Cathepsin B-Cleavable Polymeric Photosensitizer Prodrug for Selective Photodynamic Therapy: In Vitro Studies.

Cathepsin B is a lysosomal cysteine protease that plays an important role in cancer, atherosclerosis, and other inflammatory diseases. The suppression of cathepsin B can inhibit tumor growth. The overexpression of cathepsin B can be used for the imaging and photodynamic therapy (PDT) of cancer. PDT targeting of cathepsin B may have a significant potential for selective destruction of cells with high cathepsin B activity. We synthesized a cathepsin B-cleavable polymeric photosensitizer prodrug (CTSB-PPP) that releases pheophorbide a (Pha), an efficient photosensitizer upon activation with cathepsin B. We determined the concentration dependant uptake in vitro, the safety, and subsequent PDT-induced toxicity of CTSB-PPP, and ROS production. CTSB-PPP was cleaved in bone marrow cells (BMCs), which express a high cathepsin B level. We showed that the intracellular fluorescence of Pha increased with increasing doses (3-48 µM) and exerted significant dark toxicity above 12 µM, as assessed by MTT assay. However, 6 µM showed no toxicity on cell viability and ex vivo vascular function. Time-dependent studies revealed that cellular accumulation of CTSB-PPP (6 µM) peaked at 60 min of treatment. PDT (light dose: 0-100 J/cm2, fluence rate: 100 mW/cm2) was applied after CTSB-PPP treatment (6 µM for 60 min) using a special frontal light diffuser coupled to a diode laser (671 nm). PDT resulted in a light dose-dependent reduction in the viability of BMCs and was associated with an increased intracellular ROS generation. Fluorescence and ROS generation was significantly reduced when the BMCs were pre-treated with E64-d, a cysteine protease inhibitor. In conclusion, we provide evidence that CTSB-PPP showed no dark toxicity at low concentrations. This probe could be utilized as a potential imaging agent to identify cells or tissues with cathepsin B activity. CTSB-PPP-based PDT results in effective cytotoxicity and thus, holds great promise as a therapeutic agent for achieving the selective destruction of cells with high cathepsin B activity.

Double-PEGylated Cyclopeptidic Photosensitizer Prodrug Improves Drug Uptake from In Vitro to Hen's Egg Chorioallantoic Membrane Model.

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs.

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free "click" chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Correction: Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs.

Correction for 'Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs' by Jordan Bouilloux et al., Photochem. Photobiol. Sci., 2018, 17, 1728-1738.

Correction: Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher.

Correction for 'Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher' by Jordan Bouilloux et al., Photochem. Photobiol. Sci., 2018, 17, 1739-1748.

The Dark Side: Photosensitizer Prodrugs.

Photodynamic therapy (PDT) and photodiagnosis (PD) are essential approaches in the field of biophotonics. Ideally, both modalities require the selective sensitization of the targeted disease in order to avoid undesired phenomena such as the destruction of healthy tissue, skin photosensitization, or mistaken diagnosis. To a large extent, the occurrence of these incidents can be attributed to "background" accumulation in non-target tissue. Therefore, an ideal photoactive compound should be optically silent in the absence of disease, but bright in its presence. Such requirements can be fulfilled using innovative prodrug strategies targeting disease-associated alterations. Here we will summarize the elaboration, characterization, and evaluation of approaches using polymeric photosensitizer prodrugs, nanoparticles, micelles, and porphysomes. Finally, we will discuss the use of 5-aminolevulinc acid and its derivatives that are selectively transformed in neoplastic cells into photoactive protoporphyrin IX.

Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs.

Herein, we report the synthesis of a new prodrug system consisting of regioselectively addressable functionalized templates bearing multiple pheophorbide A moieties for use in photodynamic therapy. These coupling reactions were achieved using copper-free "click" chemistry, namely a strain-promoted azide-alkyne cycloaddition. This new design was used to obtain well-defined quenched photosensitizer prodrugs with perfect knowledge of the number and position of loaded photosensitizers, providing structures bearing up to six photosentitizers and two PEG chains. These conjugates are ideally quenched in their native state regarding their fluorescence emission (up to 155 ± 28 times less fluorescent for an hexasubstituted conjugate than a monosubstituted non-quenched reference compound) or singlet oxygen production (decreased 8.7-fold in the best case) when excited. After 2 h of proteolytic activation, the fluorescence emission of a tetrasubstituted conjugate was increased 17-fold compared with the initial fluorescence emission.

Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher.

Previously, we have shown that the use of a cyclopeptidic carrier could be of great interest for the design of fully characterized prodrugs for further use in photodynamic therapy. In order to further optimize the design, we decided to modify the highly quenched conjugate uPA-cPPP4/5 by co-loading a long-distance fluorescence quencher. For this purpose we tethered two black hole quenchers (BHQ3) together with two pheophorbide A moities onto the same PEGylated backbone and assessed the modified photophysical properties. In addition, to prove the reliability of our concept, we designed two analogues, uPA-cPPQ2+2/5 and CathB-cPPQ2+2/5, by using two different peptidic linkers as substrates for uPA and cathepsin B, respectively. These two conjugates proved to be much more water-soluble than their analogues bearing only Phas. These conjugates are not only highly quenched in their native state with regard to their fluorescence emission (up to 850 ± 287 times less fluorescent for CathB-cPPQ2+2/5 as compared to the unquenched monosubstituted reference uPA-cPPP1/5), but also prevent singlet oxygen production (with a total quenching of the emission when the quenchers are co-loaded with photosensitizers) when the photosentistizers are excited. After proteolytic activation, these conjugates recover their photophysical properties in the same way as occurred for uPA-cPPP4/5, with up to a 120-fold increase in fluorescence emission for uPA-cPPQ2+2/5 after two hours of incubation with uPA.

Time-resolved singlet-oxygen luminescence detection with an efficient and practical semiconductor single-photon detector.

In clinical applications, such as PhotoDynamic Therapy, direct singlet-oxygen detection through its luminescence in the near-infrared range (1270 nm) has been a challenging task due to its low emission probability and the lack of suitable single-photon detectors. Here, we propose a practical setup based on a negative-feedback avalanche diode detector that is a viable alternative to the current state-of-the art for different clinical scenarios, especially where geometric collection efficiency is limited (e.g. fiber-based systems, confocal microscopy, scanning systems etc.). The proposed setup is characterized with Rose Bengal as a standard photosensitizer and it is used to measure the singlet-oxygen quantum yield of a new set of photosensitizers for site-selective photodynamic therapy.