Cy5.5 mono nhs ester

Doxorubicin (Dox) was purchased from LC Laboratories (Woburn, MA) and functionalized with NHS-TCO-NHS linker as described below. Dox and Dox-TCO-NHS were resuspended in phosphate buffer solution (PBS, 100 mM sodium phosphate, 150 mM NaCl, pH 7.2, working buffer) for the bioconjugation studies. Tetrazine was synthesized according to previous reports.^{[4c (link),14 (link)]} The nanoparticles (NPs) with an average particle size of 32 nm were synthesized according to our previous reports^{[15 (link)]} and the brief procedure described below. The modified NPs were purified using Sephadex PD-10 columns purchased from GE Healthcare (Troy, NY). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Corning Life Sciences (CellGro, Corning, NY) and supplemented with fetal bovine serum (FBS, Gibco, Thermo Fischer Scientific, Waltham, MA) and streptomycin/penicillin (Life Tech Corp., Grand Island, NY). Cy5.5-mono NHS ester was purchased from GE Healthcare and attached to the nanoparticles as described below.

Iron (III) chloride, anhydrous (98%) was purchased from Alfa-Aesar. Oleic acid (technical grade, 90%), 1-octadecene (technical grade, 90%), chloroform (HPLC, ≥99.8%), methanol (HPLC, ≥99.9%), poly(maleic anhydride-alt-1-octadecene) (Mn 30,000–50,000), N-(3-Dimethylaminopropyl)-N ′ - ethylcarbodiimide hydrochloride (commercial grade, powder), N-Hydroxysulfosuccinimide sodium salt (≥98%, HPLC), lactoferrin from human milk (≥85%, SDS-PAGE, lyophilized powder), paraformaldehyde powder (95%), potassium hexacyanoferrate (II) trihydrate, DAPI, alminum sulfate hydrate (98%), Dulbecco's phosphate buffered saline, and dimethyl sulphoxide (DMSO) Hybri-Max™ were purchased from Sigma-Aldrich. Sodium hydroxide solution 1N, hydrochloric acid 1N solution, and sodium bicarbonate were purchased from Fisher Scientific. Dulbecco's modified Eagle medium (DMEM) and 0.25% trypsin-EDTA were purchased from Gibco. Coomassie protein assay reagent was purchased from Thermo Scientific. All the reagents were used without further purification. Amicon^® Ultra centrifugal filters ultracel^®-50K was purchased from Merck Millipore. Cy5.5™ Mono NHS Ester was purchased from GE Health Care.