Osteopontin Targeted Theranostic Nano-drug Delivery System For Dual-modal Imaging Guided Photodynamic Therapy Of Vulnerable Atherosclerotic Plaques

Background and Objective:The rupture of vulnerable atherosclerotic plaques(VASPs)and the formation of thrombus are the major causes of acute cardiovascular events and sudden death.It is of great clinical significance to achieve accurate diagnosis and targeted therapy of VASPs simultaneously in the early stage of the disease.The nano-drug delivery system which based on molecular imaging technology is capable of achieving multimodal imaging of VASPs by targeting biomarkers of the plaques and improving the therapeutic effect of anti-atherosclerotic drugs.Osteopontin(OPN)plays an important role in the process of atherosclerosis and is highly expressed in VASPs,especially in macrophage-derived foam cells and synthetic smooth muscle cells.Therefore,it can be a targer for VASPs detection.Photodynamic therapy(PDT)combines photosensitizer and laser irradiation to generate a large amount of singlet oxygen and kill cells selectively.Herein,this study constructed a human serum albumin(HSA)-based nanomedicines(NMs)by conjugating OPN peptide and MRI contrast agent Ce6-Mn2+ and encapsulating photosensitizer indocyanine green(ICG),which possessing good bio-compatibility and low toxicity,aiming at achieving precise identification of VASPs by optical/MR imaging as well as specific and efficient therapy against atherosclerosis by killing foam macrophages and synthetic smooth muscle cells through PDT,thus inhibiting plaque regression and promoting its stabilization.Methods:1.The RAW264.7 cells line and MOVAS cell line were incubated with low-density lipoprotein for 24h.The difference of OPN expression was observed by immunofluorescence and Western Blot among the model group and control group.ApoE-/-mice were fed with a high-fat diet(HFD)for 2 weeks before a perivascular cuff was placed around the right common carotid artery and then kept on a HFD for another 20-24 weeks.The establishment of the VASPs model was confirmed by oil red o staining of artery and HE,Masson,Sirius Red staining of carotid plaque while the character of OPN expression was verified by immunohistochemistry of OPN,CD68,and a-SMA.2.The NMs were prepared by surface targeting moiety of OPN peptide and conjugated with Ce6 that could be successfully developed for magnetic resonance imaging(MRI)after chelated contrast agent Mn2+.Then near infrared fluorescent dye(ICG)was encapsulated in NMs to realize fluorescence/MR dual-model imaging-guided precision PDT.The NMs were characterized by dynamic light scattering(DLS),transmission electron microscopy(TEM),ultraviolet and fluorescence spectrophotometer.3.NMs were incubated with foam macrophage and synthetic smooth muscle cells to evaluate its cellular uptake ability in vitro.For in vivo study,NMs were intravenously injected into mice and fluorescence/MR imaging signal were obtained at continuous time point to confirm whether the targeted NMs could detect VASPs accurately.4.CCK-8 assay was performed to measure the cytotoxicity and PDT effect of NMs in different concentration.Flow cytometry was conducted to confirm whether PDT could induce cell apoptosis.The size and vulnerability of plaques were evaluated after systemic administration of nanomedicines for three weeks by MR imaging.Results:1.In vitro study showed that OPN was remarkably expressed in in macrophage-derived foam cells and synthetic smooth muscle cells compared with control group,and the expression of OPN in macrophages increased more significantly after activation.Immunohistochemical results of vulnerable carotid plaques also confirmed that the expression of OPN,CD68 and a-SMA were co-localized.These results indicated that OPN could be used as a biomarker to detect VASPs.2.The TEM image showed that discoid-shaped NMs were well-prepared.DLS result indicate the NMs were stable and not prone to aggregation.The UV-Vis absorption spectra demonstrated that Ce6,ICG and HSA are all successfully constructed into NMs.The fluorescence spectra showed the NMs could generate singlet oxygen under 808 nm laser irradiation rapidly,and gradually flattened.3.Good binding sensibility and specificity of NMs were confirmed according to cellular uptake analysis.In vivo fluorescence/MR imaging revealed that the targeted NMs were abundantly accumulated in the vulnerable carotid plaques and the strongest fluorescence signal was obtained 30 minutes after intravenous injection.There was no obvious fluorescence signal was observed in the control group,indicating that targeted NMs could accurately identify VASPs.4.CCK-8 assay showed excellent biocompatibility and therapeutic effect of NMs during PDT.Flow cytometry demonstrated that PDT exerted therapeutic effect by inducing cell apoptosis.In vivo MR imaging also revealed that the carotid artery stenosis was alleviated and plaque stability was improved after PDT.Conclusion:OPN can be a molecular marker of smooth muscle cell proliferation and macrophage activation in VASPs.OPN targeted NMs can accurately identify VASPs in both in vitro and in vivo study by fluorescence/MR imaging and induce cell apoptosis to improve plaque stability through PDT.