Synthesis of water-soluble porphyrin-dendron conjugates for targeted photodynamic therapy

The targeting of photosensitisers with tumour-associated biomolecules is widely used for improved photosensitiser tumour localisation during photodynamic therapy, allowing fewer side effects in comparison to conventional cancer treatments. In particular, conjugation to antibody fragments allows exploitation of their high affinity towards tumour-associated antigens; however current methods of conjugating porphyrins to antibody fragments represent a compromise between high binding ratios and good stoichiometric and site-specific control. The work presented herein addresses this problem through the synthesis of porphyrin-dendron conjugates and their attachment at the interchain disulfide bridge of antibody fragments, allowing improved binding ratios while maintaining good structural control.Synthesis of a range of click-functionalised porphyrins and dendrons bearing complimentary peripheral functionalities was carried out, followed by click conjugation of these structures under microwave irradiation to produce a range of lipophilic and hydrophilic porphyrin-dendron conjugates with between two and four peripheral porphyrins. Photophysical evaluation demonstrated retention of UV-vis and fluorescent character of porphyrins after conjugation, with some quenching of UV-vis absorption observed due to the close proximity of the porphyrins. Singlet oxygen quantum yields showed some quenching in all conjugates, with more sterically hindered systems showing the greatest reduction in SOQY in comparison to control porphyrins.Conjugation of porphyrins to a HER2-targeted Herceptin™ Fab fragment was carried out through pre-conjugation of an alkyne-dibromomaleimide heterobifunctional linker to the Fab fragment, with two examples of cationic porphyrins conjugated via a click chemistry strategy to yield conjugates with precise 1:1 stoichiometry. Preliminary cytotoxicity studies of the targeted photosensitisers showed that both conjugates exhibited limited dark toxicity, and excellent cell killing in the HER2+ BT-474 cell line.Successful focal-point deprotection and azide functionalisation was carried out on a single porphyrin-dendron conjugate, with a successful model click reaction to an alkyne-functionalised sugar displaying the possibility of bioconjugation of porphyrindendron conjugates via a click methodology

Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: Towards mild strategies for the labelling of glycans

Fully deprotected alkynyl-functionalised mono- and oligosaccharides undergo CuAAC-based conjugation with water-soluble porphyrin azides in aqueous environments. The mild reaction conditions are fully compatible with the presence of labile glycosidic bonds. This approach provides an ideal strategy to conjugate tetrapyrroles to complex carbohydrates

PET/PDT theranostics: Synthesis and biological evaluation of a peptide-targeted gallium porphyrin

The development of novel theranostic agents is an important step in the pathway towards personalised medicine, with the combination of diagnostic and therapeutic modalities into a single treatment agent naturally lending itself to the optimisation and personalisation of treatment. In pursuit of the goal of a molecular theranostic suitable for use as a PET radiotracer and a photosensitiser for PDT, a novel radiolabelled peptide–porphyrin conjugate targeting the α6β1-integrin has been developed. 69/71Ga and 68Ga labelling of an azide-functionalised porphyrin has been carried out in excellent yields, with subsequent bioconjugation to an alkyne-functionalised peptide demonstrated. α6β1-integrin expression of two cell lines has been evaluated by flow cytometry, and therapeutic potential of the conjugate demonstrated. Evaluation of the phototoxicity of the porphyrin–peptide theranostic conjugate in comparison to an untargeted control porphyrin in vitro, demonstrated significantly enhanced activity for a cell line with higher α6β1-integrin expression when compared with a cell line exhibiting lower α6β1-integrin expression

Site-selective multi-porphyrin attachment enables the formation of a next-generation antibody-based photodynamic therapeutic

Herein we present a significant step towards next-generation antibody-based photodynamic therapeutics. Site-selective modification of a clinically relevant monoclonal antibody, with a serum-stable linker bearing a strained alkyne, allows for the controlled Cu-free “click” assembly of an in vitro active antibody-based PDT agent using a water soluble azide porpyhrin

Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy including senscence, necrosis, and autophagy, but not apoptosis

In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer

Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment

The rapidly increasing interest in the synthesis of antibody–drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin–antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case

Development of PDT/PET theranostics: synthesis and biological evaluation of an ¹⁸F-radiolabeled water soluble porphyrin

Synthesis of the first water-soluble porphyrin radiolabeled with fluorine-18 is described: a new molecular theranostic agent which integrates the therapeutic selectivity of photodynamic therapy (PDT) with the imaging efficacy of positron emission tomography (PET). Generation of the theranostic was carried out through the conjugation of a cationic water-soluble porphyrin bearing an azide functionality to a fluorine-18 radiolabeled prosthetic bearing an alkyne functionality through click conjugation, with excellent yields obtained in both cold and hot synthesis. Biological evaluation of the synthesized structures shows the first example of an 18 F-radiolabeled porphyrin retaining photocytotoxicity following radiolabeling and demonstrable conjugate uptake and potential application as a radiotracer in vivo. The promising results gained from biological evaluation demonstrate the potential of this structure as a clinically relevant theranostic agent, offering exciting possibilities for the simultaneous imaging and photodynamic treatment of tumors

Correction to: Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol

An amendment to this paper has been published and can be accessed via the original articl

Successful Drug Discovery

International audienc

Towards antibody-drug conjugates and prodrug strategies with extracellular stimuli-responsive drug delivery in the tumor microenvironment for cancer therapy

International audienc