Individually-coated Near-infrared Fluorescent Protein As A Safe And Robust Nanoprobe For In Vivo Imaging

Fluorescent imaging techniques, such as fluorophores labelling, are usedprevalent in clinical diagnosis. However, the organic fluorescence probes which usedcurrently have low intensity and are not photostable. Along with the rapiddevelopment of nanotechnology, nanoparticle (NP)-based fluorescent probes, such asC-dots, quantum dots (QDs), and inorganic fluorescent NPs and organicdye-encapsulated NP3, show high potential to replace the traditional organic dyes andrevolutionalize this area. NP-based fluorescent probes have many merits over thetraditional organic ones. Besides optical properties, NP-based fluorescent probes alsoprovide multifunctional platforms to carry molecules such as targeting groups andtherapy drugs, making it possible to deliver drug with targeting, and monitor thetherapeutic response. This kind of NP-based theranostics is expected to play animportant role in the future personalized medicine. However, a major concern forgeneral nanoprobes such as QDs is its bio-safety, especially for in vivo applications.Even after wrapped by various biocompatible shells (silica or polymers), the toxicheavy-metal in QDs is still a big biohazard, especially for long-term in vivoapplications. Safer nanoprobes are in high demand for the in vivo bioapplications.Fluorescence proteins (FPs) are a set of useful tools extensively used inbiological sciences. However, FPs are currently used as reporter genes. The purifiedFluorescence proteins are not suitable for bio-imaging for the unstable nature of theproteins. We showed formerly that encapsulation in silica NP by the common reverseemulsion method could improve the stability and fluorescence intensity of enhancedgreen FP (EGFP). However, there are still two barriers for its in vivo applications asan external probe. First, silica NPs prepared by the reverse microemulsion methodusually have large size and poor dispersibility, thus when injected into animals, theymay clog in lungs and cause harsh damage to animals. In addition, since the surfactantused in the emulsion may disrupt biomembranes, calcination of the silica NP productis usually needed to completely remove the surfactant molecules before any in vivo application. However, calcinations is not applicable for FP-encapsulated silica NP.Second, the emission wavelength of EGFP is not enough for whole body imaging.So, it is preferable to have an emission wavelengths in the near-infrared range forwhole body imaging that can penetrate the tissues and avoid autofluorescence.Fortunately, a near-infrared FP (NIRFP), eqFP650, was reported recently as thebrightest FP with emission maximum above650nm with a quantum yield of0.24.Here we reported a modified St ber method to produce silica NP wrapped NIRFP as asafe and robust near-infrared fluorescent nanoprobe. Wrapping in silica shell confersNIRFP stronger fluorescence and better stabilities against denaturant, metabolism andphotobleaching. This new nanoprobe is suitable for in vivo imaging with very brightfluorescence that can penetrate tissue. The well-proven safety of FPs and the quickclearance of NIRFP@silica NPs from mice through efficient urinary excretion, makeit a much safer in vivo nanoprobe. These biomimetic features make nanoparticlespotential tools for imaging, diagnosis and therapy.