title: Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.
abstract: The aim of this study was to synthesize a chitosan (CS) derivative, a quaternary ammonium salt crystal called N-2-hydroxypropyl trimethyl ammonium chloride chitosan (HACC), and test a series of HACC and pEGFP-DNA complexes at different weight ratios for their efficiency of gene delivery into human cells. CS was modified with cationic etherifying agent to obtain the CS derivative. Fourier transform infrared spectra were recorded on KBr pellets with a spectrometer. (1)H nuclear magnetic resonance (NMR) spectra of HACC were obtained using a spectrometer. HACC was subsequently used to prepare HACC/DNA complexes at different weight ratios by coacervation method. The resulting particle size and surface charge were assessed by laser light scattering using a zeta potential analyzer. The HACC/DNA complex formation and DNA protection in the nanoparticle complex was investigated by gel mobility shift assay and DNase I protection assay, respectively. The cytotoxicity of HACC and HACC/DNA nanoparticles was evaluated by MTT assay using (mesenchymal stem cell) MSC lines. The nanoscale structure of the particles was obtained by transmission electron microscope (TEM). The FTIR spectrum of HACC showed the characteristic quaternary ammonium group absorption band at 1475 cm(-1), which indicated the presence of quaternary ammonium group. The successful synthesis of HACC was also confirmed by (1)H NMR spectrum. HACC showed good solubility in water and was electropositive. HACC efficiently packed and protected pEGFP-DNA at a weight ratio of 10. With increased weight ratios, the surface charge of the composite particle increased from negative to positive, the average particle size increased, and HACC nanoparticle had a higher carrying efficiency. The nanoparticles released DNA in two distinct phases, and 55 % was released within the first 20 h of solubilization. The nanoparticles under TEM showed circular or oval shapes. The particles exhibited no cytotoxicity against human cells. No significant difference in gene delivery efficiency was detected between HACC/pEGFP-GDNF and liposome/pEGFP-GDNF complexes (33.8 vs. 34 %, P = 0.363). In this study, HACC was successfully synthesized, and HACC/DNA complex assembled efficiently. HACC showed strong DNA binding affinity and high protection of DNA and was non-cytotoxic to human cells. The particles had appropriate nanostructure, mean diameter, and DNA release time. The results suggest that HACC nanoparticles are a novel tool for efficient and safe gene delivery.
authors: Department of Orthopedics, Shanghai Zhongzhi Hospital, Shanghai, 200941, People's Republic of China.
authors: Guang-Feng
authors: GF
authors: Li
authors: Jing-Cheng
authors: JC
authors: Wang
authors: Xin-Min
authors: XM
authors: Feng
authors: Zhen-Dong
authors: ZD
authors: Liu
authors: Chao-Yong
authors: CY
authors: Jiang
authors: Jian-Dong
authors: JD
authors: Yang
mesh_terms: D045744:Cell Line, Tumor; D048271:Chitosan / Q000031:analogs & derivatives; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry*; D014162:Transfection
chemical_list: C495830:chitosan-N-2-hydroxypropyl trimethyl ammonium chloride; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan