Sulfonium Salts as Leaving Groups for Aromatic Labelling of Drug-like Small Molecules with Fluorine-18.

Positron emission tomography (PET) is unique in that it allows quantification of biochemical processes in vivo, but difficulties with preparing suitably labelled radiotracers limit its scientific and diagnostic applications. Aromatic [(18)F]fluorination of drug-like small molecules is particularly challenging as their functional group compositions often impair the labelling efficiency. Herein, we report a new strategy for incorporation of (18)F into highly functionalized aromatic compounds using sulfonium salts as leaving groups. The method is compatible with pharmacologically relevant functional groups, including aliphatic amines and basic heterocycles. Activated substrates react with [(18)F]fluoride at room temperature, and with heating the reaction proceeds in the presence of hydrogen bond donors. Furthermore, the use of electron rich spectator ligands allows efficient and regioselective [(18)F]fluorination of non-activated aromatic moieties. The method provides a broadly applicable route for (18)F labelling of biologically active small molecules, and offers immediate practical benefits for drug discovery and imaging with PET

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins

Drug delivery, biodistribution and anti-EGFR activity: theragnostic nanoparticles for simultaneous in vivo delivery of tyrosine kinase inhibitors and kinase activity biosensors

In vivo delivery of small molecule therapeutics to cancer cells, assessment of the selectivity of administration, and measuring the efficacity of the drug in question at the molecule level, are important ongoing challenges in developing new classes of cancer chemotherapeutics. One approach that has the potential to provide targeted delivery, tracking of biodistribution and readout of efficacy, is to use multimodal theragnostic nanoparticles to deliver the small molecule therapeutic. In this paper, we report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes. These simultaneously deliver an inhibitor of the EGFR tyrosine kinase, and plasmid DNA coding for a Crk-based biosensor, Picchu-X, which when expressed in the target cells can be used to quantify the inhibition of EGFR in vivo in a mouse colorectal cancer xenograft model. Reversible bioconjugation of a known analogue of the tyrosine kinase inhibitor Mo-IPQA to a cationic peptide, and co-formulation with peptides containing both EGFR-binding and cationic sequences, allowed for good levels of inhibitor encapsulation with targeted delivery to LIM1215 colon cancer cells. Furthermore, high levels of expression of the Picchu-X biosensor in the LIM1215 cells in vivo allowed us to demonstrate, using fluorescence lifetime microscopy (FLIM)-based biosensing, that EGFR activity can be successfully suppressed by the tyrosine kinase inhibitor, released from the lipopolyplexes. Finally, we measured the biodistribution of lipopolyplexes containing 125I-labelled inhibitors and were able to demonstrate that the lipopolyplexes gave significantly higher drug delivery to the tumors compared with free drug

Imaging of X-Ray-Excited Emissions from Quantum Dots and Biological Tissue in Whole Mouse

Optical imaging in clinical and preclinical settings can provide a wealth of biological information, particularly when coupled with targetted nanoparticles, but optical scattering and absorption limit the depth and resolution in both animal and human subjects. Two new hybrid approaches are presented, using the penetrating power of X-rays to increase the depth of optical imaging. Foremost, we demonstrate the excitation by X-rays of quantum-dots (QD) emitting in the near-infrared (NIR), using a clinical X-ray system to map the distribution of QDs at depth in whole mouse. We elicit a clear, spatially-resolved NIR signal from deep organs (brain, liver and kidney) with short (1 second) exposures and tolerable radiation doses that will permit future in vivo applications. Furthermore, X-ray-excited endogenous emission is also detected from whole mouse. The use of keV X-rays to excite emission from QDs and tissue represent novel biomedical imaging technologies, and exploit emerging QDs as optical probes for spatial-temporal molecular imaging at greater depth than previously possible

Multiparametric MR characterisation of a high-fat, high-cholesterol diet rodent model of liver disease

There is a growing interest in the development of new animal models of non-alcoholic fatty liver disease. In this study, we use T1, proton density fat fraction (PDFF) and R2* mapping to characterise hepatic parenchymal tissue and the evolution of MR properties over time in a high-fat, high-cholesterol diet model of fatty liver disease

EGFR-targeted semiconducting polymer nanoparticles for photoacoustic imaging

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV–visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles

EGFR-targeted semiconducting polymer nanoparticles for photoacoustic imaging

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV-visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles

Monitoring neovascularization and integration of decellularized human scaffolds using photoacoustic imaging

Tissue engineering is a branch of regenerative medicine that aims to manipulate cells and scaffolds to create bioartificial tissues and organs for patients. A major challenge lies in monitoring the blood supply to the new tissue following transplantation: the integration and neovascularization of scaffolds in vivo is critical to their functionality. Photoacoustic imaging (PAI) is a laser-generated ultrasound-based technique that is particularly well suited to visualising microvasculature due to the high optical absorption of haemoglobin. Here, we describe an early proof-of-concept study in which PAI in widefield tomography mode is used to image biological, decellularized human tracheal scaffolds. We found that PAI allowed the longitudinal tracking of scaffold integration into subcutaneous murine tissue with high spatial resolution at depth over an extended period of time. The results of the study were consistent with post-imaging histological analyses, demonstrating that PAI can be used to non-invasively monitor the extent of vascularization in biological tissue-engineered scaffolds. We propose that this technique may be a valuable tool for studies designed to test interventions aimed at improving the speed and extent of scaffold neovascularization in tissue engineering. With technological refinement, it could also permit in vivo monitoring of revascularization in patients, for example to determine timing of heterotopic graft transfer

An Evaluation of Alternatives for Enhancing Anaerobic Digestion of Waste Activated Sludge

Waste activated sludge (WAS) is one of the largest by-products of biological wastewater treatment. Anaerobic digestion of WAS is beneficial for several reasons. In an ever increasingly energy conscientious world the production of renewable energy resources is becoming more important, and thus the production of methane has been seen as a valuable product. To achieve efficient conversion of organic matter to methane, the biomass in the digester must be provided optimal operating conditions, as well as adequate retention times, that will allow for substrate metabolism and prevent bacteria washout. Two approaches have been taken in this research to achieve improved biodegradation. Initially microwave pretreatment was employed to improve the biodegradability of the sludge, then the addition of a submerged hollow fibre membrane separation unit was used to allow for a longer SRT while maintaining the hydraulic residence time (HRT). The impact of microwave pretreatment on WAS characteristics was assessed for both the low temperature operations and the high temperature operations. An increase due to pretreatment on the filtered to total COD ratio when comparing the feed to the microwaved feed was established to be 200 % for low temperature operations and 254 % for high temperature operations. For the low temperature operations, CODT destruction, VS destruction, and organic nitrogen destruction were all higher for the test digester than the control digester indicating that the microwaving of the WAS increased the biodegradation in the anaerobic digester. For the high temperature operation, CODT destruction and organic nitrogen destruction were improved with microwave application, however VS destruction did not support this. The measured biogas data indicated that microwaving did influence the volume of biogas produced during anaerobic digestion of WAS for both the low and high temperature operations, and hence the VS destruction data for the high temperature operations was determined to be incorrect. For the membrane operations both the CODT and the VS destruction calculations indicated that at the same SRT the test digester was capable of more biodegradation than the control digester. The control digester organic nitrogen reduction was calculated to be higher than for the test digester, suggesting that the control digester removed more organic nitrogen than the test digester, however, these results were likely due to the lower HRT of the test digester compared to those of the control digester. A greater volume of biogas was produced by the test digester than the control digester; however, the composition of the gas from both digesters was similar, although the percentage of methane produced by the test digester was higher than that produced by the control digester. The higher destruction by the test digester indicated that the presence of the membrane unit and the decoupling of the HRT and SRT improved the biodegradation capability of the digesters. The results of the membrane performance study indicated that for a hollow fibre anaerobic membrane bioreactor, stable operations could be achieved with a total solids concentration of 2.01 %+/-0.34, an HRT of 15 days and an SRT of 30 days. With a constant flux of 14 L/m2-h +/-0.68 the average TMP was 0.079 kPa/min+/-0.08. No cleaning was required to achieve this, however the operations consisted of 20 minutes of permeation followed by 5 hours and 40 minutes of relaxation. The critical flux was determined to be in the range of 18 to 22 L/m2-h

Development of Fluorine-18 Labeled Metabolically Activated Tracers for Imaging of Drug Efflux Transporters with Positron Emission Tomography

Increased activity of efflux transporters, e.g., P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier is a pathological hallmark of many neurological diseases, and the resulting multiple drug resistance represents a major clinical challenge. Noninvasive imaging of transporter activity can help to clarify the underlying mechanisms of drug resistance and facilitate diagnosis, patient stratification, and treatment monitoring. We have developed a metabolically activated radiotracer for functional imaging of P-gp/BCRP activity with positron emission tomography (PET). In preclinical studies, the tracer showed excellent initial brain uptake and clean conversion to the desired metabolite, although at a sluggish rate. Blocking with P-gp/BCRP modulators led to increased levels of brain radioactivity; however, dynamic PET did not show differential clearance rates between treatment and control groups. Our results provide proof-of-concept for development of prodrug tracers for imaging of P-gp/BCRP function in vivo but also highlight some challenges associated with this strategy