Long-carcluating SPIO-PBCA-PEG Nanoparticle: Synthesis And Application In MRI Of Rat With Liver Tumor

1,Prepare polybutylcyanoacrylate (PBCA) nanoparticles loaded with SPIO and optimize the preparative conditions.2,Compared with SPIO, to investigate the effect of SPIO-PBCA-PEG as MR specific contrast agents targeted imaging for liver in rat and debate the potential value of its clinical applications.3,establish animal model of hepatic metastases to further evaluate the value of SPIO-PBCA-PEG in the diagnosis of liver tumor.Materials and Methods1,Preparation and optimization of SPIO-PBCA-PEG(1)Determinate the preparation technics Accurate weighed a certain amount of mPEG, dissolved into the deionized water (PH 5.5-6.5). Slowly distribution droplets 10ml fat soluble SPIO and BCA into mechanical agitator (800rpm) drop by drop,up at least 5h. Terminate the reaction. Filtered with 220um membrane filter,and preserve in the refrigerator.4℃.(2) Select the optimized preparative conditions of SPIO-PBCA-PEG by orthogonal design The factors such as the concentrations of mPEG, PBCA and PH have the most important effect on the size and size distribution of SPIO-PBCA-PEG. The influence of the other parameter on the size and size distribution of SPIO-PBCA-PEG have no effect. The optimum conditions were investigated by L9 (34) orthogonal design (PH, PEG, PBCA 3 factors,3 levels).(3) Measure the content of Fe and the encapsulation efficiency of SPIO-PBCA-PEG Took the mixed liquor SPIO-PBCA-PEG 5ml centrifugal in low temperatures 20min(12000rpm),then collect the supernatant and precipitation.determinate the Fe of the supernatant, The encapsulation efficiency of SPIO-PBCA-PEG were calculated with formula described below:ER=(Wa-Ws)/Wa×100%, Wa:The content of Fe in SPIO, Ws:The content of Fe in supernatant(4) SPIO-PBCA-PEG size and size distribution measuredThe result stock solution was purified by dialysis through a semipermeable membrane with an exclusion size of 1,000,000 Da against glucose 5% solution for 48 h for removing free SPIO, mPEG.The SPIO-PBCA-PEG 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.(5)The shape morphology of SPIO-PBCA-PEGA little of the dialysis 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.(6) Determination the structure of SPIO-PBCA-PEGA little of the dialysis solution was lyophilization into powdery.then placed in the infrared instrument to measure its structure.2,The study of SPIO-PBCA-PEG targeted liver imaging(1)Animal modelsIn this experiment, twelve SPF normal Wistar rats weighted at 176.5±17.6g and were divided into two groups at random:SPIO-PBCA-PEG group:at a dose of 0.5mmolFe/kg, SPIO control group:at a dose of 0.05mmolFe/kg. All rats were buying from the animal center of NanFang hospital.(2)MR imagingAll MR imaging was performed before and after different enhancement protocols at field strength of 3.0 Tesla MR Scanner (GE Signa EXCITE HD 3.0T MR) with use of a soft coil for transmission and reception of the signal. MR sequences included T1-weighted fast spin echo images (TR/TE= 400ms/7.5ms) and T2-weighted fast spin echo images (TR/TE=2800ms/122ms). Immediately after contrast medium injection, dynamic data of different group were aqcquired at 10min,30min, 1h, 2h,4h,24h. using the same imaging parameters as for the precontrast images.(3) MR imaging analysisAverage signal intensity (SI) over region-of-interest (ROI) drawn on hepatic parenchyma and muscle were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside abdomen. Signal to noise ratio (SNR) and enhancement ratio of liver and muscles were calculated on T1WI imaging after enhanced images in three groups.(4) Statistical analysisSPSS 13.0 was used as analyzing software. RMANOVA analyzed the difference in SNR of liver among different groups. Independent-Samples T Test Analyze some strengthened from time to time between the two groups of contrast medium. P<0.05 was regarded as statistical significant difference.3,Compare MR imaging in rat tumor after administration of SPIO-PBCA-PEG and SPIO(1)AnimalsIn this experiment, twelve SPF normal wistar rats weighted at 182±15.6g.All rats were buying from the animal center of NanFang hospital. Prepare for the liver metastasis model.(2) Development of the animal modelsDevelopment of the big rat liver metastatic tumor model:form the VX2 rabbit tumor edge take fish kind tissue, shear to 0.5-1 mms lump, put into a little amount normal sodium back up pre-emergency.Test Wistar rat abdominal cavity injected 3% continal to anaesthesia, expose abdomina cavity, because the big rat liver's volume is small and sublobe more,Draw the liver leaf which near abdominal wall outside of body lightly by hand, send back the liver after use ophthalmic forceps stabbing to break the tissue where liver leaf thicker position lightly form a sinuses, taked 1-2 VX2 lump pieces into sinuses, make sure stopping bleeding, having no lump piece slipping out, then close the abdominal wall. The muscle injects penicillin.10-14 days later use MR to scan.(3) MR imagingThe liver metastatic tumor models were divided into 2 group at random: SPIO-PBCA-PEG enhanced I group, which SPIO-PBCA-PEG(0.05mol Fe/kg) was injected into the vena caudalis, and SPIO enhancedⅡgroup, which 0.05molFe/kgwas injected. The non-enhanced sequences consisted of T1WI (TR/TE=450/7.5ms), FSET2WI (TR/TE=2800/122ms).after scan contrast medium injection.then scan the enhanced imaging. (4) MR imaging analysisSizes and number were measured (Maximal diameters of lesions). Average signal intensity (SI) over region-of-interest (ROI) drawn on lesions and adjacent liver parenchyma were measured on MR images. Background noise (N) was measured in each image and ROI were placed adjacent background outside abdomen and coding direction was same to the lesions. Signal to noise ratio (SNR), contrast to noise ratio (CNR) of lesions and liver, CNR changing after enhancement were calculated on all images(5) Statistical analysisSPSS 13.0 was used as analyzing software, adopting Paired-Samples T Test and Independence-sample T Tests. P<0.05 was regarded as statistical significant difference.Results1. The optimized preparative condition:pH is 6.5, the concentrations of PEG is 1.5%, PBCA is 15%. The Fe content of SPIO-PBCA-PEG is 2.27mg/ml, the encapsulation efficiency is 86.9%.2. The morphology of SPIO-PBCA-PEG is spherical shaped with core-shell structure between particles observed by transmission electron microscopy.3. The average size of SPIO-PBCA-PEG was 177nm and the index of size distribution was 0.20measured by dynamic light scattering.4. Infrared detect the drug both with the PBCA and PEG characteristic absorption peak spectrum, determination PEG have connect to PBCA.5. The liver's SNR on T2WI were significant different at 10min,30min, 1h,2h,4h, 24h after intravenous injection of SPIO-PBCA-PEG and were lower than those injected of SPIO. After intravenous injection of SPIO-PBCA-PEG, the liver's SNR reached the lowest point at 2h post injection and rose gradually. After intravenous injection of SPIO, the liver's SNR descended quickly at the first 10min,30min, and reached the lowest point at 30min, then rose gradually.6., After intravenous injection of SPIO, the negative enhancement percentage of the liver on T2WI were respectively 21.3%,48.16% at 10 min,30min and gradually rose at 1h. After intravenous injection of SPIO-PBCA-PEG, the negative enhancement percentage of the liver on T2WI were respectively 10.7%,22.42%,56.89%,53.41%, 40.99%at 10min,30min, 1h,2h,4h,24h. Compared with the SPIO group, the SPIO-PBCA-PEG group had later peak enhancement time, higher peak enhancement ratio, and higher enhancement ratio post peak time.7. Development of the big rat liver metastatic tumor model. After intravenous injection of SPIO-PBCA-PEG, the contrast-to-noise between the lesion and the liver was significant enhanced, indicate SPIO-PBCA-PEG have effect the CNR between the lesion and the liver.Conclude1. The preparative condition:the pH is 6.5, the concentrations of PEG is 1.5%, PBCA is 15%. The average size of SPIO-PBCA-PEG was 177nm. The morphology of SPIO-PBCA-PEG was spherical shaped with core-shell structure.2. After intravenous injection of SPIO-PBCA-PEG, the liver of rats can be targeted and show negative enhancement on T2WI. The highest enhancement percentage reached at 56.89% which was higher than that injected with SPIO and the peak enhancement time occurred at about 2h post injection. The liver signal recovered slowly and may suggest that SPIO-PBCA-PEG belong to long-circulating contrast agent.3. SPIO-PBCA-PEG can increase the CNR between the lesion and liver; elevate the detection rat of tumor. SPIO-PBCA-PEG was with a better potential value than SPIO applied to detecte micro-hepatocellular carcinoma.