Photoinduced Photosensitizer-Antibody Conjugates Kill HIV Env-Expressing Cells, Also Inactivating HIV.

HIV-infected cells persist for decades in patients administered with antiretroviral therapy (ART). Meanwhile, an alarming surge in drug-resistant HIV viruses has been occurring. Addressing these issues, we propose the application of photoimmunotherapy (PIT) against not only HIV Env-expressing cells but also HIV. Previously, we showed that a human anti-gp41 antibody (7B2) conjugated to cationic or anionic photosensitizers (PSs) could specifically target and kill the HIV Env-expressing cells. Here, our photolysis studies revealed that the binding of photoimmunoconjugates (PICs) on the membrane of HIV Env-expressing cells is sufficient to induce necrotic cell death due to physical damage to the membrane by singlet oxygen, which is independent of the type of PSs. This finding persuaded us to study the virus photoinactivation of PICs using two HIV-1 strains, X4 HIV-1 NL4-3 and JR-CSF virus. We observed that the PICs could destroy the viral strains, probably via physical damage on the HIV envelope. In conclusion, we report the application of PIT as a possible dual-tool for HIV immunotherapy and ART by killing HIV-expressing cells and cell-free HIV, respectively.

Bio-Orthogonal Conjugation of a Cationic Metalloporphyrin to BSA and HSA via "Click" Chemistry.

In this study, we present a convenient method for the labelling of tyrosine residues on bovine serum albumin (BSA) and human serum albumin (HSA) and report for the first time their subsequent bio-orthogonal conjugation with porphyrins via "click" chemistry. We demonstrate that these serum proteins can be labelled with an alkyne-diazonium heterobifunctional linker and can then undergo chemo-selective bio-orthogonal conjugation with a water-soluble azido metalloporphyrin via "click" chemistry to yield protein-conjugates that retain their photodynamic properties. In our hands, this method was found to be highly reproducible, scalable, and tuneable which allows for the production of bioconjugates where the porphyrin-protein conjugate not only retains an ability to generate singlet oxygen but possess an enhanced relative singlet oxygen quantum yields relative to the porphyrin alone. Furthermore, we have investigated the photochemical properties of these conjugates through photospectrometric techniques and have determined that the porphyrin macrocycles remain appreciably photostable under light irradiation. Our phototoxic protein-photosensitizer-conjugates show excellent photodynamic activity against a human colorectal adenocarcinoma cancer cell line (HT-29) with cell viabilities of 7.7±0.5 % (IC50 8.76±2.14 µM) and 1.7±1.9 % (IC50 8.48±5.11 µM) for BSA and HAS, respectively, when irradiated with 20 J cm-2 of white-light. Importantly, neither of the conjugates was found to possess any significant "dark" toxicity even at concentrations of 100 µM. Furthermore, the natural fluorescent properties of the bioconjugates allowed for the determination of cellular uptake in vitro via fluorescence microscopy thus highlighting the potential theranostic applications of these unique protein-drug-conjugates.

RAPTA-Decorated Polyacrylamide Nanoparticles: Exploring their Synthesis, Physical Properties and Effect on Cell Viability.

In this study, we report the first successful immobilisation of a known cytoactive [Ru(η6 -arene)(C2 O4 )PTA] (RAPTA) complex to a biologically inert polyacrylamide nanoparticle support. The nanoparticles have been characterised by zetasizer analysis, UV/Vis, ATR-FTIR, TGA and ICP-MS to qualitatively and quantitatively confirm the presence of the metallodrug on the surface of the carrier. The native RAPTA complex required a concentration of 50 µM to produce a cell viability of 47.1±2.1 % when incubated with human Caucasian colorectal adenocarcinoma cells for 72 h. Under similar conditions a cell viability of 45.1±1.9 % was obtained with 0.5 µM of RAPTA complex in its immobilised form. Therefore, conjugation of the RAPTA metallodrug to our nanoparticle carriers resulted in a significant 100-fold decrease in effective concentration of ruthenium required for a near identical biological effect on cell viability.

Photoimmunotherapy Using Cationic and Anionic Photosensitizer-Antibody Conjugates against HIV Env-Expressing Cells.

Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; "Click" conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and "Lysine" conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells.

Synthesis of a porphyrin with histidine-like chelate: an efficient path towards molecular PDT/SPECT theranostics.

The goal of "personalised" medicine has seen a growing interest in the development of theranostic agents. Bifunctional, and targeted-trifunctional, theranostic water-soluble porphyrins with a histidine-like chelating group have been synthesised via copper-catalysed azide-alkyne cycloaddition (CuAAC) "click" chemistry in high yield and purity. They are capable of photodynamic treatment and [99mTc(CO)3]+ complexation for single-photon emission computed tomography (SPECT) imaging, with a radiochemical yield of >95%. The toxicity and phototoxicity were evaluated on HT-29 cells, DU145, and DU145-PSMA cell lines, with the targeted theranostic showing more potent phototoxicity towards DU145-PSMA expressing cells.

Homo- and Hetero-dinuclear Arene-Linked Osmium(II) and Ruthenium(II) Organometallics: Probing the Impact of Metal Variation on Reactivity and Biological Activity.

Dinuclear metallodrugs offer much potential in the development of novel anticancer chemotherapeutics as a result of the distinct interactions possible with bio-macromolecular targets and the unique biological activity that can result. Herein, we describe the development of isostructural homo-dinuclear OsII -OsII and hetero-dinuclear OsII -RuII organometallic complexes formed from linking the arene ligands of [M(η6 -arene)(C2 O4 )(PTA)] units (M=Os/Ru; PTA=1,3,5-triaza-7-phosphaadamantane). Using these complexes together with the known RuII -RuII analogue, a chromatin-modifying agent, we probed the impact of varying the metal ions on the structure, reactivity and biological activity of these complexes. The complexes were structurally characterised by X-ray diffraction experiments, their stability and reactivity were examined by using 1 H and 31 P NMR spectroscopy, and their biological activity was assessed, alongside that of mononuclear analogues, through MTT assays and cell-cycle analysis (HT-29 cell line). The results revealed high antiproliferative activity in each case, with cell-cycle profiles of the dinuclear complexes found to be similar to that for untreated cells, and similar but distinct profiles for the mononuclear complexes. These results indicate these complexes impact on cell viability predominantly through a non-DNA-damaging mechanism of action. The new OsII -OsII and OsII -RuII complexes reported here are further examples of a family of compounds operating via mechanisms of action atypical of the majority of metallodrugs, and which have potential as tools in chromatin research.

Synthesis and In Vitro Biological Evaluation of a Second-Generation Multimodal Water-Soluble Porphyrin-RAPTA Conjugate for the Dual-Therapy of Cancers.

In this study, we report the synthesis and biological evaluation of a novel cationic porphyrin-[Ru(η6-arene)(C2O4)PTA] (RAPTA) conjugate with potential as a multimodal dual-therapeutic agent. In the absence of high intensity light, relative to untreated cells our conjugate resulted in a 83% decrease in viable human adenocarcinoma cells at a concentration of 10 µM, which is significantly more active than the 57% decrease achieved with the same concentration of the unconjugated RAPTA complex alone. With a light dose of 20 J cm-2 (400-1200 nm) a reduction of 98% of viable cells was observed for the same concentration of conjugate. The conjugate is internalized by HT-29 cancer cells as proven by ICP-MS analysis and fluorescence microscopy; the latter result suggests that the conjugate has applications as a multimodal agent by acting as a fluorophore to obtain in vivo biodistribution data. Furthermore, the conjugate has an excellent relative singlet oxygen quantum yield, and the tetrapyrollic unit was found to be photostable under irradiation by either white light or red light.

Evaluation of a Bispidine-Based Chelator for Gallium-68 and of the Porphyrin Conjugate as PET/PDT Theranostic Agent.

In this study a bispidine ligand has been applied to the complexation of gallium(III) and radiolabelled with gallium-68 for the first time. Despite its 5-coordinate nature, the resulting complex is stable in serum for over two hours, demonstrating a ligand system well matched to the imaging window of gallium-68 positron emission tomography (PET). To show the versatility of the bispidine ligand and its potential use in PET, the bifunctional chelator was conjugated to a porphyrin, producing a PET/PDT-theranostic, which showed the same level of stability to serum as the non-conjugated gallium-68 complex. The PET/PDT complex killed >90 % of HT-29 cells upon light irradiation at 50 µm. This study shows bispidines have the versatility to be used as a ligand system for gallium-68 in PET.

c(RGDfK)- and ZnTriMPyP-Bound Polymeric Nanocarriers for Tumor-Targeted Photodynamic Therapy.

Active targeting strategies are currently being extensively investigated in order to enhance the selectivity of photodynamic therapy. The aim of the present research was to evaluate whether the external decoration of nanopolymeric carriers with targeting peptides could add more value to a photosensitizer formulation and increase antitumor therapeutic efficacy and selectivity. To this end, we assessed PLGA-PLA-PEG nanoparticles (NPs) covalently attached to a hydrophilic photosensitizer 5-[4-azidophenyl]-10,15,20-tri-(N-methyl-4-pyridinium)porphyrinato zinc (II) trichloride (ZnTriMPyP) and also to c(RGDfK) peptides, in order to target αv ß3 integrin-expressing cells. In vitro phototoxicity investigations showed that the ZnTriMPyP-PLGA-PLA-PEG-c(RGDfK) nanosystem is effective at submicromolar concentrations, is devoid of dark toxicity, successfully targets αv ß3 integrin-expressing cells and is 10-fold more potent than related nanosystems where the PS is occluded instead of covalently bound.

The Application of Reversible Intramolecular Sulfonamide Ligation to Modulate Reactivity in Organometallic Ruthenium(II) Diamine Complexes.

Metallation of biomacromolecular species forms the basis for the anticancer activity of many metallodrugs. A major limitation of these compounds is that their reactivity is indiscriminate and can, in principle, occur in healthy tissue as well as cancerous tissue, potentially leading to side effects in vivo. Here we present pH-dependent intramolecular coordination of an arene-tethered sulfonamide functionality in organometallic ruthenium(II) ethylenediamine complexes as a route to controlling the coordination environment about the central metal atom. Through variation of the sulfonamide R group and the length of the tether linking it to the arene ligand the acidity of the sulfonamide NH group, and hence the pH-region over which regulation of metal coordination occurs, can be modulated. Intramolecular sulfonamide ligation controlled the reactivity of complex 4 within the physiologically relevant pH-region, rendering it more reactive towards 5'-GMP in mildly acidic pH-conditions typical of tumour tissue compared to the mildly alkaline pH-conditions typical of healthy tissue. However, the activation of 4 by ring-opening of the chelate was found to be a slow process relative to the timescale of typical cell culture assays and members of this series of complexes were found not to be cytotoxic towards the HT-29 cell line. These complexes provide the basis for the development of analogues of increased potency where intramolecular sulfonamide ligation regulates reactivity and therefore cytotoxicity in a pH-dependent, and potentially, tissue-dependent manner.

Photosensitizer Antibody-Drug Conjugates: Past, Present, and Future.

This Review aims to highlight key aspects of tetrapyrrole-based antibody-drug conjugates (ADCs) and significant developments in the field since 2010. Many new conjugation methods have been developed and employed in the past decade, and associated with this, there has been a rising interest in theranostic conjugates. We have investigated the physicochemical properties that tetrapyrroles need to possess in order to be viable photosensitizers for conjugation to antibodies. Differences in conjugation strategies are discussed, and structure-activity relationships of tetrapyrrole-antibody conjugates are reported, where available. As the elegance of bioconjugation techniques has increased, it has paved the way for exceptionally phototoxic, yet highly selective, tetrapyrrole-antibody conjugates, with photocytotoxicities in the nanomolar range, to be synthesized and biologically evaluated.

In vitro cytotoxicity of a library of BODIPY-anthracene and -pyrene dyads for application in photodynamic therapy.

The facile synthesis and in vitro activity of a library of heavy atom-free BODIPY-anthracene, -pyrene dyads (BAD-13-BPyrD-19) and a control (BODIPY 20) are reported. We demonstrate that singlet oxygen produced from dyad triplet states formed from charge-separated states is sufficient to induce cytotoxicity in human breast cancer cells (MDA-MB-468) at micromolar concentrations. The compounds in this series are promising candidates for photodynamic therapy, especially BAD-17 which displays significant photocytotoxicity (15% cell viability) at a concentration of 5 × 10-7 M, with minimal toxicity (89% cell viability) in the absence of light.

Porphyrins and related macrocycles: Combining photosensitization with radio- or optical-imaging for next generation theranostic agents.

This review summarises recent research into combining the photosensitizing properties of porphyrins with imaging techniques such as PET and NIR fluorescence for so called "theranostic" applications, which combine biomedical imaging and therapeutic potential into a single administered substance. The photophysical mechanisms of both the therapeutic and diagnostic properties of porphyrins are discussed, as well as key characteristics that are required in order to deliver the most effective treatment.

Selective radiolabelling with 68Ga under mild conditions: a route towards a porphyrin PET/PDT theranostic agent.

A theranostic conjugate for use as a positron emission tomography (PET) radiotracer and as a photosensitiser for photodynamic therapy (PDT) has been synthesised. A water-soluble porphyrin was coupled with the bifunctional chelate, H4Dpaa.ga. This conjugate is capable of rapid 68Ga complexation under physiological conditions; with 93% and 80% radiochemical yields achieved, at pH 4.5 and pH 7.4 respectively, in 15 min at 25 °C. Photocytotoxicity was evaluated on HT-29 cells and showed the conjugate was capable of >50% cell death at 50 µM upon irradiation with light, while causing minimal toxicity in the absence of light (>95% cell survival).

Photo-induced anticancer activity and singlet oxygen production of prodigiosenes.

The photo-induced cytotoxicity of prodigiosenes is reported. One prodigiosene represents a synthetic analogue of the natural product prodigiosin, and two are conjugated to molecules that target the estrogen receptor (ER). A comparison of incubation and irradiation frameworks for the three prodigiosenes is reported, with activity against ER- and ER+ lines explored. Furthermore, the ability of the three prodigiosenes to photosensitise the production of singlet oxygen is demonstrated, shedding mechanistic light onto possible photodynamic therapeutic effects of this class of tripyrroles.

Synthesis and Characterization of Temperature-Sensitive and Chemically Cross-Linked Poly( N-isopropylacrylamide)/Photosensitizer Hydrogels for Applications in Photodynamic Therapy.

A novel poly( N-isopropylacrylamide) (PNIPAM) hydrogel containing different photosensitizers (protoporphyrin IX (PpIX), pheophorbide a (Pba), and protoporphyrin IX dimethyl ester (PpIX-DME)) has been synthesized with a significant improvement in water solubility and potential for PDT applications compared to the individual photosensitizers (PSs). Conjugation of PpIX, Pba, and PpIX-DME to the poly( N-isopropylacrylamide) chain was achieved using the dispersion polymerization method. This study describes how the use of nanohydrogel structures to deliver a photosensitizer with low water solubility and high aggregation tendencies in polar solvents overcomes these limitations. FT-IR spectroscopy, UV-vis spectroscopy, 1H NMR, fluorescence spectroscopy, SEM, and DLS analysis were used to characterize the PNIPAM-photosensitizer nanohydrogels. Spectroscopic studies indicate that the PpIX, Pba, and PpIX-DME photosensitizers are covalently conjugated to the polymer chains, which prevents aggregation and thus allows significant singlet oxygen production upon illumination. Likewise, the lower critical solution temperature was raised to ∼44 °C in the new PNIPAM-PS hydrogels. The PNIPAM hydrogels are biocompatible with >90% cell viability even at high concentrations of the photosensitizer in vitro. Furthermore, a very sharp onset of light-dependent toxicity for the PpIX-based nanohydrogel in the nanomolar range and a more modest, but significant, photocytotoxic response for Pba-PNIPAM and PpIX-DME-PNIPAM nanohydrogels suggest that the new hydrogels have potential for applications in photodynamic therapy.

Synthesis of a novel HER2 targeted aza-BODIPY-antibody conjugate: synthesis, photophysical characterisation and in vitro evaluation.

We herein report the synthesis and analysis of a novel aza-BODIPY-antibody conjugate, formed by controlled and regioselective bioconjugation methodology. Employing the clinically relevant antibody, which targets HER2 positive cancers, represents an excellent example of an antibody targeting strategy for this class of near-IR emitting fluorophore. The NIR fluorescence and binding properties were validated through in vitro studies using live cell confocal imaging.

Assembly of High-Potency Photosensitizer-Antibody Conjugates through Application of Dendron Multiplier Technology.

Exploitation of photosensitizers as payloads for antibody-based anticancer therapeutics offers a novel alternative to the small pool of commonly utilized cytotoxins. However, existing bioconjugation methodologies are incompatible with the requirement of increased antibody loading without compromising antibody function, stability, or homogeneity. Herein, we describe the first application of dendritic multiplier groups to allow the loading of more than 4 porphyrins to a full IgG antibody in a site-specific and highly homogeneous manner. Photophysical evaluation of UV-visible absorbance and singlet oxygen quantum yields highlighted porphyrin-dendron 14 as the best candidate for bioconjugation; with subsequent bioconjugation producing a HER2-targeted therapeutic with average loading ratios of 15.4:1. In vitro evaluation of conjugate 18 demonstrated a nanomolar photocytotoxic effect in a target cell line, which overexpresses HER2, with no observed photocytotoxicity at the same concentration in a control cell line which expresses native HER2 levels, or in the absence of irradiation with visible light.

Delayed release singlet oxygen sensitizers based on pyridone-appended porphyrins.

A new type of porphyrin photosensitizer capable of generating singlet oxygen upon irradiation, storing it through binding to pyridone subunits, followed by slow release at 20-40 °C, is reported. The timescale of singlet oxygen release can be varied depending on the pyridone group substitution pattern, forming endoperoxides of different stabilities. Modified tetra- and octa-substituted pyridone-porphyrins showed solubility in water, allowing for straightforward delivery into cells. The effect of delayed singlet oxygen formation due to endoperoxide decomposition was demonstrated on cancer cells in vitro.

A convenient method for multicolour labelling of proteins with BODIPY fluorophores via tyrosine residues.

Fluorescence is an essential imaging modality for labelling and visualising cells and sub-cellular structures. Multicolour labelling is especially challenging due to differences in physicochemical and photophysical behaviour of structurally unrelated fluorophores in the heterogeneous environments found in sub-cellular compartments. Herein, we report the conjugation of three azide-bearing BODIPYs with similar core structures but widely different emission wavelengths (green, red and NIR) to tyrosine residues of a model globular protein (BSA) via a common linking methodology. The resulting BODIPY-BSA conjugates have demonstrated multi-wavelength fluorescence emission for biological applications. Fluorescence imaging was performed in HeLa cells through live cell confocal microscopy imaging, with good intracellular location visualisation observed.