Study On Arginine-Functionalized Hydroxyapatite Nanoparticles As A Gene Carrier

ABSTRACT:Gene therapy is a latest strategy for malignant tumor patients, gene carrier is one of the key technology in the field of gene therapy. The safety of viral vector and non viral vector existing problems such as inefficiency seriously restricted the clinical application. Hydroxyapatite is the main component of human bone, and has a natural biological compatibility and high affinity with protein and gene, and hydroxyapatite nanoparticles itself has a function of selective inhibition for cancer cells’growing. Early research has shown that hydroxyapatite nanoparticles is a kind of extremely development prospect of gene carrier material, but need to further improve its gene transfection efficiency, and clarify its cell transduction mechanism and the relationship between the carrier and microstructure.This study chose arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles with rare earth doped as the research object by a hydrothermal method, and to clarify a mechanism of loading and releasing gene for HAP/Arg nanoparticles, to examine its gene transfection activity and its biological safety by human vascular endothelial cells and normal human Hela cells in vitro. To observe the process of HAP/Arg nanoparticles across the plasma membrane into the cell, and interpret the interaction mechanisms with cells, to establish the scientific basis of its design, synthesis and reasonable use as a nano-hydroxyapatite gene delivery system.The main content and results are summed up as following:1) The study takes liquid controllable synthesis and functionalization of HAP nanoparticles as the foundation. Crystallization degree and complete crystal shape of HAP nanoparticles with arginine functionalized are achieved by a hydrothermal method, The studies show that HAP/Arg nanoparticles has roughly the same on the structure with HAP nanoparticles without arginine functionalized. However, arginine joined inhibits the growth of HAP nanoparticles rate during the synthesis to a certain extent. Compared with HAP nanoparticles without arginine functionalized, HAP/Arg nanoparticles has less length to diameter ratio with short rod and positive surface charge. So, it laid a research foundation for HAP/Arg nanoparticles to increase the efficiency of gene transfection as gene carriers. Secondly, a small amount of rare earth ions doping such as Eu3+or Tb3+does not affect HAP/Arg nanoparticles’structure during the synthesis, its has single phase of HAP nanoparticles. What’s more, HAP/Arg nanoparticles has emission characteristic peak within the scope of the corresponding optical wavelengths, and can be a strong characteristics of the red light and green light after excitation, respectively. This leads the HAP/Arg nanoparticles to have a fluorescence property.2) To illuminate the mechanism of loading and releasing gene for HAP/Arg nanoparticles. The results show that the positively charged HAP/Arg nanoparticles binds the negatively charged plasmid DNA by electrostatic interaction and form the HAP/Arg-DNA complexes, and30μg HAP/Arg nanoparticles can bind1μg DNA at least. At the same time, the HAP/Arg nanoparticles and protect the binding DNA from being digested by human plasma protein components and Dnase I. So, the HAP/Arg nanoparticles can improve the stability of the binding DNA in vivo.3) To investigate the HAP/Arg nanoparticles transfection activity in vitro and biological safety in vitro and vivo comprehensively.The studies found that HAP/Arg-DNA complexes can transfect Hela cells effectively, but it can not do effectively for HAP nanoparticles without arginine functionalized, and its transfection efficiency is extremely low. Each dosage group of HAP/Arg nanoparticles does not affect normal growth and cell membrane structure under different duration of normal vascular endothelial cells (HAEC) and human tumor cells (Hela) by MTT and LDH methods. Experiment study on animal acute toxicity and general reproductive toxicity showed that each dosage groups of HAP/Arg nanoparticles had no significant difference compared with control group. Above results can lay the foundation to build a kind of novel efficient, safe and convenient gene delivery system for the next research step. 4) To real-time observe the process of HAP/Arg nanoparticles across the plasma membrane into the cell, and interpret the interaction mechanisms with cells. The studies have shown that HAP/Arg nanoparticles with terbium doped exists in cytoplasm, and most of distributed around the nucleus area of after cellular uptake by human umbilical vein endothelial cells (HUVEC). Furthermore, cellular uptake process of HAP/Arg nanoparticles in HUVEC is in a time and concentration dependent manner. Duration for4h and the concentration of nanoparticles for50μg/mL is the best. The cell competition test also show that HAP/Arg nanoparticles into the cells is significantly stronger than HAP nanoparticles without arginine functionalized, the results indicate that it is possibly related to the role of arginine and guanidine functional groups. Endocytosis inhibition test results demonstrated that HAP/Arg nanoparticles is internalized by HUVEC cells through an active transport and energy-dependent endocytosis process, and it was taken up by cells mainly through caveolin-mediated endocytosis although the clathrin-dependent endocytic pathway was involved.