Immunogenic cell death-inducing metal complexes: From the benchtop to the clinic.

The immune system presents a complex array of processes designed to maintain homeostasis in malignant cellular growth. Malignancy is the result of a breakdown in immune surveillance by cancer cells evading immune recognition. Significant efforts have been made in modulating immune checkpoint signaling cascades to bypass the resulting immune evasion and establish an anticancer effect. More recently, it was discovered that a form of regulated cell death can involve the stimulation of immune response as its downstream effect and subsequently re-establish immune surveillance. This mechanism, known as immunogenic cell death (ICD), is being exploited as a target to prevent tumor relapse and prevent cancer metastasis. It is now appreciated that metal-based compounds play a key role in ICD activation due to their unique biochemical properties and interactions within cancer cells. With fewer than 1% of known anticancer agents documented as ICD inducers, recent efforts have been made to identify novel entities capable of stimulating a more potent anticancer immune response. While the recent reviews by us or others focus primarily on either discussing the chemical library of ICD inducers or intricate detailing of biological pathways associated with ICD, this review aims to bridge these two topics as a concise summary. Furthermore, early clinical evidence and future directions of ICD are briefly summarized.

Lanthanide porphyrinoids as molecular theranostics.

In recent years, lanthanide (Ln) porphyrinoids have received increasing attention as theranostics. Broadly speaking, the term 'theranostics' refers to agents designed to allow both disease diagnosis and therapeutic intervention. This Review summarises the history and the 'state-of-the-art' development of Ln porphyrinoids as theranostic agents. The emphasis is on the progress made within the past decade. Applications of Ln porphyrinoids in near-infrared (NIR, 650-1700 nm) fluorescence imaging (FL), magnetic resonance imaging (MRI), radiotherapy, and chemotherapy will be discussed. The use of Ln porphyrinoids as photo-activated agents ('phototheranostics') will also be highlighted in the context of three promising strategies for regulation of porphyrinic triplet energy dissipation pathways, namely: regioisomeric effects, metal regulation, and the use of expanded porphyrinoids. The goal of this Review is to showcase some of the ongoing efforts being made to optimise Ln porphyrinoids as theranostics and as phototheranostics, in order to provide a platform for understanding likely future developments in the area, including those associated with structure-based innovations, functional improvements, and emerging biological activation strategies.

2D-ultrathin MXene/DOXjade platform for iron chelation chemo-photothermal therapy.

An increased demand for iron is a hallmark of cancer cells and is thought necessary to promote high cell proliferation, tumor progression and metastasis. This makes iron metabolism an attractive therapeutic target. Unfortunately, current iron-based therapeutic strategies often lack effectiveness and can elicit off-target toxicities. We report here a dual-therapeutic prodrug, DOXjade, that allows for iron chelation chemo-photothermal cancer therapy. This prodrug takes advantage of the clinically approved iron chelator deferasirox (ExJade®) and the topoisomerase 2 inhibitor, doxorubicin (DOX). Loading DOXjade onto ultrathin 2D Ti3C2 MXene nanosheets produces a construct, Ti 3 C 2 -PVP@DOXjade, that allows the iron chelation and chemotherapeutic functions of DOXjade to be photo-activated at the tumor sites, while potentiating a robust photothermal effect with photothermal conversion efficiencies of up to 40%. Antitumor mechanistic investigations reveal that upon activation, Ti 3 C 2 -PVP@DOXjade serves to promote apoptotic cell death and downregulate the iron depletion-induced iron transferrin receptor (TfR). A tumor pH-responsive iron chelation/photothermal/chemotherapy antitumor effect was achieved both in vitro and in vivo. The results of this study highlight what may constitute a promising iron chelation-based phototherapeutic approach to cancer therapy.

Detailed structural and spectroscopic elucidation of ferrocenium coupled N-heterocyclic carbene gold(I) complexes.

Unambiguous assignment of redox sites on ferrocene coupled N-heterocyclic carbene gold(I) complexes [(Fc-NHC)2Au(I)]+ is critical to gain a greater mechanistic understanding of their activity in a cellular environment. Such information can be garnered with isolation and detailed characterization of the oxidized version of [(Fc-NHC)2Au(I)]+. Herein we disclose a study that unambiguously illustrates redox events pertaining to [(Fc-NHC)2Au(I)]+ that stem exclusively from ferrocene sites. This work also describes novel synthetic methodologies for isolating ferrocenium coupled N-heterocyclic carbene gold(I) complexes.

Metal-based anticancer agents as immunogenic cell death inducers: the past, present, and future.

Cancer is the deadliest disease in the world behind heart disease. Sadly, this remains true even as we suffer the ravages of the Covid-19 pandemic. Whilst current chemo- and radiotherapeutic treatment strategies have significantly improved the patient survival rate, disease reoccurrence continues to pose a deadly risk for all too many patients. Incomplete removal of tumour cells from the body increases the chances of metastasis and developing resistance against current treatments. Immunotherapy represents a therapeutic modality that has helped to overcome these limitations in recent decades. However, further progress is needed. So-called immunogenic cell death (ICD) is a recently discovered and unique mode of cell death that could trigger this necessary further progress. ICD involves stimulation of a tumour-specific immune response as a downstream effect. Facilitated by certain treatment modalities, cells undergoing ICD can trigger the IFN-γ mediated immune response involving cytotoxic T cells (CTLs) and γδ T cells that eradicate residual tumour cells. In recent years, there has been a significant increase in the number of small-molecules being tested as potential ICD inducers. A large number of these ICD inducers are metal-based complexes. In fact, anticancer metal drugs based on Pt, Ru, Ir, Cu, and Au are now known to give rise to an immune response against tumour cells as the result of ICD. Advances have also been made in terms of exploiting combinatorial and delivery strategies. In favourable cases, these approaches have been shown to increase the efficacy of otherwise ICD "silent" metal complexes. Taken in concert, rationally designed novel anticancer metal complexes that can act as ICD inducers show promise as potential new immunotherapies for neoplastic disease. This Tutorial Review will allow the readers to assess the progress in this fast-evolving field thus setting the stage for future advances.

Expanded porphyrins: functional photoacoustic imaging agents that operate in the NIR-II region.

Photoacoustic imaging (PAI) relies on the use of contrast agents with high molar absorptivity in the NIR-I/NIR-II region. Expanded porphyrins, synthetic analogues of natural tetrapyrrolic pigments (e.g. heme and chlorophyll), constitute as potentially attractive platforms due to their NIR-II absorptivity and their ability to respond to stimuli. Here, we evaluate two expanded porphyrins, naphthorosarin (1) and octaphyrin (4), as stimuli responsive PA contrast agents for functional PAI. Both undergo proton-coupled electron transfer to produce species that absorb well in the NIR-II region. Octaphyrin (4) was successfully encapsulated into 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG2000) nanoparticles to afford OctaNPs. In combination with PAI, OctaNPs allowed changes in the acidic environment of the stomach to be visualized and cancerous versus healthy tissues to be discriminated.

Covalent and non-covalent albumin binding of Au(i) bis-NHCs via post-synthetic amide modification.

Recent decades have witnessed the emergence of Au(i) bis-N-heterocyclic carbenes (NHCs) as potential anticancer agents. However, these systems exhibit little interaction with serum proteins (e.g., human serum albumin), which presumably impacts their pharmacokinetic profile and tumor exposure. Anticancer drugs bound to human serum albumin (HSA) often benefit from significant advantages, including longer circulatory half-lives, tumor targeted delivery, and easier administration relative to the drug alone. In this work, we present Au(i) bis-NHCs complexes, 7 and 9, capable of binding to HSA. Complex 7 contains a reactive maleimide moiety for covalent protein conjugation, whereas its congener 9 contains a naphthalimide fluorophore for non-covalent binding. A similar drug motif was used in both cases. Complexes 7 and 9 were prepared from a carboxylic acid functionalized Au(i) bis-NHC (complex 2) using a newly developed post-synthetic amide functionalization protocol that allows coupling to both aliphatic and aromatic amines. Analytical, and in vitro techniques were used to confirm protein binding, as well as cellular uptake and antiproliferative activity in A549 human lung cancer cells. The present findings highlight a hitherto unexplored approach to modifying Au(i) bis-NHC drug candidates for protein ligation and serve to showcase the relative benefits of covalent and non-covalent HSA binding.

Rationally Designed Redox-Active Au(I) N-Heterocyclic Carbene: An Immunogenic Cell Death Inducer.

Immunogenic cell death (ICD) is a way of reengaging the tumor-specific immune system. ICD can be induced by treatment with chemotherapeutics. However, only a limited number of drugs and other treatment modalities have been shown to elicit the biomarker responses characteristic of ICD and to provide an anticancer benefit in vivo. Here, we report a rationally designed redox-active Au(I) bis-N-heterocyclic carbene that induces ICD both in vitro and in vivo. This work benefits from a synthetic pathway that allows for the facile preparation of asymmetric redox-active Au(I) bis-N-heterocyclic carbenes.

Mitochondrial relocation of a common synthetic antibiotic: A non-genotoxic approach to cancer therapy.

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most commonly prescribed synthetic antibiotics, is reported here as a new approach. Conjugating ciprofloxacin to a triphenyl phosphonium group (giving lead Mt-CFX), is used to enhance the concentration of ciprofloxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in a xenograft mouse model and proved to be well tolerated. Mitochondrial relocalization of antibiotics could emerge as a useful approach to generating anticancer leads that promote cell death via the selective induction of mitochondrially-mediated oxidative damage.

Manganese(II) Texaphyrin: A Paramagnetic Photoacoustic Contrast Agent Activated by Near-IR Light.

The NIR absorptivity of the metallotexaphyrin derivatives MMn, MGd, and MLu for photoacoustic (PA)-based imaging is explored in this study. All three complexes demonstrated excellent photostabilities; however, MMn provided the greatest PA signal intensities in both doubly distilled water and RAW 264.7 cells. In vivo experiments using a prostate tumor mouse model were performed. MMn displayed no adverse toxicity to major organs as inferred from hematoxylin and eosin (H&E) staining and cell blood count testing. MMn also allowed for PA-based imaging of tumors with excellent in vivo stability to provide 3D tumor diagnostic information. Based on the present findings and previous magnetic resonance imaging (MRI) studies, we believe MMn may have a role to play either as a stand-alone PA contrast agent or as a single molecule dual modal (PA and MR) imaging agent for tumor diagnosis.

Toward multifunctional anticancer therapeutics: post-synthetic carbonate functionalisation of asymmetric Au(i) bis-N-heterocyclic carbenes.

A post-synthetic strategy is reported that allows for functionalisation of Au(i)-bis NHCs via carbonate formation. The scope of this methodology was explored using both aromatic and aliphatic alcohols. As a demonstration of potential utility, the fluorescent Au(i)-bis NHC conjugate 5 was prepared; it was found to have enhanced stability when formulated with bovine serum albumin, localise within the mitochondria of A549 cells and do so without compromising the high cytotoxicity seen for the parent Au(i)-bis NHC system.

Oxaliplatin Pt(IV) prodrugs conjugated to gadolinium-texaphyrin as potential antitumor agents.

Described here is the development of gadolinium(III) texaphyrin-platinum(IV) conjugates capable of overcoming platinum resistance by 1) localizing to solid tumors, 2) promoting enhanced cancer cell uptake, and 3) reactivating p53 in platinum-resistant models. Side by side comparative studies of these Pt(IV) conjugates to clinically approved platinum(II) agents and previously reported platinum(II)-texaphyrin conjugates demonstrate that the present Pt(IV) conjugates are more stable against hydrolysis and nucleophilic attack. Moreover, they display high potent antiproliferative activity in vitro against human and mouse cell cancer lines. Relative to the current platinum clinical standard of care (SOC), a lead Gd(III) texaphyrin-Pt(IV) prodrug conjugate emerging from this development effort was found to be more efficacious in subcutaneous (s.c.) mouse models involving both cell-derived xenografts and platinum-resistant patient-derived xenografts. Comparative pathology studies in mice treated with equimolar doses of the lead Gd texaphyrin-Pt(IV) conjugate or the US Food and Drug Administration (FDA)-approved agent oxaliplatin revealed that the conjugate was better tolerated. Specifically, the lead could be dosed at more than three times (i.e., 70 mg/kg per dose) the tolerable dose of oxaliplatin (i.e., 4 to 6 mg/kg per dose depending on the animal model) with little to no observable adverse effects. A combination of tumor localization, redox cycling, and reversible protein binding is invoked to explain the relatively increased tolerability and enhanced anticancer activity seen in vivo. On the basis of the present studies, we conclude that metallotexaphyrin-Pt conjugates may have substantial clinical potential as antitumor agents.

Porphyrinoid Drug Conjugates.

Drawing inspiration from nature today remains a time-honored means of discovering the therapies of tomorrow. Porphyrins, the so-called "pigments of life" have played a key role in this effort due to their diverse and unique properties. They have seen use in a number of medically relevant applications, including the development of so-called drug conjugates wherein functionalization with other entities is used to improve efficacy while minimizing dose limiting side effects. In this Perspective, we highlight opportunities associated with newer, completely synthetic analogs of porphyrins, commonly referred to as porphyrinoids, as the basis for preparing drug conjugates. Many of the resulting systems show improved medicinal or site-localizing properties. As befits a Perspective of this type, our efforts to develop cancer-targeting, platinum-containing conjugates based on texaphyrins (a class of so-called "expanded porphyrins") will receive particular emphasis; however, the promise inherent in this readily generalizable approach will also be illustrated briefly using two other common porphyrin analogs, namely the corroles (a "contracted porphyrin") and porphycene (an "isomeric porphyrin").

Targeting Heterogeneous Tumors Using a Multifunctional Molecular Prodrug.

Reported here is a molecular construct (K1) designed to overcome hurdles associated with delivering active drugs to heterogeneous tumor environments. Construct K1 relies on two cancer environment triggers (GSH and H2O2) to induce prodrug activation. It releases an active drug form (SN-38) under conditions of both oxidative and reductive stress in vitro. Specific uptake of K1 in COX-2 positive aggressive colon cancer cells (SW620 and LoVo) was seen, along with enhanced anticancer activity compared with the control agent SN-38. These findings are attributed to environmentally triggered drug release, as well as simultaneous scavenging of species giving rise to intracellular redox stress. K1 serves to downregulate various cancer survival signaling pathways (AKT, p38, IL-6, VEGF, and TNF-α) and upregulate an anti-inflammatory response (IL-10). Compared with SN-38 and DMSO as controls, K1 also displayed an improved in vivo therapeutic efficacy in a xenograft tumor regrowth model with no noticeable systematic toxicity at the administrated dose. We believe that the strategy described here presents an attractive approach to addressing solid tumors characterized by intratumoral heterogeneity.

Expanding the biological utility of bis-NHC gold(i) complexes through post synthetic carbamate conjugation.

We report the synthesis of a novel hydroxyl-functionalised heteroleptic bis-NHC gold(i) complex that permits conjugation to various amines via carbamate bond formation. The resulting derivatives were studied in vitro using cell proliferation assays and fluorescent microscopic imaging of human cancer cell lines.

Hypoxia-targeted drug delivery.

Hypoxia is a state of low oxygen tension found in numerous solid tumours. It is typically associated with abnormal vasculature, which results in a reduced supply of oxygen and nutrients, as well as impaired delivery of drugs. The hypoxic nature of tumours often leads to the development of localized heterogeneous environments characterized by variable oxygen concentrations, relatively low pH, and increased levels of reactive oxygen species (ROS). The hypoxic heterogeneity promotes tumour invasiveness, metastasis, angiogenesis, and an increase in multidrug-resistant proteins. These factors decrease the therapeutic efficacy of anticancer drugs and can provide a barrier to advancing drug leads beyond the early stages of preclinical development. This review highlights various hypoxia-targeted and activated design strategies for the formulation of drugs or prodrugs and their mechanism of action for tumour diagnosis and treatment.

Activation of Platinum(IV) Prodrugs By Motexafin Gadolinium as a Redox Mediator.

Water-soluble platinum(IV) prodrugs, which proved kinetically stable to reduction in the presence of physiological concentration of ascorbate, were quickly reduced to their active form, oxaliplatin, when co-incubated with a macrocycle metallotexaphyrin (i.e., Motexafin Gadolinium (MGd)). The reduction of Pt(IV) to Pt(II) promoted by MGd occurs in cell culture as well, leading to an increase in the antiproliferative activity of the Pt(IV) species in question. The mediated effect is proportional to the concentration of MGd and gives rise to an enhancement when the prodrug is relatively hydrophilic. MGd is known to localize/accumulate preferentially in tumor tissues. Thus, the present "activation by reduction" approach may allow for the cancer-selective enhancement in the cytotoxicity of Pt(IV) prodrugs.

Discovery of Quinoline-Derived Trifluoromethyl Alcohols, Determination of Their in vivo Toxicity and Anticancer Activity in a Zebrafish Embryo Model.

In this study the rational design, synthesis, and anticancer activity of quinoline-derived trifluoromethyl alcohols were evaluated. Members of this novel class of trifluoromethyl alcohols were identified as potent growth inhibitors in a zebrafish embryo model. Synthesis of these compounds was carried out with an sp(3) -C-H functionalization strategy of methyl quinolines with trifluoromethyl ketones. A zebrafish embryo model was also used to explore the toxicity of ethyl 4,4,4-trifluoro-3-hydroxy-3-(quinolin-2-ylmethyl)butanoate (1), 2-benzyl-1,1,1-trifluoro-3-(quinolin-2-yl)propan-2-ol (2), and trifluoro-3-(isoquinolin-1-yl)-2-(thiophen-2-yl)propan-2-ol (3). Compounds 2 and 3 were found to be more toxic than compound 1; apoptotic staining assays indicated that compound 3 causes increased cell death. In vitro cell proliferation assays showed that compound 2, with an LC50 value of 14.14 µm, has more potent anticancer activity than cisplatin. This novel class of inhibitors provides a new direction in the discovery of effective anticancer agents.

A self-assembling lanthanide molecular nanoparticle for optical imaging.

Chromophores that incorporate f-block elements have considerable potential for use in bioimaging applications because of their advantageous photophysical properties compared to organic dye, which are currently widely used. We are developing new classes of lanthanide-based self-assembling molecular nanoparticles as reporters for imaging and as multi-functional nanoprobes or nanosensors for use with biological samples. One class of these materials, which we call lanthanide "nano-drums", are homogeneous 4d-4f clusters approximately 25 to 30 Å in diameter. These are capable of emitting from the visible to near-infrared wavelengths. Here, we present the synthesis, crystal structure, photophysical properties and comparative cytotoxicity data for a 32 metal Eu-Cd nano-drum [Eu(8)Cd(24)L(12)(OAc)(48)] (1). We also explored the imaging capabilities of this nano-drum using epifluorescence, TIRF, and two-photon microscopy platforms.

Lanthanide nano-drums: a new class of molecular nanoparticles for potential biomedical applications.

We are developing a new class of lanthanide-based self-assembling molecular nanoparticles as potential reporter molecules for imaging, and as multi-functional nanoprobes or nanosensors in diagnostic systems. These lanthanide "nano-drums" are homogeneous 4d-4f clusters approximately 25 to 30 Å in diameter that can emit from the visible to near-infrared (NIR) wavelengths. Here, we present syntheses, crystal structures, photophysical properties, and comparative cytotoxicity data for six nano-drums containing either Eu, Tb, Lu, Er, Yb or Ho. Imaging capabilities of these nano-drums are demonstrated using epifluorescence, total internal reflection fluorescence (TIRF), and two-photon microscopy. We discuss how these molecular nanoparticles can to be adapted for a range of assays, particularly by taking advantage of functionalization strategies with chemical moieties to enable conjugation to protein or nucleic acids.