Protease Activity-Dependent FRET-Based Turn-ON Fluorescent Nanoprobes for Image-Guided Surgery

INTRODUCTION

Metastasis to distant organs originated from a primary tumor is considered to be a major cause of cancer mortality. Thus, complete primary tumor removal, with no residual cancer cells at the incision margins of the tissue removed during surgery is crucial for patient prognosis and survival. To achieve this goal with minimal damage to healthy tissue, there is a need for diagnostic tools that will efficiently differentiate between the tumor and its normal surroundings¹.

MATERIALS AND METHODS

Novel FRET-based, polymeric Turn-ON probes were synthesized and characterized in vitro, for their hydrodynamic diameter, stability, toxicity and cellular internalization abilities. Furthermore, their quenching and enzymatically-dependent degradation properties were evaluated. Eventually, in vivo pharmacokinetics, tumor accumulation following i.v. administration and their ability to mark the tumor boundaries were evaluated utilizing an orthotopic melanoma model which spontaneously metastasizes to the brain and lungs.

RESULTS AND DISCUSSION

We recently designed, synthesized and characterized FRET-based polymeric Turn-ON probes which consist of biodegradable polyglutamic acid (PGA) nanocarrier bearing quenched Cy5 fluorescent dyes and dark quencher (Q) molecules. The probes are cathepsins-sensitive and sized to enable selective accumulation at the tumor site, utilizing the enhanced permeability and retention (EPR) effect, known to be a hallmark of tumors vasculature. This, together with specific enzymatic activation, allowed the fluorescence signal to generate in the tumor site, thus improving the signal to noise ratio (SNR). The final quenching degree was dependent on the Cy5 loading as well as on the quencher’s presence; when the conjugate bearing Cy5 in combination with a Quencher showed better signal silencing². The signal obtained from the tumor following i.v. injection of the probe to melanoma-bearing mice was consistently elevated with time suggesting its successful tumor accumulation followed by its degradation at the site of interest.

CONCLUSIONS

Our "smart" polymeric probes proposed herein can potentially assist surgeons’ decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.

REFERENCES

1. Blau R, Krivitsky A, Epshtein Y, Satchi-Fainaro R. Are nanotheranostics and nanodiagnostics-guided drug delivery stepping stones towards precision medicine? Drug Resist Update. Jul 2016;27:39-58.

2. Blau R^¥, Epshtein Y^¥, Pisarevsky E^¥, Tiram G, Israeli-Dangoor S, Yeini E, Krivitsky A, Eldar-Boock A, Ben-Shushan D, Gibori H, Scomparin A, Green O, Ben-Nun Y, Merquiol E, Doron H, Blum G, Erez N, Shabat D, Satchi-Fainaro R (2018). Image-Guided Surgery Using Near Infrared Turn-ON Fluorescent Nanoprobes for Precise Detection of Tumor Margins. Theranostics, 2018; 8(13): 3437-3460 ^¥ Equal contribution.