Preparation, Imaging And Biological Effects Of Tumor Angiogenesis-Targeted Manganese Oxide Nanoparticles

BackgroundTumor is one of the most common causes of death in the world. Accroding to reportof the World Health Organization, more than10million new cases of tumor in which thenumber of death cases is more than7million happened worldwide each year. The numberof new cases of cancer is about1.66million in the United States in2013and death causedby cancer is580000. In China, the number of new cancer cases is up to3.5million and thatof cancer death cases is2.5million by the end of2012. Early detection, early diagnosis andearly treatment are the key to improve the survival rate of cancer patients. How to detectand diagnose the tumor timely at early stage of the tumor is the basis of early treatment.Tumor angiogenesis is closely related to the occurrence, development and metastasize oftumor because of supplyment of nutrients and oxygen. It is suggested that degree of tumorangiogenesis is associated with staging or degree of some malignances. Therefore,detection quantitative research of tumor angiogenesis are not only criticle to earlydiagnosis of cancer, but also helpful to monitor and evaluate therapeutic effect. Previousmolecular biology research shows that there are integrins, belonging a catagory ofadhesion molecules, on the surface of tumor vessel or some tumor cell surface, which playan important role in the angiogenesis and metastasis of tumor. Alpha v beta3(αvβ3)receptor is the most common target for tumor angiogenesis in early diagnosis of tumor.αvβ3integrin is highly expressed in proliferated or activated endothelial cells during tumorangiogenesis. However, there are no expression of αvβ3on the stable vascular endothelialcells. It is suggested that the arginine—glycine—aspartic acid (RGD) peptide sequence inextracellular matrix is a specific recognition motif to αvβ3integrin. Development of RGDpeptide labeled，high specific MR probe for molecular imaging of tumor angiogenesis iscapable to facilitate early dectection and may help monitor tumor vascular change duringanti-angiogenesis therapy. MRI has several advantages such as high spatial resolution,multiple sequences and parameters, multi-dimensional imaging, lack of ionizing radiation,capibitily of providing the anatomical and physiological information. So MRI has becomea desired imaging tool for evaluating tumor angiogenesis. Contrast enhancement MRI,especially MRI using target contrast agent can not only improve diagnostic accuracy ofpathological changes, but also reflect biological characteristics of some disease andevaluate treatment effect. In recent years, target contrast enhancement MRI is one of hot spot in the molecular imaging research.Objective1. To develop a MR contrast agent——RGD coupled, polyethylene glycol (PEG)packaged and dopamine (DA) connected manganese oxide nanoparticles （RGD-mPEG-DA–MnO）which can target tumor blood vessels and cells and to make MR imagingmediated by integrin αvβ3availble.2. To explore and assess the feasibility of MRI contrast agent RGD-mPEG-DA-MnO for target imaging in nude mice bearing human lung adenocarcinoma.3. To evaluate the biological effect of RGD-mPEG-DA-MnO on human umbilicalvein endothelial cells (HUVEC) and A549human lung adenocarcinoma cells.Materials and Methods1. Preparation of MRI the targeted Contrast Agent and Characterization in vitro2. The toxicity of the contrast agents in vitro and relaxativity(1) MTT assay was used to detect the effect of contrast agent (RGD-mPEG-DA-MnO) on survival rate of A549cells in vitro.(2) T1Mapping method was used to detect the relaxivity of contrast agents (RGD-mPEG-DA-MnO).3. Specific and non-specific magnetic resonance contrast agent tumor xenograftsMagnetic resonance imaging(1) Study on MRI non-targeted contrast agent (mPEG-DA-MnO) imaging inBALB/C mouseTo observe signal changes of liver，kidney and muscles of BALB/C mouse in T1WIrespectively before and after the administration at different time points，MRI contrast agentmPEG-DA-MnO was administered by the mouse tail vein．(2) Study on MRI targeted contrast agent (RGD-mPEG-DA-MnO) imaging innude mouse bearing human lung adenocarcinomaThe A549lung adenocarcinoma cells were inoculated subcutaneously in nude mice toproduce human lung adenocarcinoma model. MRI target contrast agent RGD-mPEG-DA-MnO was injected by the mouse tail vein agent (RGD-mPEG-DA-MnO), signal valuechanges of tumor, liver and muscle of in nude mice bearing human lung adenocarcinoma inT1WI respectively before and after the administration at different time points wereobserved. (3) Comparion of target contrast agent (RGD-mPEG-DA-MnO) and non targetedcontrast agent (mPEG-DA-MnO) MR imaging in nude mouse bearing human lungadenocarcinomaNude mice (n=8) bearing human lung adenocarcinoma were randomly divided into2groups，including RGD-mPEG-DA-MnO target contrast agent group (n=4)，mPEG-DA-MnO non-target contrast agent group (n=4) respectively．After the injection of targeted contrast agent and non-target respctilvely through themouse tail vein，T1WI signal value changes of tumor and muscle before and after injectionat different time points were observed and compared．4. Study on biological effect of mPEG-DA-MnO on HUVECs and A549cells.A variety of cytobiology assays were conducted to explore biological effect of mPEG-DA-MnO on HUVECs and A549cells, such as proliferation, differentiation, migration,invasion, cell skeleton and the influence of the biological behavior.Results1. mPEG-DA-MnO and RGD targeting moiety with mPEG-DA-MnO weresuccessfully prepared．The nanoparticles have high purity and high affinity with αvβ3receptor.2. The toxicity of mPEG-DA-MnO MRI contrast agent is low. The relaxativity ofmPEG-DA-MnO magnetic nanoparticle is high. It is approximately three times as that ofconventional gadolinium contrast agent．3. In MRI study on the targeted nude mice bearing human lung adenocarcinoma, thereagent RGD-mPEG-DA-MnO has the accumulation trend of localizing to thetumor．The signal value peak merges in about2hours after injection and the maximumenhancement ratio is109.9％；The enhancement effect in the liver is also apparent and theenhanced peak occurs in about1hours，followed by the slow downward trend．Compared with non-target of mPEG-DA–MnO, RGD-mPEG-DA-MnO contrastagent has significant effect on tumor targeting．There is statistical difference in the signalvalues of RGD-mPEG-DA-MnO group at different time points (F=166.83，P<0．0001)；the enhanced peak Occurs in about2hours after injection；significant difference of thesignal values of non-targeting of mPEG-DA-MnO groups at different time points werealso observed；the enhanced peak occurs in about1hours after injection；There were nosignificant differences for the signal value of the muscles in both group at different scantimes． 4. The biological effects of polyethylene glycol coated MnO nanoparticles onHUVECs were investigated in terms of cell proliferation，cytoskeleton，migration／invasion and differentiation. At a concentration of equal to or more than500ug/L, mPEG-DA-MnO can inhibit HUVECs proliferation, migration and invasion; Cellproliferation,migration and invasion were inhibited in a dose dependent manner.Manganese oxide nanoparticles (MONP) can inhibite the ability of endothelial cells todifferentiate to lumen structure, cause cytoskeleton redistributed, vinculin spots diminishedand actin fiber/tubulin networks disorganized．The detachment after exposure to MONPsinduced the dysfunction of HUVECs and the manganese catalyzed free radical damagemight attribute to it．A high concentration of manganese oxide nanoparticles reduced cellmitochondrial membrane potential and induced oxidative stress reactions such as reactiveoxygen species.ConclusionThe highly binding of RGD with targeted contrast agent-MONPs (RGD-mPEG-DA-MnO) helps to intensify the effect in MR enhancement in nude mice model for tumoragiogenesis, so as to fufill for target imaging and diagnosis in MR. High concentration ofPEG coated magnetic MONS (mPEG-DA-MnO) can cause cytotoxic effect by inducedoxidative stress.