Figure 3. Co-solvent block copolymer NG formation.

(A) Co-solvent block copolymer nanogel (NG) formation begins by dissolving the polymer in 10% solvent followed by addition of an aqueous solution containing C3^KO-11 dropwise with stirring. The general NG encapsulation, crosslinking and purification procedure is then followed. (B) Encapsulation efficiencies of the block copolymers is enhanced by the co-solvent methodology (cs subscript). (C) Visualization of PEG-b-PDS NG mediated delivery of C3^KO-11 to MCF7 cells at two different doses (1.0 and 0.5 mg/mL polymer concentration). Purified C3^KO-11 was added at 0.2, 0.4, 0.8 and 1.6 μg/mL. (D) PEG-b-PDS NG prepared using co-solvents acetone (a subscript) or DMSO (d subscript) mediated delivery of WT casp-3 demonstrated cleavage of poly (ADP-ribose) polymerase 1 (PARP) indicating delivery of active casp-3 WT and apoptosis with an empty NG control prepared using the co-solvent methodology. (E) Delivery of active C3-11 by PEG-PDS NG derivatives, unfunctionalized (−) and Arg³ (R³) functionalized, and resultant PARP cleavage. These data demonstrate C3^KO-11’s ability to maintain activity against apoptotic substrates in cells and that R³ functionalization increased total protein delivered and subsequently increased the resultant PARP cleavage for various NG. Delivery of inactive C3^KO-11 used as control to demonstrate lack of apoptosis from an inactive casp-3 cargo.