Ruggero Dondi

Highly Size‐ and Shape‐Controlled Synthesis of Silver Nanoparticles via a Templated Tollens Reaction

A mild, facile one-step synthetic strategy for the preparation of size-and shape-controlled silver nanoparticles (AgNPs) is presented. The high degree of size-and shape-control of these AgNPs is achieved by the use of triazole sugar ligands scaffolded by a central resorcinol ether core. Both the triazoles and the resorcinol ether core mediate the nucleation, growth, and passivation phases of the preparation of AgNP in the presence of the Tollens reagent as the silver source. Kinetic and 1H NMR titration data is presented describing the nature of the interactions between the Tollens reagent and these ligands.

Highly efficient synthesis of DNA-binding hairpin polyamides via the use of a new triphosgene coupling strategy

A facile and highly efficient solid phase synthesis method is reported for the preparation of hairpin DNA-binding polyamides using the cost-effective triphosgene (BTC) activating agent. Difficult polyamide sequences were prepared from N-methylimidazole (Im) and N-methylpyrrole (Py) building blocks with high stepwise yields (>98%) using Boc chemistry. The versatility of the triphosgene coupling approach was also demonstrated for the first time on aryl hydrazine resins to afford biomedically relevant tail-truncated polyamides in excellent isolated yields.

Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

Highly Efficient Synthesis of DNA-Binding Polyamides Using a Convergent Fragment-Based Approach

Two advances in the synthesis of hairpin pyrrole-imidazole polyamides (PAs) are described. First, the application of a convergent synthetic strategy is shown, involving the Boc-based solid phase synthesis of a C-terminal fragment and the solution phase synthesis of the N-terminal fragment. Second a new hybrid resin is developed that allows for the preparation of hairpin PAs lacking a C-terminal β-alanine tail. Both methods are compatible with a range of coupling reagents and provide a facile, modular route to prepare PA libraries in high yield and crude purity.

Flexible synthesis of cationic peptide–porphyrin derivatives for light-triggered drug delivery and photodynamic therapy

Amphiphilic cell-penetrating peptide–porphyrin conjugates have been developed for application in light-based therapeutic techniques.

Endolysosomal targeting of a clinical chlorin photosensitiser for light-triggered delivery of nano-sized medicines

A major problem with many promising nano-sized biotherapeutics including macromolecules is that owing to their size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. Photochemical internalisation (PCI) is a technique for inducing cytosolic release of the entrapped agents that harnesses sub-lethal photodynamic therapy (PDT) using a photosensitiser that localises in endolysosomal membranes. Using light to trigger reactive oxygen species-mediated rupture of the photosensitised endolysosomal membranes, the spatio-temporal selectivity of PCI then enables cytosolic release of the agents at the selected time after administration so that they can reach their intracellular targets. However, conventional photosensitisers used clinically for PDT are ineffective for photochemical internalisation owing to their sub-optimal intracellular localisation. In this work we demonstrate that such a photosensitiser, chlorin e6, can be repurposed for PCI by conjugating the chlorin to a cell penetrating peptide, using bioorthogonal ligation chemistry. The peptide conjugation enables targeting of endosomal membranes so that light-triggered cytosolic release of an entrapped nano-sized cytotoxin can be achieved with consequent improvement in cytotoxicity. The photoproperties of the chlorin moiety are also conserved, with comparable singlet oxygen quantum yields found to the free chlorin.

Codelivery of a cytotoxin and photosensitiser via a liposomal nanocarrier: a novel strategy for light-triggered cytosolic release

Light-triggered intracellular delivery of a protein toxin was achieved by codelivery via a liposomal nanocarrier, targeted with a cell-penetrating peptide (CPP)–photosensitiser conjugate.

Abstract B38: Photochemical internalisation for the local delivery of chemotherapy in prostate cancer

• Introduction Prostate cancer is fifth leading cause of death from cancer in men. Although chemotherapy is the gold standard treatment for a wide range of cancers including the prostate, there are several limitations (systemic toxicity, multidrug resistance or inadequate cellular delivery) and there is growing interest in developing focal therapies when appropriate. Photochemical Internalisation (PCI) is a focal light-triggered drug delivery system, which combines low dose Photodynamic Therapy (PDT) with chemotherapy agents to enhance delivery of the agents to their specific subcellular target. PCI is particularly suitable for agents prone to endolysosomal sequestration, thererefore the photosensitisers (PS) used for PCI localise in endolysosomal membranes which upon visible light exposure leads to the production of reactive oxygen species (ROS), causing photooxidative disruption of membranes and subsequent cytosolic release of the entrapped agents. These will then reach their intended targets more efficiently, improving efficacy and potentially reducing the therapeutic dose. The present project focuses on the use of PCI to deliver Saporin, a ribosome inactivating (RIP) type 1 protein. RIPs are able to cause permanent damage to eukaryotic ribosomes; however, despite its intrinsic high toxicity, endolysosomal sequestration impairs its ability to inhibit protein synthesis. • Aim To assess the potential of PCI for enhanced delivery of Saporin in human and rat prostate cancer cells. In addition, a CXCR4-targeted peptide-PS conjugate was tested. • Materials & Methods Porphyrin and chlorin-based PS were co-administered with Saporin. Cells were incubated for 24hrs with different drug combinations then exposed to light, and cell viability was measured at different time points post illumination using the MTT assay. Subcellular location of Saporin and PS were observed using confocal microscopy. • Results PCI resulted in a synergistic effect between sub-lethal PDT and Saporin. Despite minimal cell kill when the toxin or PS were administered alone, PCI delivery enhanced Saporin cytotoxicity using only 20pM in the rat Mat-LyLu and 2nM in PC3 human cell lines. PCI elicits up to 1.5-fold higher efficacy in cell kill vs. PDT 48hrs after light treatment and 3.5-fold higher 96hrs after light treatment in rat and human prostate cancer cells accordingly (p Intracellular redistribution following endolysosomal release of fluorescently labelled Saporin and PS following illumination was confirmed with confocal microscopy. • Conclusions These results demonstrate the potential of PCI for prostate cancer treatment in vitro and in vivo tumour experiments are in progress. Citation Format: ALEJANDRA MARTINEZ DE PINILLOS, RUGGERO DONDI, L. WANG, ELNAZ YAGHINI, JOSEPHINE H. WOODHAMS, CAROLINE M. MOORE, IAN M. EGGLESTON, ALEXANDER J. MACROBERT. Photochemical internalisation for the local delivery of chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B38.