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The 5-year survival of glioblastoma (GBM) patients is limited to a dismal5%, highlighting the need to advance more effective therapies. GBM tissue abnormally overexpresses the L-type amino acid transporter 1 (LAT1), for which L-histidine (His) is an inexpensive and powerful targeting ligand. Thus, we propose the chemical modification of a conventional chemo-immunogenic drug, docetaxel, into a nanomedicine with surface-His (nano-DTX-His) to target GBM tissue via LAT1 adhesive binding and further augment localized cell death. Since nano-DTX-His cannot be used for IV therapies due its inability to cross the blood-brain barrier (BBB) per se, we further propose its modification with anacid-cleavable Angiopep-2 layer (nano-DTX-His-clv-Angiopep2) to favor BBBtranslocation. It is of important note that the choice of DTX is based on its IC50, which is around 20.000-times lower than the standard temozolomide (in https://www.cancerrxgene.org/).

Silica-coated Superparamagnetic Iron Nanoparticles (SiMAGs) are an exciting biomedical technology capable of targeted delivery of cell-based therapeutics and disease diagnosis. However, in order to realise their full clinical potential, their intracellular fate must be determined. The analytical techniques of super-resolution fluorescence microscopy, particle counting flow cytometry and pH-sensitive nanosensors were applied to elucidate mechanisms of intracellular SiMAG processing in human mesenchymal stem cell (hMSCs). Super-resolution microscopy showed SiMAG fluorescently-tagged nanoparticles are endocytosed and co-localised within lysosomes. When exposed simulated lysosomal conditions SiMAGs were solubilised and exhibited diminishing fluorescence emission over 7 days. The in vitro intracellular metabolism of SiMAGs was monitored in hMSCs using flow cytometry and co-localised pH-sensitive nanosensors. A decrease in SiMAG fluorescence emission, which corresponded to a decrease in lysosomal pH was observed, mirroring ex vivo observations, suggesting SiMAG lysosomal exposure degrades fluorescent silica-coatings and iron cores. These findings indicate although there is a significant decrease intracellular SiMAG loading, sufficient particles remain internalised (>50%) to render SiMAG treated cells amenable to long-term magnetic cell manipulation. Our analytical approach provides important insights into the understanding of the intracellular fate of SiMAG processing, which could be readily applied to other particle therapeutics, to advance their clinical translation.

Elucidating the intracellular fate of silica-coated superparamagnetic iron nanoparticles (SiMAGs) using fluorescent pH-sensitive nanosensors, super-resolution fluorescence microscopy and particle counting flow cytometry., Silica-coated superparamagnetic iron nanoparticles (SiMAGs) are an exciting biomedical technology capable of targeted delivery of cell-based therapeutics and disease diagnosis. However, in order to realise their full clinical potential, their intracellular fate must be determined. The analytical techniques of super-resolution fluorescence microscopy, particle counting flow cytometry and pH-sensitive nanosensors were applied to elucidate mechanisms of intracellular SiMAG processing in human mesenchymal stem cell (hMSCs). Super-resolution microscopy showed SiMAG fluorescently-tagged nanoparticles are endocytosed and co-localised within lysosomes. When exposed to simulated lysosomal conditions SiMAGs were solubilised and exhibited diminishing fluorescence emission over 7 days. The in vitro intracellular metabolism of SiMAGs was monitored in hMSCs using flow cytometry and co-localised pH-sensitive nanosensors. A decrease in SiMAG fluorescence emission, which corresponded to a decrease in lysosomal pH was observed, mirroring ex vivo observations, suggesting SiMAG lysosomal exposure degrades fluorescent silica-coatings and iron cores. These findings indicate although there is a significant decrease in intracellular SiMAG loading, sufficient particles remain internalised (>50%) to render SiMAG treated cells amenable to long-term magnetic cell manipulation. Our analytical approach provides important insights into the understanding of the intracellular fate of SiMAG processing, which could be readily applied to other particle therapeutics, to advance their clinical translation.

Silica nanoparticles (SNPs) have been used as favoured platforms for sensor, drug delivery and biological imaging applications, due to their ease of synthesis, size-control and bespoke physico-chemical properties. In this study, we have developed a protocol for the synthesis of size-tuneable SNPs, with diameters ranging from 20 nm to 500 nm, through the optimisation of experimental components required for nanoparticle synthesis. This protocol was also used to prepare fluorescent SNPs, via covalent linkages of fluorophores, to the nanoparticle matrix using 3-aminopropyltriethoxysilane (APTES). This enabled the fabrication of ratiometric, fluorescent, pH-sensitive nanosensors (75 nm diameter) composed SNPs covalently linked to two pH-sensitive fluorescent dyes Oregon Green (OG) and 5(6)-carboxyfluorescein (FAM) and a reference fluorescent dye 5-(6)-carboxytetramethylrhodamine (TAMRA), extending the dynamic range of measurement from pH 3.5 to 7.5. In addition, size-tuneable, core-shell SNPs, covalently linked to a fluorescent TAMRA core were synthesised to investigate distance-dependant fluorescence quenching between TAMRA and black hole quencher 2 (BHQ2®) using nanometre-sized silica shells as physical spacers. The results showed a significant fluorescence quenching could be observed over greater distances than that reported for the classical distance-dependent molecular fluorescence quenching techniques, e.g. the Förster (fluorescence) resonance energy transfer (FRET). The methods and protocols we have detailed in this manuscript will provide the basis for the reproducible production of size tunable SNPs, which will find broad utility in the development of sensors for biological applications.

Contractile dysfunction of smooth muscle (SM) is a feature of chronic cardiovascular, respiratory and gastro-intestinal diseases. Owing to the low availability of human ex vivo tissue for the assessment of SM contractile function, the aim of this study was to develop a novel in vitro SM model that possesses the ability to contract, and a method to measure its contractility. A range of electrospun scaffolds were produced from crosslinked gelatin and methacrylated gelatin (GelMA), generating highly aligned scaffolds with average fibre diameters ranging from 200 nm to several micrometres. Young's moduli of the scaffolds ranged from 1x105 to 1x107 Pa. Primary aortic smooth muscle cells (AoSMCs; rat) cells readily adhered to and proliferated on the fibrous scaffolds for up to 10 days. They formed highly aligned populations following the topographical cues of the aligned scaffolds and stained positive for SM markers, indicating a contractile phenotype. Cell-seeded GelMA scaffolds were able, upon stimulation with uridine 5'-triphosphate (UTP), to contract and their attachment to a force transducer allowed the force of contraction to be measured. Hence, these electrospun GelMA fibres can be used as biomimetic scaffolds for SM cell culture and in vitro model development, and enables the contractile forces generated by the aligned three-dimensional sheet of cells to be directly measured. This will supplement in vitro drug screening tools and facilitate discovery of disease mechanisms.

Our ability to tailor the electronic properties of surfaces by nanomodification is paramount for various applications, including development of sensing, fuel cell, and solar technologies. Moreover, in order to improve the rational design of conducting surfaces, an improved understanding of structure/function relationships of nanomodifications and effect they have on the underlying electronic properties is required. Herein, we report on the tuning and optimization of the electrochemical properties of indium tin oxide (ITO) functionalized with single-walled carbon nanotubes (SWCNTs). This was achieved by controlling in situ grafting of aryl amine diazonium films on the nanoscale which were used to covalently tether SWCNTs. The structure/function relationship of these nanomodifications on the electronic properties of ITO was elucidated via time-of-flight secondary ion mass spectrometry and electrochemical and physical characterization techniques which has led to new mechanistic insights into the in situ grafting of diazonium. We discovered that the connecting bond is a nitro group which is covalently linked to a carbon on the aryl amine. The increased understanding of the surface chemistry gained through these studies enabled us to fabricate surfaces with optimized electron transfer kinetics. The knowledge gained from these studies allows for the rational design and tuning of the electronic properties of ITO-based conducting surfaces important for development of various electronic applications.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period. This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Conventional airway in vitro models focus upon the function of individual structural cells cultured in a two-dimensional monolayer, with limited three-dimensional (3D) models of the bronchial mucosa. Electrospinning offers an attractive method to produce defined, porous 3D matrices for cell culture. To investigate the effects of fibre diameter on airway epithelial and fibroblast cell growth and functionality, we manipulated the concentration and deposition rate of the non-degradable polymer polyethylene terephthalate to create fibres with diameters ranging from nanometre to micrometre. The nanofibre scaffold closely resembles the basement membrane of the bronchiole mucosal layer, and epithelial cells cultured at the air–liquid interface on this scaffold showed polarized differentiation. The microfibre scaffold mimics the porous sub-mucosal layer of the airway into which lung fibroblast cells showed good penetration. Using these defined electrospinning parameters we created a biphasic scaffold with 3D topography tailored for optimal growth of both cell types. Epithelial and fibroblast cells were co-cultured onto the apical nanofibre phase and the basal microfibre phase respectively, with enhanced epithelial barrier formation observed upon co-culture. This biphasic scaffold provides a novel 3D in vitro platform optimized to mimic the different microenvironments the cells encounter in vivo on which to investigate key airway structural cell interactions in airway diseases such as asthma.