Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation

Abstract

Molecular beacons are FRET-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets, thus are useful tools for in vivo cancer imaging. Photodynamic therapy (PDT) is a cell-killing process by light activation of a photosensitizer (PS) in the presence of oxygen. The key cytotoxic agent is singlet oxygen ((1)O(2)). By combining these two principles (FRET and PDT), we have introduced a concept of photodynamic molecular beacons (PMB) for controlling the PS's ability to generate (1)O(2) and, ultimately, for controlling its PDT activity. The PMB comprises a disease-specific linker, a PS, and a (1)O(2) quencher, so that the PS's photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease. Here, we report the full implementation of this concept by synthesizing a matrix metalloproteinase-7 (MMP7)-triggered PMB and achieving not only MMP7-triggered production of (1)O(2) in solution but also MMP7-mediated photodynamic cytotoxicity in cancer cells. Preliminary in vivo studies also reveal the MMP7-activated PDT efficacy of this PMB. This study validates the core principle of the PMB concept that selective PDT-induced cell death can be achieved by exerting precise control of the PS's ability to produce (1)O(2) by responding to specific cancer-associated biomarkers. Thus, PDT selectivity will no longer depend solely on how selectively the PS can be delivered to cancer cells. Rather, it will depend on how selective a biomarker is to cancer cells, and how selective the interaction of PMB is to this biomarker.

Fig. 1.

The PMB concept and the synthesis of PP_MMP7B. (A) The concept of PMB. The synthesis and characteristics of PP_MMP7B. (B) Synthesis protocol. (C) HPLC chromatography. (D) UV-vis spectrum. (E) MALDI-TOF mass spectrum of PP_MMP7B.

Fig. 2.

The validation of MMP7-specific activation of PP_MMP7B in solution. (A) Fluorescence kinetics of 0.4 μM PP_MMP7B+MMP7 (50:1 molar ratio, blue line), 0.4 μM PP_MMP7B+MMP7+inhibitor (50:1:1,500 molar ratio, green line), 0.4 μM PP_MMP7B+MMP2 (50:1 molar ratio, red line), and 0.4 μM C-PPB+MMP7 (50:1 molar ratio, black line). (B) (1) HPLC spectrum of PP_MMP7B+MMP7 and (2) corresponding UV-vis spectra. HPLC spectra of (3) PP_MMP7B+MMP2 and (4) C-PPB+MMP7. All solutions were incubated at 37° for 2 h. (C) The relative ¹O₂ counts of PP_MMP7, PP_MMP7+BHQ3, PP_MMP7B, PP_MMP7B+MMP7, and PP_MMP7B +MMP7+inhibitor.

Fig. 3.

In vitro validation of MMP7-specific activation. (A) Confocal images of PP_MMP7B and control C-PPB in KB (MMP7⁺) and BT20 (MMP7⁻) cells showing fluorescence (Left) and bright field (Right) in each case. (1) KB cells alone, (2) KB cells plus 60 μM PP_MMP7B, (3) KB cells plus 60 μM C-PPB, (4) BT20 cells alone, (5) BT20 cells plus 60 μM PP_MMP7B, and (6) BT20 cells plus 60 μM C-PPB. (B) HPLC spectra of cell incubation media after 5-h incubation: (1) KB cells+PP_MMP7B, (2) BT20 cells+PP_MMP7B, and (3) KB cells+C-PPB.

Fig. 4.

Photodynamic cytotoxicity determined by MTT assay as a function of PS and light doses, compared with untreated cells; means ± standard errors for triplicate experiments.

Fig. 5.

Confocal images showing PP_MMP7B localization and PP_MMP7B-induced apo pto sis. (A) Confocal images of KB cells stained with 100 nM of MitoTracker Green FM for 30 min after 4-h incubation with 20 μM PP_MMP7B: (1) Pyro image, (2) differential interference contrast image, (3) DAPI image, (4) MitoTracker image, and (5) overlaid image. (B) Confocal images showing fluorescence at 488 nm (Lower) and bright field (Upper). (1) KB cells stained with Apoptag; (2) KB cells incubated with 2 μM PP_MMP7B for 3 h, kept in the dark for 3 h, and stained with Apoptag; (3) KB cells incubated with 2 μM PP_MMP7B for 3 h, treated with PDT (5 J/cm²), and stained with Apoptag 3 h after PDT.

Fig. 6.

In vivo images of mice showing bright field (A–H) and fluorescence (a–e and g). (A–E) PP_MMP7B-administered mouse (A, prescan; B, 10 min after i.v. injection; C, 3 h after i.v. injection; D, 5 h after i.v. injection and 1 h after PDT; E, 3 d after PDT). (F) Photograph of PP_MMP7B-administered mouse (30 d after PDT). Light-treated tumors are marked as “light” and nonlight-treated tumors as “dark.” (G) Drug-free mouse (prescan). (H) Photograph of drug-free mouse (30 d after PDT) with light-treated tumor marked as “light” and nonlight-treated tumor as “dark.”