The Preparation Of DCN-loaded PLGA Nanoparticles Modified By AFP Antibody And Its Activity Research In Vitro

Objective:The aims of this study were to prepare the nanoparticles consisted of decorin(DCN)and poly lactic-co-glycolic acid(PLGA) modified by anti-alpha fetoprotein(AFP)monoclonal antibody(m Ab) and to examine the conventional physical and chemical properties, the in vitro release of DCN and the targeting effect of these nanoparticles on Hep G2 cells.Method:Part11 The PLGA nanoparticles targeting Hep G2 cells were generated by compound emulsion and solvent evaporation. The morphology of the generated nanoparticles was visualized with scanning electronic microscope and their diameters were measured by laser particle size instrument. The entrapment efficiency, drug-loading rate, and the in vitro release rate of nanoparticles were investigated.2 The nanoparticles were double labeled with FAM and Rhodamine fluorescent dyes to observe the active cellular uptake in 4 and 12 hours.3 RT-PCR assay authenticate the expression of AFPm Ab PLGA- rh DCN nanoparticles in Hep G2 cells.Part21 Their inhibition rate at different concentrations of proliferation on Hep G2 cells were assessed by MTT assay.2 Flow cytometry test cell apoptosis rate and cell cycle of each group after Ig G-PLGA- rh DCN nanoparticles and AFPm Ab PLGA- rh DCN nanoparticles effection on Hep G2 cells for 48 h.3 Real-time fluorescent quantitative PCR detect the Bcl – 2 and caspase-3 expression in each group after nanoparticles effection on Hep G2 cells for 48 h.Result:Part11 The prepared nanoparticles displayed the characteristics of smooth surface and regular morphology. The mean diameter of these nanoparticles average was 216.7 + 8.2 nm with the mean Zeta electric potential of 17.4 m V. The mean entrapment efficiency was78.60 +1.03% and drug-loading rate was 3.12 + 0.17%. The encapsulated plasmid was slowly and steadily released from the nanoparticles and the release rate reached 79.3% at the time of 240 h.2 The absorb experiment results of HepG2 cells show that AFPmAb PLGA- rhDCN nanoparticles biologically targeted Hep G2 cells and were initiative taken in Hep G2 cells in high efficiency.3 According to the results of RT-PCR agarose gel electrophoresis, DCN gene in AFPm Ab PLGA- rh DCN nanoparticles can be expressed in Hep G2 cells successfully.Part21 Different concentrations of targeted PLGA nanoparticles significantly inhibited the proliferation of Hep G2 cells as compared with that of control group(P < 0.05).2 In flow cytometry results, the cell early apoptosis rate in experimental groups is higher significantly than the control group(p < 0.01) and more distinct with the concentration of nano particles rise. The percentages of S phase and G2 phase in experimental groups are significantly lower compared with the control group while relatively higher proportion of the G1 phase(p < 0.01).3 Real-time fluorescent quantitative PCR results show that the expression of Bcl- 2gene in experimental groups is decreased gradually with the increasing of targeted nanoparticles concentration, while caspase 3 gene increased(p < 0.01).Conclusions:1We successfully constructed AFPm Ab-PLGA-rhDCN nanoparticles with slow-released effect and experiments show that they were capable of specifically targeting Hep G2 cells in vitro and the DCN gene can be expressed in Hep G2 cells successfully.2 In vitro, AFPm Ab- PLGA- rh DCN nanoparticles are significantly inhibiting the proliferation of Hep G2 cells and promote the early apoptosis which its mechanism may be related to the Bcl- 2 and caspase- 3.