Engineered Macrophages Used For The Treatment And CT Imaging Of Osteosarcoma

PurposeThe target of the study is using dendrimer encased gold nanoparticles stimulating macrophage differentiation,and then the engineered macrophages with nanoparticles were used in the orthotopic transplantation model of osteosarcoma in mice for the immune cell treatment of osteosarcoma,and in the meantime,in vivo CT visual monitoring of the therapeutic effect and changes of tumor microenvironment was realized.Methods1.We synthesized and characterized dendritic macromolecule entrapped gold nanoparticles,Au DENPs.Then,we used the CT to evaluate the CT imaging performance of the nanoparticle.CCK-8 assay was used to analyze cytotoxicity and ICP to evaluate cellular uptake of quantitative analysis.2.Gold nanoparticles were used to induced macrophages to differentiate.Flow cytometry and PCR techniques were used to detect the expression of M1macrophages’ markers,CD86 and i NOS,to study the effect of gold nanoparticles on polarization of macrophages.3.Osteosarcoma cells were co-cultured with the modified macrophages,and the apoptosis of osteosarcoma cells was detected by flow cytometry and immunofluorescence.4.Balb/c mice were chosen as orthotopic animal models of osteosarcoma by injected with K7 cells under the tibial plateau.Cell therapy combined with chemotherapy was used for treatment.By detecting tumor growth and HE staining to evaluate engineered macrophages’ in vivo therapeutic effect.Results1.Gold nanoparticles encapsulated by dendrimers are successfully prepared.Transmission electron microscope was used to obtain the size of the gold particle diameter,about 3 nm.Ultraviolet spectral photometer experimental results showed that the nanoparticles have a distinct absorption peak about at 540 nm.CT imaging results displayed the nanoparticle has a good CT imaging performance.CCK-8results showed that the nanomaterial has no obvious toxicity to macrophages.2.Flow cytometry results showed after incubated 24 h with the dendrimer encapsulated gold particles,the M1 macrophages surface marker CD86 of the engineered macrophage significantly upregulated.Immunofluorescence and Western blot results showed the expression of i NOS,another marker of the M1 macrophage was significantly higher than that of unstimulated macrophages.The experimental results of PCR also obtained the same results.3.After co-culture with the engineered macrophages for 24 hours,the expression of Caspase 3 protein in osteosarcoma cells,K7 cells,can be detected by WB and immunofluorescence.Compared with K7 cells that have not been incubated with macrophages,the former expressions were increased.In addition,the results of the flow cytometry assay for apoptosis showed a higher rate of apoptosis of K7 cells incubated with engineered macrophages.Also,results of CCK-8 assay proved that cell therapy combined with chemotherapy can inhibit the proliferation of osteosarcoma cells better than chemotherapy alone.Tumor growth and H&E staining results showed that combination therapy than chemotherapy alone for the inhibition of tumor is also more pronounced.4.Macrophages that have engulfed the nanoparticles can achieve good in vitro CT imaging and in vivo CT imaging of osteosarcoma,so as to realize the imaging of osteosarcoma tumor microenvironment.ConclusionIn this study,we successfully constructed dendritic macromolecular-entrapped gold nanoparticles modified macrophages,and applied the engineered macrophages by Au DENPs into the immune cell treatment of osteosarcoma,and realized in vivo CT visual monitoring of the treatment effect and changes in the tumor microenvironment,thus providing a new idea about the treatment of osteosarcoma.