ABSTRACT
Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Fluorescent Dyes/chemistry , Hepacivirus/chemistry , Lipids/chemistry , Liver Neoplasms/metabolism , Virion/chemistry , Carcinoma, Hepatocellular/pathology , Cell Line , Humans , Liver Neoplasms/pathology , Microscopy, FluorescenceABSTRACT
Exogenous probes with far-red or near-infrared (NIR) two-photon absorption and fluorescence emission are highly desirable for deep tissue imaging while limiting autofluorescence. However, molecular probes exhibiting such properties are often hydrophobic. As an attractive alternative, we synthesized water-soluble polymer probes carrying multiple far-red fluorophores and demonstrated here their potential for live cell and zebrafish embryo imaging. First, at concentrations up to 10 µm, these polymer probes were not cytotoxic. They could efficiently label living HeLa cells, T lymphocytes and neurons at an optimal concentration of 0.5 µm. Moreover, they exhibited a high resistance to photobleaching in usual microscopy conditions. In addition, these polymer probes could be successfully used for in toto labeling and in vivo two-photon microscopy imaging of developing zebrafish embryos, with remarkable properties in terms of biocompatibility, internalization, diffusion, stability and wavelength emission range. The near-infrared two-photon absorption peak at 910 nm is particularly interesting since it does not excite the zebrafish endogenous fluorescence and is likely to enable long-term time-lapse imaging with limited photodamage.
Subject(s)
Biocompatible Materials/chemistry , Embryo, Nonmammalian/metabolism , Fluorescent Dyes/chemistry , Imaging, Three-Dimensional , Photons , Polymers/chemistry , Spectroscopy, Near-Infrared , Zebrafish/embryology , Absorption, Radiation , Animals , Cell Death , Cell Survival , Endocytosis , HeLa Cells , Humans , Jurkat Cells , Kinetics , Microscopy, Fluorescence , Photobleaching , Spectrometry, FluorescenceABSTRACT
This contribution presents a new strategy for preparing nanocapsules with a shell made of a supramolecular polymer which repeating units are held together by reversible interactions rather than covalent bonds. These nanocapsules were prepared in classical miniemulsion through interfacial addition reaction of a diisocyanate (IPDI) and a monoamine (iBA), forming low-molecular weight bis-ureas moieties which are strong self-complementary interacting molecules through hydrogen-bonding. The nanocapsules present a diameter around 100 nm, and MALDI-TOF MS and (1)H NMR analyses confirm the expected molecular characteristics for the shell. This strategy opens the scope of a new type of nanomaterials exhibiting stimuli-responsiveness due to the reversible interaction linking the repeating units.
