| Objective1. To prepare Galactose-Bovine-Serum-Albumin (Gal-BSA-SPIO) nanoparticles as Asialoglycoprotein Rceptor -directed MR contrast agent, and to test specificity of the agents in rabbit liver tissues in vitro and in vivo MR imaging experiments.2. To investigate and debate the effect and potential values of Gal-BSA-SPIO as MR specific contrast agents targeted imaging for liver VX2 tumor in rabbits.3. To study and evaluate primarily the potential values of Gal-BSA-SPIO as receptor -directed contrast agents for MR imaging with affinity and histologic receptor assay.4. Compare and analyze the enhancement features on CT or MR images with histological pathology and immunohistochemsitry of human hepatocyte nodules to know more profoundly the imaging features of its and to debate primarily whether the Gal-BSA-SPIO as MR contrast agents is more superior.Materials and Methods1. Preparation of Gal-BSA-SPIO and the affinity assays(1) Preparation of Gal-BSA-SPIO and assays By method of reductive amination, bovine serum albumin (240mg), lactose (1.2mg) and sodium cyanoborohydride (100mg) were dissolved in 30 ml of 0.2 M natrium phosphate(PH 8) and incubated at 37℃for 24 to 120 hours. The reaction mixture were dialyzed extensively (3 days) against distilled water at lower temperature. Aliquots of the dialyzed mixture were centrifuged and the supernatant was withdrawn applied to a sephadex gel chromatography column. The purified conjugates were lyophilized. Total carbohydrate and albumin were determined separately by the phenol-sulfuric acid method and coomassie brilliant blue G-250 to calculate the ratio of galactose to albumin.(2) Preparation of Gal-BSA-SPIO Smaller iron oxide particles were obtained through size fractionation of SPIO with use of a Sepharose 4B column. The separated smaller SPIO was dialyzed against buffer solution for 24 hours and then concentrated to obtain an iron concentration of 8mg/ml (PH6.5). Fractions were diluted 1:1 into a neutral buffer containing 1% W/V galactose-BSA. Thereafter, sonication was performed in an ice bath. The loosely associated BSA was removed by high salt(1M NaC1) and added by dropwise of ammonium hydroxide to PH 7.4. The iron concentration was tested and the particle size and distribution was measured by a dynamic light scattering (a Malvern Zetasizer 3000 HS). The core particle size and morphology of Gal-BSA-SPIO were examined by transmission electron microscopy.(3) Affinity assays in vivo and vitroReceptor affinity experiments in vivo Fresh liver tissues in rabbits (10g) were obtained and added with complete growth medium. And then the tissues were cut into 1-2mm3 pieces, minced through a 30 wire mesh, and incubated with 0.3 mg/ml collagenase in complete growth medium for 30 minutes at 37℃. Thereafter, the dispersion was sonicated in an ice bath to disrupt tissue and cell fragments. Samples were centrifuged, and the supematant that contained cell membranes was used for incubation assays. (1)Blocker group Samples (2ml) of the membrane suspensions were incubated for 30 minutes at 37℃with complete medium contained different doses of D(+)-galactose(0.2mg or 2mg) to block existing ASG receptor on the membrane. After this incubation, either SPIO or Gal-BSA-SPIO (contained of 101amol iron) was added to the suspensions. 30 minutes later, samples were washed twice to remove free unbound iron oxide particles. Cell and membrane fragments were then resuspended in 6ml of complete growth medium. (2) Un-blocker group Samples (2ml) of the membrane suspensions were directly incubated with either SPIO or Gal-BSA-SPIO (contained of 10μmol iron). 30 minutes later, samples were also washed twice and resuspended in 6ml of complete growth medium. Relaxation times of all samples were measured at 1.5T, and statistics were performed.Relaxation studies in vitro Six rabbits were randomly divided into two groups of blocker and un-blocker. MR imaging was separately performed before and after administrated with agents. Spin-echo(SE) images were obtained with following pulse sequences: SE TR/TE 600/20, 80, and transverse section images were obtained. The rabbits of blocker group were administrated firstly with D(+)-galactose (2mg/kg) to block ASG receptor on the hepatocyte membrane, and 30 minutes later, all were administrated with Gal-BSA-SPIO(10μmol Fe/kg). The animals of unbloked group were directly administrated with GaI-BSA-SPIO(10μmol Fe/kg). MR imaging of all animals were performed after administration of Gal-BSA-SPIO for 30 minutes, with the same sequence as before. Thereafter, T2 relaxation times pre- and post-enhancement were measured.Histological assays Liver and spleen specimens were examined 30min after injection of 20μmol Fe/kg to study the cellular distribution of Gal-BSA-SPIO or SPIO in two rabbits. After removal, the liver tissue was performed with histological specimens and electron microscopy, and the spleen tissue with histological specimens.2. Application of Gal-BSA-SPIO in VX2 tumor of rabbits(1) Model of VX2 tumor in rabbits Tumor tissue with active growth characters looked like fish was obtained from the border of VX2 tumor in breeder-rabbit. Tissue was added some physiological saline, cut into 0.5-1mm3 with ophthalmic scissor. The rabbits were anaesthetized and paunched exposed the liver. One lobe of the liver was pulled lightly outside, and pierced by ophthalmic forceps. Thereafter, a small tumor piece was put into the pierced hole. One to three pieces of tumor were put in.(2) Animal group 20 rabbits were randomly divided into two groups of SPIO and Gal-BSA-SPIO, and 10 were of each group. Each group was then separately divided into two groups of 5μmol Fe/kg and 10μmol Fe/kg.(3) MR imaging MR imaging was performed at 1.5T with a section thickness of 3-5mm, and a field of view of 87.5*100. Pulse sequences were used as below: SE T1WI: TR300ms, TE20ms,3000ms; SET2Wh TR600ms, TE20ms,80ms; TSE T2WI-TR3500ms, TE15ms,105ms; GRE T2*WI: TR600ms, TE 15ms, 18°Transverse, coronal and sagittal multisections images were obtained. Contrast scan was performed with administration of different agents by the groups.(4) Observation and measure of images Relaxation times of liver and tumor were measured before and after administration of agents. And signal intensity of liver and tumor were measured to calculate SNR of liver, the percent of the decrease of signal intensity of liver and CNR of liver to tumor.(5) Statistical analysis Paired t tests were used to evaluate data from pre- and post -contrast images. One-way ANOVA was used to evaluate PSIL and the difference of T1, T2, SNR, and CNR pre-contrast and pos-contrast. One-way ANOVA was used to evaluate pulse sequences. Multi-comparison was performed with LSD or Dunnett's T3. The significance of reported P values were defined as P＜0.05.3. Pilot study of Gal-BSA-SPIO as ASG receptor-directed agents for MR imaging in human liver(1) Affinity assays A total of 13 specimens were collected from surgery. Specimens included normal liver tissue(n=3), cirrhosis(n=4), hepatocyte carcinoma(n=6). All specimens were performed to cell and membrane suspensions by the above methods (relaxation times measure of rabbits liver in vivo). Relaxation times of T2 were measured. One-way ANOVA and Independent-samples were used to evaluate the datas. Multi-comparison was performed with LSD or Dunnett's T3. The significance of reported P values were defined as P＜0.05.(2) Distribution of ASG receptor in different fresh human tissue All specimens were cut into 2-4μm sections(-20℃). Sections were then thawed, air dried, and incubated 20 seconds(37℃) with Gal-BSA-SPIO, SPIO, or buffer solution. After incubation, slides were rinsed with saline solution to remove unbound iron. Slides were then dried for 24 hours at room temperature. Subsequent counterstaining for iron was performed with Perls Prussian blue stain.(3) Distribution of ASG receptor in different human tissue (paraffin masses) A total of 27 specimens were collected, consisted of small hepatocyte carcinoma(n=12), metastases(n=3), cholangiocarcinoma(n=2), cirrhosis nodules(n=5), focal nodular hyperplasia(n=3), hemangiomas(n=3),adenomas(n= 1). Specimens were cut into 2-41am sections( at room temperature), and then baked for 60 minutes(65℃). Sections were dewaxed extensively with xylene. Thereafter, sections were air dried, and incubated with Gal-BSA-SPIO, SPIO, or buffer solution for 10 minutes (37℃). After incubation, slides were rinsed with saline solution to remove unbound iron. Slides were then dried for 24 hours at room temperature. Subsequent counterstaining for iron was performed with Perls Prussian blue stain.(4) CD34 expression A total of 18 specimens, consisted of small hepatocyte carcinoma(n=10), cirrhosis(n=5) and focal nodular hyperplasia(n=3), were performed by CD34 stain with SABC. CD34 expression were observed and MVD were calculated by Weidner method.(5) Compare and analyze the enhancement features on CT or MR images with histological pathology and immunohistochemsitry of human hepatocyte nodules.Results1. Results of preparation of Gal-BSA-SPIO and the affinity assays(1) The ratio of galactose to albumin for 24h, 48h, 72h, 96h, and 120h were separately 8, 15, 27, 32 and 38. The iron concentration of GaI-BSA-SPIO was 2.8mg/ml. The mean volume size was 34.4nm and the index of size distribution was 0.35 measured by dynamic light scattering. The core particle size was 14.8nm and the morphology was spherical shape with slight adherence between some particles observed by transmission electron microscopy.(2) There was no significant difference between the blocker and un-bloeked group before incubation with agents(P＞0.05).After incubation with SPIO, the different blocker group showed no significant difference with the un-blocker group(P＞0.05). T2 relaxation times of membrane suspension post-incubation with GaI-BSA-SPIO were lower statistically than pre-incubation and post-incubation with SPIO(P＜0.05). After incubation with Gal-BSA-SPIO, T2 relaxation times of membrane suspension incubation of D(＋)-Galactose to block the ASG receptor on the membrane were greater statistically than without blocker (P＜0.05). And there was significant difference between the different doses of D(＋)-Galactose (P＜0.05).(3) After administration of GaI-BSA-SPIO, T2 relaxation times and signal intensity of liver decreased. The liver looked like "dark liver" since the signal intensity decreased so marked as to near the noise around. T2 relaxation times and signal intensity of liver increased if administrated firstly of D(＋)-Galactose before injected agents.(4) After administration of SPIO, many blue iron deposits were seen in kupffer cells while no in hepatocytes. And blue iron deposits were also seen in spleen tissue. After administration of Gal-BSA-SPIO, many blue iron deposits were seen in hepatocytes while only in few kupffer cells, and no in spleen tissue. Electron-dense particles were seen in lysosomes in hepatocyte after administration of Gal-BSA-SPIO by transmission electron micrograph. Electron-dense particles were seen in lysosomes in kupffer cell while no in hepatocyte after administration of SPIO.2. MR imaging of VX2 tumor in rabbits(1) All of 20 models of VX2 tumor in rabbits were successfully. A total of 28 tumors were detected by MR Imaging, 13 of them with SPIO(15 with anatomy) and 15 of them with Gal-BSA-SPIO(16 with anatomy). Tumors were hoar and spherical shape. There were seen fibrous tissues proliferation around some tumor, looked like some capsular. The minimal and maximal diameter was separately 3mm and 1.2cm.(2) T1 relaxation time of liver post-contrast with SPIO showed no significant difference with pre-contrast(P＞0.05), While T1 relaxation time post-contrast with Gal-BSA-SPIO showed significant difference with pre-contrast(P＜0.05). The difference pre-contrast and post-contrast showed no significant between different groups (F=1.059, P=0.385). T2 relaxation time of liver post-contrast showed significant differences with pre-contrast(P＜0.05), and the differences pre-contast and post-contrast showed also significant between different groups (F=19.295, P=0.000). By multi-comparison, the differences of T2 relaxation times pre-contrast and post-contrast, 10μmol Fe/kg Gal-BSA-SPIO showed significant differences with the other gourps(P＜0.05), 5μmol Fe/kg Gal-BSA-SPIO showed significant difference with 5μmol Fe/kg SPIO (P=0.001) while no significant difference with 10μmol Fe/kg SPIO(P=0.080). T1 and T2 relaxation times of tumor post-contrast with different agents showed no significant differences with pre-contrast(P＞0.05), neither the differences pre-contrast and post-contrast between different groups(P＞0.05).(3) By pulse sequence of SE 300/20, SNR of liver post-enhanced with 5μmol Fe/kg Gal-BSA-SPIO showed no significant difference with pre-enhanced (F=2.098,P=0.090). SNR of liver post-enhanced in different groups by other pulse sequences showed significant difference with pre-enhanced(P＜0.05). The difference pre-contrast and post-enhanced of liver SNR showed no significant difference (F=2.624,P=0.074) by pulse sequence of SE 300/20,while showed significant difference by other sequences. By multi-comparison, 10μmol Fe/kg GaI-BSA-SPIO showed significant difference with the other gourps by pulse sequences of SE600/80, TSE3500/105 and GRE 600/15 (P＜0.05),and 5μmol Fe/kg Gal-BSA-SPIO showed significant difference with 5μmol Fe/kg SPIO(P＜0.05). Liver SNR on different pulse sequences showed significant differences(P＜0.05).(4) There were significant differences of PSIL within different groups on all pulse sequences(P＜0.05). By multi-comparison, on pulse sequence of SE 300/20, 10μmol Fe/kg GaI-BSA-SPIO showed significant differences with 5μmol Fe/kg GaI-BSA-SPIO and 10μmol Fe/kg SPIO(P＜0.05). On pulse sequence of SE 600/80, the different groups showed significant differences (P＜0.05)except 5μmol Fe/kg GaI-BSA-SPIO with 10μmol Fe/kg SPIO(P=0.159). On pulse sequence of TSE 3500/105 and GRE 600/15, the different groups showed significant differences (P＜0.05). Different pulse sequences showed significant differences(P＜0.05). By multi-comparison, enhanced with agent of 5μmol Fe/kg SPIO, pulse sequence of GRE600/15 showed significant differences with SE 600/80 and TSE 3500/105(P＜0.05), and with agent of 10μmol Fe/kg SPIO and different dose of GaI-BSA-SPIO, the different pulse sequences showed significant differences(P＜0.05) except pulse sequence of SE600/80 with TSE3500/105(P＞0.05).(5)Tumor-liver CNR post-contrast with different agents showed significant differences with pre-contrast (P＜0.05), and the differences of pre-contrast and post-contrast showed aslo significant differences bwtween different groups(P＜.05). By multi-comparison, on pulse sequence of SE 300/20, 10μmol Fe/kg GaI-BSA-SPIO showed significant differences with different doses of SPIO(P＜0.05). On pulse sequences of SE600/80, TSE 3500/105 and GRE 600/15, there were significant differences between different groups(P＜0.05). Before contrast, there were significant differences within different pulse sequences (P＜0.05). By multi-comparison, enhanced with agent of 10pmol Fe/kg SPIO, there were significant differences within different pulse sequences (P＜0.05) except between SE300/20 and SE600/80(P=1.0). After contrast, there were also significant differences within different pulse sequences (P＜0.05). By multi-comparison, enhanced with agent of SPIO and 5μmol Fe/kg GaI-BSA-SPIO, there were significant differences within different pulse sequences (P＜0.05) except between SE600/80 and TSE 3500/105(P＞0.05). Enhanced with agent of 5μtmol Fe/kg GaI-BSA-SPIO, there were significant differences within different pulse sequences (P＜0.05).(6)Stained by Hematoxylin-Eosin, VX2 tumor was composed mainly of squamous cell carcinoma and few of adenocarcinoma. There were seen fibrous tissues proliferation around some tumor, looked like some capsular. After administration of SPIO, many blue iron deposits were seen in kupffer cells while no in hepatocyte and tumor tissue. After administration of Gal-BSA-SPIO, many blue iron deposits were seen in hepatocyte while only in few kupffer cells, and no in tumor tissue.3. Pilot study of GaI-BSA-SPIO as ASG receptor-directed agents for MR imaging in human liver(1) T2 relaxation time of the normal liver tissue incubated with Gal-BSA-SPIO showed a significant difference with SPIO (P=0.003). And T2 relaxation time of cirrhosis tissue incubated with Gal-BSA-SPIO showed a significant difference with SPIO (P=0.001). T2 relaxation time of tumor tissue incubated with Gal-BSA-SPIO showed no significant difference with SPIO (P=0.829). In addition, T2 relaxation time of the normal tissue showed a significant difference with cirrhosis pre-contrast and post-incubation with different agents (P＜0.05).(2) After administration of SPIO, many blue iron deposits were seen in kupffer cells while no in hepatocyte tissue. After administration of Gal-BSA-SPIO, many blue iron deposits were seen in hepatocyte of normal liver tissue while only in few kupffer cells. Some blue iron deposits were seen in hepatocyte cytoplast while no on membrane in low and moderate cirrhosis tissue pre-incubation or post-incubation with SPIO. Many blue iron deposits were seen both in hepatocyte cytoplast and on membrane in low-grade and moderate-grade cirrhosis tissue pre-incubation or post-incubation with GaI-BSA-SPIO. Lots of blue iron deposits were seen in hepatocyte cytoplast in high-grade cirrhosis pre-incubation or post-incubation with SPIO or Gal-BSA-SPIO. And no blue iron deposits were seen on membrane in high-grade cirrhosis post-incubation with GaI-BSA-SPIO. Few blue iron deposits were seen in carcinoma tissue post-incubation with Gal-BSA-SPIO. No blue iron deposits were seen in metastases and cholangiocarcinoma tissue pre-incubation and post-incubation. Many blue iron deposits were seen in focal nodular hyperplasia and adenomas pre-incubation and post-incubation, while no in hemangiomas(3) CD34 expression was seen mainly in small vessels inside the fibrous tissues in focal nodular hyperplasia and cirrhosis regenerating nodules. The hyperplatic hepatocyte nodules also showed diffuse positive along the sinusoid in two specimen of focal nodular hyperplasia and one specimen of cirrhosis regenerating nodules. The hepatocyte carcinoma tissue almost showed diffuse CD34 expression, except the hyaline carcinoma area showed few dispersible positive. Mean MVD of 5 specimens of full-blooded sHCC was 121.5±40.4, and that of 5 specimens of poor-blooded sHCC was 133.5±27.8.(4) 3 specimens of focal nodular hyperplasia were all hypodense on pre-contrast scan. The nodules were markedly on arterial-phase scan, turned to isodense or slightly hypodense on portal and delayed phase scans. Arteries were seen at the peripheral area, and the central scar with delayed enhanced was seen in one specimen. Another specimen of focal nodular hyperplasia were all hypodense on different phase of post-contrast. 2 specimens of cirrhosis regenerating nodules were isodense or slightly hypodense on pre-contrast scan, and turned to isodense on arterial-phase scan, isodense or slightly hypodense on portal phase scans. 1 specimen was not found on MR imaging, and it was with hepatocyte carcinoma. In addition, 2 specimens of cirrhosis regenerating nodules were isodense on pre-contrast scan, and turned to hyperdense on arterial-phase scan, isodense on portal phase scans and isodense or slightly hypodense on delayed phase scan. Full-blooded and poor-blooded sHcc were separately 5 specimens.Conclusion1. GaI-BSA-SPIO bind well with ASG receptor on hepatocyte membrane. It distributed mainly in hepatocyte and decreased markedly T2 relaxation time of normal liver that brought a good negative enhancement effect in liver.2. Gal-BSA-SPIO improved obviously tumor-liver CNR, and brought a better enhancement effect in in liver than SPIO contained same dose of iron. The pulse sequence on which there was a best enhancement effect with GaI-BSA-SPIO was GRE T2-weighted imaging, SE T2-weighted imaging and TSE T2-weighted imaging were second, and SE T1-weighted imaging was inferior.3. GaI-BSA-SPIO accumulated in normal liver tissue, low-grade and moderate-grade cirrhosis, focal nodular hyperplasia and adenomas, while decrease obviously in great or adjacent to carcinoma cirrhosis tissues. Few were seen in hepatocyte carcinoma and no in hemangiomas, metastases and cholangiocarcinoma. Gal-BSA-SPIO was with a potential value applied to detect tumor of liver and discriminate the benign tumor from malignant.4. Blood of small hepatocyte carcinoma was mainly supplied by liver artery, and many took on full-blooded features on CT or MR imagings. This hepatocyte carcinoma tissue almost showed diffuse CD34 expression. Some small hepatocyte carcinoma showed no or slight enhancement on arterial phase scan, which was because mainly of necrosis and hyaline carcinoma. Hyaline carcinoma was with few microvessel distribution and poor-blooded supplied while with some ASG receptor distribution, which was an index of gooddifferentiation.5. Although Gal-BSA-SPIO showed a potential value in diagnosis the benign tumor from malignant, it had some difficulty in discrimination some dysplastic nodules from good-differentiation hepatocyte carcinoma, especially in diagnosis and differentiation some nodules founded on cirrhosis.
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