Investigation of oxazine and rhodamine derivatives as peripheral Nerve tissue targeting contrast agent for in vivo fluorescence imaging

Abstract

Accidental nerve transection or injury is a significant morbidity associated with many surgical interventions, resulting in persistent postsurgical numbness, chronic pain, and/or paralysis. Nervesparing can be a difficult task due to patient-to-patient variability and the difficulty of nerve visualization in the operating room. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures in real time during surgery could greatly improve patient outcomes. To date, all nerve-specific contrast agents emit in the visible range. Developing a nearinfrared (NIR) nerve-specific fluorophore is poised to be a challenging task, as a NIR fluorophore must have enough "double-bonds" to reach the NIR imaging window, contradicting the requirement that a nerve-specific agent must have a relatively low molecular weight to cross the blood-nerve-barrier (BNB). Herein we report our efforts to investigate the molecular characteristics for the nerve-specific oxazine fluorophores, as well as their structurally analogous rhodamine fluorophores. Specifically, optical properties, physicochemical properties and their in vivo nerve specificity were evaluated herein.

In vivo nerve-specificity of rhodamines and Si-rhodamines

“…Previously, it was noted that the lipophilicity (LogD) of some nerve-specific fluorophores fell within an approximate range of 3-5, with several pan fluorescent compounds possessing LogD values greater than 5. 21 Here the loss of nerve specificity outside this range was notably observed to extend to compounds with LogD values less than 2. While this study further highlights the difficulty involved in designing NIR nerve-specific fluorophores as well as the need for further study into their biomolecular target, it also eliminates potential candidates by demonstrating the dramatic effect observed from such a seemingly small structural modification and serves to corroborate previously observed trends suggesting an important role lipophilicity might play in facilitating tissue uptake.…”

“…It had been previously speculated that compound lipophilicity was a potential factor contributing to nerve-specificity, noting that nerve-specific Rhodamine B and Oxazine 4 both had LogD values between 3-5, while the remaining pan-fluorescent compounds had values greater than 5. 21 Although the calculated LogD value of TMR, 2.19, is just outside this range, it was noteworthy that Si-rhodamine B and Si-TMR had much smaller values of 0.84 and −0.53, respectively. This may suggest not only an upper limit of 5 in accordance with Lipinski's rules, but also a lower limit less than two may be optimal for maintaining nerve-specificity.…”

“…90 nm in emission relative to its parent fluorophore can be elicited through replacement of the bridging oxygen at position 10 in the rhodamine structure with a silicon atom. 9,[21][22][23][24] This strategy has been successfully applied in rhodamine, fluorescein, and rhodol scaffolds with notably larger shifts observed in some cases upon replacement of a sulfur atom in the form of a sulfone. 24 These observations have been attributed to the difference in the energy gap between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), where the additionally electropositive inductive effect of the dimethylsilyl group relative to oxygen accounts for an increase in the HOMO.…”

“…A comparison was made to determine whether two previously reported compounds, Si-TMR and Si-rhodamine B, would maintain the nerve-specific properties previously observed in structurally analogous rhodamine derivatives. 20,21…”

State of the Art: Precision Surgery Guided by Intraoperative Molecular Imaging

“…These tracers fluoresce and can easily be visualized using optical imaging instrumentation, making it possible to accurately identify tumors intraoperatively, remove them with negative margins, and even identify additional lesions that were not visualized preoperatively (2). Fluorescent tracers can also help surgeons identify and avoid vital structures such as nerves and vasculature (3).…”