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Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo.

ABSTRACT: Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, racemic mixtures, and addition of heavy metals hinders FDA approval in the United States. We developed a small ultra-red fluorescent protein, named smURFP, to have optical properties similar to the small-molecule Cy5, a heptamethine subclass of cyanine dyes (Ex/Em = 642/670 nm). smURFP has a fluorescence quantum yield of 18% and expresses so well in E. coli, that gram quantities of fluorescent protein are purified from cultures in the laboratory. In this research, the fluorescent protein smURFP was combined with bovine serum albumin into fluorescent protein nanoparticles. These nanoparticles are fluorescent with a quantum yield of 17% and 12-14 nm in diameter. The far-red fluorescent protein nanoparticles noninvasively image tumors in living mice via the enhanced permeation and retention (EPR) mechanism. This manuscript describes the use of a new fluorescent protein nanoparticle for in vivo fluorescent imaging. This protein nanoparticle core should prove useful as a biomacromolecular scaffold, which could bear extended chemical modifications for studies, such as the in vivo imaging of fluorescent protein nanoparticles targeted to primary and metastatic cancer, theranostic treatment, and/or dual-modality imaging with positron emission tomography for entire human imaging.

SUBMITTER: An F

PROVIDER: S-EPMC7493049 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Two structurally related fluorescent imaging probes allow optical imaging of bacterial leg infection models in living athymic and immunocompetent mice. Structurally, the probes are comprised of a deep-red fluorescent squaraine rotaxane scaffold with two appended bis(zinc(II)-dicolylamine) (bis(Zn-DPA)) targeting ligands. The bis(Zn-DPA) ligands have high affinity for the anionic phospholipids and related biomolecules that reside within the bacterial envelope, and they are known to selectively target bacterial cells over the nearly uncharged membrane surfaces of healthy mammalian cells. Planar, whole-animal optical imaging studies showed that intravenous dosing of either probe (10 nmol) allowed imaging of localized infections of Gram-positive Staphylococcus aureus and Gram-negative Salmonella enterica serovar typhimurium. High selectivity for the infected target leg (T) over the contralateral nontarget leg (NT) was reflected by T/NT ratios up to six. The infection imaging signal was independent of mouse humoral immune status, and there was essentially no targeting at a site of sterile inflammation induced by injection of lambda-carrageenan. Furthermore, the fluorescent probe imaging signal colocalized with the bioluminescence signal from a genetically engineered strain of S. enterica serovar typhimurium. Although not highly sensitive (the localized infection must contain at least approximately 10(6) colony forming units for fluorescence visualization), the probes are remarkably selective for bacterial cells considering their low molecular weight (<1.5 kDa) and simple structural design. The more hydrophilic of the two probes produced a higher T/NT ratio in the early stages of the imaging experiment and washed out more rapidly from the blood clearance organs (liver, kidney). Therefore, it is best suited for longitudinal studies that require repeated dosing and imaging of the same animal. The results indicate that fluorescent probes based on squaraine rotaxanes should be broadly useful for in vivo animal imaging studies, and they further validate the ability of imaging probes with bis(Zn-DPA) ligands to selectively target bacterial infections in living animals.

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Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo.

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