NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics

Fluorescence probes have great potential to empower bioimaging, precision clinical diagnostics and surgery. However, current probes are limited to in vivo high-contrast diagnostics, due to the substantial background interference from tissue scattering and nonspecific activation in blood and normal tissues. Here, we developed a kind of cell endocytosis-activated fluorescence (CEAF) probe, which consists of a hydrophilic polymer unit and an acid pH-sensitive small-molecule fluorescent moiety that operates in the “tissue-transparent” second near-infrared (NIR-II) window. The CEAF probe stably presents in the form of quenched nanoaggregates in water and blood, and can be selectively activated and retained in lysosomes through cell endocytosis, driven by a synergetic mechanism of disaggregation and protonation. In vivo imaging of tumor and inflammation with a passive-targeting and affinity-tagged CEAF probe, respectively, yields highly specific signals with target-to-background ratios over 15 and prolonged observation time up to 35 hours, enabling positive implications for surgical, diagnostic and fundamental biomedical studies.

Preparation of CEAF-OMe Nanoaggregates. CEAF-OMe (5 mg) was dissolved in 1 mL of dimethyl sulfoxide. The solution was dispersed evenly under ultrasonication and then filtered with an organic filter membrane of 0.22 microns. 9 mL of deionized water was prepared in a clean glass bottle. The above CEAF-OMe solution was quickly added under rapid magnetic stirring. The reaction was stirred for five more minutes. The solution was collected and dialyzed three times (2 h per time) with dialysis tube (Mw=3500 Da) to obtain the CEAF-OMe nanoaggregates (0.5 mg mL -1 ).

Preparation of CEAF-RGD Nanoaggregates.
To the above CEAF-NHS nanoaggregates solution (1 eq), adding cRGDfK peptide (5 eq). The reaction was stirred for 12 h in the dark at room temperature.
The solution was collected and dialyzed three times (2 h per time) with dialysis tube (Mw=3500 Da) to obtain the CEAF-RGD nanoaggregates (0.5 mg mL -1 ).

Preparation of Lyso1005 micelles/boc-Lyso1005 micelles.
The nanomicelles were prepared using modified film hydration technique. In a typical procedure, Lyso1005/boc-Lyso1005 were firstly dissolved in DMSO to obtain stock solution with concentration of 5 mM. 100 μL (0.5 μmol) stock solution of Lyso1005/boc-Lyso1005 was mixed with 1.6 mL PEG-PCL (25 mg mL -1 in THF) at a mass ratio of ~1:100. The solvent was removed by vacuum rotary evaporation to form a dry dye-containing lipid film. The dried film was hydrated with 10 mL deionized water at 80 °C and sonication for 30 s to make the clear nanomicelles solution with dye concentration of 50 μM. The nanomicelle solution was S10 further concentrated if necessary by using a 30 K Amicon Ultra filter (Millipore Corporation) under centrifugation at 2000 g min-1 for 5 min.

Characterization
All 1 H-NMR and 13 C-NMR spectra were acquired on a Bruker AV-400 spectrometer. Chemicals shifts are referenced to the residue solvent peaks and given in ppm. MALDI-TOF MS analyses were performed in positive reflection mode on a 5800 proteomic analyzer (Applied Biosystems, Framingham, MA, USA) with a Nd: YAG laser. UV-Vis-NIR absorption spectra were recorded on Lamda750S PerkinElmer. NIR-II fluorescence spectra were recorded on Edinburgh Fluorescence Spectrometer FLS980 instrument or Horiba Fluorescence Spectrometer instrument (Fluorolog TM HORIBA Scientific) with external 808 nm (MDL-III-2W) and 940 nm (MDL-H-5W) semiconductor lasers (Changchun New Industries Optoelectronics Tech. Co., Ltd.) as excitation sources. In vivo NIR-II images were taken using NIRvana CCD camera (Princeton Instruments Inc.). Dynamic light scattering measurements were carried out on a Malvern Zetasizer 3600 (Malvern Instruments). Transmission electron microscopy (TEM) tests were performed using a High Contrast Transmission Electron Microscope (HT7800). NIR-II intravital imaging was carried out with an epifluorescence microscopy system with 640×512 pixel 2D InGaAs NIRvana camera. Flow cytometry analyses were performed using Beckman Coulter Gallios flow cytometer.

General procedure for absorbance and fluorescence determination
A series of standard pH buffer solutions (MeCN/H 2 O=1/1, v/v) were prepared by mixing 0.2 M Na 2 HPO 4 , 0.2 M KH 2 PO 4 and 0.2 M H 3 PO 4 at varied volume ratios, and the accurate pH values were measured by Delta 320 pH-meter. Then, 2 mL of the phosphate buffer and 5 µL of the stock solution of dyes (2 mM) in dimethyl sulfoxide solution were mixed. The resulting solution was mixed well, and an appropriate portion of the solution was transferred to a quartz cell of 1-cm optical length to measure absorbance against the corresponding reagent blank or fluorescence with λ ex = 808 nm.

S11
The calculation of pKa values was performed with Origin 2017 software (OriginLab, Northhampton, MA), using the Bolzmman fitting function: On the basis of the calculated equation of pKa, 0 corresponds to the pKa value.

Measurement of the stability of CEAF-OMe nanoaggregates
PBS, PBS with 0.015% Triton X100, simulated tissue fluid, FBS and blood of both pH=7.0 and pH=5.0 were prepared. 10 μL CEAF-OMe nanoaggregates (0.5 mg mL -1 ) was dissolved in the above solvents of 500 μL, respectively. The prepared samples were observed under 940 nm laser radiation ((laser output power density = 0.23 W cm -2 , working distance = 30 cm), 1000 nm, 1100nm and 1200nm long pass filters were used. Images were processed with the LightField imaging software, ImageJ and MATLAB.

Measurement of fluorescence quantum yield (QY)
Quantum yields ( fl ) were determined in various solvents relative to IR26 ( fl = 0.05% in DCE), [3] from plots of integrated fluorescence intensity vs. absorbance, according to the following relationship: where subscripts r and s denote standard and test sample, respectively, fl is the fluorescence quantum yield, is the slop of the integrated fluorescence intensity vs. absorbance plot, and is the refractive index of the solvent. Measurements were performed with the absorbance at 808 nm of all dye solutions＜ 0.1 in order to maximize illumination homogeneity and optical transparency. The 808 nm laser was used as the excitation source and the emission spectrum in the 850-1500 nm region was acquired in fluorescence spectrometer.

Quantum Calculations
All the quantum chemical calculations were done with the Gaussian 09 suite. [4] The geometry optimizations of the fluorophores were performed using density functional theory (DFT) with Becke's three-parameter hybrid exchange function with Lee−Yang−Parr gradient-corrected correlation functional (B3-LYP functional) and 6-31G(d) basis set. No constraints to bonds/angles/dihedral angles were applied in the calculations, and all atoms were free to optimize. The electronic transition energies and corresponding oscillator strengths were calculated with time-dependent density functional theory (TDDFT) at the B3LYP/6-311G (d, p) level.
Measurement of photo-stability S13 Lyso880, Lyso1005, Lyso855 and Lyso950 were dissolved in DMSO to obtain stock solutions of 5 mM.
ICG was dissolved in deionized water to obtain the stock solution of 5 mM. A certain amount of the above stock solutions were dissolved in the mixed solvent of acetonitrile and water (1:1, v/v) of both pH=7.0 and pH=5.0, respectively, to fixed their absorption at 808 nm to a same absorbance value (0.27).
All prepared samples were transferred to a quartz cell of 1-cm optical length to measure fluorescence with λ ex = 808 nm for 1000 s.

Measurement of chemical stability:
2 μL stock solutions (5 mM) of Lyso880, Lyso1005, Lyso855 or Lyso950 were added into the mixed solvent of acetonitrile and water (1:1, v/v, pH=7.0 or pH=5.0), respectively. Absorption spectra were recorded at 5 min after mixing with different amount of bioactive reagents solutions.

Optical setup for wield-field whole-body NIR-II imaging
NIR-II fluorescence images were acquired using a home-built imaging setup, in which the excitation light was provided by a 940 nm laser coupled to a 450-μm core metal-cladded multimode fiber (MDL-H-5W). The emitted light was directed from the imaging stage to the camera and passed through different filter sets (Thorlabs and Edmund Optics) as required by the experiments to ensure the images taken in S14 different sub-regions, and focused onto the thermo-electric cooling two-dimensional InGaAs camera (NIRvana: 640, 640 × 512 pixel; Princeton Instruments, response 900-1700 nm). The whole assembly was surrounded by a partial enclosure to eliminate excess light while enabling manipulation of the field of view during operation. Different exposure times were used to achieve sufficient signal intensities. All images were background and blemish corrected within the LightField imaging software, followed by processing with MATLAB or ImageJ software.

Optical setup for NIR-II epifluorescence microscopy
In

Flow cytometry
The mice were euthanized a day after ankle injury, and healthy mice were set as control. Then spleens and ankles of mice were separated off. Leukocytes in spleen were isolated by Mouse 1X Lymphocyte Seperation Medium (Dakewe, 7211011). The cell isolation procedures of ankles were performed according to the protocol as previous reported. [6] Then, the cells were stained with CD45, CD11b, F4/80 and CD86 antibodies (1:200, diluted with 1% FBS/PBS) for 30 min at room temperature. After staining, all cells were subjected to 300 mesh cell screens before analyzing in a Beckman Coulter Gallios flow cytometer.

HE staining
The CT26 tumor-bearing mice were euthanized after surgical resection surgery. And the traumatic ankle injury mice were dissected to isolate the ankles and spleens, which were then fixed with 4% paraformaldehyde for 24 h. Next, all samples were dehydrated with ethanol and embedded in paraffin before 6 µm sectioning. Then, sections of tissues were stained with hematoxylin and eosin.                          Figure S42. 13 C-NMR spectrum of E1 in CDCl 3 . Figure S43. 1 H-NMR spectrum of E3 in CDCl 3 . S44 Figure S44. 13 C-NMR spectrum of E3 in CDCl 3 .