Liposomes for mucosal vaccine delivery: physicochemical characterization and biological application

Liposomes are attractive vaccine carriers due to their potential to act as adjuvants, and to the fact that their composition and characteristics are virtually endlessly customizable. However, the precise physicochemical profile of an ideal carrier liposome for mucosal vaccines is still widely unknown, and how different properties affect key steps in the acquisition of protective immunity remains to be elucidated. Additionally, there is no consensus in the field regarding characterization of vaccine formulations, often with incomplete reporting of properties as a result. The focus of this work is therefore twofold: i) to contribute to a better understanding of how the physicochemical profile of vaccine carrier liposomes impacts the development of protective immunity using models at different levels of complexity, and ii) to improve and simplify the physicochemical characterization of liposomes through development and use of new analytical methods. The work in the first area consists of, firstly, an in vivo characterization of the biological response to vaccine liposomes carrying a vaccine protein and characterized by varying surface hydrophilicity (PEGylation). This study showed that non-PEGylated vaccine liposomes more efficiently induced local cell- and antibody-mediated immune responses, as well as better protection against a lethal virus challenge than both PEGylated liposomes and free vaccine protein. Secondly, in vitro studies focused on how liposome stiffness influences dendritic cells, investigating effects on uptake, antigen presentation and cellular activation. These investigations demonstrated that stiff, gel phase liposomes were able to more efficiently activate dendritic cells and induce significantly higher levels of antigen presentation and co-stimulatory signaling compared to both soft, fluid phase liposomes, and free vaccine protein. The work in the second part comprises two studies: a surface plasmon resonance-based method to characterize the influence on liposome deformation from specific multivalent interactions with supported cell membrane mimics, and a waveguide microscopy technique for characterization of optical properties of individual liposomes. While the latter method might become valuable in the context of quantifying the efficiency of dye labelling of liposomes, the surface plasmon resonance study offered information on how liposome deformation depends on membrane stiffness and ligand-receptor pair density. Taken together, the work presented in this thesis demonstrate the value of multidisciplinary approaches to complex biological and medical challenges

Nanomaterials for Biomedical and Biotechnological Applications

The need for constant improvement to reach a high standard of safety and to make nanomaterials accessible for marketing has generated a considerable number of scientific papers that highlight new important aspects to be considered, such as synthesis, stability, biocompatibility, and easy manipulation. In order to provide a comprehensive update on the latest discoveries concerning nanomaterials, this reprint presents 14 scientific papers, 10 research articles and 4 reviews, that deal with biomedical and biotechnological applications of nanomaterials

Advanced nanotechnologies for overcoming antimicrobial resistance

Multidrug-resistant pathogens are prevalent in chronic wounds. There is an urgent need to develop novel antimicrobials and formulation strategies to overcome antibiotic resistance and provide a safe alternative to traditional antibiotics. Chapter 2 aims to create a novel nanocarrier for two cationic antibiotics, tetracycline hydrochloride and lincomycin hydrochloride, overcoming antibiotic resistance. In this study, the use of surface-functionalised polyacrylic copolymer nanogels as carriers for cationic antibiotics is investigated. These nanogels can encapsulate small cationic antimicrobial molecules and act as a drug delivery system. They were further functionalised with a biocompatible cationic polyelectrolyte, bPEI, to increase their affinity towards the negatively charged bacterial cell walls. These bPEI-coated nanocarrier-encapsulated antibiotics were assessed against a range of wound isolated pathogens, which had been shown through antimicrobial susceptibility testing (AST) to be resistant to tetracycline and lincomycin. The data reveals that bPEI-coated nanogels with encapsulated tetracycline or lincomycin displayed increased antimicrobial performance against selected wound-derived bacteria, including strains resistant to the free antibiotic in solution.Next, after experimentation into the use of Carbopol nanogels against antibiotic-resistant wound-derived pathogens, in planktonic form, the work in chapter 3 investigated their use against biofilm-formed pathogens. Biofilms are prevalent in chronic wounds and once formed, are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Novel nanotechnology is applied to do this, based on protease-functionalised nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defences. This thesis's work demonstrates that they are effective against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. Additionally, it is shown that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm- forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 hours of treatment.Chapter 4 combines the work of chapter 2 (NPs against antibiotic-resistant pathogens) and chapter 3 (NPs against biofilm-forming pathogens). This concept is demonstrated by encapsulating Penicillin G and Oxacillin into shellac nanoparticles, subsequently coated Alcalase. It is shown for the first time that these active nanocarriers can destroy biofilms of S. aureus resistant to Penicillin G and are significantly more effective in killing the bacterial cells within compared to an equivalent concentration of free antibiotic. The approach of concentrating the antibiotic by encapsulating it into a nanocarrier allows a localised antibiotic delivery to the anionic cell wall, facilitated by coating the NPs with a cationic protease. This approach allowed the antibiotic to restore its effectiveness against S. aureus, characterised as resistant to the same antibiotic and cause rapid bacterial biofilm degradation. This approach could be potentially used to revive old antibiotics which have already limited clinical use due to developed resistance.Chapter 5 continued investigating the antimicrobial properties of antibiotic-loaded shellac NPs, with a cationic protease surface functionalisation, however this time on a pathogen fungal species, Candida albicans. These Amphotericin B (AmpB)‐loaded shellac NPs are fabricated by pH‐ induced nucleation of aqueous solutions of shellac and AmpB in the presence of Poloxamer 407 (P407) as a steric stabiliser (in the same fashion as penicillin G and oxacillin in chapter 4. The AmpB‐loaded shellac NPs are surface coated with the cationic protease Alcalase. The AmpB‐loaded shellac NPs show a remarkable boost of their antifungal action compared to free AmpB when applied to C. albicans in both planktonic and biofilm forms. The surface functionalisation with a cationic protease allows the NPs to adhere to the fungal cell walls, delivering AmpB directly to their membranes. Additionally, the hydrolysing activity of the protease coating degrades the biofilm matrix, thus increasing the effectiveness of the encapsulated AmpB compared to free AmpB at the same concentration.Additionally, these protease‐coated nanocarrier-based antibiotics showed no detectable cytotoxic effect against human keratinocytes. It is envisaged these antibiotic-loaded NPs. Subsequently, surface functionalised with the cationic protease could be potentially used to treat antibiotic-resistant biofilm infections in the clinic, for example, in recalcitrant chronic wounds. Chapter 6 outlines future work which could be performed using these NP formulations

Enzyme Powered Nanomotors Towards Biomedical Applications

[eng] The advancements in nanotechnology enabled the development of new diagnostic tools and drug delivery systems based on nanosystems, which offer unique features such as large surface area to volume ratio, cargo loading capabilities, increased circulation times, as well as versatility and multifunctionality. Despite this, the majority of nanomedicines do not translate into clinics, in part due to the biological barriers present in the body. Synthetic nano- and micromotors could be an alternative tool in nanomedicine, as the continuous propulsion force and potential to modulate the medium may aid tissue penetration and drug diffusion across biological barriers. Enzyme-powered motors are especially interesting for biomedical applications, owing to their biocompatibility and use of bioavailable substrates as fuel for propulsion. This thesis aims at exploring the potential applications of urease-powered nanomotors in nanomedicine. In the first work, we evaluated these motors as drug delivery systems. We found that active urease- powered nanomotors showed active motion in phosphate buffer solutions, and enhanced in vitro drug release profiles in comparison to passive nanoparticles. In addition, we observed that the motors were more efficient in delivering drug to cancer cells and caused higher toxicity levels, due to the combination of boosted drug release and local increase of pH produced by urea breakdown into ammonia and carbon dioxide. One of the major goals in nanomedicine is to achieve localized drug action, thus reducing side-effects. A commonly strategy to attain this is the use moieties to target specific diseases. In our second work, we assessed the ability of urease-powered nanomotors to improve the targeting and penetration of spheroids, using an antibody with therapeutic potential. We showed that the combination of active propulsion with targeting led to a significant increase in spheroid penetration, and that this effect caused a decrease in cell proliferation due to the antibody’s therapeutic action. Considering that high concentrations of nanomedicines are required to achieve therapeutic efficiency; in the third work we investigated the collective behavior of urease-powered nanomotors. Apart from optical microscopy, we evaluated the tracked the swarming behavior of the nanomotors using positron emission tomography, which is a technique widely used in clinics, due to its noninvasiveness and ability to provide quantitative information. We showed that the nanomotors were able to overcome hurdles while swimming in confined geometries. We observed that the nanomotors swarming behavior led to enhanced fluid convection and mixing both in vitro, and in vivo within mice’s bladders. Aiming at conferring protecting abilities to the enzyme-powered nanomotors, in the fourth work, we investigated the use of liposomes as chassis for nanomotors, encapsulating urease within their inner compartment. We demonstrated that the lipidic bilayer provides the enzymatic engines with protection from harsh acidic environments, and that the motility of liposome-based motors can be activated with bile salts. Altogether, these results demonstrate the potential of enzyme-powered nanomotors as nanomedicine tools, with versatile chassis, as well as capability to enhance drug delivery and tumor penetration. Moreover, their collective dynamics in vivo, tracked using medical imaging techniques, represent a step-forward in the journey towards clinical translation.[spa] Recientes avances en nanotecnología han permitido el desarrollo de nuevas herramientas para el diagnóstico de enfermedades y el transporte dirigido de fármacos, ofreciendo propiedades únicas como encapsulación de fármacos, el control sobre la biodistribución de estos, versatilidad y multifuncionalidad. A pesar de estos avances, la mayoría de nanomedicinas no consiguen llegar a aplicaciones médicas reales, lo cual es en parte debido a la presencia de barreras biológicas en el organismo que limitan su transporte hacia los tejidos de interés. En este sentido, el desarrollo de nuevos micro- y nanomotores sintéticos, capaces de autopropulsarse y causar cambios locales en el ambiente, podrían ofrecer una alternativa para la nanomedicina, promoviendo una mayor penetración en tejidos de interés y un mejor transporte de fármacos a través de las barreras biológicas. En concreto, los nanomotores enzimáticos poseen un alto potencial para aplicaciones biomédicas gracias a su biocompatibilidad y a la posibilidad de usar sustancias presentes en el organismo como combustible. Los trabajos presentados en esta tesis exploran el potenical de nanomotores, autopropulsados mediante la enzima ureasa, para aplicaciones biomédicas, y investigan su uso como vehículos para transporte de fármacos, su capacidad para mejorar penetración de tejidos diana, su versatilidad y movimiento colectivo. En conjunto, los resultados presentados en esta tesis doctoral demuestran el potencial del uso de nanomotores autopropulsados mediante enzimas como herramientas biomédicas, ofreciendo versatilidad en su diseño y una alta capacidad para promover el transporte de fármacos y la penetración en tumores. Por último, su movimiento colectivo observado in vivo mediante técnicas de imagen médicas representan un significativo avance en el viaje hacia su aplicación en medicina

Polymer-gold nanoparticulate formulations for combinational photochemotherapy of pancreatic cancer

Pancreatic cancer is one of the most deadly of all types of cancer, with a yearly incident that equals its mortality. Gemcitabine (Gem) is currently the first-line chemotherapeutic drug used to treat pancreatic cancer. The major deficiencies of Gem therapy are poor cell membrane permeability, short plasma half-life and toxic side effects. In order to improve the pharmacokinetic characteristics and overcome the obstacles to achieve effective drug delivery, a nanoparticulate drug delivery system can be utilised; gold nanoparticles (GNPs) have been investigated as carriers for drug delivery due to their appealing physicochemical and optical properties. This research project concerns the development of a new generation of GNPs for cancer treatment by co-delivering anti-cancer drugs in combination with laser-induced photothermal effects confined at the diseased areas. Gold nanoshells (GNShells) were synthesised with the capability to carry and deliver Gem and exert phototherapeutic properties. Protein repellent thiol capped poly (ethylene glycol) methyl ether methacrylate polymers were synthesised by RAFT polymerisation and used as efficient particle stabilising ligands. Significant stability enhancement was achieved allowing for the co-functionalisation of GNShells with Gem for applications in in vitro assays against pancreatic cancer cells. GNShells mediated strong photothermal effect owing to their strong surface plasmon absorption in the red/NIR region. This property was exploited to enhance the toxicity of Gem using laser light as the external stimulus. The concerted antitumor activity of Gem with the photothermal effect of the GNShells upon irradiation with a continuous wave laser, increase the cellular uptake and efficacy of Gem-loaded GNShells against MiaPaCa-2 cells. Therefore, the proposed nanoformulation might provide an active strategy for synergistic chemo-photothermal combined therapy

Cellulose-Based Biosensing Platforms

Cellulose empowers measurement science and technology with a simple, low-cost, and highly transformative analytical platform. This book helps the reader to understand and build an overview of the state of the art in cellulose-based (bio)sensing, particularly in terms of the design, fabrication, and advantageous analytical performance. In addition, wearable, clinical, and environmental applications of cellulose-based (bio)sensors are reported, where novel (nano)materials, architectures, signal enhancement strategies, as well as real-time connectivity and portability play a critical role

Diffusion of tin from TEC-8 conductive glass into mesoporous titanium dioxide in dye sensitized solar cells

The photoanode of a dye sensitized solar cell is typically a mesoporous titanium dioxide thin film adhered to a conductive glass plate. In the case of TEC-8 glass, an approximately 500 nm film of tin oxide provides the conductivity of this substrate. During the calcining step of photoanode fabrication, tin diffuses into the titanium dioxide layer. Scanning Electron Microscopy and Electron Dispersion Microscopy are used to analyze quantitatively the diffusion of tin through the photoanode. At temperatures (400 to 600 °C) and times (30 to 90 min) typically employed in the calcinations of titanium dioxide layers for dye sensitized solar cells, tin is observed to diffuse through several micrometers of the photoanode. The transport of tin is reasonably described using Fick\u27s Law of Diffusion through a semi-infinite medium with a fixed tin concentration at the interface. Numerical modeling allows for extraction of mass transport parameters that will be important in assessing the degree to which tin diffusion influences the performance of dye sensitized solar cells