Please cite this article in press as: Das, S., et al., Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking, DNA targeting and improved therapeutic effects in skin melanoma in vitro. Toxicol. Lett. (2013), http://dx.doi.org/10.1016/j.toxlet.2013.09.012 ARTICLE IN PRESS G Model TOXLET 8482 1–15 Toxicology Letters xxx (2013) xxx–xxx Contents lists available at ScienceDirect Toxicology Letters jou rn al hom epage: www.elsevier.com/locate/toxlet Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking, DNA targeting and improved therapeutic effects in skin melanoma in vitro Sreemanti Das a,b , Jayeeta Das a,b , Asmita Samadder a,b , Avijit Paul a,b , Q1 Anisur Rahman Khuda-Bukhsh a,b,∗ a Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India Q2 b Boiron Laboratories, Lyon, France h i g h l i g h t s • PLGA-encapsulated-nano-apigenin shows better apoptotic effects than free-apigenin. • Nano-apigenin directly targets nuclear DNA and increases ROS accumulation. • Nano-apigenin causes mitochondrial apoptosis in A375 skin melanoma. • Nano-apigenin appears to be promis- ing for further use as anticancer- drugs. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 30 July 2013 Received in revised form 13 September 2013 Accepted 16 September 2013 Available online xxx Keywords: Skin melanoma PLGA-nano-encapsulation Apigenin Drug–DNA interaction Mitochondrial dysfunction Apoptosis a b s t r a c t The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal micro- scopies. Circular dichroic spectral analysis and stability curve for Gibb’s free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activ- ity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photosta- bility studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formu- lations to have better ameliorative potentials in combating skin melanoma. © 2013 Published by Elsevier Ireland Ltd. ∗ Corresponding author at: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India. Tel.: +91 33 25828768/33 25828768. E-mail addresses: prof arkb@yahoo.co.in, khudabukhsh 48@rediffmail.com, khudabukhsh 46@rediffmail.com (A.R. Khuda-Bukhsh). 0378-4274/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.toxlet.2013.09.012 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37