Send Orders of Reprints at bspsaif@emirates.net.ae Current Pharmaceutical Biotechnology, 2012, 13, 2669-2681 2669 Nanoparticle Engineering Enhances Anticancer Efficacy of Andrographol- ide in MCF-7 Cells and Mice Bearing EAC Partha Roy a , Suvadra Das a , Anushree Mondal b , Urmi Chatterji b and Arup Mukherjee a,* a Department of Chemical Technology, University of Calcutta, Kolkata, India; b Department of Zoology, University of Calcutta, Kolkata, India Abstract: Success in cancer chemotherapy relies on efficient delivery of anti-neoplastic drugs, with minimal side-effects on non-cancerous cells. Nanoparticulation of prospective anti-cancer drugs, that were deemed unsuitable due to short bio- logical half life, poor water solubility and low cellular permeability, has been hypothesized to generate superior che- motherapeutic agents, leading to reduced non-specific action and fewer side-effects. In lieu of the above, different syn- thetic modulations on the putative anti-cancer compound andrographolide (AG) were explored to improve its therapeutic efficiency. Our results indicated that PLGA-nanoparticulation of andrographolide diterpenoid enhanced its anti-cancer properties three fold. Chitosan coating of AG nanoparticles further accentuated cellular localization, induced G 1 cell cycle arrest and increased cellular toxicity and apoptosis in MCF-7 cells. The charge modulated nanoparticles were seen to trav- erse more efficiently through the cytoplasm and accumulate in the nucleus, thus enhancing their anti-proliferative effi- cacy. In vivo studies confirm that the nanoparticles reduced tumor weight by 68.21% as compared to 24.7% by AG, and increased the life span of mice infected with Ehrlich ascites carcinoma (EAC) by 78.08% as compared to 23.5% for AG alone. This was achieved through development of slow release-type nanoparticle cargo delivery devices, and enhanced the efficiency of AGnps for targeting cancer cells. AG nanoparticles also showed sufficient promise as safe anti-cancer drugs since they had minimal impact on animal hematology. Hence, we successfully prepared non-toxic and delivery-efficient andrographolide nanoparticles, and established for the first time that PLGA-nanoparticulation of andrographolide and ad- ditional chitosan coating increased its anti-cancer efficacy in human breast cancer cells and mouse EAC model. Keywords: Andrographolide, apoptosis, chitosan, Ehrlich Ascites Carcinoma, G 1 cell cycle arrest, human breast cancer cells, nanoparticles. INTRODUCTION Till date, cancer chemotherapy remains a challenge [1], and development of anticancer agents with low toxicity and increased specificity of action is therefore a priority. Andro- grapholide (AG) is a labdane diterpenoid, extracted from the leaves of Andrographis paniculata. AG, popularly known as the ‘King of Bitters’, is a pleiotropic agent and interacts with a diverse range of biomolecular targets. AG is known to ex- ert immunomodulatory effects by inhibiting NF-kB [2], and interferes with NFAT activation and ERK1/ ERK5 phos- phorylation in T-cells. AG is a potent inducer of mitochon- drial-mediated apoptosis in a variety of cancer cell lines [3, 4], and alters reactive oxygen species (ROS) levels in HepG2 cell lines. Andrographis paniculata extracts have been used in Indian and Chinese traditional medicine as an anti- infective, hepatoprotectant and anti-inflammatory agent [5]. Anti-tumor activities of AG have also been established in cancer models [6, 7]. Like curcumin and resveratrol, AG modulates the Pl3K/Akt signaling pathway and induces G 1 cell cycle arrest through induction of p27 and suppression of cyclin-dependent kinase (CDK 4). However, like most plant terpenoids, AG has significant solubility and biopharmaceu- tical limitations, and a short biological half life of 2 hours *Address correspondence to this author at the Department of Chemical Technology, University of Calcutta, 92 A. P. C. Road, Kolkata-700 009, India; Tel: 91-33-23508387; Fax: 91-33-23519755; E-mail: arupm1234@gmail.com [8]. These limitations deter all therapeutic applications of AG for which different synthetic modifications of AG were explored to improve its therapeutic efficiency [6]. Nanopar- ticle engineering was aimed as an appropriate solution for specific anti-cancer applications of AG in vitro and in vivo. In the present study we report nanoparticulation of AG within a narrow Gaussian size range. US FDA approved bio- polymer PLGA [poly(DL-lactide-co-glycolic acid)] was pre- ferred because of its established biodegradability and stabili- zation in circulation [9]. It is known that surface charges of nanoparticles often modulate cellular internalization, particu- larly in cancer cells [10]. Consequently, polycationic deposi- tion on the PLGA-nanoparticles was carried out to produce cationic nanoparticles, in order to increase cellular localiza- tion. Finally, AG nanoparticle-induced cellular effects were studied in MCF-7 human breast cancer cells, and addition- ally confirmed in ME-180 human cervical cancer cell lines. The efficacy of AG and AG nanoparticles was also estab- lished for the first time in the mouse Ehrlich Ascites Carci- noma (EAC) model. MATERIALS AND METHODS Materials All solvents and water used were of HPLC grade (E. Merck or Spectrochem). PLGA (50:50, MW 40,000– 75,000), chitosan (MW Medium), dialysis tubing (MW cut 1 - 1 /12 $58.00+.00 © 2012 Bentham Science Publishers