Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies

Abstract

Endotoxin (lipooligosaccharide, LOS, and lipopolysaccharide, LPS) is the major molecular component of Gram-negative bacteria outer membrane, and very potent proinflammatory substance. Visualizing and tracking the distribution of the circulating endotoxin is one of the fundamental approaches to understand the molecular aspects of infection with subsequent inflammatory and immune responses, LPS also being a key player in the molecular dialogue between microbiota and host. While fluorescently labeled LPS has previously been used to track its subcellular localization and colocalization with TLR4 receptor and downstream effectors, our knowledge on lipopolysaccharide (LOS) localization and cellular activity remains almost unexplored. In this study, LOS was labeled with a novel fluorophore, Cy7N, featuring a large Stokes-shifted emission in the deep-red spectrum resulting in lower light scattering and better imaging contrast. The LOS-Cy7N chemical identity was determined by mass spectrometry, and immunoreactivity of the conjugate was evaluated. Interestingly, its application to microscopic imaging showed a faster cell internalization compared to LPS-Alexa488, despite that it is also CD14-dependent and undergoes the same endocytic pathway as LPS toward lysosomal detoxification. Our results suggest the use of the new infrared fluorophore Cy7N for cell imaging of labeled LOS by confocal fluorescence microscopy, and propose that LOS is imported in the cells by mechanisms different from those responsible for LPS uptake.

About the origin of the large Stokes shift in aminoalkyl substituted heptamethine cyanine dyes

“…19,20 In particular, aminosubstituted heptamethine cyanine dyes obtained by substitution of the chlorine atom show a huge Stokes shift (larger than 0.3 eV), 13 maintaining or even improving the fluorescence properties typical of heptamethine dyes. These molecules have been exploited as fluorescent probes for bioimaging 21,22 and in artificial light harvesting antennas for solar energy conversion. [23][24][25] The mechanism at the basis of the spectroscopic effects observed upon substitution of the chlorine atom with an aminoalkyl group has not been fully addressed in the literature.…”

Impact of Ion‐Pairing Effects on Linear and Nonlinear Photophysical Properties of Polymethine Dyes**

“…[4][5][6][7][8][9] Commercially available indocyanine green, is largely used for in vivo bio-imaging applications or further bioconjugation. [11,12] Some polymethines have also been extensively studied for nonlinear optical applications, such as alloptical signal processing or optical limiting, in the telecommunication wavelengths range [1.3-1.5 μm]. [13][14][15][16][17][18][19] The development of such applications requires the optimization of the optical properties of the material through a precise molecular-level manipulation of the electronic structure of the polymethine dyes, by controlling the nature of the counterion.…”

“…Within the cyanine family, pentamethines and heptamethines with five‐ and seven‐carbon bridges, respectively, have attracted much attention for applications in bio‐imaging and material science [4–9] . Commercially available indocyanine green, is largely used for in vivo bio‐imaging applications or further bioconjugation [11,12] . Some polymethines have also been extensively studied for nonlinear optical applications, such as all‐optical signal processing or optical limiting, in the telecommunication wavelengths range [1.3–1.5 μm] [13–19] .…”

Stimuli-Responsive Polymeric Nanocarriers for Drug Delivery, Imaging, and Theragnosis

“…In order to mollify these limitations, a wide variety of cyanines were incorporated into different polymer formulations with the goal of enhancing its bioavailability and durability. There are several studies of NIR fluorescent probes insertion in lipooligosaccharides [ 225 ], water-soluble carboxylated N-acylated poly(amino ester)-based comb polymers [ 226 ], supramolecular nanodiscs self-assembled [ 227 ], polymer micelle [ 228 ] and mitochondrion- and nucleus-acting polymeric nanoagents [ 229 ]. For instance, Yang et al (2020) constructed a multi- and cascaded switchable polymer nanocarrier that self-assembled from nano polymers for imaging and anticancer treatment.…”