Effect of morpholine and charge distribution of cyanine dyes on cell internalization and cytotoxicity

Sirilak Wangngae,Pornthip Piyanuch,Montarop Yamabhai,Kantapat Chansaenpak,Parinya Noisa,Rung-Yi Lai,Oratai Weeranantanapan,Thitima Sumphanapai,Anyanee Kamkaew

doi:10.1038/s41598-022-07533-5

Abstract

To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and synthesized two novel Hcyanines based theranostic probes, IR794-Morph and IR794-Morph-Mpip, to enhance cancer cell internalization and targeting. In acidic conditions that resemble to tumour environment, both IR794 derivatives exhibited broad NIR absorption band (704‒794 nm) and fluorescence emission (798‒828 nm) that is suitable for deep seated tumour imaging. Moreover, in vitro study revealed that IR794-Morph-Mpip exhibited better cancer targetability towards various cancer cell lines under physiological and slightly acidic conditions compared to normal cells. IR794-Morph-Mpip was fast internalized into the cancer cells within the first 5 min and mostly localized in lysosomes and mitochondria. In addition, the internalized signal was brighter when the cells were in the hypoxic environment. Furthermore, cellular uptake mechanism of both IR794 dyes, investigated via flow cytometry, revealed that endocytosis through OATPs receptors and clathrin-mediated endocytosis were the main routes. Moreover, IR794-Morph-Mpip, displayed anti-cancer activity towards all tested cancer cell types with IC50 below 7 μM (at 6 h incubation), which is approximately three times lower than that of the normal cells. Therefore, increasing protonated cites in tumour environment of Hcyanines together with incorporating morpholine in the molecule can enhance structure-inherent targeting of these dyes.

Highlights

To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and synthesized two novel Hcyanines based theranostic probes, IR794-Morph and IR794-MorphMpip, to enhance cancer cell internalization and targeting
There is no clear reason why they tend to accumulate in solid tumors, uptake into cancer cells via organic anion transporting polypeptides (OATPs)[10–12] and roles of albumin in Hcyanines accumulation and persistence in solid tumor were d iscussed[6,13,14]
If the particles could enter the cells via endocytosis, it has a high possibility to accumulate at lysosomes and trigger cellular cascades that can be harmful to the cells[29]

Summary

Introduction

To improve the potency of Heptamethine cyanines (Hcyanines) in cancer research, we designed and synthesized two novel Hcyanines based theranostic probes, IR794-Morph and IR794-MorphMpip, to enhance cancer cell internalization and targeting. In vitro study revealed that IR794-Morph-Mpip exhibited better cancer targetability towards various cancer cell lines under physiological and slightly acidic conditions compared to normal cells. Increasing protonated cites in tumour environment of Hcyanines together with incorporating morpholine in the molecule can enhance structure-inherent targeting of these dyes. The uptake of Hcyanines in cancer cells was concerted actions exerted by hypoxia and activation of HIF1alpha/OATPs signalling leading to enhance dye uptake[9,15]. An idea of developing structure–inherent targeting (SIT) NIR fluorescent dyes was proposed[5,21,22]. This offers a new opportunity to realize targeting delivery with no need of extra conjugations. If the particles could enter the cells via endocytosis, it has a high possibility to accumulate at lysosomes and trigger cellular cascades that can be harmful to the cells[29]

Methods

Results

Conclusion