Preparation method and application of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance

Abstract

The present invention provides a preparation method and application of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance, which relates to the field of biomedicine technology, wherein the preparation method comprises the following steps: preparing a nanoreactor, extracting neutrophils, and preparing a bionic nanoreactor. The bionic nanoreactor prepared by the above preparation method contains a disulfide-crosslinked BSA nanoskeleton, a Fenton reagent Fe(III), an oxidant DTNB, and a targeted delivery carrier neutrophils. Wherein Fe(III) and DTNB are wrapped in the BSA nanoskeleton to form a nanoreactor and loaded on the surface of neutrophils. Guided by tumor inflammation signals, neutrophils can target and deliver the nanoreactor to tumor tissues. After being phagocytosed by the cells, the bionic nanoreactor can use disulfide bonds, Fe(III) and DTNB to coordinately consume intracellular GSH in a triple extreme manner, and release acidic products to immediately regulate pH, thereby improving the efficiency of the Fenton reaction and inhibiting the loss of product ROS, thereby exerting an anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method and application of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance. Background Art

[0002] Glioma is the most common primary central nervous system tumor with poor prognosis and high mortality. As an important treatment for glioma, chemotherapy has poor targeting and strong toxic side effects, which limit its clinical application. With the deepening of the understanding of tumors, the tumor microenvironment has attracted much attention as a target for drug therapy. Chemodynamic therapy is a new tumor treatment model developed by utilizing the intracellular microenvironment. Its mechanism is to use the characteristic of tumor overexpression of H2O2 to deliver Fenton reagents to tumor tissues, and then produce highly cytotoxic reactive oxygen species (ROS)-hydroxyl radicals (·OH) through the Fenton reaction in the acidic microenvironment, mediating cell apoptosis to achieve tumor treatment. Obviously, the Fenton reaction is difficult to occur in alkaline conditions and non-tumor areas with insufficient H2O2, so it has good tumor selectivity and low systemic toxicity. In addition, compared with traditional chemotherapy and photothermal therapy, ROS-based treatment models generally do not cause tumor tolerance. Therefore, thanks to its unique mechanism of action, chemodynamic therapy shows great potential for anti-tumor applications.

[0003] However, despite its many advantages, the anti-tumor efficacy of chemodynamics is not ideal. On the one hand, the Fenton reaction can only efficiently produce ROS under the condition of pH ≈ 2-4, while the intracellular overexpressed GSH and the slightly acidic tumor environment can not only directly consume ROS, but also significantly reduce the efficiency of Fenton reaction to produce ROS, thereby inhibiting the efficacy of chemodynamics. On the other hand, due to the presence of physiological barriers such as the blood-brain barrier and the extracellular matrix, traditional nanodrug delivery systems often find it difficult to effectively deliver Fenton reagents to the tumor site, further reducing the efficacy of chemodynamics. Therefore, there is an urgent need to develop a method for intratumoral Fenton chemical targeted regulation to enhance the efficacy of the Fenton reaction and improve the chemodynamic treatment effect of gliomas. Summary of the invention

[0004] Limited by the intracellular overexpression of GSH, the slightly acidic tumor environment and physiological barriers, it is still challenging to efficiently regulate intratumoral Fenton chemistry and enhance the chemokinetic efficacy of glioma. To this end, the present invention provides a bionic nanoreactor with intratumoral Fenton chemistry targeted regulation performance. The bionic nanoreactor in the present invention contains a disulfide-crosslinked BSA nanoskeleton, a Fenton reagent Fe (III), an oxidant DTNB, and a targeted delivery carrier neutrophil. Among them, DTNB is a thiol reaction reagent, which can not only mediate the loading of protein nanocarriers on the surface of neutrophils through a thiol-disulfide exchange reaction, but also release the acidic product thionitrobenzoic acid (TNB) as an oxidant while rapidly consuming GSH, and has great potential for Fenton chemical regulation. In this study, Fenton reagent Fe (III) and DTNB are encapsulated in a disulfide-crosslinked BSA nanoskeleton to form a nanoreactor and loaded on the surface of neutrophils. Guided by tumor inflammatory signals, neutrophils can target and deliver nanoreactors to tumor tissues. After being phagocytosed by the cells, the bionic nanoreactors can use disulfide bonds, Fe(III) and DTNB to synergistically perform triple extreme consumption of intracellular GSH, while releasing acidic products to instantly regulate pH, thereby improving the efficiency of the Fenton reaction and inhibiting the loss of product ROS, thereby enhancing the chemokinetic efficacy of gliomas.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance comprises the following steps:

[0007] S1: Preparation of nanoreactor:

[0008] BSA, SDS and DTT were mixed and dissolved in deionized water to obtain reduced BSA;

[0009] diluting the reduced BSA to obtain diluted reduced BSA, and adding DTNB under stirring to obtain a mixed solution;

[0010] After 12 minutes, FeCl3 solution and diluted reducing BSA were added to the mixed solution, and the mixture was stirred continuously and ultrafiltered to obtain a nanoreactor;

[0011] S2: Extract neutrophils;

[0012] S3: Preparation of biomimetic nanoreactor:

[0013] The nanoreactor obtained in S1 was resuspended with PBS; the resuspended nanoreactor was mixed with the neutrophils in S2, and reacted for 7 minutes after slight resuspending, and then centrifuged and the supernatant was discarded to obtain a bionic nanoreactor.

[0014] Preferably, the specific steps of S2 are as follows:

[0015] A1: BALB / c nude mice were selected and intraperitoneally injected with 3 mL of sterile thioglycolate;

[0016] A2: After thioglycollate stimulation, the peritoneal fluid was collected, and then the peritoneal cavity was lavaged twice with pre-cooled PBS. The peritoneal fluid and lavage fluid were mixed and centrifuged to collect the cell pellet;

[0017] A3: Add red blood cell lysis buffer to the cell pellet and resuspend it. Let it stand for 5 minutes and then centrifuge it. Wash the obtained cell pellet with PBS and collect the cell pellet by centrifugation.

[0018] A4: Add 1640 medium containing 10% FBS to the cell pellet in A3, resuspend and place in a cell culture dish, and incubate in a cell culture incubator for 30 minutes;

[0019] A5: Gently shake the cell culture dish to absorb the cells that are not attached to the wall, centrifuge and discard the supernatant to obtain neutrophils.

[0020] Preferably, the centrifugal conditions are 2000 rpm and 5 min.

[0021] Preferably, the thioglycollate is sterilized before injection, the concentration of the thioglycollate is 3%, and the stimulation is performed for 6 hours after injection.

[0022] Preferably, the mass ratio of BSA, SDS, DTT and deionized water in the reducing BSA in S1 is 40:20:1.49:1000, and the reaction conditions are 70° C. and reaction time for 2 h.

[0023] Preferably, the reduced BSA in S1 is diluted with MES buffer at pH 4.4 to a concentration of 1 mg mL -1 , the concentration of the MES buffer is 0.05M.

[0024] Preferably, the concentration of the FeCl3 solution in S1 is 2 mM.

[0025] Preferably, in the step of resuspending the nanoreactor in PBS in S3, the concentration of the nanoreactor dissolved in PBS is 0.1 mg mL -1 , 0.2mg mL -1 , 0.3mg mL -1 One of them.

[0026] The present application also provides a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance, which is prepared using the preparation method described above.

[0027] Based on the preparation method of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance obtained by the above-mentioned preparation method, the present application also provides an application of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance in enhancing the chemokinetic efficacy of gliomas.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] 1. In the bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance prepared in the present application, the bionic nanoreactor is loaded on the surface of neutrophils, which not only has little effect on the physiological function of the cells, ensuring the inflammatory targeting characteristics of neutrophils, but also avoids the degradation of drugs by cells, ultimately promoting the enrichment of bionic nanoreactors in tumors and improving the efficacy of tumor chemical dynamics;

[0030] 2. After reaching the tumor, the bionic nanoreactor prepared in the present application with intratumoral Fenton chemical targeted regulation performance is internalized by tumor cells, and can triple-extremely consume intracellular GSH through disulfide bonds, Fe(III), and DTNB, inhibit the loss of ROS, enhance the yield of Fenton reaction ·OH, and improve the tumor chemical dynamics efficacy;

[0031] 3. After reaching the tumor, the bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance prepared in the present application is internalized by the tumor cells. The loaded drug DTNB can react rapidly with GSH after release to generate TNB, thereby instantly regulating the intracellular pH to acidity, improving the efficiency of Fenton reaction to produce ·OH, and enhancing the tumor chemokinetic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a characterization diagram of the nanoreactor in Example 1 of the present invention;

[0033] in, Figure 1 a: Water and particle size and transmission electron microscopy image of the nanoreactor; Figure 1 b: Stability of the nanoreactor in culture medium containing 10% FBS; Figure 1 c: GSH responsiveness of the nanoreactor; Figure 1 d: GSH-responsive release characteristics of DTNB in ​​the nanoreactor.

[0034] Figure 2 This is a diagram showing the anti-tumor effect of the nanoreactor in Example 2 of the present invention;

[0035] in, Figure 2 a: Effect of nanoreactor on the activity of C6 glioma cells; Figure 2 b: Evaluation of nanoreactor-induced apoptosis of C6 glioma cells.

[0036] Figure 3 This is a diagram showing the effect of Fenton chemical regulation in tumor cells of the nanoreactor in Example 3 of the present invention;

[0037] in, Figure 3 a: Consumption of GSH in C6 glioma cells by nanoreactor; Figure 3 b, c: Nanoreactor increases the intracellular acidity of C6 glioma cells; Figure 3 d, e: Nanoreactor induced the production of ROS in C6 glioma cells; Figure 3 f: The content of malondialdehyde (MDA) in C6 glioma cells after nanoreactor treatment; Figure 3 g: Changes in the activity of C6 glioma cells after treatment with the nanoreactor and introduction of Fenton reaction inhibitors.

[0038] Figure 4 This is a characterization diagram of the bionic nanoreactor in Example 4 of the present invention and an inflammatory chemotaxis effect diagram.

[0039] in, Figure 4 a: Confocal microscopy observation of FITC-labeled nanoreactors loaded on the surface of neutrophils; Figure 4 b: Flow cytometry analysis of the enrichment of FITC-labeled biomimetic nanoreactors in the inflamed peritoneal cavity after intravenous injection (Dio-labeled normal neutrophils were used as a control). DETAILED DESCRIPTION

[0040] Partial English interpretation:

[0041] SDS Sodium Lauryl Sulfate DTT Dithiothreitol DTNB 5,5'-Dithiobis(2-nitrobenzoic acid) FBS Fetal bovine serum GSH Glutathione MDA Malondialdehyde ROS Active oxygen

[0042] Specifically, the present application provides a method for preparing a biomimetic nanoreactor having intratumoral Fenton chemical targeted regulation performance, comprising the following steps:

[0043] S1: Preparation of nanoreactor;

[0044] 1) 40 mg BSA, 20 mg SDS and 1.49 mg DTT were mixed and dissolved in 1 mL deionized water, and stirred at 70°C for 2 hours to prepare reduced BSA.

[0045] 2) Dilute the reduced BSA in step 1) to 1 mg mL with MES buffer at pH 4.4. -1 Obtain diluted reduced BSA, and add 11 μL of DTNB to 1 mL of the diluted reduced BSA solution under stirring to obtain a mixed solution;

[0046] The concentration of the MES buffer is 0.05M.

[0047] 3) After 12 minutes, add 75 μL of FeCl 3 solution and 1 mL of the reducing BSA solution in step 1) to the mixed solution in step 2).

[0048] The concentration of the FeCl3 solution is 2 mM, and the reduced BSA solution is diluted to 1 mg mL in step 2). -1 of reducing BSA solution.

[0049] 4) Continue stirring for 5 hours and ultrafiltration to obtain a nanoreactor.

[0050] S2: Extract neutrophils;

[0051] 1) sterilizing a thioglycollate solution, wherein the thioglycollate concentration is 3%;

[0052] 2) BALB / c nude mice were selected and intraperitoneally injected with 3 mL of sterile thioglycolate;

[0053] 3) After the nude mice were stimulated with thioglycollate for 6 hours, they were disinfected with 75% ethanol and the peritoneal fluid was carefully collected. After the peritoneal fluid was collected, the peritoneal cavity was lavaged twice with pre-cooled PBS and the lavage fluid was collected;

[0054] In one embodiment, the method for collecting peritoneal fluid is as follows: opening the abdominal skin without opening the peritoneum, and extracting the peritoneal fluid;

[0055] The peritoneal fluid was mixed with the collected lavage fluid and centrifuged at 2000 rpm for 5 min to collect the cell pellet;

[0056] 4) Add 2 mL of red blood cell lysis buffer to the cell pellet and resuspend it. After standing for 5 minutes, centrifuge at 2000 rpm for 5 minutes. Wash the obtained cell pellet once with PBS and collect the cell pellet by centrifugation.

[0057] 5) Add 4 mL of 1640 medium containing 10% FBS to the above cell pellet, resuspend and place in a cell culture dish, and incubate in a cell culture incubator for 30 minutes;

[0058] 6) Gently shake the cell culture dish to absorb the cells that are not attached to the wall, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant to obtain neutrophils.

[0059] S3: Preparation of biomimetic nanoreactor.

[0060] 1) Resuspend the nanoreactor with PBS;

[0061] In one embodiment, the dilution concentration of the nanoreactor is preferably 0.1 mg mL -1 , 0.2mg mL -1 , 0.3mg mL -1One of;

[0062] 2) the nanoreactor is rapidly mixed with the neutrophils obtained in S2 after the addition of FBS. In one embodiment, the final concentration of the initially added FBS in the nanoreactor solution is 3%, and the nanoreactor is mixed with the neutrophils within 30 seconds after the addition of FBS;

[0063] After slight resuspension, the mixture was reacted for 7 minutes, and FBS was added again to terminate the reaction. The final concentration of FBS added again in the nanoreactor solution was 10%, and then centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded to obtain a bionic nanoreactor.

[0064] The present application also provides a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance, which is prepared using the preparation method described above.

[0065] Based on the preparation method of the above-mentioned bionic nanoreactor, the present application also provides an application of a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance in the preparation of drugs that enhance the chemokinetic efficacy of gliomas.

[0066] The following is an explanation of the above content in combination with specific verification experiments:

[0067] Example 1: Characterization of Nanoreactor:

[0068] Experimental methods:

[0069] 1. Stability evaluation of nanoreactor

[0070] 1 mg of the nanoreactor was dispersed in 1 mL of a culture medium containing 10% FBS, and incubated at 37° C. The particle size of the nanoreactor was measured at 0, 24, and 72 hours, and the change in the particle size of the nanoreactor was observed.

[0071] 2. GSH responsiveness of nanoreactor

[0072] 0.5 mg of the nanoreactor was dispersed in 1 mL of PBS containing GSH (10 mM), and the particle size of the nanoreactor was measured at 0 and 24 hours to observe the change in the particle size of the nanoreactor.

[0073] 3. GSH-responsive release of DTNB in ​​nanoreactor

[0074] 10 mg of the nanoreactor was dispersed in 5 mL of PBS solution containing GSH (10 mM), and then the mixed solution was placed in a dialysis bag, 20 mL of PBS was placed outside the dialysis bag and stirred continuously at 37 ° C. At 0.5, 1, 2, 3, 4, and 5 hours, 1 mL of the solution outside the dialysis bag was taken and the release of DTNB was detected by UV spectrophotometer. Due to the reaction characteristics of DTNB and GSH, the total release of DTNB is equal to the DTNB in ​​the solution plus the DTNB consumed by the reaction (the product is TNB). The maximum UV absorption peaks of DTNB and TNB are 324 nm and 412 nm, respectively. PBS release medium without GSH was used as a control.

[0075] Experimental results:

[0076] See also Figure 1 a, b, the nanoreactor has a spherical morphology and uniform size. In addition, the size of the nanoreactor does not change significantly within 72 hours, indicating good stability.

[0077] like Figure 1 As shown in c, the nanoreactor becomes smaller in size under the action of GSH, which proves the existence of disulfide bonds in the particles, thereby giving the nanoreactor GSH response characteristics.

[0078] like Figure 1 As shown in (d), in the presence of GSH, the release rate of DTNB in ​​the nanoreactor was faster, indicating the GSH-responsive release characteristics of the nanoreactor.

[0079] Example 2: Verification of the anti-tumor effect of the nanoreactor:

[0080] Experimental methods:

[0081] 1. CCK-8 method to detect tumor cell survival rate

[0082] C6 glioma cells were cultured at 5×10 3 / well were inoculated in a 96-well plate, and after overnight incubation, different concentrations of nanoreactors (DTNB / Fe(III)@BNP) and control samples (the control samples included FeCl3, DTNB, BNP and Fe(III)@BNP) were added. After incubation for 24 hours, the material solution was discarded, and then 100 μL of culture medium containing CCK-8 was added to each well, and incubation continued for 30 minutes. Finally, the absorbance at 450 nm was measured to calculate the cell activity. The amount of FeCl3 and DTNB added was the same as the drugs loaded in the nanoreactor at the corresponding concentration.

[0083] 2. Apoptosis and necrosis kit to detect tumor cell apoptosis

[0084] C6 glioma cells were cultured at 2×10 5The density of 100 μg / mL was inoculated into 6-well plates. After 24 hours, 60 μg mL -1 The nanoreactor (DTNB / Fe(III)@BNP) and its control sample were added to the well plate. After incubation for 4 hours, the cells were stained with apoptosis and necrosis detection reagent, and the cell apoptosis was analyzed by flow cytometry.

[0085] Experimental results:

[0086] See also Figure 2 a, The nanoreactor (DTNB / Fe(III)@BNP) showed concentration-dependent cytotoxicity. Compared with other control groups, the nanoreactor had the strongest cytotoxicity at each concentration.

[0087] like Figure 2 As shown in b, at the same concentration, the nanoreactor (DTNB / Fe(III)@BNP) showed the strongest pro-apoptotic effect, which was consistent with the results of cell activity detection, indicating that the nanoreactor has good anti-tumor application potential.

[0088] Example 3: Verification of Fenton chemical regulation in tumor cells by nanoreactor:

[0089] Experimental methods:

[0090] 1. Consumption of GSH in tumor cells by nanoreactor

[0091] C6 glioma cells were cultured at 4×10 6 The density of 100 μg mL -1 The nanoreactor (DTNB / Fe(III)@BNP) and its control sample were added into the culture dish. After continued cultivation for 1, 2, and 4 hours, the intracellular GSH content was determined using a GSH detection kit.

[0092] 2. Nanoreactors increase the acidity in tumor cells

[0093] The changes in acidity in tumor cells after each sample treatment were detected using a fluorescent pH probe (BCECF-AM). C6 glioma cells were cultured at 2×10 5 / well in a 6-well plate or confocal dish. After 24 hours, 100 μg mL -1 The nanoreactor (DTNB / Fe(III)@BNP) and its control sample were added to the well plate or confocal dish. After continued culture for 1, 2, and 4 hours, pH fluorescent probes were added respectively, and the changes in acidity in tumor cells were detected and analyzed by confocal microscopy and flow cytometry.

[0094] 3. Nanoreactor induces ROS production in tumor cells

[0095] After each sample was treated, ROS in tumor cells was detected using DCFH-DA fluorescent probe. C6 glioma cells were cultured at 2×10 5 / well in a 6-well plate or confocal dish. After 24 hours, 100 μg mL -1 The nanoreactor (DTNB / Fe(III)@BNP) and its control sample were added to the well plate or confocal dish. After continued culture for 2 and 4 hours, DCFH-DA fluorescent probe was added respectively, and the generation of ROS in tumor cells was detected and analyzed by confocal microscopy and flow cytometry.

[0096] Malondialdehyde (MDA) is an intermediate product of lipid peroxidation. The detection of MDA content can reflect the generation of ROS. After the above treatment (4h), C6 glioma cells were lysed, the supernatant was collected, and then the MDA content in the supernatant was detected using an MDA detection kit. The MDA content per unit protein is used to characterize the MDA content of tumor cells after treatment.

[0097] 4. Rescue experiment

[0098] C6 glioma cells were cultured at 5×10 3 / well in a 96-well plate. 24 hours later, 80 μg mL -1 The nanoreactor (DTNB / Fe(III)@BNP) was added to the well plate. After incubation for 4 hours, the material solution was discarded and various Fenton reaction inhibitors (GSH: 5mM; deferoxamine DFO: 100μM; vitamin VE: 100μM) were added. After incubation for 24 hours, the activity of each tumor cell was determined by CCK-8.

[0099] Experimental results:

[0100] See also Figure 3 a, Compared with the control group, the nanoreactor (DTNB / Fe(III)@BNP) has the strongest ability to consume intracellular GSH.

[0101] like Figure 3 As shown in b and c, compared with the control group, the BCECF fluorescence in the cytoplasm was significantly weakened after the nanoreactor (DTNB / Fe(III)@BNP) treatment, indicating that the acidity was enhanced. Flow cytometry also showed that the nanoreactor increased the intracellular acidity.

[0102] like Figure 3 As shown in d and e, compared with the control group, the DCF fluorescence in cells treated with the nanoreactor (DTNB / Fe(III)@BNP) was significantly enhanced, indicating that a large amount of ROS was generated. Flow cytometry also showed that the nanoreactor induced an increase in ROS generation in cells.

[0103] like Figure 3 As shown in (f), the MDA content increased significantly after nanoreactor treatment, further indicating the generation of ROS.

[0104] like Figure 3 As shown in Figure g, after adding different Fenton reaction inhibitors, the activity of tumor cells was restored to a certain extent, which once again proved that the nanoreactor induced ROS generation through the Fenton reaction to mediate cytotoxicity.

[0105] Example 4: Characterization of biomimetic nanoreactor and verification of inflammatory chemotaxis effect:

[0106] Experimental methods:

[0107] Nanoreactors labeled with different concentrations of FITC (0.1, 0.2, 0.3 mg mL -1 ) were co-incubated with neutrophils to prepare biomimetic nanoreactors with different loading rates (DTNB / Fe(III)@BNP-NEs). Each biomimetic nanoreactor was intravenously injected into mice (6×10 6 cells / mouse), and then sterile thioglycollate was injected intraperitoneally to recruit neutrophils. The inflammatory chemotaxis ability of the biomimetic nanoreactor was analyzed by flow cytometry. Dio-labeled neutrophils were injected and recruited as controls.

[0108] For experimental results, see Figure 4 a, b, The nanoreactor can be successfully loaded on the surface of neutrophils, and there is no obvious difference in the inflammatory chemotaxis ability of bionic nanoreactors with different loading rates, indicating that the nanoreactor is expected to be efficiently delivered to inflammatory tumor tissues by loading on the surface of neutrophils.

[0109] Through the above verification experiments, it is proved that the bionic nanoreactor provided in the present application has good inflammation targeting ability and effective intracellular Fenton chemical regulation characteristics, and can therefore be used for chemodynamic treatment of gliomas.

[0110] In summary, the biomimetic nanoreactor with intratumoral Fenton chemical targeted regulation performance provided in the present application comprises a disulfide-crosslinked BSA nanoskeleton, a Fenton reagent Fe (III), an oxidant DTNB, and a targeted delivery carrier neutrophil. Among them, DTNB is a thiol-reactive reagent, which can not only mediate the loading of protein nanocarriers on the surface of neutrophils through a thiol-disulfide exchange reaction, but also release the acidic product thionitrobenzoic acid (TNB) as an oxidant while rapidly consuming GSH, and has great Fenton chemical regulation potential. In the present application, Fenton reagent Fe (III) and DTNB are wrapped in a disulfide-crosslinked BSA nanoskeleton to form a nanoreactor and loaded on the surface of neutrophils. Guided by tumor inflammatory signals, neutrophils can target and deliver nanoreactors to tumor tissues. After being phagocytosed by cells, nanoreactors can use disulfide bonds, Fe(III) and DTNB to coordinate triple extreme consumption of intracellular GSH, while releasing acidic products to regulate pH in real time, thereby improving the efficiency of Fenton reaction and inhibiting product ROS loss, thereby enhancing the chemokinetic efficacy of gliomas. In summary, the bionic nanoreactor provided in this application has the performance of targeted regulation of intratumoral Fenton chemistry and the efficacy of enhancing the chemokinetic efficacy of gliomas.

Claims

1. A method for preparing a biomimetic nanoreactor with intratumoral Fenton chemical targeted regulation performance, characterized in that: The following steps are involved: S1: Preparation of nanoreactor: BSA, SDS and DTT were mixed and dissolved in deionized water to obtain reduced BSA; diluting the reduced BSA to obtain diluted reduced BSA, and adding DTNB under stirring to obtain a mixed solution; After 12 min, FeCl3 solution and diluted reduced BSA were added to the mixed solution, and the mixture was stirred and ultrafiltered to obtain a nanoreactor; S2: Extract neutrophils; S3: Preparation of biomimetic nanoreactor: The nanoreactor obtained in S1 was resuspended with PBS; the resuspended nanoreactor was mixed with the neutrophils in S2, slightly resuspended and reacted for 7 min, then centrifuged and the supernatant was discarded to obtain a biomimetic nanoreactor.

2. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 1, characterized in that: The specific steps of S2 are as follows: A1: BALB / c nude mice were selected and intraperitoneally injected with 3 mL of sterile thioglycolate; A2: After thioglycollate stimulation, the peritoneal fluid was collected, and then the peritoneal cavity was lavaged twice with pre-cooled PBS. The peritoneal fluid and lavage fluid were mixed and centrifuged to collect the cell pellet; A3: Add red blood cell lysis buffer to the cell pellet and resuspend it. Let it stand for 5 minutes and then centrifuge it. Wash the obtained cell pellet with PBS and collect the cell pellet by centrifugation. A4: Add 1640 medium containing 10% FBS to the cell pellet in A3, resuspend and place in a cell culture dish, and incubate in a cell culture incubator for 30 min; A5: Gently shake the cell culture dish to absorb the cells that are not attached to the wall, centrifuge and discard the supernatant to obtain neutrophils.

3. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 2, characterized in that: The centrifugal conditions are 2000 rpm and 5 min.

4. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 2, characterized in that: The thioglycollate was sterilized before injection. The concentration of the thioglycollate was 3%, and the stimulation lasted for 6 hours after injection.

5. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 1, characterized in that: The mass ratio of BSA, SDS, DTT and deionized water in the reduced BSA in S1 is 40:20:1.49:1000, and the reaction conditions are 70°C and the reaction time is 2 h.

6. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 1, characterized in that: The reduced BSA in S1 was diluted with MES buffer at pH 4.4 to a concentration of 1 mg mL -1 , the concentration of the MES buffer is 0.05 M.

7. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 1, characterized in that: The concentration of the FeCl3 solution in the S1 is 2 mM.

8. The method for preparing a bionic nanoreactor with intratumoral Fenton chemical targeted regulation performance according to claim 1, characterized in that: In the step of resuspending the nanoreactor in PBS in S3, the concentration of the nanoreactor dissolved in PBS was 0.1 mg mL -1 , 0.2 mg mL -1 , 0.3 mg mL -1 One of them.

9. A biomimetic nanoreactor with intratumoral Fenton chemistry targeted regulation performance, characterized in that: The method is used to prepare the product according to any one of claims 1 to 8.

Images