Structural Investigations, Cellular Imaging, and Radiolabeling of Neutral, Polycationic, and Polyanionic Functional Metalloporphyrin Conjugates.

Over the past decade, porphyrin derivatives have emerged as invaluable synthetic building blocks and theranostic kits for the delivery of cellular fluorescence imaging and photodynamic therapy. Tetraphenylporphyrin (TPP), its metal complexes, and related derivatives have been investigated for their use as dyes in histology and as components of multimodal imaging probes. The photophysical properties of porphyrin-metal complexes featuring radiometals have been a focus of our attention for the realization of fluorescence imaging probes coupled with radioimaging capabilities and therapeutic potential having "true" theranostic promise. We report hereby on the synthesis, radiochemistry, structural investigations, and preliminary in vitro and in vivo uptake studies on a range of functionalized porphyrin-based derivatives. In pursuit of developing new porphyrin-based probes for multimodality imaging applications, we report new functionalized neutral, polycationic, and polyanionic porphyrins incorporating nitroimidazole and sulfonamide moieties, which were used as targeting groups to improve the notoriously poor pharmacokinetics of porphyrin tags. The resulting functional metalloporphyrin species were stable under serum challenges and the nitroimidazole and sulfonamide derivatives remained fluorescent, allowing in vitro confocal studies and visualization of the lysosomal uptake in a gallium(III) sulfonamide derivative. The molecular structures of selected porphyrin derivatives were determined by single crystal X-ray diffraction using synchrotron radiation. We also investigated the nature of the emission/excitation behavior of model functional porphyrins using in silico approaches such as TD DFT in simple solvation models. The conjugation of porphyrins with the [7-13] and [7-14] fragments of bombesin was also achieved, to provide targeting of the gastrin releasing peptide receptor (GRPR). Depending on the metal, probe conjugates of relevance for single photon emission computed tomography (SPECT) or positron emission tomography (PET) probes have been designed and tested hereby, using TPP and related functional free base porphyrins as the bifunctional chelator synthetic scaffold and 111In[In] or 68Ga[Ga], respectively, as the central metal ions. Interestingly, for simple porphyrin conjugates good radiochemical incorporation was obtained for both radiometals, but the presence of peptides significantly diminished the radio-incorporation yields. Although the gallium-68 radiochemistry of the bombesin conjugates did not show radiochemical incorporation suitable for in vivo studies, likely because the presence of the peptide changed the behavior of the TPP-NH2 synthon taken alone, the optical imaging assays indicated that the conjugated peptide tags do mediate uptake of the porphyrin units into cells.

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

Endosomal entrapment is a key issue for the intracellular delivery of many nano-sized biotherapeutics to their cytosolic or nuclear targets. Photochemical internalisation (PCI) is a novel light-based solution that can be used to trigger the endosomal escape of a range of bioactive agents into the cytosol leading to improved efficacy in pre-clinical and clinical studies. PCI typically depends upon the endolysosomal colocalisation of the bioactive agent with a suitable photosensitiser that is administered separately. In this study we demonstrate that both these components may be combined for codelivery via a novel multifunctional liposomal nanocarrier, with a corresponding increase in the biological efficacy of the encapsulated agent. As proof of concept, we show here that the cytotoxicity of the 30 kDa protein toxin, saporin, in MC28 fibrosarcoma cells is significantly enhanced when delivered via a cell penetrating peptide (CPP)-modified liposome, with the CPP additionally functionalised with a photosensitiser that is targeted to endolysosomal membranes. This innovation opens the way for the efficient delivery of a range of biotherapeutics by the PCI approach, incorporating a clinically proven liposome delivery platform and using bioorthogonal ligation chemistries to append photosensitisers and peptides of choice.

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.

SERS enhancement of silver nanoparticles prepared by a template-directed triazole ligand strategy.

Two advances in the development of a one-pot method to prepare silver nanoparticles (AgNPs) using the Tollens' reagent are described. First, a template-directed process of AgNP synthesis using resorcinol triazole ligands bearing two pendent galactose sugars is shown. Second, the conversion of these AgNPs into SERS nanotags is demonstrated using malachite green isothiocyanate as the Raman reporter molecule.

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 4kDa fluorescent dextran but not 70kDa 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 70kDa 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 ß-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.

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 (1)H NMR titration data is presented describing the nature of the interactions between the Tollens reagent and these ligands.

Discovery of a novel series of CXCR3 antagonists.

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC(50) 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.

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.

Substituents on quinone methides strongly modulate formation and stability of their nucleophilic adducts.

Electronic perturbation of quinone methides (QM) greatly influences their stability and in turn alters the kinetics and product profile of QM reaction with deoxynucleosides. Consistent with the electron-deficient nature of this reactive intermediate, electron-donating substituents are stabilizing and electron-withdrawing substituents are destabilizing. For example, a dC N3-QM adduct is made stable over the course of observation (7 days) by the presence of an electron-withdrawing ester group that inhibits QM regeneration. Conversely, a related adduct with an electron-donating methyl group is very labile and regenerates its QM with a half-life of approximately 5 h. The generality of these effects is demonstrated with a series of alternative quinone methide precursors (QMP) containing a variety of substituents attached at different positions with respect to the exocyclic methylene. The rates of nucleophilic addition to substituted QMs measured by laser flash photolysis similarly span 5 orders of magnitude with electron-rich species reacting most slowly and electron-deficient species reacting most quickly. The reversibility of QM reaction can now be predictably adjusted for any desired application.