A carbohydrate-grafted nanovesicle with activatable optical and acoustic contrasts for dual modality high performance tumor imaging

High-performance illumination of subcutaneous tumor and liver tumor foci was achieved with sialic acid-targeted acid-responsive nanovesicles which become fluorescent and photoacoustic upon internalization into tumor lysosomes.

Changsha Red was syntheszied following a published procedure. 4 pNIR was prepared following a reported procedure. 1 Briefly, Changsha Red (5 g) was added to a flask containing methanol (100 ml). To the solution in ice bath was added thionyl chloride (10 ml) dropwise. The resultant solution was heated at 70℃ for 12 h. The solution was concentrated by rotary evaporation to remove the solvent and and the resulting residue was dissolved in methanol (20 ml). To the solution was added ethylenediamine (10 ml). The reaction solution was heated at 70℃ for 1 h and then concentrated by evaporation. The residue was purified on silica gel chromatography using dichloromethane/hexanes/triethylamine (10:10:1) as the eluent to give pNIR as a pale yellow solid in 36% yield. The analytical data of pNIR are identical to reported values. 1  To a flask containing anhydrous dimethylformamide (DMF, 10 ml) was added poly[(styrene-alter-(maleic anhydride)] (700 mg, mw: 10, 000) in the presence of triethylamine (1 ml). The solution of pNIR (100 mg) in anhydrous DMF (1 ml) was dropwise added and the reaction was stirred for 2 h. After that, the solution was divided into 2 equal portions to which was respectively added 9-Amino-9deoxy-5-N-acetylneuraminic acid (9-Amino-SA, 175 mg) or no addition. The mixtures were stirred at rt for overnight followed by addition of aqueous Na 2 CO 3 solution (1 M, 5 ml). The mixtures were first stirred at rt for 1 h and then extensively dialyzed against distilled water using a dialysis tube (MWCO 3500) to remove excess reagents and DMF. The solutions were respectively lyophilized and the resultant solids were dissolved in distilled water and then ultrasonicated for 30 min to afford pNIR@P@SA or pNIR@P. The aqueous solutions of these micelles (1 mg ml -1 ) were respectively characterized by Zetasizer Nano ZS for their hydrodynamic sizes and zeta potentials. The aqueous solutions of these micelles (10 mg ml -1 ) were used for the in vivo experiment. In parallel experiment, to a flask containing anhydrous dimethylformamide (DMF, 10 ml) was added poly[(styrene-alter-(maleic anhydride)] (700 mg, mw: 10, 000) in the presence of triethylamine (1 ml). and then 9-Amino-SA (175 mg). The mixtures were stirred at rt for overnight followed by addition of aqueous Na 2 CO 3 solution (1 M, 5 ml). The mixtures were first stirred at rt for 1 h and then extensively dialyzed against distilled water using a dialysis tube (MWCO 3500) to remove excess reagents and DMF. The solutions were respectively lyophilized and the resultant solids were dissolved in distilled water and then ultrasonicated for 30 min to afford P@SA.

pH titration of pNIR@P@SA and pNIR@P
Aliquots of stock solution of (10 l, 10 mg ml -1 in distilled water) or @SA (10 l, 10 mg ml -1 in distilled water) were respectively added to sodium phosphate buffers (100 mM, 1 ml) of various pH containing 10 % acetonitrile (V/V). The fluorescence emission of the solutions was recorded as a function of buffer pH using λex@715 nm. The titration curves were plotted by fluorescence emission intensities@745 nm versus pH. UV-vis-NIR absorption of the solutions was recorded as a function of buffer pH. The titration curves were plotted by absorbance@720 nm versus buffer pH.

Cytotoxicity of pNIR@P@SA and pNIR@P
HeLa cells were cultured in DMEM containing various levels (0, 25, 50, 75, 100 g ml -1 ) of pNIR@P@SA or pNIR@P for 24 h at 37 ℃ with 5% CO 2 . The cells were stained with trypan blue. Cell number and cell viability were determined using the trypan blue exclusion test. In an separate assays, sodium phosphate buffer (100 mM, pH 4.5) was respectively spiked with or without P@SA (1 mg ml -1 ) and pNIR (0.1 mg ml -1 ). The solutions were irradiated with NIR laser for 10 min (660 nm, 0.5 W cm -2 ) and the temperature of the solutions was recorded over irradiation time. pH titration of optoacoustic response of pNIR@P@SA pNIR@P@SA was spiked into a serial of sodium phosphate buffer (100 mM, pH 4.5, 5.5, 6.5, 7.5, 8.5

Fluorescence imaging of subcutaneous tumors in mice with pNIR@P@SA
and 9.5) to a final concentration of 1 mg ml -1 . The solutions were imaged for optoacoustic signal intensity.

Photoacoustic imaging of subcutaneous tumors with pNIR@P@SA
Nude mice were xenografted by subcutaneous injections of H22 cells (1x10 6 ). At 5-10 days after the transplantation, a cohort of tumor-bearing mice were injected intravenously via the tail vein with pNIR@P@SA (40 mg kg -1 ) or PBS (100 l). The mice were imaged for photoacoustic signals before and 24 h post-injection.

Cytotoxicity of pNIR@P@SA
For cell toxicity: HeLa cells were respectively cultured for 24 h in DMEM medium containing various levels of pNIR@P@SA (0-100 µg ml -1 ) or pNIR@P (0-100 µg ml -1 ). The cell number and cell viability were determined by trypan blue exclusion assay.
For systemic toxicity: A healthy mice was intravenously injected with pNIR@P@SA (160 mg kg -1 ) via tail vein. The mice was monitored regularly for whole body NIR fluorescecne and adverse physiological effects. At 7 days post-injection, the mice was sacrificed and the tumor and selected organ was harvested and examined for ex vivo NIR fluorescence.