Dual Cathepsin B and Glutathione-Activated Dimeric and Trimeric Phthalocyanine-Based Photodynamic Molecular Beacons for Targeted Photodynamic Therapy

Abstract

Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC 50 values of 0.21−0.39 μM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

Specific Activation of Photosensitizer with Extrinsic Enzyme for Precisive Photodynamic Therapy

“…While the absorption spectrum of 9 exhibited an intense absorption at 651 nm, this absorption band was split, broadened, and red-shifted (to 710 nm for the longest-wavelength absorption) for gal-DSBDP as observed previously for related DSBDPs . Upon excitation at 610 nm, 9 showed a strong fluorescence band at 685 nm with a fluorescence quantum yield (Φ f ) of 0.14 relative to zinc­(II) phthalocyanine [Φ F = 0.28 in N , N -dimethylformamide (DMF)] . In contrast, gal-DSBDP was essentially nonemissive (Φ F = 0.01), showing that the fluorescence of gal-DSBDP was almost completely quenched by caging the meso carboxylate of the DSBDP core.…”

“…Apart from fluorescence emission, the singlet oxygen generation of gal-DSBDP could also be restored upon treatment with β-Gal. Using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger, the singlet oxygen generation efficiency of gal-DSBDP was studied both before and after the treatment with β-Gal. As shown in Figure e, the absorbance of the DPBF’s absorption at 417 nm was essentially unchanged for gal-DSBDP upon irradiation (λ > 610 nm), showing that the singlet oxygen generation of this dye was largely inhibited.…”

“…Similarly, a series of matrix metalloproteinase-activated photosensitizers have been reported, which show preferential activation in tumors. , Although these enzymes are overexpressed in pancreatic, colon, breast, and non-small-cell lung cancer cells, they are also present in most epithelial cells . In addition, while cathepsin B has also been used to activate various prodrugs, fluorescent probes, and photosensitizers, , it is also overproduced in the lysosomes of many types of cells . To the best of our knowledge, all of the enzyme-activated photosensitizers reported so far require the intrinsic enzymes for activation.…”

A Tumor‐Targeting Dual‐Stimuli‐Activatable Photodynamic Molecular Beacon for Precise Photodynamic Therapy

“…Owing to the superior response of the aforementioned phthalocyanine trimer toward GSH and cathepsin B, [10] we maintained its basic skeleton that involved three zinc(II) phthalocyanine (ZnPc) units each substituted with a GSH‐responsive 2,4‐dinitrobenzensulfonate (DNBS) quencher, which were connected via two cathepsin B‐cleavable GFLG peptide linkers. This short peptide sequence has been commonly used in the design of cathepsin B‐responsive drug delivery systems [10,11] . This trimer was found to be completely quenched by the strong photoinduced electron transfer (PET) effect of the DNBS moieties and the strong self‐quenching of the ZnPc units.…”

“…All of the protecting groups on the peptide sequence and the resin were then removed upon treatment with a mixture of trifluoroacetic acid (TFA) and water (95 : 5 v/v) to afford the tetrazine‐substituted peptide 13 . It is worth mentioning that while rink amide resin was used previously as the solid support in the preparation of the non‐tetrazine‐modified counterpart, [10] it could not lead to the formation of 13 . It is likely that the tetrazine moiety was susceptible to triisopropylsilane, which was used to detach the peptide from the resin in the final step.…”