Specific Activation of Photosensitizer with Extrinsic Enzyme for Precisive Photodynamic Therapy

Delivery of functional proteins into the intracellular space has been a challenging task that could lead to a myriad of therapeutic applications. We report herein a novel bioconjugation strategy for enzyme modification and selective delivery into cancer cells for lock-and-key-type activation of photosensitizers. Using a bifunctional linker containing a bis(bromomethyl)phenyl group and an o-phthalaldehyde moiety, it could induce cyclization of the peptide sequence Ac-NH-CRGDfC-CONH2 through site-specific dibenzylation with the two cysteine residues and further coupling with beta-galactosidase via the phthalaldehyde-amine capture reaction. This facile two-step one-pot procedure enabled the preparation of cyclic RGD-modified beta-galactosidase readily, which could be internalized selectively into alpha(v)beta(3) integrin-overexpressed cancer cells. Upon encountering an intrinsically quenched distyryl boron dipyrromethene-based photosensitizer conjugated with a galactose moiety through a self-immolative linker inside the cells, the extrinsic enzyme induced specific cleavage of the beta-galactosidic bond followed by self-immolation to release an activated derivative, thereby restoring the photodynamic activities and causing cell death effectively. The high specificity of this extrinsic enzyme-activated photosensitizing system was also demonstrated in vivo using nude mice bearing an alpha(v)beta(3) integrin-positive U87-MG tumor. The specific activation at the tumor site resulted in lighting up and complete eradication of the tumor upon laser irradiation, while by using the native beta-galactosidase, the effects were largely reduced. In contrast to the conventional activation using intrinsic enzymes, this extrinsic enzyme activatable approach can further minimize the nonspecific activation toward precisive photodynamic therapy.

Automated summary

This study presents a novel bioconjugation strategy for the selective delivery of modified enzymes into cancer cells, enabling the activation of photosensitizers for therapeutic applications. The activation of photosensitizers inside the cancer cells resulted in cell death, demonstrating the potential of this approach for efficient photodynamic therapy.