The synthesis of C′ dots was modified from previously reported protocols (25). More specifically, maleimide-functionalized dye was first dissolved in dimethyl sulfoxide (DMSO) under nitrogen at a concentration of around 0.01 M. The dye/DMSO solution was then mixed with (3-mercaptopropyl)trimethoxysilane (MPTMS) under nitrogen at a dye:MPTMS molar ratio 1:25. The mixture was left under nitrogen overnight to conjugate the dye to the silane group via a thiolene click reaction. Afterward, around 100 μl of the obtained dye-silane precursor was added together with 68 μl of tetramethyl orthosilicate into 10 ml of DI water at pH around 8. The reaction mixture was stirred at room temperature overnight, and 100 μl of PEG-silane (Gelest, molecular weight of around 500) was then added. The reaction mixture was further stirred at room temperature overnight, followed by heat treatment at 80°C overnight. Before particle characterization, the reaction solution was lastly cooled to room temperature, purified by GPC, and sterile filtered. Four types of dyes were used in this study, including Cy5.5, DY782, DY800, and CW800, for the synthesis of C′ dots with different NIR fluorescence characteristics. The synthesis conditions, e.g., reaction time and reagent concentrations, of C′ dots with different types of dyes were slightly varied to optimize both reaction yield and product performance (details not included).

For the synthesis of C′ dots that are further functionalized with targeting peptides, cysteine-functionalized targeting peptide and heterobifunctional PEG (mal-dPEG@12-NHS, Quantum Biodesign) were first separately dissolved in DMSO under nitrogen at a concentration of around 0.021 and 1.2 M, respectively. Mal-dPEG@12-NHS/DMSO solution was then mixed with 3-triethoxysilylpropylamine (APTES, liquid) under nitrogen at a mal-dPEG@12-NHS:APTES molar ratio of 1:0.9. The mixture was left under nitrogen overnight to conjugate the mal-dPEG@12-NHS molecule with a silane group via amine-NHS ester reaction. After that, peptide/DMSO solution was further added into the mixture under nitrogen at peptide:mal-dPEG@12-NHS:APTES molar ratios of 1.1:1:0.9. The mixture was then left under nitrogen overnight to further conjugate the mal-PEG-silane molecule with a targeting peptide via thiol-ene click reaction. Last, around 100 μl of the obtained peptide-PEG-silane precursor was added into the C′ dot synthesis solution right before the addition of PEG-silane to functionalize C′ dots with different types of targeting peptides. Two types of targeting peptides were used in this study, i.e., αMSH (23) and RGDY (22).

Each batch of the obtained NIR C′ dots was characterized by GPC, FCS, ultraviolet-visible (UV-vis) absorption and emission spectroscopy, and TEM. A BioLogic LP system (Bio-Rad) equipped with a 275-nm UV detector and a self-packed column (Superdex 200 resin, GE Healthcare Life Sciences) were used for GPC purification and characterization. A home-made multichannel FCS setup was used for the characterization of concentration, hydrodynamic size, number of dyes per C′ dot, and number of targeting ligand per C′ dot. A 635-nm solid-state laser was used for the FCS characterization of C′ dots labeled with Cy5.5, while a 780-nm solid-state laser was used for the FCS characterization of C′ dots labeled with either DY782, DY800, or CW800. UV-vis absorbance spectra were measured by a Cary 5000 spectrophotometer (Agilent), and emission spectra were measured by a QuantaMaster spectrofluorometer (Photon Technologies International). TEM images were taken using a T12 Spirit electron microscope (FEI).

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