A Prefabricating And Tagging Study Of Patent-blue Liposomes On Lymphography In Vitro

OBJECTIVE To prepare Patent-blue liposomes and to develop a method for determining its entrapment efficiency,to investigate distribution and migratory route of Patent-blue liposomes in lymph nodes of rats,and to investigate the feasibility and clinical significance of using Patent-blue liposomes violet-directed mapping for sentinel lymph nodes(SLN)detection in gastric cancer.METHODS Patent-blue was encapsulated in the liposomes using the concentration gradient-dependent remote loading technique.Free Patent-blue and liposomes were separated by Sephadex and centrifugalization was applied to determine the entrapment efficiency.The experiment involved 60 female Wistar rats that were injected subcutaneously with the Patent-blue liposomes,Patent-blue,blank liposomes or isotonic Na chloride at the heel soles,after which,at 1,6,12,24 and 48 hours respectively,the lymph nodes were dissected for observing gross morphology and colour.With immobilization,the lymph nodes were sliced to HE stained pathological sections for investigating distribution of the Patent-blue liposomes or Patent-blue in the lymph nodes.Choose thirty eight patients with gastric cancer,who underwent standard radical gastrectomy with lymphadenectomy, were enrolled in this study.After resection,Patent-blue liposomes violet was intraoperatively injected into subserosal or submucosal layer adjacent to the tumor.Blue stained nodes were defined as SLNs and analyzed by routine HE preparations.Those negative SLNs were examined with cytokeratin immunohistochemical staining.The incidence of metastasis in SLNs was compared with that in non-SLNs.The diagnostic accuracy and false negative rate of regional lymph node status on the basis of SLN status were calculated, respectively.RESULTS The entrapment efficiency of Patent-blue liposomes prepared by concentration gradient method equal to the following numbers:81.50%(lower concentration group),80.98%(middle concentration group),51.86%(high concentration group).The centrifugalization method was validated and Patent-blue was well separated.The standard curve was linear over the range of 15-80μg/ml with the correlation coefficient of 1.000,0.937,0.835.The lymph nodes at injection sites turned obviously blue staining,with their sequence coinciding with the direction of lymphatic return.After entrance into the lymph nodes,the Patent-blue liposomes particles were distributed in the marginal sinus first and then entered the central sinus,with no particles in the blood vessels,trabeculae or folliculus lymphaticus of the lymph nodes.SLNs were detected in 36 of 38 patients with a success rate of 94.7 percent.The number of SLNs ranged from 1 to 3 in one case,with a mean value of 1.8 per case.The incidence of metastasis was significantly higher in SLNs than that in non-SLNs(55.38%＞13.16%).The diagnostic accuracy and false negative rate were 94.44 and 6.67 percent,respectively.Focused analysis of SLN upstaged 2 cases of patients with gastric cancer.CONCLUSION The Patent-blue liposomes,a lymph-mapping developer,can be successfully prefabricated with a high entrapment efficiency and stable on jugation by a simple concentration gradient technique.The Patent-blue liposomes can be used as a useful lymphatic tracer and drug carrier,for it is characterized by avirulence,high lymphatic tropism,long residence time than Paten-blue in the lymph nodes.Patent-blue liposomes violet-directed mapping of SLN in gastric cancer is technically feasible and may provide an accurate diagnostic procedure for detecting lymph nodes metastasis in patients with gastric cancer.Focused analysis of SLN may upstage gastric cancer.