Hydrophilic Nanogels | EPFL Graph Search

Hydrophilic nanocarriers, formed by electrostatic interaction between their oppositely charged components, are recognized as nanomaterials with an enormous potential as vectors in biomedical and pharmaceutical applications. However, comprehensive information about their fate in various biological environments is still lacking. In general, due to their size, nanoparticles are able to enter and transport molecules into cells via pathways forbidden for bigger structures of even identical chemical nature. Research under defined conditions aiming at a better understanding of nanoparticle-cell interactions is crucial for the development of safe and successful nanocarriers for biomedical and pharmaceutical applications. Nanogels produced by electrostatic interaction of two types of the polysaccharide chitosan and pentasodium triphosphate were the subject of the research of this thesis. Additional surface coating with sodium alginate conferred to the nanogels a negatively charged surface enabling sufficient stability under physiological and in vivo relevant conditions. Of particular interest was the comparison of the two chitosan types, the first one obtained from usual animal sources while the second one became newly available from fungal sources. Prerequisite for the comparison in terms of nanogel formation, stability, interaction with cells and potential applications was the detailed analysis and characterization of the chitosan samples. The identification of almost identical degree of deacetylation, very similar molar mass distribution and solution behavior, but a somewhat higher molar mass of the animal chitosan as the only difference, allowed for a direct comparison. The animal and fungal derived chitosan samples yielded similar nanogels in relation to formation, size distributions and zeta potential. The nanogels were spherical with a hydrodynamic diameter in the range of 450 nm and a zeta potential of approx. -65 mV. The physical characteristics of the nanogels were clearly influenced by the ionic strength and temperature of the suspension. High deformability of the nanogel network was demonstrated by filtration through pores much narrower than the gel diameter. Temperature cycles in the range of 4°C to 37°C proved reversibility of size and zeta potential. The nanogels were stable in both simulated in vivo environment and in conditioned cell culture media. Exposure of seven human cancerous and non-cancerous cell lines to the nanogels, to identify the influence on the cell metabolic activity, revealed cell-type, nanogel dosage and exposure time dependence. However, nanogels prepared from animal and fungal chitosan showed similar behavior in vitro in all cell lines tested. The results obtained so far confirm fungal derived chitosan as an alternative to the animal derived chitosan. Nanogels carrying model protein antigen cargoes were taken up by porcine monocyte-derived dendritic cells, and kinetic analysis using an auto-quenched protein revealed a time-dependent endocytic processing of the nanogel cargo. These in vitro analyses were elaborated with an in vivo murine immunization experiment. Nanogels carrying ovalbumin (OVA) induced a dose-dependent antibody response over 6 weeks post-vaccination. An effective memory response was induced in nanogel-OVA immunized mice following a 0.1 µg OVA boost injection. Nanogels were also prepared carrying recombinant vaccine antigen against Neospora caninum. These induced a protective immune response following both intranasal and intraperitoneal vaccination in mice challenged with the tachyzoite form of the parasite. Overall, the results obtained in the framework of this research extend the knowledge on chitosan-based nanogels intended as nanocarriers for biomedical/pharmaceutical applications and contribute to further development of effective nanogel-based delivery systems.

Tissue engineering complex and mechanically active microenvironments for studies in asthma and cancer

Alice Anna Tomei

The goals of tissue engineering include recapitulating specific tissue functions for regenerative medicine and developing in vitro models of human tissues to study human physiology and pathophysiology and for testing and screening drugs before expensive clinical trials. Success of these goals relies on the capability to recapitulate the complex interactions between cells and their microenvironments in vitro, which requires interdisciplinary approaches. Tissue engineering has evolved greatly in the past two decades, with notable progress in replacing function in tissues such as cartilage, bone, arteries, skin, and pancreatic islets. What is lacking, however, are examples of, and design principles for, engineering complex microenvironments to understand disease progression. The two particular examples addressed in this thesis are (1) airway remodeling in asthma and (2) tumor invasion and metastasis in cancer. These diseases involve complex interactions between different cell types as well as extracellular matrix remodeling, which, together with the mechanical environment that affects cell-cell and cell-matrix interactions and that can change in a diseased state (or in some cases induce the disease state), collectively cause the pathology. Such disease states are difficult to study in vivo because (a) one cannot easily tease out mechanisms or have much control over these complex interactions in an animal model, and (b) there are many differences between animal and human cells. The aim of this thesis was to engineer mechanically dynamic microenvironments for the study of two disease processes: viral infection in asthmatic remodeled airways and cancer metastasis to the lymph node. These involved multidisciplinary approaches and drew tools and methods from mechanobiology, materials science, cell biology, and molecular biology. First, we developed and characterized a relevant 3D in vitro model of the bronchial mucosa with a functional epithelial barrier, including improved epithelial differentiation, ciliogenesis, and mucociliary function and showed for the first time that dynamic compression, such as that seen in asthmatic bronchoconstriction, enhances viral infection of epithelial and subepithelial cells using lentiviral technology and may help explaining why asthmatic patients are more susceptible to viral infection. However, the strain device had deficiencies in reproducibility, ease of use, time to assemble, and most importantly, homogeneous distribution of strain (because collagen and other natural extracellular matrices are mechanically weak since they have >99% water and cannot retain residual strain). To address these problems, we engineered a completely different dynamic strain device, which includes an elastic porous superstructure to contain the collagen and which can modulate stress/strain to the entire culture system. We characterized this device and showed that we could impose dynamic strains to 3D soft, hydrated cultures over relatively long periods of time and with variable amplitude and frequency in a uniform way. The device has the potential to be used in a wide spectrum of applications, including the possibility to study the correlation between mechanostimulation and metastatic potential of tumor cells in the context of lung metastasis of many cancer types. Finally, we used this material as a supporting structure for tissue-generated stress such as that generated by myofibroblasts, and in particular the ones that build up the lymph node stroma in the paracortex. The importance of these cells relies on their ability to form conduits for chemokine transport to high endothelial venules, where lymphocytes enter the node, the ability to form a platform for directing immune cell migration and interactions, and the ability to secrete two chemokines that are important to regulate lymphocyte adhesion and migration: CCL19 and CCL21. Specifically, we found that lymph node stromal cells use this supporting composite to remodel and reorganize their surrounding matrix into physiologically relevant bundles. By giving these cells the relevant stimuli (matrix microenvironment and flow), we could recapitulate certain features of the lymph node microenvironment, including a physiological stromal structure and chemokine profile, that may be critical for understanding immune and cancer cell interactions there. Using this model we demonstrated for the first time that slow flow through the lymph node stroma can itself drive cell and stromal organization and upregulates CCL21 production. Finally, we incorporated this engineered lymph node model into a larger model of the tumor-lymphatic-lymph node pathway to model the relative modulation of tumor growth, migration within a lymphatic "channel" towards the lymph node, and behavior within this lymph node environment. We used human lymphatic endothelial cells and a mass of transduced tumor cells with engineered chemokine profiles. We showed how CCR7 signaling correlates with the ability of tumor cells to metastasize to the lymph node and to interact with lymph node stromal cells once they reach the node. Our findings demonstrate how a multidisciplinary tissue engineering approach allows creating complex microenvironments in vitro that include both physical and chemical cues and allows recapitulating tissue function in vitro and investigating dynamic pathophysiological diseases like asthma and cancer metastasis. It combines both the development of these model systems and some examples of their usefulness to gain insight into asthma pathophysiology and cancer biology.

EPFL2008

Oxidative Stress on Human Cells in the Presence of Nano-Sized Titanium Dioxide

Katarzyna Pierzchala

Nanosized TiO2 (nanoTiO2) is one of the most widely used nanomaterials, with applications ranging from paints, self-cleaning coatings, pharmaceuticals, to food and cosmetics. In spite of this massive use of nanoTiO2, its biological activity and toxicity remains subject of the intense debate. In particular, there is still considerable uncertainty in the current understanding of the relationship between physico-chemical parameters of nanoTiO2, such as crystalline phase, size, aspect ratio, surface properties, surface defects and surface chemistry and its potential toxicological effects. Motivated by this general problem, this thesis provides a multidisciplinary experimental insight into to the toxicity and photo-toxicity of various forms of nanoTiO2. Firstly, since all of the widely accepted models of nanoTiO2 toxicity involve reactive oxygen species (ROS), electron spin resonance (ESR) in combination with spintrapping was used to measure ROS formation efficiency for the two most industrially important polymorphs of TiO2, anatase and rutile. The study was performed in an unprecedentedly broad range of particle sizes: 3.8 nm to 150 nm and 5 nm to 215 nm, for anatase and rutile nanoTiO2, respectively. Moreover, the photocatalytic and toxic properties of custom-made anatase-based TiO2 nanowires (with a diameter of 35 nm and a length of 0.5-1 µm) were characterized for the first time. For pure anatase nanoTiO2, the maximum ROS generation efficacy was found for nanoparticle sizes in the range of 25 – 30 nm. The ROS generation efficacy of the custom-made TiO2 nanowires was ca. 30% lower, being close to that of the commercial anatase nanoTiO2 with primary grain sizes of 5.3 nm. Secondly, this thesis addressed challenging, complex and still poorly understood processes occurring when nanoTiO2 particles are brought into contact with living cells. In particular, the comparative nanotoxicity study towards human melanoma Lu1205 and WM793 cell lines was performed for three selected nanomaterials: the custom-made anatase-based TiO2 nanowires, the commercial anatase nanoTiO2 with a primary particle size of 5.3 nm and similar in vitro ROS formation efficacy, and the industrial photocatalytic standard, P25 Degussa (a formulation consisting of 80% anatase and 20% rutile, with primary grain sizes of 25 nm). This comparative nanotoxicity study was performed using very low concentrations of nanoTiO2 (2 – 2.5 µg/mL), considerably lower than that applied in the majority of previous ex vivo cell culture studies. A wide palette of spectroscopic and microscopic methods was applied to verify the cellular response to the presence of selected nanoTiO2-based particles, both in the dark and under illumination with UV-A light of low intensity (λ = 365 nm, 1 mW/cm2). In particular, to detect intracellular ROS in cells incubated with the selected nanoTiO2, ESR spin-trapping with an intracellular spin-trap (ACP) and histochemical detection with a ROS-sensitive dye (NBT) were used. These studies were completed by optical microscopy visualization of ROS-induced changes in cell morphology and actin filament organization (FITC-phalloidin staining). Moreover, atomic force microscopy (AFM) was used to follow the changes in the cell topology and elastic properties (AFM force spectroscopy) of cells incubated with nanoTiO2 and exposed to photo-oxidative stress under UV-A illumination. All of these techniques revealed, either directly or indirectly, deleterious effects induced by incubation of living cells with nanoTiO2 and by photo-oxidative stress under illumination with UV-A. Primarily, optical microscopy visualization of selectively stained actin stress fibers and AFM topography images pointed to a rapid disorganization of the actin cortex for cells exposed to photo-oxidative stress in the presence of nanoTiO2. These results corroborated the changes in the cell shape and elasticity measured by AFM. The cell elasticity measurements, quantitatively described in terms of local Young's modulus values, pointed to an internal remodeling of the cytoskeleton in cells exposed to the presence of nanoTiO2 and photo-oxidative stress. The custom made anatase-based TiO2 nanowires revealed generally stronger cytotoxic effects than the anatase nanoTiO2 with a particle size of 5.3 nm, despite having similar photocatalytic properties in vitro. These observations were confirmed by AFM measurements of the cellular elasticity, histochemical detection with a ROS-sensitive dye (NBT) and by intracellular detection of ROS with ESR spin-trapping. The latter technique provided also a new important observation pointing to the enhanced ROS formation in cells incubated with nanoTiO2 in the dark. Overall, the findings stemming from cell toxicity studies were much more complex than those obtained from measurements of ROS generation efficacy in the presence of nanoTiO2 in vitro. In contrast to a marked evolution of ROS generation efficacy as a function of the primary grain size in vitro, the cellular work pointed to the lack of direct and simple correlations between the physical parameters of nanoTiO2, such as particles size, their aspect ratio, and aggregation state, and cell toxicity.

EPFL2010

Caged luciferin probes for the bioluminescence imaging of nitroreductase and hydrogen sulfide in living animals

Anzhelika Vorobyeva

Molecular imaging advances our understanding of cellular and molecular functions within complex biological systems. The development of novel imaging probes that can be applied to answer fundamental biological questions, diagnose and monitor disease progression and the efficacy of therapy in vivo is essential to the field of molecular imaging. Bioluminescence imaging is a powerful technique that allows to study and follow biological processes of interest noninvasively in real-time in living animals. Activatable bioluminescent probes can be designed according to the target of interest and can provide imaging signal in response to the specific stimuli. A strategy called "caged luciferin" can be used to design new bioluminescent probes for imaging of enzymatic activity, the presence of small molecules or metabolite uptake in vivo. In Chapter 1 we present an overview of bioluminescence imaging, with a focus on firefly luciferase-luciferin system. The caged luciferin approach is discussed and examples of existing caged luciferin probes are presented. In Chapter 2 we describe the development of a bioluminescent probe for imaging of bacterial nitroreductase activity and show the potential for its application in different areas of research. Bacterial nitroreductases have been widely utilized in the gene-directed enzyme prodrug therapy (GDEPT) approach for cancer therapy that reached clinical trials. However, both preclinical and clinical development of NTR-based GDEPT systems has been hampered by the lack of imaging tools that allow in vivo evaluation of transgene expression. We developed the NTR caged luciferin (NCL) probe and demonstrated its application for sensitive bioluminescence imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. We anticipate that this probe would significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where evaluation of NTR expression is important. In Chapter 3 we describe the development of a bioluminescent probe for imaging of hydrogen sulfide (H2S) using the caged luciferin approach. The objective of the research was to develop a probe that can detect endogenously produced H2S noninvasively in real-time in living animals. We present a series of caged luciferin probes that were evaluated on their ability to rapidly and selectively detect H2S in in vitro assays, in cells and in bacterial culture. The two selected probes were applied for imaging of exogenous H2S in the gastrointestinal tract in living mice. We anticipate further applications of the best performing probe (Az-CL) for imaging of H2S production by gut microbiota in mice. In Chapter 4 we present the overall conclusion followed by the outlook and future perspectives.

EPFL2016