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Cancer cell-selective, nuclear targeting is expected to enhance the therapeutic efficacy of a myriad of antineoplastic drugs, particularly those whose pharmacodynamic site of action is the nucleus. In this study, a steroid-macromolecular bioconjugate based on PEG-linked 17β-Estradiol (E2) was appended to intrinsically cell-penetrable multiwalled carbon nanotubes (MWCNTs) for intranuclear drug delivery and effective breast cancer treatment, both in vitro and in vivo. Taking Doxorubicin (DOX) as a model anticancer agent, we tried to elucidate how E2 appendage influences the cell internalization, intracellular trafficking, and antitumor efficacy of the supramolecularly complexed drug. We observed that the combination of DOX with E2-PEG-MWCNTs not only facilitated nuclear targeting through an estrogen receptor (ER)-mediated pathway but also deciphered to a synergistic anticancer response in vivo. The antitumor efficacy of DOX@E2-PEG-MWCNTs in chemically breast cancer-induced female rats was approximately 18, 17, 5, and 2 times higher compared to the groups exposed to saline, drug-deprived E2-PEG-MWCNTs, free DOX, and DOX@m-PEG-MWCNTs, respectively. While free DOX treatment induced severe cardiotoxicity in animals, animals treated with DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs were devoid of any perceivable cardiotoxicity, hepatotoxicity, and nephrotoxicity. To the best of our knowledge, this is the first instance in which cancer cell-selective, intranuclear drug delivery, and, subsequently, effective in vivo breast cancer therapy has been achieved using estrogen-appended MWCNTs as the molecular transporter.

The present study reports the design, synthesis, and biological evaluation of a novel, intravenously injectable, theranostic prodrug based on multiwalled carbon nanotubes (MWCNTs) concomitantly decorated with a fluorochrome (Alexa-fluor, AF488/647), radionucleide (Technitium-99m), tumor-targeting module (folic acid, FA), and anticancer agent (methotrexate, MTX). Specifically, MTX was conjugated to MWCNTs via a serum-stable yet intracellularly hydrolyzable ester linkage to ensure minimum drug loss in circulation. Cell uptake studies corroborated the selective internalization of AF-FA-MTX-MWCNTs (1) by folate receptor (FR) positive human lung (A549) and breast (MCF 7) cancer cells through FR mediated endocytosis. Lysosomal trafficking of 1 enabled the conjugate to exert higher anticancer activity as compared to its nontargeted counterpart that was mainly restricted to cytoplasm. Tumor-specific accumulation of 1 in Ehlrich Ascites Tumor (EAT) xenografted mice was almost 19 and 8.6 times higher than free MTX and FA-deprived MWCNTs. Subsequently, the conjugate 1 was shown to arrest tumor growth more effectively in chemically breast tumor induced rats, when compared to either free MTX or nontargeted controls. Interestingly, the anticancer activities of the ester-linked CNT-MTX conjugates (including the one deprived of FA) were significantly higher than their amide-linked counterpart, suggesting that cleavability of linkers between drug and multifunctional nanotubes critically influence their therapeutic performance. The results were also supported by in silico docking and ligand similarity analysis. Toxicity studies in mice confirmed that all CNT-MTX conjugates were devoid of any perceivable hepatotoxicity, cardiotoxicity, and nephrotoxicity. Overall, the delivery property of MWCNTs, high tumor binding avidity of FA, optical detectability of AF fluorochromes, and radio-traceability of (99m)Tc could be successfully integrated and partitioned on a single CNT-platform to augment the therapeutic efficacy of MTX against FR overexpressing cancer cells while allowing a real-time monitoring of treatment response through multimodal imaging.

The present study reports the optimized synthesis, physicochemical characterization, and biological evaluation of a novel, multiwalled carbon nanotube-hyaluronic acid (MWCNT-HA) conjugate, complexed with an anticancer agent, Doxorubicin (DOX) via π-π stacking interaction. The therapeutic conjugate was concomitantly labeled with a near-infrared fluorescent dye, Alexa-Flour-647 (AF-647), and radiotracer Technetium-99m ((99m)Tc) to track its whereabouts both in vitro and in vivo via optical and scintigraphic imaging techniques. Covalent functionalization of MWCNTs with HA facilitated their internalization into human lung adenocarcinoma, A549 cells via hyaluronan receptors (HR) mediated endocytosis. Internalized nanotubes showed lysosomal trafficking, followed by low pH-triggered DOX release under endolysosomal conditions. Consequently, DOX-loaded HA-MWCNTs exhibited 3.2 times higher cytotoxicity and increased apoptotic activity than free DOX in equivalent concentrations. Organ distribution studies in Ehlrich ascites tumor (EAT) bearing mice model indicated that tumor specific localization of (99m)Tc-MWCNT-HA-DOX is significantly higher than both free drug and nontargeted MWCNTs. Pharmacodynamic studies in chemically breast-cancer-induced rats showed that the tumor-growth inhibitory effect of HA-MWCNT-DOX was 5 times higher than free DOX in equivalent concentration. DOX delivered through HA-MWCNTs was devoid of any detectable cardiotoxity, hepatotoxicity, or nephrotoxicity. All these promising attributes make HA-MWCNTs a "smart" platform for tumor-targeted delivery of anticancer agents.

The present study investigates the effect of functionalization density on the toxicity and cellular uptake of oxidized multiwalled carbon nanotubes (f-MWCNTs) in vitro. The toxicity of f-MWCNTs at varying degrees of carboxylation was assessed in a murine macrophage RAW 264.7 cell line, a model for liver Kupffer cells. In vitro cytotoxicity of oxidized MWCNTs was directly proportional to their functionalization density. The increased cytotoxicity was associated with a concurrent increase in the number of apoptotic cells and production of reactive nitrogen species (RNS). In contrast, reactive oxygen species (ROS) generation was the highest in the case of pristine MWCNTs and decreased with increased functionalization density. Quantitative cellular uptake studies indicated that endogenous ROS production was independent of the concentration of CNTs internalized by a specific cell population and was directly proportional to their surface hydrophobicity. Mechanistic studies suggested that cellular uptake of CNTs was critically charge-dependent and mediated through scavenger receptors, albeit the involvement of nonscavenger receptor mechanisms at low CNT concentrations and their saturation at the experimental concentration cannot be ruled out. A mathematical model was established to correlate between the cellular uptake of CNTs with their length and zeta potential. In an attempt to correlate the results of in vitro toxicity experiments with those of the in vivo toxicity in the mouse model, we found that the toxicity trends in vitro and in vivo are rather opposing. The apparent anomaly was explained on the basis of different experimental conditions and doses associated with cells under in vivo and in vitro culture conditions.

The present investigation reports an extensive evaluation of in vitro and in vivo anticancer efficacy of orally administered doxorubicin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Dox-NPs) in a breast cancer induced animal model. Spherically shaped Dox-NPs were prepared with an entrapment efficiency and particle size of 55.40 ± 2.30% and 160.20 ± 0.99 nm, respectively, and freeze-dried with 5% trehalose using stepwise freeze-drying. Cytotoxicity, as investigated on C127I cell line, revealed insignificant differences between the IC(50) of free Dox and Dox-NPs treated cells in the first 24 h, while higher cytotoxicity was demonstrated by Dox-NPs, following 72 h of incubation. Confocal laser scanning microscopy (CLSM) imaging corroborated that nanoparticles were efficiently localized into the nuclear region of C127I cells. The cellular uptake profile of Dox-NPs revealed both time and concentration dependent increases in the Caco-2 cell uptake as compared to the free Dox solution. Further, Dox-NPs significantly suppressed the growth of breast tumor in female Sprague-Dawley (SD) rats upon oral administration. Finally, orally administered Dox-NPs showed a marked reduction in cardiotoxicity when compared with intravenously injected free Dox as also evident by the increased level of malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine phosphokinase (CK-MB) and reduced levels of glutathione (GSH) and superoxide dismutase (SOD). The reduced cardiotoxicity of orally administered Dox-NPs was also confirmed by the major histopathological changes in the heart tissue after the treatments of intravenously injected free Dox and orally delivered Dox-NPs.