A dermal sludge for targeted genetic auto-inflammatory skin disorders

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Pharmacy. 2016Genetic auto-inflammatory inflammatory skin disorders (GAISDs) are a group of inherited disorders which are characterized by seemingly unprovoked recurrent episodes of fever and severe localised inflammation. GAISDs are associated with abnormal activation of the innate immune system, leading to clinical inflammation and high levels of acute-phase reactants. The most common disorder is Familial Mediterranean Fever (FMF), followed by Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS). TRAPS episodes generally last longer than FMF and FMF patients tend to respond well with colchicine while TRAPS management seems to be challenging. Hence this work is directed towards improving TRAPS diseases management. A definitive treatment for TRAPS has yet to be identified, and current corticosteroid treatment is mainly limited by the long-term side-effects due to high systemic drug exposure, and the poor availability of drugs at the site of action. A number of measures were taken in order to overcome the limitations of corticosteroids.Herein a novel stimuli responsive nanocolloidal gel system was developed. A nanoliposomal gel was the stimuli responsive gel system of choice due to its advantages of skin penetration enhancement in transdermal drug delivery system. In this research, a phospholipid based system with Eudragit® E100 (EuE100) chemically modified into EuE100-cystamine derivative for dual pH/redox responsive delivery of [Copper-glycylglycine-prednisolone succinate] ([Cu(glygly)(PS)]) was developed. The rationale of using [Cu(glygly)(PS)] complex instead of the pure PS corticosteroid was supported by comparing the biological activities of these two compounds. Results indicated a high inflammatory/oxidant inhibitory activity of [Cu(glygly)(PS)] in comparison to the free PS drug. The [Cu(glygly)(PS)] complex exhibited a significant free radical-scavenging activity (60.1±1.2%) and lipoxygenase (LOX-5) inhibitory activity (36.6±1.3%) in comparison to PS which gave activity of 4.4±1.4% and inhibition of 6.1±2.6% respectively. The [Cu(glygly)(PS)] loaded NLs showed a low level of [Cu(glygly)(PS)] release of 22.9±5.4% in 6h at pH 7.4, in comparison to a significant accelerated release at pH 5 in a reducing environment of 75.9±3.7%in 6h. Thereafter optimized [Cu(glygly)(PS)]-loaded NLs were dispersed in hydroxypropyl methylcellulose (HPMC)/Polyvinyl alcohol(PVA) gel resulting in a [Cu(glygly)(PS)]-loaded nanoliposomal gel termed asdermal sludge.A dermal sludge is defined as a viscous gel suspended with solid particles ([Cu(glygly)(PS)]-loaded nanoliposomes). The sludge was characterized using ex vivo permeation, in vitro release, cytotoxicity and in vivo studies, and compared to the conventional PS formulations. The results indicated that the novel dual redox/pH responsive nanoliposomal dermal sludge holds great potential for targeted bioactive delivery in TRAPS through the transdermal route, hence improving the therapeutic outcome.MT201

Lattice-Boltzmann simulations of cerebral blood flow

Computational haemodynamics play a central role in the understanding of blood behaviour in the cerebral vasculature, increasing our knowledge in the onset of vascular diseases and their progression, improving diagnosis and ultimately providing better patient prognosis. Computer simulations hold the potential of accurately characterising motion of blood and its interaction with the vessel wall, providing the capability to assess surgical treatments with no danger to the patient. These aspects considerably contribute to better understand of blood circulation processes as well as to augment pre-treatment planning. Existing software environments for treatment planning consist of several stages, each requiring significant user interaction and processing time, significantly limiting their use in clinical scenarios. The aim of this PhD is to provide clinicians and researchers with a tool to aid in the understanding of human cerebral haemodynamics. This tool employs a high performance fluid solver based on the lattice-Boltzmann method (coined HemeLB), high performance distributed computing and grid computing, and various advanced software applications useful to efficiently set up and run patient-specific simulations. A graphical tool is used to segment the vasculature from patient-specific CT or MR data and configure boundary conditions with ease, creating models of the vasculature in real time. Blood flow visualisation is done in real time using in situ rendering techniques implemented within the parallel fluid solver and aided by steering capabilities; these programming strategies allows the clinician to interactively display the simulation results on a local workstation. A separate software application is used to numerically compare simulation results carried out at different spatial resolutions, providing a strategy to approach numerical validation. This developed software and supporting computational infrastructure was used to study various patient-specific intracranial aneurysms with the collaborating interventionalists at the National Hospital for Neurology and Neuroscience (London), using three-dimensional rotational angiography data to define the patient-specific vasculature. Blood flow motion was depicted in detail by the visualisation capabilities, clearly showing vortex fluid ow features and stress distribution at the inner surface of the aneurysms and their surrounding vasculature. These investigations permitted the clinicians to rapidly assess the risk associated with the growth and rupture of each aneurysm. The ultimate goal of this work is to aid clinical practice with an efficient easy-to-use toolkit for real-time decision support

Microfluidics for assessing the behaviour of deformable biological objects

Biological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated.Biological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated

Plasma methionine metabolic profile is associated with longevity in mammals

Methionine metabolism arises as a key target to elucidate the molecular adaptations underlying animal longevity due to the negative association between longevity and methionine content. The present study follows a comparative approach to analyse plasma methionine metabolic profile using a LC-MS/MS platform from 11 mammalian species with a longevity ranging from 3.5 to 120 years. Our findings demonstrate the existence of a species-specific plasma profile for methionine metabolism associated with longevity characterised by: i) reduced methionine, cystathionine and choline; ii) increased non-polar amino acids; iii) reduced succinate and malate; and iv) increased carnitine. Our results support the existence of plasma longevity features that might respond to an optimised energetic metabolism and intracellular structures found in long-lived species.This work was supported by the Spanish Ministry of Science, Innovation and Universities (RTI2018-099200-B-I00), and the Generalitat of Catalonia (Agency for Management of University and Research Grants (2017SGR696) and Department of Health (SLT002/16/00250)) to R.P. This study has been co-financed by FEDER funds from the European Union (“A way to build Europe”). IRBLleida is a CERCA Programme/Generalitat of Catalonia. M.J. is a ‘Serra Hunter’ Fellow. N.M.M. received a predoctoral fellowship from the Generalitat of Catalonia (AGAUR, ref 2018FI_B2_00104)

Scalable strategies for tumour targeting of magnetic carriers and seeds

With the evolving landscape of medical oncology, focus has shifted away from nonspecific cytotoxic treatment strategies toward therapeutic paradigms more characteristic of targeted therapies. These therapies rely on delivery vehicles such as nano-carriers or micro robotic devices to boosts the concentration of therapeutics in a specific targeted site inside the body. The use of externally applied magnetic field is suggested to be a predominant approach for remote localisation of magnetically responsive carriers and devices to the target region that could not be otherwise reached. However, the fast decline of the magnetic fields and gradients with increasing distances from the source is posing a major challenge for its clinical application. The aim of this thesis was to investigate potential magnetic delivery strategies which can circumvent some of the typical limitations of this technique. Two different approaches were explored to this end. The first approach was to characterise the ability of a conventional permanent magnet on targeting individual nano-carriers and develop novel magnetic designs which improve the targeting efficiency. The second approach was evaluating the feasibility of a magnetic resonance imaging system to move a millimetre-sized magnetic particle within the body. Phantom and in vivo magnetic targeting experiments illustrated the significant increase in effective targeting depth when our novel magnetic design was used for targeting nano-carriers compared with conventional magnets. In the later part of the thesis, the proof of concept and characterisation experiments showed that a 3 mm magnetic particle can be moved in ex vivo brain tissue using a magnetic resonance imaging system using clinically relevant gradient strengths. The magnetic systems introduced in this thesis provide the potential to target nano-carriers and millimetre-sized thermoseeds to tumours located at deep regions of human body through vasculature and soft tissue respectively

Three dimensional optofluidic devices for manipulation of particles and cells

Optical forces offer a powerful tool for manipulating single cells noninvasively. Integration of optical functions within microfluidic devices provides a new freedom for manipulating and studying biological samples at the micro scale. In the pursuit to realise such microfluidic devices with integrated optical components, Ultrafast Laser Inscription (ULI) fabrication technology shows great potential. The uniqueness and versatility of the technique in rapid prototyping of 3D complex microfluidic and optical elements as well as the ability to perform one step integration of these elements provides exciting opportunities in fabricating novel devices for biophotonics applications. The work described in this thesis details the development of three dimensional optofluidic devices that can be used for biophotonics applications, in particular for performing cell manipulation and particle separation. Firstly, the potential of optical forces to manipulate cells and particles in ULI microfluidic channels is investigated. The ability to controllably displace particles within a ULI microchannel using a waveguide positioned orthogonal to it is explored in detail. We then prototype a more complex 3D device with multiple functionalities in which a 3D optofluidic device containing a complex microchannel network and waveguides was used for further investigations into optical manipulation and particle separation. The microfluidic channel network and the waveguides within the device possess the capability to manipulate the injected sample fluid through hydrodynamic focusing and optically manipulate the individual particles, respectively. This geometry provided a more efficient way of investigating optical manipulation within the device. Finally, work towards developing a fully optimised 3D cell separator device is presented. Initial functional validation was performed by investigating the capability of the device to route particles through different outlet channels using optical forces. A proof of concept study demonstrates the potential of the device to use for cell separation based on the size of the cells. It was shown that both passive and active cell separation is possible using this device

Inferring Geodesic Cerebrovascular Graphs: Image Processing, Topological Alignment and Biomarkers Extraction

A vectorial representation of the vascular network that embodies quantitative features - location, direction, scale, and bifurcations - has many potential neuro-vascular applications. Patient-specific models support computer-assisted surgical procedures in neurovascular interventions, while analyses on multiple subjects are essential for group-level studies on which clinical prediction and therapeutic inference ultimately depend. This first motivated the development of a variety of methods to segment the cerebrovascular system. Nonetheless, a number of limitations, ranging from data-driven inhomogeneities, the anatomical intra- and inter-subject variability, the lack of exhaustive ground-truth, the need for operator-dependent processing pipelines, and the highly non-linear vascular domain, still make the automatic inference of the cerebrovascular topology an open problem. In this thesis, brain vessels’ topology is inferred by focusing on their connectedness. With a novel framework, the brain vasculature is recovered from 3D angiographies by solving a connectivity-optimised anisotropic level-set over a voxel-wise tensor field representing the orientation of the underlying vasculature. Assuming vessels joining by minimal paths, a connectivity paradigm is formulated to automatically determine the vascular topology as an over-connected geodesic graph. Ultimately, deep-brain vascular structures are extracted with geodesic minimum spanning trees. The inferred topologies are then aligned with similar ones for labelling and propagating information over a non-linear vectorial domain, where the branching pattern of a set of vessels transcends a subject-specific quantized grid. Using a multi-source embedding of a vascular graph, the pairwise registration of topologies is performed with the state-of-the-art graph matching techniques employed in computer vision. Functional biomarkers are determined over the neurovascular graphs with two complementary approaches. Efficient approximations of blood flow and pressure drop account for autoregulation and compensation mechanisms in the whole network in presence of perturbations, using lumped-parameters analog-equivalents from clinical angiographies. Also, a localised NURBS-based parametrisation of bifurcations is introduced to model fluid-solid interactions by means of hemodynamic simulations using an isogeometric analysis framework, where both geometry and solution profile at the interface share the same homogeneous domain. Experimental results on synthetic and clinical angiographies validated the proposed formulations. Perspectives and future works are discussed for the group-wise alignment of cerebrovascular topologies over a population, towards defining cerebrovascular atlases, and for further topological optimisation strategies and risk prediction models for therapeutic inference. Most of the algorithms presented in this work are available as part of the open-source package VTrails