Koatale Palesa C, Welling Mick M, Mdanda Sipho, Mdlophane Amanda, Takyi-Williams John, Durandt Chrisna, van den Bout Iman, Cleeren Frederik, Sathekge Mike M, Ebenhan Thomas
Department of Nuclear Medicine, University of Pretoria, Pretoria 0001, South Africa.
Nuclear Medicine Research Infrastructure (NuMeRI), Steve Biko Academic Hospital, Pretoria 0001, South Africa.
Pharmaceuticals (Basel). 2024 Aug 31;17(9):1150. doi: 10.3390/ph17091150.
The ability of bacteria to recycle exogenous amino acid-based peptides and amino sugars for peptidoglycan biosynthesis was extensively investigated using optical imaging. In particular, fluorescent AeK-NBD was effectively utilized to study the peptidoglycan recycling pathway in Gram-negative bacteria. Based on these promising results, we were inspired to develop the radioactive AeK conjugate [Ga]Ga-DOTA-AeK for the in vivo localization of bacterial infection using PET/CT. An easy-to-implement radiolabeling procedure for DOTA-AeK with [Ga]GaCI followed by solid-phase purification was successfully established to obtain [Ga]Ga-DOTA-AeK with a radiochemical purity of ≥95%. [Ga]Ga-DOTA-AeK showed good stability over time with less protein binding under physiological conditions. The bacterial incorporation of [Ga]Ga-DOTA-AeK and its fluorescent Aek-NBD analog were investigated in live and heat-killed () and (). Unfortunately, no conclusive in vitro intracellular uptake of [Ga]Ga-DOTA-AeK was observed for or live and heat-killed bacterial strains ( > 0.05). In contrast, AeK-NBD showed significantly higher intracellular incorporation in live bacteria compared to the heat-killed control ( < 0.05). Preliminary biodistribution studies of [Ga]Ga-DOTA-AeK in a dual-model of chronic infection and inflammation revealed limited localization at the infection site with non-specific accumulation in response to inflammatory markers. Finally, our study demonstrates proof that the intracellular incorporation of AeK is necessary for successful bacteria-specific imaging using PET/CT. Therefore, Ga-68 was not a suitable radioisotope for tracing the bacterial uptake of AeK tripeptide, as it required chelation with a bulky metal chelator such as DOTA, which may have limited its active membrane transportation. An alternative for optimization is to explore diverse chemical structures of AeK that would allow for radiolabeling with F or C.
利用光学成像广泛研究了细菌将外源氨基酸基肽和氨基糖循环利用以进行肽聚糖生物合成的能力。特别是，荧光AeK-NBD被有效地用于研究革兰氏阴性菌中的肽聚糖循环途径。基于这些有前景的结果，我们受到启发开发了放射性AeK共轭物[Ga]Ga-DOTA-AeK，用于使用PET/CT对细菌感染进行体内定位。成功建立了一种易于实施的用[Ga]GaCl对DOTA-AeK进行放射性标记的程序，随后进行固相纯化，以获得放射化学纯度≥95%的[Ga]Ga-DOTA-AeK。[Ga]Ga-DOTA-AeK在生理条件下随时间显示出良好的稳定性，且蛋白质结合较少。研究了[Ga]Ga-DOTA-AeK及其荧光Aek-NBD类似物在活的和热灭活的（）和（）中的细菌摄取情况。不幸的是，对于活的和热灭活的细菌菌株，未观察到[Ga]Ga-DOTA-AeK有确凿的体外细胞内摄取（>0.05）。相比之下，与热灭活对照相比，AeK-NBD在活细菌中显示出明显更高的细胞内掺入（<0.05）。[Ga]Ga-DOTA-AeK在慢性感染和炎症双模型中的初步生物分布研究表明，在感染部位的定位有限，且会因炎症标志物而出现非特异性积累。最后，我们的研究证明，AeK的细胞内掺入对于使用PET/CT成功进行细菌特异性成像至关重要。因此，Ga-68不是追踪AeK三肽细菌摄取的合适放射性同位素，因为它需要与诸如DOTA这样的大分子金属螯合剂螯合，这可能限制了其主动膜转运。优化的另一种方法是探索AeK的多种化学结构，以便能够用F或C进行放射性标记。
