Preparation Of Carboxymethyl-chitosan Nanoparticles And The Effect On Proliferation Of Keloid Fibroblasts

Keloids are excessive scarring caused by the abnormal produce of collagen after theskin injury. The main reason is the enhanced ability of keloid fibroblast to synthesisand secretion collagen or the increased number of keloid fibroblasts.carboxymethyl-chitosan (CMCS) is a water soluble chitosan (CS) derivative withgood biocompatibility and histological affinity. In addition, CMCS can promotetheproliferation of the normal skin fibroblast and have a good application in skinparenchyma repairing. In this paper, CMCS nanoparticles were prepared as a noveldisperse system. Using keloid tissue of yellow race, we successfully cultured keloidfibroblasts invitro. And then the effect and mechanism of CMCS nanoparticles onproliferation of keloid fibroblasts was preliminary evaluated.1. Preparation, Characteristics of CMCS nanoparticlesTwo carboxymethyl-chitosan(CMCS) samples with different molecular weights(MWs)(196kDa,6.3kDa) were prepared from the carboxymethylation reaction ofchitosan under soft conditions. The obtained CMCS sample with MW196kDa wasdegraded with hydrogen peroxide to prepare CMCS with different MWs (16kDa,7.5kDa).The four samples of CMCS had good water solubility. The MW among themwas different, but degree of deacetylation (DD) values and degree of substitution (DS)values on the chitosan’s backbone wereal most similar(DD92%±2%，DS78%±3%).FT-IR suggested the chemical structure of CMCS and CMCS nanoparticles.By usingdynamic light scattering (DLS) and transmission electron microscope (TEM), theCMCS nanoparticles with various MWs all showeda spherical morphology withdiameters in the range of150-200nm.2. Keloid fibroblasts culture in vitroBy using keloid tissues of yellow race, we cultured keloid fibroblasts in vitro. After 10-15d in primary culture, keloid fibroblasts and epithelial cell emigrated from tissueblocks. After3-4generations, epithelialcells disappeared and only keloid fibroblastswere present. The growth curve of keloid fibroblasts suggested keloid fibroblastsreached logarithmic growth phase after two days culture.3. The effect of CMCS nanoparticles onproliferation of keloidfibroblastsEffects of concentration, exposure time and MW of CMCS nanoparticles on theproliferation of the keloid fibroblast were studied by MTT method. With the increaseof CMCS nanoparticls esconcentration from2to200μg/mL, the growth inhibitioneffects on the keloid fibroblast increased. At the concentration of100μg/mL,CMCS nanoparticles withMW6.3kDa had a significant inhibitory effect (inhibitionratio48.79%) of the proliferation of keloid fibroblast. Compared with CMCSnanoparticles, the growth inhibition of CMCS solution was higher than CMCSnanoparticles in thefirst four days (P<0.05), but similar on the sixth day (inhibitionrate48.79%for CMCS nanoparticles and49.69%for CMCS solution, P=0.844). Byanalyzingthe different effects of CS, CMCS solutionand CMCS nanoparticles onproliferation of keloid fibroblast, we have found that the carboxylmethyl groups ofCMCS play an important role in inhibition of proliferation of keloid fibroblasts.In this paper, we prepared CMCS nanoparticles disperse system and evaluate theeffect and mechanism of CMCS nanoparticles on proliferation of keloid fibroblasts.These experiments provided certain theoretical basis for skin parenchyma repairingand prevention and treatment of scar.