Light-triggered chemical amplification to accelerate degradation and release from polymeric particles

We describe a means of chemical amplification to accelerate triggered degradation of a polymer and particles composed thereof.


Degradation of polymer 1
Degradation of polymer 1 followed 1 H NMR. A concentrated solution of polymer 1 (12.5 mg/ mL) was prepared in d 6 -DMSO. The DMSO stock was divided and an appropriate amount of deuterated sodium phosphate buffer at pH 7.4 (0.1 M) and sodium phosphate solution at pH 5 were added to make 9:1 solutions of d 6 -DMSO:PBS. The solution was irradiated for 1 to 20 min in a 1.7 mm Bruker NMR tube in a Luzchem photoreactor. The samples were then incubated at 37 o C for the specified times. Spectra were taken on a 600 MHz Bruker spectrometer after the prescribed incubation times.   Figure S6: PDI of nanoparticles after irradiation 5 min (35 mW/cm 2 , λ = 320-480 nm) by DLS. The initial rapid increase in PDI in irradiated samples is related to swelling and degradation caused by irradiation.
Later changes are the result of slower subsequent hydrolysis and aggregation.The PDI of non-irradiated particles also slowly increases over time due to slow hydrolysis and subsequent aggregation.
Nile Red quenching. Freeze-dried Nile Red-containing particles were re-suspended in 1x PBS pH 7.4 buffer (1 mg/mL). Quenching of Nile Red fluorescence upon degradation and solubility change was followed by the decrease in the 620 nm fluorescence peak (Horiba Jobin Yvon FL-1000). Suspensions were irradiated for 1, 3, or 5s intervals (1.5 mW/cm 2 , 300-400 nm, λ max = 365 nm), incubating 10 min in between each consecutive irradiation. cells were seeded on a tissue culture treated 96-well plate (Corning) at a density of 20000 cells/well in DMEM media. The cells were washed twice with 100 µL PBS at 37 °C, and then incubated with the polymer/particle suspensions in triplicates for 24 h at 37 °C, in 5% CO 2 . Following incubation with particles, cells were again washed twice with 100 µL PBS. Following the MTT assay kit instructions, the cells were then incubated at 37°C, in 5% CO 2 for 3 h in 100 µL DMEM containing 0.5 mg/mL 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT agent, Sigma-Aldrich, USA). Triton-X (1% w/v, Sigma-Aldrich) was used as a positive apoptosis control. After the incubation, 100 µL of MTT solution (Sigma-Aldrich) was added to each well and the solution was thoroughly triturated to fully solubilize formazan crystals. To quantify mitochondrial activity, absorbance at 570 nm normalized to background absorbance at 690 nm was measured using a plate reader (Molecular Devices SpectraMax M5). S13 Figure S7: MTT assay of polymer 1.
Compound 8. Compound 7 (1.65 g, 3.78 mmol) was dissolved in DCM (18 mL) and TFA (18 mL) and stirred for 1.2 h. The reaction mixture was concentrated. The resulting colorless oil was dissolved in DCM (40 mL) and concentrated 3 more times. The oil was then dissolved in DCM (37 mL) and Et 3 N (4.32 mL, 30.98 mmol) was dripped into the solution slowly and the solution was chilled to 0 o C.

Degradation of control polymer 9
Deprotection of polymer 9 via hydrogenation. Polymer 9 (15 mg) was dissolved in THF (4 mL) under argon. Pd/C 10% (10 mg) was added and the reaction was put under an H 2 atmosphere. The reaction was allowed to proceed for 15 h then was flushed with argon, filtered through celite, then concentrated. The resulting oil was used directly in 1 H NMR experiments. The hydrogenation appeared to remove roughly 50% of the protecting group ( Figure S8). This is likely due to poisoning of the palladium catalyst with residual thiols. S18 Figure S10: Hydrogenated polymer 9.
Degradation of polymer 9 followed 1 H NMR. A concentrated solution of polymer 9 (12.5 mg/ mL) was prepared in d 6 -DMSO. An appropriate amount of deuterated sodium phosphate buffer at pH 7.4 (0.1 M) was added to make 9:1 solutions of d 6 -DMSO:PBS and was separated to two samples. One sample was irradiated for 20 min in a 1.7 mm Bruker NMR tube in a Luzchem photoreactor. One sample of polymer 9 was not irradiated. Hydrogenated polymer 9 was prepared in d 6 -DMSO (22 mg/ mL) and an appropriate amount of deuterated sodium phosphate buffer at pH 7.4 (0.1 M) was added to make a 9:1 solutions of d 6 -DMSO:PBS solution. The samples were then incubated at 37 o C for the specified times. Spectra were taken on a 600 MHz Bruker spectrometer after the prescribed incubation times. S19 Figure S11: ( irradiated for 20 min (red squares), and hydrogenated polymer 9 (green triangles).