Article pubs.acs.org/bc
Fluorescent, Bioactive Protein Nanoparticles (Prodots) for Rapid, Improved Cellular Uptake Inoka K. Deshapriya,† Bobbi S. Stromer,† Ajith Pattammattel,† Christina S. Kim,† Ramiro Iglesias-Bartolome,‡ Laura Gonzalez-Fajardo,§ Vyomesh Patel,‡ J. Silvio Gutkind,‡ Xiuling Lu,§ and Challa V. Kumar*,† †
Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States ‡ Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4340, United States § Department of Pharmaceutics, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092, United States S Supporting Information *
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.
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INTRODUCTION
Most prominent examples of nanoparticles used in cell imaging include but are not limited to semiconducting fluorescent nanocrystals or quantum dots (QD),14 plasmonic nanoparticles such as gold and silver,15 inorganic nanoparticles such as silicon,16 and magnetic nanoparticles.17 These nanosized probes are designed to produce bright fluorescence detectable by optical methods. However, these probes require biocompatible surface coatings and appropriate modifications to reduce their toxicity and improve their fluorescence/ solubility in aqueous media. Hence, nanoparticles synthesized
Synthesis of protein nanoparticles (Prodots) that are highly fluorescent, stable, and versatile, and their rapid cellular uptake by cells is reported here. Currently, nanoparticles are extensively being used in a wide range of applications including biocatalysis,1−3 drug delivery,4−6 biosensing,7−9 and bioimaging.10−12 Nanoparticles are being tested for a variety of biological applications essentially due to their attractive properties and their amenability for surface modification.13 However, many of them are toxic and their toxicity has not been fully evaluated. Protein-based nanoparticles that are stable, biocompatible, benign, and also strongly fluorescent for cellular imaging are highly desirable as alternatives. © XXXX American Chemical Society
Received: May 17, 2014 Revised: January 18, 2015
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DOI: 10.1021/bc500621h Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Imaging with fluorescent Prodots reported here is advantageous in some ways, because specific chromophores of desired optical characteristics can be embedded or incorporated within the particles and the choice of chromophore is not restricted by limited access to naturally occurring fluorescent proteins. A general approach is described here for the synthesis of fluorescein-labeled nanoparticles directly from several proteins, as a proof-of-concept. Specifically, fluorescently labeled nanoparticles of bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) are described here. Data indicated small size (
