Research on cell membrane-coated drug delivery nanovesicles in inducing the polarization of tumor-associated macrophages and treating osteosarcoma / 中华骨科杂志

ABSTRACT

Objective:To prepare cell membrane-coated nanovesicles with targeted delivery of toll-like receptor 4 (TLR4) agonist, and to explore the effect and mechanism of inducing the polarization of tumor-associated macrophages (TAMs) and treating osteosarcoma.Methods:TLR4 agonist loaded nanovesicles were prepared by polycarbonate membrane extruders. The morphology and size of nanovesicles were detected by transmission electron microscopy (TEM) and particle size analyzer, and the drug loading performance of the nanovesicles to TLR4 agonist was investigated. TLR4 agonist loaded nanovesicles were co-incubated with macrophages in vitro, and the targeting ability of nanovesicles to macrophages and its role in regulating the function of macrophages were detected by confocal fluorescence microscopy. In vitro experiments, a cell co-culture system was established. After the upper layer macrophages were treated by the control group, the TLR4 agonist group and the TLR4 agonist loaded nanovesicle group, the lower layer osteosarcoma cells were collected for CCK-8 and cloning formation experiments to evaluate their effects on the proliferation and migration of osteosarcoma cells. In vivo experiments, an osteosarcoma subcutaneous graft tumor model was established, and mice were randomly divided into the control group, the TLR4 agonist group, and the TLR4 agonist loaded nanovesicle group. After the treatment by caudal vein, the tumor targeting ability of nanovesicles in vivo was explored through the in vivo imaging system, and the volume of tumor tissue was continuously detected. The subcutaneous tumors were stained to detect macrophage-related markers, and their effect on the polarization of macrophages was evaluated. The TUNEL fluorescence of tumor tissues was further detected.Results:TEM showed the round shape of TLR4 agonist loaded nanovesicle and the size was about 200 nm. The co-incubation of 0.05 mg TLR4 agonist with 0.1 mg nanovesicles was the best condition for the preparation of drug-loaded nanovesicles. The drug loading efficiency was about 35% and the drug loading content was about 0.11 mg/mg. The membrane-coated nanovesicles could efficiently load and deliver TLR4 agonist. TLR4 agonist loaded nanovesicles were labeled with DiD red fluorescent dye, and then the labeled nanovesicles were co-incubated with macrophages. It was found by confocal fluorescence microscopy that DiD labeled TLR4 agonist loaded nanovesicles significantly accumulated in macrophages, and the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced, which could induce M1 polarization of macrophages. In vitro experiments, it was found that the number of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced under the light microscope, and the cell morphology was wrinkled and rounded. CCK-8 and cloning formation experiments showed that the proliferation and migration ability of osteosarcoma cells in the TLR4 agonist loaded nanovesicle group was significantly reduced compared with the control group and the TLR4 agonist group. A subcutaneous graft tumor model was established. In vivo imaging experiments showed that TLR4 agonist loaded nanovesicles locally accumulated in tumor tissues in vivo, but were not distributed in other organs. The growth of tumor tissue was significantly inhibited in the TLR4 agonist loaded nanovesicle group. Moreover, the fluorescence of M1-type macrophage marker (iNOS) was significantly enhanced (relative fluorescence intensity 3.27±0.19), while the fluorescence of M2-type macrophage marker (CD163) was significantly decreased (relative fluorescence intensity 0.14±0.04). TUNEL fluorescence staining showed that the apoptosis level of osteosarcoma cells was significantly increased (relative fluorescence intensity 9.53±0.21).Conclusion:Membrane-coated nanovesicles could targeted deliver TLR4 agonist to osteosarcoma, induce TAMspolarization, remodel tumor immunosuppressive microenvironment, promote cell apoptosis, and effectively kill osteosarcoma.