Lysosome directed red light photodynamic therapy using glycosylated iron-(III) conjugates of boron-dipyrromethene

The impetus in the medicinal chemistry of metal-based compounds in cancer chemotherapy started with the discovery of cytotoxicity of cisplatin by B. Rosenberg in 1965 and iron-based bleomycin by H. Umezawa in 1966 [[1], [2], [3]]. Metal complexes are amenable to structural modifications, that allow us to tune their kinetic properties that cannot otherwise be realized using conventional carbon-based compounds [4]. Metal complexes having coordinated ligands in a three-dimensional configuration allow for tailored functionalization for desired molecular targets. In a parallel development, Photodynamic Therapy (PDT) has shown great potential as an alternate non-invasive mode of cancer treatment wherein low doses of photosensitizers (PS) are used to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) and/or radicals by type-I or type-II photo-pathways, and the ROS thus generated damage the tumor cells irreversibly in a localized manner, therefore leading to selectivity [5]. PDT is known to significantly reduce the toxic side effects that are commonly observed among the clinically approved chemotherapeutic drugs that, in general, do not distinguish between cancer and normal cells [6]. PDT has extensively been used for localized and superficial cancers and its application has been expanded to solid tumors in recent times [7]. Hematoporphyrin based photosensitizers like the clinically approved Photofrin®, however, suffer from adverse side-effects like skin sensitivity and hepatotoxicity [8]. In addition, their efficacy often gets reduced due to poor aqueous solubility, aggregation-induced photobiological deactivation, and low molar extinction coefficient values at longer wavelengths of light. These shortcomings have led to the emergence of boron-dipyrromethene (BODIPY) based dyes as alternatives. BODIPY dyes are known for their intense absorption spectral characteristics, besides chemical stability, photostability and emissive properties [9]. Compared to its porphyrin counterpart, synthetic modifications in the BODIPY core can be done with ease to design them as highly emissive cellular imaging agents and/or as PS with good singlet oxygen quantum yield for applications in PDT [10]. The poor aqueous solubility of BODIPY dyes, which limits their direct use, can be circumvented by designing transition metal complexes having appended BODIPY moiety. This makes them function as a potent photocytotoxic agent in PDT [11]. Our choice of iron as the metal in designing new PS for its importance in medicinal chemistry as an essential component of metabolism [12]. Bleomycin, an antitumor antibiotic is based on a glycopeptide that chelates iron which is used extensively in the treatment of lymphomas and testicular cancer. Ferrocifen, a tamoxifen derivative, is also an iron-based therapeutic agent [13]. Due to their biocompatible nature, the U.S. Food and Drug Administration (FDA) has approved the use of iron oxide nanoparticles as an MRI contrast agent [14]. Being an element of bio-essential significance, iron does not possess the systemic toxicity often encountered with heavy metal (Pt/Ru) based photosensitizers [15]. Iron in its stable +3 oxidation state significantly reduces the possibility of metal induced dark toxicity which is observed in some copper(II) complexes [16]. This can be achieved in dipicolylamine (dpa) complexes having catechol as the iron(III) binding ligand as this O,O-donor dianionic ligand effectively stabilizes hard Lewis acidic Fe3+ center. Catecholates are well known to stabilize iron(III) in biological systems such as siderophores and enterobactins, where they have high binding constants up to 1039 [17]. Thus, any leaching of the metal ion for non-redox active iron complexes within the biological potential window is circumvented. In addition, the use of a BODIPY dye opens the possibility of using the drug for live cell fluorescence imaging, offering a low cost, non-radioactive tool for accurate tumor diagnosis. We have earlier reported the PDT activity of analogous iron(III)-BODIPY conjugates that showed photoactivity in green light [18]. Herein, we have explored new iron(III) complexes [Fe(L1/2)(L3)Cl] (1–3), where L1, L2 and L3 are benzyl-dipicolylamine (bz-dpa), its glycosylated analogue and dianionic catecholate-BODIPY ligand, having a BODIPY photosensitizer showing significant red light PDT activity and selectively targeting the cancer cells over normal ones. Complex 1 was used as a control to study the effect of glycosylation. The combination of iron in its +3-oxidation state, BODIPY-appended catecholate ligand and glycosylated-dpa ancillary ligand results in a highly cytotoxic red light (600–800 nm) active PDT agent [Fe(L2)(L3)Cl] (2) with deep tissue penetration ability (lowered scattering and background emissions) and is appreciable for its benign nature in dark and for non-cancerous cells [19]. Schematic drawing of the iron(III) complexes presented is this work are shown in Fig. 1.

Targeted drug delivery is an important aspect in medicinal chemistry over conventional cytotoxicity of commonly used chemo-drugs. The necessity for a glycosylated appendage is based on the recent focus on designing targeted anticancer drugs [20]. Otto Warburg had observed that owing to a higher rate of aerobic glycolysis, the requirement of glucose is higher in cancerous tissues compared to non-transformed tissue [21,22]. The insulin-independent glucose transporter GLUT-1 as well as glycolytic enzymes are notably overexpressed in human cancers to keep up with their higher levels of energy requirement. Glyco-conjugation of the metal complexes improves both aqueous solubility as well as targeted delivery [23]. The iron(III) catecholates (1,2) having d-glucose appended dipicolylamine ligand and an emissive catecholate BODIPY unit have been designed to achieve cellular imaging and photo-induced cytotoxicity mediated by ROS and their PDT activity has been studied. Complex 1 as a control species has benzyl-dipicolylamine base to ascertain the role of glycosylation in complex 2 on the overall PDT activity and cellular uptake. Complex 2 is the active species having both BODIPY and glucose moieties.

The noteworthy results of this work include remarkable PDT activity of the iron(III) complexes giving sub-micromolar IC50 values in cancer cells upon red light irradiation while remaining significantly less toxic in normal cells and in dark. Singlet oxygen (1O2) was observed to be the primary reactive oxygen species that is produced via the type-II pathway [24]. In addition, complex 2 showed specific localization in the lysosomes. Any non-nuclear accumulation of the PDT agent is of importance because nucleotide excision repair mechanism (NER) can be avoided inside the cells to enhance the drug efficacy and lower resistance. Some mitochondria targeting photosensitizers (PSs) are also known to produce dark toxicity due to sudden depolarization of the mitochondrial membrane potential [25]. Therefore, PSs that can target cellular organelles like the endoplasmic reticulum, golgi apparatus or lysosomes are of potential therapeutic interests [26].

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