Glycopolymers for targeted delivery applications

Currently, the area of polymeric drug delivery is one of intense international and interdisciplinary research. Many of the applications of materials derived from this research are focussed on therapeutic applications ultimately to be used in whole body systems. Herein, the research described focuses on a novel application of multicomponent glycopolymers. The majority of pig breading is conducted via artificial insemination. Whilst this offers many advantages, one of the major drawbacks is the adverse effect of oxidation on the spermatozoa during storage (which can be several days) prior to use. The supplementation of vitamin E is highly desirable, however due to its lypophilic character it cannot be added directly to the largely aqueous boar ejaculate mixture. Polymethacrylate derivatives were synthesised carrying carbohydrate residues, vitamin E residues and solubility enhancing amino residues. These vitamin E bearing glycopolymers were found to be soluble in aqueous media. The interactions of these materials with boar spermatozoa were thoroughly investigated. It was demonstrated that supplementation with these glycopolymers significantly reduced cellular oxidation under induced oxidative conditions. Analogous polymers were also synthesised containing a fluorescent dye. Confocal microscopy images were obtained of boar spermatozoa incubated with dye-labelled polymers with and without carbohydrate moieties in order to demonstrate that the internalisation of the polymers was enhanced by carbohydrate-lectin interactions

Magnetic reconance imaging characterization of porous substrates and models of soil and biofilms

Bacteria and biofilms are frequently found growing in sponges and are a common cause of malodour in the sponge and cross-contamination of humans and surfaces with the risk of causing bacterial infections. Bacterial infection generates high annual healthcare costs and are related to high mortality rates. Removal of bacteria and biofilms from sponges is thus of critical importance to avoid infections, food poisoning, and surface contamination. To tackle biofilm development in sponges, and thus reduce cross contamination, fast-moving consumer goods (FMCGs) companies have focused on developing detergents which can remove food from sponges. However, due to their intrinsic method’s limitation, current existing techniques, such as X-Ray micro-CT, confocal laser scanning microscopy, scanning and transmission electron microscopy, do not allow a complete understanding and visualization on how detergents act in removing food and biofilms from sponges. To fulfil this gap, new methodologies need to be developed to increase knowledge on biofilm development, particularly within sponges, and its relationship with food residues, as well as on fluid dynamics within sponges. Thanks to their nature, nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) could allow to image food residues and biofilm development in porous media in-situ and in-vivo. Moreover, NRM and MRI enable the characterization of the 3D internal structure of porous media and the visualization of fluid flow within them. Thus, NMR and MRI could potentially allow to study how the physical parameters of sponges affect fluid flow, and how this is related to food residues removal and biofilm development in sponges, in-situ and without the need of pre-treatments. In this work, novel nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) have been developed to characterize polyurethane open-cell porous media, and directly visualize food and biofilms within, without the need of sample pre-treatment or the addition of contrast agents. A combination of NMR relaxometry, MRI T2 relaxation maps and X-ray micro computed tomography (µCT), has highlighted the dependence of T2 relaxation times of water, within the foam, on the pore sizes. This has enabled the creation of new protocols to characterize polymeric open-cell porous media directly by MRI, which can be combined with MRI visualization of composition and flow. MRI Chemical Shift Selective (CHESS) imaging has been employed to visualize food residues within polyurethane (PU) sponges, enabling the selective mapping of hydrophobic and hydrophilic residues within the sponge without additional contrast agent. Finally, MRI chemical exchange saturation transfer (CEST) experiments have been successfully developed to image polysaccharide-based (alginate) materials within sponges, demonstrating their potential to visualise the exopolysaccharide matrix of biofilms without the need for additional contrast agents

Vat 3D printable materials and post-3D printing procedures for the development of engineered devices for the biomedical field

L'abstract è presente nell'allegato / the abstract is in the attachmen

DEVELOPING ELECTROOSMOTIC-PUMP-BASED MINIATURIZED DEVICES: TOWARD A LIQUID CHROMATOGRAPHY CARTRIDGE COUPLED WITH MASS SPECTROMETER AND A SUB-PICOLITER PIPETTOR FOR SINGLE CELL SURGERY

There has been increasing interest on miniaturizing high performance liquid chromatography (HPLC), since it can reduce the costs of instrument manufacturing and perform onsite analysis. The electroosmotic pump (EOP) is one of the most promising micropumps that can be used for HPLC due to its compact size, and low fabrication cost. We have recently reported an electroosmotic pump (EOP), consisting of monolithic columns, capable of producing more than 1200 bar output pressure. However, when it pumps organic solutions, the flow rate becomes unstable. Hence, we here report two ways to address the issue. In the second chapter, we describe the first method. A ten-port valve was used to create a gradient profile by inserting it between an EOP and an injection valve (Figure 2 1). In the position as shown in Figure 2 1, an eluent in loop 1 was delivered to the separation column, while a higher concentration eluent was loaded into loop 2. Once the valve was switched, loop 2 was connected to the separation column, while the next gradient eluent was loaded into loop 1. Repeating these steps formed a gradient profile that was close to linear. In the second method (Chapter 3), we added a mixing chamber after the EOP (Figure 3 1). When a high voltage was applied, the EOP withdrew a series of gradient eluents having different elution strengths via a selection valve. The eluents went through the mixing chamber and towards to EOP. Once an opposite high voltage was applied, the EOP pushed the eluents out, through the mixing chamber again, and towards to the separation column. During the movements of these eluents (i.e., back and forth in the mixing chamber, as well as inside of capillary assembly), a smooth gradient profile was formed. Since it is possible to change the size of mixing chamber, the number of eluents, as well as the volume, concentration, and the movement pattern (i.e., duration, velocity, and movement style), any desired gradient profile can be generated. Finally, we incorporated either approach with HPLC and a mass spectrometer for the separation and analysis of peptides and proteins. In the fourth chapter, we described the application of EOP on single cell analysis. There has been increasing interest and requirement of single cell analysis with the development of bioanalytical and biochemical methods, since average data from population eliminate the heterogeneity of single cells. However, there are few methods currently available that can transfer mass into, and especially out of single cells under precise control. In this study, we developed a monolith-based EOP-driven pipettor (EDP), which was able to inject solutions into and withdraw cellular contents out of single cells down to ~250 fL. We subsequently demonstrated an application of the EDP in real-world samples by quantitative analysis of cholesterol from zebrafish embryos, and 89.3%-91.7% of the embryos survived after surgery that was performed at different developmental stages. Since the compatibility of the pump solution, an EDP has potential to be directly coupled with mass spectrometry. Taken together, our results showed an EDP can be used for living-single-cell analysis

Nanocomposite systems based on metal nanoparticles and polysaccharides for biomedical applications

2008/2009Questo lavoro riguarda lo sviluppo di materiali nanocompositi per applicazioni biomediche e si configura all’ interno del progetto europeo “Newbone” (EU-FP6); in particolare, lo scopo principale della tesi era realizzare un rivestimento biocompatibile e dotato di proprietà antibatteriche per protesi ortopediche. Sono stati preparati sistemi nanocompositi basati su un polisaccaride derivato dal chitosano (Chitlac) che permette di ottenere soluzioni colloidali di nanoparticelle (argento e oro) con proprietà antibatteriche. Parallelamente, è stato studiato un particolare meccanismo chimico di riduzione degli ioni argento ad opera dei residui di lattitolo del Chitlac; le proprietà ottiche delle nanoparticelle ottenute attraverso questo meccanismo sono state valutate attraverso spettroscopia Raman, evidenziando la possibilità di avere un incremento del segnale grazie al verificarsi dell’ effetto SERS. Essendo state riscontrate migliori proprietà biologiche del sistema a base di argento (Chitlac-nAg) rispetto a quello a base di oro in termini di efficacia antimicrobica e biocompatibilità, Chitlac-nAg è stato scelto per i successivi studi di realizzazione del rivestimento per la protesi. Test sul meccanismo antimicrobico della soluzione ChitlacnAg hanno dimostrato l’interazione tra le nanoparticelle e la membrana batterica. Allo stesso tempo, poiché la mancanza di barriere fisiche può favorire la diffusione delle nanoparticelle all’ interno delle cellule eucariote con rischio di effetti citotossici causati dalla loro internalizzazione, si è voluto realizzare delle strutture tridimensionali a base di Chitlac in grado di intrappolare le nanoparticelle. A questo scopo, sono state sfruttate le proprietà di gelificazione del polisaccaride alginato in modo da ottenere un sistema semi-solido in miscela con Chitlac-nAg; il materiale ottenuto possiede marcate proprietà antibatteriche senza però risultare tossico per le cellule eucariote, come dimostrato da test in vitro e in vivo. Questo risultato è particolarmente importante in relazione allo stato dell’ arte sull’ argomento. Poiché la parte portante della protesi è costituita da un polimero metacrilico, al fine di rivestire questo materiale di substrato è stata messa a punto una tecnica basata sull’ attivazione della superficie e successiva deposizione del rivestimento a base di Chitlac. Questa tecnica permette di ottenere un rivestimento nanocomposito costituito da nanoparticelle di argento incorporate nella matrice di Chitlac. Grazie a questo strato bioattivo la superficie della protesi acquisisce un’ efficace attività antibatterica che si manifesta quando i batteri entrano in diretto contatto con il materiale. Inoltre, test in vitro hanno dimostrato che le cellule eucariote aderiscono e proliferano sul rivestimento nanocomposito, suggerendo quindi una buona integrazione del materiale nei tessuti attorno all’ impianto. La combinazione di tali proprietà ha determinato la scelta di questo rivestimento per il test in vivo su “minipig” a conclusione del progetto europeo: questo test è al momento in via di svolgimento e da esso ci si può attendere una conferma degli incoraggianti risultati ottenuti dagli studi in vitro.The present work is focused on the development of nanocomposite systems for biomedical applications and has been carried out in the framework of the European Project called “Newbone” (EU-FP6); in particular, the main goal of the thesis was to realize biocompatible coatings for orthopedic prosthesis endowed with antimicrobial properties. Nanocomposite systems based on a chitosan-derived polysaccharide (Chitlac) that stabilizes metal nanoparticles (silver and gold) have been prepared in colloidal solutions which possess broad spectrum antibacterial properties. As a complementary work, it was studied and defined a particular chemical mechanism of silver ions reduction carried out by the lactose moieties of Chitlac; the optical properties of the metallic nanoparticles obtained through this mechanism were tested by means of Raman spectroscopy, thus detecting considerable enhancements of the signal due to the SERS effect (Surface Enhanced Raman Scattering). Given the better biological properties of silver-based systems (Chitlac-nAg) with respect to gold in terms of antimicrobial efficacy and biocompatibility, only the former metal was chosen in the following steps towards the preparation of the nanocomposite coating for the prosthesis. Studies on the biocidal mechanism of the Chitlac-nAg solution ascribed the activity to the interaction metal-bacteria membrane. On the other hand, since the lack of physical barriers to nanoparticle diffusion into eukaryotic cells determines the risk of a massive uptake with cytotoxic outcomes, we focused our attention toward the preparation of Chitlac-based threedimensional structures entrapping silver nanoparticles. To this end, the gel forming properties of the polysaccharide alginate were exploited allowing the production of a semi-solid system in a mixture with Chitlac-nAg: this material displays potent antibacterial properties without showing cytotoxic effects towards eukaryotic cells, as verified by in vitro and in vivo tests. Such result was particularly important in relation to the state of the art in this research field. Since the core material of the prosthesis is made of methacrylic thermosets, in order to coat this substrate material we have devised a technique based on surface activation followed by deposition of the Chitlac-based layer. Such technique allows obtaining a nanocomposite coating where silver nanoparticles are entrapped within the Chitlac matrix. This bioactive layer endows the thermoset surface with considerable antimicrobial properties, as bacteria are rapidly killed upon direct contact with the material. At the same time, in vitro tests proved that eukaryotic cells adhere and proliferate on the nanocomposite coating, which indicates the possibility to have good integration of the material in the tissues surrounding the implant. The combination of these properties determined the choice of our coating for the final in vivo test in a minipig model as a conclusion of the European project; this test is in progress at the moment and it will hopefully confirm the encouraging studies in vitro.XXII Ciclo198

Cementos óseos acrílicos con capacidad de estimular la regeneración de tejido óseo.

Los cementos óseos acrílicos (ABC) son materiales ampliamente usados en ortopedia en la fijación de prótesis articulares, estabilización de fracturas, como material de relleno de defectos óseos, entre otros. Desde el punto de vista d la primera aplicación, las funciones principales de los cementos son la transferencia de las cargas de servicio y del peso corporal de la prótesis al hueso y la inmovilización inmediata de la prótesis. A pesar de su uso extenso, los ABC presentan algunas desventajas relacionadas con la falta de bioactividad debido a su naturaleza inerte y la susceptibilidad a la generación de infecciones por bacterias, todo lo cual causa aflojamiento de las prótesis en el tiempo. Con el fin de aportar a la solución de algunos de los inconvenientes presentados por los ABC, en este Trabajo de Investigación Doctoral se planteó la incorporación de quitosano en porcentajes entre 0 ¿ 20% en peso con respecto a la fase sólida del cemento y óxido de grafeno en porcentajes entre 0 ¿ 0.5% en peso de la fase líquida. El GO fue sintetizado mediante el método de Hummers modificado y posteriormente caracterizado fisicoquímicamente mediante técnicas Espectroscópicas (FTIR, XPS y Raman), Difracción de rayos-X (DRX), Dispersión de Luz Dinámica (DLS) y Microscopia de Fuerza Atómica (AFM). El estudio del efecto de la incorporación del CS y el GO en los cementos sobre sus propiedades físicas, químicas, térmicas, mecánicas y biológicas en condiciones in vitro e in vivo, mostró que el CS le confiere a los cementos propiedades bioactivas además de incrementar la porosidad y la humectabilidad, favoreciendo la biocompatibilidad. Sin embargo, también se presenta la desventaja de que reduce las propiedades mecánicas de compresión y flexión. Por otro lado, la adición de GO incrementó las propiedades mecánicas y le confirió actividad antibacteriana al cemento. Asimismo, tanto el CS como el GO redujeron la temperatura máxima durante la reacción de polimerización e incrementaron el tiempo de curado y la rugosidad y el contenido de monómero residual. Cuando se incorporaron el CS y el GO juntos en la formulación del cemento, el efecto combinado de las cargas mostró una mayor osteointegración después de 3 meses de implantación en hueso parietal de ratas Wistar y las desventajas mecánicas incorporadas por el CS fueron compensadas gracias al refuerzo alcanzado con las nanopartículas de GO. A partir de los resultados obtenidos en esta investigación se aporta al estado del arte del conocimiento asociado a los ABC cargados con bajo contenido de CS y con GO, consiguiendo principalmente conferir propiedades bioactivas y antibacterianas a los ABC, manteniendo el cumplimiento de las propiedades mecánicas exigidas por la norma ISO 5833- 02. Estos resultados evidencian que las formulaciones desarrolladas son susceptibles de ser empleadas en aplicaciones en aplicaciones como artroplastias, relleno de defectos óseos, y demás usos donde se requiera un desempeño superior en el materiaDoctoradoDOCTOR(A) EN INGENIERÍ

Preparation and characterization of silica, reduced graphene oxide and composite aerogels

Ph. D. Thesis.In this thesis, experimental results are reported on the synthesized of waterglass-based silica aerogels monoliths by mixing graphene oxide. The new ambient pressure approach is used to produce aerogels by using a mixture of sodium bicarbonate and water instead of low surface tension solvent. Furthermore, to enhance mechanical properties graphene oxide as nanofillers is added into silica matrix to synthesize composite aerogels based on sol-gel technology. Furthermore, we report a simple method for obtaining reduced graphene oxide aerogels doped with different metal oxide nanoparticles, germanium dioxide (GeO2), cobalt ferrite (CoFe2O4), and iron(III) oxide (Fe3O4) by using supercritical drying method. Firstly, all metal oxide nanoparticles are synthesized. Then metal oxide nanoparticles are added into graphene oxide solution and sodium bisulfide is utilised as a cheap reducing agent. Lastly, prepared aerogels’ properties such as surface area, morphology, the chemical composition, and electrochemical measurements as an anode material for lithium-ion batteries are investigated. Finally, ambient pressure dried graphene-based aerogels are produced with common devices by rewording a partially reduced graphene oxide hydrogel with an ice-template method. This work can be cost effective of high-performance graphene aerogels. Scanning electron microscope (SEM), X-ray diffraction (XRD), uniaxial compression test, fourier transform infrared spectroscopy (FTIR), raman spectroscopy, Brunauer - Emmett - Teller (BET) theory and Barret - Joyner - Halenda (BJH) method are utilised to characterise the produced aerogels.Turkish Governmen

Targeting Cell Wall Formation in the Oomycete Phytophthora cinnamomi for Disease Control

The oomycete Phytophthora genus comprises microorganisms that cause devastating plant diseases, such as late blight and root rot diseases, leading to significant agricultural economic losses, and causing extensive damages to ecosystems. To date, no practical method is available to prevent these diseases. Furthermore, current strategies to control Phytophthora-induced diseases are ineffective in the long term. These strategies currently rely on different classes of chemicals. However, repeated use of the same chemicals can lead to development of pesticide resistance in phytopathogens. This concern, combined with an increased awareness of alternative approaches that have minimal impact on biodiversity and human health, highlights that efficient methods for controlling diseases caused by Phytophthora are urgently required. Targeting cell wall biosynthesis is a promising strategy to combat these pathogens. Indeed, the inhibition of enzymes involved in carbohydrate biosynthesis affects the growth and survival of these pathogens, offering a promising avenue for the development of effective treatments. In recent years, plant antimicrobial peptides (AMPs) have been found to be effective against different phytopathogens. Well-known AMPs are plant defensins, a family of small cysteinerich peptides that can bind to chitin and cell wall glucans in fungi. However, knowledge about the inhibitory role of plant defensins in oomycetes is limited. As such, this work investigates the effects of the plant defensin NaD1 (Nicotiana alata defensin 1) on Phytophthora species, which may reveal novel opportunities for controlling plant diseases. Our findings demonstrate that NaD1 effectively inhibits the mycelial growth of Phytophthora cinnamomi, Phytophthora cambivora, Phytophthora nicotianae, and Phytophthora citricola. Exposure to NaD1 induced alterations in the growth and structure of P. cinnamomi, leading to suppressed apical dominance, hyper-branching, and changes in cell wall composition, likely due to disruption of calcium homeostasis. Transcriptomic analyses confirmed altered expression of genes involved in cellulose synthesis and calcium transport (Chapter 2), and uncovered changes in the transcriptome across the entire genome in hyphal cells exposed to NaD1, shedding light on the mechanism of action of this AMP. These differentially expressed genes can serve as candidates to study the efficacy of NaD1 against Phytophthora species (Chapter 4). In addition to NaD1, the effects of a chitin synthase inhibitor, nikkomycin Z, were also investigated. This study shows that nikkomycin Z causes strong growth inhibition of four Phytophthora species and induces abnormal hyphal growth. Exposure to this inhibitor decreases cellulose levels and affects the expression of genes related to vital functions such as cell wall biosynthesis, hexosamine biosynthesis and chitin deacetylation (Chapter 3). Altogether, the present work reveals critical information about the fundamental inhibitory mechanisms of NaD1 and nikkomycin Z on Phytophthora species, with a focus on cell wall biosynthesis. This work paves the way for the development of novel effective targets for oomycete disease control.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 202