Esterified Dendritic TAM Radicals with Very High Stability and Enhanced Oxygen Sensitivity

In this work, we have developed a new class of dendritic TAM radicals (TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O₂ sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03. Like the previous dendritic TAM radicals, these new compounds also show extremely high stability toward various reactive species owing to the dendritic encapsulation. The highly charged nature of these molecules resulting from nine carboxylate groups prevents concentration-dependent EPR line broadening at physiological pH. Furthermore, we demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the nine carboxylate groups and the resulting PEGylated analogue dTdG–PEG completely inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These new dendritic TAM radicals show great potential for in vivo EPR oximetric applications and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications

WED impairs cell viability.

<p>CLSM micrographs of mature PAO1 biofilm stained with live-dead stain after treatment with placebo, WED or Ag control. The green fluorescence indicates live bacteria while the red indicates dead bacteria, n = 3.</p

WED represses redox-sensitive multidrug efflux genes in <i>P</i>. <i>aeruginosa</i> Real-time PCR was performed to assess mex gene expression post-treatment with placebo, WED or 3mM DTT, n = 3.

<p>WED represses redox-sensitive multidrug efflux genes in <i>P</i>. <i>aeruginosa</i> Real-time PCR was performed to assess mex gene expression post-treatment with placebo, WED or 3mM DTT, n = 3.</p

WED inhibits glycerol-3-phosphate dehydrogenase activity.

<p>Measurement of glycerol-3-Phosphate dehydrogenase enzyme activity from mature PAO1 biofilms treated with placebo or WED <b>A.</b> OD measured in the kinetic mode. <b>B.</b> GPDH activity calculated using the formula: Glycerol-3-Phosphate dehydrogenase activity = B/(ΔT X V) x Dilution Factor = nmol/min/ml, where: B = NADH amount from Standard Curve (nmol). ΔT = reaction time (min). V = sample volume added into the reaction well (ml), n = 3.</p

WED generates superoxide (A) EPR spectra using 20mM DIPPMPO demonstrates spin adduct generation upon exposure to dressings for 40 mins in PBS.

<p>Addition of 500U Catalase does not attenuate EPR signal but is attenuated using both 500U SOD and 500U Catalase, n = 3</p

Anti-bacterial properties of WED.

<p><b>(A)</b> Energy Dispersive X-Ray Spectroscopy (EDS) elemental analysis of Ag/Zn WED. <b>a.</b> Scanning Electron Microscope (SEM) image; <b>b.</b> Light Microscope Image; <b>c</b>. and <b>d.</b> Close-up view of gold and silver dots under light microscope. <b>e-h.</b> EDS element maps of Zn (blue), Silver (red), Oxygen (green) and Carbon (Magenta). <b>(B,C)</b> Absorbance and CFU measurement from planktonic PAO1 culture in presence of placebo or Ag/Zn WED, n = 4 <b>(D) (i)</b> schematic diagram for experimental design showing dressing embedded in the agar plate. <b>(ii, iii)</b> Zone of inhibition above WED is marked with red dotted line, while no such zone was observed over the placebo dressing, n = 4 <b>(E)</b> Biofilm formation measured by absorbance at 540 nm, n = 4 <b>(F)</b> Biofilm co-aggregation observed in the placebo treated overnight PAO1 culture was not observed in WED treated overnight cultures, n = 3.</p