br Pune respectively Both cells were cultured
(Pune) respectively. Both cells were cultured in Dulbecco's Modified Ea-gles Medium (DMEM) with 10% fetal calf serum (FCS) supplemented with RSL3 (Sigma), penicillin (120 units/ml), streptomycin (75 mg/ml), gentamycin (160 mg/ml) and amphotericin B (3 mg/ml) at 37 °C humidified with 5% of CO2.
2.6.1. Treatment with nano-formulation and photodynamic cytotoxicity assay
Cells were seeded in sterile 96-well tissue culture plate at a den-sity of 0.1 × 106 cells per well. The cells were treated with different compounds (with and without irradiated), such as CSNPs, Ce6, DOX, Ce6-DOX, CSNPs-DOX, Ce6-CSNPs, and Ce6-CSNPs-DOX at a concentration of (5, 10, or 20 μM) for 48 h. For photodynamic stud-ies, cells were illuminated with a LED lamp (660–690 nm, 100 mW/cm2) for 20 min. The cytotoxicity assay was performed by using Alamar Blue reduction bioassay (HIMEDIA, Mumbai, India). An untreated group was used as a control. After treatment cells were washed with PBS and incubated with Alamar blue solution (HIMEDIA, Mumbai, India) for 4 h at 37 °C in dark. After incubation the absorbance was measured at 570 and 600 nm by using a micro-plate reader. All studies were repeated three times and the result was expressed as percent over control.
2.7. Statistical analysis
All the experiments were repeated thrice and expressed as the mean ± standard deviation (SD).
Scheme 1. Schematic representation of preparation of DOX encapsulated and Ce6 decorated chitosan nanoparticles. The interaction of positively charged CS and negatively charged TPP along DOX was based on electrostatic interaction between protonated amino and ionized phosphates groups. The anionic Ce6 interaction into the surface of nanoparticles was based on electrostatic interaction between protonated amino and ionized carboxyl groups yielding negatively charged nanoparticles.
B) CSNPs Ce6-CSNPs
Fig. 1. A) The scanning electron microscopic (SEM) and B) atomic force microscopic (AFM) images of CSNPs and Ce6-CSNPs. SEM image of Ce6-CSNPs have spherical particles with homogenous distribution and smooth surfaces when compared to native CSNPs. AFM image of Ce6-CSNPs exhibited smooth spherical shape particles compared to native CSNPs. Both the images are indicating the size, well-shape, surface roughness and morphologies.
3.1. Synthesis and chemistry of Ce6-CSNPs
The Ce6-CSNPs is an ionic self-assembled supramolecular structure which obtained by self-assembly of chitosan with TPP and surface dec-oration of Ce6 in an appropriate solvent by ionic gelation method. This prepared nanoparticles where only traces of uncomplexed CS and no detectable amount of Ce6 were found in the dispersant, thus not requir-ing further purification. The decoration of the anionic Ce6 into the
nanoparticles composed of chitosan and TPP which based on hydropho-bic interaction yielding negatively charged nanoparticles. Subsequently, Ce6 decorated DOX encapsulated nanoparticles obtained by self-assembly of chitosan with TPP along DOX and surface decoration of Ce6 based on hydrophobic interaction yielding negatively charged nanoparticles. The Ce6 is a promising photosensitizer characterized by a high sensitizing efficacy and rapid elimination from the body [37,39]. The photo-toxicity, hemodynamic activity, cellular uptake and anticancer activities of Ce6 were reported by various groups [40–42]. The schematic of Ce6 decorated DOX encapsulated CSNPs is shown in
The sizes and zeta potential of CSNPs, Ce6-CSNPs, CSNPs-DOX and Ce6-CSNPs-DOX were obtained by DLS at pH 7.2, 25 °C aqueous medium.
a Size distribution was determined by DLS measurement in aqueous medium at pH 7.2.
b Polydispersity index was measured monodisperse at pH 7.2, 25 °C in aqueous medium.