Abstract Body:
Gliomas, the most prevalent adult brain tumors1, and non-small cell lung cancer (NSCLC), the leading cause of cancer-related death in the US2, present significant surgical challenges due to difficulties in accurately delineating tumor margins. The objective of this research was to synthesize and characterize low molecular weight (MW) NIR-II fluorophores to enhance the efficacy of fluorescence-guided surgery (FGS) in the treatment of both glioma and NSCLC. Our hypothesis is that NIR-II fluorophores will provide sufficient tissue penetration and minimal background interference needed to offer precise tumor delineation. This is particularly critical in glioma surgeries, where the distinction between healthy and cancerous tissue is vital for successful patient outcomes.
A series of four 4,4’-quinocyanines (QCy) dyes were synthesized employing Knoevenagel condensations of N-alkyl lepidonium salts and purified through recrystallization. The quinolinium head group moiety extends the polymethine conjugation system allowing for the synthesis of NIR-II fluorophores with high water solubility and low cation MWs of <500 Da. The QCys exhibited broad absorption range (600-1000 nm), with absorption maxima at 942-970 nm, and maximum emission at 979-1010 nm, effectively situating them in the NIR-II region.The longest wavelength dye, JAM317 (λex= 970 nm, λem= 1010 nm, MW=480 Da), exhibited the highest fluorescence intensity and was chosen for further studies. JAM317, when encapsulated in liposomes showed high photostability when continuously exposed to a 970 nm laser at a power density of 165 mW/mm2 for one hour.
Tissue penetration analysis was performed using a Photon etc IR VIVO Scanner by covering the dyes with varying thickness of chicken breast tissue. The NIR-II emissions of JAM317 could be seen through 6 mm of chicken breast compared to NIR-I dyes JAS239 and ICG which could only be seen through 3 mm of chicken breast tissue. Cell studies revealed that both cultured U-87 MG glioma cells and KLN 205 NSCLC cells were able to uptake and retain JAM317.
In vivo studies were performed on the IR VIVO using athymic Nu/Nu mice injected with 40 nmol of JAM317 imaged with a 970 nm excitation laser and a 1000 or 1250 nm long pass emission filter. Enhanced vascular details were observed with the 1250 nm filter demonstrating the advantage of NIR-II dyes for deeper tissue penetration, lower light scattering and lower autofluorescence in vivo.
Fresh human glioma tumor tissue samples were obtained at surgery and treated ex vivo with 20 nmol of JAM317 using fresh normal mouse brain as a control. JAM317 exhibited an average tumor-to-normal fluorescence ratio of 14.5 (Figure 1), demonstrating its potential in improving tumor margin identification.
QCy dyes have low molecular weight and Blood-Brain Barrier (BBB) scores of 3.61-4.61 indicating their potential effectiveness in BBB penetration.3 These properties suggest that QCy dyes have strong potential for imaging deep brain tissue diseases and NSCLC within the NIR-II optical window.
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Image/Figure Caption:
Figure 1: Normal mouse brain and Human Glioma tissues were stained ex vivo with 20 nmol of JAM317. JAM317 preferentially stains human glioma samples over normal mouse brain tissue, with observed tumor-to-normal ratios of 14.55, 15.40, and 13.41, respectively in above image.
Author
Ritesh K. Isuri, B.A Chemistry and Biochemistry
University of Pennsylvania