Li et al. Nanoscale Res Lett 2011, 6:29 http://www.nanoscalereslett.com/content/6/1/29 been widely used in medical research such as DNA binding delivery with PLA/PEG nanoparticles, poorly soluble Ethaselen’s delivery with mPEG-PLA copoly- mers, prostheses for tissue replacements, supporting surgical operation and artificial organs for temporary or permanent assistance [17-19]. Some biocompatible poly- mer can also act as drug carriers by controlling the release rate of the loaded drug [20-23]. Meanwhile, the blends of the biodegradable polymers have been explored for the potential applications in biomedical field such as the drug release/implants for orthopedic surgery or blood vessels due to their good biocompat- ibility, low cost, safe elimination, lightweight and high performance [24-29]. Thus, on the basis of these obser- vations, the biodegradable poly(lactic acid) (PLA) nano- fibers have been fabricated in this study by using electrospinning and then adopted to blend with Au nanoparticles to form a new nanocomposites with the good biocompatibility. Afterward, the PLA/Au nano- composites were further conjugated with anticancer drug daunorubicin to efficiently facilitate the intracellu- lar accumulation of the anticancer agents inside drug- sensitive and drug-resistant leukemia cells. Experimental Section Reagents Daunorubicin was purchased from Nanjing Pharmacy Factory (analytical grade) and freshly prepared with phosphate buffer solution (PBS, 0.1 M, pH 7.2). Au nanoparticles were freshly prepared according to the previous report [30], in which 100 mL of 0.01% HAuCl4 was heated to boiling with uninterruptedly agitating and 3.0 mL of 1% trisodium citrate was dropped into the above solution. The system was continually agitated about 30 min until the reaction color did not change. Ultrapure water was added in which the final volume was 100 mL and the diameter of Au nanoparticle was about 15 nm. The other reagents were analytical grade. For the following studies, all experimental measure- ments were performed at least three times in parallel. The Preparation of Poly(lactic acid) Nanofibers Poly(lactic acid) nanofibers were fabricated by electro- spinning. First, the poly(lactic acid) (Mn = 340,000) was dissolved in the solvent of chloroform (10%, wt) and stirred for 2 h. Next, it was loaded into a syringe con- nected with anodal voltage. Aluminum foil as a collect- ing substrate was connected with cathodal voltage. Electrospinning was performed at room temperature with a gap between the substrate electrode and the tip of the capillary of 10 cm at driving voltages of 10 kV. The PLA nanofibers were characterized by scanning electron microscopy (Magnification: 10,000) as shown in our previous report [31]. Page 2 of 8 The Preparation of PLA/Au Nanocomposites Conjugated with Dauborubicin The aqueous suspension of PLA nanofibers was pre- pared in double-distilled water by ultrasonic treatment for about 20 min. Then, Au (2.80 × 10-7 mol/L) and PLA nanofiber gel aqueous solutions (1.3 × 10-3 g/L) were mixed and incubated together for more than 12 h to form the PLA/Au nanocomposites. The solution of daunorubicin (3.3 × 10-4 mol/L) was mixed into the nanocomposites and stored in the dark at 4°C for more than 12 h to form the PLA/Au nanocomposites conju- gated with dauborubicin. Atomic Force Microscopy Study In a relevant atomic force microscopy study (AFM), 5 μL from the aforementioned different solutions was deposited onto freshly cleaved mica (already glued on a steel disk) and incubated for 5 min. After the sample dried under a nitrogen stream, imaging was performed in tapping mode, by using a Nanoscope IIIa Multimode AFM (Digital Instruments, Santa Barbara, CA, USA) operating in air at room temperature. MTT Assay For cell culture, human leukemia cells (K562 and K562/ AO2) were cultured in a flask in an RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 mU/mL) and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere con- taining 5% CO2, while 1 μg/mL doxorubicin was con- tained in the culture to maintain K562/AO2 cells’ drug resistance in its daily culture. The inhibition of cell growth was measured by MTT (Microculture Tetrazolium) assay. Initially, the respec- tive leukemia cells (K562 and K562/AO2) in the log phase were seeded in a 96-well plate at a concentration of 1.0 × 104 cells) well. The target cells were treated with different concentration of PLA/Au nanocomposites, daunorubicin or daunorubicin conjugated with PLA/Au nanocomposites, respectively. Controls were cultivated with the respective solvent under the same conditions. Each culture was incubated for 48 h in a 5% CO2 incu- bators at 37°C, and then 20 μL of 5 mg/mL MTT was added to the wells and incubated for an additional 4 h. Subsequently, it was centrifuged at 1,000 rpm for 10 min and the supernatant was discarded, followed by the addition of 150 μL of dimethyl sulfoxide (DMSO) into each well and then incubated in the shaker at 37°C with gentle shaking about 5 min. Then, the optical den- sity (OD) was read at a wavelength of 492 nm. Laser Confocal Fluorescence Microscopy The cell culture conditions were similar to those described above. Initially, the different cells were