DL-Lactide (LA) and glycolide (GA) were purchased from Boehringer Ingelheim (Germany). They were recrystallized twice from ethyl acetate and dried under high vacuum at room temperature before use. Polyethylene glycol (PEG, molecular weight: 1000) was obtained from Sigma Chemical Co. (St. Louis, MO, USA) and dried under high vacuum at room temperature. Stannous octoate was also obtained from Sigma.
Synthesis and characterization of PLGA-PEG copolymers
PLGA-PEG copolymers of different composition were prepared by a melt polymerization process under nitrogen, using stannous octoate as catalyst (Beletsi et al 1999
). They were characterized by FT-IR (Shimadzu 8400) and 1
H-NMR (Bruker AC-80) and their molecular weight and molecular weight distribution (polydispersity index, Mw/Mn) was determinedly gel permeation chromatography (GPC, Walters, 515 HPMC pump).
Preparation of nanoparticles
PLGA-PEG nanoparticles were prepared using a precipitation-solvent evaporation technique (Avgoustakis et al 2003
). Briefly, a solution of the polymer in acetone was transferred drop wise to a stirred aqueous solution of poly vinyl alcohol (PVA, molecular weight: 10000, 0.5% w/v). The mixture was kept under stirring until acetone had been evaporated, and the nanoparticle dispersion formed was condensed in a rotary evaporator and filtered through a 1.2 μm filter (Millex AP, Millipore). Finally, the nanoparticles were collected by centrifugation at 25000×g for 25 min and washed twice with water before lyophilization (Christ Alpha 1–4).
FT-IR (cm−1): 3010, 2955, 2880, 1765, 1185-1070.
H-NMR (ppm); δ=3.7 (m, 4H, methylene groups of PEG), δ=1.6 (d, 3H, methyl group of the lactide), δ=4.8 (d, 2H, methylene group of glycolide), δ= 5.2 (m, 1H, CH of lactide).
Observation of transmission electron microscope (TEM)
The morphology of the polymeric nanoparticles was observed using a TEM (LEO 960). A drop of drug loaded nanoparticle suspension in aqueous solution was placed on a carbon film coated on a copper grid for TEM and freez-dried. Observation was done at 80 kV.
Loaded drug quantity was determined according to the following procedure: after nanoparticles were formed, unbound adriamycin was separated by centrifugation at 30000×g at 25º C for 10 min and the precipitate was discarded. The precipitate was then lyophilized and resulting powder containing the loaded nanoparticles was dissolved in ethanol to obtain a clear solution and analyzed UV spectrophotometrically (Shimadzu UV-160) at 479 nm. Loading capacity was expressed in terms of entrapped drug quantity, and entrapment efficiency.
In vitro drug release
The in vitro release experiments were carried out as follows: 5g of adriamycin-loaded polymeric nanoparticles and 1 ml of phosphate buffer saline (PBS 0.1 M, pH 7.4) were put into a dialysis tube and then was introduced into a vial with 10 mL of PBS. At specific time interval, the whole medium was taken and replaced with fresh PBS. The concentration of the released adriamycin was determined by UV spectrophotometer at 479 nm.