Preparation Of UPAR Targeting Superparamagnetic Iron Oxide Nanoparticles And Their Application In The Detection Of Atherosclerotic Plaque And Breast Cancer

Objective:With the development of medical imaging,more and more imaging technologies have been applied into the detection and diagnosis of diseases.Magnetic resomance imaging(MRI)is a safe,non-invasive diagnostic technology.With its obvious advantages,MRI has been widely used in the detection of all kinds of diseases such as tumors and obtained increasing recognitions.Superparamagnetic iron oxide nanoparticles(SPIO)is a kind of effective MRI contrast agent.With a large specific surface area,SPIO can be introduced different targeting ligands through chemical coupling method to help realize targeting diagnosis of diseases and improve detection rate of small lesions.Atherosclerosis remains one of the health-threatening diseases and the leading cause of morbidity and mortality worldwide.Atherosclerotic plaque rupture with thrombosis leads to many adverse events,such as stroke and acute coronary syndrome.Therefore,there is an urgent need to detect plaques prior to their sudden rupture to improve treatment strategies.Besides,breast cancer is one of the most common malignancies in women.It is a highly recurrent disease that is often difficult to cure.Patients with advanced breast cancer usually have systemic metastasis,which is associated with its high morality.Thus,faced with the clinical treatment challenges of metastasis and high recurrent rate of breast cancer,it is of great significance to develop specific molecular contrast agents to realize the early diagnosis of primary and secondary breast cancer and improve patients'long-term survival rate.Urokinase-type plasminogen activator receptor(uPAR)is one kind of membrane anchored glycoprotein and can bind to the amino-terminal fragment(ATF)of the receptor binding domain of urokinase plasminogen activator(uPA),the native ligand of uPAR.Studies have shown that the expression of uPAR in ruptured plaques is higher than that in non-ruptured plaques and it is mainly located in the macrophage-rich area of plaques.Therefore,uPAR is a promising target biomarker for the detection and evaluation of atherosclerotic plaques and there is no related studies have been reported.uPAR is lowly expressed in normal tissues and highly expressed in most tumors.Different from the tumor specific antigen,uPAR is not only highly expressed on the tumor cells,but also on tumor-associated cells.Thus,uPAR-targeted contrast agents present an invaluable potential in the tumor early diagnosis.In this work,we developed an uPAR-targeted MRI contrast agent.For one thing,we hope it can be used for the non-invasive imaging of macrophages in plaques and the targeting diagnosis of AS.For another thing,mouse breast cancer cell line 4T1 was used to evaluate its targetability to tumor on the purpose of improving the detection rate in the diagnosis of early breast cancer.Methods:First,with the conjugation of nitrilotriacetic acid(NTA)to the surface of SPIO,an universal imaging platform was prepared that can load different ligands attached with.Next,we conjugated ATF to SPIO-NTA through the interaction between histidine tags(His tags)on ATF and NTA to obtain the targeted MRI agent(SPIO-ATF).The morphology of SPIO was observed using transmission electron microscope(TEM).The iron content of SPIO was determined using the o-phenanthroline method and the dextran content was obtained by deducting the weight of Fe2O3 from the total weight of SPIO after lyophilization.The hydrodynamic diameters and Zeta potentials of SPIO-ATF were determined by dynamic light scattering(DLS).The introduction of ATF was evaluated by protein gel electrophoresis with Coomassie brilliant blue staining and BCA Protein Quantification Kit.A co-precipitation experiment and western blot were performed to confirm the binding capability of SPIO-ATF to uPAR protein.The cell binding capability,cellular uptake and cytotoxicity of SPIO-ATF were investigated on Raw 264.7 cell line and 4T1 cell line.Herein,the cell binding capability was investigated by Prussian blue staining,o-phenanthroline method and western blot.The cellular uptake was measured by Prussian blue staining and o-phenanthroline method.CCK-8 assay was performed to detect the effects of SPIO-ATF on cell proliferation.The atherosclerosis model was induced in apolipoprotein E deficient(ApoE-/?)mouse by high-fat diet for 3 months.The plaque was detected by oil red O staining,masson trichrome staining and alizarin red staining to conform the establishment of atherosclerosis model.In vivo MRI studies of ApoE-/-atherosclerosis model mice were carried out on a 7.0 T MRI system before and 24 h after the intravenous injection of SPIOs.Successive sections of the plaque tissues were treated by immunohistochemistry staining and Prussian blue staining to visulize the distributions of uPAR,macrophages and SPIO-ATF.The biodistributions of SPIO and SPIO-ATF were detected by Prussian blue staining.Results:TEM showed that the diameter of the y-Fe2O3 core was less than 10 nm.The iron content of SPIO was 24.67 ± 0.37 mg/mL and the dextran content was 98.73 ± 2.83 mg/mL.The DLS results revealed that SPIO had a hydrodynamic diameter of 67.13 ±1.43 nm,which slightly increased to 76.59 ± 1.67 nm after the conjugation of ATF.The Zeta potentials of SPIO and SPIO-ATF were-37.13 ± 2.54 mV and-13.00 ± 1.55 mV,respectively.The decrease in Zeta potential may be attributed to the charge screen effect of ATF introduced on the surface of SPIO.The introduction of ATF was further confirmed by protein gel electrophoresis with Coomassie brilliant blue staining.The molar ratio of dextran,Fe2O3 and ATF was 4:250:9 in SPIO-ATF,indicating that two to three ATF peptides were attached to each dextran molecule on average.The co-precipitation experiment and western blot results showed that SPIO-ATF can bind to uPAR protein via the ATF segment.It was indicated in vitro that after 4-h incubation,the binding amount of SPIO-ATF to Raw 264.7 cells was significant higher than that of SPIO and pre-treatment of cells with excess free ATF almost completely blocked the binding capability of SPIO-ATF to Raw 264.7 cells.The expression of uPAR in Raw 264.7 cells was up-regulated by phorbol ester(PMA)or down-regulated by SB203580 before incubation with SPIO-ATF.Both the Prussian blue staining and iron quantification results showed a positive relationship between the SPIO-ATF bound with cells and relative expression of uPAR,indicating the specific binding capability of SPIO-ATF to Raw 264.7 cells via the interaction between ATF and uPAR.After 24-h incubation with different concentrations of SPIO-ATF,the cellular uptake of SPIO-ATF by Raw 264.7 cells was increased with the increase in the incubation concentration of SPIO-ATF.CCK-8 assay showed that no cytotoxicity was observed under the experimental conditions regardless of whether SPIO or SPIO-ATF was used.As a kind of highly-expressing uPAR cell,4T1 cells can be specifically bound by SPIO-ATF.The cellular uptake of SPIO-ATF by 4T1 cells was also dependent on the incubation concentration of SPIO-ATF.After a 3-month exposure of ApoE-/-mice to a high-fat diet,multiple areas on the aortic lining were stained red with oil red O staining.Additionally,lipid had accumulated in the vessel walls,and atherosclerotic plaques had formed.The collagen mainly produced by smooth muscle cells(SMCs)was stained blue by Masson trichrome staining,indicating the migration and proliferation of medial SMCs.Calcification in the plaque was stained red with alizarin red staining,indicating that the plaque had developed to a late-stage plaque.In vivo MRI results showed that after the intravenous injection of SPIO-ATF,the relative T2 signal intensity of the arterial wall was decreased by 53.2%,displaying a distinct enhanced contrast,while SPIO injection only led to a 26.5%reduction in the T2 signal.Prussian blue staining of the plaque tissues showed that ATF modification can increase the accumulation of SPIOs in the plaque significantly.Immunohistochemistry staining showed that the uPAR-positive areas and CD68-positive areas were highly consistent,indicating that macrophages are the major cell component expressing uPAR in the plaque.The co-localization of SPIO-ATF and CD68-positive areas indicated the targetability to macrophages of SPIO-ATF.SPIO-ATF may be largely taken into macrophages mediated by the interaction between ATF and uPAR,inducing a high accumulation of SPIO-ATF in the plaque.Prussian blue staining of major organs sectioned after MR imaging showed the preferential accumulation of SPIO/SPIO-ATF in the liver and spleen rather than in the heart,brain,lung and kidney.The introduction of ATF may slightly reduce the nonspecific trapping of SPIO-ATF in the liver and spleen and SPIO-ATF may be more likely to accumulate in the plaques.Conclusion:This work presents a novel MRI contrast agent--SPIO-ATF.On the one hand,with targetability to macrophages mediated by the interaction between ATF and uPAR,SPIO-ATF may accumulate in the macrophage-rich area of atherosclerotic plaques and displays distinct contrast in in vivo T2-weighted MR images,thereby exhibiting potential for the detection and diagnosis of vulnerable atherosclerotic plaques.On the other hand,SPIO-ATF can specifically bind to 4T1 cells and be taken in via the interaction between ATF and uPAR,presenting a potential in the targeting detection of breast cancer and providing a new strategy for the early MRI diagnosis of the small lesions of breast cancer.