46 research outputs found
Progress towards ultrasound-mediated targeted drug delivery in the small intestine
The intestinal mucosa acts as a selective barrier to permeation of material. Small molecules can usually pass the barrier provided they fulfil specific physicochemical requirements, but many macromolecular biotherapeutics cannot cross it. This restricts the delivery of biologics to injections, which can be associated with administration-related injuries and often require administration by a healthcare professional. However, the oral route is the drug administration route best accepted by patients. Ultrasound (US) and microbubbles (MBs) may allow this route to be used in future for biotherapeutics. Hence, an ingestible capsule incorporating an US transducer has the potential to offer a method for oral delivery of drugs to the small intestine. This capsule could both protect the drug payload against the destructive action of low stomach pH and facilitate delivery of a drug once it reaches the intestine.
The thesis describes the development and testing of a proof of concept tethered therapeutic capsule for delivering medication to the small intestine. The first experimental chapters present in vitro work, progressing to ex vivo tests on porcine tissue and in vivo tests in pigs. Preliminary studies suggested how to increase the relevance of in vitro tests for in vivo systems, in order to help reduce the number of animals used for research. To this end, classical in vitro systems for testing US-mediated drug delivery were refined to include stem cell-derived cell layers. US combined with either MBs or nanodroplets decreased the barrier function of these improved cell layers to different extents. The in vitro pathway also identified suitable transducer types and US parameters to facilitate the delivery of compounds across relevant cell layers. It was found that the effect of focused single-element transducers was surpassed by unfocused single-element and phased array transducers, with the latter allowing most control over barrier permeation. Array transducers were therefore integrated into test capsules. The US parameters tested in vitro were frequency, cycle number, pulse repetition frequency, duty cycle, mechanical index and surface area insonated. A decrease in barrier function was associated with lower frequencies, more cycles, more pulses, a higher duty cycle, a higher mechanical index and insonating a larger surface area.
The results obtained determined the insonation parameters for the in vivo test with capsules able to deliver insulin, MBs and fluorescent quantum dots (qDots). One control pig was administered insulin and qDots alone and two test pigs were administered insulin and qDots, with US and MBs. Blood glucose tests showed that the two test pigs displayed a smaller increase in glucose levels than the control pig, suggesting that more insulin reached the systemic circulation. This early result suggests US combined with MBs can facilitate drug delivery through the small intestine
Molecular mechanisms of sonoporation in cancer therapy : Optimization of sonoporation parameters and investigations of intracellular signalling
Background: Sonoporation, which is treatment with ultrasound (US) and microbubbles (MB), has shown great potential for enhancing the therapeutic efficacy of chemotherapy in cancer therapy. However, there is still very little consensus regarding the mechanism or optimal experimental and therapeutic parameters. The original assumption was that pore formation in the cell membrane was responsible for the increased uptake of drugs, but it is currently understood that the mechanisms are far more complex. The field combines US physics, MB formulation and physics, (cell) biology, pharmacology, pharmacokinetics and the biodistribution of both drugs and MBs. Hence, there is an almost endless range of experimental parameters and potential bioeffects. The current literature includes a plethora of experimental setups and parameters, which complicates the clinical translation of sonoporation. Aims and methodology: In this thesis, the effects of low-intensity US and MB parameters were investigated in vitro using custom-made ultrasound chambers and correlating commonly used measures as uptakes of impermeable dye (i.e. flow cytometry) and viability to detect intracellular signalling responses to sonoporation in different cell types. Intracellular signalling responses to sonoporation are largely unknown, and their influence on key proteins in important signalling pathways have been elucidated using phosphoflow cytometry. To gain the understanding and translatability of US + MB parameters, three commercially available MB formulations were characterized, and important parameters, such as dose and formulation, were investigated in vitro and the in vivo enhancement of chemotherapy in a mural model of pancreatic ductal adenocarcinoma (PDAC). Results and conclusions: Effective sonoporation was achieved using commercial microbubbles and low-intensity US in the diagnostic range, both in vitro and in vivo. In the low-intensity US regimen, effective sonoporation required MBs, and the efficacy increased as US intensity and MB concentrations increased. The choice of optimal MBs depended on the US parameters used, and must be carefully chosen based on the therapeutic context. The findings in vivo were correlated to those in the in vitro experiments and to simulations on MB behaviour. Sonoporation induced the immediate, transient activation of intracellular signalling (MAPK-kinases; p38, ERK1/2, CREB, STAT3, Akt) as well as changes in the phosphorylation status of the proteins involved in protein translation (i.e. ribosomal protein S6, 4E-BP1 and eIF2α). The intracellular signalling response resembles cellular recovery after pore formation by electroporation and pore-forming toxins. Based on this observation, we hypothesize that sonoporation induces a cellular stress response that is related to the membrane repair and restoration of cellular homeostasis, and it may be exploited therapeutically. Varying responses in different cell types better represent the variability within a tumour, and they indicate that the effects on the tumour microenvironment may be important for sonoporation efficacy. In the present work, cellular stress was induced using low-intensity US below the intensity limit approved for diagnostic imaging, and healthy blood peripheral cells were minimally affected
Magnetic Hybrid-Materials
Externally tunable properties allow for new applications of suspensions of micro- and nanoparticles in sensors and actuators in technical and medical applications. By means of easy to generate and control magnetic fields, fluids inside of matrices are studied. This monnograph delivers the latest insigths into multi-scale modelling, manufacturing and application of those magnetic hybrid materials
Sonobactericide
This thesis proposes sonobactericide, which is the use of ultrasound and exogenous cavitation nuclei, such as microbubbles, either alone or as a therapeutic complement for biofilm eradication or as a theragnostic for bacterial infections. Using infective endocarditis clinical isolates of _Staphylococcus aureus_, three different translational _in vitro_ biofilm models were developed and used in this thesis. Observed sonobactericide effects that can enhance therapy were biofilm disruption with bacterial dispersal, sonoporation (membrane permeabilization), fibrin strand manipulation (bending, recovery, and breaking), and synergistic responses when a thrombolytic and/or antibiotic were present. This thesis paves the way towards the clinical translation of sonobactericide as an effective treatment, diagnostic, or theranostic strategy employed in infectious disease care
Preclinical MRI of the kidney : methods and protocols
This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers
Characterizing Disease with Contrast-Enhanced Ultrasound: Applications in Oncology and the Kidney
Early assessment of disease progression and response to therapy play an important role in patient health and contribute positively towards desired treatment outcomes. For cancer patients, imaging techniques that provide rapid feedback detailing therapeutic efficacy, particularly in the case of non-responsive or recurring tumors, allow clinicians to appropriately modify treatment regimens on a faster timescale. Current standards rely on changes in tumor volume, measured using computed tomography, to determine treatment response, an effective strategy against cytotoxic therapies. However, for targeted therapies, tumor volume may inadequately classify response while changes in molecular expression occur quite rapidly after treatment and may provide faster feedback regarding therapeutic response. Similarly, early detection strategies capable of identifying the presence and progression of kidney disease faster than current clinical markers could facilitate faster intervention and more effectively mitigate long-term tissue damage. With chronic kidney disease (CKD) progression, changes in renal perfusion in response to structural and functional kidney has potential as a strategy for early detection. Contrast-enhanced ultrasound (CEUS) imaging offers a portable, widely accessible, inexpensive, and safe method for quantifying vascular changes. Ultrasound contrast agent (microbubble) destruction imaging techniques used to measure changes in disease biomarkers such as blood perfusion or vascular endothelial receptor expression are widely-available and easily implemented on clinical systems. This dissertation focuses on the clinical adaptation of CEUS for disease detection and monitoring. The first hypothesis is that CEUS can monitor response to different cancer therapies more accurately than tumor volume, both in vivo and in clinically relevant populations. Enhancements such as acoustic radiation force, buried ligand architecture, and microbubble size-selection were applied to improve the sensitivity of ultrasound molecular imaging and aid in the clinical translation of this imaging technique. The second hypothesis is that CEUS can be used to diagnose kidney health through the early detection of kidney disease and can diagnose subsequent complications that arise from CKD. Here, flash-replenishment perfusion imaging was evaluated as a method for identifying kidney disease and characterizing kidney lesions in the presence of CKD. Together, the results presented in this work aid in the clinical advancement of CEUS imaging techniques for disease characterization.Doctor of Philosoph
Micromechanics of particle-coated bubbles: deformation from quasistatic to millisecond timescales
Particles adsorbed at fluid-fluid interfaces confer stability to dispersed systems such
as foams and emulsions. The emergent properties associated with the interfacial
microstructure underpins the creation of functional materials. In the design, synthesis
and application of such materials, it is essential to understand the dynamic behaviour of
structured interfaces at deformation timescales that are relevant in practical scenarios.
In this experiment-driven study, a bubble is used as a probe to understand the
stability mechanisms and dynamics of fluid-fluid interfaces coated with particles.
First, in a model wax-based oil foam, or oleofoam, bubble dissolution time, under
controlled conditions, is used as a parameter to assess the bulk and interfacial rheological
contributions responsible for the remarkable stability observed. Focus is then drawn
to interfacial phenomena, by removing bulk effects, through microscopic observations
of crystal-coated bubbles undergoing deformation due to either bubble dissolution
or ultrasound-driven volumetric oscillations. In this way phenomena at two extreme
timescales, 10,000 s and 0.0001 s, are observed and interpreted. Finally, the effect of
unsteady, fast deformation on a complex interface is systematically studied using a well
characterised model interface, comprising of bubbles coated with optically resolvable,
monodisperse latex microspheres. The bubbles are subjected to acoustic forcing, leading
to the rapid cyclic compression and expansion of the colloidal monolayer. Effects of
pressure amplitude, particle size and surface coverage on bubble excursions are studied.
The results signify the importance of local mesoscopic phenomena in explaining
the stability of oleofoams, where invoking macroscopic rheological reasoning alone is
somewhat inadequate. Experimental timescales strongly influence the nature, integrity
and response of complex interfaces to imposed stresses. Further, a bubble driven by
ultrasound has potential in studying time-dependent interfacial mechanics.Open Acces
Multi-functional chromatography materials: new designs & applications
Process chromatography proves to be a continual bottleneck in bioprocessing, especially when considered against constraints such as rocketing product titres, complexity and size of emerging bio-products and a drive to reduce cost of goods and waste generation. Incremental changes in mono-functional chromatography media do not appear to be sufficient in coping with these increased demands and opens the door for novel ‘multifunctional’ beaded media, designed from the ground up, to posit a solution.
This work will, in part, demonstrate the creation of bi-layered chromatography resins with size-exclusion and anion exchange functionality and subsequently assess their performance against benchmarked commercial beads of similar structure. Three size exclusion chromatography matrices, differing in agarose content, cross-linker chemistry, particle and pore size distributions, were transformed into bi-layered supports featuring anion exchange functionalised cores and exterior size excluding shells.
The media were evaluated with respect to loss of surface and core binding, utilising finite bath studies allied with confocal scanning laser microscopic imaging. Plasmid DNA, bovine serum albumin and bovine serum albumin nanoparticles were utilised as surface and core binding probes, and the binding selectivities of different bi-layered support materials were compared by means of a simple selectivity ratio (μg of pDNA (or NP) bound per mg of BSA bound); the lower the number the more selective the support. The best performing bi-layered matrix was Superose 6 Prep Grade, when challenged with both plasmid DNA and BSA nanoparticles. Results for other base matrices was dependent on the nanoplex challenge as well as the bi-layering technique employed.
A second challenge addressed in this thesis was the acquisition of representative virus feedstock to road-test chromatography media, which can pose several practical issues with respect to requirements of particle concentration, volume and methods for tracking the species through chromatography media. A major focus has been developing a reliable platform in characterising commercial and in-house manufactured mono-functional and multi-functional chromatography resins, especially in the context of purifying large nanoplexes.
Against this backdrop, this work details a method for the reproducible manufacture of protein nanoparticles using a modified desolvation method; the nanoparticles function as surrogate mimics of virus products with a view to characterising viral chromatographic media.
Initially, BSA nanoparticles were explored and their manufacture optimised to develop a robust and reproducible system for creating defined nanoparticles within a defined size range. The nanoparticles were subsequently characterised for efficiency of manufacture, their morphological appearance, and secondary protein structure composition as well as how well they represented a bona fide viral species, namely adenovirus type V. The approach was then translated to other readily available proteins (e.g. lysozyme, bovine haemoglobin and ovalbumin) to determine the flexibility of the desolvation/coacervation approach and to target a wider range of viral species in terms of their physiochemical properties. The concept here was to develop a “toolbox approach” to develop custom-made nanoparticles to target a variety of viral species. Similar characterisation techniques were applied to the newly formed nanoparticles, as well as novel strategies applied to develop “second-generation nanoparticles” using alternative cross-linking mechanisms to produce multi-component nanoparticles.
The final chapter brings the work full-circle and looks to road-test the chromatography materials manufactured in Chapter II, with a range of sizes of BSA nanoparticles described in Chapter III. The nanoparticles are qualitatively assessed using confocal microscopy alongside quantitative assessment to determine bead performance. As in Chapter II, BSA protein is utilised as a core-binding probe. The nanoparticles are also applied to commercial resins, Q-Sepharose Fast Flow and Capto™ Core 700, both in batch and column scenarios. Of the bi-layered materials, Superose 6 Prep Grade generally outperformed both Sepharose 6 Fast Flow and Superose 12 Prep Grade, revealing differences between the media not shown by the plasmid DNA binding studies. This was generally supported by the complimentary confocal data, which provided an insight into the spatial location and binding kinetics when challenged onto the beads.
It is envisioned that the chromatographic supports manufactured here may be utilised in large scale nanoplex purification, exploiting their utility derived from their bi-functional modality. Furthermore, it is hoped that the protein nanoparticle-viral mimic methodology may be employed in the characterisation of a wider range of purification materials, taking advantage of their low cost and rapid manufacture, as well as their ability to provide valuable purification data which may inform important bioprocessing decisions