Sulfo cy5.5 nhs ester

Gold chloride trihydrate (HAuCl₄·3H₂O, G4022), FITC–dextran (20 kDa, ~3.3 nm, FD20S), cell culture-grade dimethyl sulfoxide (DMSO, D4540) and d-glucosamine (2DG, G4875) were purchased from Sigma-Aldrich. High-purity bovine α-LA (Bos taurus) was provided by Agropur (Ca²⁺ < 0.055%). The purity was further verified by SDS–PAGE on a Novex NuPAGE (NP0335PK2, Invitrogen) 4−12% Bis-Tris precast protein gel using SimplyBlue SafeStain solution (LC6065, Invitrogen) for Coomassie Brilliant Blue G-250 staining. EIPA (3378) and cytochalasin D (1233) were obtained from Tocris Bioscience. NVP-BEZ235 (SYN-1018), wortmannin (AC32859) and staurosporine (ALX-380-014) were bought from AdipoGen, Acros Organics and Enzo Life Sciences, respectively. Sulfo-Cy5.5 NHS ester was purchased from Lumiprobe. Methanol-free paraformaldehyde (PFA; 16%, 0219998380) was bought from MP Biomedicals. All of the chemicals were analytical grade unless otherwise noted. Ultrapure 18.2 MΩ-cm Milli-Q water was used throughout the study. All PBS used in the study was 1× and diluted from 10× stock (BP3994, Thermo Fisher Scientific) as Ca²⁺ and Mg²⁺ free 11.9 mM pH 7.4 with 137 mM NaCl and 2.7 mM KCl, unless otherwise specified.

Tumor-specific uptake of nanoparticles was enabled by fluorescent labeling of CP-PTX at the N-terminal amine on the micelle corona. SulfoCy5.5 NHS ester (Lumiprobe) was first dissolved in 500 μL of DMSO in 4.5 mL of PBS. The fluorophore was then reacted at an 8:1 molar ratio of CP-PTX for 12 h at 4°C. The reaction mixture was purified using 10 kDa MWCO Amicon- Ultracentrifugation filter units (Millipore). Final formulation concentration was evaluated by measuring the relative absorbance at 280 nm (ELP, $\mathrm{\epsilon }=5690$ ) and 675 nm (sulfoCy5.5, $\mathrm{\epsilon }=195000$ ). SulfoCy5.5 concentration was determined to be 113.6 μM.
BxPc3-luc2 tumors were grown subcutaneously on the hind flank of 15 athymic nu/nu mice. Upon reaching a target size of 100 mm³, mice were randomized into three groups $(n=5)$ and received either ¹³¹I-ELP treatment at 10 μCi/mm³, hypofractionated EBRT therapy, or remained untreated. SulfoCy5.5-CP-PTX was injected i.v. at ~25 mg/kg on the initial day of treatment with a subsequent dose 7 days later. Fluorescent flux was tracked at the tumor site over time using the IVIS Lumina XR (Perkin Elmer) and normalized to individual tumor sizes as measured with calipers.