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Off-target effects of drugs severely limit cancer therapy. Targeted nanocarriers are promising to enhance the delivery of therapeutics to tumors. Among many approaches for active tumor-targeting, arginine-rich cell penetrating peptides (AR-CPP) and ligands specific to target over-expressed receptors on cancer-cell surfaces, are popular. Earlier, we showed that the attachment of an AR-CPP octaarginine (R8) to the surface of DOXIL® (Doxorubicin encapsulated PEGylated liposomes) improved cytoplasmic and nuclear DOX delivery that enhanced the cytotoxic effect in vitro and improved therapeutic efficacy in vivo. Here, we report on DOX-loaded liposomes, surface-modified with, R8 and transferrin (Tf) (Dual DOX-L), to improve targeting of A2780 ovarian carcinoma cells via the over-expressed transferrin receptors (TfRs) with R8-mediated intracellular DOX delivery. Flow cytometry analysis with fluorescently labeled DualL (without DOX) showed two-fold higher cancer-cell association than other treatments after 4 h treatment. Blocking entry pathways of R8 (macropinocytosis) and Tf (receptor-mediated endocytosis, RME) resulted in a decreased cancer-cell association of DualL. Confocal microscopy confirmed involvement of both entry pathways and cytoplasmic liposome accumulation with nuclear DOX delivery for Dual DOX-L. Dual DOX-L exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an A2780 ovarian xenograft model compared to other treatments. A pilot biodistribution study showed improved DOX accumulation in tumors after Dual DOX-L treatment. All results collectively presented a clear advantage of the R8 and Tf combination to elevate the therapeutic potential of DOX-L by exploiting TfR over-expression imparting specificity followed by endosomal escape and intracellular delivery via R8. [ABSTRACT FROM AUTHOR]

Purpose: To develop a multifunctional nanoparticle system carrying a combination of pro-apoptotic drug, NCL-240, TRAIL [tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand] and anti-survivin siRNA and to test the combination preparation for anti-cancer effects in different cancer cells. Methods: Polyethylene glycol-phosphoethanolamine (PEG-PE) - based polymeric micelles were prepared carrying NCL-240. These micelles were used in combination with TRAIL-conjugated micelles and anti-survivin siRNA-S-S-PE containing micelles. All the micelles were characterized for size, zeta potential, and drug encapsulation efficiency. Different cancer cells were used to study the cytotoxicity potential of the individual as well as the combination formulations. Other cell based assays included cellular association studies of transferrin-targeted NCL-240 micelles and study of cellular survivin protein downregulation by anti-survivin siRNA-S-S-PE containing micelles. Results: NCL-240 micelles and the combination NCL-240/TRAIL micelles significantly increased cytotoxicity in the resistant strains of SKOV-3, MCF-7 and A549 as compared to free drugs or single drug formulations. The NCL-240/TRAIL micelles were also more effective in NCI/ADR-RES cancer cell spheroids. Anti-survivin siRNA micelles alone displayed a dose-dependent reduction in survivin protein levels in A2780 cells. Treatment with NCL-240/TRAIL after pre-incubation with anti-survivin siRNA inhibited cancer cell proliferation. Additionally, a single multifunctional system composed of NCL-240/TRAIL/siRNA PM also had significant cytotoxic effects in vitro in multiple cell lines. Conclusion: These results demonstrate the efficacy of a combination of small-molecule PI3K inhibitors, TRAIL, and siRNA delivered by micellar preparations in multiple cancer cell lines. [ABSTRACT FROM AUTHOR]

We prepared and evaluated transferrin (Tf) and monoclonal antibody (mAb) 2C5-modified dual ligand-targeted poly(ethylene glycol)-phosphatidylethanolamine micelles loaded with a poorly soluble drug, R547 (a selective adenosine triphosphate-competitive cyclin-dependent kinase inhibitor) for enhancement of targeting efficiency and cytotoxicity in vitro and in vivo to A2780 ovarian carcinoma compared to single ligand-targeted micelles. Micellar solubilization significantly improved the solubility of R547 from 1 to 800 μg/mL. The size of modified and non-modified micelles was 13-16 nm. Flow cytometry indicated significantly enhanced cellular association of dual ligand-targeted micelles compared to single ligand-targeted micelles. Confocal microscopy confirmed the Tf receptor-mediated endocytosis of rhodamine-labeled Tf-modified micelles after staining the micelle-treated cells with the endosomal marker Tf-Alexa488. The optimized dual-targeted micelles enhanced cytotoxicity in vitro against A2780 ovarian cancer cells compared to plain and single ligand-targeted micelles. Interestingly, in vivo anti-tumor efficacy was more pronounced for the preparation with a single-targeting ligand (Tf). The specific combination Tf and mAb 2C5 did not yield the expected increase in efficacy as was observed in vitro. This observation suggests that the relationships between targeting ligands in vivo could be more complex than in simplified in vitro systems, and the results of the optimization process should always be verified in vivo. [ABSTRACT FROM AUTHOR]