CN-121971404-A - PD-L1 targeted synergistic chemotherapy parabacteroides gullet nano-preparation and application thereof

CN121971404ACN 121971404 ACN121971404 ACN 121971404ACN-121971404-A

Abstract

The invention discloses a nano preparation of a Paecilomyces griseus vesicle for PD-L1 targeted synergistic chemotherapy and a preparation method and application thereof, wherein the nano preparation takes a membrane vesicle derived from Paecilomyces griseus as a carrier, an internal loading drug Patinib is named Apatinib-MVs aPD‑L1 , an anti-PD-L1 antibody is modified on the surface through chemical coupling, the nano preparation can cooperatively deliver the Patinib and the PD-L1 antibody to a breast cancer focus to realize the synergistic interaction of chemotherapy and immune check point blocking, in-vitro experiments show that the nano preparation can remarkably inhibit proliferation of breast cancer cells and induce apoptosis of the breast cancer cells, in-vivo experiments show that the nano preparation can effectively inhibit tumor growth, remarkably increase infiltration of CD8+T cells and TNF‑α+CD8+T cells in a tumor microenvironment, has no toxic or side effect, and provides a novel safe and efficient drug delivery platform for combined treatment of breast cancer.

Inventors

DUAN XIANGGUO
SU CHUNXIA
YANG XIAOJUAN
YAN YAJUAN

Assignees

宁夏医科大学

Dates

Publication Date: 20260505
Application Date: 20260201

Claims (6)

1. A nano-preparation of a vesicle of a pair of pseudo-bacillus of Goodyear of PD-L1 targeted synergistic chemotherapy is characterized in that the nano-preparation takes an outer membrane vesicle of a pair of pseudo-bacillus of Goodyear source as a carrier, and is internally coated with a chemotherapeutic drug, namely apatinib, and the surface is covalently connected with an anti-PD-L1 antibody through chemical crosslinking and is named Apatinib-MVs aPD-L1 .
2. The nanofabricated formulation of claim 1, wherein the chemical crosslinking is achieved by the crosslinker DSPE-PEG-NHS.
3. A method for preparing a nano-preparation according to any one of claims 1 to 2, comprising the steps of culturing Paramycolatopsis, collecting culture supernatant, performing ultracentrifugation and purification to obtain membrane vesicles, loading apatinib into the membrane vesicles to obtain drug-loaded vesicles, adding DSPE-PEG-NHS into drug-loaded vesicle suspension to enable DSPE to be embedded into vesicle lipid membranes, finally adjusting the pH of a reaction system to 8.0-8.5, adding an anti-PD-L1 antibody, stirring and incubating at 4 ℃ for 2 hours, enabling the antibody to be covalently coupled to the surface of the vesicles, and purifying to remove unreacted substances to obtain Apatinib-MVs aPD-L1 .
4. Use of a nano-formulation according to any one of claims 1-2 for the preparation of a medicament for the treatment of breast cancer.
5. The use of claim 4, wherein the nanofabric is capable of inhibiting breast cancer cell proliferation and increasing the ratio of CD8 + T cells to TNF- α + CD8 + T cells in a tumor microenvironment.
6. The use of claim 5, wherein the nanoformulation comprises a pharmaceutically acceptable excipient comprising one or more of a diluent, a stabilizer, a buffer, a preservative, or a carrier.

Description

PD-L1 targeted synergistic chemotherapy parabacteroides gullet nano-preparation and application thereof Technical Field The invention belongs to the technical field of biological medicines and nano medicines, and particularly relates to a nano preparation of a Paenibacillus archaea source-entrapped apatinib-linked PD-L1 antibody, and a preparation method and application of the nano preparation. Background The apoptosis ligand 1 (PD-L1) is highly expressed on the surfaces of various tumor cells, and inhibits anti-tumor immune response through combining with PD-1 on T cells, and the anti-PD-L1 antibody has been clinically successful in various cancers as an immune checkpoint inhibitor, but the single drug response rate in breast cancer is less than 20 percent, and is mainly limited by immunosuppressive tumor microenvironment and lack of effective antigen release. Bacterial outer membrane vesicles (Outer Membrane Vesicles, OMVs) are natural nano-particles (20-250 nm) secreted by gram-negative bacteria, are rich in Lipopolysaccharide (LPS), lipoprotein and other pathogen related molecular patterns (PAMPs), can activate TLR signaling pathways, promote dendritic cell maturation, and have potent adjuvant activity, however, traditional OMVs are mostly derived from pathogenic bacteria (such as escherichia coli) and have toxicity risks. The parabacteroides guli (Parabacteroides goldsteinii) is a human intestinal symbiotic bacterium, and in recent years, research discovers that the metabolite of the parabacteroides guli has anti-inflammatory and insulin resistance improving effects, but the outer membrane vesicle of the parabacteroides guli is not yet developed for drug delivery, and in addition, the existing combined immune-chemotherapy strategy relies on artificial synthesis nanoparticles (such as liposome and polymer micelle) and has the problems of complex preparation, low immunogenicity and the like. Therefore, development of a novel nano platform which is derived from probiotics and has the functions of immune activation, targeting blocking and chemotherapy is needed to break through the bottleneck of breast cancer treatment. Disclosure of Invention The invention aims to overcome the defects of the prior art, provide a multifunctional nano preparation which is safe, efficient and capable of being prepared in a large scale, and solve the technical problems of limited curative effect, large toxic and side effects of chemotherapeutics and lack of a natural immune activating carrier of the prior PD-L1 antibody. Another object of the present invention is to provide a method for preparing the above nano-formulation. It is a further object of the present invention to provide the use of the above nano-formulations. A nanometer preparation of a vesicle of a pair of pseudo-bacillus of Goodyear of PD-L1 targeted cooperative chemotherapy takes an outer membrane vesicle of a pair of pseudo-bacillus of Goodyear source as a carrier, is internally encapsulated with a chemotherapeutic drug, and is covalently connected with an anti-PD-L1 antibody through chemical crosslinking, and is named Apatinib-MVs aPD-L1. Preferably, the chemical crosslinking is achieved by the crosslinker DSPE-PEG-NHS. A preparation method of a nano-preparation of a vesicle of the Paecilomyces griseus for PD-L1 targeted synergistic chemotherapy comprises the following steps of firstly culturing the Paecilomyces griseus, collecting culture supernatant, carrying out ultracentrifugation and purification to obtain membrane vesicles, then loading an apatinib package into the membrane vesicles to obtain drug-loaded vesicles, adding DSPE-PEG-NHS into a drug-loaded vesicle suspension to enable DSPE to be embedded into a vesicle lipid membrane, finally adjusting the pH of a reaction system to 8.0-8.5, adding an anti-PD-L1 antibody, stirring and incubating at 4 ℃ for 2 hours to enable the antibody to be covalently coupled to the surface of the vesicles, and purifying to remove unreacted matters to obtain Apatinib-MVs aPD-L1. Application of a nano preparation of a vesicle of a Paenibacillus guli in PD-L1 targeted synergistic chemotherapy in preparation of a medicament for treating breast cancer. Preferably, the nano-preparation can inhibit proliferation of breast cancer cells and increase the ratio of CD8 + T cells to TNF-alpha +CD8+ T cells in the tumor microenvironment, Preferably, the nanoformulation includes a pharmaceutically acceptable excipient including one or more of a diluent, stabilizer, buffer, preservative, or carrier. The apatinib-immune synergic nano-drug based on the bacterial source vesicle has the beneficial effects that (1) a triple synergic mechanism is adopted for activating an innate immunity + PD-L1 antibody by the bacterial vesicle to relieve T cell inhibition + apatinib to promote tumor antigen release and enhance tumor immunogenicity, (2) targeting enrichment is adopted for facilitating high permeability and retention of solid tumors by nano-