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

Photodynamic therapy (PDT) involves the combined action of light, oxygen and a photosensitizer (PS). It offers unique control in the PS's action because the key cytotoxic agent, singlet oxygen (1O2), is only produced in situ upon irradiation. The 1O2 production can be controlled in three levels. The first level involves the judicious use of fiber optics to selectively deliver light to disease tissues. The second level is to exert control over the PS's localization by selectively delivering PS to cancer cells. The third level is to exert control of the PS's ability to generate 1O2 in responding to specific cancer biomarkers. Here, we present two PDT agents based on the latter two levels of 1O2 control. The first PDT agent contains a PS (Pyro) and a tumor homing molecule (folate) and a peptide linker. PPF was found to be selectively accumulated in cancer cells via folate receptor (FR) pathway. The second PDT agent is a matrix metalloproteinase-7 (MMP7)-triggered photodynamic molecular beacon (PMB) containing a PS (Pyro), a 1O2 quencher (BHQ3) and a MMP7-cleavable peptide linker. Thus, the 1O2 production of PPMMP7B is highly sequence-specific and its photodynamic cytotoxicity is MMP7-dependent. Since these agents are designed to share functional modules (PS and peptide linker) and common cancer cell model (KB cells overexpress both FR and MMP7), it forms the basis for rational design of receptor-targeted PMB for achieving a multi-level control of 1O2 production in cancer cells, which in term, could provide a much higher level of PDT selectivity.

We recently introduced the concept of photodynamic molecular beacons (PMB) for selective control of photodynamic therapy (PDT). The PMB consists of a peptide linker that is sequence specific to a cancer-associated protease. A photosensitizer (PS) and a singlet oxygen (1O2) quencher are conjugated to the opposite ends of this linker. Proximity of the PS and quencher can efficiently inhibit 1O2 generation. In the presence of a targeted protease, the substrate sequence is cleaved and the PS and quencher will separate so that the PS can be photo-activated. There are two ways to optimize the PMB selectivity to cancer cells. The first is to increase the protease specificity to targeted cells and the second is to minimize the phototoxicity of intact (uncleaved) PMBs in non-targeted (normal) cells. Carotenoids (CARs) are well known in nature for their role in quenching excited states of PS and in directly scavenging 1O2. The purpose of this study is to evaluate whether the CAR with dual quenching modes (PS excited states deactivation and 1O2 scavenging) can be used to minimize the photodamage of intact PMBs to non-targeted cells. Thus, we synthesized a beacon (PPC) with a caspase-3 cleavable peptide linking a PS and a CAR quencher. It was confirmed that CAR deactivates the PS excited states and also directly scavenges 1O2. Moreover, the in vitro PDT response showed that CAR completely shuts off the photodynamic effect in non-targeted HepG(2) cells, while PS without CAR (control) remains highly potent even at a much lower (30-fold) dose.

New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT

Photodynamic molecular beacon (PMB) is a novel photodynamic therapy (PDT) concept featuring the precise control of the ability of photosensitizer (PS) to produce singlet oxygen in response to specific cancer-associated biomarkers. It comprises a disease-specific linker, a PS and a singlet oxygen quencher so that the PS's photodynamic toxicity is silenced until the linker interacts with a tumor-associated biomarker. The development of PMB depends on two key factors. The first is the design of a suitable PS-quencher pair to achieve an effective singlet oxygen quenching, minimizing phototoxicity of native PMB in non-targeted (normal) cells. The second is the design of a suitable linker for the choice of target biomarker to achieve a specific photodynamic activation, resulting in selective PDT efficacy in targeted (tumor) cells. These two factors make PMB designs versatile and customizable. In this report, we will focus on the new directions on PMB linker design utilizing two "on-and-off" activation mechanisms. The first one uses a "cleavable" linker that is triggered by fibroblast activation protein or phospholipase. The second one uses an "openable" linker that can hybridize with a tumor-specific mRNA.

: We have developed breast cancer-targeted photodynamic molecular beacons (PMB) using two different activation mechanisms: mRNA-triggered (openable) PMB and protease-triggered (cleavable) PMB. We have validated the core principle of PMB concept: the ability of photosensitizer (PS) to produce singlet oxygen (1O2) can be precisely controlled in response to specific breast cancer-associated biomarkers. For the first time, using mouse models and on separate cells, we have shown that it is possible to limit the collateral damage to surrounding normal cells using this approach, thus achieved the unprecedented tumor selectivity for breast cancer PDT. In addition, we also demonstrated the versatility of the PMB design by developing PDT beacons with tailored functions such as the PDT agents with a built-in apoptosis sensor for in situ and real time monitoring of the therapeutic outcome.

Aggregation Turns BODIPY Fluorophores into Photosensitizers: Reversibly Switching Intersystem Crossing On and Off for Smart Photodynamic Therapy

Emerging Designs of Aggregation-Induced Emission Agents for Enhanced Phototherapy Applications

Facile synthesis and biological evaluation of [a]-benzannulated BODIPY derivatives as novel heavy-atom-free photosensitizers for photodynamic anticancer therapy.

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

In this research, a new axial phthalocyanine complex compound was synthesized. This study involved the synthesis of the novel silicon phthalocyanine prepared by reacting SiPcCl2, 9-phenyl-9H-xanthen-9-ol and K2CO3 in dry toluene. The novel silicon phthalocyanine was characterized by using different spectroscopic techniques including 1H NMR, UV-Visible, FT-IR and mass spectroscopy. The electronic properties of the compound for example the aggregation and solubility parameters investigated. Aggregation is a crucial parameter dictating the functional performance of phthalocyanines. Aggregation is closely related to temperature, concentration, type of ligand attached to the phthalocyanine ring, substituents attached to peripheral positions and polarity of the solvent. Ligands attached to the phthalocyanine ring disrupt the planarity of the ring, resulting in reduced aggregation. For these reasons, in this study, we focused on the aggregation characteristics of phthalocyanines and how axial ligand modifications regulate this behavior. Axial ligand (especially those with metals like Si, Ge, Sn) coordination increases steric bulk and reduces stacking. The low aggregation tendency of phthalocyanines provides significant advantages, especially in photodynamic therapy (PDT), optoelectronics and sensor applications. Photodynamic therapy (PDT) is a light based therapeutic modality that requires the coordinated presence of three key components: a photosensitizer (PS), a light source, and molecular oxygen. Phthalocyanine based photosensitizers (Pcs) are among the most promising candidates for photodynamic therapy (PDT) due to their favorable photophysical and photochemical properties. An ideal photosensitizer should exhibit strong absorption (600–800 nm), where tissue penetration of light is optimal. Phthalocyanines meet this criterion through their intense Q band absorption, usually around 670–700 nm. Moreover, they are capable of efficiently generating singlet oxygen (ΦΔ), a key cytotoxic agent in PDT. In addition to their photodynamic efficiency, phthalocyanines demonstrate excellent chemical and photostability. Preliminary research was conducted for photosensitizer properties of the compound. After researches, the results have showed that singlet oxygen quantum yield has increased in the photodynamic study (ΦΔ=0.19) compare to the Std-SiPc (Φ∆= 0.15)

Photodynamic molecular beacons (PMBs) are highly appealing for activatable photodynamic therapy (PDT), but their applications are hindered by limited therapeutic efficacy. Here, by molecular engineering of enzyme-responsive units in the loop region of DNA-based PMBs, we present for the first time the modular design of an enzyme/microRNA dual-regulated PMB (D-PMB) to achieve cancer-cell-selective amplification of PDT efficacy. In the design, the "inert" photosensitizers in D-PMB could be repeatedly activated in the presence of both tumor-specific enzyme and miRNA, leading to amplified generation of cytotoxic singlet oxygen species and therefore enhanced PDT efficacy in vitro and in vivo. By contrast, low photodynamic activity could be observed in healthy cells, as D-PMB activation has been largely avoided by the dual-regulatable design. This work presents a cooperatively activated PDT strategy, which enables enhanced therapeutic efficacy with improved tumor-specificity and thus conceptualizes an approach to expand the repertoire of designing smart tumor treatment modality.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Enhancing Intersystem Crossing by Intermolecular Dimer-Stacking of Cyanine as Photosensitizer for Cancer Therapy

Photodynamic therapy (PDT) is a cancer treatment modality involving the combination of light, a photosensitizer (PS) and molecular oxygen, which results in the production of cytotoxic reactive oxygen species (ROS). Singlet oxygen ((1)O(2)) is one of the most important of these ROS. Because the lifetime and diffusion of (1)O(2) is very limited, a controllable singlet oxygen generation with high selectivity and localization would lead to more efficient and reliable PDT. The lack of selective accumulation of the PS within tumour tissue is a major problem in PDT. Targeted PDT would offer the advantage to enhance photodynamic efficiency by directly targeting diseased cells or tissues. Many attempts have been made to either selectively deliver light to diseased tissues or increase the uptake of the photoactive compounds by the target cells. The review will survey the literature regarding the multi-level control of (1)O(2) production for PDT applications. The mechanisms of ROS formation are described. The different strategies leading to targeted formation of (1)O(2) are developed. Some active PDT agents have been based on energy transfer between PS by control of the aggregation/ disaggregation. The concept of molecular beacon based on quenching-dequenching upon protease cleavage is capable of precise control of (1)O(2) by responding to specific cancer-associated biomarkers.

Cancer is one of the main causes of death worldwide. There are several different types of cancer recognized thus far, which can be treated by different approaches including surgery, radiotherapy, chemotherapy or a combination thereof. However, these approaches have certain drawbacks and limitations. Photodynamic therapy (PDT) is regarded as an alternative noninvasive approach for cancer treatment based on the generation of toxic oxygen (known as reactive oxygen species (ROS)) at the treatment site. PDT requires photoactivation by a photosensitizer (PS) at a specific wavelength (λ) of light in the vicinity of molecular oxygen (singlet oxygen). The cell death mechanisms adopted in PDT upon PS photoactivation are necrosis, apoptosis and stimulation of the immune system. Over the past few decades, the use of natural compounds as a photoactive agent for the selective eradication of neoplastic lesions has attracted researchers’ attention. Many reviews have focused on the PS cell death mode of action and photonanomedicine approaches for PDT, while limited attention has been paid to the photoactivation of phytocompounds. Photoactivation is ever-present in nature and also found in natural plant compounds. The availability of various laser light setups can play a vital role in the discovery of photoactive phytocompounds that can be used as a natural PS. Exploring phytocompounds for their photoactive properties could reveal novel natural compounds that can be used as a PS in future pharmaceutical research. In this review, we highlight the current research regarding several photoactive phytocompound classes (furanocoumarins, alkaloids, poly-acetylenes and thiophenes, curcumins, flavonoids, anthraquinones, and natural extracts) and their photoactive potential to encourage researchers to focus on studies of natural agents and their use as a potent PS to enhance the efficiency of PDT.