Francesca Bryden

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.

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.

Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy include senescence, 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.

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.

Duramycin-porphyrin conjugates for targeting of tumour cells using photodynamic therapy

Duramycin, through binding with phosphatidylethanolamine (PE), has been shown to be a selective molecular probe for the targeting and imaging of cancer cells. Photodynamic therapy aims to bring about specific cytotoxic damage to tumours through delivery of a photosensitising agent and light irradiation. Conjugation to biological molecules that specifically target cancer has been shown to increase photosensitiser (PS) selectivity and decrease damage to surrounding normal tissue. The aim of this study was to target tumour cells with a PE-specific PS therefore duramycin was conjugated to a porphyrin based PS which was achieved via direct reaction with the ε-amino group on the lysine residue near duramycins N-terminal. The compound was subsequently purified using RP-HPLC and confirmed using mass spectrometry. Binding of the conjugate to ovarian and pancreatic cancer cell lines was assessed by flow cytometry. Light irradiation with a light fluence of 7.5J/cm(2) was delivered to conjugate treated cancer cells and cell proliferation analysed by MTT assay. The conjugate detected PE on all 4 cancer cell lines in a concentration dependent manner and conjugate plus irradiation effectively reduced cell proliferation at concentrations ≥0.5μM, dependent on cancer cell line. Reduction in cell proliferation by the irradiated conjugate was enhanced over unconjugated duramycin in A2780, AsPC-1 and SK-OV-3 (p<0.05). In this study we have shown that a duramycin-porphyrin conjugate retained good binding affinity for its target and, following irradiation, reduced cell proliferation of pancreatic and ovarian cancer cell lines.

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.

Poly-(D,L-lactide-co-glycolide) nanoparticles with covalently-bound porphyrins for efficient singlet oxygen photosensitization

With the aim of assessing the role of the chemical structure of the photosensitizer on the photophysical and photochemical properties of the final nanoparticle suspension, we have investigated a series of poly-(ethylene glycol)-poly-(D,L-lactide-co-glycolide) nanoparticles containing a hydrophobic or a hydrophilic porphyrin covalently conjugated to the nanoparticle. Covalent conjugation responded to the objective of trying to improve photosensitizer loading in these nanoparticles, especially for hydrophilic photosensitizers, but also enabled the porphyrins to remain attached to the nanoparticle without necessarily being inside the poly-(D,L-lactide-co-glycolide) core. This strategy has provided valuable information about the dependence of the photophysical and singlet oxygen photosensitizing properties of the suspensions on the nature of the photosensitizer. It is concluded that poly-(D,L-lactide-co-glycolide) nanoparticles with covalently-bound hydrophilic porphyrins show superior singlet oxygen photosensitizing ability.