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

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Studying the influence of the physicochemical properties of lipid nanoparticles for mucosal vaccine delivery

Lipid-based nanoparticles have attracted attention as promising pharmaceutical carriers. Reports of them having inherent adjuvant properties make them particularly interesting as vaccine vectors; however, the physicochemical profile of an ideal nanoparticle for mucosal vaccine delivery remains unknown. The aim of this thesis work is to contribute a better understanding of the connection between physicochemical properties of lipid nanoparticles used as vaccine carriers and the activation of the immune response at several different levels of complexity. As combined antigen and adjuvant, we used a novel fusion protein comprising the Cholera toxin A1 subunit, combined with either the M2e or Ealpha peptide and a dimer of the D subunit of Staphylococcus aureus protein A. This fusion protein was coupled to liposomes and lipodisks with systematically varied poly(ethylene glycol) (PEG) content, protein load, rigidity and size/shape. Firstly, a detailed characterization of the biological response in vitro and in vivo, in a mouse model, to two types of fusion protein-carrying lipid particles was performed. Compared with the free fusion protein, which is in itself already an effective vaccination compound, the result showed that the non-PEGylated liposomes more efficiently induce both cell- and antibody-mediated immune responses as well as protection against a lethal virus challenge than both free fusion protein and the PEGylated liposomes. Secondly, an in vitro study was performed, focusing on elucidating the effect of the physicochemical properties of the carrier particle on processing, in particular the antigen presentation in major histocompatibility complex class II (MHC II), by dendritic cells. Out of 6 different formulations, which varied with respect to PEGylation, fusion protein load, membrane rigidity, size and shape it was found that only the DSPC-based liposome formulation, the only liposome formulation in gel phase, was able to increase antigen presentation compared to free fusion protein. Additionally, this formulation lead to an increased amount of surface-bound MHC II, indicating that the liposomes themselves might have an immunostimulatory effect, making them a promising candidate for further evaluation as a vaccine carrier with inherent adjuvant properties

Föräldrar som trampar i rabatten - är det så farligt? : Föräldrar med ADHD om föräldraskap och samhällets familjebilder.

This study examined parenting in relation to ADHD. The key point of the study was to let parents who experience an ADHD diagnosis share their view on parenting. The research required a comprehensive literature search, partly to examine earlier research in the particular field but also to gather information on parenting. One focus group interview and three semi structured interviews were used to gather relevant information. Informants were recruited from a project for individuals with neuropsychiatric disabilities. Aspects that were not taken in consideration in this study were gender differences in the view on parenting. Neither was the aim of the study to examine the children’s point of view. The study revealed that the informants’ view of parenting of today is all about status and reaching results. From their own experience of parenting, focus was on confirming their children and respect them as individuals. They considered themselves different than other parents, in areas as taking part of their children’s life and not to fit in according to clothing or appearance. Partly, they also felt that they did not get any understanding for their differences from the academic part of society

High School Students' Experiences in School Toilets or Restrooms

Previous research about school toilets is based on studies of children in elementary school. Thus, the aim of this study was to explore the experiences when using the school toilets reported by students aged 16-18 years. Qualitative interviews with 21 students were conducted and analyzed using content analysis. The data revealed that the toilets were considered insecure, dirty, and unpleasant. Additionally, students refrained from drinking during school hours and remained in constant movement or jumped up and down to withhold urine and stool. This was illustrated in the following categories: assessing the toilet environment, coping with the situation, and feeling exposed. Hence, there is an urgent need to improve the school toilet environment in order to respect the rights of all students to void or defecate when necessary, a process which will require involvement of students, teachers, and other school staff as well as the School Health Service

Optical properties of amorphous tungsten oxide films : Effect of stoichiometry

The optical properties of sputter deposited amorphous tungsten oxide films have been measured in-situ during slow electrochemical cycling in a lithium containing electrolyte. Amorphous films exhibit coloration under Li insertion and bleaching under Li extraction. Substoichiometric films show almost reversible optical changes already in the first electrochemical cycle and are completely reversible thereafter. Tungsten oxide films sputtered in a large excess of oxygen were found to be slightly overstoichiometric, as determined by Rutherford Backscattering Spectrometry. They exhibit irreversible charge transfer and coloration in the first cycle. Thereafter they cannot be completely bleached and exhibit transmittance contrast between coloured and partially bleached states. The irreversible colouration of the stoichiometric films is associated with a feature at 2.6 to 2.9 eV vs. Li in electrochemical measurements. Possible chemical reactions giving rise to this behaviour are discussed

Optical properties of amorphous tungsten oxide films : Effect of stoichiometry

The optical properties of sputter deposited amorphous tungsten oxide films have been measured in-situ during slow electrochemical cycling in a lithium containing electrolyte. Amorphous films exhibit coloration under Li insertion and bleaching under Li extraction. Substoichiometric films show almost reversible optical changes already in the first electrochemical cycle and are completely reversible thereafter. Tungsten oxide films sputtered in a large excess of oxygen were found to be slightly overstoichiometric, as determined by Rutherford Backscattering Spectrometry. They exhibit irreversible charge transfer and coloration in the first cycle. Thereafter they cannot be completely bleached and exhibit transmittance contrast between coloured and partially bleached states. The irreversible colouration of the stoichiometric films is associated with a feature at 2.6 to 2.9 eV vs. Li in electrochemical measurements. Possible chemical reactions giving rise to this behaviour are discussed

Mucosal Vaccine Development Based on Liposome Technology

Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen. Hence, there is an increasing demand for vaccine formulations that can induce strong protective immunity following oral, respiratory, or genital tract administration. At present, only few mucosal vaccines are found on the market, but recent technological advancements and a better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimistic view on the future of mucosal vaccines. Compared to live attenuated vaccines, subcomponent vaccines, most often protein-based, are considered safer, more stable, and less complicated to manufacture, but they require the addition of nontoxic and clinically safe adjuvants to be effective. In addition, another limiting factor is the large antigen dose that usually is required for mucosal vaccines. Therefore, the combination of mucosal adjuvants with the recent progress in nanoparticle technology provides an attractive solution to these problems. In particular, the liposome technology is ideal for combining protein antigen and adjuvant into an effective mucosal vaccine. Here, we describe and discuss recent progress in nanoparticle formulations using various types of liposomes that convey strong promise for the successful development of the next generation of mucosal vaccines

Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance

The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding