Subsequently, the interactions between the NPs with the individual substrates and enzymes were explored by measuring changes to the intrinsic CD spectra (Figure , Supporting Information). The NPs induced CD spectral shifts for HA (Figure ) and ACAN (Figure ), but not for COL (Figure , Supporting Information). NP-induced spectral shifts were also observed for ADA (Figure ), but not HYA (Figure , Supporting Information) or COL (Figure , Supporting Information). To simplify the effect comparisons, we plotted the normalized spectral changes for the substrates (Figure ), and the enzymes (Figure ). This revealed that HA was significantly altered by all NPs likely suggesting a binding interaction, whereas ACAN structural alterations were apparent with NP0 and NP5000, but not the larger PLGA-based NPs (Figure ), and ADA was most significantly affected by NP0 and PLGA-PEIL (Figure ).