title: Regulation of stem cell signaling by nanoparticle-mediated intracellular protein delivery.
abstract: 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-3β (GSK-3β) - 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 β-catenin levels due to GSK-3β inhibition. Accumulation of β-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.
authors.affiliation: Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Dhiral A
authors.initials: DA
authors.lastname: Shah
authors.forename: Seok-Joon
authors.initials: SJ
authors.lastname: Kwon
authors.forename: Shyam S
authors.initials: SS
authors.lastname: Bale
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Banerjee
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.forename: Ravi S
authors.initials: RS
authors.lastname: Kane
mesh_terms: D000818:Animals; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D058953:Neural Stem Cells / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015398:Signal Transduction; D012822:Silicon Dioxide / Q000737:chemistry; D014162:Transfection; D051176:beta Catenin / Q000378:metabolism
chemical_list: D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D051176:beta Catenin; D049452:Green Fluorescent Proteins; D012822:Silicon Dioxide; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3