Liposomal Gd-DTPA As MR Contrast Agents Targeted Reticuloendothelial System: Synthesis And Study In Hepatic Metastases And MR Lymphography

Objective1. To prepare liposomal Gd-DTPA and measurc particle size and distribution;The morphology of the particle was cxamined by transmission electron microscopy.2. Compared to Gd-DTPA,to investigate the cffect of intravenous injection of liposomal Gd-DTPA as MR specific contrast agents imaging for hepatic mctastascs aftcr Devclopping rabbit model of hcpatic metastascs3. To further evaluate the effect of liposomal Gd-DTPA targetcd lymphatic tissue and the value of the MR lymphography to differentiate mctastascs from bcnigh lymph nodes after interstitial administration.Reactive hypcrplasia lymph nodes and metastatic lymph nodes were developed.Materials and Methods1. Preparation and exosyndrome of liposomal Gd-DTPA1.1 Preparationsmall unilamellar vesicles were prepared by combining 0.92 g of distearoylphosphatidylcholinc (DSPC) with 23 mL of distilled watcr.The mixture underwent sonication for 1 hour.A contrast material-ethanol mixture was prepared by combining Gd-DTPA,distillcd water(6.7 mL),and ethanol(16.3mL).The mixture was combined with cqual amounts of the previously prepared small unilameller vesicles to produce a translucent gel.The gel was allowed to stand at room temperature overnight. The mixture was then incubated in a hot-water bath for approximately I hour and sparged with a slow stream of nitrogen gas.The resulting material was allowed to cool to room temperature and mixed with 100 mL of tris(hydroxymethyl)aminomethane (Tris) buffer (pH, 7.1; 20 mmol/L Tris, 0.1 g/L sodium calcium edetic acid in 0.9% saline solution).To isolate the liposomes,the mixture was centrifuged at 4,000 rpm for about 5 minutes.The supernatant was removed and saved, and the liposome-containing pellet was resuspended in fresh buffer. This isolation procedure was repeated until the total counts in the wash were less than 1% of the total added to the original mixture (the washing procedure was usually repeated five times).The final, washed liposome suspension can be brought to any desired volume with buffer.1.2 Small unilamellar vesicles 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±0.05.1.3 The shape morphology of Small unilamellar vesiclesThe morphology of Gd-PBCA-NP 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 microscopy1.4 IFvs size and size distribution measured1.5 The shape morphology of IFvs1.6 Measureing the encapsulation efficiency and loading capacity of Liposomal Gd-DTPAA sample volume of 5ml was sent to analyze (Zhongshan University Analytical Center, Guangzhou).The concentration of Gd-DTPA in dialysed solution were measured by inductively coupled plasma atomic emission spectrophotometry (ICP-AES).2. The study of liposomal Gd-DTPA targeted hepatic metastases2.1 AnimalsIn this experiment, 18 New Zealand White Rabbits weighted at 2.0～2.5kg were used. All the animals were divided into 3 groups at random:0.2mmolGd/kg liposome group(n=6); 0.5mmolGd/kg liposome group(n=6);Gd-DTPA control group(n=6).All rabbits were buying from the animal center of NanFang hospital.2.2 The development of modelsA certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces.Three pieces of tumor tissue were implanted into the different site of the liver.2.3 UltrasoundTwo animal models were studied by conventional ultrasound2.4 MR imagingAll MR imaging was performed before and after different enhancement protocols at field strength of 1.5 Tesla MR scanner (Magnetom vision plus 4, VB33A; Siemens, Germany) with use of a head coil for transmission and reception of the signal. MR sequences included Tl-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 500/15);Two animals had other sequences included fat suppression(FS)TlWI(539/14)and gradient-echo(GRE)TlWI(150/14,flip angle,70°),double SE(T2-weighted:1800/70;PDWI:1800/20)and T2-weighted fast SE images(4100/90).Immediately after contrast medium injection, dynamic data of different group were aqcquired at 5min, 15min, 30min, lh, 2h using the Tl-weighted spin echo images.2.5 MR imaging analysisAverage signal intensity (SI) over region-of-interest (ROI) drawn on hepatic metastases and liver were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside abdomen. Contrast to noise ratio(CNR)of lesions and liver were calculated on all images.2.6 Pathological examination:After MRI,the liver were transected into slices with 2.0mm and taken count of the lesions.All liver tissues were fixed in neutral formalin solution and embedded with paraffin and sliced up.These slices were preformed with HE staining.2.7 Statistical analysis SPSS 10.0 was used as analyzing software. Repeated measures ANOVA analyzed the difference in CNR of liver metasteses among different groups. P＞0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.3. MR lymphography after interstitial administration of liposomal Gd-DTPA3.1 Animals18 New Zealand White Rabbits weighted at 2.0-2.5kg were used. All the animals were divided into 3 groups at random:1.control group(n=6);2.reactive hyperplasia group(n=6);3.endometrial neoplasms-bearing group(n=6).3.2 The development of models3.2.1 Reactive hyperplasia modelEach rabbit was injected 0.5ml of egg-yolk emulsion bilaterally in the femoral muscles and subcutaneously in the flank.The injections were repeated 3-4 days later.Magnetic resonance imaging was performed 3-4 days after the second administration.3.2.2 Endometrial neoplasms-bearing modelA certain quantity of fresh tumor tissue was obtained from the tumor-bearing rabbit and was skived to little pieces,VX2 tumor grafts were established by orthotopic embedding endometrium of rabbits.3.3 MR imaging3.3.1 The non-enhanced sequences consisted of TlWI (TR/TE=539/14ms), T₁WI FS(TR/TE=539/14ms) ,T₂WI(TR/TE=2234/85ms).3.3.2 Liposomal Gd-DTPA(0.25mol Gd/L) were injected into both thighs(0.5ml,each).Following administration,massage was performed at the injection sites for 5min to assist the lymph flow of the anesthetized animals.MR imaging was performed 5,15,30,45min and lh after administration of the agents using the same imaging parameters as for the precontrast images.3.4 MR imaging analysis3.4.1 Size of lymph nodes were measured on T1WI3.4.2 The signal intensity of iliac medial,superficial inguinal and popliteal lymph nodes were measured on different sequences of images before and after contrast enhancement.Background noise(N)was measured in each image.Signal to noise ratio(SNR)were calculated on all images.The enhanced ratio was calculated on T₁WI images after injected liposomal Gd-DTPA in the reactive hyperplasia lymph node group.3.5 Histopathologic examinationAfter the completion of MR imaging, all animals were deeply anesthetized and then were sacrificed by exasnguinations. Iliac medial lymph 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.3.6 Statistical analysisSPSS 10.0 was used as analyzing software. One-way ANOVA analyzed the difference in size and SNR of lymph nodes on plain images, on liposomal Gd-DTPA enhanced images of lymph nodes between groups which was performed 30min after administration. P＜0.05 was regarded as statistical difference.Results1. Exosyndrome of liposomal Gd-DTPA1.1 The average size of SUVs and IFvs were 235nm and 2.2μm,and the index of size distribution of SUVs was 0.08.1.2 The morphology of liposomal Gd-DTPA was spherical shape and without adherence between particles observed by transmission electron microscopy.1.3 The average encapsulation efficiency and loading capacity of liposomal Gd-DTPA were respective 22 %, 51.23 %.2. The study of liposomal Gd-DTPA targeted hepatic metastases2.1 The development of models and histopathologyHepatic metastases were successfully induced in all rabbits in which 35 lesions were founded.The tumor growth rate was 65%. Histopathology show that the implanted tuomrs had imfiltrated into the rabbit liver tissue, similar as the biological features of squamous cell carcinoma being transplanted in other sites of rabbits. 2.2 MR imaging on different sequences before enhancedOn T1WI,hepatic metastases were hypointensity or iso-hypointensity.The signal intensity of the hepatic parenchyma was comparatively higher so that the edge and inside of tumor was clear.On FS T1WI, the signal intensity of the hepatic parenchyma and hepatic metaseses were higher relative to T1WI,so the contrast of lesions were no better comparable to T1WI.2.3 MR imaging after enhancedAfter intravenous injection of liposomal Gd-DTPA, the difference in the CNR of lesions were significant at 5 different times.The CNR of 0.5mmolGd/kg liposome group were higher than that of 0.2mmolGd/kg liposome group.The summit were all lh after enhanced in two groups.After intravenous injection of Gd-DTPA, the difference in the CNR of lesions were significant between non-enhanced images and enhanced images at all 5 different times.After intravenous injection of PBCA-NP, the difference in the signal-to-noise ratios of liver and muscle between before and after enhanced images in all times.There were significant difference in enhanced style between three groups.3. MR lymphography after interstitial administration of liposomal Gd-DTPA3.1 On non-enhanced images, the size of iliac medial lymph nodes had significant difference between control groups and reactive,tumor-bearing groups.3.2 On non-enhanced images ,the signal-to-noise ratios had no significant difference in three sequences of images of three groups,3.3 On liposomal Gd-DTPA enhanced T₁WI, T₁WI FS images ,the reactive hyperplasia lymph nodes demonstrated obviously homogenous enhancement while metastatic lymph nodes showed heterogeneous or no enhancement, there were significant difference in the signal-to-noise ratios of lymph nodes3.4 After interstitial administration of liposomal Gd-DTPA ,the signal-to-noise ratios of the reactive hyperplasia iliac medial lymph nodes increased quickly, and the maximal enhancement occurred at 30min.3.5 Histopathological findings showed that the structure of the metastatic lymph nodes were destroyed at varied extent by tumor tissue. Conclusions1. Interdigitation-fusion as a new method for procducing lipod vesicles of high internal volume is feasible.2. The average size of liposomal Gd-DTPA was 2.2 um and the average encapsulation efficiency and loading capacity of Gd-PBCA-NP were respective22%, 51.23 %.The morphology of liposomal Gd-DTPA was spherical shape.3. On Tl-weighted spin echo images,the contrast of hepatic metastases was better than that of other sequences.4. After intravenous injection of liposomal Gd-DTPA, the contrast of hepatic metastases was better than that before enhanced.So liposomal Gd-DTPA can be taken as MRI contrast agents targeted for reticuloendothelial system.5. Liposomal Gd-DTPA has characteristic of delayed release.6. The lymph of the two models resemble the MR appearance of hyperplastic and metastatic lymph nodes in humans.Therefore,these models are well suited for testing the efficacy of MR-tomographic techniques in the detection of lymph-node metastasis.7. Reactive and tumor-bearing iliac medial lymph nodes were larger than control.8. Plain MR scan can not differentiate metastatic lymph nodes from normal and reactive hyperplasia nodes accurately.9. After interstitial administration of liposomal Gd-DTPA, the maximal enhancement of the reactive iliac medial hyperplasia lymph nodes occurred at 30min, so MR lymphography may be carried out at this time.10. Metastatic lymph nodes showed different enhancing characteristics from those of reactive hyperplasia nodes in MR lymphography after interstitial administration of liposomal Gd-DTPA, so they can be differentiated accurately.