MR Imaging Evaluation Of Angiogenesis And Lymphangiogenesis In Breast Cancer Using Dual Targeting CL-PEG-MnFe₂O₄Nanomicelles

Background and Objective：Hematogenous and lymphatic metastasis is the most common form of metastasis inpatients with malignant tumor. The angiogenesis and anti-angiogenesis therapy is alwaysthe hot point in cancer research since Folkman first proposed the hypothesis of tumordevelopment was dependent vascular in1971. In recent years, lymphangiogenesisgradually in depth study at the molecular level with a series of lymphatic endothelial cells（LECs） markers found on tumor. Many tumors （especially tumors of epithelial origin, suchas breast cancer, et al.） likely to simultaneously led to hematogenous and lymphaticmetastasis. Microvessel density （MVD） and lymphatic vessel density （LVD） are closelyrelated to the metastasis and prognosis of tumor. So, it is difficult to determine whether thetumor has occurred metastasis or not if we only focus on one metastatic pathway. At thesame time, In order to evaluate the angiogenesis and lymphangiogenesis accurately, wemust differentiate the tumor neoangiogenesis and neonatal lymphatic vessels from themicrovessels in the normal tissue.Endoglin, also known as CD105, is a glycoprotein expressed in endothelial cellmembrane, is one of the transforming growth factor-β （TGF-β） receptor complex. Endoglinregulate the signal transduction between endothelial cell and interstitial tissue through theTGF-β, and promote the angiogenesis. Endoglin overexpressed in neonatal vascularendothelial cells in regenerated tissue, inflammation and tumor tissue. The expression levelsand degree of Endoglin is closely related to the endothelial cell proliferation. The MVDbased on Endoglin staining is closely related to the tumor prognosis. Endoglin is also theproliferation marker of LECs, which is overexpressed in neonatal lymphatic vessel, but notexpressed in the LECs of normal tissue. Thus, it is expected to achieve dual targeting imaging of breast cancer angiogenesis and lymphangiogenesis if Endoglin was used as amolecular target in magnetic resonance （MR） molecular imaging.To our knowledge, there is no relevant report on simultaneous detection of tumormicrovessel and microlymphatic vessel using a MR contrast agent. In this study, MnFe₂O₄nanoparticles were synthesized using thermal decomposition method. MnFe₂O₄nanoparticles were self-assembly with two block polymer of PEG-PCL to constructwater-soluble PEG-PCL-MnFe₂O₄nanomicelles. Endoglin specifically targeted polypeptideCL-1555was then bound to the surface of PEG-PCL-MnFe₂O₄nanomicelles,whosephysical properties were then studied. Using these nanomicelles, tumor angiogenesis andlymphangiogenesis were evaluated with MR imaging in vitro and in vivo, and the feasibilityof evaluating tumor angiogenesis and lymphangiogenesis in vivo were investigated in thisstudy.Contents and Methods：1. Synthesis and characterization of PEG-PCL-MnFe₂O₄nanomicellesMnFe₂O₄nanoparticles were synthesized using thermal decomposition method.MnFe₂O₄nanoparticles were self-assembled with two block polymer of PEG-PCL toconstruct water-soluble PEG-PCL-MnFe₂O₄nanomicelles. The characteristics of theMnFe₂O₄nanoparticles and PEG-PCL-MnFe₂O₄nanomicelles were tested usingtransmission electron microscope （TEM）, zeta-particle analyzer, infrared absorptionspectrum and inductively coupled plasma atomic emission spectrometry. The relaxation rateof MnFe₂O₄nanomicelles were measured with MR scanner.2. Synthesis of CL-PEG-MnFe₂O₄nanomicelles and in vitro experimentEndoglin specifically targeted polypeptide CL-1555was synthesized, which wasbound to the surface of PEG-PCL-MnFe₂O₄nanomicelles to constructed CL-PEG-MnFe₂O₄nanomicelles by crosslinking reaction. In order for the VECs and LECs to acquire thecharacteristics of breast cancer, VECs and LECs were incubated with breast cancer cells inMillicell cell culture. The incubated VECs and LECs were co-cultured with CL-PEG-MnFe₂O₄nanomicelles at molar iron concentration of0mmol/L、0.01mmol/L、0.02mmol/L、0.03mmol/L、0.04mmol/L、0.06mmol/L、0.08mmol/L、0.1mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L and0.8mmol/L. The intracytoplasmic nanoparticleswere confirmed with Prussian blue iron staining,fluorescence microscopy and TEM. Non- incubated HDLECs and PEG-PCL-MnFe₂O₄nanomicelles were used as control. Labeledcells were suspended in PBS in EP tubes and axial MR imaging was performed on a3.0TMR scanner using an eight-channel phased-array head coil, with PBS as a control. Themorphologic features of cells in each concentration were observed to assess the toxicity ofCL-PEG-MnFe₂O₄nanomicelles on VECs. CCK-8assay kit was used to test the affect ofCL-PEG-MnFe₂O₄nanomicelles on cell proliferation activity. The labeled cells werepassaged, and the labeling efficiencies of labeled cells were observed. The contents ofmanganese and iron in passaged cells were measured using ICP-AES instrument, and theMR imaging were performed to observe the variation of MR signals.3. Establishment of breast cancer xenograft and optimization of MR imagingMR imaging were performed with small joints coil and dedicated small animal coil, andthe parameters for MR imaging were optimized to evaluate the practical value ofconventional MR scanner in nude mice. Superparamagnetic iron oxide （SPIO） nanoparticleswere intravenous through tail vein and retrobulbar vein. The value of retrobulbar veininjection of magnetic nanoparticles was evaluated using MR imaging.4. MR molecular imaging of breast cancer xenograft based on CL-PEG-MnFe₂O₄nanomecellesCL-PEG-MnFe₂O₄nanomicelles were intravenous administrated by retrobulbar vein.MR imaging was performed on a3.0T MR scanner using dedicated small animal coil. TheMR imaging sequences included spin echo （SE） T1-weighted imaging （T1WI）, fast spinecho （FSE） T2-weighted imaging （T2WI）, gradient echo （GRE） T2*-weighted imaging andFSE T2mapping with16echos, with PEG-PCL-MnFe₂O₄as a control. Rats were sacrificedafter MR imaging. Pathological and immunohistochemical analysis were performed in thearea consistent with the ROI of the MR images. Finally, the value of Endoglin-targeted MRimaging in detecting the angiogenesis and lymphangiogenesis in breast cancer wasevaluated by comparatively analyzing the MR images and pathological andimmunohistochemical results.Results：1. MnFe₂O₄nanoparticles appeared as round under TEM. The average size ofGoldMag particles was11nm with good monodisperse, zeta-particle size was11.18±1.72nm. The molar ratio of iron/manganese was2.13:1. After MnFe₂O₄nanoparticles were self-assembled with two block polymer of PEG-PCL, the size of PEG-PCL-MnFe₂O₄nanomicelles was enlarged, ranging from52nm to86nm, with a mean size of78.8±12.4nm. The absorption peak of the C=O around1710cm-1under the IR indicates the presenceof PEG. With increasing iron concentration, signal intensity （SI） decreasing after increasingfirst in SE T1WI, and decreased in FSE T2WI and GRE T2*WI. The signal intensity changesin FSE T2WI and GRE T2*WI were significantly stronger than that in SE T1WI, especiallyin GRE T2*WI （P<0.05）.2. The human umbilical vein endothelial cells （HUVECs） were isolated successfully.Incubated VECs were positive for CD105immunofluorescence staining, and the incubatedLECs showed red and green fluorescence after double staining with podoplanin and CD105.The fluorescence intensity of incubated VECs and LECs was significantly stronger than thatof non-incubated VECs and LECs. The cell labeling ratio with CL-PEG-MnFe₂O₄nanomicelles was significantly higher than that of PEG-PCL-MnFe₂O₄nanomicelles. Atiron concentrations of0ug/mL,0.5ug/mL,1ug/mL,2ug/mL,5ug/mL,10ug/mL,the labelingratios were, respectively,0%,26.95±4.38%,62.73±3.07%,82.34±3.67%,100%and100%forincubated HDLECs, and0%,15.61±3.42%,36.35±2.26%,52.31±3.23%,87.53±4.62%and100%for non-incubated HDLECs. Under the TEM, high electron density particles werefound in cytoplasm and lysosomes. SI gradually increased in SE T1WI, and decreased inFSE T2WI and GRE T2*WI. The signal intensity changes in GRE T2*WI were significantlystronger than that in FSE T2WI （P<0.05）. The T2relaxation time decreased with increasingiron concentration. The SI of labeling cells with CL-PEG-MnFe₂O₄nanomicelles was lowerthan that with PEG-PCL-MnFe₂O₄nanomicelles, and the SI of incubated cell suspensionswas lower than that of non-incubated cells. The proliferative activity of VECs was affectedlittle when the iron concentration was lower than20ug/mL. When the iron concentrationreached50ug/mL, the proliferative activity of VECs was affected to some degree, and cellproliferation significantly affected when iron concentration reached100ug/mL, the activecells decreased significantly. As the cell passaging, labeling efficiency decreased. SIgradually increased in FSE T2WI. In SE T1WI, the SI of passage1（P1） cells was lower thanthat of PBS. However, the SI of P2and P3cells was higher than that of PBS. The P4cellsshowed isointense both in T1WI and T2WI. As the labeled cell passaging, the amount of irondecreased faster than manganese, and the molar ratio of iron/manganese gradually increased.3. The establishment of breast cancer xenograft model by subcutaneous inoculationcell suspension was a simple operation. The tumor formation rate was100%, and the tumorincubation period was about1-2weeks. The characteristics of xenograft were similar to thatof human breast cancer. The signal to noise ratio （SNR）, resolution and contrast withdedicated small animal coil were higher than that with small joints coil. The size ofxenograft in two weeks is about1cm3, and the SI of tumor is homogeneous. The tumordisplayed slightly hypointense in T1WI and hyperintense in T2WI images. The boundary ofthe tumor was clear, and no adhesion with adjacent structures. Necrosis occurred in largertumor （usually after3weeks）, and the necrotic area showed hyperintense in T2WI images.The means of retrobulbar vein puncture is simple and easy. After injection of magneticnanoparticles by retrobulbar vein, nanoparticles accumulated in liver consistent with theclearance of nanoparticles in the plasma.4. The enhancement degree at peripheral area of the tumor is higher than that in thecentral area after intravenous administration of CL-PEG-MnFe₂O₄nanomicelles. After60min clearance, the tumor displayed patchy or spot-like enhancement, which was mainlylimited to the peripheral areas of the tumors. However, the SI returned to the baseline after a60min recovery when the contrast agent was PEG-PCL-MnFe₂O₄nanomicelles. Under themicroscope, the blue-stained iron particles were observed around the cancer cell nest withPrussian blue iron staining. In the same area, blood vessels and lymphatics with brownstaining were observed, corresponding to immunohistochemical staining for CD105, CD34and podoplanin, respectively, indicating that the nanoparticles were combined with bloodvessels and lymphatic vessels of breast cancer. After intravenous injection ofCL-PEG-MnFe₂O₄nanomicelles, SI decreased immediately and the relative SI on T2WIweighted images were16%, thereafter, relative SI returned to about48%of the peak valueafter a60min recovery. However, the SI returned to the baseline after a30min recoverywhen the contrast was PEG-PCL-MnFe₂O₄nanomicelles.Conclusion：1. High quality MnFe₂O₄nanoparticles can be synthesized by thermal decomposition.The self-assembly PEG-PCL/MnFe₂O₄nanomicelles coated with amphiphilic blockcopolymer are more sensitive contrast agent for T2WI, which have stronger T2relaxivity than PEG-PCL/Fe3O4nanomicelles.2. The separation method of HUVECs is convenient, simple, and can get many cells inone time. The HUVECs is a reliable cell for characterized study of endothelial cell. Themethod is a reliable way for gaining tumor-derived endothelial cells by co-culture ofendothelial cells and tumor cells. After sequence optimization, conventional clinical MRscanner could be used to image nude mice combining with a dedicated small animal coils.3. Establishment of breast cancer xenograft by subcutaneous inoculation of cellsuspension in nude mice is simple, and the tumor formation rate is higher. The constructedbreast cancer is a reliable animal model for research of angiogenesis and lymphangio-genesis.4. Venipuncture in the retrobulbar vein is more simple than the tail vein puncture,which is reliable route for intravenous administration of magnetic nanoparticles.5. The CL-PEG-MnFe₂O₄nanomicelles combined with Endoglin targeting peptidescan specifically bind to tumor-derived VECs and LECs, which can bind to neovascularendothelial cells and lymphatic endothelial cells after intravenous injection, which could bedetected by MR imaging. The CL-PEG-MnFe₂O₄nanomicelles show no toxicity to cellswithin a certain concentration, which have potential application for target MR imaging oftumor neovascular and lymphatic vessels.6. The paramagnetic manganese ions （Mn2+） can be released from MnFe₂O₄nanoparticles metabolized in cytoplasm, which can stay longer in the cell and could bedetected with T1WI imaging in a certain concentration. That is to say, the MnFe₂O₄nanoparticles are expected to apply for double-contrast agent of MR imaging.