Study Of Cytobiological Effects And Translocation Through Blood-brain Barrier In Vitro Of Silver Nanoparticles

As an antibacterial materials of long-effective and good weatherability, silver had been applied to a broad range of products from health care to medical instruments. These agents were applied directly to living tissue or plasma, their cytotoxicity should be investigated, whereas the interactions between nano- biomaterial and target cell, and its influences on the cellular structures and functions were poorly studied. On the other hand, silver particles would give rise to some specific physical and chemical interactions for their super micro scales, such as high surface activity and chemical activity, and high adsorption. Because of those properties, nanoparticles may penetrate through the cellular gaps into the cell and organelles more easily than regular materials. Even, it could probably went through those physiological barrier, and accumulated in some deep tissue which regular materials could never reach. During the animal test, silver nanoparticles were given to SD rats as same as the clinical trail. Results showed that the blood-brain barrier (BBB) was injured , the nerve cell of the experiment group was pyknosis and nuclide was formed more signifficant than the control negative group. However there were so many unstable factors that would impact the in vivo test, whether the nanoparticles could penetrate through the BBB was still unknown. Still, more tests were needed to investigate wether nanoparticles would cause cytotoxicity to brain microvessel vescular endothelial cell (BMVEC) and astrocyte cell (AC) , wether it could lead nonreversible damage to the tight junction (TJ).In this dissertation, we measured the diameter distributions of several kinds of nano-scale and micro-scale silver particles by SEM , and investigate the in vitro cytotoxity by test of extract and test of indirect contact, which were demonstrated in the International Standard for Biological Evaluation of Medical Devices-part 5: test for invitro cytotoxicity. No significant toxic effect were found in all the experiment group during those tests. Then we made a modification to the test of direct contact method. Cellular morphology, infrastructure changes, mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase(LDH leakage assay) were assessed under control and exposed conditions. By studying the relationship between the cytotoxicity and the concentration/diameter of the silver particles, we can determine the dose of silver which would cause toxic effects, and make clear the biocompatibility differences between nano-materials and regular biomaterials. The results demonstrated that silver nanoparticles caused more serious damage to L929 cell than silver nanoparticles under the same silver concentration, which meaned the property of the specimen must be considerated fisrtly during the in vitro cytotoxicity test. There was little influence to the cell fonction when the silver concentration was lower than 100μg/ml in nanoparticles group and 500μg/ml in microparticles group. It made a reference to the silver concentration limit of nano-silver products.In this dissertation a method of coculture of BMVEC and AC of rats was used to establish an BBB model in vitro and evaluate BBB transcytosis and toxicity at the endothelial tight junction. Atomic Absorption Spectroscopy (AAS) methods and Inductively coupled plasma Mass Spaectrometer (ICP-MS) methods were used to determine the concentration of silver in the cell culture media. Silver distribution inside and outside the cytoplasm was determined and the permeation ratio of those two silver particles were calculated to raveal the different biological effects between nanoparticles and regular biomaterials. This in vitro experimental model of rat BBB was close to resemble the in vivo situation for examination of the permeability of nanoparticle and toxicity evaluation. The resullts showed silver nanoparticle was more easier to penetrate through the in vitro BBB model. In the nanoparticles group, the percentage of particles entering into cells were higher and the TJ damage was more signifficant than microparticles. Therefore, the penetration through BBB inferred to the both transcytosis process via transendothelial transport and paracellular permeation. Although the nanopartiles provided a great prospect to diagnosis and treatment of central nervous system (CNS) disease, its nonreversible damage to TJ should be considered as well.