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WIREs Nanomed Nanobiotechnol
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Protein nanoparticles in molecular, cellular, and tissue imaging

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Abstract The quest to develop ideal nanoparticles capable of molecular, cellular, and tissue level imaging is ongoing. Since certain imaging probes and nanoparticles face drawbacks such as low aqueous solubility, increased ROS generation leading to DNA damage, apoptosis, and high cellular/organ toxicities, the development of versatile and biocompatible nanocarriers becomes necessary. Protein nanoparticles (PNPs) are one such promising class of nanocarriers that possess most of the desirable properties of an ideal nanocarrier for bioimaging applications. PNPs demonstrate high aqueous solubility, minimal cytotoxicity, and multi‐cargo loading capacity. They are also amenable to surface‐functionalization, as well as modulation of their hydrophobicity and hydrophilicity. The use of PNPs for bioimaging applications has made rapid advancements in the past two decades. Being comparatively less explored, the field opens up a plethora of opportunities and focus areas to engineer ideal bioimaging protein nanocarriers. The use of PNPs as carriers of their natural ligands as well as other heavy metals and fluorescent probes, along with drug molecules for combined theranostic applications has been reported. In addition, surface functionalization to impart specificity of targeting the PNPs has been shown to reduce nonspecific cellular interactions, thus reducing systemic toxicity. PNPs have been explored for their application in imaging of numerous cancers, cardiovascular diseases as well as imaging of the brain using near infrared fluorescence (NIRF) imaging, magnetic resonance imaging (MRI), X‐ray computed tomography (CT), positron emission tomography (PET), single‐photon emission computed tomography (SPECT), ultrasound (US), and photoacoustic (PA) imaging. This article is categorized under: Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Use of protein nanoparticles (PNPs) for MR, NIRF, CT, SPECT, PET, and US imaging. PNPs have been used as multifunctional nanocarriers and can be employed for simultaneous bioimaging and drug delivery for a wide range of imaging modalities. The property of surface functionalization enables the PNPs to be used for specific targeting. SELF indicates targeting of natural receptors (e.g., transferrin) using unmodified PNP (e.g., ferritin) surface
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Representative images of various imaging applications of PNPs using multiple imaging modalities. (a) The 3D PAI of U87MG glioblastoma tumor, preinjection and postinjection of CuS‐Fn (top); PET images of tumor bearing mice post 50 μCi 64CuS‐Fn nanocage i.v. injection, after 2, 4, 8, 20, and 24 h (bottom). Adapted with permission from Wang et al. (2016). Copyright (2016) American Chemical Society; (b) In vivo MRI of Gd(III)‐DTPA and Hsp 16.5 4‐Nanocage conjugated to gadolinium(III)‐chelated contrast agents measured with 9.4 T MRI. The 4‐Nanocage showed selective and high contrast enhancement of tumor regions represented by red dashed circles. Adapted with permission from Kawano et al. (2018). Copyright (2018), with permission from Elsevier; (c) GV enhancement of transcranial functional ultrasound signals. Activation maps overlaid on power Doppler images of the mouse brain with and without bolus injection of saline, microbubbles, and GVs, respectively, have been represented in the figure. Adapted with permission from Maresca et al. (2020), from Elsevier; (d) SPECT–CT images of wild type and Apoe−/− mice using 99mTc‐HFn over a period of 12, 20, and 33 weeks. The red circles indicate atherosclerotic (AS) plaques. Adapted with permission from Liang et al. (2018). Copyright (2018) American Chemical Society; (e) in vivo NIRF imaging measured in real‐time post i.v. injection of Alexa Fluor 750‐labeled HspG41C‐CTT nanocages in HT1080 and HT29 (negative control) tumor‐bearing mice. The dotted squares indicate solid tumor growths of subcutaneously injected cancer cells. Adapted with permission from Kawano et al. (2015)
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Transmission electron microscope images of (a) ironfree ferritin nanocages. Adapted with permission from Wang et al. (2016); Copyright (2016) American Chemical Society; (b) VLP‐QDs assembled from wild type VP1 protein and quantum dots. Adapted with permission from Sun et al. (2016). Copyright (2016) American Chemical Society; (c) HspG41C PNPs where the white arrowheads point to the nanocages. Adapted with permission from Kawano et al. (2014). Systemic delivery of protein nanocages bearing CTT peptides for enhanced imaging of MMP‐2 expression in metastatic tumor models, open access); (d) structure of GVs from Anabaena flos‐aquae in its intact form, and (e) in its collapsed form, used for ultrasound contrast. The part figures (d,e) have been adapted with permission from Lakshmanan et al. (2017). Copyright (2017) Springer Nature. Scale bars are (a) 100 nm, (b) 50 nm, (c) 20 nm, (d) 200 nm, and (e) 200 nm
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Crystal structures of PNP representatives (downloaded from RCSB PDB and displayed using PyMOL). (a) Human H chain ferritin (PDB ID 2FHA) (Hempstead et al., 1997). (b) A small heat shock protein from Methanococcus jannaschii (PDB ID 1SHS) (Kim et al., 1998). (c) A closed form of the Archaeoglobus fulgidus ferritin obtained by site directed mutagenesis (PDB ID 3KX9) (Sana et al., 2013). (d) Horse spleen apoferritin (PDB ID 2W0O) (de Val et al., 2012). (e) Structure of free SV40 VP1 pentamer (PDB ID 3BWQ) (Neu et al., 2008). And (f) crystal structures of a novel open pore ferritin from the hyperthermophilic archaeon Archaeoglobus fulgidus (PDB ID 1SQ3) (Johnson et al., 2005). These images were created using PyMOL 2.4 (The PyMOL Molecular Graphics System, Version 2.4.1 Schrödinger, LLC). The α helices, β sheets, and loops have been represented in blue, gray, and red colors, respectively
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Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Biology-Inspired Nanomaterials > Protein and Virus-Based Structures

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