Highly Monodisperse Chitosan Nanoparticles Prepared by a Combined Triple-Method for Potential Use as Drug Carriers

Chitosan (CS) as a biodegradable polymer with unique bio-attachment properties that makes it favorable to be used in biomedical applications. Insolubility in water is the problem with use of CS. The purpose of this study was to prepare low molecular weight, water-soluble CS nanoparticles that exhibit excellent water solubility and biological, chemical, and physical functions. Oxidative degradation technique using hydrogen peroxide (H2O2) was used to decrease chitosan molecular weight. Then ultrasonication and ionic gelation method using sodium tripolyphosphate (TPP) were used to prepare CS nanoparticles. Molecular weight of chitosan determined by Ubbelohde viscometry and it decreased by approximately 100%. From the spectral information (FTIR) it was observed that the cross-linking between CS and TPP was taking place while the main structure of chitosan was the same. The dynamic light scattering results showed that water-soluble nanoparticles had a multimodal size distribution pattern, while low molecular particles yielded monodisperse particle distribution with an average size of ~45 nm, which was directly ascribed to the role of ultrasonication process. The morphology of nanoparticles was observed by SEM and TEM techniques. The mean diameter of nanoparticles was obtained in a range of 30 nm to 45 nm with a narrow size distribution and polydispersity index smaller than 0.2. Cytotoxicity of CS nanoparticles on human umbilical valve endothelial cells (HUVEC) was assessed by CCK-8 assay, as well as “Live/Dead” assay and subsequent fluorescent imaging. The results showed no to minimal cytotoxicity for CS solutions up to 50 µg/ml, while sporadic dead cells observed for 100 µg/ml solutions. This suggested that our monodisperse nanoparticle systems are great candidates to be used for drug delivery systems.