Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications

Robertson, Kelly L.; Liu, Jinny L.

doi:10.1002/wnan.1177

Cited by 13 publications

(14 citation statements)

References 77 publications

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“…Discussion T4 capsids display a large surface area and have a large cargo capacity for carrying protein and DNA molecules for cell delivery. Previously, we demonstrated that more than 10 4 dye molecules per capsid were chemically conjugated to both the inner and outer surfaces of the T4 capsid, yielding highly fluorescent T4 NPs that were used for imaging and flow cytometry applications after internalization by cancer cells (2,6). In this study, we further demonstrated the imaging of highly fluorescent T4 capsids that result from encapsidating multiple modified bases conjugated to reactive dyes in dsDNA synthesized by reverse transcriptase.…”

Section: Assessment Of Cell Viability Of Cre Procapsid-treated A549 Cmentioning

confidence: 66%

“…terminase | DNA packaging | capsid decoration proteins | Soc | Hoc V iral-based nanoparticles (NPs) that deliver cargo to specific targets continue to be of widespread interest for drug-and gene-delivery applications in human diseases and other areas of technology, such as diagnostic and cellular imaging (1)(2)(3)(4). NPs derived from bacteriophage capsids are among the most attractive for these purposes (5-7).…”

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confidence: 99%

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Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

Liu

Dixit

Robertson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein-DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications.terminase | DNA packaging | capsid decoration proteins | Soc | Hoc V iral-based nanoparticles (NPs) that deliver cargo to specific targets continue to be of widespread interest for drug-and gene-delivery applications in human diseases and other areas of technology, such as diagnostic and cellular imaging (1-4). NPs derived from bacteriophage capsids are among the most attractive for these purposes (5-7). As NPs for medical applications, bacteriophage capsids have the advantages of (i) biocompatibility and demonstrated stability in the circulatory system and widespread delivery to tissues; (ii) absence of eukaryotic cell toxicity and of a preexisting immune response; (iii) ease and flexibility of molecular manipulation of the capsids and the encapsidated cargo; and (iv) easy accessibility of large amounts of material from fully characterized and nonpathogenic bacteria. Bacteriophage capsids and bacteriophages themselves vary tremendously in size and shape as well as complexity. The T4-like phages, which are among the largest and most complex of such bacteriophages, are particularly attractive because they are exceedingly well studied at the molecular and structural level. Thus, the large bacteriophage T4 capsid can serve in vaccine development, as displayed by surface decoration of full-length and active proteins (8-10), or for affinity studies using bipartite randomized peptide display libraries (Fig. 1) (11).The phage T4 capsid has an additional unusual feature among bacteriophages: It is able to pack m...

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Section: Assessment Of Cell Viability Of Cre Procapsid-treated A549 Cmentioning

confidence: 66%

mentioning

confidence: 99%

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

Liu

Dixit

Robertson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The attachment of fluorescent dyes to VNPs can facilitate flow cytometry, confocal microscopy, and in vivo imaging experiments because a large number of dye molecules can be incorporated per particle and the sensitivity of detection can be modulated by controlling the spacing of the molecules to optimize individual particle detection (125). Dye molecules are usually attached by conjugation to the surface of the VNP (126, 127), but some investigators have used genetic engineering to incorporate polypeptides such as green fluorescent protein (GFP) into the coat protein, thus creating stable virus chimeras that are fluorescent.…”

Section: Virus-based Materials As Imaging Probesmentioning

confidence: 99%

Virus-Based Nanoparticles as Versatile Nanomachines

et al. 2015

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Nanoscale engineering is revolutionizing the way we prevent, detect, and treat diseases. Viruses have played a special role in these developments because they can function as prefabricated nanoscaffolds that have unique properties and are easily modified. The interiors of virus particles can encapsulate and protect sensitive compounds, while the exteriors can be altered to display large and small molecules in precisely defined arrays. These properties of viruses, along with their innate biocompatibility, have led to their development as actively targeted drug delivery systems that expand on and improve current pharmaceutical options. Viruses are naturally immunogenic, and antigens displayed on their surface have been used to create vaccines against pathogens and to break self-tolerance to initiate an immune response to dysfunctional proteins. Densely and specifically aligned imaging agents on viruses have allowed for high-resolution and noninvasive visualization tools to detect and treat diseases earlier than previously possible. These and future applications of viruses have created an exciting new field within the disciplines of both nanotechnology and medicine.

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“…In the field of biomedicine, plant and bacterial viruses (bacteriophages) are favoured as they are nonpathogenic to animals, which limits safety concerns (Soto and Ratna ). Cowpea mosaic virus and other virus‐based protein cage architectures have been engineered for viral delivery of tissue‐ or cell‐specific fluorescent reporters and drugs (Robertson and Liu ; Niikura et al . ).…”

Section: Introductionmentioning

confidence: 99%

“…In the field of biomedicine, plant and bacterial viruses (bacteriophages) are favoured as they are nonpathogenic to animals, which limits safety concerns (Soto and Ratna 2010). Cowpea mosaic virus and other virus-based protein cage architectures have been engineered for viral delivery of tissue-or cell-specific fluorescent reporters and drugs (Robertson and Liu 2012;Niikura et al 2013). In some instances, chemical modifications were added to the virus to increase evasion of the host immune system, for increasing circulation time, and for increasing tissue distribution (Kim et al 2008).…”

Section: Introductionmentioning

confidence: 99%

SMV1, an extremely stable thermophilic virus platform for nanoparticle trafficking in the mammalian GI tract

et al. 2017

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Aims Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract. Methods and Results A major challenge of using nano-vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo-acidophilic archaeal virus SMV1 as a candidate therapeutic nano-vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium. Conclusion Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract. Significance and Impact of the Study This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.

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Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications

Cited by 13 publications

References 77 publications

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

Virus-Based Nanoparticles as Versatile Nanomachines

SMV1, an extremely stable thermophilic virus platform for nanoparticle trafficking in the mammalian GI tract

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