The supernatant collected were centrifuged at 10,000 rpm for 4 min and the concentration of Dox in the cell lysates was measured in

a fluorometer (FLx800, BioTek) at an excitation wavelength of 485 nm and an emission wavelength of 590 nm. Results are expressed as micrograms of Dox per milligrams of cellular protein. Protein concentration of the cell lysates was determined using Coomassie plus protein assay reagent and bovine serum albumin as standards (Pierce, Rockford, IL, USA). Confocal fluorescence click here microscopy was used to observe the intracellular uptake and distribution of Dox from PST-Dox nanoparticles and the standard Dox. Adherent cancer cells (HCT116 and MCF-7) were grown overnight in 12 mm circular glass coverslips with 10 % DMEM for 24 hours. Cells were incubated with PST-Dox nanoparticles (1 μg/ml) for 2 h and 6 h or Dox (1 μg/ml) for 6 h. The cells in the cover slips were fixed with 4% paraformaldehyde, counterstained with DAPI and mounted with DPX on a clean glass slide. Slides were observed under a fluorescence confocal microscope (NIKON A1R, USA) and were analyzed using NIS Elements software. The confocal microscopy settings were kept the same between samples. Doxorubicin excitation and

emission occurred at 485 nm and 595 nm whereas for DAPI, excitation and emission occurred at 405 nm and 450 nm respectively. Images were acquired in 60x optical zoom (Plan Apo VC 60x Oil DIC N2 DIC N2). Female BIBF 1120 purchase BALB/c mice were maintained in well-ventilated cages with free access to normal mouse food and water provided ad libitum. Temperature (25 ± 2°C) and humidity (50 ± 5%) was regulated and the illumination cycle was set to 12 h light/dark. Animal protocols were reviewed and approved by Institutional Animal Ethics Committee (IAEC) and Committee for the Purpose of Control and Supervision Parvulin of Experiments on Animals (CPCSEA), India and the experiments were performed as summarized in Figure 1. Briefly, animals were divided into four groups.

All groups had mice inoculated with either DLA or EAC on Day 1, except for group 4, where the cells were injected on Day 8. Group 1 was treated only once (day 2) with compounds. In group 2, compounds were administered on days 2 to 15. Group 3 had compounds administered on days 9 to 22. Group 4 received prophylactic treatment of compounds from day 1 to 7. Each of these groups had four treatment protocols – PBS (vehicle or control), PST001 (100 mg/kg), PST-Dox nanoparticles (2.25 mg/kg) and Dox (2.25 mg/kg) under subgroups (n = 12/sub group). Six animals from the group were used for survival analysis. Vehicle and the compounds were administered once daily by intraperitoneal (i.p.) injection. The mean survival time and percentage of increment in life span (% ILS) was calculated as previously reported [25] and [32]. EAC cells (1×106 cells) were injected subcutaneously with a fine needle (31G) to develop solid tumors in the hind limb of mice (n = 6/group).