wells per group, and a blank group were set up at the same time. PBS buffer was added to the outermost wells to reduce the impact of evaporation, and then the cells were placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. After the culture, the nanoparticles were aspirated and slowly rinsed with PBS twice, and the culture medium containing 10% CCK-8 solution was prepared and added to the well plate. Avoid bubbles during the addition process, and place the culture plate with CCK-8 in a 37°C, 5% CO2 incubator for further incubation for 1.5-2 hours. The 96-well plate was taken out, and the OD value of each well at a wavelength of 450nm was detected using an enzyme marker. The effects of different concentrations of materials on cell proliferation were compared with buffer PBS as a control, and the cell viability was calculated based on this value.

[0091] Cytotoxicity test results

[0092] Reference Manual Attached Figure 8 , in SPN NE and SPNP NE When the concentrations of SPN were 0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL, they were co-incubated with Gl261-luc cells for 24 h, and the cell survival rates were all above 90%, indicating that SPN NE and SPNP NE Both materials showed little toxicity to cells.

[0093] Example 9

[0094] Transwell simulates blood-brain barrier penetration:

[0095] Add 1×10 6 After incubation for 48 h, 5 × 10 5 After incubation for 24 h, the culture medium was removed with a 1 mL gun, and the upper and lower chambers were washed three times with sterile PBS buffer. NE and SPNP NE Prepare a solution with a concentration of 70 μg / mL using sterile DMEM. Take 1 mL and add it to a solution containing 1×10 6Place a bEnd.3 cell in the upper chamber of the Transwell, add 500μL DMEM culture medium to the lower chamber, and incubate for 24 hours. Aspirate the liquid in the upper and lower chambers, detect with a fluorescence chromatograph, and calculate the permeability. Wash the lower chamber repeatedly three times with sterile PBS buffer. Take 3μL of Hoechst dye and 3mL of DMEM culture medium to prepare a nuclear dye and add it to the lower chamber, 500μL, incubate for 15 minutes, aspirate the dye, and wash the lower chamber repeatedly three times with sterile PBS buffer. Add 500μL of tissue fixative to each well, fix for 15 minutes, aspirate the fixative, and wash the lower chamber repeatedly three times with sterile PBS buffer. Wrap the cell plate with tin foil and take pictures with a confocal microscope. Results of Transwell simulating penetration of the blood-brain barrier

[0096] Reference Manual Attached Fig. 9 As shown, the Tranwell chamber was used to simulate the blood-brain barrier to study the penetration ability of nanoparticles. NE and SPNP NE The red fluorescence signal of the nanoparticles can be observed in the group, and 70 μg / mL of SPN NE and SPNP NE The permeability reaches 20.0%, showing a certain penetration effect.

[0097] Example 10

[0098] Singlet oxygen generation capacity assessment:

[0099] The SPN obtained in Example 1 NE and SPNP NE Use sterile DMEM to prepare a solution with a concentration of 25 μg / mL. Take a volume of 500 μL, containing 8×10 4 The Gl261-luc cell suspension was seeded in a 24-well plate and cultured for 24 h. Sterile PBS, SPN NE and SPNP NE Three solutions, PBS group as blank control, the materials and cells were incubated for 12h. The materials in the 24-well plate were slowly aspirated with a pipette, washed 3 times with sterile PBS, and DCFH-DA fluorescent probe diluted with DMEM medium was added. After incubation for 30min, X-rays (4Gy) were irradiated in the dark, the well plate was washed 3 times with PBS, and 300μL PBS was added. The fluorescence of DCFH-DA fluorescent probe was immediately photographed using an inverted fluorescence microscope.

[0100] Singlet oxygen generation test results

[0101] Reference Manual Attached Fig.10 As shown, X-ray (4 Gy) was used to treat SPN NE and SPNPNE After irradiation, a singlet oxygen green fluorescent probe was added to detect the generation of singlet oxygen. As shown in the figure, after X-ray treatment, SPN NE and SPNP NE The group showed obvious fluorescence, indicating that singlet oxygen was generated and SPNP NE The singlet oxygen generation ability of the group is slightly stronger, and the fluorescence intensity is SPN NE The CD95 death domain has a certain ability to produce ROS.

[0102] Embodiment 11

[0103] Immunofluorescence of CD95 death domain:

[0104] The SPN obtained in Example 1 NE and SPNP NE Use sterile PBS buffer to prepare a solution with a concentration of 25 μg / mL. Take a volume of 500 μL, containing 5×10 4 The Gl261-luc cell suspension was seeded in a 24-well plate and cultured for 24 h. Sterile PBS, SPN NE and SPNP NE Three solutions, divided into the following six groups: PBS–X-ray, SPN NE –X-ray, SPNP NE –X-ray, PBS+X-ray, SPN NE +X-ray and SPNP NE +X-ray, and the PBS-X-ray group was used as a blank control. After the materials were co-incubated with cells for 12 h, the treatment group was irradiated with X-rays (4 Gy) and incubated for another 4 h. The materials in the 24-well plate were slowly aspirated with a pipette, washed three times with sterile PBS, and then immunofluorescence staining was performed.

[0105] Immunofluorescence results of CD95 death domain

[0106] Reference Manual Attached Fig.11 As shown, SPNP NE +X-ray group PPE enters the cell and hydrolyzes the transmembrane protein CD95 receptor on the cell membrane, releasing the CD95 death domain into the cytoplasm matrix; PBS-X-ray group, SPN NE –X-ray group, SPNP NE – X-ray group, PBS+X-ray group and SPN NE The +X-ray group did not release the CD95 death domain due to the absence of PPE.

[0107] Example 12

[0108] Fluorescence imaging of drug enrichment:

[0109] The volume was 6 μL and the number of cells was 2 × 10 6 The Gl261-luc cell suspension was injected into the brain of C57 mice to construct an orthotopic glioma model. NE and SPNP NE A solution with a concentration of 300 μg / mL was prepared with sterile PBS and injected into mice through the tail vein. At 0h, 4h, 8h, 12h, 24h, 36h and 48h after injection, the mice were imaged in vivo with near-infrared fluorescence using an IVIS imaging system with excitation and emission wavelengths of 520nm and 700nm, and the collected images were analyzed using Living Image software.

[0110] In vivo fluorescence imaging results

[0111] Reference Manual Attached Fig.12 As shown, an orthotopic brain glioma model was established in C57BL / 6 mice, and the mice were randomly divided into two groups (n=3) and injected with SPN NE and SPNP NE (0.2mL, 0.3mg / mL) PBS solution. Real-time fluorescence imaging of mice was performed using an IVIS spectral imaging system with excitation and emission wavelengths of 520 and 700nm, respectively, at 0, 4, 8, 12, 24, 36, and 48 hours after injection. SPN NE and SPNP NE After being injected into mice, the fluorescence intensity of the tumor area gradually increased over time and reached the maximum value 24 hours after injection. The fluorescence intensity of the tumor area was measured using Living Image 4.3 software. The results showed that at 24 hours, SPN NE and SPNP NE The fluorescence intensity reached the highest level, indicating that the nanoparticles were effectively accumulated in the tumor.

[0112] Example 13

[0113] Mouse survival rate and weight monitoring results:

[0114] The SPN obtained in Example 1 NE and SPNP NE The nanoparticles were prepared into a solution with a PFODBT concentration of 300 μg / mL using sterile PBS, and 200 μL was injected into Gl261-luc tumor-bearing mice through the tail vein. After 24 hours, the mouse brain glioma site was irradiated with an X-ray device (6 Gy). NE –X-ray, SPNP NE –X-ray, PBS+X-ray, SPNNE +X-ray and SPNP NE +X-ray six groups, and then record the mouse body weight for 14 days and the mouse survival rate for 65 days.

[0115] Mouse weight and survival rate monitoring results

[0116] SPN NE and SPNP NE Injected into mice through the tail vein, and X-ray (6Gy) treatment was performed 24 hours after injection. Fig.13 -a, PBS–X-ray group, SPN NE –X-ray group, SPNP NE – The body weight of the X-ray group and the PBS+X-ray group showed a downward trend; SPN NE +X-ray group and SPNP NE The weight of mice in the +X-ray group showed a steady increase trend, and SPNP NE +X-ray group SPN NE The weight gain of the +X-ray group was slightly higher. Fig.13 -b, after 65 days of treatment, SPNP NE The survival rate of mice in the +X-ray group was still 100%, much higher than that in other groups.

[0117] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An X-ray activated biomimetic neutrophil nanoparticle based on a conjugated polymer, characterized in that: A nano-delivery system is constructed by coating PPE with a singlet oxygen-responsive shell as a carrier and encapsulating a layer of neutrophil membrane; the singlet oxygen-responsive shell is DSPE-TK-PEG; DSPE-TK-PEG@BSA @PFODBT@PPE is added to the broken neutrophil membrane; the liposome extruder is used to extrude it repeatedly for 20 times, and ultrafiltration is performed for 3-5 times with an ultrafiltration tube with a molecular cutoff of 50,000 to obtain the final material DSPE-TK-PEG@BSA @PFODBT@PPE@NM; the radiotherapy sensitizer is composed of PFODBT, PPE and neutrophil membrane.

2. A method for preparing the conjugated polymer-based X-ray activated biomimetic neutrophil nanoparticles according to claim 1, characterized in that: The following steps are involved: Step (1) dissolving the radiosensitizer PFODBT in tetrahydrofuran; Step (2) dissolving BSA in water; Step (3) adding the solutions of step (1) and step (2) into the aqueous solution under ultrasonic conditions, and ultrasonicating; Step (4) using a shaking table to fully volatilize tetrahydrofuran; Step (5) dissolving PPE in water and mixing it with the volatilized BSA@PFODBT; Step (6) dissolving DSPE-TK-PEG, HSPC and cholesterol in chloroform; Step (7) volatilization using a rotary evaporator; After volatilization in step (8), water is added and ultrasonication is performed; Step (9) adding water and hydrating the product of step (8) in a 60°C water bath; Step (10) mixing the products of step (5) and step (9) under ultrasonic conditions; Step (11) extruding using a liposome extruder; Step (12) extracting neutrophils from BALB / c mice using a Percoll gradient method and activating the neutrophils using a medium containing lipopolysaccharide (LPS); Step (13) using an ultrasonic disruptor to disrupt the activated neutrophils; Step (14) The products of step (11) and step (13) are mixed and extruded using a liposome extruder; then the mixture is passed through a membrane and ultrafiltered.

3. The preparation method according to claim 2, characterized in that: The step (1) dissolves the radiosensitizer PFODBT in tetrahydrofuran, the step (2) dissolves BSA in water, the step (5) dissolves PPE in water, and the step (6) dissolves DSPE-TK-PEG, HSPC and cholesterol in chloroform. The mass ratio of radiosensitizer PFODBT: BSA: PPE: DSPE-TK-PEG: HSPC: cholesterol is 0.5:1:0.5: 10:20:3; aqueous solution of tetrahydrofuran, the ratio of tetrahydrofuran to ultrapure water is 1:9, the total volume is 10 mL; the volume of chloroform is 3 mL.

4. The preparation method according to claim 2, characterized in that: The ultrasonication time in step (3) is 30 minutes.

5. The preparation method according to claim 2, characterized in that: In the step (7), a rotary evaporator is used; the mixture is rotated for 10 min.

6. The preparation method according to claim 2, characterized in that: In the step (9), hydration is performed in a water bath for 30 minutes.

7. The preparation method according to claim 2, characterized in that: In the steps (11) and (14), a liposome extruder is used to extrude back and forth 20 times.

8. The preparation method according to claim 2, characterized in that: In the step (12), bone marrow is extracted from the hind legs of BALB / c mice; the cell suspension is transferred to a 200-mesh nylon mesh and filtered into a 15 mL centrifuge tube, and centrifuged at 500 g, 4°C for 5 min; 100% Percoll, 10×PBS and Percoll stock solution are prepared in a ratio of 1:9; 100% Percoll is diluted with 1×PBS to three concentration gradients of 80% Percoll, 65% Percoll and 55% Percoll; 2 mL 80% Percoll, 1.5 mL 65% Percoll and 1.5 mL 55% Percoll are added to the 15 mL centrifuge tube from bottom to top, and the mixture is allowed to stand for 5 min; bone marrow cell suspension is added to the top layer, and the mixture is centrifuged at 1200 g, 4°C for 30 min; cells between the 80% and 65% Percoll layers are collected in a new 15 mL centrifuge tube, the liquid is supplemented to 14 mL with 1×PBS, the mixture is thoroughly mixed, and centrifuged at 500 g, 4°C for 5 min. min; add 3 mL of pre-cooled red blood cell lysis buffer, and after 5 min, add 1× PBS to make up the liquid to 14 mL, 500 g, 4 ℃, centrifuge for 5 min; discard the supernatant; add LPS (100 ng / mL) to the separated neutrophil culture medium, and the neutrophils are further cultured in the incubator for 4 hours to obtain activated neutrophils, and then washed with 1× PBS.

9. The preparation method according to claim 4, characterized in that: In the step (13), an ultrasonic disruptor is used to disrupt neutrophils at a power of 40 W for 20 min.

10. A nanomedicine obtained by using the X-ray activated biomimetic neutrophil nanoparticles based on conjugated polymers as claimed in claim 1.

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