FRET Quenching of Photosensitizer Singlet Oxygen Generation

Abstract

The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-alpha (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J > or = 5.1 x 10(13) M(-1) cm(-1) nm4) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J > or = 3.7 x 10(15) M(-1) cm(-1) nm4) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.

Tumor-homing photosensitizer-conjugated glycol chitosan nanoparticles for synchronous photodynamic imaging and therapy based on cellular on/off system

“…The light irradiation causes the photosensitizer to generate cytotoxic singlet oxygen that destroys tumor cells through apoptosis or necrosis [6]. In addition, the selective accumulation of photosensitizers like protoporphyrin IX (PpIX) in tumor tissues provides an intense fluorescence signal that also can be employed in photodynamic imaging (PDI) [7].…”

“…It has been reported that various photosensitizer-encapsulated nano-sized carriers could enhance the tumor target specificity and therapeutic efficacy in cancer treatment, compared to free photosensitizer [2,3,11]. On the other hand, there are some efforts to control the photosensitizer's activity using quenching/dequenching system, which show the specific recovery of the photosensitizer's activity in target tumor tissue [7]. For this purpose, the quenched nano-sized drug carriers have been developed to recover the quenched photosensitizer's activity under tumor-specific stimuli such as tumoral acidic pH or tumor-specific enzymes [12,13], whereas the carriers at normal state presented the 'off' state with no fluorescence signal and phototoxicity with light exposure, due to the quenching effect.…”

Optically Controlled Pore Formation in Self‐Sealing Giant Porphyrin Vesicles

“…Porphyrin fl uorescence self-quenching has been shown to be highly correlated with self-quenching of singlet oxygen quantum yield. [ 16 ] Although less quenched, 1% porphyrin-lipid GPVs generated substantially more fl uorescence and thus more singlet oxygen (hundreds of fold more, based on the quenching in porphysomes of similar composition [ 10 ] ), they did not form pores in response to laser irradiation, indicating that pore formation is not a result of singlet oxygen interaction with the membrane. This is consistent with previous examination of singlet oxygen generation of porphyrins anchored in low molar percentages (1-10%) in phospholipid giant unilamellar vesicles, which did not produce visible membrane poration in response to irradiation.…”

Application of Chitosan-Based Nanocarriers in Tumor-Targeted Drug Delivery

“…Protoporphyrin IX (PpIX) is another photo-sensitizer that its accumulation in tumor tissues provides an intense fluorescence signal that also can be employed in photodynamic imaging (PDI) [102]. Lee et al designed the PpIX-conjugated GC nanoparticles (PpIXeGCe NPs) based on cellular on/off system for synchronous photodynamic imaging (PDI) and PDT in cancer treatment [97].…”