bc500621h_si_002.avi (71.91 MB)
Fluorescent, Bioactive Protein Nanoparticles (Prodots) for Rapid, Improved Cellular Uptake
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posted on 2015-03-18, 00:00 authored by Inoka
K. Deshapriya, Bobbi S. Stromer, Ajith Pattammattel, Christina S. Kim, Ramiro Iglesias-Bartolome, Laura Gonzalez-Fajardo, Vyomesh Patel, J. Silvio Gutkind, Xiuling Lu, Challa V. KumarA 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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Keywords
hydrogen peroxideprotein nanoparticlesFITCcontrol studiesmembrane permeabilityCellular UptakeAgel electrophoresisProdotchemical triggersDLSenzyme activitiesplasma membraneTEMtransmission electron microscopyCalorimetric studiesBioactive Protein NanoparticlesnGOdenaturation temperaturescell uptakeserum albumin
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