Iron oxide nanoparticles for diagnosis and therapy of lymph node metastases

This thesis presents the synthesis, coating and characterization of iron oxide nanoparticles (IONPs) with properties especially designed for the ultimate simultaneous MRI detection and hyperthermia treatment of lymph node metastases, a combination called theranostics. This demanding aim requires to fulfill two different challenges at the same time: (i) synthesis of IONPs with exceptional properties for MRI and hyperthermia obtained with a synthesis method transferable to manufacturing facilities, and (ii) coating of IONPs with molecules, which simultaneously allow their dispersion and ideally have higher affinity to lymph node metastases than to other tissues. We therefore developed a novel simple aqueous method to synthesize IONPs by combining the standard co-precipitation method with a hydrothermal treatment. We studied the influence of the synthesis parameters, especially the hydrothermal treatment duration, on the IONPs' size, morphology, structure, crystallinity, as well as their magnetic, relaxometric and heating properties. As a result, we were able to control simultaneously the studied properties and to obtain IONPs with optimal properties for both MRI and hyperthermia. Subsequently, IONPs were coated in aqueous conditions with nine different molecules considered as biocompatible and non-toxic. In order to preserve the high relaxometric and heating properties of IONPs, we used small molecules to reduce the coating to a minimum. In that case, dispersion of IONPs with the chosen molecules were obtained by electrostatic stabilization. One of the major constraints was to obtain coated IONPs with hydrodynamic diameters smaller than 100 nm and with negative surface charges, both needed to access and be retained in the lymphatic system that is required for the targeting of lymph node metastases. In order to avoid to further enlarge the coating, we also used the available active groups of the coating molecules to target lymph node metastatic cells. In order to confirm the possibility to target such cells, we investigated the uptake of coated IONPs in four tumor cell lines from different cancer stages. Finally, the platform with optimal properties necessary for the targeting, detection and treatment of lymph node metastases was found to be IONPs coated with folic acid or adenosine triphosphate, which are targeting the prostate specific membrane antigen receptors or the highly metabolic nature of tumor cells, respectively. In the last part of this thesis, the interaction of IONPs with blood and lymph, especially the formation of the protein corona around IONPs, was investigated. Our study took into account the complex environment, which IONPs are subjected to in vivo. So far unconsidered in vitro conditions were examined, such as the dynamic variations of the flow or the transition of IONPs from one body fluid to the other.

Biophysical Regulation of Lymphatic Vessel Function

Dimana Miteva

Lymphatic capillaries collect interstitial fluid and dendritic cells from the periphery and deliver them to the lymph nodes for immune surveillance and tolerance maintenance. Upon injury and inflammation, lymphatic drainage can increase rapidly, and thus the lymphatic capillaries experience a broad range of fluid stresses and need to respond rapidly to such changes. Our understanding of their functional biology, adaptive ability and response to pathological changes in the biophysical environment is still very limited. This thesis focuses on how the lymphatic capillary endothelium senses and responds to changes of its biophysical environment with respect to its function, including fluid and cell transport, in the context of physiological and pathophysiological conditions. Using in vitro and in vivo models, we demonstrate that lymphatic endothelium is exquisitely sensitive to transmural flow, modulating both fluid and cell transport functions even in the absence of inflammatory cytokines. Flow increased lymphatic permeability and dendritic cell (DC) transmigration, which were coincident with changes in gene and protein expression of factor known to be involved in these, including chemoattractant chemokines, adhesion molecules and junctional proteins. These data provide the first evidence that lymphatic endothelium can regulate its transport functions in response to flow cues. They also show for the first time that transmural flow can, in the absence of inflammatory cues like TNF-a, drive expression of DC adhesion molecules like ICAM-1 and chemokines like CCL21 by lymphatic endothelium. Based on these findings we suggest that lymphatic flow is an important mediator of lymphatic function, particularly with respect to DC recruitment and trafficking to the lymph node. Furthermore, the response of the lymphatic endothelium to flow may be specific to the type of low, as lymphatic endothelial cells (LECs) might react differently to luminal shear flow, at physiological and pathophysiological levels, compared to transmural flow. To address this we used laminar shear stress chambers to expose only the apical surface of the lymphatic endothelial cells (LECs) to shear stress and demonstrated that lymphatic endothelium is indeed sensitive to luminal flow, fine-tuning its expression of adhesion molecules to modulate DC adhesion according to the shear stress. Specifically, at extremely low shear, adhesion was enhanced, unlike at higher shear, adhesion was downregulated. This is consistent with the notion that DC adhesion to LECs is only needed for entry but not transport in conducting vessels, since only the absorbing capillaries have low shear stresses. The interactions between DCs and LECs were strongly mediated by CCL21 and its receptor CCR7. These findings suggest that luminal shear stress acts as an active mechanosignal in LECs to potentially facilitate the traffic of immune cells inside of the lymphatic vessel to reach the lymph node and trigger an immune response. Additionally, we demonstrated that lymphatic vessels respond differentially to immunogenic and tolerogenic inflammatory stimulus. We used in vitro models to evaluate lymphatic uptake and lymphatic participation in dendritic cell transmigration in the presence of various inflammatory stimuli. We found that lymphatic endothelium exposed to TNF-a or complement activation were more conductive to dendritic cell transmigration, while LPS and TGF-β had opposite effect, affecting DCs to decrease DC transmigration. We further presented some insights in similarities in tumor and dendritic cells intravasation into inflamed lymphatic capillaries. These findings imply that lymphatic activation and participation in immune cells transport is not a universal phenomenon, but is strongly dependent on the nature of the inflammation. In conclusion, this work highlights the critical role of biophysical environment in lymphatic endothelial function, and how exquisitely sensitive the lymphatic capillaries are to their extracellular environment and how they use multiple cues to sense inflammation and danger, modulating their transport functions accordingly to regulate the delivery of antigens and immune cells to the lymph nodes.

EPFL2010

Engineering of chitosan-based derivatives for non-viral gene delivery

Laura Camille Louise Nicolle

Gene therapy offers the possibility to treat or even cure diseases originating from genetic defects by introducing a therapeutic gene in target cells or correcting the initial defective gene. Amongst different options, non-viral vectors rely on the delivery of such genes using vehicles composed of lipids, inorganic nanoparticles or polymers. These systems are designed to protect the genetic material, while efficiently delivering it to the cells of interest. The initial backbone can be complemented with various components to help it overcome the many physiological barriers it may encounter during its journey. Amongst polymers, chitosan is a polysaccharide presenting many advantages for such purpose. Besides its biocompatibility and biodegradability, its potential for functionalization, thanks to numerous primary amines and hydroxyl groups, make it a backbone of choice for the preparation of a polymeric delivery vehicle. This work describes the covalent functionalization of chitosan with other polymers, peptides, and small molecules to later form polymeric nanoparticles condensing therapeutic DNA. Each component brings additional features to the initial system, thus finely tuning it for biological applications. The final system aims at treating liver inherited diseases such as ornithine transcarbamylase deficiency and phenylketonuria.This manuscript first exposes the preparation of a chitosan-based cationic system able to properly condense anionic DNA through electrostatic interactions. Due to its excellent DNA condensation properties, polyethylenimine was grafted to chitosan via a succinyl linker. The first in vitro investigations confirmed the potential of the resulting core system, but in vivo experiments highlighted toxicity coming from the remaining cationic charge on the nanoparticles. In order to better shield this cationic charge, bifunctional polyethylene glycol chains terminated by carboxylic acid moieties were grafted to chitosan amines. The resulting polymer was used to coat the first polymeric system through electrostatic interactions between the carboxylate groups and the remaining free amines of the chitosan-polyethylenimine derivative. The newly formed core-shell complex showed sustained cell viability in vitro and induced higher transfection efficiency of the gene as compared to the core system alone. However, its in vivo evaluation via retrograde intrabiliary infusion did not confirm such results: although transfection efficiency was retained, the core-shell complex did not significantly decrease the toxicity previously reported.The last part of this work includes the decoration of the core-shell system with targeting ligands making possible to perform in vivo assays via intravenous injection, for which the developed polymeric systems did not induce toxicity in preliminary experiments. So far, only a proof-of-concept with folic acid, an anti-cancer targeting ligand, was achieved. To do so, the shell polymer was further functionalized with folic acid-derived polyethylene glycol chains through strain-promoted azide-alkyne cycloaddition. The same strategy shall be applied in the near future with a liver-specific targeting ligand. Lastly, the improvement of the system's cell-penetration features was tackled by the grafting asparagusic acid-derived polyethylene glycol chains following the same strategy than for folic acid.

EPFL2022

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