Ultrabright NIR‐II Emissive Polymer Dots for Metastatic Ovarian Cancer Detection

Abstract Intraoperative diagnosis of metastatic tumors is of significant importance to the treatment of ovarian cancer. NIR‐II fluorescence imaging holds great promise for facile detection of tumor in situ with high sensitivity and resolution. Herein, a kind of NIR‐II fluorescent polymer dots (NIR‐II Pdots) with high brightness is developed for real‐time detection of metastatic ovarian cancer via NIR‐II fluorescence imaging. The NIR‐II Pdots are constructed via the self‐assembly of NIR‐II emissive aggregation induced emission luminogens (NIR‐II AIEgens) and poly (styrene)‐graft‐poly(ethylene glycol) in water. Such NIR‐II Pdots show very high fluorophore contents of nearly 30% and high quantum yield of 5.4% at emission maximum near 1020 nm. Further modification of the NIR‐II Pdots with targeting peptides yields NIR‐II Pdots‐GnRH, which can afford enhanced affinity of NIR‐II Pdots to ovarian cancer. Upon intravenous injection of the NIR‐II Pdots, whole‐body organs and vessels, peritoneal and lymphatic metastases of ovarian cancer are clearly visualized by NIR‐II fluorescence imaging. Under the guidance of NIR‐II fluorescence imaging, the metastatic foci with the diameter down to ≈2 mm can be facilely eliminated. The results indicate preclinical potential value of the NIR‐II Pdots for metastatic ovarian cancer detection.


Calculation of single probe mass
For inorganic nanoparticle: M NPs = xM A +yM B +zM C … (1) Ratio of mass content = xM A : yM B : zM C … (2) V NPs = xV A +yV B +zV C …, means that r NPs 3 = xr A 3 +yr B 3 +zrC 3

… (3)
Where M NPs is the single particle mass of inorganic nanoparticle; M A , M B , M C ... are the single atom mass of different element which doped in nanoparticle (can be obtained by molar mass to N A -6.0210 23 of different element); x, y, z are the average number of different element atoms in single nanoparticle; V NPs is the single particle volume of inorganic nanoparticle; V A , V B , V C are the single atom volume of different element which doped in nanoparticle; r NPs is the single particle semidiameter of inorganic nanoparticle (can be determined by TEM and DLS characterization, the morphology of nanoparticles are seen as sphere); r A , r B , rC are the single atom semidiameter of different element which doped in nanoparticle. According to (2) and (3), the value of x, y, z can be achieved, then the value of M NPs can be obtained.
For organic nanoparticle: M ONPs ≈ ρV ONPs = ρ4πr 3 ONPs /3 (4) Where M ONPs is the single particle mass of organic nanoparticle; ρ is the density of organic nanoparticles (the value of ρ is nearly to the density of water, 1.00 g/mL); V ONPs is the single particle volume of organic nanoparticle; r ONPs is the single particle semidiameter of organic nanoparticle (can be determined by TEM and DLS characterization, the morphology of nanoparticles are seen as sphere).

Calculation of the dye contents in organic nanoparticles
A certain volume of aqueous solution contained organic nanoparticles are dried. The weight of the nanoparticles powder is measured for mass concentration calculation, termed C NPs . The nanoparticles powder is then dissolved in a certain volume of tetrahydrofuran (THF), and BBTD are extracted in THF. The absorbance of BBTD at 760 nm in THF was measured.
According to the extinction coefficient of BBTD in THF at 760 nm (1.510 4 M -1 cm -1 ), the molar concentration of BBTD (C dye-molar ) in THF can be obtained. Then the dye content of different organic nanoparticles can be calculated by following equation: Where m dye is the mass of BBTD in THF solution; m NPs is the mass of the organic nanoparticles in THF solution; M dye is the molar mass of BBTD; C dye-molar is the molar concentration of BBTD in THF solution; C NPs-mass is the mass concentration of organic nanoparticles in THF solution (according to the C NPs ).

Calculation of single probe brightness
Where is the extinction coefficient of a single luminescent probe; is the luminescence quantum yield of luminescent probe; when the probe is an organic nanoparticle, is the number of dyes in a single luminescent organic nanoprobe; is the extinction coefficient of a single dye; N A is the avogadro's number-. For organic nanoparticles, when the dye contents and diameter of organic nanoparticles have been determined, can be calculated by following equation.

[ ]
Where is the molar mass of organic nanoparticle; is the molar mass of doped dyes.

Fluorescence quantum yield measurement
To measure the quantum yield of NIR-II Pdots, the reference fluorophore is IR26 in DCE (QY = 0.5%), Ex = 730 nm. The quantum yield was calculated in the following manner.
Difference concentrations at or below OD 0.1 were measured and the integrated fluorescence was plotted against absorbance for every fluorescent molecular. Comparison of the slopes led to the determination of the quantum yield of NIR-II Pdots.

Cytotoxicity test
The in vitro cytotoxicity was measured using a standard methyl thiazolyl tetrazolium (MTT, reader was used to measure the OD570 (absorbance value) of each well referenced at 490 nm.
The following formula was used to calculate the viability of cell growth: Viability (%) = (mean of absorbance value of treatment group / mean of absorbance value of control) × 100 Table S1. Photophysical properties of the reported NIR-II luminescent probes.  Figure S1. Absorption and emission spectra of BBTD in toluene.