| Objective:The hyaluronic acid-modified silver nanoparticles was prepared for improving tumor targeting delivery and antitumor efficacy.The mechanisms of the anti-tumor effect were explored,providing helpful insight into cancer therapy of silver nanoparticles for clinical application. Methods:The carboxyl group of hyaluronic acid (Hyaluronic Acid, HA) was chemically conjugated with the amino group of pentaethylene hexamine for prepared the aminated HA. AgNP was synthesized by a reduction method based on the reducibility of HA, which also functioned as the polymer stabilizer for AgNP. The thus-formed HA-AgNP was characterized. The particle size and electric potential of HA-AgNP was measured by malvern particle size analyzer; the morphology of HA-AgNP was analyzed by transmission electron microscope(TEM);[Ag+] was determined by using O-phenylendiamine method. The drug-loading rate of HA-AgNP was carried out using TGA assay. The stability of AgNP and HA-AgNP in ultrapure water, PBS and culture medium was investigated by turbidimetry method. The cellular uptake of HA-AgNP was observed by fluorescence microscopy and quantified by fluorescence-activated cell sorting (FACS). The inhibitory efficiency of HA-AgNP against tumor cells was analyzed by MTT assay. Cell cycle analysis was conducted by FACS with PI labeling. The cell mitochondrial membrane potential was detected by using the JC-1staining method. Autophagic vacuoles in the cells were observed by staining with MDC.The AgNP-induced apoptosis of the tumor cell was monitored with Hoechest33258/PI staining. The interaction between HA-AgNP and CD44in tumor cells was observed by laser scanning confocal microscopy. Results:Aminated hyaluronic acid was successfully prepared by chemical modification. The content of amino group content was measured to be1mMol/g by the TNBS method, the modification rate reached40%. The HA-AgNP was synthesized by reducing silver nitrate with aminated HA, which functioned as both a reducing agent and stabilizer. The HA-AgNP with a mean size of about300nm, zeta potential of17.2mV. The drug-loading rate of HA-AgNP reached13.1%. HA-AgNP was stable in ultrapure water, PBS and culture medium. In vitro experiments showed that HA-AgNP had significant cytotoxicity on CD44+SW480cells and MCF-7cells, but with better antitumor effect on the SW480cell compared to the MCF-7cell line. HA-AgNP induced the decline of mitochondrial membrane potential, cell-cycle arrest, pyknosis and cell cycle inhibition, resulting in apoptosis and autophagy. Conelusions:The prepared HA-AgNP was demonstrated its strong antitumor and CD44targeting effects in vitro. The antitumor mechanism of HA-AgNP could be accounted for the declination of mitochondrial membrane potential, cycle arrest, pyknosis. Our results showed HA-AgNP effectively induced to apoptosis and autophagy, and the apoptosis could be the mitochondrion pathwany. |
