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Cyclic RGDfK Peptide Functionalized Polymeric Nanocarriers for Targeting Gemcitabine to Ovarian Cancer Cells Hitesh Kulhari, Deep Pooja, Raju Kota, Srinivasa Reddy Telukutla, Rico F. Tabor, Ravi Shukla, David J. Adams, Ramakrishna Sistla, and Vipul Bansal Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00935 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 9, 2016
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Molecular Pharmaceutics
Cyclic RGDfK Peptide Functionalized Polymeric Nanocarriers for Targeting Gemcitabine to Ovarian Cancer Cells Hitesh Kulhari,¥,†,‡,#Deep Pooja,¥,#Raju Kota,# Srinivasa Reddy Telukutla,¥,†,‡,# Rico F. Tabor,§ Ravi Shukla,¥,† David J. Adams,† Ramakrishna Sistla,#,*Vipul Bansal,¥,†,* ¥
Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of
Applied Sciences, RMIT University, Melbourne, VIC, 3001, Australia †
Health Innovations Research Institute, RMIT University, Melbourne, VIC, 3083 Australia
‡
IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad,
Telangana, 500007, India #
Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad, Telangana, 500007, India §
School of Chemistry, Monash University, Clayton, VIC, 3800, Australia
*E-mail:
[email protected] (V.B.);
[email protected] (S.R.)
D.J. Adams current affiliation: Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522 Australia. Email:
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Molecular Pharmaceutics
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Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; MTT, 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; NHS, N-hydroxysuccinimide; PLGA, poly(lactic-co-glycolic acid); cRGDfK, cyclo(Arg-Gly-Asp-D-Phe-Lys);MMP, mitochondrial membrane potential; ROS, reactive oxygen species; PVA, polyvinyl alcohol; DCFDA, 2,7-dichlorofluorescin diacetate; DCF, 2’,7’-dichlorofluorescein; GEM, gemcitabine hydrochloride; GEM-PLGA, GEM-loaded PLGA nanoparticles; PEO, polyethylene oxide; PPO, polypropylene oxide; W/O, water-in-oil; W/O/W, water-in-oil-in-water; EE, entrapment efficiency; DL, drug loading; DLS, dynamic light scattering; PDI, polydispersity index; TEM, transmission electron microscopy; AFM, atomic force microscopy; FTIR, Fourier transform infrared; XRD, x-ray diffraction; SANS, small-angle neutron scattering; FDA, Food and Drug Administration; RBCs, red blood cells; RhB, rhodamine-B.
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Molecular Pharmaceutics
Abstract Current cancer chemotherapies commonly suffer from non-specificity, drug resistance, poor bioavailability and narrow therapeutic indices. To achieve the optimum drug efficacy, we designed a polymeric drug delivery system for targeted intracellular delivery of a clinicallyapproved, water-soluble anticancer drug, gemcitabine hydrochloride (GEM). We utilised the unique ability of a cyclic pentapeptide cRGDfK to specifically target α v β 3 integrin receptors that are over-expressed on SKOV-3 human ovarian cancer cells. This significantly increased the effective intracellular drug concentration even at low doses, thereby remarkably improving the chemotherapeutic potential of GEM. cRGDfK-conjugated, GEM-loaded nanoparticles reduced the nonspecific hemolytic cytotoxicity of the drug, simultaneously influencing intracellular processes such as mitochondrial membrane potential (DΨm), reactive oxygen species (ROS) levels and apoptosis, thereby favourably influencing drug anti-proliferative efficacy.
Keywords: Targeted drug delivery, gemcitabine, integrin, cRGDfK, ovarian cancer
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Molecular Pharmaceutics
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INTRODUCTION
Design and development of new drug delivery systems to achieve controllable targeted delivery of clinically approved anticancer drugs is an area of active investigation.1,2 It is well recognised that most of the clinically approved anticancer drugs present with major issues around lack of biological target specificity.3,4 This leads to uncontrolled bio-distribution of anticancer drugs to non-specific tissues and severe unwanted side effects. To ensure safety and efficacy, anticancer drugs need to be delivered to their target site selectively and at an optimal rate. In contrast to conventional drug delivery systems, a carefully designed targeted drug delivery system has the potential to deliver a therapeutic agent to the tissue of interest at higher dosages, while reducing the relative drug concentration in non-target tissues. Such delivery systems may overcome problems of drug non-specificity and improve therapeutic indices.5-8 Gemcitabine hydrochloride (GEM) is a clinically-approved (United States Food and Drug Administration: US-FDA) chemotherapeutic agent that is used to treat various cancers including breast, non-small cell lung, pancreatic and platinum-resistant ovarian cancer.9-10 Being a pseudonucleotide, its efficacy relies upon incorporation into cellular DNA. This blocks DNA chain elongation, leading to cell arrest in the G1 phase with cytostatic effects.11,12 Although GEM offers an efficient chemotherapy option, its short plasma half-life (
