Copper sulfate pentahydrate

Mesyl chloride (MsCl), sodium azide, triphenylphosphine (PPh 3 ), 2-iminothiolane hydrochloride, sucrose, N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), methoxy carboxy polyethylene glycol (mPEG-COOH), sodium ascorbate, Dulbecco's phosphate buffered saline (DPBS) buffer, DMF, pyridine, and methanol were obtained from Sigma-Aldrich (Steinheim, Germany). Chloroform was purchased from VWR (Radnor, PA, USA), celite, sulfur trioxide pyridine complex (SO 3 Py), and Tween20 from Acros Organics (Geel, Belgium), THF from Roth (Karlsruhe, Germany), N,N-diisopropylethylamine (DIPEA) from Fluka (Buchs, Switzerland), copper sulfate pentahydrate, N-ethyl-N 0 -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and ethylendiamine-tetraacetic acid (EDTA) from Merck (Darmstadt, Germany), deuterated solvents from Deutero (Kastellaun, Germany), soluble bovine collagen type II from abcam (Cambridge, UK), and ICC-labeled collagen II antibody from biorbyt (Cambridge, UK). All chemicals were reagent grade and used without further purification. Reactions sensitive to air or moisture were carried out under argon atmosphere using flame-dried glassware and anhydrous solvents. Ultrafiltration was performed in solvent-resistant stirred cells obtained from Millipore (Billerica, Massachusetts, USA) with PLAC-regenerated cellulose membranes (MWCO 1000 g mol À1 ). Dialysis was performed in benzoylated cellulose tubing purchased from Sigma-Aldrich (MWCO 2000 g mol À1 ) changing the solvent at least 5 times over a period of 48 h. SEC was performed with Sephadext G-25 superfine (Sigma Aldrich, Steinheim, Germany) in distilled water under room temperature and pressure. 1 H-, 13 C-, and 31 P-NMR spectra were recorded on a Jeol ECX 400 spectrometer or on a Bruker Biospin Avance 700 spectrometer. Chemical shifts d were reported in ppm using the deuterated solvent peak as the internal standard (CDCl 3 : The average polymer-to-dye ratio was calculated by UV-Vis spectra from a 6 mM solution in DPBS at pH 7.4 recorded on a LAMBDA 950 UV/Vis/NIR spectrometer (PerkinElmer, Waltham, MA, USA) at 25 1C by applying a molar extinction coefficient of 120 000 M À1 cm À1 for ICC l (552 nm) and of 100 000 M À1 cm À1 for 6S-ICG (l = 785 nm). IR measurements were recorded on a Nicolet Avatar 320 FT-IR equipped with a DTGS detector from 4000 to 650 cm À1 and evaluated with the software EZ OMNIC ESP. Wavenumbers n max were reported in cm À1 ; the intensities of the absorption bands were assigned as strong (s), medium (m), and weak (w). Combustion analysis was performed on a VARIO EL III instrument (Elementar, Hanau, Germany) using sulfanilic acid as the standard. DLS and z-potential measurements were carried out on a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, U.K.) equipped with a 4 mW He-Ne laser (l = 633 nm, NIBS) that operated with a 1731 scattering angle (backscatter). Hydrodynamic diameters were determined in UV-transparent disposable cuvettes (UltraVette, 8.5 mm, Brand, Wertheim, Germany) at 25 1C. Samples were dissolved in DPBS (DPBS, 1Â, without Ca 2+ , Mg 2+ , pH = 7.4, Sigma-Aldrich, Steinheim, Germany) at a concentration of 2 mg mL À1 . The stated values are the mean of at least 15 independent measurements. z-Potential measurements were conducted at a concentration of 2 mg mL À1 in phosphate buffer (PB, 10 mM, pH 7.4) at 25 1C in folded DTS 1060 capillary cells (Malvern Instruments Ltd, Worcestershire, U.K.). Data evaluation was performed with Malvern Zetasizer Software 6.12. The stated values and standard deviations are the mean of at least five independent measurements with 15 scans each and are based on the Smoluchowski model. ICP-MS measurements were conducted on an Element 2 (Thermo Scientific, Waltham, MA, USA).