title: Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.
abstract: A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.
authors: †Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
authors: Inoka K
authors: IK
authors: Deshapriya
authors: †Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
authors: Bobbi S
authors: BS
authors: Stromer
authors: †Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
authors: Ajith
authors: A
authors: Pattammattel
authors: †Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
authors: Christina S
authors: CS
authors: Kim
authors: ‡Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4340, United States.
authors: Ramiro
authors: R
authors: Iglesias-Bartolome
authors: §Department of Pharmaceutics, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092, United States.
authors: Laura
authors: L
authors: Gonzalez-Fajardo
authors: ‡Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4340, United States.
authors: Vyomesh
authors: V
authors: Patel
authors: ‡Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4340, United States.
authors: J Silvio
authors: JS
authors: Gutkind
authors: §Department of Pharmaceutics, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092, United States.
authors: Xiuling
authors: X
authors: Lu
authors: †Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.
authors: Challa V
authors: CV
authors: Kumar
mesh_terms: D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism; D010316:Particle Size; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate