9 research outputs found
Comparison of the In Vitro and In Vivo Behavior of a Series of NIR-II-Emitting Aza-BODIPYs Containing Different Water-Solubilizing Groups and Their Trastuzumab Antibody Conjugates
The development of new fluorescent organic probes effective
in
the NIR-II region is currently a fast-growing field and represents
a challenge in the domain of medical imaging. In this study, we have
designed and synthesized an innovative series of aza-boron dipyrromethenes
emitting in the NIR-II region. We have investigated the effect of
different water-solubilizing groups not only on the photophysical
properties of the compounds but also on their in vitro and in vivo
performance after bioconjugation to the antibody trastuzumab. Remarkably,
we discovered that the most lipophilic compound unexpectedly displayed
the most favorable in vivo properties after bioconjugation. This underlines
the profound influence that the fluorophore functionalization approach
can have on the efficiency of the resulting imaging agent
Comparison of the In Vitro and In Vivo Behavior of a Series of NIR-II-Emitting Aza-BODIPYs Containing Different Water-Solubilizing Groups and Their Trastuzumab Antibody Conjugates
The development of new fluorescent organic probes effective
in
the NIR-II region is currently a fast-growing field and represents
a challenge in the domain of medical imaging. In this study, we have
designed and synthesized an innovative series of aza-boron dipyrromethenes
emitting in the NIR-II region. We have investigated the effect of
different water-solubilizing groups not only on the photophysical
properties of the compounds but also on their in vitro and in vivo
performance after bioconjugation to the antibody trastuzumab. Remarkably,
we discovered that the most lipophilic compound unexpectedly displayed
the most favorable in vivo properties after bioconjugation. This underlines
the profound influence that the fluorophore functionalization approach
can have on the efficiency of the resulting imaging agent
PVL staining of breast tumoral tissues.
<p>A. A Breast tumor TMA (formalin-fixed) was stained and imaged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128190#pone.0128190.g008" target="_blank">Fig 8A</a>. Tumors were all positive with a vast majority of cancer cells stained. Labeling intensity was estimated on a scale ranging from 1 to 4. Representative examples of tumors of each staining level are shown. B. Staining level distributions according to tumor molecular characteristics. (HR = Hormone Receptors, TN = Triple Negative).</p
PVL staining of healthy tissues.
<p>A tissue microarray (TMA) comprising healthy tissues from respiratory, digestive and genital origin was stained with 0.7 μg ml<sup><b>-1</b></sup> rPVL-biot in presence of 0.1 M Fucose or 0.1 M GlcNAc followed by Streptavidin-HRP. AEC was used as a peroxidase substrate to reveal the PVL staining and counterstaining was performed using hematoxylin. Slides were imaged using a NanoZoomer slide scanner with a 20x magnification. 40x digital magnifications are also shown as insets.</p
PVL binding of tumor cell lines.
<p>A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml<sup>-1</sup> for 30 mn; red line: rPVL-alexa 488 5 μg ml<sup>-1</sup> in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml<sup>-1</sup> after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml<sup>-1</sup> rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.</p
Determination of affinity characteristics and thermodynamic contributions for the binding of rPVL with different oligosaccharides.
<p><sup>a</sup> only one experiment by ITC</p><p><sup>b</sup> stoichiometry value fixed during fitting procedure.</p><p>Standard deviation lower than 20% were obtained for ITC experiments.</p><p>Determination of affinity characteristics and thermodynamic contributions for the binding of rPVL with different oligosaccharides.</p
Representation of glycoconjugates and binding of rPVL to glycans on chips.
<p>A. Examples of normal and truncated oligosaccharides that can be found on normal or cancer tissues. Coding for schematic representation of monosaccharides is in the lower part of the figure. The heptasaccharide used in binding experiments is indicated as “hepta”. B. Synthesis of heptasaccharide azide 2 corresponding to oligosaccharide “hepta” in panel A.</p
PVL binds to tumor tissue with a mixed Neu5Ac and GlcNAc specificity.
<p>A. Sections from ethanol-fixed colon carcinoma and adjacent healthy tissue from 2 different patients (# 5345 and 5378) were stained with 1 μg ml<sup><b>-1</b></sup> rPVL-biot in presence of 0.1 M Fucose or 0.1 M GlcNAc followed by Streptavidin-HRP. AEC was used as a peroxidase substrate to reveal the rPVL staining and counterstaining was performed using hematoxylin. Slides were imaged using a NanoZoomer slide scanner with a 20x magnification.40x digital magnifications are also shown as insets. B. Canine breast tumor sections (formalin fixed) were treated or not with glycosidases and then stained with 2 μg ml<sup><b>-1</b></sup> rPVL-biot and imaged as in A.</p
PVL binding in the presence of glycosylation inhibitors or after treatment with glycosidases.
<p>A. Biotinylated rPVL (1 μg ml<sup><b>-1</b></sup>) or MAH (5 μg ml<sup><b>-1</b></sup>) were incubated with A549 cells grown for 4 days in presence of DMSO (green line) or of either 400 μM 2-Fluoro-Fucose or 100 μM Fluoro-Neu5Ac (pink line). Lectin binding was revealed by PE conjugated Streptavidin. Percentage of inhibitions are indicated according to the mean fluorescence intensities. B. Similar experiment than in A but with A549 cells grown in the presence of 5 μM Kifunensine for 4 days or in the presence of either 6 mM Benzyl-GalNAc or 10 μM PPMP for 48h. C. Similar experiment than in A and B but with cells treated with 2.5 U (pink line) or 12.5 U ß-D-N-acetyl-hexosaminidase (blue line).</p