Long-circulating SPIO Liposomes: Synthesis And Application In MR Lymphography Compared With SPIO

Objective1. To prepare Long-circulating SPIO liposomes and optimize the preparative conditions and measure particle size and distribution; The morphology of the particle was examined by transmission electron microscopy.2. The reactive hyperplasia and VX2 tumor-bearing lymph node in New Zealand White Rabbits were developed. To further evaluate the effect of Long-circulating SPIO liposomes targeted lymphatic tissue and the value of the MR lymphography to differentiate metastases from benign lymph nodes was compared between Long-circulating SPIO liposomes and SPIO as specific contrast agents for reticuloendothelial system after interstitial administration of Long-circulating SPIO liposomes and SPIO.Materials and Methods一,Preparation and exosyndrome of Long circulating liposomes SPIO Nanoparticles Synthesis(一)Determination the preparation technicsFeso4 and Fecl3 were combined under alkalinity conditions, using 1g lauric acid, got the Synthesis of lauric acid-stabilized SPIO nanopaticles. Preparation of long-circulation blank liposomes by film dispersion method, using DSPG and DSPE-PEG2000 with a molar ratio of 20:1. The two products above were dialyzed for 3 days and then were stored at 4℃.(二)blank liposomesand Long-circulating SPIO liposomes size and size distribution measuredThe size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5ul was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.(三)The shape morphology of blank liposomes,lauric acid-stabilized SPIO and Long circulating liposomes SPIO Nanoparticles SynthesisThe shape morphology of blank liposomes and Long-circulating SPIO liposomes was examined by transmission electron microscopy. A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was freeze-dried and examined by transmission electron microscopy(四)Ferri concentration of Long-circulating SPIO liposomesMeasurement for ferri concentration of Long-circulating SPIO liposomes using the lassic method of phenanthroline.(五)XRD analysisDrayed lauric acid-stabilized SPIO and Long-circulating SPIO liposomes were analyzed by the X-ray diffractometer.二,MR lymphography after intravenous administration of Long-circulating SPIO liposomes and SPIO(一)AnimalsIn this experiment, twenty four New Zealand White Rabbits weighted at 2.0-2.5kg were used and divided into 2 groups at random. Reactive hyperplasia lymph nodes group(n=12) and metastatic lymph nodes group(n=12). All rats were buying from the animal center of kongjun hospital..(二)The development of modelsReactive hyperplasia lymph nodes were developed by intramuscular injection of 0.5 ml egg yolk emulsion into the lateral upper hind limb of New Zealand White Rabbits. A certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to prepare the tumor cell suspension. The metastatic lymph nodes were developed by intramuscular injection of 0.5ml tumor cell suspension into the lateral upper hind limb of New Zealand White Rabbits.(三)MR imaging1.The non-enhanced sequences consisted of T1WI (TR/TE=539/14ms) T2WI(TR/TE=2234/85ms).2. The reactive hyperplasia lymph nodes were subdivided into 2 group: Long-circulating SPIO liposomes I group and SPIO enhanced II group, which 0.5ml of Long-circulating SPIO liposomes was injected intravenously. And SPIO enhanced II group:which 0.5ml of SPIO was injected intravenously. MR imaging was performed 15,30,60min,4,24h and 48h after administration of the agents using the same imaging parameters as for the precontrast images.(四)MR imaging analysis1. Size of lymph nodes were measured on T1WI.2. The signal intensity of popliteal lymphv nodes were measured on different sequences of images before and after contrast enhancement.Background noise was measured in each image. Signal to noise ratio(SNR)were calculated on all images.The enhanced ratio was calculated on T2WI images after injected Long circulating liposomes SPIO Nanoparticles Synthesis in the reactive hyperplasia lymph node group.(五)Histopathologic examinationAfter the completion of MR imaging, all animals were deeply anesthetized and then were sacrificed by exasnguinations. popliteal lymphv nodes were exposed to determine their positions and then were carefully removed. The size of each lymph node was measured and the amount of lymph nodes was calculated. Removed lymph nodes were fixed and subjected to H-E staining so that benign or malignant lymph node could be assessed.(六)Statistical analysisSPSS 13.0 was used as analyzing software. Paired t-test analyzed the difference in size and SNR of lymph nodes on plain images, on Long-circulating SPIO liposomes enhanced images of lymph nodes between groups which was performed 24h after administration. P<0.05 was regarded as statistical difference.Results一,Exosyndrome of Long-circulating SPIO liposomes1. Under the transmission electron microscopy, the average size of blank liposomes is 20nm. The average size of lauric acid-stabilized SPIO is 12nm.and Long-circulating SPIO liposomes is 60nm.2. The morphology of blank liposomes was spherical shape, lauric acid-stabilized SPIO is Long-circulating SPIO liposomes was spherical shape with core-shell structure and without adherence between particles observed by transmission electron microscopy.3.XRD analysis:lauric acid-stabilized SPIO and Long-circulating SPIO liposomes are face-concentration cubic spinel structure of Fe3o4,and the crystalline particles are in good form.4. With the Malvern-3000HS, the measurement of Long-circulating SPIO liposomes were:di=78.7nm, dv=76.7nm, dn=73.5nm, peak area:100%, peak number is one, polydispersity coefficient:0.212.5.Standard curve of iron:A=0.1947C+0.008, R2=0.9982. The Fe concentration of Long-circulating SPIO liposomes was 6.7213mg/ml.二,The development of models and histopathology1.The reactive hyperplasia and metastatic lymph node models in New Zealand White Rabbits were successfully developed. On non-enhanced images, the signal-to-noise ratios and the size of popliteal lymph nodes had no significant difference in two sequences of images of two groups, but the size of metastatic popliteal lymph nodes were larger than that of reactive hyperplasia lymph nodes.2. On non-enhanced images,the signal-to-noise ratios had no significant difference in two sequences of images of two groups,.3.After intravenous injection of Long-circulating SPIO liposomes, the reactive hyperplasia lymph nodes demonstrated obviously homogenous enhancement while metastatic lymph nodes showed heterogeneous enhancement, there were significant difference in the signal-to-noise ratios of lymph nodes and no significant difference on T2WI. After intravenous injection of SPIO, the reactive hyperplasia lymph nodes and metastatic lymph nodes showed no-enhancement.Conclusions1. The average size of long-circulating SPIO liposomes are with suitable size,, The Long-circulating SPIO liposomes was spherical shape with core-shell structure.2. Compared with SPIO, MR lymphography after intravenous administration of long-circulating SPIO liposomes have higher accuracy in differentiating benign from malignant lymph nodes which can provide valuable reference for the diagnosis between benign and malignant lymph nodes.3.becaus of the half time of SPIO is too short, intravenous injection was not suitable to MR lymphography.4. Plain MR scan can not differentiate metastatic lymph nodes from normal and reactive hyperplasia nodes accurately.5. After intravenous administration of long-circulating SPIO liposomes the maximal enhancement of the reactive hyperplasia lymph nodes occurred at 24 hours.6.Metastatic lymph nodes showed different enhancing characteristics from those of reactive hyperplasia nodes in MR lymphography after intravenous administration of long-circulating SPIO liposomes and SPIO, so they can be differentiated accurately.