| At present, the best method of cancer threpy is to remove metastasis regional lymph nodes. However, lymph nodes are usually hidden in the adipose tissue, accompanied with large blood vessels, and it’s difficult to completely resecte lymph nodes in the surgery. The lymph tracer can stain lymph nodes in vivo, and help doctors resecte lymph nodes. Thus, to develop safe and effective lymph tracer is one of the most important means to improve the quality of patients’life and prolong their life span.Methylene Blue (MB) is a reductant-oxidant, which is often used as antidote in the clinical cyanide poisoning. Recent studies show that it possesses some analgesic effects. With the development and consummation of Sentinel Lymph Node Biopsy (SLNB), methylene blue has been widely used as a lymph tracer in SLNB. There are more lipophilic components in MB water-in-oil microemulsion (MB-ME) and MB multiple microemulsion (MB-MME), which have a certain affinity with lymph resulting in the character of natural lymphatic targeting. MB capsuled in ME and MME can only penetrate the lymphatic capillary but not the blood capillary, avoiding the blood capillary and surrounding tissues blue-stained resulting in more accurate and effective lymphatic tracing in SLNB.The method of high performance liquid chromatography (HPLC) was developed to determine the concentration of MB and related substances in vitro. The stability of MB in different mediums was investigated, and the solubility and oil/water partition coefficient of MB in different media were determined, respectively, providing a basis to prescription and technology design.Separately, The co-surfactant concentration titration method and the Km value titration method were employed to draw pseudo-ternary phase diagrams of MB-ME and MB-MME. With the criteria of microemulsion region, factors affecting the formation of MB-ME and MB-MME were studied, such as the aqueous phase, the oil phase, surfactants, co-surfactants, the adding of the drug, the temperature and technical factors. The optimum conditions of MB-ME and MB-MME for process were performed by orthogonal design experiments.The physicochemical properties of MB-ME and MB-MME were investigated. Types of MB-ME and MB-MME were distinguished by the staining and the conductivity method. Their viscosities were 102.75 mPa-S and 14.93 mPa-S, respectively. Zeta potentials were both about 0 mV. The average diameter of MB-ME and MB-MME were 74.11 nm and 100.7 nm. The particles are spherical in shape and well dispersed by transmission electron microscopy (TEM), and the pH value were 6.08 and 5.68. MB-ME and MB-MME were stable at centrifugation and freezing-heating condition, but were not stable at 121℃15 min sterilization condition. In order to ensure the sterilization efficiency,115℃30 min sterilization condition was determined.An HPLC method was developed for the determination of MB in the plasma and biological samples. MB solution (MB-S), MB-ME and MB-MME were prepared with the aim to evaluate whether the three formulations carry MB target to regional lymph nodes and show lymphatic tropism after subcutaneous administration. MB concentrations in plasma, lymph nodes and limb soles were quantitatively analyzed using HPLC. Results show that MB-ME and MB-MME can’t enter the blood circulation, thereby avoid blood capillaries and surrounding tissues blue-colored. MB-S and MB-MME show better lymphatic tropism compared with MB-ME. The results indicate that blue colored lymph nodes could be observed, only when the concentration of MB reach 0.456μg/mg. MB-MME is a potential lymph tracer resulting in more accurate and effective lymphatic tracing. |
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