Zwitterionic near infrared fluorescent agents for noninvasive real-time transcutaneous assessment of kidney function

Abstract

We developed novel zwitterionic near infrared (NIR) fluorescent agents (ABZWCY-HPβCD and AAZWCY-HPβCD), which exhibit favorable hydrophilicity, low plasma protein binding, high stability and non-toxicity. These attractive characteristics ensure that they are excreted rapidly, without any skin accumulation or metabolism in vivo. More importantly, zwitterionic HPβCD based agents can be efficiently filtrated by the glomerulus and completely excreted through the kidneys into urine without reabsorption or secretion in the kidney proximal tubule. Relying on these novel zwitterionic NIR agents and a transcutaneous device, we demonstrate a rapid, robust and biocompatible approach for assessing kidney function in rat models of both healthy rats and those with kidney disease, without the need for time-consuming blood/urine sample preparation. Our work provides a promising tool for in vivo real-time non-invasive kidney function assessment in preclinical applications.

Scheme 1. Structures of cyanine dyes and HPβCD based NIR agents.

Fig. 1. Absorption and emission spectra of (a) ZWCY (0.05 mg mL^–1), (b) ABZWCY (0.02 mg mL^–1), and (c) ABZWCY-HPβCD (2 mg mL^–1) in PBS. (d and e) High performance liquid chromatography (HPLC, monitored at 710 nm) spectra of ABZWCY-HPβCD and AAZWCY-HPβCD incubated with porcine liver esterase (PLE) for 24 h. (f and g) Cell viability of HK-2 human proximal tubular cells incubated with ABZWCY-HPβCD and AAZWCY-HPβCD in MTT assays.

Fig. 2. Elimination curves of ABANCY-HPβCD (a and b, n = 3), ABZWCY (c and d, n = 6), ABZWCY mixed with HPβCD (e, n = 3) and IRDye800CW (f, n = 2) by transcutaneous measurements in healthy rats in the absence and presence of probenecid, n means the number of rats. (g) Clearance half-life for ABANCY-HPβCD and ABZWCY in the absence and presence of probenecid. **P < 0.01.

Fig. 3. Elimination curves of ABZWCY-HPβCD (a and b, n = 8) and AAZWCY-HPβCD (c and d, n = 6; e, n = 2) by transcutaneous measurements in healthy rats in the absence and presence of probenecid or cimetidine, n means the number of rats. (f) Clearance half-life for ABZWCY-HPβCD and AAZWCY-HPβCD in the absence and presence of probenecid or cimetidine.

Fig. 4. (a) Urinary recovery for each marker in healthy rats within 24 h. Urinary recovery-time curves of (b) ABZWCY (n = 3), (c) ABZWCY mixed with HPβCD (n = 3), (d) ABZWCY-HPβCD (n = 3) and (e) AAZWCY-HPβCD (n = 3), n means the number of rats.

Fig. 5. In vivo biodistribution and clearance of zwitterionic HPβCD based agents in healthy rats. Saline (a and b), ABZWCY-HPβCD (c and d) and AAZWCY-HPβCD (e and f) were injected intravenously into rats 5 h prior to imaging. Abbreviations: Bl, bladder; He, heart; Ki, kidney; Li, liver; Lu, lung; Sp, spleen. (g) Relative fluorescence intensity of organs in panels b, d and f.

Fig. 6. Urinary albumin excretion (a, ***P < 0.001), protein excretion (b, ***P < 0.001) and clearance half-life of ABZWCY-HPβCD (c, **P < 0.01) in wild-type rats and AT1R transgenic rats. Elimination curves of ABZWCY-HPβCD by transcutaneous measurements in wild-type rats (d) and AT1R transgenic rats (e).