A GFP complementation system for monitoring and directing nanomaterial mediated protein delivery to human cellular organelles.

Protein-based therapeutics are gaining importance for their biocompatibility and activity toward specific targets. When these targets are intracellular, it is critical to deliver biomolecules to sites in the cell cytoplasm while retaining biomolecule activity in the complex cellular milieu. However, intracellular protein delivery is not viable unless accompanied by an active uptake mechanism or carrier mediated delivery. Moreover, once entry into the cell is achieved, detection of the biomolecule requires laborious techniques that lack real-time measurement. We have developed a fluorescence-based complementary protein delivery sensing system using split green fluorescence protein (GFP(1-10) and GFP(11)) fragments, which can be used as an indicator for protein delivery and retention of activity, and as a means to pinpoint subcellular localization. We demonstrate in vitro localized delivery by expressing the GFP(11) fragment onto the mitochondrial outer membrane of human cells, and using a model carrier (15 nm silica nanoparticles) to deliver GFP(1-10) and image trafficking and mitochondrial localization of protein delivery. Our results indicate that nanoscale materials can be used as protein carriers for targeting cell constituents including functional molecules, signaling pathways, and organelles. We envision that this GFP complementation system is ideally suited for directing nanoparticle-based delivery of drugs and other bioactive molecules into subcellular locations within cells, which can impact protein-protein interactions, signal transduction pathways, and organelle function in vitro within the context of high-throughput screening protocols.

High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening.

Differential expression of various drug-metabolizing enzymes (DMEs) in the human liver may cause deviations of pharmacokinetic profiles, resulting in interindividual variability of drug toxicity and/or efficacy. Here, we present the 'Transfected Enzyme and Metabolism Chip' (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1 and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Regulation of stem cell signaling by nanoparticle-mediated intracellular protein delivery.

Intracellular delivery of specific proteins and peptides may be used to influence signaling pathways and manipulate cell function, including stem cell fate. Herein, we describe the delivery of proteins attached to hydrophobically modified 15-nm silica nanoparticles to manipulate specifically targeted cell signaling proteins. We designed a chimeric protein, GFP-FRATtide, wherein GFP acts as a biomarker for fluorescence detection, and FRATtide binds to and blocks the active site of glycogen synthase kinase-3ß (GSK-3ß) - a protein kinase involved in Wnt signaling. The SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, resulting in an elevation of ß-catenin levels due to GSK-3ß inhibition. Accumulation of ß-catenin led to increased transcription of Wnt target genes, such as c-MYC, which instruct the cell to actively proliferate and remain in an undifferentiated state. The results presented here suggest that functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. This ability is critical for the design of in vitro screens for gain/loss of pathway function, and may also prove to be useful for in vivo delivery applications.

Nanoparticle-mediated cytoplasmic delivery of proteins to target cellular machinery.

Despite recent advances in nanomaterial-based delivery systems, their applicability as carriers of cargo, especially proteins for targeting cellular components and manipulating cell function, is not well-understood. Herein, we demonstrate the ability of hydrophobic silica nanoparticles to deliver proteins, including enzymes and antibodies, to a diverse set of mammalian cells, including human cancer cells and rat stem cells, while preserving the activity of the biomolecule post-delivery. Specifically, we have explored the delivery and cytosolic activity of hydrophobically functionalized silica nanoparticle-protein conjugates in a human breast cancer cell line (MCF-7) and rat neural stem cells (NSCs) and elucidated the mechanism of cytosolic transport. Importantly, the proteins were delivered to the cytosol without extended entrapment in the endosomes, which facilitated the retention of biological activity of the delivered proteins. As a result, delivery of ribonuclease A (RNase A) and the antibody to phospho-Akt (pAkt) resulted in the initiation of cell death. Delivery of control protein conjugates (e.g., those containing green fluorescent protein or goat antirabbit IgG) resulted in minimal cell death, indicating that the carrier-mediated toxicity was low. The results presented here provide insight into the design of nanomaterials as protein carriers that enable control of cell function.

Structure, function, and stability of enzymes covalently attached to single-walled carbon nanotubes.

We describe the structure, activity, and stability of enzymes covalently attached to single-walled carbon nanotubes (SWNTs). Conjugates of SWNTs with three functionally unrelated enzymes-horseradish peroxidase, subtilisin Carlsberg, and chicken egg white lysozyme-were found to be soluble in aqueous solutions. Furthermore, characterization of the secondary and tertiary structure of the immobilized proteins by circular dichroism and fluorescence spectroscopies, respectively, and determination of enzyme kinetics revealed that the enzymes retained a high fraction of their native structure and activity upon attachment to SWNTs. The SWNT-enzyme conjugates were also more stable in guanidine hydrochloride (GdnHCl) and at elevated temperatures relative to their solution counterparts. Thus, these protein conjugates represent novel preparations that possess the attributes of both soluble enzymes-high activity and low diffusional resistance-and immobilized enzymes-high stability-making them attractive choices for applications ranging from diagnostics and sensing to drug delivery.