In Vivo Tracing Study Of Macrophages With Different Phenotypes And Design Of Novel Inflammatory Targeted Delivery Drugs

Objective:The purpose of this study was to establish PET imaging in vivo tracking technology of macrophages with different phenotypes,compare the recruitment ability of different phenotypes of macrophages to inflammatory sites in vivo.Then a macrophage drug delivery system targeting inflammatory diseases was established.Methods:Undifferentiated naive macrophages RAW264.7（naive）were induced to differentiate into M1-type macrophages by Interferon（IFN-γ）and Lipopolysaccharide（LPS）or into M2-type macrophages by IL-4 induction in vitro.The phenotypic markers of naive/M1/M2 cells were determined by real-time quantitative polymerase chain reaction（RT-q PCR）and immunofluorescence staining,and then M1 and M2 macrophages were successfully induced.By synthesizing ⁸⁹Zr-oxine,naive,M1 and M2 macrophages were labeled,and the phenotype,survival rate and intracellular retention rate of ⁸⁹Zr after labeling were evaluated.The mouse model of local inflammation of turpentine was established,and the distribution of⁸⁹Zr labeled M0,M1 and M2 macrophages in vivo was monitored by positron emission tomography（PET）.The distribution of three macrophages was verified by cell migration and cell adhesion experiments in vitro.Liquid nitrogen-treated M1-type macrophages（LNT M1）were prepared in vitro,and LNT M1 was evaluated for cell size,cytokine secretion capacity,adhesion capacity,and cell targeting capacity.The drug loading,drug release and in vivo retention of LNT M1-loaded dexamethasone sodium phosphate（LNT M1-DSP）were studied.Finally,the lung dry and wet weight of mice with acute lung injury（ALI）pneumonia were measured after LNT M1-DSP treatment.The expression levels of inflammatory factors TNF-α,IL-1βand IL-6 in lung tissue were detected by ELISA.The therapeutic effect of LNT M1-DSP was evaluated by H&E staining of lung tissue sections.Results:M1 and M2 macrophages were successfully induced by naive macrophages in vitro.The phenotype and survival rate of naive,M1 and M2 macrophages labeled with radionuclide⁸⁹Zr did not change.The retention rate of ⁸⁹Zr in cells decreased with time,and about 25%of⁸⁹Zr remained in cells on the 7th day.The PET imaging results showed that naive,M1 and M2macrophages injected intravenously could rapidly migrate to the liver,spleen and inflammatory tissue of the local inflammatory mouse model.Inflammatory tissue uptake injected with M1macrophages was significantly higher than that of naive and M2 macrophages.In vitro experiments showed that the three types of macrophages have targete migration ability to inflammatory sites;M1 macrophages have stronger adhesion,which can make them better remain in the inflammatory site.The cell size of LNT M1 cells treated with liquid nitrogen was slightly smaller than that of untreated M1 cells,which lost the ability to secrete pro-inflammatory cytokines.However,LNT M1 still had targeted migration and strong retention ability to inflammatory lesions.LNT M1-DSP can continuously release drugs in the body and stay for a longer time;LNT M1-DSP can reduce pulmonary edema in ALI mice,significantly reduce the secretion level of lung inflammatory factors and reduce lung damage.Conclusion:This study successfully established ⁸⁹Zr labeling method for macrophages with different phenotypes,and verified the feasibility of PET imaging in vivo tracking.It was revealed that macrophages were rapidly recruited in inflammatory lesions,and the ability of M1-type macrophages to migrate and stay in inflammatory tissues was significantly higher than that of naive and M2-type macrophages.Furthermore,a novel inflammatory targeted delivery drug LNT M1-DSP using M1 macrophages as a carrier was prepared,which has a good therapeutic effect on pneumonia caused by acute lung injury in mice.