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  • FluoDots
    May 22, 2019 | ACS MATERIAL LLC

    FluoDots are single protein nanoparticles labeled with particular fluorescent dyes for imaging and other applications. They are composed of a central protein core surrounded by a covalently attached lipid layer. FluoDots retain the structure and functions of the protein embedded in the core, however the particles have increased thermal stability and a longer shelf life. Numerous carboxylic groups present on the surface of FluoDots are available for further chemical modification of the particles. FluoDot size is tunable at 2.5 Å resolution and the color is tuned independently of the size, which is a major advantage over quantum dots. FluoDots are water-soluble, non-toxic, biocompatible, biodegradable and can be potentially used in LED coatings as biophosphors, in cell imaging or solar cell applications, cancer drug delivery and other biological applications.

    Introduction

    Protein nanoparticles have been widely used for various applications, including drug delivery1, cell imaging2, or biological sensors.3,4 Single protein nanoparticles are a type of nanoparticle that only have one protein molecule in each particle, wrapped in a thin layer of polymers or small molecules. The thin layer is about 3-5 nm thick and is highly porous. The structure of the protein does not change after chemical modification and the binding sites on the protein remain active.5

    Fig.1Figure 1. FluoDot Structure

    Bovine serum albumin (BSA) is an inexpensive protein and a waste product in the meat industry. It has about 59 primary amines and most of them are reactive6. This provides a good platform for attaching other small molecules onto the surface of BSA via carbodiimide chemistry. The attachment does not disrupt the secondary structure of the protein7, which plays an important role in the binding of fluorescent dyes. Compared to quantum dots, fluorescent dye-labeled protein nanoparticles are advantageous because they are synthesized more easily, have better solubilities, and are both biocompatible and biodegradable. Unlike quantum dots, the size and color of FluoDots can be tuned independently.

    FluoDots are synthesized by combining a suitable lipid molecule with the protein core via carbodiimide chemistry and the particle size is tuned by increasing the length of the carbon chain in the lipid. Thus, the size of a FluoDot is controlled at the angstrom level. The color of FluoDots is tunable based on the chromophores attached to the particles. Similarly, the emission from the particles is also controlled by attaching appropriate fluorophores. These novel single protein nanoparticles with precise size and narrow size distribution can be potentially used in BioLEDs, cell imaging, solar cell coatings, and protein detection, etc..

    Size and Shape of FluoDots

    Dynamic light scattering (DLS) and transmission electron microscopy (TEM) are used to characterize FluoDots. These are labeled based on the number of carbons in the lipid segment and the absorption maximum of the dye used. Thus, FluoDot24-N represents the particles made from C12 lipid and has not been labeled with a dye. 

                                         A. Fig.2AB. Fig.2B C. Fig.2C

     Figure 2. A. DLS showing a hydrodynamic radius of about 10 nm for FluoDOT24-N. B. TEM image of Fluodot24-N, in agreement with the DLS data, showing spherical morphology. C. Circular dichroism spectra showing minimal change in protein secondary structure.

    The DLS of FluoDot24-N and FluoDot28-N indicate their sizes to be about 10 and 11 nm, respectively (Figure 2A) and these are bigger than that of BSA, but smaller than the size of two BSA molecules, which indicates the formation of single protein nanoparticles.

    The TEM of FluoDot24-N (Figure 2B) indicates nearly spherical particles of nearly uniform size consistent with the DLS data. Circular dichroism (CD) indicates the retention of the structure of BSA in the particles (Figure 2C). The shape and peak intensity of the CD spectrum of FluoDot24-N in the UV region (260 nm-190 nm) is similar to that of BSA. Based on this, it can be concluded that the secondary protein structure was not significantly affected.

    FluoDot28-494

    Agarose gel electrophoresis was also used to characterize the particles. For example, the agarose analysis of FluoDot28-494 indicated a single band, distinct from that of the protein (Figure 3A).  After fluorescent dye labeling, FluoDot24-494 had a similar size to FluoDot24-N about10-11 nm. The absorbance spectrum showed two peaks at 280 and 494 nm, corresponding to the protein and that of the fluorescent dye (Figure 3B). When FluoDot24-494 was excited at 480 nm, it had the emission peak at 520 nm.

                          A. Fig.3A B. Fig.3B

    Figure 3. A. Agarose gel analysis of FluoDot24-494 showing the purity of the sample and its labeling with the dye 494. B. UV-Visible absorbance and fluorescence spectra of FluoDot24-494. Excitation at 480 nm resulted in the emission peak at 520 nm.

    Conclusions

    A novel single protein nanoparticle has been created and its particle size is tunable on an angstrom scale.  Its color and emission over a wide wavelength range are under strict control via appropriate synthetic methodology. The particles were labeled with single or multiple fluorescent dyes to form specific FluoDots with one or multiple labels. FluoDots are completely biodegradable, biocompatible and they can be applied as a general platform for sensing, imaging or drug delivery.

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    References

    1. Hawkins, Michael J., Patrick Soon-Shiong, and Neil Desai. "Protein nanoparticles as drug carriers in clinical medicine." Advanced drug delivery reviews 60.8 (2008): 876-885.

    2. Stromer, Bobbi S., and Challa Vijaya Kumar. "White‐Emitting Protein Nanoparticles for Cell‐Entry and pH Sensing." Advanced Functional Materials 27.3 (2017).

    3. Gu, Zhen, et al. "Probing protease activity by single-fluorescent-protein nanocapsules." Chemical Communications 46.35 (2010): 6467-6469.

    4. Yang, Zhengpeng, and Chunjing Zhang. "Single-enzyme nanoparticles based urea biosensor." Sensors and Actuators B: Chemical 188 (2013): 313-317.

    5. Hegedüs, Imre, et al. "Biomedical Applications of Single Protein Nanoparticles." Journal of Pharmacy and Pharmacology 2 (2014): 652-659.

    6. Bujacz, Anna, Kamil Zielinski, and Bartosz Sekula. "Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen." Proteins: Structure, Function, and Bioinformatics82.9 (2014): 2199-2208.

    7. Conde, João, et al. "Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine." Frontiers in chemistry 2 (2014): 48.