Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process.

Current strategies for the discovery and bioconjugation of smaller, targetable drug conjugates tailored for solid tumor therapy.

: Antibody-Drug Conjugates (ADCs) have undergone a recent resurgence with 5 product approvals over the last 2 years but for those close to the field, it's been repeated cycles of setbacks and new innovations. A new wave of innovation is in the type of format used to deliver the cytotoxic payloads, with smaller bio-molecules being designed to have more optimal penetration and elimination properties tailored for solid tumors.: In this review, the authors cover many of the recently described smaller-format drug conjugates (including formats such as diabodies, Fabs, scFvs, domain antibodies) with an emphasis on the types of conjugation technologies used to attach the chemical linker-payload.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations.

Targeted fluorescence lifetime probes reveal responsive organelle viscosity and membrane fluidity.

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction.

Antibody Fragment and Affibody ImmunoPET Imaging Agents: Radiolabelling Strategies and Applications.

Antibodies have long been recognised as potent vectors for carrying diagnostic medical radionuclides, contrast agents and optical probes to diseased tissue for imaging. The area of ImmunoPET combines the use of positron emission tomography (PET) imaging with antibodies to improve the diagnosis, staging and monitoring of diseases. Recent developments in antibody engineering and PET radiochemistry have led to a new wave of experimental ImmunoPET imaging agents that are based on a range of antibody fragments and affibodies.

Using antibody directed phototherapy to target oesophageal adenocarcinoma with heterogeneous HER2 expression.

Early oesophageal adenocarcinoma (OA) and pre-neoplastic dysplasia may be treated with endoscopic resection and ablative techniques such as photodynamic therapy (PDT). Though effective, discrete areas of disease may be missed leading to recurrence. PDT further suffers from the side effects of off-target photosensitivity.

Small-Format Drug Conjugates: A Viable Alternative to ADCs for Solid Tumours?

Antibody-Drug Conjugates (ADCs) have been through multiple cycles of technological innovation since the concept was first practically demonstrated ~40 years ago. Current technology is focusing on large, whole immunoglobulin formats (of which there are approaching 100 in clinical development), many with site-specifically conjugated payloads numbering 2 or 4. Despite the success of trastuzumab-emtansine in breast cancer, ADCs have generally failed to have an impact in solid tumours, leading many to explore alternative, smaller formats which have better penetrating properties as well as more rapid pharmacokinetics (PK).

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate.

Background: Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett's epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1.

Results: MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively.

A HER2 selective theranostic agent for surgical resection guidance and photodynamic therapy.

In many cancers early intervention involves surgical resection of small localised tumour masses. Inadequate resection leads to recurrence whereas overzealous treatment can lead to organ damage. This work describes production of a HER2 targeting antibody Fab fragment dual conjugated to achieve both real time near-infrared fluorescent imaging and photodynamic therapy.

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function.

The role of membrane cholesterol as a crucial regulator in the structure and function of membrane proteins and receptors is well documented. However, there is a lack of consensus on the mechanism for such regulation. We have previously shown that the function of an important neuronal receptor, the serotonin1A receptor, is modulated by cholesterol in hippocampal membranes.

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.

Background: Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections.

Emerging formats for next-generation antibody drug conjugates.

Introduction: Antibody drug conjugates now make up a significant fraction of biopharma's oncology pipeline due to great advances in the understanding of the three key components and how they should be optimised together. With this clinical success comes innovation to produce new enabling technologies that can deliver more effective antibody-drug conjugates (ADCs) with a larger therapeutic index.

Areas Covered: There are many reviews that discuss the various strategies for ADCs design but the last 5 years or so have witnessed the emergence of a number of different antibody formats compete with the standard whole immunoglobulin.

Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions.

Fluorescence lifetime imaging of molecular rotors in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level. While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge.

Fluorescence anisotropy of molecular rotors.

We present polarization-resolved fluorescence measurements of fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-dicyanovinyl)julolidine (DCVJ), and a meso-substituted boron dipyrromethene (BODIPY-C(12)). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore.

Novel photosensitisers derived from pyropheophorbide-a: uptake by cells and photodynamic efficiency in vitro.

Photodynamic Therapy (PDT) is a minimally invasive procedure used for treating a range of neoplastic diseases, which utilises combined action of light and a PDT drug called a photosensitiser. The efficiency of this treatment depends crucially on the properties of the photosensitiser used, namely on its efficient uptake by cells or by the surrounding vasculature, intracellular localisation, minimal dark toxicity and substantial phototoxicity. In this report we compare the spectroscopic properties, cell uptake and in vitro phototoxicity of two novel hydrophilic photosensitisers derived from pyropheophorbide-a (PPa).

Monitoring sol-to-gel transitions via fluorescence lifetime determination using viscosity sensitive fluorescent probes.

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths.

Singlet oxygen in a cell: spatially dependent lifetimes and quenching rate constants.

Singlet molecular oxygen, O(2)(a(1)Delta(g)), can be created in a single cell from ground-state oxygen, O(2)(X(3)Sigma(g)(-)), upon focused laser irradiation of an intracellular sensitizer. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a(1)Delta(g) --> X(3)Sigma(g)(-)) with subcellular spatial resolution. The singlet oxygen lifetime determines its diffusion distance and hence the intracellular volume element in which singlet-oxygen-initiated perturbation of the cell occurs.

Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging.

The fluorescence intensity and lifetime of the 4,4'-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the local microviscosity in biological systems, based on the determination of the fluorescence lifetime of 1. Fluorescence lifetime imaging (FLIM) performed on live cells incubated with 1 demonstrates the distinct intracellular lifetime of the molecular rotor of 1.

Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments.

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs.

Fluorescence characterisation of multiply-loaded anti-HER2 single chain Fv-photosensitizer conjugates suitable for photodynamic therapy.

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution ( View Article and Find Full Text PDF

Molecular control of recombination dynamics in dye sensitised nanocrystalline TiO2 films.

Modification of the structure of a porphyrin dye shows a significant change in the rate of charge recombination between injected electrons in the TiO2 and the oxidized dye anchored to it following optical excitation, offering an insight into fundamental understanding of processes occurring at the dye/semiconductor interface.

Redox behaviour of phenolic porphyrins in basic solutions: a reappraisal.

Phenolic porphyrins 1, 4, and 5 form stable phenoxyl radicals 3, 6, and 7 in deoxygenated as well as oxygenated basic solutions. Mechanistic schemes are presented, involving coupled disproportionation and conproportionation reactions, that do not require oxygen involvement in the redox processes leading to phenoxyl radicals.