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CN-121987817-A - Bispecific bionic nano material and preparation method and application thereof

CN121987817ACN 121987817 ACN121987817 ACN 121987817ACN-121987817-A

Abstract

The application discloses a bispecific bionic nanomaterial and a preparation method and application thereof, and belongs to the field of biological medicine, wherein the bispecific bionic nanomaterial comprises an enzyme-carrying porphyrin-based covalent organic framework modified by a bone marrow stroma cell membrane, an anti-CD 3 antibody and an anti-PD-L1 antibody, wherein the enzyme-carrying porphyrin-based covalent organic framework carries glucose oxidase, and the antibody is anchored on a phospholipid bilayer of the enzyme-carrying porphyrin-based covalent organic framework modified by the cell membrane through physical action; the bispecific bionic nano material prepared by the application can block CXCR4/CXCL12 axis and inhibit leukemia cells from migrating and adhering; the bi-specific bionic nano material converts hydrogen peroxide generated by glucose decomposition into hydroxyl free radicals by utilizing the peroxidase-like activity of the porphyrin-based covalent organic framework for chemical kinetics treatment, and redirects T cells to leukemia cells for killing.

Inventors

  • BAI HUIYUAN

Assignees

  • 南通大学

Dates

Publication Date
20260508
Application Date
20251229

Claims (10)

  1. 1. The preparation method of the bispecific bionic nano material is characterized by comprising the following specific steps of: s1, preparing a cell membrane by combining hypotonic treatment with repeated freeze thawing, wherein the preparation method comprises the following steps: s11, suspending bone marrow stromal cells in hypotonic solution, and placing the cells in a liquid nitrogen tank for freezing for 8S after the cells are swelled; s12, taking out the cells, melting the cells at room temperature to obtain a cell suspension, and continuously putting the cells in a liquid nitrogen tank for freezing for 8S when the cell suspension is completely in a liquid state; s13, repeating the freeze thawing operation of the step S12 for 6 times to obtain a cell suspension after the freeze thawing operation for 6 times; s14, centrifuging to remove cell contents in the cell suspension, and adding sterile pure water for resuspension to obtain a cell membrane; s15, measuring the protein concentration of a cell membrane by a BCA method, subpackaging, and preserving at-80 ℃ in a refrigerator; S2, preparation of FeC and FeC-G: S21, weighing 20-40 mg of 5,10,15, 20-tetra (4-pyridyl) -21H, 23H-porphyrin and 300-400 mg of polyvinylpyrrolidone, and dissolving the mixture into 12-18 mL of 1-methyl-2-pyrrolidone; s22, adding 30-50 mg of p-dibromoxylene, stirring until the p-dibromoxylene is fully dissolved, continuously introducing nitrogen for 10-20 min, and placing the mixture in a water bath at 75-85 ℃ for reaction for 20-30 h; S23, after the reaction is finished, centrifuging, discarding the supernatant, adding 30-50 mg of ferrous chloride and 4-8 mL of methanol, and continuously stirring for 32-38 hours; s24, after stirring, centrifuging, discarding the supernatant, and washing the precipitate with methanol to obtain the porphyrin-based covalent organic framework FeC; S25, mixing glucose oxidase GOX and porphyrin-based covalent organic frameworks FeC according to the mass ratio of 1:0.8-1.2, and stirring at room temperature for 2-4 hours to load the GOX with the FeC, so as to prepare enzyme-carrying porphyrin-based covalent organic frameworks FeC-G; s3, performing cell membrane bionic modification on FeC-G and anchoring an antibody to prepare FeC-G@M-C & P: S31, incubating FeC-G and cell membranes together under ice bath conditions and performing ultrasound to obtain an enzyme-carrying porphyrin-based covalent organic framework FeC-G@M modified by bone marrow stroma cell membranes; s32, preparing a DSPE modified antibody, namely a phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS modified antibody by utilizing an amide reaction between NHS and amino; S33, feC-G@M and DSPE modified antibody are incubated at 37 ℃ to enable the DSPE end to be inserted into a phospholipid bilayer on the surface of the nanomaterial, so that FeC-G@M-C & P, namely the bispecific bionic nanomaterial, is obtained.
  2. 2. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the hypotonic solution in S11 is 0.1 XPBS buffer, the mass ratio of bone marrow stromal cells to hypotonic solution is 1.2-1.5:1.2-1.5, the centrifugal speed in S14 is 11000-14000 rpm, and the centrifugal time is 15-25 min.
  3. 3. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the concentration of the protein packaged in the S15 is 0.4-0.8 mg/mL.
  4. 4. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the stirring speed in S22 is 500-1000 rpm, and the stirring time is 15-25 min.
  5. 5. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the centrifugal speed in S23 is 8000-11000 rpm, the stirring speed is 500-1000 rpm, and the stirring speed in S24 is 8000~11000 rpm;S25 and 500-1000 rpm.
  6. 6. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the ultrasonic power in S31 is 35-45 w, and the ultrasonic time is 4-8 min.
  7. 7. The preparation method of the bispecific bionic nanomaterial of claim 1, wherein the specific steps of preparing the DSPE modified antibody by S32 are that the DSPE-PEG-NHS and the antibody are mixed according to a molar ratio of 4-8:1 under a stirring state, sodium bicarbonate is added to adjust the pH of a reaction solution to 8.0, the reaction is carried out at room temperature for 1.5-3 hours, after the reaction is finished, the reaction solution is transferred to an MWCO 10 kDa ultrafilter tube, centrifugation is carried out at 3500-4500 rpm for 15-25 min, pure water is used for washing, and an inner tube solution is collected, so that the antibody is the anti-CD 3 antibody and the anti-PD-L1 antibody.
  8. 8. The method for preparing the bispecific bionic nanomaterial of claim 1, wherein the incubation time in S33 is 1-3 hours.
  9. 9. A bispecific bionic nanomaterial prepared by the preparation method of any one of claims 1 to 8 is characterized by comprising enzyme-carrying porphyrin-based covalent organic frameworks FeC-G@M modified by bone marrow stromal cell membranes and antibodies, wherein the enzyme-carrying porphyrin-based covalent organic frameworks FeC-G comprise glucose oxidase GOX and porphyrin-based covalent organic frameworks FeC, the antibodies are anti-CD 3 antibodies and anti-PD-L1 antibodies, the antibodies are anchored on phospholipid bilayer of FeC-G@M through physical action, and the antibodies are modified by phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS in advance.
  10. 10. Use of the bispecific bionic nanomaterial prepared by the preparation method of any of claims 1-8 in preparation of acute myeloid leukemia therapeutic drugs.

Description

Bispecific bionic nano material and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a bispecific bionic nanomaterial and a preparation method and application thereof. Background Acute Myeloid Leukemia (AML) is a tumor of the blood system caused by abnormal proliferation of hematopoietic stem cells. Chemotherapy, targeted therapy, and hematopoietic stem cell transplantation are the clinically significant therapeutic approaches. The remission rate of most patients after being treated is improved, however, prognosis of the aged patients is not ideal, and with the continuous progress of anti-tumor technology, many new therapies including chemotherapy, hunger therapy, immunotherapy and the like are advanced in the anti-tumor treatment field, however, because the occurrence and development of tumors involve multiple pathological mechanisms, the therapy of a single action mechanism is difficult to remove tumor cells efficiently. Drug resistance recurrence has been a significant challenge in the clinic. Therefore, there is a need to develop a multi-pathway combination therapeutic strategy to enhance AML treatment. Bone marrow microenvironment plays an important role in AML drug resistance recurrence. CXCR4/CXCL12 is an important biological axis regulating the return of AML cells to the bone marrow microenvironment. AML cells migrate to the bone marrow through recognition of CXCR4, which is highly expressed on their own surface, and CXCL12, which is secreted by bone marrow stromal cells, avoiding killing of the drug, forming tiny residues, leading to disease recurrence. Interference with the combination of the two is an effective way to block disease resistance recurrence for the mechanisms described above. CXCR4 is an important therapeutic target for a variety of diseases, and development of related antagonists has been attracting attention, and in recent years, small molecule compounds, polypeptides, monoclonal antibodies, and the like have been developed successively. The bionic nano-drug prepared by the human body in the art by utilizing the mouse breast cancer cell membrane with CXCR4 over-expression can keep the CXCR4 expression effect of the source cell, effectively combine with CXCL12 and block the tumor cell metastasis. The biological axis is interfered by targeting of a specific biological film, so that the problem of drug resistance recurrence is hopefully solved. The chemical kinetics therapy is an emerging tumor treatment method, and the therapy utilizes the characteristics of weak acidity and excessive hydrogen peroxide in tumor microenvironment, and the hydrogen peroxide is catalyzed to generate a strong toxic hydroxyl free radical (active oxygen with very damaging property) through Fenton or Fenton-like reaction, so that oxidative stress of tumor cells is initiated, and the tumor cells are specifically killed. The nano enzyme is a nano material with enzyme-like catalytic activity, can overcome the defects of harsh catalytic environment, poor stability, low production cost and the like of natural enzyme, and can simulate the catalytic process of the natural enzyme to generate a large amount of active oxygen to induce the death of tumor cells. In recent years, nano-enzyme-based chemotherapy plays an important role in the field of tumor treatment, but limited hydrogen peroxide and weak acidity in tumor microenvironment limit the generation of sufficient active oxygen, so that the treatment effect is not ideal. Glucose oxidase can convert glucose (glucose) in tumor cells into gluconic acid and hydrogen peroxide, providing an acidic environment and sufficient substrate for the Fenton reaction. Therefore, the carried glucose oxidase can be used as an enhancement strategy for the chemical kinetics therapy. Immunotherapy is a powerful anti-tumor technology, and brings new breakthrough for the treatment of blood tumor. Monoclonal antibodies become an efficient anti-tumor mode due to the advantages of good specificity, strong affinity, small toxic and side effects and the like. However, due to the complex pathological mechanisms of tumors, which involve multiple signaling pathways, monoclonal antibodies directed against single targets are not able to completely inhibit tumors. Compared with monoclonal antibodies, bispecific antibodies can bind to two different antigens or link T cells, exhibiting better therapeutic efficacy. T cell bispecific antibodies are a common bispecific antibody that is capable of binding both the T cell surface molecule CD3 and a tumor-associated antigen, recruiting T cells and directing their directed killing of tumor cells. T cell bispecific antibodies aiming at targets such as CD19, CD20, EGFR and the like show good treatment prospects, but have the defects of complex preparation process, high blood clearance speed and the like. The nano technology has obvious advantages in im