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MR Imaging Study of Lymphatic Endothelial Cell Labeled by Molecular Probe Via podoplanin in Vitro

Author: ZhangSong
Tutor: ZouLiGuang
School: Third Military Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: magnetic resonance imaging molecular imaging GoldMag podoplanin lymphatic endothelial cell
CLC: R445.2
Type: Master's thesis
Year: 2011
Downloads: 34
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Abstract


ObjectiveTo study the feasibility of applying GoldMag in magnetic resonance （MR） molecular imaging （MI） by detecting the physical, chemical and biological features of GoldMag nanoparticles. The podoplanin targeted molecular probe （GoldMag-pod） was synthesized by using GoldMag as carrier. The labelled specificity of GoldMag-pod and lymphatic endothelial cell （LEC） was evaluated to provide experiment and theory basis for studying the tumor lymphangiogenesis via GoldMag-pod.Materials and methodsSynthesis of molecular probe and the evaluation of physical and chemical features: （1） molecular probe （GoldMag-pod or GoldMag-IgG） was synthesized in a single-step process by using GoldMag as a carrier. The non-specific binding sites of the molecular probe surface were blocked by bovine serum albumin （BSA）; （2） UV/Vis spectrophotometry was applied to detect the absorption value of each sample at 280 nm. The antibody encapsulation efficiency and antibody loading capacity of GoldMag was measured; （3） the morphology and size of USPIO, GoldMag, GoldMag-pod and GoldMag-IgG nanoparticles were observed by transmission electron microscopy （TEM）; （4） the Z-average diameters and Zeta potentials of the 4 kinds nanoparticles were measured by Zeta grainsize analyzer; （5） enzyme linked immunosorbent assay （ELISA） method was applied to evaluate the antibody （IgG and anti-podoplanin antibody） immunoactivity before and after coupling with GoldMag; （6） FSE T1WI, FSE T2WI and GRE T2*WI sequenses were used to scan a series concentrations of USPIO, GoldMag, and GoldMag-BSA solutions to study the MRI signal features of nanoparticles.MR imaging study of LEC labeled by GoldMag-pod in vitro: （1） cultivated and generated human dermal lymphatic endothelial cell （HDLECs） and human umbilical vein endothelial cells （HUVECs）; （2） the positive labelling rate of LEC was detected by Prussian blue staining to study the correlation of GoldMag-pod concentration with the coupling rate; （3） the LEC label status of GoldMag-pod was observed by immunofluorescence staining and immunocytochemical staining; （4） the cytotoxicity of GoldMag was assessed by MTT assay; （5） the metabolism of GoldMag in LEC was observed by TEM; （6） series of concentrations of LEC solution labeled by GoldMag, LEC solution labeled by GoldMag-pod, GoldMag-pod solution and GoldMag solution were scanned by GRE T2*WI to evaluate the MR signal changing situation.ResultsSynthesis of molecular probe and the evaluation of physical and chemical features: （1） GoldMag can couple with anti-podoplanin antibody or IgG antibody by the non-covalent coupling effect of colloidal gold; （2） the antibody encapsulation efficiency of GoldMag-pod was measured as 52.94%, and the antibody loading capacity was 79.41μg anti-podoplanin antibody per milligram of GoldMag; the antibody encapsulation efficiency of GoldMag was measured as 58.74%, and the antibody loading capacity was 88.11μg IgG antibody per milligram of GoldMag; （3） TEM results for USPIO, GoldMag, GoldMag-pod, and GoldMag-IgG showed that the nanoparticles were oval-like in shape. The particle sizes of the four kinds of nanoparticles were 14.7±1.4, 47.7±4.1, 66.8±4.9, and 67.3±5.5 nm, respectively; （4） Dynamic light scattering results showed that the Zave values of USPIO, GoldMag, GoldMag-pod, and GoldMag-IgG were 17.4, 49.9, 70.2, and 72.5 nm, respectively; and the potentials of the four kinds of nanoparticles were 10.1±4.1, 13.7±5.6, -17.5±4.0, and -16.1±3.9 mV, respectively; （5） the immunoactivity of GoldMag-pod was estimated to be 42.05% of that of the original anti-podoplanin antibody. And the immunoactivity of GoldMag-IgG was estimated to be 43.34% of that of the original IgG antibody. （6） MRI results showed that the changing of FSE T1WI signal intensity was not obvious, FSE T2WI signal intensity slightly declined as the concentrations of the nanoparticles raising up, GRE T2*WI signal intensity declined obviously as the concentrations of the nanoparticles raising up.MR imaging study of LEC labeled by GoldMag-pod in vitro: （1） HDLECs and HUVECs grew well in vitro cultivated circumstance; （2） the result of Prussian blue staining showed that the positive label rate of LEC increased as the concentration of GoldMag-pod raising up; （3） the results of immunofluorescence staining and immunocytochemical staining both showed that GoldMag-pod can specifically label LEC, but the immunoactivity of GoldMag-pod was lower thant that of anti-podoplanin antibody; （4） the result of MTT method showed that it has only little influence on LEC; （5） TEM results show that GoldMag-pod was found in lysosomes after swallowed by LEC, but the cell morphology and function of LEC did not change significantly; （6） the GRE T2*WI signal intensity of LEC solution labeled by GoldMag, LEC solution labeled by GoldMag-pod, GoldMag-pod solution and GoldMag solution declined gradually as the concention of each solution raising up, and the lower degree increased among the group’s order.ConclusionGoldMag is a kind of bi-functional nanoparticle, composed of a gold nanoshell and an iron oxide core. GoldMag can couple with antibody （anti-podoplanin antibody or IgG antibody） in a single-step process. The high antibody encapsulation efficiency, the antibody loading capacity, and the immunoactivity of molecular probe ensure it can be used in immunology experiment. And the particle size of GoldMag is small, which is conducive to penetrate the vascular basement membrane to reach the target tissue and target cells. GoldMag-pod can specifically label with LEC, and the positive label rate of LEC increased as the concentration of GoldMag-pod raised up. GoldMag has only little influence on LEC. And the GRE T2*WI signal intensity of LEC solution significantly dropped after labeling by GoldMag-pod. All the results showed that GoldMag nanoparticles are suiable for MR molecular imaging, and GoldMag-pod can specifically label with LEC. The research provides a certain experimental basis and theoretical foundation for MR imaging of tumor lymphangiogenesis in vivo.

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CLC: > Medicine, health > Clinical > Diagnostics > Diagnostic Imaging > Magnetic resonance imaging
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