Gas and particle dynamics of a contoured shock tube for pre-clinical microparticle drug delivery

We investigate the gas-particle dynamics of a device designed for biological pre-clinical experiments. The device uses transonic/supersonic gas flow to accelerate microparticles such that they penetrate the outer skin layers. By using a shock tube coupled to a correctly expanded nozzle, a quasi-one-dimensional, quasi-steady flow (QSF) is produced to uniformly accelerate the microparticles. The system utilises a microparticle cassette (a diaphragm sealed container) that incorporates a jet mixing mechanism to stir the particles prior to diaphragm rupture. Pressure measurements reveal that a QSF exit period - suitable for uniformly accelerating microparticles - exists between 155 and 220 mus after diaphragm rupture. Immediately preceding the QSF period, a starting process secondary shock was shown to form with its (x,t) trajectory comparing well to theoretical estimates. To characterise the microparticle, flow particle image velocimetry experiments were conducted at the nozzle exit, using particle payloads with varying diameter (2.7-48 mu m), density (600-16,800 kg/m(3)) and mass (0.25-10 mg). The resultant microparticle velocities were temporally uniform. The experiments also show that the starting process does not significantly influence the microparticle nozzle exit velocities. The velocity distribution across the nozzle exit was also uniform for the majority of microparticle types tested. For payload masses typically used in pre-clinical drug and vaccine applications (

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16 +/- 0.27 (mean +/- S.D.), 1.35 +/- 0.285 and 2.72 +/- 0.47 per 1000 mu m(2) resulted in percent deaths of 3.96 +/- 5.22, 45.91 +/- 10.89, 90.52 +/- 12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death. (c) 2005 Elsevier Ltd. All rights reserved.

Numerical analysis of gas and micro-particle interactions in a hand-held shock-tube device

A unique hand-held gene gun is employed for ballistically delivering biomolecules to key cells in the skin and mucosa in the treatment of the major diseases. One of these types of devices, called the Contoured Shock Tube (CST), delivers powdered micro-particles to the skin with a narrow and highly controllable velocity distribution and a nominally uniform spatial distribution. In this paper, we apply a numerical approach to gain new insights in to the behavior of the CST prototype device. The drag correlations proposed by Henderson (1976), Igra and Takayama (1993) and Kurian and Das (1997) were applied to predict the micro-particle transport in a numerically simulated gas flow. Simulated pressure histories agree well with the corresponding static and Pitot pressure measurements, validating the CFD approach. The calculated velocity distributions show a good agreement, with the best prediction from Igra & Takayama correlation (maximum discrepancy of 5%). Key features of the gas dynamics and gas-particle interaction are discussed. Statistic analyses show a tight free-jet particle velocity distribution is achieved (570 +/- 14.7 m/s) for polystyrene particles (39 +/- 1 mu m), representative of a drug payload.

The efficiency of encapsulation within surface rehydrated polymersomes

The key to the use of polymersomes as effective molecular delivery systems is in the ability to design processing routes that can efficiently encapsulate the molecular payload. We have evaluated various surface rehydration mechanisms for encapsulation, in each case characterizing the morphologies formed using DLS and confocal microscopy as well as determining the encapsulation efficiency for the hydrophilic dye Rhodamine B. In contrast to bulk methods, where the encapsulation efficiencies are low, we find that higher efficiencies can be obtained by the rehydration of thin films. We relate these results to the non-equilibrium mechanisms that underlie vesicle formation and discuss how an understanding of these mechanisms can help optimize encapsulation efficiencies. Our conclusion is that, even considering the good encapsulation efficiency, surface methods are still unsuitable for the massive scale-up needed when applied to commercial mass market molecular delivery scenarios. However, targeting more specialized applications for high value ingredients (like pharmaceuticals) might be more feasible.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-(n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm2) caused the release of liposome-entrapped CaCl2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production. © 2007 Elsevier B.V. All rights reserved.

Effects of crosstalk in WDM optical label switching networks due to wavelength switching of a tunable laser

Crosstalk caused by switching events in fast tunable lasers in an optical label switching (OLS) system is investigated for the first time. A wavelength-division-multiplexed OLS system based on subcarrier multiplexed labels is presented which employs a 40-Gb/s duobinary payload and a 155-Mb/s label on a 40-GHz subcarrier. Degradation in system performance as the transmitters switch between different channels is then characterized in terms of the frequency drift of the tunable laser.

Conjugated Polymer Nanoparticles for Biological Labeling and Delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.

Towards the design of monolithic decoupled XYZ compliant parallel mechanisms for multi-function applications

This paper deals with the monolithic decoupled XYZ compliant parallel mechanisms (CPMs) for multi-function applications, which can be fabricated monolithically without assembly and has the capability of kinetostatic decoupling. At first, the conceptual design of monolithic decoupled XYZ CPMs is presented using identical spatial compliant multi-beam modules based on a decoupled 3-PPPR parallel kinematic mechanism. Three types of applications: motion/positioning stages, force/acceleration sensors and energy harvesting devices are described in principle. The kinetostatic and dynamic modelling is then conducted to capture the displacements of any stage under loads acting at any stage and the natural frequency with the comparisons with FEA results. Finally, performance characteristics analysis for motion stage applications is detailed investigated to show how the change of the geometrical parameter can affect the performance characteristics, which provides initial optimal estimations. Results show that the smaller thickness of beams and larger dimension of cubic stages can improve the performance characteristics excluding natural frequency under allowable conditions. In order to improve the natural frequency characteristic, a stiffness-enhanced monolithic decoupled configuration that is achieved through employing more beams in the spatial modules or reducing the mass of each cubic stage mass can be adopted. In addition, an isotropic variation with different motion range along each axis and same payload in each leg is proposed. The redundant design for monolithic fabrication is introduced in this paper, which can overcome the drawback of monolithic fabrication that the failed compliant beam is difficult to replace, and extend the CPM’s life.

Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells

Admittance Control for Robotic Loading: Design and Experiments with a 1-Tonne Loader and a 14-Tonne LHD

This paper describes the design, tuning, and extensive field testing of an admittance-based Autonomous Loading Controller (ALC) for robotic excavation. Several iterations of the ALC were tuned and tested in fragmented rock piles—similar to those found in operating mines—by using both a robotic 1-tonne capacity Kubota R520S diesel-hydraulic surface loader and a 14-tonne capacity Atlas Copco ST14 underground load-haul-dump (LHD) machine. On the R520S loader, the ALC increased payload by 18 % with greater consistency, although with more energy expended and longer dig times when compared with digging at maximum actuator velocity. On the ST14 LHD, the ALC took 61 % less time to load 39 % more payload when compared to a single manual operator. The manual operator made 28 dig attempts by using three different digging strategies, and had one failed dig. The tuned ALC made 26 dig attempts at 10 and 11 MN target force levels. All 10 11 MN digs succeeded while 6 of the 16 10 MN digs failed. The results presented in this paper suggest that the admittance-based ALC is more productive and consistent than manual operators, but that care should be taken when detecting entry into the muck pile

Towards Mapping of Rock Walls Using a UAV-Mounted 2D Laser Scanner in GPS Denied Environments

In geotechnical engineering, the stability of rock excavations and walls is estimated by using tools that include a map of the orientations of exposed rock faces. However, measuring these orientations by using conventional methods can be time consuming, sometimes dangerous, and is limited to regions of the exposed rock that are reachable by a human. This thesis introduces a 2D, simulated, quadcopter-based rock wall mapping algorithm for GPS denied environments such as underground mines or near high walls on surface. The proposed algorithm employs techniques from the field of robotics known as simultaneous localization and mapping (SLAM) and is a step towards 3D rock wall mapping. Not only are quadcopters agile, but they can hover. This is very useful for confined spaces such as underground or near rock walls. The quadcopter requires sensors to enable self localization and mapping in dark, confined and GPS denied environments. However, these sensors are limited by the quadcopter payload and power restrictions. Because of these restrictions, a light weight 2D laser scanner is proposed. As a first step towards a 3D mapping algorithm, this thesis proposes a simplified scenario in which a simulated 1D laser range finder and 2D IMU are mounted on a quadcopter that is moving on a plane. Because the 1D laser does not provide enough information to map the 2D world from a single measurement, many measurements are combined over the trajectory of the quadcopter. Least Squares Optimization (LSO) is used to optimize the estimated trajectory and rock face for all data collected over the length of a light. Simulation results show that the mapping algorithm developed is a good first step. It shows that by combining measurements over a trajectory, the scanned rock face can be estimated using a lower-dimensional range sensor. A swathing manoeuvre is introduced as a way to promote loop closures within a short time period, thus reducing accumulated error. Some suggestions on how to improve the algorithm are also provided.

Spectral Signature Secured Retrodirective Array

A novel retrodirective array (RDA) architecture is proposed which utilises a special case spectral signature embedded within the data payload as pilot signals. With the help of a pair of phase-locked-loop (PLL) based phase conjugators (PCs) the RDA’s response to other unwanted and/or unfriendly interrogating signals can be disabled, leading to enhanced secrecy performance directly in the wireless physical layer. The effectiveness of the proposed RDA system is experimentally demonstrated.

Simulation and analysis of an emergency lowering system for crane applications

An emergency lowering system for use in safety critical crane applications is discussed. The system is used to safely lower the payload of a crane in case of an electric blackout. The system is based on a backup power source, which is used to operate the crane while the regular supply is not available. The system enables both horizontal and vertical movements of the crane. Two different configurations for building the system are described, one with an uninterruptible power source (UPS) or a diesel generator connected in parallel to the crane’s power supply and one with a customized energy storage connected to the intermediate DC-link in the crane. In order to be able to size the backup power source, the power required during emergency lowering needs to be understood. A simulation model is used to study and optimize the power used during emergency lowering. The simulation model and optimizations are verified in a test hoist. Simulation results are presented with non-optimized and optimized controls for two example applications: a paper roll crane and a steel mill ladle crane. The optimizations are found to significantly reduce the required power for the crane movements during emergency lowering.

SPACE-DEPLOYED, THIN-WALLED ENCLOSURE FOR A CRYOGENICALLY-COOLED HIGH TEMPERATURE SUPERCONDUCTING COIL

The interaction of magnetic fields generated by large superconducting coils has multiple applications in space, including actuation of spacecraft or spacecraft components, wireless power transfer, and shielding of spacecraft from radiation and high energy particles. These applications require coils with major diameters as large as 20 meters and a thermal management system to maintain the superconducting material of the coil below its critical temperature. Since a rigid thermal management system, such as a heat pipe, is unsuitable for compact stowage inside a 5 meter payload fairing, a thin-walled thermal enclosure is proposed. A 1.85 meter diameter test article consisting of a bladder layer for containing chilled nitrogen vapor, a restraint layer, and multilayer insulation was tested in a custom toroidal vacuum chamber. The material properties found during laboratory testing are used to predict the performance of the test article in low Earth orbit. Deployment motion of the same test article was measured using a motion capture system and the results are used to predict the deployment in space. A 20 meter major diameter and coil current of 6.7 MA is selected as a point design case. This design point represents a single coil in a high energy particle shielding system. Sizing of the thermal and structural components of the enclosure is completed. The thermal and deployment performance is predicted.