Research ArticleAPPLIED SCIENCES AND ENGINEERING

Albumin-chaperoned cyanine dye yields superbright NIR-II fluorophore with enhanced pharmacokinetics

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Science Advances  13 Sep 2019:
Vol. 5, no. 9, eaaw0672
DOI: 10.1126/sciadv.aaw0672
  • Fig. 1 Optimization of the assembly between cyanine dyes and albumin produced an efficient NIR-II complex.

    (A) Simplified design for the formation of the fluorophore@BSA complex. (B) Kinetic binding assay of IR-783 to albumin was measured by biolayer interferometry with Kd value ~1 nM. (C) Brightness enhancement after heating for 10 min of dyes premixed with BSA (100 μM). (D) Absorption and NIR-I and NIR-II emission of IR-783 in 100 μM BSA solution. Ab., absorption spectra; Si Em., emission spectra recorded on silicon camera; InGaAs Em., emission spectra recorded on InGaAs camera. (E) Fluorescence enhancement of the fluorophore (IR-783, IR-12N3, and ICG) @BSA complex (1:1 ratio) at 60°C. The IR-783:BSA complex is the most stable, with a fluorescence ratio closest to 1. GTD, glutaraldehyde. (F) Electrophoresis analysis of IR-783@BSA complexes (C1 to C4 were synthesized with 10-min posttreatment at 60°C). C2 and C3 have a large population close to the top compared with C1 and C4. (G) Electrophoresis gel analysis of IR-783@BSA complexes [prepared from C2: glutathione (GSH) and heat] with 10 min posttreatment at different temperatures [room temperature (RT), 50°, 60°, and 70°C]. At higher temperatures, a higher–molecular weight complex was observed.

  • Fig. 2 The possible interaction mechanism between IR-783 and albumin is critical to guide efficient nanocomplex synthesis and fluorescence enhancement.

    (A) Docking modeling for the IR-783@albumin complex. (B) Details of binding residues and active pocket between IR-783 and albumin. (C and D) The intermolecular stacking of IR-783 and ICG. ICG showed relatively higher tendency to be self-assembling compared with IR-783. (E) Docking modeling for IR-783 and albumin with partial disulfide bond cleavage, affording strong interaction between IR-783 and the Trp residue of albumin. (F) Schematic illustration of the transformation of TICT induced NIR-I and NIR-II states of IR-783, including the HOMOs and LUMOs and the ESP maps that are plotted using the color range from red [−0.119 electron (e), negative] to blue (0.119 electron, positive). S0, ground state; S1, excited state. (G) The LUMO (top), HOMO (middle), and HOMO-1 (bottom) are the three major molecular orbitals involved in the absorption (HOMO-1 → LUMO) and emission (LUMO→HOMO) processes. HOMO-1 lies on the indole ring of Trp214, while LUMO and HOMO are lying on the closer and further side of IR-783 with respect to Trp214. (H) The absorption and emission form a cycle with three steps: (1) vertical excitation (HOMO-1 → LUMO), (2) charge transfer from IR-783 to Trp214 (HOMO → HOMO-1), and (3) emission (LUMO → HOMO).

  • Fig. 3 The IR-783@BSA complex enables high-contrast, real-time NIR-II vessel imaging.

    (A) The recorded NIR-II video of hindlimb vessels in mouse at several time points p.i. of the IR-783@BSA complex at 1300-nm long-pass (LP) filters. (B) PCA analysis from the real-time video imaging distinguished both vein and artery vessels, as well as small vessels and background. (C) The monitor of fluorescence intensity profile of five selected vessels in NIR-II video imaging of (A). High contrast between the vessel and background is maintained for up to ~30 min in the NIR-II imaging window. The red dotted square is the ROI of vessel, and the cyan circle is the ROI of muscle. a.u., arbitrary units. (D) Vessel-to-muscle signal ratio at 30 min p.i. showed high-contrast value up to 6. (E) The high brightness of the IR-783@BSA complex affords low-power LED-excited hindlimb imaging at wide NIR-II subwindows. The low-power LED light (10 to 50 mW/cm2) is more suitable for potential clinical use. The dotted line shows a representative cross-sectional profile of hindlimb. SP, short-pass. (F) The cross-sectional intensity profile of hindlimb in (E) (the dotted line). BKG, background. (G) LED-excited imaging shows equal vessel-to-muscle signal ratio [ROI 1 to 4 in (E)] with 785-nm laser excited imaging. (H) Scheme of LED-excited NIR-II imaging system. (I) Scheme of prolonged vessel circulation of the IR-783@BSA complex compared with free IR-783.

  • Fig. 4 NIR-II microscope imaging of the IR-783@BSA complex affords high-performance vessel imaging with both high resolution and contrast.

    (A) Ex vivo NIR-II microscope imaging of mouse brains at 1 hour p.i. of IR-783@BSA at both NIR-I and NIR-II windows. (B) Cross-sectional intensity profile of both NIR-I and NIR-II images at the same position. NIR-II imaging affords two times enhancement of SBR. (C) In vivo NIR-II whole-body (2.5×), vessel scheme and ex vivo microscope imaging of shaved/sectioned mouse head/brains after IR-783@BSA complex administration over 1300- and 1200-nm long-pass filters, respectively. (D) Cross-sectional intensity profile of both IR-783@BSA complex and free IR-783 images. (E) Vessel–to–normal tissue ratio statistics of both IR-783@BSA complex and free IR-783 images indicated that the IR-783@BSA complex exhibited super vessel imaging capacity.

  • Fig. 5 The complex of antibody and dye can afford to target imaging in the NIR-II window.

    (A) Cell assay (lysate) test of the IR-783@Erbitux complex at both positive SCC cell and negative U87 cell. (B) SCC cell staining of IR-783@Erbitux described the reserved targeting ability of an antibody-based complex. DAPI, 4′,6-diamidino-2-phenylindole. (C) In vivo NIR-II imaging of an SCC tumor–bearing mouse with tail vein injection of the IR-783@Erbitux complex. (D) In vivo NIR-II imaging of an SCC tumor–bearing mouse with tail vein injection of free IR-783 under the same imaging conditions as the IR-783@Erbitux case. (E) NIR-II tumor signal of both IR-783@Erbitux and free IR-783 at increasing time points p.i. (significant difference between two groups was shown as *P < 0.05, **P < 0.01, and ***P < 0.001). Insets show tumors from different treatment cohorts with a lower z scale. (F) Ex vivo biodistribution of the IR-783@Erbitux complex at 168-hour time points p.i. MFI, mean fluorescence intensity.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw0672/DC1

    Fig. S1. Relative QYs of IR-783, IR-12N3, and ICG in BSA/FBS, depicted by the slope of fluorescence intensity versus absorption.

    Fig. S2. Method optimization for the fluorophore@BSA complex.

    Fig. S3. The optimization for ratio and reaction concentration of BSA and IR-783.

    Fig. S4. Stability, brightness, and size of the IR-783@BSA complex at 1:0.5, 1:1, and 1:2 ratios.

    Fig. S5. Docking simulation and computational modeling demonstrating the binding poses and the mechanism of increased QY for complexation.

    Fig. S6. The free IR-12N3/ICG/IR-783 has quick hepatobiliary clearance and short imaging window compared to their complex with albumin.

    Fig. S7. Comparison of the vessel imaging time window of free IR-783 with the IR-783@BSA complex.

    Fig. S8. Further imaging comparison of IR-783@BSA from different conditions (C2, C3, and C4).

    Fig. S9. NIR-II QY of the IR-783@BSA complex and NIR-II vessel imaging in whole-body mode.

    Fig. S10. The IR-783@Erbitux complex afforded an efficient conjugate and decent targeting ability for molecular imaging.

    Table S1. The excitation energies for both vertical excitation and emission computed using TDDFT/IEFPCM in complex mode.

    References (4152)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Relative QYs of IR-783, IR-12N3, and ICG in BSA/FBS, depicted by the slope of fluorescence intensity versus absorption.
    • Fig. S2. Method optimization for the fluorophore@BSA complex.
    • Fig. S3. The optimization for ratio and reaction concentration of BSA and IR-783.
    • Fig. S4. Stability, brightness, and size of the IR-783@BSA complex at 1:0.5, 1:1, and 1:2 ratios.
    • Fig. S5. Docking simulation and computational modeling demonstrating the binding poses and the mechanism of increased QY for complexation.
    • Fig. S6. The free IR-12N3/ICG/IR-783 has quick hepatobiliary clearance and short imaging window compared to their complex with albumin.
    • Fig. S7. Comparison of the vessel imaging time window of free IR-783 with the IR-783@BSA complex.
    • Fig. S8. Further imaging comparison of IR-783@BSA from different conditions (C2, C3, and C4).
    • Fig. S9. NIR-II QY of the IR-783@BSA complex and NIR-II vessel imaging in whole-body mode.
    • Fig. S10. The IR-783@Erbitux complex afforded an efficient conjugate and decent targeting ability for molecular imaging.
    • Table S1. The excitation energies for both vertical excitation and emission computed using TDDFT/IEFPCM in complex mode.
    • References (4152)

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