Preparation And Evaluation In Vivo And In Vitro Of Amoitone B-Loaded Long Circulating Nanostructured Lipid Carriers

Nur77 (also known as TR3, TIS1 and NGFI-B) receptor is a vital participant in various biological processes such as cell proliferation, metabolism, differentiation and apoptosis. Once activated, it transfers from caryon to mitochondria, resulting in the release of cytochrome C which can kill tumor cells. It has been proved to be over expressed in numerous cancer cells including stomach, colon, ovarian as well as lung. Moreover, Nur77 plays a key role in decreasing inflammatory reaction in atherogenesis and facilitating gluconeogenesis. Thus, Nur77 is an ideal target for the therapy of cancer and other diseases. Amoitone B (n-amyl-2-[3,5-dihydroxy-2-(1-nonanoyl) phenyl] acetate), a powerful agonist of Nur77 receptor, is artificially synthesized as a derivative of the nature antitumor compound Cytosporone B (also named as Amoitone A). It is confirmed as the most effective derivative and it has surpassed anticancer activity than Cytosporone B due to the enhanced Nur77-binding and activating properties. Furthermore, it can strengthen the anticancer activity of Nur77 by inhibiting the function of BRE, an anti-apoptotic protein which is able to suppress the mitochondrial apoptotic pathway. Therefore, it is regarded as a potential anticancer agent. Nevertheless, the water-insolubility and short biological half-time confine its successful application. It is urgent to develop an efficient formulation to improve the bioavailability.In this study, PEG-modified Amoitone-loaded nanostructured lipid carriers (AmB-PEG-NLC) were prepared with the purpose of prolonging circulation time in body and enhancing bioavailability of Amoitone B.The NLC were prepared by the method of emulsion-evaporation and low temperature-solidification. Briefly,200 mg of lipid (PEG-SA:GMS:MCT=2.5:2.5:1), 12 mg drug and 200 mg soybean lecithin (SL) were mixed and melted at 75℃ and then dispersed in 10 mL aqueous solution containing 250 mg F68 (heated at 75℃) under a mechanical stirrerwith 1000 rpm in a water bath for 2.5 h. The resultant thermal nanoemulsion was dispersed rapidly into 20 mL of distilled water (0-2℃) in ice bath with stirring at 1000 rpm for 2 h. Finally, the AmB-PEG-NLC dispersions were achieved from the supernatant after centrifugation at 3500 rpm for 10 min. The entrapment efficiency and the drug loading of the nanoparticles were 68.17±0.94% and 4.09±0.06%, respectively. The particle size was 217.2±0.95 nm. The Zeta potential was -12.7±0.49 mV.The AMB-PEG-NLC dispersions were freeze-dried. Mannitol (5%, m/v) was utilized as cryoprotectant in the freeze-drying process. Firstly, the NLC dispersions were pre-frozen with an ultra cold freezerat -80℃ for 24 h. Then, the samples were freeze-dried at -50℃ for 48 h utilizing a freeze dryer (FD-1000, EYELA, Japan). The various characteristics of AmB-PEG-NLC in vitro and in vivo were expounded in detail. DSC and XRD measurements displayed the amorphous or less ordered form of Amoitone B in NLC. In vitro release test revealed that AmB-PEG-NLC had a biphasic drug release patterns with burst release incipiently and sustained release afterwards. The initiatory stability research showed that the freeze-dried AMB-PEG-NLC could be stored at 25℃ or 4℃ for 3 months.In vivo pharmacokinetic evaluation demonstrated the prolonged MRT in body and higher AUC values of Amoitone B in AmB-PEG-NLC compared with Amoitone B solution and AmB-NLC, AUC12.549 h mg L-1, MRT=8.879 h,t1/2=3.731 h, CL=0.638 L·h-1·kg-1.MTT assay showed that AmB-PEG-NLC was more cytotoxic to human colon cancer SW620 cells and liver cancer HepG2 cells than AmB-NLC and AmB-Sol in a time- and dose- dependent manner. Observation of fluorescent morphology changes and apoptosis examination revealed that AmB-PEG-NLC significantly promoted apoptosis of SW620 cells. There was an improved uptake of AmB-PEG-NLC compared with AmB-NLC and AmB-Sol in SW620 cells, and the increased uptake offered sound explanation to the enhanced cytotoxicity of AmB-PEG-NLC.It is the first report on the preparation of AmB-loaded long circulating nanostructured lipid carriers, the results of our studies contributes to the development of injection of poorly soluble drugs, and plays a very important role in clinical application of AmB.