Monosodium phosphate

The polymer PEG-b-PLGA (MW: 5k–10kDa) and suite of PTX-silicate prodrugs (see Table 1) were synthesized as previously reported.^{13 (link), 15 (link)} Paclitaxel was obtained from Phytogen Life Sciences. Water (H₂O, HPLC grade), tetrahydrofuran (THF, HPLC grade), and acetonitrile (ACN, HPLC grade) were purchased from Sigma-Aldrich and used as received. CDCl₃ was purchased from Cambridge Isotope Laboratories, Inc. Ethanol (anhydrous) was purchased from Decon Labs, Inc. Monosodium phosphate and disodium phosphate were purchased from Fisher Scientific. Phosphate buffered saline was prepared by dissolving 3.12 g of Monosodium phosphate, 20.74 g of disodium phosphate, and 9 g of sodium chloride in 1 L of water to achieve a pH = 7.4. The 10 mM acetate buffer was prepared by dissolving 0.21 g of glacial acetic acid, 0.52 g of anhydrous sodium acetate, and 9 g of sodium chloride in 1 L of water to achieve a pH of 5.0. Slide-A-Lyzer dialysis mini capsules (MWCO: 10 kDa, 0.5–1 mL) were purchased from Thermo Scientific.

All chemicals were high purity analytical or HPLC grade. The following
commercially available materials were used as received: calpain-1 (from human
erythrocytes) was from Calbiochem (La Jolla, CA). Calpain-1 (from human
erythrocytes) was from Calbiochem (La Jolla, CA). N-Acetylglycyllysine methyl
ester was from Bachem (Torrance, CA), β-amyloid(11-17) peptide EVHHQKL
(β-amyloid residues 11-17) was from Peptide 2.0 Inc (Chantilly, VA),
N-ethylmorpholine (NEM) was from Aldrich (Milwaukee, WI). Sodium acetate,
ammonium bicarbonate, disodium hydrogen phosphate and monosodium phosphate were
from Fisher Scientific Co. Chromatography was performed with ACS HPLC grade
solvents.

The aldehyde-functionalized dextran resin was prepared starting from CM Sephadex C-50 resin (GE Healthcare Life Sciences) with a carboxylic acid substitution of approximately 0.45 mmol/g of dry resin, using the general protocol (Villain, et al., 2001 (link)). 1 g of resin (1 equiv., 4.5 mmol –COOH functionality) was swelled according to the manufacturer’s instructions: 24 hours in 200mM monosodium phosphate (Fisher), pH 6.5. 1 g of resin swelled to approximately 30 mL of wet volume. The carboxylic acid was activated by the addition of a water solution containing 1.42 g of N-hydroxysuccinimide (NHS, Oakwood) (2 equiv., 9 mmol) and 3.47 g of 1′-ethyl-(3′-dimethylaminopropyl)-carbodiimide-HCl (EDC, Bachem) (4 equiv., 18 mmol) for 10 minutes. The resin was then washed thoroughly with water and then incubated for 1 hour with a water solution of 1 M 2-amino-acetaldehyde diethyl acetal (Sigma Aldrich) and 200 mM 2-morpholino-ethanesulfonic acid (MES, Fisher), adjusted to pH 6.4 with HCl (Fisher). The resin was then washed thoroughly with water, after which any unreacted sites were blocked by adding a solution of 1 M ammonium acetate (Fisher), pH 7.5 for 10 minutes. Following another wash with water, the resin was rinsed thoroughly with 10 mM HCl to liberate the aldehyde functionality. The finished resin was then equilibrated with 0.1 M acetic acid (Fisher) prior to storage at 4°C.