Correlation Between The Size Of Carbamazepine Nanoparticles And Blood-brain Barrier Penetration And Brain Accumulation In Mice

Objective: Prepare drug-loaded nanoparticles of different diameters and explore the effects of the different particle sizes on their BBB penetration and the accumulation in the brain.Methods:（1）Nanoparticles with different diameters loaded with anti-epileptic drugs CBZ and fluorescent dye Di R were prepared using the nanoprecipitation method and characterized using DLS and TEM.（2）Using UV-visible spectrophotometer to measure the LE% and EE% of nanoparticles.Determining the in vitro release of the nano-drugs using HPLC.（3）Evaluating the cytotoxicity,biocompatibility,and biosafety of nano-drugs through MTT experiment,hemolysis experiment,serum biochemical analysis,blood routine analysis,and tissue HE staining.（4）Evaluating the impact of nanoparticles with different sizes on the endocytosis and their penetration of the BBB and accumulation in the brain through endocytosis experiment,lysosome co-localization experiment,in vitro imaging analysis of animals,brain tissue section analysis,and CBZ distribution in brain tissue of mice.Results:（1）Successfully prepared three nanoparticles with different sizes（60 nm,90 nm,and 120 nm）,and the prepared nanoparticles have uniform particle size and good dispersibility.（2）The prepared nanoparticles would not cause cytotoxicity and hemolysis of red blood cells and caused no change in the liver and kidney functions of animals and the number of blood cells,and cause no damage to the main organs（heart,liver,spleen,lung,kidney）.（3）The results of flow cytometry and CLSM showed that the endocytosis of nanoparticles with different sizes gradually increased with the extension of incubation time.And among the three nanoparticles,the endocytosis efficiency of the nanoparticles with the smallest size（60 nm）was higher than the other two groups at each time point,and the endocytosis efficiency with the largest particle size（120 nm）was the lowest.（4）The lysosome co-localization experiment showed that the nanoparticles uptake by HCMEC/D3 cells by endocytosis.（5）It is shown that nanocarriers could effectively carry drugs and accumulated in the brain,and the smallest particle size（60 nm）nanoparticles displayed the strongest brain tissue fluorescence distribution and the highest brain drug concentration,the largest particle size（120 nm）nanoparticles displayed the weakest brain tissue fluorescence distribution and the lowest brain drug concentration through in vitro imaging analysis of animals,semi-quantitative data,brain tissue slice analysis and quantitative analysis of drugs in the brain of mice.Conclusions:（1）The prepared CBZ-loaded nanoparticles have uniform size distribution,good water solubility and significantly reduced the cytotoxicity of carbamazepine,and have good biocompatibility and biosafety.（2）The size of nanoparticles is an important factor affecting endocytosis,and nanoparticles with smaller sizes can be uptake by HCMEC/D3 cells more easily.（3）The size of nanoparticles is an important factor affecting their BBB penetration and nanoparticles with smaller particle sizes more easily penetrate the BBB,thereby enhancing the effective accumulation of drugs in the brain.