PD-L1 Immune Checkpoint Targeted Photoactivable Liposomes (iTPALs) Prime the Stroma of Pancreatic Tumors and Promote Self-Delivery.

Desmoplasia in pancreatic ductal adenocarcinoma (PDAC) limits the penetration and efficacy of therapies. It has been previously shown that photodynamic priming (PDP) using EGFR targeted photoactivable multi-inhibitor liposomes remediates desmoplasia in PDAC and doubles overall survival. Here, bifunctional PD-L1 immune checkpoint targeted photoactivable liposomes (iTPALs) that mediate both PDP and PD-L1 blockade are presented. iTPALs also improve phototoxicity in PDAC cells and induce immunogenic cell death. PDP using iTPALs reduces collagen density, thereby promoting self-delivery by 5.4-fold in collagen hydrogels, and by 2.4-fold in syngeneic CT1BA5 murine PDAC tumors. PDP also reduces tumor fibroblast content by 39.4%. Importantly, iTPALs also block the PD-1/PD-L1 immune checkpoint more efficiently than free α-PD-L1 antibodies. Only a single sub-curative priming dose using iTPALs provides 54.1% tumor growth inhibition and prolongs overall survival in mice by 42.9%. Overall survival directly correlates with the extent of tumor iTPAL self-delivery following PDP (Pearson's r = 0.670, p = 0.034), while no relationship is found for sham non-specific IgG constructs activated with light. When applied over multiple cycles, as is typical for immune checkpoint therapy, PDP using iTPALs promises to offer durable tumor growth delay and significant survival benefit in PDAC patients, especially when used to promote self-delivery of integrated chemo-immunotherapy regimens.

Immunofluorescence profiling of collagen subtypes is a predictor of treatment outcomes in pancreatic cancer.

Desmoplasia in pancreatic ductal adenocarcinoma (PDAC) is characterized by elevated levels of tumor collagen. Desmoplasia restricts drug delivery in PDAC, contributes to treatment resistance, and is associated with poor survival outcomes. We have previously shown that photodynamic therapy (PDT)-based treatment remediates desmoplasia in orthotopic PDAC tumors by reducing second harmonic generation signals from collagen by >90% and by reducing collagen alignment by >103-fold [19]. Remediating desmoplasia correlated with improved survival outcomes in mice. To understand this phenomenon at a fundamental level, it is important to dissect the impact of therapy on collagen subtypes. In this study, we demonstrate that immunofluorescence profiling of collagen subtypes I, II, III and IV in PDAC tumors 72 h following multiple treatment regimens is predictive of long-term outcomes. Treatment regimens include nanoliposomal irinotecan chemotherapy (nal-IRI; akin to ONIVYDE™), a combination of nal-IRI chemotherapy with PDT encapsulated in a single photoactivable multi-inhibitor liposome (PMIL) and an EGFR-targeted PMIL construct (TPMIL). Results show that the relative tumor content of collagen I, II and III was inversely correlated with overall survival (P ≤ 0.0013, P ≤ 0.0001, P ≤ 0.0011, respectively), while, surprisingly, the relative tumor content of collagen IV was directly correlated with overall survival (P ≤ 0.0001). Similar relationships were observed between the relative tumor content of collagen subtypes and the residual tumor volume at day 88 following treatment. Considering that the relationship between collagen subtypes and treatment outcomes is observed across multiple treatment regimens, immunofluorescence profiling at 72 h following treatment appears to be predictive of tumor growth inhibition and survival in PDAC. Early immunofluorescence collagen subtype profiling may therefore aid in treatment personalization and may inform the dosimetry and scheduling of combination regimens for PDAC, such as chemotherapy and emerging PDT-based combinations, to maximize patient survival benefit.

Establishing a Robust and Reliable Response from a Potent Osmium-Based Photosensitizer Via Lipid Nanoformulation†.

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 EC50 values as low as 20 pm. ML18J03 also exhibits low dark toxicity and submicromolar light EC50 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 PEG-modified DPPC liposomes (138 nm diameter) and DSPE-mPEG2000 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 EC50 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.

Osmium (Os)-based photosensitizers (PSs) exhibit unique broad, red-shifted absorption, favoring PDT activity at greater tissue depths. We recently reported on a potent Os(II) PS, rac-[Os(phen)2(IP-4T)](Cl)2 (ML18J03) with submicromolar hypoxia activity. ML18J03 exhibits a low luminescence quantum yield of 9.8 × 10-5 in PBS, which limits its capacity for in vivo luminescence imaging. We recently showed that formulating ML18J03 into 10.2 nm DSPE-mPEG2000 micelles (Mic-ML18J03) increases its luminescence quantum yield by two orders of magnitude. Here, we demonstrate that Mic-ML18J03 exhibits 47-fold improved accumulative luminescence signals in orthotopic AT-84 head and neck tumors. We show, for the first time, that micellar formulation provides up to 11.7-fold tumor selectivity for ML18J03. Furthermore, Mic-ML18J03 does not experience the concentration-dependent quenching observed with unformulated ML18J03 in PBS, and formulation reduces spectral shifting of the emission maxima during PDT (variance = 6.5 and 27.3, respectively). The Mic-ML18J03 formulation also increases the production of reactive molecular species 2-3-fold. These findings demonstrate that micellar formulation is a versatile and effective approach to enable in vivo luminescence imaging options for an otherwise quenched, yet promising, PS.

Towards Photodynamic Image-Guided Surgery of Head and Neck Tumors: Photodynamic Priming Improves Delivery and Diagnostic Accuracy of Cetuximab-IRDye800CW.

Fluorescence image-guided surgery (IGS) using antibody conjugates of the fluorophore IRDye800CW have revolutionized the surgical debulking of tumors. Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, conjugated to IRDye800CW (Cet-IRDye800) is the first molecular targeted antibody probe to be used for IGS in head and neck cancer patients. In addition to surgical debulking, Cetuximab-targeted photodynamic therapy (photoimmunotherapy; PIT) is emerging in the clinic as a powerful modality for head and neck tumor photodestruction. A plethora of other photoactivable agents are also in clinical trials for photodynamic-based therapies of head and neck cancer. Considering the vascular and stromal modulating effects of sub-therapeutic photodynamic therapy, namely photodynamic priming (PDP), this study explores the potential synergy between PDP and IGS for a novel photodynamic image-guided surgery (P-IGS) strategy. To the best of our knowledge, this is the first demonstration that PDP of the tumor microenvironment can augment the tumor delivery of full-length antibodies, namely Cet-IRDye800. In this study, we demonstrate a proof-of-concept that PDP primes orthotopic FaDu human head and neck tumors in mice for P-IGS by increasing the delivery of Cet-IRDye800 by up to 138.6%, by expediting its interstitial accumulation by 10.5-fold, and by increasing its fractional tumor coverage by 49.5% at 1 h following Cet-IRDye800 administration. Importantly, PDP improves the diagnostic accuracy of tumor detection by up to 264.2% with respect to vicinal salivary glands at 1 h. As such, PDP provides a time-to-surgery benefit by reducing the time to plateau 10-fold from 25.7 h to 2.5 h. We therefore propose that a pre-operative PDP regimen can expedite and augment the accuracy of IGS-mediated surgical debulking of head and neck tumors and reduce the time-to-IGS. Furthermore, this P-IGS regimen, can also enable a forward-looking post-operative protocol for the photodestruction of unresectable microscopic disease in the surgical bed. Beyond this scope, the role of PDP in the homogenous delivery of diagnostic, theranostic and therapeutic antibodies in solid tumors is of considerable significance to the wider community.

Membrane composition is a functional determinant of NIR-activable liposomes in orthotopic head and neck cancer.

Near-infrared (NIR)-activable liposomes containing photosensitizer (PS)-lipid conjugates are emerging as tunable, high-payload, and tumor-selective platforms for photodynamic therapy (PDT)-based theranostics. To date, the impact that the membrane composition of a NIR-activable liposome (the chemical nature and subsequent conformation of PS-lipid conjugates) has on their in vitro and in vivo functionality has not been fully investigated. While their chemical nature is critical, the resultant physical conformation dictates their interactions with the immediate biological environments. Here, we evaluate NIR-activable liposomes containing lipid conjugates of the clinically-used PSs benzoporphyrin derivative (BPD; hydrophobic, membrane-inserting conformation) or IRDye 700DX (hydrophilic, membrane-protruding conformation) and demonstrate that membrane composition is critical for their function as tumor-selective PDT-based platforms. The PS-lipid conformations were primarily dictated by the varying solubilities of the two PSs and assisted by their lipid conjugation sites. Conformation was further validated by photophysical analysis and computational predictions of PS membrane partitioning (topological polar surface area [tPSA], calculated octanol/water partition [cLogP], and apparent biomembrane permeability coefficient [Papp]). Results show that the membrane-protruding lipo-IRDye700DX exhibits 5-fold more efficient photodynamic generation of reactive molecular species (RMS), 12-fold expedited phototriggered burst release of entrap-ped agents, and 15-fold brighter fluorescence intensity as compared to the membrane-inserting lipo-BPD-PC (phosphatidylcholine conjugate). Although the membrane-inserting lipo-BPD-PC exhibits less efficient photo-dynamic generation of RMS, it allows for more sustained phototriggered release, 10-fold greater FaDu cancer cell phototoxicity, and 7.16-fold higher tumor-selective delivery in orthotopic mouse FaDu head and neck tumors. These critical insights pave the path for the rational design of emerging NIR-activable liposomes, whereby functional consequences of membrane composition can be tailored toward a specific therapeutic purpose.