Synthesis and its targeting effect in vitro of disulfide-bonded hyaluronic acid-functionalized sodium-meter probe for hepatocellular carcinoma

Abstract

Objective To explore the construction method and physicochemical properties of disulfide-bonded hyaluronic acid-functionalized sodium-meter probe for hepatocellular carcinoma,and its biological evaluation in vitro and feasibility of MRI. Methods Synthesis of hyaluronic acid-disulfide-bonded-poly e-caprolactone (HA-SS-PCL) by disulfide-bonded alkynyl-terminated polycaprolacton (alkyne-SS-PCL) and azido-terminated hyaluronic acid (HA-N3) by clicking chemical reaction,then doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) were encapsulated in HA-SS-PCL core by dialysis method.HA-SS-PCL@DOX/SPIO was prepared and its particle size,DOX and SPIO loading rate were measured. With PBS as control group,the safety of HA-SS-PCL on human hepatocellular carcinoma cells HepG2 and normal liver cells LO2 was evaluated by the methylthiazolyl tetrazolium (MTT) assay, and the cytotoxicity of HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO on human hepatocellular carcinoma cells HepG2 was evaluated. Immunofluorescence and flow cytometry were used to observe the expression of CD44 receptor on the surface of HepG2 cells in HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups.Through in vitro MRI, PBS was used as the control group to observe the changes of T2 signal intensity of HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups when SPIO concentration was 10,20,40,80 μg/ml. One way ANOVA test and t test were used. Results HA-SS-PCL@DOX/SPIO sodium-meter probes were successfully constructed. The particle size of HA-SS-PCL@DOX/SPIO was (126.9±6.3) nm,and they were spherical with uniform size. The loading rates of DOX and SPIO were 61.4% and 58.7%. MTT assay showed that the survival rate of HepG2 and LO2 cells was more than 80% even at 500 μg/ml of HA-SS-PCL,66.6% in HA-PCL@DOX/SPIO group and 55.2% in HA-SS-PCL@DOX/SPIO group. Immunofluorescence and flow cytometry showed that HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups all have strong fluorescence,and the latter has stronger fluorescence intensity the former fluorescence intensity was 139.70±8.52,less than the latter 245.06±13.21. In vitro MRI showed that the T2 signal intensity of HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO was significantly lower than that of the control group (F values were 613.591 and 569.234,P=0.000), the latter decline rate was more significant. Conclusion The disulfide-bonded hyaluronic acid-functionalized sodium-meter probe for hepatocellular carcinoma has excellent physicochemical properties,good targeting and MRI functions on human hepatoma cell HepG2 at the cellular level in vitro. Key words: Magnetic resonance imaging; Disulfide; Hyaluronicacid; Sodium-meter probe