The Study Of Self-assembled Tumor-targeted Novel Drug Carrier Derived From Pullulan Conjugate

Folate receptors (FRs), overexpressed on the surface of a variety of tumor cells, are potential targets for tumor targeting therapy. This study aimed to develop folate conjugated pullulan acetate as a FRs -mediated drug delivery system to target chemotherapeutic agents to FRs-overexpressed tumor cells and tumor in vitro and in vivo.Folate was coupled to pullulan acetate (PA) by N, N'-Dicyclohexylcarbodiimide (DCC) and 4-Dimethylamino-pyridine (DMAP) mediated ester formation. The product was characterized by proton nuclear magnetic resonance (1H NMR) spectroscopy. The degree of folate substitution (DS) was caculated by ultraviolet spectrometry is 0.235 per sugar residues of PA. Folate conjugated pullulan acetate nanoparticles (FPANs) and Epirubicin-loaded FPANs (FPA/EPI) were prepared by dialysis method. Various physicochemical properties of FPA and FPA/EPI including size, surface charge and morphology were characterized by dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM). FPANs and FPA/EPI had the nearly spherical shape with a size range of (204.2±10.9) nm and (273.4±11.0) nm respectively, they had lowζpotentials both in distill water and in 10%FBS. The storage stability of FPA and FPA/EPI were observed in distilled water, the size andζpotential were determined once a month. The nanoparticles were stable for at least three months. Flow cytometry test revealed that FPA/EPI NPs exhibited a dose dependent cellular uptake against human epithelial cervical cancer (Hela) cells with over-expressing folate receptors on the surface. Ensemble-averaged measurements with flow cytometry indicate that FPA/EPI can be internalized by HeLa cells in a dose dependent manner through receptor-mediated endocytosis, as confirmed by competitive inhibition assays.An in vivo toxicity study was performed to establish the safety of the prepared blank FPANs and PANs after i.v. administration in ICR mice. The study indicated that the NPs were well tolerated at 125mg/kg and 200mg/kg, respectively. The in vivo pharmacokinetics was investigated after an i.v. administration at 10mg EPI/kg in rats. Promisingly,1.57-fold increase in the half-life (t1/2),1.33-fold increase in the mean retention time (MRT0-24h) and 3.95-fold increase in the area under the curve (AUC0-24h) of EPI were achieved for the FPA/EPI compared with the free EPI. Moreover, the in vivo biodistribution and anticancer effects were investigated using a model of nude mice xenografted with Hela cells in the study. The drug level in stomach and gastric was significantly reduced, which is an indication of reduced side effects. The antitumor test demonstrated that when systemically administered at dose of 5.0mg/kg and 7.5mg/kg, the anti-cancer effects of FPA/EPI appear to be dose-dependent and the highest dose completely inhibits tumor growth through inducing the tumor cell apoptosis, the related rate of reproduction were 81.38% and 37.72%, and the rate of tumor inhibition were 32.37% and 61.19% respectively.All results suggested that FPANs were prepared easily, stable, safe and showed a promising potential on reducing the toxicity and improving the anticancer efficiency of the encapsulated drug. The conjugate showed great potential to be a carrier of nanodrug.