Establishing a Robust and Reliable Response from a Potent Osmium‐Based Photosensitizer Via Lipid Nanoformulation^†

Abstract

Osmium (Os) based photosensitizers (PSs) are a unique class of nontetrapyrrolic metal-containing PSs that absorb red light. We recently reported a highly potent Os(II) PS, rac-[Os (phen) 2 (IP-4T)](Cl) 2 , referred to as ML18J03 herein, with light EC 50 values as low as 20 pM. ML18J03 also exhibits low dark toxicity and submicromolar light EC 50 values in hypoxia in some cell lines. However, owing to its longer oligothiophene chain, ML18J03 is not completely water soluble and forms 1-2 μm sized aggregates in PBS containing 1% DMSO. This aggregation causes variability in PDT efficacy between assays and thus unreliable and irreproducible reports of in vitro activity. To that end, we utilized PEGmodified DPPC liposomes (138 nm diameter) and DSPE-mPEG 2000 micelles (10.2 nm diameter) as lipid nanoformulation vehicles to mitigate aggregation of ML18J03 and found that the spectroscopic properties important to biological activity were maintained or improved. Importantly, the lipid formulations decreased the interassay variance between the EC 50 values by almost 20-fold, with respect to the unformulated ML18J03 when using broadband visible light excitation (P = 0.0276). Herein, lipid formulations are presented as reliable platforms for more accurate in vitro photocytotoxicity quantification for PSs prone to aggregation (such as ML18J03) and will be useful for assessing their in vivo PDT effects.

Enabling In Vivo Optical Imaging of an Osmium Photosensitizer by Micellar Formulation

“…We previously showed that DSPE-mPEG 2000 micelles are an efficient and robust platform that stably encapsulate a hydrophobic conjugate of the PS benzoporphyrin derivative and cholesterol, even when liposomes fail to do so [18]. Our recent study has shown that ML18J03, when formulated in 10.2 nm DSPE-mPEG 2000 micelles, exhibits low dark toxicity, submicromolar EC 50 values in hypoxia, retains its photoactivity in normoxia, and provides a significant reduction in inter-assay variability of ML18J03 [10]. Of particular significance to this study, the formulation of ML18J03 into DSPE-mPEG 2000 micelles increases its luminescence quantum yield in PBS from 9.8 × 10 −5 to 1.3 × 10 −3 , Figure 1 [10].…”

“…ML18J03 was synthesized as previously described [8]. Aqueous Mic-ML18J03 preparations were obtained by formulating the PS in DSPE-mPEG 2000 micelles according to our previously published protocol [10]. Briefly, 0.085 µmol of ML18J03 (1 mg/mL stock in methanol) was mixed with 0.71 µmol DSPE-mPEG 2000 (25 mg/mL stock in chloroform; NOF America Coorporation, White Plains, NY, USA) to provide 11 mol% PS with respect to total lipid content.…”

“…We have shown that ML18J03 exhibits excellent in vivo tolerability with maximum tolerated doses (MTD) exceeding 200 mg/kg [9]. Despite these attractive features, ML18J03 is not suitable for in vivo optical imaging because its luminescence is almost completely suppressed in an aqueous solution due to aggregation-induced quenching [10]. Novel approaches are therefore required to harness the luminescence imaging capabilities of ML18J03 as well as other promising Os(II) complexes.…”

“…Our recent study has shown that ML18J03, when formulated in 10.2 nm DSPE-mPEG 2000 micelles, exhibits low dark toxicity, submicromolar EC 50 values in hypoxia, retains its photoactivity in normoxia, and provides a significant reduction in inter-assay variability of ML18J03 [10]. Of particular significance to this study, the formulation of ML18J03 into DSPE-mPEG 2000 micelles increases its luminescence quantum yield in PBS from 9.8 × 10 −5 to 1.3 × 10 −3 , Figure 1 [10]. As such, in this study, we explore the capability of micellar formulation to enable luminescence imaging of ML18J03 in vivo in orthotopic AT-84 head and neck tumors and assess their tumor selectivity following intravenous administration.…”

Nanoformulation of Lipophilic Osmium Photosensitizers in Liposomes and Micelles as a General Strategy for Improving Reproducibility and Reducing Quenching†

“…Both TLD-1433 and TLD-1633 have significant batch-to-batch variability in cellular phototoxicity studies (1). The current highlighted paper by McFarland et al (2) addresses this last point by finding a means of improving run-torun consistency for highly potent and lipophilic drugs that are prone to aggregation. This work demonstrates the utility of their methods with highly potent Os photosensitizers (PSs), but the work is relevant to other metallodrugs (3) and is also relevant to lipophilic organic photosensitizer drugs as well.…”