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Cancer diagnosis and its stage-wise assessment are determined through invasive solid tissue biopsies. Conversely, cancer imaging is enriched through emission tomography and longitudinal high-resolution analysis for the early detection of cancer through altered cell morphology and cell-deformation. Similarly, in post multiple chemo-cycle exposures, the tumor regression and progression thereafter are not well understood. Here, we report chemo-cycles of doxorubicin (Dox) carrying nanoparticles (NPs) to be highly indicative of cell deformation and a progressive indicator of phenotypic expressions of acquired drug resistance (ADR). We designed graphene (G) based nanocarriers by chemically conjugating multiple components: (i) G; (ii) iron oxide (Fe3O4) NPs; and (iii) Dox through a cysteine (Cys) linker (G-Dox and G-Cys-Fe3O4-Dox). Although Dox underwent cell diffusion, the G-based nanocarriers followed a receptor-mediated endocytosis which created a profound impact on the cell membrane integrity. ADR owing to Dox and G-based nanocarriers was analyzed through a cytotoxicity assay, cell morphology deformation parameters and cellular uptake kinetic patterns. Interestingly, after the third chemo-cycle, G-Dox incubated cells showed the greatest decrease in the alteration of the nuclear surface area (NSA) of ∼28%, a ∼40% reduction of the cell surface area (CSA) and a ∼32% increase in the cell roundness (CRd). Our results suggested that the G-based nanocarriers induced the cell deformation process, subsequently resulting in ADR. Although the G-based nanocarriers initiated ADR, G-Dox was most cytotoxic to cancer cells and induced the maximum cell morphology deformation within our scope of study. This outcome implies caution is needed when using G-based nanocarriers and other multi-component nanosystems for Dox delivery as they lead to possible phenotypic expressions of drug resistance in cancer cells.

Efficacy of anticancer drug is limited by the severe adverse effects induced by drug; therefore the crux is in designing delivery systems targeted only to cancer cells. Toward this objectives, we propose, synthesis of poly(ethylene glycol) (PEG)-doxorubicin (DOX) prodrug conjugates consisting N-acetyl glucosamine (NAG) as a targeting moiety. Multicomponent system proposed here is characterized by (1)H NMR, UV spectroscopy, and HPLC. The multicomponent system is evaluated for in vitro cellular kinetics and anticancer activity using MCF-7 and MDA-MB-231 cells. Molecular modeling study demonstrated sterically stabilized conformations of polymeric conjugates. Interestingly, PEG-DOX conjugate with NAG ligand showed significantly higher cytotoxicity compared to drug conjugate with DOX. In addition, the polymer drug conjugate with NAG and DOX showed enhanced internalization and retention effect in cancer cells, compared to free DOX. Thus, with enhanced internalization and targeting ability of PEG conjugate of NAG-DOX has implication in targeted anticancer therapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: To evaluate a novel targeted anticancer prodrug consisting of several copies of sialic acid (SA, targeting moiety), doxorubicin (DOX), citric acid (multifunctional spacer) and poly(ethylene glycol) (PEG, carrier)., Methods: alpha, omega bis carboxyl PEG was covalently conjugated with multiple copies of SA and DOX through a citric acid spacer and characterized by proton nuclear magnetic resonance ((1)HNMR), matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF), and high-performance liquid chromatography (HPLC). The molecular models of conjugates were established using ChemDraw software. Stability, spontaneous and esterase-stimulated drug release was analyzed by HPLC. Cellular internalization (fluorescence microscopy) and cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay] of free DOX and prodrugs were evaluated., Results: (1)HNMR, MALDI/TOF, and HPLC showed the formation of the PEG prodrug conjugates. More than 40% of the drug was released from its conjugate in the presence of esterase enzyme, whereas the conjugate was stable at pH 7.4 in the absence of enzyme. Molecular modeling studies showed stable conformations of conjugates. The targeted prodrug conjugates with two copies of SA and DOX showed enhanced cytotoxicity when compared with non-targeted prodrugs and free DOX., Conclusions: Targeting of the conjugate to cancer cells by SA with increased copies of targeting moiety and anticancer drug enhanced prodrug uptake by cancer cells and cytotoxicity of the prodrug.