Synthesis, characterization and mechanistic-insight into the anti- proliferative potential of PLGA-gemcitabine conjugate $ Vaibhav Khare a , Smit Kour b, c , Noor Alam a , Ravindra Dharr Dubey a , Ankit Saneja a, b , Mytre Koul b, c , Ajai Prakash Gupta d , Deepika Singh b, e , Shashank K. Singh b, c, **, Ajit K. Saxena b, c , Prem N. Gupta a, b, * a Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India b Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India d Quality Control & Quality Assurance Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India e Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India A R T I C L E I N F O Article history: Received 18 March 2014 Received in revised form 2 May 2014 Accepted 3 May 2014 Available online 6 May 2014 PubChem classifications: Gemcitabine hydrochloride (PubChem CID: 60749) poly(DL-lactic-co-glycolic acid) (PubChem CID: 23111554) Dicyclohexylcarbodiimide (PubChem CID: 10868) N-hydroxysuccinimide (PubChem CID: 80170) Dipyridamole (PubChem CID: 3108) Triethylamine (PubChem CID: 8471) Keywords: Gemcitabine PLGA Apoptosis Cytotoxicity Polymer drug conjugate A B S T R A C T Gemcitabine, a nucleoside analogue, is used in the treatment of various solid tumors, however, its efficacy is limited by rapid metabolism by cytidine deaminase and fast kidney excretion. In this study, a polymeric conjugate of gemcitabine was prepared by covalent coupling with poly(lactic-co-glycolic) acid (PLGA), in order to improve anticancer efficacy of the drug. The prepared conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, in vitro cytotoxicity determined in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), indicated that the cytotoxic activity of gemcitabine was retained following conjugation with polymeric carrier. In the nucleoside transportation inhibition assay, it was found that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporter- deficient resistance cancer. Further, the cell cycle analysis showed that the sub-G1 (G 0 ) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis. In addition, there was a marked increase in PARP cleavage and P-H2AX expression with PLGA gemcitabine conjugate as compared to native gemcitabine indicating improved apoptotic activity. The findings demonstrated the potential of PLGA gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer. ã 2014 Elsevier B.V. All rights reserved. 1. Introduction Gemcitabine (2 0 ,2 0 -difluoro-2 0 -deoxycytidine) is a nucleoside analogue approved by FDA for treatment of various solid tumors including pancreatic (Carmichael et al.,1996), breast (Yardley, 2005), ovarian (Ozols,2005) and non-small lung cancers (Hoanget al., 2003). Unfortunately, rapid enzymatic metabolism and fast kidney excretion of gemcitabine compromised its efficient use. Gemcitabine is metabolized to its inactive uracil derivative (2 0 ,2 0 -difluorodeoxyur- idine) by an enzyme, cytidine deaminase (CDA), present in plasma, kidney and liver, and over-expression of CDA is also associated with gemcitabine resistance (Heinemann et al., 1992; Neff and Blau,1996). The uptake of gemcitabine occurs mainly through nucleoside transporters (hENT1, hCNT1) (Mackey et al., 1998b). After entry into the cells, gemcitabine is phosphorylated to its monophosphate by deoxycytidine kinase and to its active triphosphate by pyrimidine nucleotide kinases (Abbruzzese et al., 1991; Plunkett et al., 1995). Due to short half-life (8–17 min) of gemcitabine, its frequent $ IIIM communication number: IIIM/1621/2013. * Corresponding author: Formulation & Drug Delivery Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. ** Corresponding author: Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India. E-mail addresses: sksingh@iiim.ac.in (S.K. Singh), pngupta@iiim.ac.in, pngupta10@gmail.com (P.N. Gupta). http://dx.doi.org/10.1016/j.ijpharm.2014.05.005 0378-5173/ ã 2014 Elsevier B.V. All rights reserved. International Journal of Pharmaceutics 470 (2014) 51–62 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsev ier.com/locate /ijpharm