The total weight of the feed was about 3 g. Stannous octoate was dissolved in hexane and added at a concentration of 0.03% by the weight of the feed. Then, the tubes were heated at 190��C for the 2 h. The resulting copolymer was purified by dissolving in chloroform and then precipitating in an excess methanol. The purified copolymer was dried under vaccum.5 http://www.selleckchem.com/products/MLN-2238.html Preparation of non-PEGylated nanoparticles Drug loaded Non-PEGylated nanoparticles were prepared using emulsification solvent evaporation method (Avgoustakis, 2004) which is widely used for the encapsulation of hydrophobic drugs. Briefly, temozolomide (5 mg) and PLGA (50 mg) were dissolved in acetone (5 ml) (organic phase). The organic phase was added at a constant flow rate (0.

3 ml/min) into 20 ml of aqueous phase containing 1% of PVA under intense shear using probe sonicator (Lark Innovative Fine Tecknowledge). The resultant mixture was further stirred for 2 h using magnetic stirrer. The organic solvent was then evaporated off under vacuum using a rotavapor (Steroglass). Then NPs were lyophilized (Heto Drywiner) at -46��C, 0.001 atm for 24 h with 5% w/w mannitol which was used as cryoprotectant (Yin et al., 2006).6 Preparation of PEGylated nanoparticles PEGylated nanoparticles were also prepared by emulsification solvent evaporation method. All the parameters were kept same except the PLGA polymer was replaced with PEG PLGA Copolymer. These Non-PEGylated and PEGylated nanoparticles were stored in a tightly closed container until further evaluation.

Characterization of non-PEGylated and PEGylated nanoparticles Shape morphology The Non-PEGylated and PEGylated nanoparticles were characterized for the shape by Scanning Electron Microscopy (SEM, Philips XL 30 scanning microscope, Philips). The nanoparticles were coated with gold-palladium alloy (150�C250 ?) using a sputter coater. The coater was operated at 2.2 kV, 20 mV, 0.1 torr (argon) for 90 sec at an accelerating voltage of 15 kv. The samples were viewed under a scanning electron microscope. Particle size The size of the nanoparticles was determined with the help of laser diffraction particle size analyzer (Cilas 1604L). Non-PEGylated and PEGylated nanoparticles were suspended in the chamber of particle size analyzer containing milli-Q water and the vesicles size were determined using the software provided with the instrument.

Percentage drug entrapment (PDE) The amount of drug encapsulated in nanoparticles was determined using the method reported by Khuller and Pandey.5 The lyophilized non-PEGylated and PEGylated nanoparticles were digested in 5 ml of 0.1 M NaOH at 50��C GSK-3 for 10 min to release the drug content and resultant mixture was filtered. The volume was adjusted to 10 ml with 0.1M NaOH. The amount of drug was quantified by HPLC method. Drug encapsulation efficiency (%) = Amount of drug released from the lysed NPs/amount of drug initially taken to prepare the NPs �� 100.