N-(2-hydroxypropyl)-methacrylamide (
HPMA) copolymers have shown promise for application in the detection and staging of
cancer. However, non-target accumulation, particularly in the liver and spleen, hinders the detection of resident or nearby metastatic lesions thereby decreasing diagnostic effectiveness. Our laboratory has pursued the development of
cathepsin S susceptible linkers (CSLs) to reduce the non-target accumulation of diagnostic/radiotherapeutic
HPMA copolymers. In this study, we ascertain if the length of the linking group impacts the cleavage and clearance kinetics, relative to each other and a non-cleavable control, due to a reduction in steric inhibition. Three different CSLs with linking groups of various lengths (0, 6 and 13 atoms) were conjugated to
HPMA copolymers. In vitro cleavage studies revealed that the longest linking group (13 atoms) led to more rapid cleavage when challenged with
cathepsin S. The CSL incorporated
HPMA copolymers demonstrated significantly higher levels of excretion and a significant decrease in long-term hepatic and splenic retention relative to the non-cleavable control. Contrary to in vitro observations, the length of the linking group did not substantially impact the non-target in vivo clearance. In the case of HPAC
tumor retention, the CSL with the null (0 atom) linker demonstrated significantly higher levels of retention relative to the other CSLs. Given these results, we find that the length of the linking group of the CSLs did not substantially impact non-target clearance, but did influence
tumor retention. Overall, these results demonstrate that the CSLs can substantially improve the non-target clearance of
HPMA copolymers thereby enhancing clinical potential.