Optical Imaging of Bacterial Infection in Living Mice Using Deep-Red Fluorescent Squaraine Rotaxane Probes

Abstract

Two structurally related fluorescent imaging probes allow optical imaging of bacterial leg infection models in living athymic and immunocompetent mice. Structurally, the probes are comprised of a deep-red fluorescent squaraine rotaxane scaffold with two appended bis(zinc(II)-dicolylamine) (bis(Zn-DPA)) targeting ligands. The bis(Zn-DPA) ligands have high affinity for the anionic phospholipids and related biomolecules that reside within the bacterial envelope, and they are known to selectively target bacterial cells over the nearly uncharged membrane surfaces of healthy mammalian cells. Planar, whole-animal optical imaging studies showed that intravenous dosing of either probe (10 nmol) allowed imaging of localized infections of Grampositive Staphylococcus aureus and Gram-negative Salmonella enterica serovar typhimurium. High selectivity for the infected target leg (T) over the contralateral non-target leg (NT) was reflected by T/NT ratios up to six. The infection imaging signal was independent of mouse humoral immune status, and there was essentially no targeting at a site of sterile inflammation induced by injection of λ-carrageenan. Furthermore, the fluorescent probe imaging signal colocalized with the bioluminescence signal from a genetically engineered strain of S. enterica serovar typhimurium. Although not highly sensitive (the localized infection must contain at least 10 6 colony forming units for fluorescence visualization) the probes are remarkably selective for bacterial cells considering their low molecular weight (<1.5 kDa) and simple structural design. The more hydrophilic of the two probes produced a higher T/NT ratio in the early stages of the imaging experiment and washed out more rapidly from the blood clearance organs (liver, kidney). Therefore, it is best suited for longitudinal studies that require repeated dosing and imaging of the same animal. The results indicate that fluorescent probes based on squaraine rotaxanes should be broadly useful for in vivo animal imaging studies, and they further validate the ability of imaging probes with bis(Zn-DPA) ligands to selectively target bacterial infections in living animals.

Bacterial vectors for imaging and cancer gene therapy: a review

“…This is exemplified by the decrease in luminescence that has been observed when many luxexpressing bacterial species, such as Bifidobacterium, 38 enter stationary phase during in vitro growth. [46][47][48] Bioluminescent bacteria have been used extensively to study a diverse range of biological processes such as infection, 49 quorum sensing 50 and gene expression. 51 However, the employment of luciferase or lux tagged bacterial strains as gene delivery vehicles for the treatment of cancer is an emerging field.…”

PET imaging of sterile inflammation with a 18F-labeled bis(zinc(II)-dipicolylamine) complex

“…During apoptosis, phosphatidylserine (PS) externalized to the surface of neutrophils provides a specific biomarker for the detection of infection and inflammation [12,23]. Previous studies have reported that synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes possess a selective affinity for biomembranes enriched with PS [10], and fluorescence-labeled bis(zinc(II)-dipicolylamine) coordination complexes have been used for in vivo optical imaging of bacterial abscesses with a contribution from PS fluorescence [12,13,24]. However, this method presents significant challenges for imaging those lesions that are deeply seated in the body, since emitted light photons may not penetrate the tissue.…”

“…Synthetic bis(zinc (II)-dipicolylamine) coordination complexes (DPAZn2) are the best known compounds with the potential to binding PS, and some fluorescent DPAZn2 analogues have been used for optical imaging of cell death with the same target as Annexin V [10,11]. In addition, optical imaging with fluorescent DPAZn2 analogues has also been used to detect the infectious tissues induced with bacterial or nonbacterial pathway [12,13]. However, optical imaging has poor tissue penetration, which limits the use of optical probes for in vivo imaging applications.…”

Targeting of anionic membrane species by lanthanide(iii) complexes: towards improved MRI contrast agents for apoptosis

“…Smith has developed a range of bis-ZnDPA complexes as probes able to recognise apoptotic and bacterial cells, which they have demonstrated with considerable success both in vitro (liposomal models and cells) 14,15 and in small animal tumour and bacterial infection models, using NIR optical imaging. 16,17 In a recently-published study we showed that bis-ZnDPA, lanthanide complexes [Ln(L)Zn 2 ](NO 3 ) 4 (Ln = Gd, Eu; see Scheme 1), bind strongly to polyanionic species, including Fmoc-phosphoserine. 18 This prompted us to study whether this complex might also bind bisanionic phosphoserine head groups at membrane surfaces.…”

“…This selectivity is consistent with the high selectivity for apoptotic cells previously reported in a wide and structurally diverse range of bis-(Zn-DPA) complexes. 14,15,17 As such…”