Drug Loaded Magnetic Liposome As A Dual-Targeting Delivery System For Idiopathic Pulmonary Fibrosis Treatment

Idiopathic pulmonary fibrosis（IPF）is a chronic,progressive and interstitial lung disease characterized by progressive pulmonary scar formation and histological manifestations of common interstitial pneumonia.Although the mechanism of IPF is still unclear,it is generally believed that the occurrence and development of IPF are driven by abnormally activated alveolar epithelial cells.The pathogenesis involves repeated injury of the alveolar epithelium and the release of abundant cytokines and growth factors that promote the recruitment,proliferation and differentiation of pulmonary fibroblasts into myofibroblasts which resulting in excessive collagen deposition,scar formation,and destruction of lung structure,which eventually lead to irreversible damage of lung function.If untreated after diagnosis,the average life expectancy is 3-5 years.In the past decades,many studies have shown that macrophages play a significant role in the pathogenesis of IPF.Macrophages can be polarized into classical activation M1 or alternative activation M2 phenotype.M1 macrophages,as a pro-inflammatory phenotype,are responsible for wound healing after alveolar epithelial injury.M2 macrophages stimulate fibroblast proliferation and collagen production,which play a crucial role in wound healing in the early stage of injury.However,the increased M2 macrophages will facilitate the development of IPF by forming a fibrous microenvironment.Therefore,M2 macrophages are also considered to be a fibrogenic phenotype.Targeting M2 macrophages and regulating their polarization may be a potential therapeutic strategy for IPF.Here,we designed mannose-modified magnetic nanoparticles（MAN-MNPs）that are loaded on the surface of the liposome（MAN-MNPs@LP）,which can both accumulate in the lung in response to a static magnetic field（SMF）and the targeting of M2 macrophages via mannose receptor-dependence internalization.Firstly,theα-Fe₂O₃based MNPs were prepared by hydrothermal method.After mannose was modified on the surface of MNPs,the MAN-MNPs with positive charge were modified on the outer surface of LP with negative charge through electrostatic interaction to form a mannose modified magnetic liposome（MAN-MNPs@LP）.The surface charge and particle size were measured by Dynamic Light Scattering（DLS）,the surface charge of MAN-MNPs@LP was between MAN-MNPs and LP,and the particle size was larger than MAN-MNPs and LP.The Transmission Electron Microscope（TEM）,Atomic Force Microscope（AFM）,and Scanning Electron Microscope（SEM）were intuitively observed that the MAN-MNPs were successfully modified on the outer surface of LP.The modification of MAN in MAN-MNPs and the content of MAN-MNPs in MAN-MNPs@LP were analyzed quantitatively by Thermogravimetric Analysis（TGA）.The functional groups and element types on the surface and inside of MAN-MNP@LP were detected by using X-Ray Photoelectron Spectroscopy（XPS）,Energy Dispersive X-Ray Spectroscopy（EDX）,and Fourier Transform Infrared Spectrometer（FT-IR）.The X-Ray Diffraction Spectrum（XRD）determined that MNPs wereα-Fe₂O₃structure,and it was clear that the modification of MAN and LP would not damage its crystal structure.The Saturation Magnetization（Ms）of MNPs,MAN-MNPs,and MAN-MNP@LP measured by the Vibrating Sample Magnetometer（VSM）was 3.84,3.12,and 1.61 emu/g,respectively,which was much higher than the average Ms ofα-Fe₂O₃（Ms=0.3 emu/g）.The increased Ms was more conducive to response to the SMF for the magnetic targeting.Finally,the encapsulation efficiency（EE%）of Dex in MAN-MNPs@LP was determined to be 21.26±3.72%by using Ultra high performance liquid chromatography（UPLC）.The release curve of Dexamethasone（Dex）was time-dependent,with about released 63.59±3.98%of Dex at 10 h and 73.98±3.08%at 48 h.Subsequently,it was determined that MAN-MNPs@LP and MNPs@LP had low cytotoxicity.RAW 264.7 cell lines were induced by 20 ng/m L Interleukin-4（IL-4）for 48h,the M2 macrophages model was successfully constructed in vitro.High content cell imaging analysis system and flow cytometry were used to detect the uptake of MAN-MNPs@LP and MNPs@LP that were modified with fluorescent lipid by M2 macrophages per unit time.The results showed that M2 macrophages could absorb more MAN-MNPs@LP after modifying mannose.Next,the mouse with the IPF model was constructed by intratracheal instillation of bleomycin（BLM）.In Vivo Imaging System（IVIS）and Prussian blue staining was used to compare the accumulation of MAN-MNPs@LP in the lungs of IPF mice with or without SMF and mannose modification.It was found that dual-targeting MAN-MNPs@LP can rapidly and passively accumulate in the lung when SMF is applied.Subsequently,mannose exerted the targeting function of M2 macrophages and targeted M2 macrophages.Finally,the therapeutic effect of MAN-MNPs@LP that loaded with Dex（MAN-MNPs@LP-Dex）on IPF mice was investigated.The degree of pulmonary fibrosis was detected by Micro Computed Tomography（Micro-CT）.The morphological changes in lung tissues were observed by H&E staining.Masson’s trichrome staining detected collagen deposition.Furthermore,Immunohistochemical staining was used to detect the expression ofα-SMA,which is the activation marker of lung fibroblasts.Immunofluorescence staining was used to detect the infiltration of total macrophages and M2 macrophages.MAN-MNPs@LP and MNPs@LP were given to unmodeled healthy mice,then H&E staining found that MAN-MNPs@LP and MNPs@LP showed good biocompatibility in vivo.The results showed that MAN-MNPs@LP-Dex could significantly improve the survival rate of IPF mice after applying SMF and delay the development of IPF by reducing collagen deposition,inhibiting myofibroblast activation,and reducing macrophage inflammatory infiltration and inhibiting M2 macrophage polarization.In this study,a mannose-modified drug-loaded magnetic liposome was constructed.Using the magnetic targeting of MNPs and the macrophage targeting of MAN,it could rapidly aggregate in the lung in response to SMF and then targeted to the M2 macrophages for drug delivery to retarded the development of IPF.The dual-targeting drug delivery system can effectively deliver Dex to the lung.It is expected to become a potential treatment system for IPF treatment and provide ideas for treating pulmonary diseases.